ph20 polypeptide variants, formulations and uses thereof

专利摘要:
  PH20 POLYPEPTIDE VARIANTS, FORMULATIONS AND USES OF THE SAME.Modified PH20 hyaluronidase polypeptides, including polypeptides that exhibit increased stability and / or increased activity, are provided. Arecompositions and formulations and uses thereof are also provided.
公开号:BR112014016195A2
申请号:R112014016195-0
申请日:2012-12-28
公开日:2020-10-27
发明作者:Ge Wei；H. Michael Shepard；Qiping Zhao；Robert James Connor
申请人:Halozyme, Inc.；
IPC主号:

专利说明:

[0001] [0001] Priority benefit is claimed in US provisional order no. 61 / 631,313, filed on December 30, 2011, and to provisional application US no. 61 / 796,208 filed on November 1, 2012, each entitled “PH Polypeptide variants, formulations and uses thereof.”
[0002] [0002] This order relates to US order number 13 / 684,731, filed on the same day as the present, entitled “Polypeptide PHβ0 variants, formulations and uses thereof,” which claims priority to US provisional order no. 61 / 631,313 and to provisional application US no. 61 / 796,208.
[0003] [0003] Where permitted, the subject of each of the aforementioned related requests is incorporated by reference in its entirety. INCORPORATION BY LISTING REFERENCE OF SEQUENCES PROVIDED ELECTRONICALLY
[0004] [0004] An electronic version of the Sequence Listing is deposited with the present, the content of which is incorporated as a reference in its entirety. The electronic file was created on December 28, 2012, has a size of 3.48 megabytes and is entitled 3087seqPC1.txt. FIELD OF THE INVENTION
[0005] [0005] Modified PH20 hyaluronidase polypeptides, including modified polypeptides that exhibit increased stability and / or increased activity, are provided. Compositions and formulations and uses are also provided. BACKGROUND
[0006] [0006] Hyaluronan (hyaluronic acid; HA) is a polypeptide that is found in the extracellular matrix of many cells, especially in soft connective tissues. HA is also found predominantly in skin, cartilage and synovial fluid in mammals. Hyaluronan is also the main constituent of the vitreous of the eye. HA has a role in several physiological processes, such as plasma and water protein homeostasis (Laurent TC et al. (1992) FASEB J 6: 2397-2404). Certain diseases are associated with the expression and / or production of hyaluronan. Hyaluronan-degrading enzymes, such as hyaluronidases, are enzymes that degrade hyaluronan. By catalyzing the degradation of HA, enzymes that degrade hyaluronan (eg, hyaluronidases) can be used to treat diseases or disorders associated with the accumulation of HA or other glycosaminoglycans. Also, since HA is a major component of the interstitial barrier, enzymes that degrade hyaluronan (e.g., hyaluronidase) increase tissue permeability and therefore can be used to increase the dispersion and delivery of therapeutic agents. Several hyaluronidases have been used therapeutically (for example, Hydase ™, Vitrase ™ and Wydase ™), typically as dispersing and spreading agents in combination with other therapeutic agents. Many of these are ovine or bovine forms, which can be immunogenic for the treatment of humans. Enhanced hyaluronan-degrading enzymes, such as hyaluronidases and compositions thereof that can be used for treatment are necessary. SUMMARY
[0007] [0007] Modified PH20 polypeptides which have an altered property or properties compared to PH20 polypeptide which do not have the modification (s) are provided. The modifications include substitution, deletion and / or amino acid insertions. The detailed function / structure of virtually each amino acid in a PH20 polypeptide is provided here, as well as the identification of residues and locations that contribute to altering a property, such as stability under specific conditions, is provided. Consequently, modified PH20 polypeptides are provided that contain one or more amino acid substitutions that result in a PH20 polypeptide that retains activity and / or has increased or altered stability under a variety of conditions. The retained activity can be, for example, hyaluronidase activity which is at least approximately 40% or more of the PH20 polypeptide which does not include substitution. Exemplary modifications are amino acid substitutions. For the purposes of the present invention, amino acid substitutions are indicated by the single amino acid letter followed by the corresponding amino acid position in SEQ ID NO: 3 in which the substitution occurs. Unique amino acid abbreviations for residues are well known to a skilled person (see, for example, table 1) and are used here from beginning to end of the description and examples. For example, substitution with P in a position corresponding to position 204 in a PH20 polypeptide with reference to amino acid residue positions exposed in SEQ ID NO: 3 means that the substitution covers P204P in a PH20 polypeptide exposed in SEQ ID NO: 3 or the same substitution in the corresponding position in another PH20 polypeptide.
[0008] [0008] Modified PH20 polypeptides containing at least one amino acid substitution in a PH20 polypeptide are provided, whereby the modified PH20 polypeptide exhibits increased stability compared to PH20 polypeptide not containing the amino acid substitution. Increased stability can be manifested as increased resistance to one or more protein conditions that are denaturing to proteins. The stability of modified and unmodified PH20 is comparable under the same conditions. Exemplary protein denaturation conditions (or denaturing, used interchangeably here) include, but are not limited to, elevated temperature, greater than 30 ° C or approximately 30 ° C, agitation, low salt content, including essentially or substantially or no salt, and presence of excipients that tend to denature proteins. Examples of such non-stick excipients (s), binders (s), coating (s), filler (s) and diluent (s), aroma (s), color (s), lubricant (s), glidant (s), condom (s), detergent (s), sorbent (s) and combinations thereof.
[0009] [0009] The modified PH20 polypeptide may be one in which the unmodified form thereof has at least approximately 68% sequence identity to SEQ ID NO: 3 and further contains modifications that alter stability and / or may be a PH20 polypeptide that includes as much as approximately 100, 110, 120, 130, 150 amino acid differences from PH20 but retains enzymatic activity, particularly at least approximately 40% of the activity of the unmodified PH20 polypeptide and has increased stability, such as stability under denaturation conditions. Thus, modified PH20 polypeptides are included that have at least 68% or approximately 68% amino acid sequence identity to the amino acid sequence exposed in SEQ ID NO: 3. Modified PH20 polypeptides that are at least 70% are included, 75 %, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to the amino acid sequence exposed in SEQ ID NO: 3. Examples of such modified PH20 polypeptides are polypeptides that contain amino acid substitution (s) in a PH20 polypeptide that contains the amino acid residue sequence as set out in any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32 -66, 69, 72, 857, 859, 861, 870 or an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857 , 859, 861, or 870.
[00010] [00010] For example, a modified PH20 polypeptide is provided here which exhibits increased stability containing an amino acid substitution in a PH20 polypeptide which confers increased stability, where the increased stability is manifested as increased resistance to denaturation in the presence of one or more protein denaturation conditions, stability is increased compared to PH20 polypeptide not containing amino acid substitution, and unmodified PH20 polypeptide consists of the sequence of amino acids exposed in SEQ ID NOS: 7 or is a C-terminal truncated fragment of even if it is a soluble PH20 polypeptide or has at least 85% sequence identity to it. As above, the modified PH20 polypeptide that exhibits increased stability exhibits increased stability at a denaturation condition that is temperature greater than or approximately 30 ° C; agitation; low salt content or no salt; or presence of an excipient or denaturing agent, such as a non-stick (s), binder (s), coating (s), filler (s) and diluent (s), flavor (s), color (s), lubricant ( s), glidante (s), preservative (s), detergent (s), sorbent (s) or sweetener (s) and a combination thereof, and in particular a condom. In some examples of such modified PH20 polypeptides that have increased stability, the denaturation condition is higher than 30ºC, and the modified PH20 polypeptide has higher hyaluronidase activity at temperature compared to unmodified PH20 polypeptide containing no (s) amino acid substitution (s) where activities are compared under the same conditions. In other examples, the protein denaturation condition is the presence of low salt concentrations less than 100 mM, and the modified PH20 polypeptide exhibits hyaluronidase activity in the presence of low salt concentrations compared to the unmodified PH20 polypeptide containing ( s) amino acid substitution (s) where activities are compared under the same conditions.
[00011] [00011] In any of the above examples of a modified PH20 polypeptide that exhibits increased stability, stability can be assessed based on a variety of parameters including hyaluronidase activity, solubility, aggregation and / or crystallization. Stability can be assessed in the presence of a denaturation condition. When stability of two or more polypeptides is compared, stability is assessed under the same conditions. In some cases, among the PH20 polypeptides provided here, the modified PH20 polypeptide has at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250% , 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% or more of the PH20 polypeptide hyaluronidase activity not containing the amino acid substitution (s) ).
[00012] [00012] In any of the above examples of a modified PH20 polypeptide that exhibits increased stability, denaturation conditions include the presence of excipients that denature proteins.
[00013] [00013] In examples of modified PH20 polypeptide that exhibits increased stability to a phenolic condom, increased stability in a phenolic condom can be presented under temperature conditions that include any temperature between, for example, 0 ° C to 40 ° C, as between or approximately between 0 ° C to 40 ° C, 2 ° C to 6 ° C, 24 ° C to 32 ° C and 35 ° C to 40 ° C. Exemplary polypeptides have increased stability at temperatures between or approximately between ° C to 45 ° C, 35 ° C to 45 ° C, 30 ° C to 37 ° C, 35 ° C to 37 ° C or 37 ° C to 42 ° C, each inclusive. The specific modified PH20 polypeptide and conditions depend on the intended formulation, conditions to which the formulation will be exposed and / or intended applications.
[00014] [00014] Exemplary and specific modified PH20 polypeptides that exhibit increased stability, such as increased stability to a phenolic condom, include those that contain a single amino acid modification, as a substitution, and combinations of modifications, such as at least or 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50,
[00015] [00015] Thus, modified PH20 polypeptides that have increased stability in the presence of a phenolic preservative containing an amino acid substitution in a PH20 polypeptide that gives increased stability are provided here, in unmodified PH20 polypeptide has the sequence of amino acids exposed in SEQ ID NO: 7 is either a C-terminal truncated fragment thereof which is a soluble PH20 polypeptide or has at least 85% sequence identity thereto.
[00016] [00016] Among modified PH20 polypeptides provided here that exhibit increased stability are those that exhibit increased hyaluronidase activity at elevated temperature compared to PH20 polypeptide not containing the amino acid substitution (s), such as at least 110%, 120% , 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% or more of hyaluronidase activity for at least 4 hours compared to PH20 polypeptide not containing the amino acid substitution (s). Also among the polypeptides are those that have activity, but they also typically have increased stability or other property at elevated temperatures, such as a modified PH20 polypeptide that has at least 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300%, 400%, 500% of hyaluronidase activity for at least 4 hours at a temperature between or approximately 32 ° C to 37 ° C compared to the hyaluronidase activity of the PH20 polypeptide modified at a temperature between or approximately 2 ° C to 8 ° C, where activity is compared with the same conditions except for differences in temperature .
[00017] [00017] Modified PH20 polypeptides are also provided which have increased stability in low salt conditions, such as, for example, NaCl concentrations less than 100 mM, but not limited to NaCl concentrations less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. Among PH20 polypeptides, hyaluronidase activity increased at lower salt concentrations compared to PH20 polypeptide not containing the amino acid substitution (s). Such activity includes, for example, at least more than 100%, or at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 300% , 400%, 500% or more hyaluronidase activity compared to PH20 polypeptide not containing the amino acid substitution (s). Examples of such modified PH20 polypeptides are those that exhibit at least 60% of hyaluronidase activity in low salt concentrations, such as between or approximately between 10 mM NaCl and 100 mM NaCl, inclusive (or comparable concentrations of other salts or mixtures of salts) , in comparison with the hyaluronidase activity of the PH20 polypeptide modified in 150 mM NaCl, where activities are compared under the same conditions except for the difference in salt concentration. In specific examples provided here, any such modified PH20 polypeptides contain a single amino acid modification, such as a substitution and combinations of modifications such as at least or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications. The modification, as a substitution, can be on an unmodified PH20 polypeptide that has the amino acid sequence exposed in SEQ ID NO: 7 or is a C-terminal truncated fragment of it that is a soluble PH20 polypeptide as exposed in any of the SEQ ID NOS: 3 or 32- 66, or have at least 85% sequence identity to the same. For example, any of such modified PH20 polypeptides have at least 85% sequence identity with SEQ ID NO: 3.
[00018] [00018] Modified PH20 polypeptides that contain at least one amino acid substitution in a PH20 polypeptide are also provided, where the modified PH20 polypeptide has increased hyaluronidase activity compared to PH20 polypeptide not containing the amino acid substitution. When comparing activity between polypeptides, activity is compared with the same conditions. Among these are polypeptides, where unmodified PH20 has at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity of the amino acids exposed in SEQ ID NO: 3, or the resulting modified PH20 shows such sequence identity with the amino acid sequence exposed in SEQ ID NO: 3. Examples of such modified PH20 polypeptides are any containing an amino acid substitution (s) in the amino acid sequence exposed in any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32-66, 69, or 72, or an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% identical to any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32-66, 69, or 72. Amino acid substitution (s) can also be done in the amino acid sequence exposed in any of SEQ ID NOS: 857, 859, 861 or 870, or an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any of SEQ ID NOS: 857, 859, 861 or 870. In particular, modified PH20 polypeptides containing a substitution are provided of amino acid in the amino acid sequence exposed in SEQ ID NOS: 3, 7, 32-66, 69 or
[00019] [00019] Among the polypeptides that have increased hyaluronidase activity are those that have at least 2.0 times the hyaluronidase activity of the PH20 polypeptide that does not contain amino acid substitution. For example, these include modified PH20 polypeptides that contain at least one amino acid substitution at an amino acid position corresponding to a position selected from 24, 29, 31, 48, 58, 69, 70, 75, 84, 97, 165, 166, 271, 278, 317, 320, 325 and 326 with reference to positions exposed in SEQ ID NO: 3, where corresponding amino acid positions are identified by aligning PH20 polypeptide with the polypeptide exposed in SEQ ID NO: 3, as modified PH20 polypeptides containing at least one amino acid substitution selected from substitution with: E in a position corresponding to position 24; And in a position corresponding to position 29; V in a position corresponding to position 31; N in a position corresponding to position 48; K in a position corresponding to position 58; Q in a position corresponding to position 58; A in a position corresponding to position 69; F in a position corresponding to position 69; G in a position corresponding to position 69; P in a position corresponding to position 69; R in a position corresponding to position 69; A in a position corresponding to position 70; F in a position corresponding to position 70; G in a position corresponding to position 70; H in a position corresponding to position 70; H in a position corresponding to position 70; N in a position corresponding to position 70; R in a position corresponding to position 70; T in a position corresponding to position 70; V in a position corresponding to position 70; L in a position corresponding to position 75; T in a position corresponding to position 75; G in a position corresponding to position 84; G in a position corresponding to position 97; D in a position corresponding to position 165; L in a position corresponding to position 166; R in a position corresponding to position 166; T in a position corresponding to position 166; L in a position corresponding to position 271; H in a position corresponding to position 278; R in a position corresponding to position 278; K in a position corresponding to position 317; K in a position corresponding to position 320; And in a position corresponding to position 325, with G in a position corresponding to position 325; K in a position corresponding to position 325; N in a position corresponding to position 325; Q in a position corresponding to position 325; and V in a position corresponding to position 326; with reference to amino acid positions exposed in SEQ ID NO: 3.
[00020] [00020] Among any of the polypeptides provided here that exhibit hyaluronidase activity, any such modified PH20 polypeptides contain a unique amino acid modification, as a substitution and combinations of modifications such as at least or 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 and more modifications. The modification, as a substitution, may be in an unmodified PH20 polypeptide that has the amino acid sequence exposed in SEQ ID NO: 7 or is a C-terminal truncated fragment of it that is a soluble PH20 polypeptide, as exposed in any of the SEQ ID NOS: 3 or 32-66, or has at least 85% sequence identity to it. For example, any of such modified PH20 polypeptides have at least 85% sequence identity to SEQ ID NO: 3.
[00021] [00021] Modified PH20 polypeptides containing at least one amino acid substitution in the PH20 polypeptide whose sequence is set out in SEQ ID NO: 7, a C-terminal truncated fragment thereof, a soluble fragment thereof, or in a PH20 polypeptide that has an amino acid sequence that is at least 91% identical to the amino acid sequence exposed in SEQ ID NO: 7, where at least one amino substitution (s) is at an amino acid position corresponding to a position selected from 1 , 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 58, 59, 60, 61, 63 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 77, 79, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93 , 94, 96, 97, 98, 99, 102, 103, 104, 105, 106, 107, 108, 110, 114, 117, 118, 119, 120, 122, 124, 125, 127, 128, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 1 43, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 186, 192, 193, 195, 196, 197, 198, 200, 202, 204, 205, 206, 208, 209, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 224, 226, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 242, 245, 247, 248, 251, 253, 255, 256, 257, 258, 259, 260, 261, 263, 264, 265, 266, 267, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 297, 298, 300, 301, 302, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 323, 324, 325, 326, 327, 328, 331, 334, 335, 338, 339, 342, 343, 347, 348, 349, 351, 353, 356, 357, 358, 359, 360, 361, 367, 368, 369, 371, 373, 374, 375, 376, 377, 378, 379, 380, 381, 383, 385, 387, 388, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 401, 403, 404, 4 05, 406, 407, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 425, 426, 427, 428, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 and
[00022] [00022] Included among these polypeptides are those that contain an amino acid substitution in the amino acid sequence exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 and 72, or in an amino acid sequence that has at least 91 % sequence identity to any of SEQ ID NOS: 3, 7, 32-66, 69, or
[00023] [00023] In particular, modified PH20 polypeptides that contain at least one amino acid substitution are provided in a PH20 polypeptide exposed in SEQ ID NO: 7, a C-terminal truncated fragment thereof, or in a PH20 polypeptide that has a sequence of amino acids that is at least 91% identical to the amino acid sequence exposed in SEQ ID NO: 7 or a corresponding truncated fragment, where: the modified PH20 polypeptides have less than 20% of the hyaluronidase activity of the PH20 polypeptide without containing the amino acid substitution, where activities are compared with the same conditions; the amino acid substitution (s) is (are) in an amino acid position corresponding to a position selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 98 , 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 121, 122, 123, 124 , 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 143, 144, 145, 149, 150, 152, 153, 154, 155, 156 , 157, 158, 159, 161, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 178, 179, 180, 181, 182, 183, 184 , 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326,
[00024] [00024] (i) if the modified PH20 polypeptide contains an amino acid substitution in a position corresponding to position 200, 333, 358 or 393 the substitution is not substitution with an Alanine (A).
[00025] [00025] (ii) if the modified PH20 polypeptide contains an amino acid substitution in a position corresponding to position 111 or 249 the substitution is not a substitution with an asparagine (N);
[00026] [00026] (iii) if the modified PH20 polypeptide contains an amino acid substitution in a position corresponding to position 113 the substitution is not a substitution with a glutamine (Q);
[00027] [00027] (iv) if the modified PH20 polypeptide contains an amino acid substitution in a position corresponding to position 176 the substitution is not substitution with a glycine (G); and
[00028] [00028] (v) if the modified PH20 polypeptide contains an amino acid substitution in a position corresponding to position 252 the substitution is not a substitution with a threonine (T).
[00029] [00029] Examples of such modified PH20 polypeptides are any containing amino acid substitution (s) in a PH20 polypeptide that has the amino acid sequence exposed in any of SEQ ID NOS: 3, 7, 32-66, 69, or 72, or in an amino acid sequence that has at least 91% sequence identity to any of SEQ ID NOS: 3, 7, 32-66, 69, or 72. For example, the modified PH20 polypeptide contains amino acid substitution (s) in SEQ ID NOS: 3, 7, 32-66, 69, or 72, which are polypeptides that are a C-terminal truncated fragment of SEQ ID NO: 7 or a PH20 polypeptide that has an amino acid sequence that is at least 91% identical to the amino acid sequence shown in SEQ ID NO: 7. In the examples of such modified PH20 polypeptides provided here, the modified PH20 polypeptides may exhibit activity similar to or equal to PH20 without modification, or may exhibit increased activity or activity that is less than 15%, 10%, 9%, 8% , 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4 %, 0.3%, 0.2%, 0.1%, 0.05% or less than the hyaluronidase activity of the PH20 polypeptide not containing the amino acid substitution. Examples of such modified PH20 polypeptides are any shown in Table 5.
[00030] [00030] Among all and any of the modified PH20 polypeptides provided here and above, the modified PH20 polypeptide is one that does not consist of the amino acid sequence exposed in any of SEQ ID NOS: 3, 6-66, 69-72, 856-861 , 869 or 870. In particular, among any of the modified PH20 polypeptides provided here above or elsewhere in the present invention are any that contain an amino acid substitution (s) in a PH20 polypeptide having the amino acid sequence exposed in any of the SEQ IDs NO: 3, 7, 69 or 72 with the proviso that: (i) where the modified PH20 polypeptide includes only a single amino acid substitution, the substitution does not correspond to amino acid substitutions V12A, N47A, D111N, E113Q, N131A, R176G, N200A, N219A, E249Q, R252T, N333A or N358A, with reference to amino acid positions exposed amino acid positions exposed in SEQ ID NO: 3; (ii) where the modified PH20 polypeptide includes only two amino acid substitutions the substitutions do not correspond to amino acid substitutions P13A / L464W, N47A / N131A, N47A / N219A, N131A / N219A or N333A / N358A with reference to positions exposed in SEQ ID NO : 3; and (iii) where the modified PH20 polypeptide includes only three amino acid substitutions, the substitutions do not correspond to N47A / N131A / N219A amino acid substitutions, with reference to amino acid positions exposed in SEQ ID NO: 3.
[00031] [00031] Any of the modified PH20 polypeptides above and any provided here and described above and below may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or more of the amino acid substitutions. The modified PH20 polypeptides can include a signal sequence, including the native sequence or a heterologous sequence or a modified sequence, and also include a mature PH20 polypeptide that lacks the signal sequence.
[00032] [00032] Among any of the modified PH20 polypeptides provided here above or described below are modified PH20 polypeptides that contain or have the amino acid sequence exposed in any of SEQ ID NOS: 73-855 or an amino acid sequence that has at least 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity for an amino acid sequence exposed in any of SEQ ID NOS: 73-855 and which contains at least one amino acid substitution, as described above or elsewhere herein, with reference to positions compared to the amino acid sequence set out in SEQ ID NO: 3. In any of the examples of the modified PH20 polypeptides provided here, the modified PH20 polypeptide does not have or contain the amino acid sequence exposed in any of SEQ ID NOS: 8-31, 69-72, 856-861, 869 or 870.
[00033] [00033] The modified PH20 polypeptides provided here can be substantially purified or isolated, can show neutron pH catalytic activity, can be secreted after expression from cells and are soluble in the supernatant, and / or can include modified amino acids, such as a modification selected from glycosylation, sialization, albumination, farnisylation, carboxylation, hydroxylation, conjugation with a polymer such as PEGylation or conjugation with dextran, conjugation with another fraction, such as a multimerization domain, toxin, label or detectable drug, and phosphorylation. The modified PH20 polymer can be glycosylated, as it contains at least a fraction of N-acetyl glucosamine attached to each of at least three asparagine residues (N) where, for example, the three asparagine residues correspond to amino acid residues 200, 333 and 358 of SEQ ID NO: 3. Multimerization domains include Fc domains.
[00034] [00034] Nucleic acid molecules that encode any of the modified PH20 polypeptides provided here are also provided. Vectors, eukaryotic and prokaryotic, which contain the nucleic acid molecules are provided. The vectors include expression vectors and include mammalian vectors including viral vectors. Viral vectors include adenovirus vectors, retrovirus vectors, vaccinia virus vectors, herpes simplex viruses and cytomegalovirus vectors and other such viral vectors. Of interest are oncolytic vectors that accumulate in or are targeted to tumors. Cells containing the nucleic acid molecules and cells containing the vectors are also provided. The cells can be prokaryotic or eukaryotic, particularly mammalian cells, such as Chinese hamster ovary (CHO) cells.
[00035] [00035] A modified PH20 polypeptide is also provided that is produced by any of the cells provided. Accordingly, methods are provided here for producing a modified PH20 polypeptide by cultivating any of the cells provided here under conditions whereby a modified PH20 encoded polypeptide is produced and secreted by the cell, and recovering the expressed polypeptide. Also provided here is a method of producing a modified PH20 polypeptide by introducing any of the nucleic acids provided here or any of the vectors provided here into a cell capable of incorporating N-linked sugar fractions into the polypeptide, cultivating the cell with conditions whereby a polypeptide Modified encoded PH20 is produced and secreted by the cell, and retrieves the expressed polypeptide. In such examples, the nucleic acid is operably linked to a promoter. The cultured cell can be a eukaryotic cell, such as a mammalian cell, for example, a Chinese hamster ovary (CHO) cell.
[00036] [00036] Compositions are also provided that contain any of the modified PH20 polypeptides provided here or any of the nucleic acids or vectors provided here. The compositions can be formulated with other agents and / or with other components, such as preservatives. The compositions can be formulated so that the components, particularly the PH20 and any other active agent, remain active or are stable under pre-selected conditions. In addition, as described here, PH20 polypeptides are modified so that they exhibit increased stability under various conditions. For example, compositions are provided in which the modified PH20 polypeptide is stable (ie, retains activity as described here) at a temperature of or approximately 2 ° C to 8 ° C inclusive, for at least 1 month or is stable at a temperature of or approximately 30ºC to 42ºC, inclusive, for at least 3 days. Compositions are provided here in which the PH20 polypeptide modified in the composition is stable at a temperature of or approximately 2ºC to 8ºC, including for at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, months, 26 months, 27 months, 28 months, 29 months or 30 months. Compositions are also provided in which the PH20 polypeptide modified in the composition is stable at a temperature of or approximately 30 ° C to 42 ° C, including, for at least 3 days, at least 4 days, 5 days, 6 days, 7 days , 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days or more. The pharmaceutical compositions can contain a pharmaceutically acceptable excipient.
[00037] [00037] The conditions, formulations, components and modified PH20 polypeptide are chosen to obtain a desired stability. Pharmaceutical compositions can be formulated for direct administration or may require dilution. They can be formulated for administration in single or multiple dosage. Exemplary compositions include concentrations of modified PH20 between or approximately between 0.1 µg / mL and 100 µg / mL, 1 µg / mL and 50 µg / mL or 1 µg / mL and 20 µg / mL, or 10 U / mL and 5000 U / ml, 50 U / ml and 4000 U / ml, 100 U / ml and 2000 U / ml, 300 U / ml and 2000 U / ml, 600 U / ml and 2000 U / ml, or 100 U / ml and 1000 U / ml. Exemplary salts include NaCl in a concentration, for example, less than or approximately or 200 mM, 180 mM, 150 mM, 140 mM, 130 mM, 120 mM, 110 mM, 100 mM, 90 mM, 80mM, 70mM, 60 mM , 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less, or between or approximately between 0.1 mM and 200 mM, 0.1 mM and 100 mM, 120 mM and 200 mM, 10 mM and 50 mM, 10 mM and 90 mM, 80 mM and 200 mM, 80 mM and 140 mM, 50 mM and 100 mM, 80 mM and 100 mM, 50 mM and 80 mM, 100 mM and 140 mM or 120 mM and 140 mM.
[00038] [00038] Pharmaceutical compositions may contain an antimicrobial effective amount of a condom or mixture of condoms, such as one, two, three, fourth or more of a phenolic condom (s), a non-phenolic condom (s) ) or a phenolic condom (s) and a non-phenolic condom (s) such as, but not limited to, phenol, m-cresol, methylparaben, benzyl alcohol, thimerosal, benzalkonium chloride, 4-chlorine -1-
[00039] [00039] The pharmaceutical compositions may contain an additional therapeutically active agent. The active agent can be formulated in the composition or supplied as a combination with the composition containing PH20, but in a separate composition for administration separately, sequentially, intermittently, simultaneously or together. Therapeutically active agents include, for example, an agent selected from a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amebicidal agent, a trichomonic agent, an anti-parkinson agent, an antimalarial agent, an anticonvulsant, an antidepressant agent, an antiarthritic agent, an antifungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasitic agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocking agent, an alpha blocking agent anesthetic, a bronchial dilating agent, a biocidal agent, a bactericidal agent, a bacteriostatic agent, a beta-adrenergic blocking agent, a calcium channel blocking agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent , a depressant agent, a diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone agent, a h iperglycemic, muscle relaxant, muscle contraction agent, ophthalmic agent, parasympathomimetic agent, psychic energizing agent, sedative agent, sympathomimetic agent, tranquilizer, urinary agent, vaginal agent, viricidal agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibiting agent, a polypeptide, a protein, a nucleic acid, a drug, an organic molecule and a sleep inducer.
[00040] [00040] In particular, a pharmaceutical composition containing any of the modified PH20 polypeptides provided here that have increased stability to a phenolic condom and an insulin, such as a fast-acting insulin, is provided here. The modified PH20 polypeptides and insulin can be supplied in therapeutically effective amounts. For example, a pharmaceutical composition is provided here that contains any of the modified PH20 polypeptides provided here that has increased stability to a phenolic condom in an amount that is approximately 100 U / mL to 1000 U / mL and a fast-acting insulin in a amount that is or approximately 10 U / mL to 1000 U / mL. For example, fast-acting insulin can be an insulin analog, like insulin lispro,
[00041] [00041] Other therapeutic agents in any of the pharmaceutical compositions provided here include, but are not limited to, Adalimumabs, Agalsidase Betas, Alefacepts, Ampicillins, Anakinras, Polypolymyelitic vaccines, Anti-Thymocytes, Azithromycin, Becaplermins, Caspofungins, Cefazolins, Cefazolins, Cefazolins, Cefazolins, Cefazolim, , Ceftazidimas, Ceftriaxones, Cetuximabs, Cilastatinas, Clavulanic acids, Clindamycins, Darbepoetin Alfas, Daclizumabs, Diphtheria, Diphtheria antitoxins, Diphtheria Toxoides, Efalizumabs, Epinephrines, Erythropoietin Alfas, Flushings, Ethanes, Ethans Folitropin Betas, Phosphenitoines, Gadodiamides, Gadopentetates, Gatifloxacins, Glatirameros, GM-CSF's, Goserelins, Goserelin acetates, Granisetrons, Haemophilus Influenza B's, Haloperidols, hepatitis vaccines, hepatitis A vaccines, Hepatitis vaccines, Hepatitis vaccines, Tiuxetans, Immunoglobulins, Hemophilus influenza vaccines, vac flu virus, Infliximabs, Insulin lispro, 75% neutral protamine (NPL) / 25% insulin lispro, 50% neutral protamine (NPH) / 50% regular insulin, 70% NPH / 30% insulin regular; Regular insulin, NPH insulin, Ultra insulin, Ultralent insulin, and Insulin Glargines, Interferons, Interferon alfa, Interferon Betas, Interferon ranges, Interferon alfa-2a, Interferon alfa 2-b, Interferon alpha-n, Interferon alfa-n, Interferon Betas, Interferon Beta-1a's, Interferon Ranges, Interferon alfa-con, Iodixanois, Iohexois, Iopamidois, Ioversols, Ketorolacs,
[00042] [00042] The modified PH20 compositions and polypeptides can be used to treat any condition normally treated by the PH20 polypeptide or the therapeutically active agent. These include, for example, conditions in which hyaluronan plays a role or is associated with the etiology of the disease due, for example, to the accumulation or excessive production of hyaluronan. Accordingly, methods, uses of the modified PH20 compositions and polypeptides are provided to treat a disease or condition associated with hyaluronan by administering any of the modified PH20 polypeptides or compositions provided herein. Diseases and conditions associated with hyaluronan include, for example, inflammatory disease and tumors or cancers, including late-stage cancer, metastatic cancers and undifferentiated cancers, such as ovarian cancer, carcinoma at the site (ISC), squamous cell carcinoma (SCC ), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer and colon cancer. PH20 polypeptide can be modified to have an increased half-life for such treatments. For example, PH20 polypeptide can be modified with a polymer as a PEG fraction for such treatments.
[00043] [00043] Methods are also provided to increase delivery of a therapeutic agent to a subject by: administering to any subject any of the modified PH20 polypeptides or compositions provided herein, and administering the therapeutic agent. The therapeutic agent can be administered in the same composition or separately, and can be administered before or after, simultaneously or intermittently, with administration of the PH20 polypeptide (s). Administration includes any route, including intravenous and subcutaneous administration, such as simultaneously with, intermittently with, or subsequent to the administration of the therapeutic agent. Therapeutic agents include any of those set out above, elsewhere herein and / known to those skilled in the art.
[00044] [00044] Methods are also provided to treat an excess of glycosaminoglycans; to treat a tumor; to treat glycosaminoglycan build-up in the brain; to treat a cardiovascular disorder; to treat an ophthalmic disorder; to treat lung disease; to increase the penetration of chemotherapeutic agents in solid tumors; to treat cellulite; to treat a proliferative disorder; or to increase bioavailability of drugs and other therapeutic agents to deliver the modified PH20 polypeptides or compositions provided here.
[00045] [00045] Pharmaceutical compositions for use in the treatment of a disease or disorder associated with hyaluronan are also provided; for use in providing a therapeutic agent to a subject; to treat an excess of glycosaminoglycans; to treat a tumor; to treat glycosaminoglycan build-up in the brain; to treat a cardiovascular disorder; to treat an ophthalmic disorder; to treat lung disease; to increase the penetration of chemotherapeutic agents in solid tumors; to treat cellulite; to treat a proliferative disorder; or to increase the bioavailability of drugs and other therapeutic agents; and for any other use of compositions containing PH20 polypeptides.
[00046] [00046] A method is provided here to identify or select a modified hyaluronan degrading enzyme that presents stability under a denaturing condition that includes the steps of: a) testing the activity of a modified hyaluronan degrading enzyme in a composition containing an agent denaturing and / or under a denaturing condition; b) test the activity of the enzyme to degrade modified hyaluronan in the same composition and / or under the same conditions as a) except without the condition or denaturing agent; and c) selecting or identifying a modified hyaluronan degrading enzyme that has activity in a) that is at least 5% of the activity in b). in such an example, the activity is hyaluronidase activity. In some examples of the methods, a modified hyaluronan degrading enzyme is selected or identified if the activity in a) is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50 %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the activity in b), for example, a modified hyaluronan degrading enzyme is selected or identified if the activity in a) is at least 40% or more of the activity in b). The method may also include the steps of: d) comparing the activity of the modified hyaluronan degrading enzyme in a) with the activity of the unmodified hyaluronan degrading enzyme tested under the same conditions and e) identifying or selecting a modified hyaluronan degrading enzyme that at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200%, 250%, 300%, 350%, 400%, 500%, 1500% , 2000%, 3000%, 4000%, 5000% or more of hyaluronidase activity compared to the unmodified hyaluronan degrading enzyme.
[00047] [00047] A method is also provided here to identify or select a modified hyaluronan degrading enzyme that has stability, such as increased stability, under a denaturation condition, which includes the steps of: a) testing the activity of a hyaluronan degrading enzyme modified into a composition containing a denaturing agent and / or under a denaturing condition; b) test the activity of the corresponding unmodified hyaluronan degrading enzyme in a composition containing the same denaturing agent and / or under the same denaturing condition as a), so the activity is tested under the same conditions as a); and c) selecting or identifying a modified hyaluronan degrading enzyme that exhibits greater activity than the unmodified hyaluronan degrading enzyme, thereby identifying or selecting a modified hyaluronan degrading enzyme that has increased stability under a condition of denaturation. In such an example, the activity may be a hyaluronidase activity. In the examples of the method, a modified hyaluronan degrading enzyme is selected or identified if the activity is at least 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200% , 250%, 300%,
[00048] [00048] In any of the methods provided here to identify or select a modified hyaluronan degrading enzyme, the denaturing agent or condition is caused by temperature, agitation, no salt or low salt content or the presence of an excipient. For example, the denaturing agent or condition is caused by an elevated temperature that is from or approximately 30 ° C to 42 ° C, as greater than or greater than approximately 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 ° C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C or 42 ° C. in other examples, the denaturing agent or condition is the absence of salt or low salt content less than 100 mM, such as low salt content less than 90 mM, 80mM, 70mM, 60 mM, 50 mM, 40 mM, 30 mM, mM, 20 mM, 15 mM, 10 mM, 5 mM. In additional examples, the denaturing agent or condition is a denaturing excipient selected from a non-stick, binder, coating, filler and diluent, flavors, colors, lubricants, glidants, preservatives, sorbents and sweeteners.
[00049] [00049] In specific examples of any of the methods provided here to identify or select a modified hyaluronan degrading enzyme, the denaturing agent or condition is a condom (s), for example, a phenolic condom (s). The phenolic condom (s) can be a phenol, metacresol (m-cresol), benzyl alcohol or a paraben. For example, the denaturing agent or condition is a preservative (s) that is phenol and / or m-cresol. In such examples, the total amount of phenolic preservative in the composition, as a percentage (%) of mass concentration (weight / v) is or approximately 0.05% to 0.6%, 0.1% to 0, 4%, 0.1% to 0.3%, 0.15% to 0.325%, 0.15% to 0.25%, 0.1% to 0.2%, 0.2% to 0.3% or 0.3% to 0.4% inclusive.
[00050] [00050] In any of the methods provided here to identify or select a modified hyaluronan degrading enzyme, prior to testing the activity of a hyaluronan degrading enzyme composition in a) and / or b), the hyaluronan degrading enzyme is exposed to denaturing condition or denaturing agent for a predetermined time. The predetermined time is a period of time that is selected by the user depending on the specific hyaluronan degrading enzyme being developed or selected, the specific denaturing condition or denaturing agent, the amount or extent of the denaturing condition or denaturing agent, the application or use of the enzyme to degrade hyaluronan and other similar factors. For example, the predetermined time can be approximately 1 minute to 1 month, 1 minute to 3 weeks, 1 minute to 2 weeks, 1 minute to 1 week, 1 minute to 24 hours, 1 minute to 12 hours, 30 minutes to 6 hours or 1 hour to 4 hours, such as at least or approximately at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, two days, three days, four days, five days, six days, 7 days, two weeks or a month.
[00051] [00051] In any of the methods provided here to identify or select a modified hyaluronan degrading enzyme, the modified hyaluronan degrading enzyme is one that contains an amino acid substitution, insertion or deletion of amino acids compared to an unmodified hyaluronan degrading enzyme . For example, the modified hyaluronan degrading enzyme contains an amino acid substitution as a single amino acid substitution or two, three, four, five, six, seven, eight, nine or more amino acid substitutions compared to an unmodified form of the enzyme to degrade hyaluronan. In specific aspects of the method, a library or collection of modified hyaluronan degrading enzymes is selected to develop or identify or select a modified hyaluronan degrading enzyme that presents stability, such as increased stability, in a condition of denaturation. Thus, in examples of the methods of the present invention, a plurality of modified hyaluronan degrading enzymes is modified compared to the corresponding unmodified hyaluronan degrading enzyme to generate a collection of modified hyaluronan degrading enzymes, so that each modified protein in the enzyme degrading modified hyaluronan contains a single amino acid substitution compared to the unmodified form of the degrading enzyme hyaluronan such that the plurality of modified enzymes is such that the amino acid in each modified position is replaced by up to 1-19 other different amino acids of the original amino acid in the position, so that each modified hyaluronan-degrading enzyme contains a different amino acid substitution and every amino acid along the length of the hyaluronan-degrading enzyme, or a selected portion thereof, is replaced.
[00052] [00052] In any of the methods provided here, the modified hyaluronan degrading enzyme is modified compared to the unmodified hyaluronan degrading enzyme by insertion, deletion or substitution of an amino acid (s). the unmodified hyaluronan degrading enzyme can be a chondroitinase or it can be a hyaluronidase. In the examples of the present invention, unmodified hyaluronidase is a PH20 hyaluronidase or truncated form thereof without a C-terminal glycosyl phosphatidylinositol (GPI) anchor attachment site or a portion of the GPI anchor attachment site, whereby the form truncated has hyaluronidase activity. Hyaluronidase PH20 can be a human, monkey, bovine, sheep, rat, fox, mouse or guinea pig PH20. In specific examples, hyaluronidase PH20 is a human PH20 or a C-terminal truncated form thereof. For example, the unmodified hyaluronan degrading enzyme is one that has the amino acid sequence exposed in any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, 870 or an amino acid sequence that is at least 80% sequence identity for any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861 .870, as at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any of SEQ ID NOS: 3, 7, 10, 12, 14, 24, 32-66, 69, 72, 857, 859, 861, or 870. In specific examples, the unmodified hyaluronan degrading enzyme is a PH20 hyaluronidase having the amino acid sequence as set out in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72, or an amino acid sequence that has at least 85% sequence identity to any of SEQ ID NOS: 3, 7, 32-66, 69 or 72, as an amino acid sequence that presents at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or more sequence identity for any of SEQ ID NOS: 3, 7, 32-66, 69 or 72.
[00053] [00053] In any of the methods provided here to identify or select a modified hyaluronan degrading enzyme that has stability, the method is performed in vitro. Any of the methods that are iterative are also provided, so the steps of the method are repeated a plurality of times, with each repeat, additional modified modified hyaluronan degrading enzymes from a selected modified hyaluronan degrading enzyme are generated and tested, so the modified hyaluronan degrading enzyme is developed to show increased stability under a condition of denaturation. A modified hyaluronan degrading enzyme identified by any of the methods provided here is also provided here. BRIEF DESCRIPTION OF THE FIGURES
[00054] [00054] Figure 1 represents the full length PH20 amino acid sequence (set out in SEQ ID
[00055] [00055] Figure 2 represents exemplary alignments of human soluble PH20 exposed in SEQ ID NO: 3 with other PH20 polypeptides. An "*" means that the aligned residues are identical, an ":" means that the aligned residues are not identical but are similar and contain conservative amino acid residues in the aligned position, and a "." means that the aligned residues are similar and contain semiconservative amino acid residues in the aligned position. Corresponding non-limiting positions, examples for amino acid substitutions are indicated to be highlighted. For example, figure 2A represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with chimpanzee PH20 exposed in SEQ ID NO: 10. Figure 2B represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with Rhesus monkey PH20 exposed in SEQ ID NO: 12. Figure 2C represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with monkey PH20 Cynomolgus exposed in SEQ ID NO: 14. Figure 2D represents the alignment of soluble human PH20 exposed in SEQ ID NO: 3 with bovine PH20 exposed in SEQ ID NO: 16. Figure 2E represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with mouse PH20 exposed in SEQ ID NO: 20. Figure 2F represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with rat PH20 exposed in SEQ ID NO: 22. Figure 2G represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with rabbit PH20 exposed in SEQ ID NO: 24. Figure 2H represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with guinea pig PH20 exposed in SEQ ID NO: 29. Figure 2I represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with fox PH20 exposed in SEQ ID NO: 31. Figure 2J represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with Gibão PH20 exposed in SEQ ID NO: 857. Figure 2K represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with PH20 of Marmoset exposed in SEQ ID NO: 859. Figure 2L represents the alignment of a soluble human PH20 exposed in SEQ ID NO: 3 with orangutan PH20 exposed in SEQ ID NO: 861. DETAILED DESCRIPTION
[00056] [00056] Outline
[00057] [00057] A. Definitions
[00058] [00058] B. PH20 hyaluronidase
[00059] [00059] 1. Structure
[00060] [00060] 2. Function
[00061] [00061] 3. Soluble PH20 polypeptides
[00062] [00062] C. Modified PH20 polypeptides
[00063] [00063] 1. Active mutants
[00064] [00064] A. increased activity
[00065] [00065] B. increased stability
[00066] [00066] i. phenophiles
[00067] [00067] ii. Thermophiles
[00068] [00068] iii. Absence of salt
[00069] [00069] 2. Inactive mutants
[00070] [00070] 3. Additional modifications
[00071] [00071] A. decreased immunogenicity
[00072] [00072] B. conjugation with polymers
[00073] [00073] D. Methods for identifying modified hyaluronan degrading enzymes with altered properties or activities
[00074] [00074] 1. Hyaluronan-degrading enzymes and modified hyaluronan-degrading enzyme libraries
[00075] [00075] 2. Selection or testing in relation to a desired property or activity
[00076] [00076] 3. Selection or identification
[00077] [00077] 4. Iterative methods
[00078] [00078] E. production of modified polypeptides and encoding nucleic acid molecules
[00079] [00079] 1. Isolation or preparation of nucleic acids encoding PH20 polypeptides
[00080] [00080] 2. Generation of modified or mutant nucleic acid and coding polypeptides
[00081] [00081] 3. Vectors and cells
[00082] [00082] 4. Expression
[00083] [00083] A. prokaryotic cells
[00084] [00084] B. yeast cells
[00085] [00085] C. insects and insect cells
[00086] [00086] D. mammalian expression
[00087] [00087] E. plants and plant cells
[00088] [00088] 5. Purification
[00089] [00089] 6. Modification of polypeptides by PEGylation
[00090] [00090] F. Pharmaceutical Compositions and Formulations, Dosages and Administration
[00091] [00091] 1. Formulations (liquids, injectables, solutions and emulsions)
[00092] [00092] A. lyophilized
[00093] [00093] B. exemplary formulations
[00094] [00094] i. NaCl
[00095] [00095] ii. pH and buffer
[00096] [00096] iii. Condoms
[00097] [00097] iv. Stabilizers
[00098] [00098] 2. Compositions for other routes of administration
[00099] [00099] 3. Dosages and administration
[000100] [000100] 4. Exemplary insulin-PH20 co-formulations
[000101] [000101] 5. Packaging, industrial products and kits
[000102] [000102] G. Methods of evaluating hyaluronidase activity
[000103] [000103] 1. Hyaluronidase activity
[000104] [000104] 2. Solubility
[000105] [000105] 3. Purity, crystallization or aggregation
[000106] [000106] 4. Pharmacodynamics / pharmacokinetics
[000107] [000107] H. Methods of treatment and combination therapy
[000108] [000108] 1. Methods of delivering therapeutic agents - Delivering insulin
[000109] [000109] 2. Methods of treating conditions and illness associated with hyaluronan
[000110] [000110] 3. Other uses
[000111] [000111] 4. Contraception
[000112] [000112] I. Examples A. DEFINITIONS
[000113] [000113] Unless otherwise stated,
[000114] [000114] As used here, a hyaluronan-degrading enzyme refers to an enzyme that catalyzes the cleavage of a hyaluronan polymer (also referred to as hyaluronic acid or HA) into fragments of lower molecular weight. Exemplary hyaluronan degrading enzymes are hyaluronidases, and specific chondroitinases and lyases that have the ability to depolymerize hyaluronan. Exemplary chondroitinases which are hyaluronan-degrading enzymes include, but are not limited to, chondroitin ABC lyase (also known as chondroitinase ABC), chondroitin AC lyase (also known as chondroitin sulfate lyase or chondroitin sulfate eliminate) and chondroitin C lyase. Chondroitin ABC lyase contains two enzymes, chondroitin-sulfate-ABC endolysis (EC 4.2.2.20) and chondroitin-sulfate-ABC exolysis (EC 4.2.2.21). Chondroitin-sulfate-ABC endoliases and chondroitin-sulfate-
[000115] [000115] As used here, hyaluronidase refers to a class of enzymes that degrade hyaluronan. Hyaluronidases include, but are not limited to, bacterial hyaluronidases (EC 4.2.2.1 or EC 4.2.99.1), leeches hyaluronidases, other parasites and crustaceans (EC 3.2.1.36), and mammalian type hyaluronidases (EC 3.2.1.35) . Hyaluronidases include any non-animal origin, including, but not limited to, murine, canine, feline, cleft, avian, bovine, sheep, porcine, equine, fish, ranin, bacterial, and any of leeches, other parasites and crustaceans. Exemplary human hyaluronidases include HYAL1, HYAL2, HYAL3, HYAL4, and PH20. Also included among hyaluronidases are soluble hyaluronidases, including, sheep and bovine PH20, and soluble PH20. Exemplary hyaluronidases include any exposed in SEQ ID NOS: 6, 7-
[000116] [000116] As used here, PH20 refers to a type of hyaluronidase that occurs in sperm and is neutrally active. PH-20 occurs on the sperm surface, and on the lysosome-derived acrosome, where it is attached to the internal acrosomal membrane. PH20 include those of any origin including, but not limited to, human, chimpanzee, Cynomolgus monkey, Rhesus monkey, murine, bovine, sheep, guinea pig, rabbit and rat. Exemplary PH20 polypeptides, including precursor and mature forms, include those from human (SEQ ID NO: 6 and 7), chimpanzee (SEQ ID NO: 8, 9, 10, 869 and 870), Rhesus monkey (SEQ ID NO: 11 and 12), Cynomolgus monkey (SEQ ID NO: 13 and 14), cow (for example, SEQ ID NOS: 15-18); mouse (SEQ ID NO: 19 and 20); rat (SEQ ID NO: 21 and 22); rabbit (SEQ ID NO: 23 and 24); sheep (SEQ ID NOS: 25-27), guinea pig (SEQ ID NO: 28 and 29); fox (SEQ ID NO: 30 and 31); Gibbon (SEQ ID NO: 856 and 857), Marmoset (SEQ ID NO: 858 and 859) and orangutan (SEQ ID NO: 860 and 861). References to PH20 include precursor PH20 polypeptides and mature PH20 polypeptides (such as those in which a signal sequence has been removed), truncated forms of which have activity, and include allelic variants and species variants, variants encoded by junction variants, and others variants, including polypeptides that have at least 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the precursor polypeptides shown in SEQ ID NO: 7, or the mature forms thereof. PH20 polypeptides also include those that contain chemical or post-translational modifications and those that do not contain chemical or post-translational modifications. Such modifications include, but are not limited to, PEGylation, albumination, glycosylation, farnisylation, carboxylation, hydroxylation, phosphorylation, and other polypeptide modifications known in the art. Examples of commercially available soluble bovine or sheep hyaluronidases are Vitrase® hyaluronidase (sheep hyaluronidase) and Amphadase® hyaluronidase (bovine hyaluronidase).
[000117] [000117] As used here, a soluble PH20 refers to a polypeptide characterized by its solubility under physiological conditions. Generally, a soluble PH20 lacks all or a portion of a glycophosphatidyl anchor fixation sequence (GPI), or does not otherwise sufficiently attach to the cell membrane. For example, a soluble PH20 can be a truncated C-terminal variant of a PH20 without having a contiguous amino acid sequence that corresponds to all or a portion of a glycophosphatidyl anchor fixation sequence (GPI). Consequently, after expression of a cell, a soluble PH20 is secreted in the medium. Soluble PH20 proteins can be distinguished, for example, by their division in the aqueous phase of a solution of Triton X-114 heated to 37 ° C (Bordier et al., (1981) J. Biol. Chem., 256: 1604-7 ). Membrane-fixed hyaluronidases, as fixed in lipid, will divide into the detergent-rich phase, but will divide into the detergent-poor or aqueous phase after treatment with Phospholipase-C. Included among soluble PH20 hyaluronidases are membrane-fixed hyaluronidases in which one or more regions associated with fixation of hyaluronidase to the hyaluronidase membrane has been removed or modified, where the soluble form retains hyaluronidase activity. Soluble hyaluronidases include recombinant soluble hyaluronidases and those contained in or purified from natural sources, such as extracts from sheep or ox testicles. They are examples of such soluble human PH20 hyaluronidases (SEQ ID NO: 3 or 32-66). Other soluble hyaluronidases include sheep PH20 (SEQ ID NO: 25-27) and bovine PH20 (SEQ ID NO: 16 or 18).
[000118] [000118] As used here, soluble human PH20 (sHuPH20) includes human PH20 polypeptides that do not have a contiguous amino acid sequence from the human PH20 C-terminus that includes all or a portion of the glycosylphosphatidylinositol (GPI) anchor sequence ( PH20 truncated C-terminal polypeptides) such that after expression, the polypeptides are soluble under physiological conditions. For example, soluble human PH20 polypeptides are C-terminal truncated polypeptides of human PH20 exposed as SEQ ID NO: 6 in its precursor form or in SEQ ID NO: 7 in its mature form having no signal sequence, or allelic variants thereof. (for example, exposed in any of SEQ ID NOS: 68-72). The solder will be evaluated by any appropriate method that demonstrates solubility under physiological conditions. An example of such a method is the Triton® X-114 assay, which assesses the division in the aqueous phase and which is described above. In addition, a soluble human PH20 polypeptide is, if produced in CHO cells, such as CHO-S cells, a polypeptide that is expressed and secreted in the cell culture medium. Soluble human PH20 polypeptides, however, are not limited to those produced in CHO cells, but can be produced in any cell or by any method, including recombinant expression and polypeptide synthesis. Reference to secretion in CHO cells is by definition.
[000119] [000119] As used here, "native" or "wild type" with reference to PHβ0 polypeptide refers to a PH20 polypeptide encoded by a naturally occurring or native PH20 gene, including allelic variants, that is present in an organism, including a human being and other animals, in nature. Reference to PH20 of the wild type without reference to a species is intended to cover any species of a PH20 of the wild type. Included in the wild type PH20 polypeptides are the encoded precursor polypeptide, fragments thereof and processed forms thereof, as a Mature form that lack the signal peptide as well as any pre- or post-translated processed or modified forms. Also included among native PH20 polypeptides are those that are modified post-translating including, but not limited to, those that are modified by glycosylation, carboxylation and / or hydroxylation. The amino acid sequences of exemplary wild-type human PH20 are set out in SEQ ID NOS: 6 and 7 and those of allelic variants, including mature forms thereof, are set out in SEQ ID NOS: 68-72. Other animals produce native PH20, including, but not limited to, native or wild-type sequences exposed in any of SEQ ID NOS: 8-31, 856-861, 869 or 870.
[000120] [000120] As used herein, modification is in reference to the modification of an amino acid sequence of a polypeptide or a nucleotide sequence in a nucleic acid molecule and includes deletions, insertions and substitutions of amino acids and nucleotides, respectively. Modifications can also include post-translational modifications or other changes in the molecule that may occur due to conjugation or bonding, directly or indirectly, with another fraction. The methods of modifying a polypeptide are routine for those skilled in the art, such as using recombinant DAN methodologies.
[000121] [000121] As used herein, a "modified hyaluronan degrading enzyme" refers to a hyaluronan degrading enzyme that contains a modification compared to an unmodified or reference hyaluronan degrading enzyme. The modification can be an amino acid substitution (substitution), insertion (addition) or deletion of one or more amino acid residues. The amino acid residue can be a natural or unnatural amino acid. In some cases, the modification may be a post-translational modification. A modified hyaluronan-degrading enzyme can have up to 150 amino acid differences compared to an unmodified or reference hyaluronan-degrading enzyme, as long as the resulting modified hyaluronan-degrading enzyme has hyaluronidase activity. Typically, a modified hyaluronan-degrading enzyme contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, or 50 amino acid modifications.
[000122] [000122] As used herein, an unmodified hyaluronan degrading enzyme refers to a starting polypeptide that is selected for modification as provided here.
[000123] [000123] As used here, “modified PHβ0 polypeptide” or “variant PHβ0 polypeptide” refers to a PH20 polypeptide that contains at least one amino acid modification, such as at least one amino acid substitution as described here, in its amino acid sequence in compared to a reference unmodified PH20 polypeptide. A modified PH20 polypeptide can have up to 150 amino acid substitutions, as long as the resulting modified PH20 polypeptide exhibits hyaluronidase activity. Typically, a modified PH20 polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 , 48, 49, or 50 amino acid substitutions. It is understood that a modified PH20 polypeptide can also include any one or more other modifications, in addition to at least one amino acid substitution as described herein.
[000124] [000124] As used here, an unmodified PH20 polypeptide refers to a starting PH20 polypeptide that is selected for modification as provided here. The starting polypeptide can be a naturally occurring form of a wild-type polypeptide. In addition, the starting polypeptide can be altered or changed in such a way that it differs from a native wild-type isoform, however it is nevertheless mentioned here as an unmodified starting polypeptide in relation to the subsequently modified polypeptides produced here.
[000125] [000125] As used here, an N-linked fraction refers to an asparagine (N) amino acid residue of a polypeptide that is capable of being glycosylated by post-translational modification of a polypeptide. Exemplary N-linked fractions of human PH20 include amino acids N47, N131, N200, N219, N333, N358 and N365 of the amino acid sequence shown in SEQ ID NO: 3 or 7 (corresponding to amino acid residues N82, N166, N235, N254, N368, N393 and N490 of human PH20 exposed in SEQ ID NO: 6).
[000126] [000126] As used herein, an N-glycosylated polypeptide refers to a PH20 polypeptide containing oligosaccharide binding of at least three N-linked amino acid residues, for example, N-linked fractions corresponding to N200, N333 and N358 amino acid residues of SEQ ID NO: 3 or 7. An N-glycosylated polypeptide can include a polypeptide where three, fourth, five and even all N-linked fractions are linked to an oligosaccharide. N-linked oligosaccharides are linked to an oligosaccharide. N-linked oligosaccharides can include oligosaccharides, complex, hybrid or sulfated oligosaccharides, or other monosaccharides and oligosaccharides.
[000127] [000127] As used herein, a partially glycosylated N- polypeptide refers to a polypeptide that minimally contains an N-acetyl glucosamine glycan linked to at least three fractions linked by N. a partially glycosylated polypeptide can include various forms of glycan, including monosaccharides , oligosaccharides and branched forms of sugar, including those formed by treating a polypeptide with EndoH, EndoF1, EndoF2 and / or EndoF3.
[000128] [000128] As used here, "conditions" refers to any parameter that may influence the activity or properties of a protein or agent. For the purposes of the present invention, conditions generally refer to the presence, including quantity of excipients, vehicles or other components in a formulation other than that of the active agent (for example, modified PH20 hyaluronidase); temperature; time (for example, storage or exposure time); storage container; storage properties (eg, agitation) and / or other conditions associated with exposure or use.
[000129] [000129] As used herein, "denaturation" or "denaturing" or grammatical variations thereof with reference to a protein refers to a biochemical change in a protein so that a property or activity of the protein is decreased or eliminated. The biochemical change can be a change in the tertiary structure of the protein to unfold. The property or activity can be completely abolished or can be reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.
[000130] [000130] As used here, property refers to a physical or structural property, such as three-dimensional structure, pI, half-life, conformation and other such physical characteristics. For example, a change in a property can be manifested as the solubility, aggregation or crystallization of a protein.
[000131] [000131] As used herein, activity refers to an activity or functional activities of a polypeptide or portion thereof associated with a full-length (full) protein. Functional activities include, but are not limited to, biological activity, catalytic or enzymatic activity, antigenicity
[000132] [000132] As used here, hyaluronidase activity refers to the ability to enzymatically catalyze the cleavage of hyaluronic acid. United States Pharmacopeia (USP) Assay XXII for hyaluronidase determines hyaluronidase activity indirectly by measuring the amount of higher molecular weight hyaluronic acid, or hyaluronan substrate (HA) that remains after the enzyme is allowed to react with HA for 30 min At 37 ° C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, MD). A standard reference solution can be used in an assay to determine the relative activity, in units, of any hyaluronidase. In vitro assays to determine hyaluronidase activity of hyaluronidases, such as PH20, including modified PH20 polypeptides, are known in the art and described here. Exemplary assays include the microturbity assay described here which measures cleavage of hyaluronic acid by hyaluronidase indirectly by detecting the insoluble precipitate formed when uncleaved hyaluronic acid binds with serum albumin. Reference standards can be used, for example, to generate a standard curve to determine the activity in Units of the hyaluronidase being tested.
[000133] [000133] As used here, neutron active refers to the ability of a PH20 polypeptide to enzymatically catalyze the cleavage of hyaluronic acid at neutron pH, such as at a pH between or approximately between pH 6.0 and pH 7.8.
[000134] [000134] As used here, “increased activity” with reference to a modified PH20 hyaluronidase means that when tested under the same conditions, the modified PH20 hyaluronidase shows greater hyaluronidase activity compared to an unmodified PH20 hyaluronidase not containing (s ) amino acid substitution (s). For example, a modified PH20 hyaluronidase has at least or approximately at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the unmodified or reference hyaluronidase PH20 activity.
[000135] [000135] As used here, "solubility" with reference to a protein refers to a protein that is homogeneous in an aqueous solution, so that protein molecules diffuse and do not settle spontaneously. Consequently, a soluble protein solution is one in which there is no visible or discrete particle in a solution containing the protein, such that the particles cannot be easily filtered. Generally, a protein is soluble if there are no visible or discrete particles in the solution. For example, a protein is soluble if it does not contain or contains few particles that can be removed by a filter with a pore size of 0.22 µm.
[000136] [000136] As used here, aggregation or crystallization with reference to a protein refers to the presence of visible or discrete particles in a solution containing the protein. Typically, the particles are larger than 10 µm, such as greater than 15 µm, 20 µm, µm, 30 µm, 40 µm, 50 µm or larger. Aggregation or crystallization can result due to reduced solubility, increased denaturation of a protein or the formation of covalent bonds.
[000137] [000137] As used herein, "denaturing condition" or "denaturation condition" refers to any condition or agent that, when exposed to a protein, affects or influences protein degradation or denaturation, generically as a result of a loss or partial loss of the tertiary or secondary structure of the protein. Denaturation conditions can result in effects such as loss or reduction in activity, loss or reduction of solubility, aggregation and / or crystallization. The denaturation condition does not need to be one that is totally deadly to the protein, however it is nevertheless one that leads to a reduction in the activity of the protein over time. Thus, a condition is denaturation if the activity of the protein is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more in presence of the condition than in its absence. A denaturation condition may be due to external stress or physical condition (eg, agitation, temperature, shelf life, absence of a stabilizer) or it may be due to the presence of a denaturing agent. For example, the denaturation condition can be caused by heat, acid or a chemical denaturant. Exemplary denaturing conditions include, but are not limited to, the presence of a strong acid or base, a concentrated inorganic salt, an organic solvent (eg, alcohol or chloroform), urea, high or low pH (pH extremes), temperature high (for example, heat), the presence of excipients that can be denaturing (For example, phenolic preservatives or detergent) and low or substantially no stabilizing agent that is otherwise necessary for protein stability (for example, (NaCl )).
[000138] [000138] As used here, "denaturing agent" or "denaturing" refers to any substance, molecule or compound that causes denaturation. For example, a denaturing agent can include a strong acid or base, a concentrated inorganic salt, an organic solvent (for example, alcohol or chloroform), a preservative, detergent or other excipient.
[000139] [000139] As used herein, "resistance to the denaturation condition" refers to any amount of decreased reduction or elimination of a protein property or activity associated with or caused by denaturation. For example, denaturation is associated with or causes crystallization or increased aggregation, reduced solubility or decreased activity. Consequently, resistance to denaturation means that the protein exhibits decreased aggregation or crystallization, increased solubility or increased or greater activity (eg, hyaluronidase activity) when exposed to a denaturation condition compared to a reference protein (eg, enzyme unmodified). Resistance to a denaturation condition does not need to be absolute or permanent, but can be achieved because the denaturation of the modified hyaluronan degrading enzyme occurs more slowly than the unmodified enzyme in the denaturation condition in such a way that an activity or property of the enzyme to degrade modified hyaluronan is obtained longer. For example, a modified hyaluronan-degrading enzyme, such as modified PH20 hyaluronidase, is resistant to a denaturation condition if, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8 %, 9%, 10%,… β0%,… 30%,… 40%,… 50%,… 60%,…, 70%,… 80%,… 90%, 91%, 9β%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% more resistance to denaturation in the presence of a denaturing condition or denaturing agent than an unmodified polypeptide. In some cases, a modified polypeptide exhibits 105%, 110%, 120%, 130%, 140%, 150%, 200%, 300%, 400%, 500%, or more Increased resistance to denaturation compared to a non-polypeptide modified.
[000140] [000140] As used herein, stability of a modified PH20 hyaluronidase means that it has resistance to denaturation caused by a denaturing condition or denaturing agent. A modified PH20 polypeptide is stable if it retains some activity in the presence of a denaturing condition or denaturing agent, such as at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the original or initial hyaluronidase activity prior to exposure to the denaturation condition (s). Generally, a modified PH20 hyaluronidase is stable if it retains at least 50% or more of the hyaluronidase activity under a denaturation condition compared to the absence of the denaturation condition. Assays to assess hyaluronidase activity are known to a person skilled in the art and described here. It is understood that the stability of the enzyme does not need to be permanent or long-term, but it is manifested by a length of time in which the activity is desired. For example, a modified PH20 hyaluronidase is stable if it has been active for at least 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours, one day, two days, three days, four days, five days, six days, a week, a month, six months or a year after exposure, or during exposure, to one or more denaturing condition (s) or agent (s), (for example, presence of a denaturing excipient as a condom) . For example, a modified PH20 hyaluronidase is stable if it exhibits an activity after or during exposure to one or more denaturing condition (s) or agent (s) (for example, presence of a denaturing excipient as a condom) for at least 1 month at temperatures of or approximately 2 ° C to 8 ° C, inclusive or for at least 3 days at a temperature of or approximately 30 ° C to 42 ° C, inclusive.
[000141] [000141] Consequently, "stable" or "stability", with reference to a formulation or a co-formulation provided here, refers to one in which a modified hyaluronan degrading enzyme, such as a modified PH20 hyaluronidase, is still stable after exposure to one or more denaturing condition (s) or agent (s) in it (for example, presence of a denaturing excipient as a condom) for at least 1 month at temperatures of or approximately 2 ° C to 8 ° C , inclusive or for at least 3 days at a temperature of or approximately 30 ° C to 42 ° C, inclusive.
[000142] [000142] As used herein, “increased stability” with reference to a modified PHβ0 hyaluronidase means that, in the presence of the same condition (s) of denaturing or denaturation (for example, presence of a denaturing excipient as a condom), PH20 modified hyaluronidase has higher hyaluronidase activity compared to an unmodified PH20 hyaluronidase that does not contain the amino acid substitution (s). For example, a modified PH20 hyaluronidase has increased stability if it is at least or approximately at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250% , 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the unmodified or reference hyaluronidase PH20 activity in the presence of a denaturing or denaturation condition (s) example, in the presence of a denaturing excipient such as a condom).
[000143] [000143] As used here, "elevated temperatures" refers to temperatures that are higher than room temperature. Generally, an elevated temperature is a temperature that is at least greater than, or approximately 30 ºC, such as 30 ºC to 42 ºC, and generically 32 ° C to 37 ° C or 35 ° C to 37 ° C, inclusive.
[000144] [000144] As used here, room temperature refers to a range generally from approximately or 18 ºC to approximately or 32 ºC. those skilled in the art recognize that ambient temperature varies by location and prevailing conditions. For example, ambient temperatures can be higher in warmer climates like Italy or Texas.
[000145] [000145] As used herein, the mention that proteins are "compared under the same conditions" means that different proteins are treated identically or substantially identically in such a way that any one or more conditions that may influence the activity or properties of a protein or agent do not vary or do not vary substantially between test agents. For example, when the hyaluronidase activity of a modified PH20 polypeptide is compared with an unmodified PH20 polypeptide any one or more conditions such as the amount or concentration of the polypeptide, presence, including quantity, of excipients, vehicles or other components in a different formulation the active agent (for example, modified hyaluronidase PH20); temperature; storage time; storage container; storage properties (e.g., agitation) and / or other conditions associated with exposure or use are identical or substantially identical among the compared polypeptides.
[000146] [000146] As used here, "predetermined time" refers to a time that is established or decided in advance. For example, the predetermined time may be a time chosen in advance that is associated with the desired duration of activity of a hyaluronan degrading enzyme depending on the application or desired use of the protein. A predetermined time can be hours, days, months or years. For example, a predetermined time can be at least approximately or approximately 2 hours, 3 hours, 4 hours, five hours, six hours, 12 hours, 24 hours, 2 days, three days, four days, five days, six days, one week, two weeks, three weeks, a month, six months, a year or more.
[000147] [000147] As used herein, "storage" means that a formulation is not immediately administered to a subject after preparation, but is maintained for a period of time under specific conditions (for example, specific temperature; time, and / or form (for example, liquid or lyophilized form)) before use. For example, a liquid formulation can be stored for days, weeks, months or years, before administration to a subject under varying temperatures such as refrigerated (0 ° C to 10 ° C, such as 2 to 8 ° C), room temperature (eg temperature up to 32 ° C, like 18 ° C at or approximately 32 ° C), or elevated temperature (for example, 30 ° C to 42 ° C, such as 32 ° C to 37 ° C or 35 ° C to 37 ° C).
[000148] [000148] As used herein, an "excipient" refers to a compound in an active agent formulation that does not prove the biological effect of the active agent when administered in the absence of the active agent. Exemplary excipients include, but are not limited to, salts,
[000149] [000149] As used herein, a stabilizing agent refers to a compound added to the formulation to protect the modified PH20 polypeptide or other active agent against degradation, if necessary, as due to denaturation conditions to which a formulation of the present invention is exposed when manipulated, stored or used. In this way, agents are included that prevent protein degradation from other components in the compositions. Examples of such agents are amino acids, derivatives of amino acids, amines, sugars, polyols, salts and buffers, surfactants, inhibitors or substrates and other agents as described herein.
[000150] [000150] As used here, an antimicrobial effectiveness test or condom effectiveness test (PET) demonstrates the effectiveness of the condom system in a product. A product is inoculated with a controlled amount of specific organisms. The test then compares the level of microorganisms found in a control sample versus the test sample over a 28 day period. Generally, target markets have different PET requirements. For example, the PET requirements of the United States Pharmacopoeia (USP) and European Pharmacopoeia (EP) differ. The parameters for performing an antimicrobial effectiveness test, including in different markets, are known to a person skilled in the art as described here.
[000151] [000151] As used herein, an effective antimicrobial or antimicrobial amount of a condom refers to an amount of the condom that kills or inhibits the spread of microbial organisms in a sample that can be introduced for storage or use.
[000152] [000152] As used herein, “preservative” refers to a naturally occurring substance or produced synthetically or recombinantly that, when added to a protein or molecule composition, prevents microbial growth, including growth of bacteria or fungi, in the composition .
[000153] [000153] As used here, a "phenolic condom" refers to a condom that contains a hydroxyl group attached to an aromatic carbon ring, such as a benzene ring. Exemplary phenolic condoms include, but are not limited to, phenol, m-cresol, p-hydroxybenzoic acid, methylparaben, ethylparaben, and propylparaben. For example, cresols, including meta-cresol (m-cresol), have a methyl group substituted on the benzene ring of a phenol molecule.
[000154] [000154] As used here, a "phenophile" refers to a protein, such as a modified PH20 polypeptide, which exhibits stability in the presence of an antimicrobial effective amount of a condom (s). the term "phenolphile" can be used interchangeably here with "phenophile" and has the same meaning. For example, a modified PH20 polypeptide that is a phenophile or phenolphyl typically has increased stability compared to an unmodified PH20 hyaluronidase that does not contain the amino acid substitution (s) when tested under the same condition (s) of denaturation containing a phenolic preservative (s). for example, a modified PH20 hyaluronidase has at least or approximately at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the reference or unmodified hyaluronidase PH20 activity in the presence of a phenolic condom (s).
[000155] [000155] As used here, a "thermophile" refers to a protein, such as a modified PH20 polypeptide, which exhibits stability at elevated temperatures greater than or approximately 30 ° C, such as 30 ° C to 42 ° C and generally 32 ° C to 37 ° C or 35 ° C to 37 ° C. For example, a modified PH20 polypeptide that is a thermophile typically exhibits increased stability compared to an unmodified PH20 hyaluronidase that does not contain the amino acid substitution (s) when tested under the same condition (s) high temperature denaturation. For example, a modified PH20 hyaluronidase has at least or approximately at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the reference or unmodified hyaluronidase PH20 activity at elevated temperatures.
[000156] [000156] As used here, the term "detergent" is used interchangeably with the term "surfactant" or "surface acting agent". Surfactants are typically organic compounds that are amphiphilic, that is, containing both hydrophobic groups
[000157] [000157] As used here, a "buffer" refers to a substance, generally a solution, that can maintain its pH constant, despite the addition of strong acids or strong bases and external influences of temperature, pressure, volume or redox potential. A buffer prevents changes in the concentration of another chemical, for example, proton donor and acceptor systems that prevent sharp changes in hydrogen ion concentration (pH). The pH values of all buffers are dependent on concentration and temperature. The choice of buffer to maintain a pH value or range can be empirically determined by a person skilled in the art based on the known buffering capacity of known buffers. Exemplary buffers include, but are not limited to, bicarbonate buffer, cacodylate buffer, phosphate buffer or Tris buffer. For example, Tris buffer (tromethamine) is an amine based buffer that has a pKa of 8.06 and has an effective pH range between 7.9 and 9.2. For Tris buffers, the pH increases approximately 0.03 units by decreasing the temperature in ºC, and decreases 0.03 to 0.05 units per ten-fold dilution.
[000158] [000158] As used here, naturally occurring α - amino acid residues are the residues of those α-amino acids found in nature that are incorporated into protein by specific recognition of the tRNA molecule loaded with its cognate mRNA codon in humans.
[000159] [000159] As used herein, nucleic acids include DNA, RNA and analogs thereof, including peptide nucleic acids (PNA) and mixtures thereof. Nucleic acids can be single or double stranded. When referring to probes or primers, which are optionally labeled, as with a detectable label, such as a fluorescent or radio label, single-stranded molecules are considered. Such molecules are typically of such a length that their target is statistically unique or low copy number (typically less than 5, preferably less than 3) to probe or start a library. Generally, a probe or primer contains at least 14, 16 or 30 contiguous nucleotides of a sequence complementary to or identical to a gene of interest. Probes and primers can be 10, 20, 30, 50, 100 or more nucleic acids in length.
[000160] [000160] As used here, a peptide refers to a polypeptide that is 2 to 40 amino acids in length.
[000161] [000161] As used here, the amino acids that occur in the various amino acid sequences provided here are identified according to their known three-letter or one-letter abbreviations (Table 1). The nucleotides that occur in the various nucleic acid fragments are designated with the standard single letter designations used routinely in the art.
[000162] [000162] As used here, an "amino acid" is an organic compound containing an amino group and a carboxylic acid group. A polypeptide contains two or more amino acids. For the purposes of the present invention, amino acids include the twenty naturally occurring amino acids, unnatural amino acids and amino acid analogs (i.e., amino acids in which the α-cabono has a side chain).
[000163] [000163] As used here, "amino acid residue" refers to an amino acid formed after chemical digestion (hydrolysis) of a polypeptide in its peptide bonds. The amino acid residues described here are assumed to be in the "L" isomeric form. residues in the isomeric form "D" which are thus designated, can be replaced by any L-amino acid residue as long as the desired functional property is retained by the polypeptide. NH2 refers to the group of free amino present at the amino end of a polypeptide. COOH refers to the group of free carboxy present at the carboxyl end of a polypeptide. In maintaining the standard polypeptide nomenclature described in J. Biol. Chem., 243: 3557-3559 (1968), and adopted 37 C.F.R. §§ 1.821-1.822, abbreviations for amino acid residues are shown in table 1: Table 1 - Correspondence table
[000164] [000164] It should be noted that all the amino acid residue sequences represented here by the formulas have a left-to-right orientation in the conventional direction from amino end to carboxyl end. In addition, the phrase “amino acid residue is broadly defined as including the amino acids listed in the Correspondence Table (Table 1) and modified and unusual amino acids, such as those mentioned in 37 C.F.R. §§ 1.821-1.822, and incorporated herein by reference. In addition, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond with an additional sequence of one or more amino acid residues, to an amino-terminal group such as NH2 or to a terminal group - carboxyl as COOH.
[000165] [000165] As used here, "naturally occurring amino acids" refer to β0 L-amino acids that occur in polypeptides.
[000166] [000166] As used here, "unnatural amino acid" refers to an organic compound that has a structure similar to a natural amino acid but has been structurally modified to simulate the structure and reactivity of a natural amino acid. Non-naturally occurring amino acids thus include, for example, amino acids or amino acid analogs other than the 20 naturally occurring amino acids and include, but are not limited to, D-stereoisomers of amino acids. Exemplary unnatural amino acids are described here and are known to those of skill in the art.
[000167] [000167] As used here, an isokinetic mixture is one in which the molar ratios of amino acids have been adjusted based on their reported reaction rates (see, for example, Ostresh et al., (1994) Biopolymers 34: 1681).
[000168] [000168] As used herein, appropriate conservative amino acid substitutions are known to those skilled in the art and can be made generically without altering the biological activity of the resulting molecule. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th edition, 1987, The Benjamin / Cummings Pub. Co., P.224). Such substitutions can be made in accordance with those set out in Table 2 as follows: TABLE 2 Original waste Exemplary conservative substitution Ala (A) Gly; Ser Arg (R) Lys Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) Asp Gly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile Phe (F) Met; Read; Tyr Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Read
[000169] [000169] Other substitutions are also permissible and can be determined empirically or according to known conservative substitutions.
[000170] [000170] As used here, a DNA construct is a linear or circular DNA molecule, single or double stranded that contains DNA segments combined and juxtaposed in a way not found in nature. DNA constructions exist as a result of human manipulation, and include clones and other copies of manipulated molecules.
[000171] [000171] As used here, a segment of DNA is a portion of a larger DNA molecule having specified attributes. For example, a segment of DNA encoding a specified polypeptide is a portion of a longer DNA molecule, such as a plasmid or plasmid fragment, which, when read from the 5 'to 3' direction, encodes the amino acid sequence of the specified polypeptide.
[000172] [000172] As used herein, the term poly nucleotide means a single or double-stranded polymer of deoxyribonucleotides or ribonucleotide bases read from the 5 'to 3' end. Polynucleotides include RNA and DNA, and can be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. The length of a polynucleotide molecule is given here in terms of nucleotides (abbreviated "nt") or base pairs (abbreviated "bp"). The term nucleotides is used for single and double-stranded molecules where context allows. When the term is applied to double-stranded molecules it is used to indicate overall length and will be understood to be equivalent to the term base pairs. It will be recognized by those skilled in the art that the two strands of a double-stranded polynucleotide may differ slightly in length and that their ends may be spread out; therefore, all nucleotides in a double-stranded polynucleotide molecule cannot be paired. Such unpaired ends, in general, will not exceed 20 nucleotides in length.
[000173] [000173] As used here, "in a position corresponding to" or mention that nucleotides or amino acid positions "correspond to" nucleotides or amino acid positions in a revealed sequence, as set out in the Sequences listing, refers to nucleotides or positions of amino acids identified after alignment with the revealed sequence to maximize identity using a standard alignment algorithm, such as the GAP algorithm.
[000174] [000174] As used herein, "sequence identity" refers to the number of identical or similar amino acids or nucleotide bases in a comparison between a test and a reference polypeptide or polynucleotide. Sequence identity can be determined by nucleic acid sequence alignment or protein sequences to identify regions of similarity or identity. For purposes of the present invention, sequence identity is generally determined by alignment to identify identical residues. The alignment can be local or global, but for the purposes of the present invention the alignment is generally a global alignment where the total length of each sequence is compared. Weddings, mismatches and breaks can be identified between compared strings. Gaps are null amino acids or nucleotides inserted between the residues of aligned sequences so that identical or similar characters are aligned. Generally, there may be internal and terminal clearances. The sequence identity can be determined by taking into account clearances such as the number of identical residues / length of the shortest sequence x 100. When using clearance penalties, sequence identity can be determined without penalty for final clearances (for example, unused end clearances) penalized). Alternatively, the sequence identity can be determined without taking into account gaps such as the number of identical positions / length of the total aligned sequence x 100.
[000175] [000175] As used here, an “overall alignment” is an alignment that aligns two strings from the beginning to the end, aligning each letter in each sequence only once. An alignment is produced,
[000176] [000176] As used here, a "local alignment" is an alignment that aligns two sequences, but only aligns those portions of the sequences that share similarity or identity. Consequently, a local alignment determines whether subsegments of one sequence are present in another sequence. If there is no similarity, no alignment is returned. Local alignment algorithms include BLAST or Smith-Waterman algorithm (Adv. Appl. Math. 2: 482 (1981)). For example, 50% sequence identity based on “local alignment”
[000177] [000177] For the purposes of the present invention, sequence identity can be determined by standard alignment algorithm programs used with default slack penalties set by each supplier. Default parameters for the GAP program may include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) and the weight comparison matrix of Gribskov and others. Nucl. Acids Res. 14: 6745 (1986), as described by Schwartz and Diahoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, p. 353-358 (1979); (2) a 3.0 penalty for each clearance and an additional 0.10 penalty for each symbol in each clearance; and (3) no penalty for final clearances. The fact that any two nucleic acid molecules have nucleotide sequences or any two polypeptides have amino acid sequences that are at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% " identical, "or other similar variations mentioning a percentage of identity, can be determined using known computer algorithms based on local or global alignment (see, for example, wikipedia.org/wiki/Sequence_alignment_software, providing links to dozens of databases and publicly available and known alignment programs). Generally, for the purposes of the present invention, sequence identity is determined using computer algorithms based on global alignment, such as the
[000178] [000178] Therefore, as used here, the term "identity" represents a comparison or alignment between a test and a reference polypeptide or polynucleotide. In a non-limiting example, “at least 90% identical to” refers to the percentage of identities from 90 to 100% relative to the reference polypeptide or polynucleotide. Identity at a level of 90% or more is indicative of the fact that assuming for reference purposes a reference and test nucleotide polypeptide or poly length of 100 amino acids or nucleotides are compared, no more than 10% (ie 10 of 100) of amino acids or nucleotides in the test polypeptide or polynucleotide differs from that of the reference polypeptides. Such differences can be represented as point mutations randomly distributed over the total length of an amino acid sequence or they can be grouped in one or more locations of variable length up to the maximum allowable, for example, 10/100 amino acid difference (approximately 90% identity). The differences may also be due to deletions or truncations of amino acid residues. Differences are defined as amino acid or nucleic acid substitutions, insertions or deletions. Depending on the length of the sequences compared, at the level of homologies or identities above approximately 85-90%, the result may be independent of the set of slack and program parameters; such high levels of identity can be readily assessed, often without being based on software.
[000179] [000179] As used here, an allelic variant or allelic variation references any of two or more alternative forms of a gene that occupies the same chromosomal site. Allelic variation originates naturally through mutation, and can result in phenotypic polymorphism in populations. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides having an altered amino acid sequence. The term "allelic variant" is also used here to indicate a protein encoded by an allelic variant of a gene. Typically, the reference form of the gene encodes a wild-type and / or predominant form of a polypeptide from a population or unique reference number of a species. Typically, allelic variants, which include variants between species, typically have at least 80%, 90% or greater of amino acid identity with a wild type and / or predominant form of the same species; the degree of identity depends on the gene and whether the comparison is interspecies or intraspecies. Generally, intraspecies allelic variants have at least approximately 80%, 80%, 85%, 90% or 95% identity or wild type and / or predominant form including 96%, 97%, 98%, 99% or greater identity with a wild type and / or predominant form of a polypeptide, reference to an allelic variant here generically refers to variations in proteins between elements of the same species.
[000180] [000180] As used here, "allele" which is used interchangeably here with "allelic variant" refers to alternative forms of a gene or portions of it. Alleles occupy the same location or position on homologous chromosomes. When a subject has two identical alleles of a gene, the subject is said to be heterozygous for the gene. Alleles of a specific gene can differ from one nucleotide to several nucleotides, and may include modifications such as nucleotide substitutions, deletions and insertions. A gene allele can also be a form of a gene containing a mutation.
[000181] [000181] As used here, species variants refer to variants in polypeptides between different species, including different mammalian species, such as mouse and human. Examples of species of primate species provided here are PH20, but not limited to humans, chimpanzees, monkeys, cynomolgus monkeys, gibbons, orangutans or marmosets. Generically, species variants have 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence identity. Corresponding residues between species variants can be determined by comparing and aligning sequences to maximize the number of nucleotides or matched residues, for example, such that the identity between the sequences is equal to or greater than 95%, equal to or greater than 96%, equal to or greater than 97%, equal to or greater than 98% or equal to or greater than 99%. The position of interest is then assigned the number assigned to the reference nucleic acid molecule. Alignment can be performed manually or by eye, particularly where the sequence identity is greater than 80%.
[000182] [000182] As used here, substantially pure means sufficiently homogeneous to appear free of readily detectable impurities, as determined by standard methods of analysis such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those skilled in the art to assess such purity, or sufficiently pure so that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purifying the compounds to produce substantially chemically pure compounds are known to those skilled in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers or isomers. In such cases, further purification could increase the specific activity of the compound.
[000183] [000183] As used herein, isolated or purified polypeptide or protein or biologically active portion thereof is substantially free of cellular material or other proteins from contamination from the cell or tissue from which the protein is derived, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preparations can be determined to be substantially free if they appear free of easily detectable impurities as determined by standard methods of analysis, such as thin layer chromatography (TLC), gel electrophoresis and high performance liquid chromatography (HPLC), used by those skilled in the art. in the art to assess such purity, or sufficiently pure such that further purification would not detectably alter the physical and chemical properties, such as enzymatic and biological activities, of the substance. Methods for purifying the compounds to produce substantially chemically pure compounds are known to those skilled in the art. A substantially chemically pure compound, however, can be a mixture of stereoisomers. In such cases, further purification can increase the specific activity of the compound.
[000184] [000184] Consequently, reference to a substantially purified polypeptide, such as a substantially purified PH20 polypeptide refers to PH20 protein preparations that are substantially free of cellular material, includes protein preparations in which the protein is separated from cellular components of the cells from which is isolated or recombinantly produced. In one embodiment, the term substantially free of cellular material includes enzyme protein preparations having less than approximately 30% (dry weight) of non-enzyme proteins (also referred to here as contamination proteins), generally less than approximately 20 % non-enzyme proteins or 10% non-enzyme proteins or less than approximately 5% non-enzyme proteins. When the enzyme protein is recombinantly produced, it is also substantially free of culture medium, i.e., culture medium represents less than approximately or in 20%, 10% or 5% of the volume of the enzyme protein preparation.
[000185] [000185] As used herein, the term substantially free of chemical precursors or other chemicals includes enzyme protein preparations in which the protein is separated from chemical precursors or other chemicals that are involved in protein synthesis. The term includes enzyme protein preparations having less than approximately 30% (dry weight), 20%, 10%, 5% or less of chemical precursors or non-enzyme components or chemicals.
[000186] [000186] As used herein, synthetic with reference to, for example, a synthetic nucleic acid molecule or a synthetic gene or a synthetic peptide refers to a nucleic acid molecule or polypeptide molecule that is produced by recombinant and / or methods by chemical synthesis methods.
[000187] [000187] As used here, production by recombinant means or using recombinant DNA methods means the use of well-known methods of molecular biology to express proteins encoded by cloned DNA.
[000188] [000188] As used here, vector (or plasmid) refers to discrete elements that are used to introduce a heterologous nucleic acid into cells for expression or replication. Vectors typically remain episomal, but can be designed to integrate a gene or portion of it into a chromosome of the genome. They are also considered vectors that are artificial chromosomes, such as artificial yeast chromosomes and artificial mammal chromosomes. The selection and use of such vehicles are well known to those skilled in the art.
[000189] [000189] As used here, an expression vector includes vectors capable of expressing DNA that is operably linked with regulatory sequences, such as promoter regions, that are capable of effecting expression of such DNA fragments. Such additional segments can include promoter and terminator sequences, and optionally can include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal and the like. Expression vectors are generically derived from plasmid or viral DNA, or can contain elements of both. Thus, an expression vector refers to a recombinant RNA or DNA construct, such as a plasmid, phage, recombinant virus or other vector that, after introduction into an appropriate host cell, results in expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic cells and / or prokaryotic cells and those that remain episomal or those that integrate into the host cell genome.
[000190] [000190] As used here, vector also includes "virus vectors" or "viral vectors". Viral vectors are constructed viruses that are operatively linked to exogenous genes to transfer (as vehicles or to and fro) exogenous genes to cells. Viral vectors include, but are not limited to, adenoviral vectors, retroviral vectors, and vaccinia virus vectors.
[000191] [000191] As used here “operatively” or “operably linked” when referring to DNA segments means that the segments are arranged so that they work in combination for their intended purposes, for example, transcription starts downstream of the promoter and upstream of any transcribed strings. The promoter is usually the domain to which the transcription machinery connects to initiate transcription and proceeds through the coding segment to the terminator.
[000192] [000192] As used herein, a conjugate refers to a modified PH20 polypeptide linked directly or indirectly to one or more other polypeptides or chemical fractions. Such conjugates include fusion proteins, those produced by chemical conjugates and those produced by any other method whereby at least one modified PH20 polypeptide is linked, directly or indirectly, to another polypeptide or chemical fraction as long as the conjugate retains hyaluronidase activity. Examples of conjugates provided here include PH20 polypeptides linked directly or indirectly to a multimerization domain (for example, an Fc fraction), a toxin, a label or a drug.
[000193] [000193] As used herein, a fusion protein refers to a polypeptide encoded by a nucleic acid sequence containing a coding sequence from one nucleic acid molecule and the coding sequence from another nucleic acid molecule in the which the coding sequences are in the same reading frame such that when the fusion construct is transcribed and translated into a host cell, the protein is produced containing the two proteins. The two molecules can be adjacent in the construction or separated by a linker polypeptide that contains 1, 2, 3 or more, but typically less than 10, 9, 8, 7 or 6 amino acids. The protein product encoded by a fusion construct is referred to as a fusion polypeptide. Examples of fusion polypeptides include Fc fusions.
[000194] [000194] As used herein, a polymer that is conjugated to a modified PH20 polypeptide refers to any polymer that is covalently or otherwise stably linked, directly or through a linker, to such a polypeptide. Such polymers typically increase the serum half-life, and include, but are not limited to, sialic fractions, polyethylene glycol (PEG) fractions, dextran, and sugar and other fractions, as for glycosylation.
[000195] [000195] As used here, the term evaluate or determine intends to include quantitative or qualitative determination in order to obtain an absolute value for the activity of a product and also to obtain an index, ratio, percentage, visual or other value indicative of the level of activity. The assessment can be direct or indirect.
[000196] [000196] As used here, a "composition" refers to any mixture of two or more products or compounds. It can be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
[000197] [000197] As used herein, a formulation refers to a composition containing at least one active pharmaceutical or therapeutic agent and one or more excipients.
[000198] [000198] As used herein, a co-formulation refers to a composition containing two or more active or pharmaceutical or therapeutic agents and one or more excipients. For example, a co-formulation of a fast-acting insulin and a hyaluronan-degrading enzyme contains a fast-acting insulin, a hyaluronan-degrading enzyme and one or more excipients.
[000199] [000199] As used here, "a combination" refers to any association between two or more items or elements. Exemplary combinations include, but are not limited to, two or more pharmaceutical compositions, a composition containing two or more active ingredients, such as two modified PH20 polypeptides, a modified PH20 polypeptide and an anti-cancer agent, such as a chemotherapeutic compound, a modified PH20 polypeptide and a therapeutic agent (e.g., an insulin), a modified PH20 polypeptide and a plurality of therapeutic agents and / or imaging, or any combination thereof. Such combinations can be packaged as kits.
[000200] [000200] As used here, a kit is a packaged combination, optionally including instructions for using the combination and / or other reactions and components for such use.
[000201] [000201] As used herein, "disease or disorder" refers to a pathological condition in an organism resulting from a cause or condition including, but not limited to, infection, acquired conditions, genetic conditions, and characterized by identifiable symptoms.
[000202] [000202] As used herein, a disease, disorder or condition associated with hyaluronan refers to any disease or condition in which levels of hyaluronan are elevated as a cause, consequence or otherwise observed in the disease or condition. Diseases and conditions associated with hyaluronan are associated with increased expression of hyaluronan in a tissue or cell, increased interstitial fluid pressure, decreased vascular volume, and / or increased water content in a tissue. Diseases, disorders or conditions associated with hyaluronan can be treated by administering a composition containing a hyaluronan-degrading enzyme, such as a hyaluronidase, for example, a soluble hyaluronidase; individually or in combination with or in addition to another treatment and / or agent. Exemplary diseases and conditions include, but are not limited to, hyaluronan-rich cancers, for example tumors, including solid tumors such as late stage cancers, metastatic cancers, undifferentiated cancers, ovarian cancer, carcinoma in situ (ISC), carcinoma squamous cell (SCC), prostate cancer, pancreatic cancer, non-small cell lung cancer, breast cancer, colon cancer and other cancers. Exemplary diseases and conditions associated with hyaluronan are also diseases that are associated with high interstitial fluid pressure, such as diseases associated with disc pressure, and edema, for example, edema caused by organ transplantation, stroke, brain trauma or other injury. Exemplary diseases and conditions associated with hyaluronan include diseases and conditions associated with high interstitial fluid pressure, decreased vascular volume, and / or increased water content in a tissue, including cancers, disc pressure and edema. In one example, treatment of the condition, disease or disorder associated with hyaluronan includes improvement, reduction or other beneficial effect on one or more of increased interstitial fluid pressure (IFP), decreased vascular volume and increased water content in a tissue.
[000203] [000203] As used here, "treatment" of a subject with a disease or condition means that the subject's symptoms are partially or totally relieved, or remain static after treatment. Consequently, treatment includes prophylaxis, therapy and / or cure. Prophylaxis refers to the prevention of a potential disease and / or the prevention of worsening symptoms or advancing a disease. The treatment also covers any pharmaceutical use of a modified interferon and compositions provided here.
[000204] [000204] As used herein, a pharmaceutically effective agent or therapeutic agent includes any bioactive agent that may have a therapeutic effect in treating a disease or disorder. Exemplary therapeutic agents are described here. Therapeutic agents include, but are not limited to, anesthetics, vasoconstrictors, dispersing agents, conventional therapeutic drugs, including small molecule drugs, including, but not limited to, bisphosphonates, and therapeutic proteins, including, but not limited to,
[000205] [000205] As used here, "insulin" refers to a hormone, precursor or a synthetic or recombinant analogue of it that acts to increase glucose absorption and / or storage and / or decrease endogenous glucose production. Insulin and its analogues are well known to a person skilled in the art, including human beings and allelic variants and species thereof. Insulin is translated as a precursor polypeptide called preproinsulin (110 amino acids for human insulin), containing a signal peptide that guides the protein to the endoplasmic reticulum (ER) into which the signal sequence is cleaved, resulting in proinsulin. Proinsulin is further processed to release a connecting chain peptide or –C (a 31 amino acid C-chain in human insulin). The resulting insulin contains an A-chain (21 amino acids long in human insulin; exposed in SEQ ID NO: 862) and a B-chain (30 amino acids long in human insulin; exposed in SEQ ID NO: 863) which are cross-linked by disulfide bonds. A fully cross-linked human insulin contains three disulfide bonds: one between position 7 of the A-chain and position 7 of the B-chain, a second between position of the A chain and position 19 of the B-chain, and a third between positions 6 and 11 of chain A. Reference to an insulin includes monomeric and multimeric insulins, including hexameric insulins, as well as humanized insulins. Exemplary insulin polypeptides are those of mammalian origin, including humans. Insulin reference includes preproinsulin, proinsulin and insulin polypeptides in single-stranded or two-stranded forms, truncated forms of which have activity, and includes allelic variants and human insulin species variants, variants encoded by junction variants and other variants, as insulin analogs. An exemplary insulin is human insulin having a sequence of amino acids from human insulin chains A and B are shown in SEQ ID NOS: 862 and 863, respectively, and variants or analogues thereof that present at least 80%, 85%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity with the same with one or both of the A-chain or B-chain and acting for increase glucose absorption and storage and / or decrease endogenous glucose production. An additional exemplary insulin is porcine insulin having an amino acid sequence for preproinsulin as set out in SEQ ID NO: 864, whereby the A chain corresponds to amino acid residue positions 88-108 and the B chain corresponds to amino acid, and variants or analogues thereof that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of sequence identity with the even with one or both A-chain or B-chain and which acts to increase glucose absorption and storage and / or decrease endogenous glucose production.
[000206] [000206] As used herein, "fast-acting insulin" refers to any insulin that has peak insulin levels within or approximately no more than four hours after subcutaneous administration to a subject. Fast-acting insulins include any insulin or any fast-acting insulin composition for acute administration in a diabetic subject in response to an effective, perceived or predicted hyperglycemic condition in the subject that arises at or approximately four hours after administration of the fast-acting insulin (as a resulting hyperglycemic dining condition or expected to result from eating a meal), so fast-acting insulin is able to prevent, control or improve the acute hyperglycemic condition. Fast-acting insulins include recombinant insulins and isolated insulins (also referred to as “regular” insulins) such as insulin sold as human insulin, porcine insulin and bovine insulin, as well as fast-acting insulin analogs (also called insulin analogs of action here) designed to be fast-acting due to amino acid changes. Exemplary regular insulin preparations include, but are not limited to, regular human insulins, such as those sold under the trademarks Humulin® R, Novolin® R and Velosulin®, Human Insulin, USP and Human Insulin Injection, USP, as well as formulations insulin acid, such as Toronto Insulin, Old Insulin, and Clear Insulin, and regular pork insulins like Iletin II® (porcine insulin). Regular insulins typically have an onset of action between 30 minutes to an hour, and a peak insulin level of 2 hours after administration.
[000207] [000207] As used here, fast acting insulin analogs (also called fast acting insulin analogs) are insulins that have a fast onset of action. Rapid insulins are typically insulin analogs that have been prepared, such as by introducing one or more amino acid substitutions, to be faster acting than regular insulins. Rapid-acting insulin analogs typically have an onset of action of 10-30 minutes after injection, with peak insulin levels seen 30-90 minutes after injection. Exemplary fast-acting insulin analogs are human insulin analogs containing one or more amino acid changes in the A and / or B chain of human insulin exposed in SEQ ID NO: 862 or 863, respectively, and showing an onset of action 10 -30 minutes after injection with peak insulin levels seen 30-90 minutes after injection. Exemplary fast-acting insulin analogues include, but are not limited to, for example, insulin lispro (eg, insulin Humalog®), insulin aspart (eg, insulin NovoLog®), and insulin glulisine (eg insulin Apidra® ) the fast-acting insulin composition sold as VIAject® and VIAtab® (see, for example, US Patent No. 7,279,457). The amino acid sequence of exemplary fast-acting insulin analogs has an A chain with an amino acid sequence exposed in SEQ ID NO: 862 and a B chain having an amino acid sequence exposed in any of SEQ ID NOS: 865-867. Also included are any other insulins that have an onset of 30 minutes or less and a peak level before 90 minutes, typically 30-90 minutes, after injection.
[000208] [000208] As used herein, a human insulin refers to an insulin that is synthetic or produced recombinantly based on the human polypeptide, including allelic variants and analogues thereof.
[000209] [000209] As used here, human fast-acting insulin or human fast-acting insulin compositions include any human insulin or human insulin composition that is fast-acting, but excludes non-human insulin, such as regular pig insulin.
[000210] [000210] As used here, the terms "basal-acting insulin" or "basal insulin" refer to insulin administered to maintain a basal insulin level as part of a general treatment regimen to treat a chronic condition such as diabetes. Typically, a basal-acting insulin is formulated to maintain an approximately steady state insulin level by the controlled release of insulin when administered periodically (for example, once or twice a day). Basal-acting insulins include crystalline insulins (eg, NPH and Lente®, protamine insulin, surfen insulin), basal insulin analogs (insulin glargine, HOE 901, NovoSol Basal) and other chemical insulin formulations (eg, gum arabic, lecithin or oil suspensions) that slow the regular insulin absorption rate. As used here, basal-acting insulin may include insulins that are typically understood to be long-acting (typically reaching a relatively low peak concentration, while having a maximum duration of action in approximately 20-30 hours) or intermediate action (typically causing peak insulin concentrations approximately 4-12 hours after administration).
[000211] [000211] As used herein, treatment means any way in which the symptoms of a condition, disorder or disease or other indication, are improved or otherwise beneficially altered.
[000212] [000212] As used here, therapeutic effect means an effect resulting from the treatment of a subject that changes, typically improves the symptoms of a disease or condition or that cures a disease or condition. A therapeutically effective amount refers to the amount of a composition, molecule or compound that results in a therapeutic effect after administration to a subject.
[000213] [000213] As used herein, the term "subject" refers to an animal, including a mammal, as a human being.
[000214] [000214] As used here, a patient refers to a human subject who presents symptoms of a disease or disorder.
[000215] [000215] As used here, improvement of the symptoms of a specific disease or disorder by a treatment, such as by administration of a pharmaceutical or other therapeutic composition, refers to any decrease, whether permanent or temporary, lasting or transient, of the symptoms that may be assigned to or associated with composition or therapeutic administration.
[000216] [000216] As used here, prevention or prophylaxis refers to methods in which the risk of developing a disease or condition is reduced.
[000217] [000217] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to the amount of an agent, compound, material or composition containing a compound that is at least sufficient to produce a therapeutic effect. Consequently, it is the amount needed to prevent, cure, improve, stop or partially stop a symptom of a disease or disorder.
[000218] [000218] As used herein, unit dose form refers to physically distinct units suitable for human and animal subjects and individually packaged as known in the art.
[000219] [000219] As used herein, a single dosage formulation refers to a formulation containing a single dose of therapeutic agent for direct administration. Single dosage formulations generally do not contain any preservatives.
[000220] [000220] As used herein, a multi-dose formulation refers to a formulation that contains multiple doses of a therapeutic agent and that can be directly administered to deliver several single doses of the therapeutic agent. Doses can be administered over the course of minutes, hours, weeks, days or months. Multidose formulations may allow dose adjustment, dose pooling and / or dose division. As multi-dose formulations are used over time, they generally contain one or more preservatives to prevent microbial growth.
[000221] [000221] As used here, an "industrial product" is a product that is made and sold. As used throughout this application, the term is intended to cover a therapeutic agent with a soluble PH20, such as esPH20, or an esPH20 individually, contained in the same or separate packaging items.
[000222] [000222] As used here, fluid refers to any composition that can flow. Fluids thus comprise compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other compositions.
[000223] [000223] As used here, a “control” or “standard” refers to a sample that is substantially identical to the test sample, except that it is not treated with a test parameter, or, if it is a plasma sample, it can be a normal volunteer unaffected by the condition of interest. A control can also be an internal control. For example, a control can be a sample, such as a virus, that has a known property or activity.
[000224] [000224] As used here, the singular forms "one", "one" and "o, a" include plural referents unless the context clearly determines otherwise. Thus, for example, reference to "an" agent includes one or more agents.
[000225] [000225] As used here, the term "or" is used to mean "and / or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
[000226] [000226] As used here, ranges and quantities can be expressed as "approximately" a specific value or range. It also includes the exact amount. Consequently, "approximately 5 bases" means "approximately 5 bases" and also "5 bases".
[000227] [000227] As used here, "optional" or "optionally" means that the subsequently described event or circumstance occurs or does not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.
[000228] [000228] As used here, the abbreviations for any protecting groups, amino acids and other compounds are, unless otherwise indicated, according to their common usage, recognized abbreviations, from the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11: 1726).
[000229] [000229] For clarity of disclosure, and not as a limitation, the detailed description is divided into the following subsections. Hyaluronidase PH20
[000230] [000230] Modified PH20 polypeptides are provided here. PH20 (also known as sperm surface protein, sperm adhesion molecule 1 or SPAM1) is a hyaluronidase that hydrolyzes hyaluronan (also called hyaluronic acid, hyaluronate or HA) found in connective tissues such as the extracellular matrix. Hyaluronan polymers are composed of repeating disaccharide units, D-glucuronic acid (GlcA) and N-acetyl-D-
[000231] [000231] PH20 cDNA has been cloned from numerous mammal species. Exemplary PH20 polypeptide precursors include, but are not limited to human PH20 polypeptides (SEQ ID NO: 6), bovine (SEQ ID NOS: 15 or 17), rabbit (SEQ ID NO: 23), Cynomolgus monkey (SEQ ID NO: 13 ), guinea pig (SEQ ID NO: 28), rat (SEQ ID NO: 21), mouse (SEQ ID NO: 19), chimpanzee (SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 869 ) Rhesus monkey (SEQ ID NO: 11), fox (SEQ ID NO: 30), Gibbon (SEQ ID NO: 856), marmoset (SEQ ID NO: 858) or orangutan (SEQ ID NO: 860). The mRNA transcript is typically translated to generate a precursor protein containing a 35 amino acid signal sequence at the N-terminus. After transport to the ER, the signal peptide is removed to provide a mature PH20 polypeptide. Exemplary mature PH20 polypeptides include, but are not limited to, human PH20 polypeptides (SEQ ID NO: 7), bovine (SEQ ID NOS: 16 or 18), rabbit (SEQ ID NO: 24), Cynomolgus monkey (SEQ ID NO: 14), guinea pig (SEQ ID NO: 29), rat (SEQ ID NO: 22), mouse (SEQ ID NO: 20), chimpanzee (SEQ ID NO: 10 or SEQ ID NO: 870), monkey Rhesus (SEQ ID NO: 12), Fox (SEQ ID NO: 31), Gibbon (SEQ ID NO: 857), Marmoset (SEQ ID NO: 859) or orangutan (SEQ ID NO: 861). For example, the human PH20 mRNA transcript is normally translated to generate a 509 amino acid precursor protein (SEQ ID NO: 6) containing a sequence of 35 amino acid signals at the N-terminus (amino acid residue positions 1-35 of SEQ ID NO: 6) thus, after transport to the ER and removal of the signal peptide, a mature 474 amino acid polypeptide with an amino acid sequence exposed in SEQ ID NO: 7 is produced, PH20 sequences from sheep are also known (see, for example, SEQ ID NOS: 25-27).
[000232] [000232] In particular, human PH20 has the amino acid sequence exposed in SEQ ID NO: 6. Mature human PH20 that does not have a signal sequence is exposed in SEQ ID NO: 7. Allele variants and other PH20 variants are known . Other PH20 sequences have been reported. For example, a PH20 variant is known to be exposed in the precursor sequence exposed in SEQ ID NO: 68 that contains Ala at position 48 and a Trp at position 499, or the mature sequence of it exposed in SEQ ID NO: 69 containing the corresponding differences in positions 13 and 464, respectively, compared to the sequence shown in SEQ ID NO: 7 (see, for example, Gmachl et al. (1993) FEBS Lett., 336: 545-548; GenBank accession No. AAC60607 ). In addition, a natural PH20 variant has been identified containing a Glutamine (Gln; Q) at position 5 as compared to the amino acid precursor sequence exposed in SEQ ID NO: 6 (see for example, SEQ ID NO: 70, see also Varela et al. (2011) Nature, 469: 539-542). Another natural variant contains an Alanine (Ala; A) at position 47 compared to the amino acid sequence shown in SEQ ID NO: 6 (as shown in SEQ ID NO: 71) and corresponding to position 12 compared to the exposed amino acid sequence in SEQ ID NO: 3 or 7 (as set out in SEQ ID NO: 72).
[000233] [000233] The sequence and structure of PH20 polypeptides is highly conserved. The sequence identity between PH20 proteins from various species is approximately 50% to 90%. The hydrophobic N-terminal signal sequence of 35 amino acids in length is generally conserved among PH20 hyaluronidase polypeptides. PH20 hyaluronidases contain a common core hyaluronidase domain region of approximately 340 amino acids in length that corresponds to amino acid residues 38-374 of the precursor human PH20 sequence exposed in SEQ ID NO: 6. A mature PH20 polypeptide that lacks the signal sequence and contains a contiguous amino acid sequence having a C-terminal amino acid residue corresponding to amino acid residue 464 of SEQ ID NO: 6 (for example, amino acid residues corresponding to positions 36- 464 of the amino acid sequence set out in SEQ ID NO: 6) is the minimum sequence required for hyaluronidase activity (see, for example, US patent application No. 10 / 795,095, which is issued as US patent No. 7,767,429; see also US publication No. US20100143457).
[000234] [000234] In the common hyaluronidase domain region, at least 57 amino acids are conserved between species (see for example, Arming et al. (1997) Eur. J. Biochem., 247: 810-814; ten Have and others (1998) Reprod. Fertil. Dev., 10: 165-72; Chowpongpang et al. (2004) Biotechnology Letters, 26: 1247-1252). For example, PH20 hyaluronidases contain 12 conserved cysteine residues corresponding to amino acid residue 25, 189, 203, 316, 341, 346, 352, 400, 402, 408, 423 and 429 of the amino acid sequence of a mature PH20 without signal sequence as shown in SEQ ID NO: 3 or 7 (corresponding to amino acid residues 60, 224, 238, 351, 376, 381, 387, 435, 437, 443, 458 or 464 of full-length human PH20 exposed in SEQ ID NO: 6). Cysteine residues corresponding to 25 and 316 and cysteine residues corresponding to 189 and 203 form disulfide bonds. The other cysteine residues also form disulfide bonds, are involved in post-translational protein maturation and / or activity modulation. For example, four additional disulfide bonds are formed between the C376 and C387 cysteine residues; between C381 and C435; between C437 and C443; and between C458 and C464 of the polypeptide exemplified in SEQ ID NO: 6 (corresponding to positions C341 and C352; between C346 and C400; between C402 and C408; and between C423 and C429 of the mature polypeptide exposed in SEQ ID NO: 3 or 7, respectively).
[000235] [000235] Amino acid residues corresponding to amino acid residue D111, E113 and E249 of the amino acid sequence exposed in SEQ ID NO: 3 or 7 are acid residues part of the active enzyme site and are conserved among PH20 species. Amino acid residues R176, R246, R252 of the amino acid sequence exposed in SEQ ID NO: 3 or 7 are also conserved between species and contribute to substrate binding and / or hyaluronidase activity. Amino acid mutations D111N, E113Q, R176G, E249N and R252T result in enzymes that have no detectable enzyme activity or enzyme activity (see, for example, Arming et al. (1997) Eur. J. Biochem., 247: 810-814).
[000236] [000236] The results here confirm the requirement for PH20 amino acid residues corresponding to positions 25, 111, 113, 176, 189, 203, 246, 249, 252, 316, 341, 346, 352, 400, 402, 408, 423 and 429 of the amino acid sequence exposed in a mature PH20 that lacks the signal sequence as shown in SEQ ID NO: 3 or 7 for hyaluronidase activity, since mutagenesis of these residues results in an enzyme that is not active (for example, example, is not expressed or is inactive when expressed, see, for example, tables 5 and 10). The exception is that the amino acid substitution corresponding to R176K and C316D resulted in mutants that generated some residual hyaluronidase activity.
[000237] [000237] Glycosylation is also required for PH20 hyaluronidase activity based on the NxS or NxT recognition motive. There are six N-linked oligosaccharides in the amino acid residues corresponding to positions N47, N131, N200, N219, N333 and N358 of the amino acid sequence exposed in SEQ ID NO: 3 or 7 (corresponding to amino acid residues N82, N166, N235, N254 , N368 and N393 of human PH20 exposed in SEQ ID NO: 6). In particular, at least N-linked glycosylation sites corresponding to amino acid residues N200, N333 and N358 are required for enzyme secretion and / or activity (see for example, US publication No. US20100143457). For example, a PH20 polypeptide containing amino acid mutations N200A, N333A, N358A or N333A / N393A results in inactive proteins. Single mutations of N47A, N131A, N219A, N47A / N131A, N47A / N219A, N131A / N291A glycosylation sites retain activity. The N-linked glycosylation site corresponding to human PH20 amino acid residue N368 exposed in SEQ ID NO: 6 is conserved between species (see for example, Chowpongpang et al. (2004) Biotechnology Letters, 26: 1247-1252). Hyaluronidases PH20 also contains O-linked glycosylation sites. For example, human PH20 has an O-linked oligosaccharide at the amino acid residue corresponding to amino acid T440 of the amino acid sequence set out in SEQ ID NO: 3 or 7 (corresponding to amino acid residue T475 in SEQ ID NO: 6).
[000238] [000238] In addition to the catalytic sites, PH20 also contains a hyaluronan binding site. This site is located in the peptide 2 region, which corresponds to amino acid positions 205-235 of the precursor polypeptide exposed in SEQ ID NO: 6 and positions 170-200 of the mature polypeptide exposed in SEQ ID NO: 3 or 7. This region is highly conserved among hyaluronidases and is similar to the motive for heparin binding. Mutation of the arginine residue at position 176 (corresponding to the mature PH20 polypeptide exposed in SEQ ID NO: 3 or 7) to a glycine results in a polypeptide with only approximately 1% of the hyaluronidase activity of the wild type polypeptide (Arming and others , (1997) Eur. J. Biochem. 247: 810-814).
[000239] [000239] PH20 polypeptides contain a glycosyl phosphatidylinositol (GPI) anchor attached to the C-terminus of the protein that anchors the protein to the extracellular leaflet of the plasma cell membrane. At least PH20 human, monkey, mouse and guinea pig are strongly linked to the plasma membrane through the GPI anchor, which can be released by treating with phosphatidylinositol C specific phospholipase (PI-PLC; see for example, Lin et al. ( 1994) Journal of Cell Biology, 125: 1157-1163; Lin et al (1993) Proc. Natl. Acad. Sci., 90: 10071-10075). Other PH20 enzymes, such as bovine PH20, are loosely attached to the plasma membrane and are not anchored through a phospholipase-sensitive anchor. As discussed below, soluble active forms that, when expressed, are not bound to the membrane but are secreted, can be generated by removing an entire portion of the GPI anchor binding site (see also US patent No. 7,767,429; US publication No. US20100143457). These include, for example, soluble PH20 Polypeptides exposed in any of SEQ ID NOS: 3 or 32-66, or precursor forms thereof containing a signal sequence.
[000240] [000240] GPI-anchored proteins, for example, human PH20, are translated with a cleavable N-terminal signal peptide that guides the protein to the endoplasmic reticulum (ER). At the C-terminus of these proteins is another sequence of signals that guides the addition of a preformed GPI anchor for the polypeptide in the ER lumen. The addition of the GPI anchor occurs after cleavage of the C-terminal portion at a specific amino acid position, called the  site (typically located approximately 20-30 amino acids from the C-terminus). Although there appears to be no consensus sequence to identify the location of the  site, GPI-anchored proteins contain a C-terminal or anchor-GPI fixation signal sequence that typically contains a predominantly hydrophobic region of 8-20 amino acids, preceded by a hydrophilic spacer region of 8-12 amino acids immediately downstream of the sítio-site. This hydrophilic spacer region is often rich in charged amino acids and proline (White et al. (2000) J. Cell Sci. 113 (Pt.4): 721-727). There is generally a region of approximately 11 amino acids before the ω-1 position that is characterized by a low amount of predicted secondary structure, a region around the cleavage site (ω-site), from ω-1 to ω + β by the presence of small side chain residues, the spacer region between positions ω + 3 and ω + 9, and a hydrophobic tail of ω + 10 at the C-terminus (Pierleoni et al., (2008) BMC Bioinformatics 9: 392).
[000241] [000241] Although there is no consensus GPI anchor signal signal sequence, several in silico methods and algorithms have been developed that can be used to identify such sequences in polypeptides (see, for example, Udenfriend et al. (1995) Methods Enzymol. 250: 571-582; Eisenhaber et al (1999) J. Mol. Chem. 292: 741-758; Kronegg and Buloz, (1999), “Detection / prediction of GPI cleavage site (GPI-anchor) in a protein (DGPI ), ”
[000242] [000242] The covalent attachment of a GPI anchor to the C-terminus of human PH20 and, therefore, the membrane-bound nature of PH20, has been confirmed using studies of phosphatidylinositol C specific phospholipase (PI-PLC) (see for example, Lin et al. (1994) J. Biol. Chem. 125: 1157-1163). Phosphatidylinositol specific phospholipase C (PI-PLC) and D (PI-PLD) hydrolyze the GPI anchor, releasing the PH20 polypeptide from the cell membrane. The prior art literature reports that a human PH20 de site cleavage site is identified between Ser-490 and Ala-491 and for monkey PH20 it is identified between Ser491 and Thr492 (Lin et al (1993) Proc. Natl. Acad Sci, (1993) 90: 10071-10075). Thus, the literature reports that a human PH20 GPI anchor-fixing signal sequence is located at amino acid positions 491-509 of the precursor polypeptide exposed in SEQ ID NO: 6, and the -site is amino acid position 490 Thus, in this modeling of human PH20, amino acids 491-509 are cleaved after transport to the ER and a GPI anchor is covalently attached to the serine residue at position 490.
[000243] [000243] PH20 is normally expressed in sperm from a specific single testis gene. PH20 is a protein associated with sperm involved in fertilization. PH20 is normally located on the sperm surface, and on the lysosome-derived acrosome, where it is attached to the internal acrosomal membrane. PH20 is multifunctional and has hyaluronidase activity, cell signaling activity mediated by hyaluronan (HA), and acts as a sperm receptor for the pellucid zone that surrounds the oocyte when present in sperm reacted with acrosome (AR). For example, PH20 is naturally involved in egg-sperm adhesion and assists sperm penetration of the cumulus cell layer by digesting hyaluronic acid. In addition to being a hyaluronidase, PH20 also appears to be a receptor for HA-induced cell signaling, and a receptor for the pellucid zone surrounding the oocyte. Due to the role of PH20 in fertilization, PH20 can be used as an antigen for immunocontraception.
[000244] [000244] PH20 is a neutral active hyaluronidase, although it may show active acid activity in some cases. PH20 hyaluronidase activity is shown by PH20 associated with internal acrosomal membrane and plasma membrane. The plasma membrane PH20 shows hyaluronidase activity only at neutral pH, while the PH20 associated with the internal acrosomal membrane shows active enzyme activity with acid. The structural basis for these differences is due to the presence of two catalytic sites in PH20. A first catalytic site is called the Peptide 1 region, corresponding to amino acid residues 142-172 of SEQ ID NO: 6, which is involved in PH20 enzyme activity at neutral pH. A second catalytic site is called the peptide 3 region, corresponding to amino acid residues 277-297 of SEQ ID NO: 6, which is involved in enzyme activity at a lower pH. A change in the PH20 structure associated with the internal acrosomal membrane occurs after the acrosome reaction, so PPH20 is endoproteolytically cleaved, but retained together by disulfide bonds. The result of endoproteolysis is that the peptide 3 region is activated and can thus perform acidic and neutral activity for PH20 (see for example, Cherr et al. (2001) Matrix Biology, 20: 515-525.) Also, after acrosome reaction, lower molecular weight forms are generated by release from the internal acrosomal membrane (for example, a soluble 53 kDa form of PH20 is generated in ape). The lower molecular weight form (s) is also active in acid.
[000245] [000245] The hyaluronidase activity of PH20 accounts for the dispersion activity seen in extracts from animal testicles that have been used clinically for decades to increase drug dispersion and absorption
[000246] [000246] PH20 can exist in membrane-associated or membrane-bound form, or it can be secreted into the medium when expressed from cells, and thus can exist in soluble form. Soluble PH20 can be detected and discriminated from membrane-bound, insoluble PH20 using methods well known in the art, including, but not limited to, those using a Triton® X-114 assay. In this test, soluble PH20 hyaluronidases divide into the aqueous phase of a solution of Triton® X-114 heated to 37 ° C (Bordier et al., (1981) J. Biol. Chem., 256: 1604-7) while PH20 hyaluronidases anchored by membrane divide into the detergent-rich phase. Thus, in addition to using algorithms to assess whether a PH20 polypeptide is naturally anchored by GPI and consequently membrane bound, solubility experiments can also be performed.
[000247] [000247] Soluble PH20 enzymes include hyaluronidases that contain a GPI anchor fixation signal sequence, but are loosely attached to the membrane in such a way that they do not contain a phospholipase sensitive anchor. For example, soluble PH20 polypeptides include ovine or bovine PH20. Various forms of such soluble PH20 hyaluronidases have been prepared and approved for therapeutic use in subjects, including humans. For example, animal-derived hyaluronidase preparations include Vitrase® (ISTA Pharmaceuticals), a purified sheep testicular hyaluronidase and Amphadase® (Amphastar Pharmaceuticals), a bovine testicular hyaluronidase. Soluble PH20 enzymes also include truncated forms of PH20 hyaluronidases associated with a human or non-human membrane that lack one or more amino acid residues from a glycosylphosphatidylinositol (GPI) anchor fixation signal sequence and that retain hyaluronidase activity (see for example US patent No. 7,767,429; US publication No. US20100143457). Thus, instead of having a GPI anchor covalently attached to the C end of the protein in the ER and being anchored to the extracellular leaflet of the plasma membrane, these polypeptides are secreted when expressed in cells and are soluble. In instances where the soluble hyaluronan-degrading enzyme retains a portion of the GPI anchor fixing signal sequence, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residues in the signal sequence anchor-fixing devices can be retained, with the proviso that the polypeptide is soluble (that is, secreted when expressed from cells) and active.
[000248] [000248] Exemplary soluble hyaluronidases that are C-terminally truncated and lack all or a portion of the GPI anchor fixation signal sequence include, but are not limited to, PH20 polypeptides of primate origin, such as PH20 polypeptides humans and chimpanzees. For example, soluble PH20 polypeptides can be made by C-terminal truncation of a polypeptide exposed in SEQ ID NOS: 7, 10, 12, 14, 69, 72, 857, 859, 861 or 870 or variants thereof that exhibit at least minus 80%, 85%, 90%, 95% or more of sequence identity to any of SEQ ID NO: 7, 10, 12, 14, 69, 72, 857, 859, 861 or 870, where the polypeptide The resulting is active, soluble and lacks all or a portion of amino acid residues from the anchor-GPI signal sequence.
[000249] [000249] Exemplary soluble PH20 polypeptides are C-terminal truncated human PH20 polypeptides that are mature (do not have a signal sequence), soluble and have neutral activity, and that contain a contiguous amino acid sequence exposed in SEQ ID NO: 6 or SEQ ID NO: 7 which minimally has a C-terminal truncated amino acid residue on or after amino acid residue 464 of the amino acid sequence exposed in SEQ ID NO: 6. For example, soluble PH20 polypeptides include C-terminal truncated polypeptides that minimally contain a contiguous amino acid sequence 36-464 of SEQ ID NO: 6, or include an amino acid sequence that has at least
[000250] [000250] In particular, a soluble human PH20 polypeptide is a polypeptide that is truncated after amino acid 482 of the sequence shown in SEQ ID NO: 6. Such a polypeptide can be generated from a nucleic acid molecule containing a signal sequence and encoding amino acids 36-482, for example, as set out in SEQ ID NO: 1 (containing an IgG kappa signal sequence) or SEQ ID NO: 67 (containing the native signal sequence). Post-translational processing removes the signal sequence, leaving a recombinant human PH20 soluble in 447 amino acids (SEQ ID NO: 3). A product produced after expression of a vector exposed in SEQ ID NO: 4 or 5, and containing a nucleic acid molecule exposed in SEQ ID NO: 67, results in a secreted product, called rHuPH20, in the culture medium that has heterogeneity in the C-terminus such that the product includes a mixture of species that can include any one or more of SEQ ID NOS: 3 and 44-48 in varying abundance. Typically, rHuPH20 is produced in cells that facilitate correct N-glycosylation to retain activity, such as mammalian cells, for example, CHO cells (for example, DG44 CHO cells). Hylenex® (Halozyme) is a recombinant human hyaluronidase produced by genetically engineered Chinese hamster ovary (CHO) cells containing nucleic acid that encodes a truncated human PH20 polypeptide (designated rHuPH20). C. Modified PH20 polypeptides
[000251] [000251] Variant or modified PH20 polypeptides are provided here. The modified PH20 polypeptides provided here have altered activities or properties compared to a wild-type, native or reference PH20 polypeptide. Included among the modified PH20 polypeptides provided here are PH20 polypeptides that are active mutants, so the polypeptides exhibit at least 40% of the hyaluronidase activity of the corresponding PH20 polypeptide without containing the amino acid modification (for example, amino acid substitution). In particular, PH20 polypeptides which exhibit hyaluronidase activity and which show increased stability compared to PH20 without containing the amino acid modification are provided here. Modified PH20 polypeptides are also provided which are inactive, and which can be used, for example, as antigens in contraceptive vaccines.
[000252] [000252] The modifications may be a single amino acid modification, such as single amino acid substitutions, insertions or deletions, or multiple amino acid modifications, such as multiple amino acid substitutions, insertions or deletions. Exemplary modifications are amino acid substitutions, including single or multiple amino acid substitutions. The amino acid substitution can be a conservative substitution, as set out in Table 2, or a non-conservative substitution, like any described here. Modified PH20 polypeptides provided here may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more modified positions compared to PH20 polypeptide not containing the modification.
[000253] [000253] The modifications described here can be in any PH20 polypeptide, including, mature C-terminal truncated forms, or precursor, provided that the modified form has hyaluronidase activity.
[000254] [000254] In particular, PH20 polypeptides are provided here that contain modifications compared to a PH20 polypeptide exposed in SEQ ID NO: 3, 7, 32-66, 69 or 72, or a polypeptide that has an amino acid sequence that is at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% identical to any of SEQ ID NOS: 3, 7, 32-66, 69 or 72. For example, the modifications provided here can also be made on a PH20 polypeptide exposed as SEQ ID NO: 10, 12 , 14, 24, 857, 859, 861 or 870.
[000255] [000255] In particular, modified soluble PH20 polypeptides are provided here which are PH20 polypeptides containing a modification provided here, and which when expressed from cells are secreted in the medium as a soluble protein. For example, modifications are made to a soluble PH20 polypeptide that is truncated at the C-terminus at or near the C end portion containing the GPI anchor signal sequence of a PH20 polypeptide that contains a GPI anchor signal sequence. The C-terminal truncation can be a truncation or deletion of 8 contiguous amino acids at the C-terminus, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
[000256] [000256] Modifications can also be made in the corresponding precursor form containing a signal peptide of any of SEQ ID NOS: 3, 7, 10, 12, 14, 16, 18, 20, 22, 24-27, 29, 31, 32-66, 69, 72, 857, 859, 861 or
[000257] [000257] In the examples of modified PH20 polypeptides provided here, the modified PH20 polypeptide does not contain the amino acid sequence exposed in any of SEQ ID NOS: 3-66, 68-72, 856-861, 869 or 870. Typically, the polypeptide Modified PH20 is a human PH20 polypeptide and does not contain the amino acid sequence exposed in any of SEQ ID NOS: 8-31, 856- 861, 869 or 870.
[000258] [000258] Generically, any modification such as amino acid substitution, deletion or substitution, can be made on a PH20 polypeptide, with the proviso that the modification is not an amino acid substitution where the only modification is substitution of a single amino acid which is V12A , N47A, D111N, E113Q, N131A, R176G, N200A, N219A, E249Q, R252T, N333A or N358A. Also, where the modified PH20 polypeptide contains only two amino acid substitutions, the amino acid substitutions are not P13A / L464W, N47A / N131A, N47A / N219A, N131A / N219A or N333A / N358A. In an additional example, where the modified PH20 polypeptide contains only three amino acid substitutions, the amino acid substitutions are not N47A / N131A / N219A. Exemplary modifications provided here are described in detail below.
[000259] [000259] For purposes of the present invention, reference to positions and amino acids for modification in the present invention, including amino acid substitution or substitutions, are with reference to the PH20 polypeptide set out in SEQ ID NO: 3. It is understood at the level of a person skilled in the art to make any of the modifications provided here in another PH20 polypeptide by identifying the corresponding amino acid residue in another PH20 polypeptide, like any exposed in SEQ ID NOS: 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24-27, 28, 29, 30, 31, 32-66, 68-72, 856, 857 , 858, 859, 860, 861, 869 or 870 or a variant thereof that has at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity for any of SEQ ID NOS: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24-27, 28, 29, 30, 31, 32-66, 68-72, 856, 857, 858, 859, 860, 861, 869 or 870. Corresponding positions in another PH20 polypeptide can be identified by aligning the PH20 polypeptide with reference to the PH20 polypeptide exposed in SEQ ID NO: 3. For example, Figure 2 represents alignment of P polypeptides H20 specimens with SEQ ID NO: 3, and identification of corresponding specimen positions.
[000260] [000260] Modifications to a PH20 polypeptide can also be made to a PH20 polypeptide which also contains other modifications, including modifications to the primary sequence and modifications not to the primary polypeptide sequence. For example, modifications described here can be on a PH20 polypeptide that is a fusion polypeptide or chimeric polypeptide. The modified PH20 polypeptides provided here also include polypeptides that are conjugated to a polymer, such as a PEG reagent.
[000261] [000261] Nucleic acid molecules encoding any of the modified PH20 polypeptides provided here are also provided here. In specific examples, the nucleic acid sequence can be codon-optimized, for example, to increase expression levels of the encoded sequence. The use of a specific codon depends on the host organism in which the modified polypeptide is expressed. A person skilled in the art is familiar with optimal codons for expression in mammalian or human cells,
[000262] [000262] The modified polypeptides and coding nucleic acid molecules provided here can be produced by standard recombinant DNA techniques known to a person skilled in the art. Any method known in the art to mutate any one or more amino acids in a target protein can be employed. The methods include random or site-directed mutagenesis of standard encoding nucleic acid molecules, or solid phase polypeptide synthesis methods. For example, nucleic acid molecules that encode a PH20 polypeptide may undergo mutagenesis, such as random mutation of the encoding nucleic acid, error-prone PCR, site-directed mutagenesis, overlapping PCR, gene mixing or other recombinant methods. The nucleic acid encoding the polypeptides can then be introduced into a host cell to be expressed heterologously. Consequently, by also providing here nucleic acid molecules that encode any of the modified polypeptides provided here. In some examples, modified PH20 polypeptides are produced synthetically, as using solid phase or solution phase peptide synthesis.
[000263] [000263] In the subsections below, the exemplary modified PH20 polypeptide showing altered properties and activities, and encoding nucleic acid molecules, provided here are described.
[000264] [000264] Modified PH20 polypeptides which contain one or more amino acid substitutions in a PH20 polypeptide and which exhibit hyaluronidase activity are provided here. Modified PH20 polypeptides can exhibit 40% to 5000% of the hyaluronidase activity of a PH20 polypeptide of the wild type or reference, such as the polypeptide shown in SEQ ID NOS: 3 or 7. For example, modified PH20 polypeptides provided here have at least 40% of hyaluronidase activity, such as at least 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190 %, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%, 2000%, 3000% or more of the hyaluronidase activity of a reference PH20 polypeptide or wild type , such as the corresponding polypeptide not containing the amino acid modification (for example, amino acid substitution), for example, a polypeptide shown in SEQ ID NO: 3 or 7. For example, exemplary positions that can be modified, for example, by substitution of amino acids, include, but are not limited to any of the positions corresponding to position 1, 2, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
[000265] [000265] To retain hyaluronidase activity, modifications are typically not made in those positions that are less tolerant of change or required for hyaluronidase activity.
[000266] [000266] Exemplary amino acid substitutions in any of the corresponding positions above are set out in table 3. Reference to the corresponding amino acid position in table 3 is with reference to positions set out in SEQ ID NO: 3. It is understood that substitutions can be made in corresponding position in another PH20 polypeptide by aligning it with the sequence shown in SEQ ID NO: 3 (see, for example, figures 1 and 2), so the corresponding position is the aligned position. In specific examples, the amino acid substitution (s) may be in the corresponding position on a PH20 polypeptide as set forth in any of SEQ ID Nos: 2, 3, 6-66, 68-72, 856-861 , 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity thereto, as long as the resulting modified PH20 polypeptide exhibits at least 40% hyaluronidase activity of the corresponding PH20 polypeptide not containing the amino acid substitution. In particular, the substitution (s) may be in a corresponding position in a human PH20 polypeptide, for example, any exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72, or a variant thereof that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity for any of the SEQ ID NOS : 3, 7, 32-66, 69 or 72. In one example, one or more of the substitutions are in SEQ ID NO: 3, provided that the resulting modified PH20 polypeptide exhibits at least 40% of the hyaluronidase activity of the exposed PH20 polypeptide in SEQ ID NO: 3. TABLE 3: Active mutants Substitution position Substitution position Substitution position o o cor rente spondent pondente teente 1 A C E F G H K 2 A C G I L P Q 3 E H L Y N P Q R S T V S T V
[000267] [000267] In specific examples, a modified PH20 polypeptide containing an amino acid substitution or substitutions in one position or positions corresponding to 1, 6, 8, 9, 10, 11, 12, 14, 15, 20, 22 is provided here 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 46, 47, 48, 49, 50, 52, 58, 59, 63, 67, 68, 69, 70, 71, 72, 73, 74, 75, 79, 82, 83, 84, 86, 87, 89, 90, 92, 93, 94, 97, 102, 104, 107, 114, 118, 120, 127, 128, 130, 131, 132, 135, 138, 139, 140, 141, 142, 143, 144, 146, 147, 148, 149, 150, 151, 152, 155, 156, 158, 160, 162, 163, 164, 165, 166, 167, 169, 170, 172, 173, 174, 175, 178, 179, 193, 195, 196, 198, 204, 205, 206, 209, 212, 213, 215, 219, 220, 221, 222, 232, 233, 234, 235, 236, 237, 238, 240, 247, 248, 249, 257, 258, 259, 260, 261, 263, 267, 269, 271, 272, 273, 274, 276, 277, 278, 279, 282, 283, 285, 287, 289, 291, 292, 293, 298, 305, 307, 308, 309, 310, 313, 314, 315, 317, 318, 320, 321, 324, 325, 326, 328, 335, 347, 349, 351, 353, 356, 359 , 367, 368, 369, 371, 373, 374, 375, 376, 377, 380, 381, 383, 385, 389, 392, 393, 395, 396, 399, 401, 404, 405, 406, 407, 409 , 410, 412, 416, 418, 419, 421, 425, 427, 428, 431, 433, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446 or 447 with reference to positions of amino acids exposed in SEQ ID NO: 3. For example, amino acid positions can be substitutions at positions corresponding to the substitution of Leucine (L) at position 1 (L1), P6, V8, I9, P10, N11, V12, F14, L15, A20, S22, F24, L26 , G27, K28, F29, D30, E31, P32, L33, D34, M35, S36, L37, F38, S39, F40, I41, I46, N47, A48, T49, G50, G52, V58, D59, Y63, I67 , D68, S69, I70, T71, G72, V73, T74, V75, I79, K82, I83, S84, G86, D87, L89, D90, A92, K93, K94, T97, V102, N104, M107, E114, T118 , A120, D127, V128, K130, N131, R132, E135, Q138, Q139, Q140, N141, V142, Q143, L144, L146, T147, E148, A149, T150, E151, K152, Q155, E156, E158, A160 , K162, D163, F164, L165,
[000268] [000268] Exemplary amino acid substitutions in the modified PH20 polypeptides provided here include, but are not limited to, substitution with: histidine (H) in a position corresponding to position 1; A in a position corresponding to position 1; And in a position corresponding to position 1; G in a position corresponding to position 1; K in a position corresponding to position 1; Q in a position corresponding to position 1; R in a position corresponding to position 1; A in a position corresponding to position 6; M in a position corresponding to position 8; Q in a position corresponding to position 9; G in a position corresponding to position 10; H in a position corresponding to position 10; S in a position corresponding to position 11; And in a position corresponding to position 12; I in a position corresponding to position 12; K in a position corresponding to position 12; T in a position corresponding to position 12; V in a position corresponding to position 14; V in a position corresponding to position 15; M in a position corresponding to position 15; S in a position corresponding to position 20; T in a position corresponding to position 22; And in a position corresponding to position 24; H in a position corresponding to position 24; R in a position corresponding to position 24; A in a position corresponding to position 26; And in a position corresponding to position 26; K in a position corresponding to position 26; M in a position corresponding to position 26; Q in a position corresponding to position 26; R in a position corresponding to position 26; D in a position corresponding to position 27; K in a position corresponding to position 27; R in a position corresponding to position 27; R in a position corresponding to position 28; And in a position corresponding to position 29; I in a position corresponding to position 29; K in a position corresponding to position 29; L in a position corresponding to position 29; M in a position corresponding to position 29; P in a position corresponding to position 29; R in a position corresponding to position 29; S in a position corresponding to position 29; T in a position corresponding to position 29; V in a position corresponding to position 29; G in a position corresponding to position 30; H in a position corresponding to position 30; K in a position corresponding to position 30; L in a position corresponding to position 30; M in a position corresponding to position 30; R in a position corresponding to position 30; S in a position corresponding to position 30; A in a position corresponding to position 31; C in a position corresponding to position 31; G in a position corresponding to position 31; H in a position corresponding to position 31; I in a position corresponding to position 31; K in a position corresponding to position 31; L in a position corresponding to position 31; P in a position corresponding to position 31; R in a position corresponding to position 31; S in a position corresponding to position 31; T in a position corresponding to position 31; V in a position corresponding to position 31; W in a position corresponding to position 31; C in a position corresponding to position 32; F in a position corresponding to position 32; G in a position corresponding to position 32; H in a position corresponding to position 32; W in a position corresponding to position 33; G in a position corresponding to position 33; W in a position corresponding to position 34; Q in a position corresponding to position 35; V in a position corresponding to position 35; H in a position corresponding to position 36; N in a position corresponding to position 36; F in a position corresponding to position 37; M in a position corresponding to position 37; Y in a position corresponding to position 38; A in a position corresponding to position 39; L in a position corresponding to position 39; N in a position corresponding to position 39; T in a position corresponding to position 39; L in a position corresponding to position 40; T in a position corresponding to position 41; L in a position corresponding to position 46; R in a position corresponding to position 46; D in a position corresponding to position 47; F in a position corresponding to position 47; T in a position corresponding to position 47; W in a position corresponding to position 47, with F in a position corresponding to position 48; H in a position corresponding to position 48; K in a position corresponding to position 48; N in a position corresponding to position 48; R in a position corresponding to position 49; D in a position corresponding to position 50; S in a position corresponding to position 50; M in a position corresponding to position 50; N in a position corresponding to position 52; Q in a position corresponding to position 52; R in a position corresponding to position 52; S in a position corresponding to position 52; T in a position corresponding to position 52; C in a position corresponding to position 58; K in a position corresponding to position 58; L in a position corresponding to position 58; P in a position corresponding to position 58; Q in a position corresponding to position 58; R in a position corresponding to position 58; H in a position corresponding to position 58; N in a position corresponding to position 58; Y in a position corresponding to position 58; N in a position corresponding to position 59; K in a position corresponding to position 63; L in a position corresponding to position 63; M in a position corresponding to position 63; R in a position corresponding to position 63; W in a position corresponding to position 63; V in a position corresponding to position 67; H in a position corresponding to position 68; P in a position corresponding to position 68; Q in a position corresponding to position 68; A in a position corresponding to position 69; C in a position corresponding to position 69; And in a position corresponding to position 69; F in a position corresponding to position 69; G in a position corresponding to position 69; I in a position corresponding to position 69; L in a position corresponding to position 69; M in a position corresponding to position 69; P in a position corresponding to position 69; R in a position corresponding to position 69; T in a position corresponding to position 69; W in a position corresponding to position 69; Y in a position corresponding to position 69; A in a position corresponding to position 70; C in a position corresponding to position 70; F in a position corresponding to position 70; G in a position corresponding to position 70; H in a position corresponding to position 70; K in a position corresponding to position 70; L in a position corresponding to position 70; N in a position corresponding to position 70; P in a position corresponding to position 70; R in a position corresponding to position 70; S in a position corresponding to position 70; T in a position corresponding to position 70; V in a position corresponding to position 70; Y in a position corresponding to position 70; G in a position corresponding to position 71; N in a position corresponding to position 71; R in a position corresponding to position 71; S in a position corresponding to position 71; K in a position corresponding to position 72; M in a position corresponding to position 72; Q in a position corresponding to position 72; A in a position corresponding to position 73; H in a position corresponding to position 73; K in a position corresponding to position 73; L in a position corresponding to position 73; Q in a position corresponding to position 73; R in a position corresponding to position 73; T in a position corresponding to position 73; W in a position corresponding to position 73; A in a position corresponding to position 74; C in a position corresponding to position 74; And in a position corresponding to position 74; F in a position corresponding to position 74; G in a position corresponding to position 74; H in a position corresponding to position 74; K in a position corresponding to position 74; L in a position corresponding to position 74; M in a position corresponding to position 74; N in a position corresponding to position 74; P in a position corresponding to position 74; R in a position corresponding to position 74; S in a position corresponding to position 74; V in a position corresponding to position 74; W in a position corresponding to position 74; F in a position corresponding to position 75; L in a position corresponding to position 75; M in position corresponding to position 75; R in a position corresponding to position 75; T in a position corresponding to position 75; L in a position corresponding to position 79; L in a position corresponding to position 82; N in a position corresponding to position 82; V in a position corresponding to position 83; Q in a position corresponding to position 83; S in a position corresponding to position 83; G in a position corresponding to position 83; And in a position corresponding to position 84; F in a position corresponding to position 84; G in a position corresponding to position 84; N in a position corresponding to position 84; R in a position corresponding to position 84; A in a position corresponding to position 86; H in a position corresponding to position 86; K in a position corresponding to position 86; N in a position corresponding to position 86; S in a position corresponding to position 86; T in a position corresponding to position 86; W in a position corresponding to position 86; C in a position corresponding to position 87; G in a position corresponding to position 87; L in a position corresponding to position 87; M in a position corresponding to position 87; R in a position corresponding to position 87; S in a position corresponding to position 87; T in a position corresponding to position 87; V in a position corresponding to position 87; Y in a position corresponding to position 87; C in a position corresponding to position 89; A in a position corresponding to position 90; And in a position corresponding to position 90; H in a position corresponding to position 90; K in a position corresponding to position 90; N in a position corresponding to position 90; R in a position corresponding to position 90; C in a position corresponding to position 92; L in a position corresponding to position 92; I in a position corresponding to position 93; L in a position corresponding to position 93; Q in a position corresponding to position 93; R in a position corresponding to position 93; S in a position corresponding to position 93; T in a position corresponding to position 93; D in a position corresponding to position 94; Q in a position corresponding to position 94; R in a position corresponding to position 94; A in a position corresponding to position 97; C in an amino acid residue corresponding to position 97; D in a position corresponding to position 97; And in a position corresponding to position 97; G in a position corresponding to position 97; L in a position corresponding to position 97; S in a position corresponding to position 97; S in a position corresponding to position 102; T in a position corresponding to position 102; R in a position corresponding to position 104; L in a position corresponding to position 107; A in a position corresponding to position 114; Q in a position corresponding to position 118; H in a position corresponding to position 120; F in a position corresponding to position 120; I in a position corresponding to position 120; S in a position corresponding to position 120; V in a position corresponding to position 120; Y in a position corresponding to position 120; And in a position corresponding to position 127; H in a position corresponding to position 127; N in a position corresponding to position 127; Q in a position corresponding to position 127; R in a position corresponding to position 127; I in a position corresponding to position 128; R in a position corresponding to position 130; G in a position corresponding to position 131; I in a position corresponding to position 131; M in a position corresponding to position 131; Q in a position corresponding to position 131; R in a position corresponding to position 131; V in a position corresponding to position 131; N in a position corresponding to position 132; L in a position corresponding to position 132; D in a position corresponding to position 135; G in a position corresponding to position 135; R in a position corresponding to position 135, with L in a position corresponding to position 138; T in a position corresponding to position 139; K in a position corresponding to position 140; H in a position corresponding to position 141; R in a position corresponding to position 141; S in a position corresponding to position 141; W in a position corresponding to position 141; Y in a position corresponding to position 141; D in a position corresponding to position 142; G in a position corresponding to position 142; K in a position corresponding to position 142; N in a position corresponding to position 142; P in a position corresponding to position 142; Q in a position corresponding to position 142; R in a position corresponding to position 142; S in a position corresponding to position 142; T in a position corresponding to position 142; G in a position corresponding to position 143; K in a position corresponding to position 143; R in a position corresponding to position 144; T in a position corresponding to position 144; P in a position corresponding to position 146; R in a position corresponding to position 146; A in a position corresponding to position 147; F in a position corresponding to position 147; L in a position corresponding to position 147; R in a position corresponding to position 147; S in a position corresponding to position 147; V in a position corresponding to position 147; H in a position corresponding to position 148; K in a position corresponding to position 148; Q in a position corresponding to position 148; T in a position corresponding to position 149; V in a position corresponding to position 149; A in a position corresponding to position 150; D in a position corresponding to position 150; G in a position corresponding to position 150; N in a position corresponding to position 150; S in a position corresponding to position 150; W in a position corresponding to position 150; Y in a position corresponding to position 150; A in a position corresponding to position 151; H in a position corresponding to position 151; K in a position corresponding to position 151; L in a position corresponding to position 151; M in a position corresponding to position 151; Q in a position corresponding to position 151; R in a position corresponding to position 151; S in a position corresponding to position 151; T in a position corresponding to position 151; V in a position corresponding to position 151; W in a position corresponding to position 151; Y in a position corresponding to position 151; R in a position corresponding to position 152; T in a position corresponding to position 152; W in a position corresponding to position 152; D in a position corresponding to position 155; G in a position corresponding to position 155; K in a position corresponding to position 155; R in a position corresponding to position 155; D in a position corresponding to position 156; Q in a position corresponding to position 158; S in a position corresponding to position 158; S in a position corresponding to position 160; And in a position corresponding to position 162; A in a position corresponding to position 163; And in a position corresponding to position 163; K in a position corresponding to position 163; Q in a position corresponding to position 163; R in a position corresponding to position 163; S in a position corresponding to position 163; M in a position corresponding to position 164; V in a position corresponding to position 164; D in a position corresponding to position 165; F in a position corresponding to position 165; N in a position corresponding to position 165; S in a position corresponding to position 165; V in a position corresponding to position 165; A in a position corresponding to position 166; And in a position corresponding to position 166; F in a position corresponding to position 166; H in a position corresponding to position 166; L in a position corresponding to position 166; Q in a position corresponding to position 166; R in a position corresponding to position 166; T in a position corresponding to position 166; W in a position corresponding to position 166; Y in a position corresponding to position 166; D in a position corresponding to position 167; L in a position corresponding to position 169; R in a position corresponding to position 170; A in a position corresponding to position 172; R in a position corresponding to position 173; G in a position corresponding to position 174; K in a position corresponding to position 174; N in a position corresponding to position 174; R in a position corresponding to position 174; T in a position corresponding to position 174; T in a position corresponding to position 175; K in a position corresponding to position 178; R in a position corresponding to position 178; K in a position corresponding to position 179; Q in a position corresponding to position 193; T in a position corresponding to position 195; N in a position corresponding to position 195; with E in a position corresponding to position 196; R in a position corresponding to position 196; with D in a position corresponding to position 198; P in a position corresponding to position 204; A in a position corresponding to position 205; And in a position corresponding to position 205; L in a position corresponding to position 205; T in a position corresponding to position 205; I in a position corresponding to position 206; K in a position corresponding to position 206; L in a position corresponding to position 206; R in a position corresponding to position 206; R in a position corresponding to position 209; N in a position corresponding to position 212; S in a position corresponding to position 212; A in a position corresponding to position 213; M in a position corresponding to position 213; N in a position corresponding to position 213; H in a position corresponding to position 215; M in a position corresponding to position 215; A in a position corresponding to position 219; I in a position corresponding to position 219; K in a position corresponding to position 219; S in a position corresponding to position 219; H in a position corresponding to position 220; I in a position corresponding to position 220; L in a position corresponding to position 220; V in a position corresponding to position 220; Q in a position corresponding to position 221; G in a position corresponding to position 222; F in a position corresponding to position 232; G in a position corresponding to position 233; K in a position corresponding to position 233; R in a position corresponding to position 233; M in a position corresponding to position 234; A in a position corresponding to position 235; R in a position corresponding to position 236; C in a position corresponding to position 237; And in a position corresponding to position 237; H in a position corresponding to position 237; Q in a position corresponding to position 237; T in a position corresponding to position 237; And in a position corresponding to position 238; H in a position corresponding to the amino acid position 238; S in a position corresponding to position 238; A in a position corresponding to position 240; Q in a position corresponding to position 240; I in a position corresponding to position 247; A in a position corresponding to position 248; V in a position corresponding to position 249; G in a position corresponding to position 257; T in a position corresponding to position 257; R in a position corresponding to position 257; N in a position corresponding to position 258; S in a position corresponding to position 258; P in a position corresponding to position 259; M in a position corresponding to position 260; Y in a position corresponding to position 260; A in a position corresponding to position 261; K in a position corresponding to position 261; N in a position corresponding to position 261; K in a position corresponding to position 263; R in a position corresponding to position 263; T in a position corresponding to position 267; A in a position corresponding to position 269; L in a position corresponding to position 271; M in a position corresponding to position 271; D in a position corresponding to position 272; T in a position corresponding to position 272; H in a position corresponding to position 273; Y in a position corresponding to position 273; F in a position corresponding to position 274; D in a position corresponding to position 276; H in a position corresponding to position 276; M in a position corresponding to position 276; R in a position corresponding to position 276; S in a position corresponding to position 276; Y in a position corresponding to position 276; A in a position corresponding to position 277; And in a position corresponding to position 277; H in a position corresponding to position 277; K in a position corresponding to position 277; M in a position corresponding to position 277; N in a position corresponding to position 277; Q in a position corresponding to position 277; R in a position corresponding to position 277; S in a position corresponding to position 277; T in a position corresponding to position 277; And in a position corresponding to position 278; F in a position corresponding to position 278; G in a position corresponding to position 278; H in a position corresponding to position 278; K in a position corresponding to position 278; N in a position corresponding to position 278; R in a position corresponding to position 278; S in a position corresponding to position 278; T in a position corresponding to position 278; Y in a position corresponding to position 278; H in a position corresponding to position 279; M in a position corresponding to position 282; S in a position corresponding to position 283; H in a position corresponding to position 285; T in a position corresponding to position 287; S in a position corresponding to position 289; S in a position corresponding to position 291; V in a position corresponding to position 291; C in a position corresponding to position 292; F in a position corresponding to position 292; H in a position corresponding to position 292; K in a position corresponding to position 292; R in a position corresponding to position 292; V in a position corresponding to position 292; A in a position corresponding to position 293; C in a position corresponding to position 293; D in a position corresponding to position 293; F in a position corresponding to position 293; K in a position corresponding to position 293; M in a position corresponding to position 293; P in a position corresponding to position 293; Q in a position corresponding to position 293; V in a position corresponding to position 293; Y in a position corresponding to position 293; G in a position corresponding to position 298; And in a position corresponding to position 305; G in a position corresponding to position 307; D in a position corresponding to position 308; G in a position corresponding to position 308; K in a position corresponding to position 308; N in a position corresponding to position 308; R in a position corresponding to position 308; And in a position corresponding to position 309; G in a position corresponding to position 309; H in a position corresponding to position 309; L in a position corresponding to position 309; M in a position corresponding to position 309; N in a position corresponding to position 309; Q in a position corresponding to position 309; R in a position corresponding to position 309; S in a position corresponding to position 309; T in a position corresponding to position 309; V in a position corresponding to position 309; A in a position corresponding to position 310; G in a position corresponding to position 310; Q in a position corresponding to position 310; S in a position corresponding to position 310; A in a position corresponding to position 313; G in a position corresponding to position 313; H in a position corresponding to position 313; K in a position corresponding to position 313; P in a position corresponding to position 313; R in a position corresponding to position 313; T in a position corresponding to position 313; Y in a position corresponding to position 313; with S in a position corresponding to position 314; Y in a position corresponding to position 314; A in a position corresponding to position 315; H in a position corresponding to position 315; Y in a position corresponding to position 315; A in a position corresponding to position 317; I in a position corresponding to position 317; K in a position corresponding to position 317; N in a position corresponding to position 317; Q in a position corresponding to position 317; R in a position corresponding to position 317; S in a position corresponding to position 317; T in a position corresponding to position 317; W in a position corresponding to position 317; D in a position corresponding to position 318; H in a position corresponding to position 318; K in a position corresponding to position 318; M in a position corresponding to position 318; R in a position corresponding to position 318; H in a position corresponding to position 320; K in a position corresponding to position 320; R in a position corresponding to position 320; R in a position corresponding to position 321; S in a position corresponding to position 321; N in a position corresponding to position 324; R in a position corresponding to position 324; A in a position corresponding to position 325; D in a position corresponding to position 325; And in a position corresponding to position 325; G in a position corresponding to position 325; H in a position corresponding to position 325; K in a position corresponding to position 325; M in a position corresponding to position 325; N in a position corresponding to position 325; Q in a position corresponding to position 325; S in a position corresponding to position 325; V in a position corresponding to position 325; L in a position corresponding to position 326; V in a position corresponding to position 326; C in a position corresponding to position 328; G in a position corresponding to position 328; I in a position corresponding to position 328; K in a position corresponding to position 328; L in a position corresponding to position 328; S in a position corresponding to position 328; Y in a position corresponding to position 328; S in a position corresponding to position 335; A in a position corresponding to position 347; G in a position corresponding to position 347; S in a position corresponding to position 347; M in a position corresponding to position 349; R in a position corresponding to position 349; S in a position corresponding to position 351; V in a position corresponding to position 353; with H in a position corresponding to position 356; S in a position corresponding to position 356; And in a position corresponding to position 359; H in a position corresponding to position 359; T in a position corresponding to position 359; A in a position corresponding to position 367; G in a position corresponding to position 367; K in a position corresponding to position 367; S in a position corresponding to position 367; A in a position corresponding to position 368; And in a position corresponding to position 368; K in a position corresponding to position 368; L in a position corresponding to amino acid position 368; M at a position corresponding to amino acid position 368; R in a position corresponding to position 368; T in a position corresponding to the amino acid position 368; H in a position corresponding to position 369; R in a position corresponding to position 369; F in a position corresponding to position 371; H in a position corresponding to position 371; K in a position corresponding to position 371; L in a position corresponding to position 371; R in a position corresponding to position 371; S in a position corresponding to position 371; M in a position corresponding to position 373; H in a position corresponding to position 374; P in a position corresponding to position 374; A in a position corresponding to position 375; G in a position corresponding to position 375; K in a position corresponding to position 375; R in a position corresponding to position 375; D in a position corresponding to position 376; And in a position corresponding to position 376; Q in a position corresponding to position 376; R in a position corresponding to position 376; T in a position corresponding to position 376; V in a position corresponding to position 376; Y in a position corresponding to position 376; D in a position corresponding to position 377; And in a position corresponding to position 377; H in a position corresponding to position 377; K in a position corresponding to position 377; P in a position corresponding to position 377; R in a position corresponding to position 377; S in a position corresponding to position 377; T in a position corresponding to position 377; W in a position corresponding to position 380; Y in a position corresponding to position 380; S in a position corresponding to position 381; I in a position corresponding to position 383; K in a position corresponding to position 383; L in a position corresponding to position 383; S in a position corresponding to position 383; A in a position corresponding to position 385; Q in a position corresponding to position 385; V in a position corresponding to position 385; A in a position corresponding to position 389; G in a position corresponding to position 389; L in a position corresponding to position 389; K in a position corresponding to position 389; Q in a position corresponding to position 389; S in a position corresponding to position 389; A in a position corresponding to position 392; F in a position corresponding to position 392; M in a position corresponding to position 392; Q in a position corresponding to position 392; R in a position corresponding to position 392; V in a position corresponding to position 392; F in a position corresponding to position 393; M in a position corresponding to position 393; A in a position corresponding to position 395; H in a position corresponding to position 395; R in a position corresponding to position 395; A in a position corresponding to position 396; H in a position corresponding to position 396; Q in a position corresponding to position 396; S in a position corresponding to position 396; K in a position corresponding to position 399; M in a position corresponding to position 399; T in a position corresponding to position 399; V in a position corresponding to position 399; W in a position corresponding to position 399; A in a position corresponding to position 401; And in a position corresponding to position 401; A in a position corresponding to position 404; G in a position corresponding to position 405; F in a position corresponding to position 406; N in a position corresponding to position 406; A in a position corresponding to position 407; D in a position corresponding to position 407; And in a position corresponding to position 407; F in a position corresponding to position 407; H in a position corresponding to position 407; Q in a position corresponding to position 407; P in a position corresponding to position 407; A in a position corresponding to position 409; Q in a position corresponding to position 409; T in a position corresponding to position 410; Q in a position corresponding to position 412; R in a position corresponding to position 412; V in a position corresponding to position 412; L in a position corresponding to position 416; And in a position corresponding to position 418; L in a position corresponding to position 418; P in a position corresponding to position 418; R in a position corresponding to position 418; V in a position corresponding to position 418; F in a position corresponding to position 419; H in a position corresponding to position 419; I in a position corresponding to position 419; K in a position corresponding to position 419; R in a position corresponding to position 419; S in a position corresponding to position 419; Y in a position corresponding to position 419; A in a position corresponding to position 421; H in a position corresponding to position 421; K in a position corresponding to position 421; N in a position corresponding to position 421; Q in a position corresponding to position 421; R in a position corresponding to position 421; S in a position corresponding to position 421; G in a position corresponding to position 425; K in a position corresponding to position 425; Q in a position corresponding to position 427; T in a position corresponding to position 427; L in a position corresponding to position 428; A in a position corresponding to position 431; G in a position corresponding to position 431; And in a position corresponding to position 431; H in a position corresponding to position 431; K in a position corresponding to position 431; L in a position corresponding to position 431; N in a position corresponding to position 431; Q in a position corresponding to position 431; R in a position corresponding to position 431; S in a position corresponding to position 431; V in a position corresponding to position 431; A in a position corresponding to position 433; H in a position corresponding to position 433; I in a position corresponding to position 433; K in a position corresponding to position 433; L in a position corresponding to position 433; R in a position corresponding to position 433; T in a position corresponding to position 433; V in a position corresponding to position 433; W in a position corresponding to position 433; K in a position corresponding to position 436; I in a position corresponding to position 437; M in a position corresponding to position 437; A in a position corresponding to position 438; D in a position corresponding to position 438; And in a position corresponding to position 438; L in a position corresponding to position 438; N in a position corresponding to position 438; T in a position corresponding to position 438; A in a position corresponding to position 439; C in a position corresponding to position 439; K in a position corresponding to position 439; P in a position corresponding to position 439; Q in a position corresponding to position 439; T in a position corresponding to position 439; V in a position corresponding to position 439; D in a position corresponding to position 440; H in a position corresponding to position 440; M in a position corresponding to position 440; P in a position corresponding to position 440; R in a position corresponding to position 440; S in a position corresponding to position 440; A in a position corresponding to position 441; F in a position corresponding to position 441; C in a position corresponding to position 442; G in a position corresponding to position 442; R in a position corresponding to position 442; A in a position corresponding to position 443; And in a position corresponding to position 443; F in a position corresponding to position 443; G in a position corresponding to position 443; M in a position corresponding to position 443; N in a position corresponding to position 443; And in a position corresponding to position 444; H in a position corresponding to position 444; V in a position corresponding to position 444; H in a position corresponding to position 445; M in a position corresponding to position 445; N in a position corresponding to position 445; P in a position corresponding to position 445; Q in a position corresponding to position 445; S in a position corresponding to position 445; T in a position corresponding to position 445; V in a position corresponding to position 445; W in a position corresponding to position 445; A in a position corresponding to position 446; M in a position corresponding to position 446; W in a position corresponding to position 446; D in a position corresponding to position 447; And in a position corresponding to position 447; G in a position corresponding to position 447; I in a position corresponding to position 447; N in a position corresponding to position 447; P in a position corresponding to position 447; Q in a position corresponding to position 447; T in a position corresponding to position 447, and / or substitution with V in a position corresponding to position 447, each with reference to amino acid positions exposed in SEQ ID NO: 3.
[000269] [000269] Examples of such modified PH20 polypeptides are any having the amino acid sequence exposed in any of SEQ ID NOS: 74-855, or having an amino acid sequence that has at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of SEQ ID NOS: 74- 855 and contains the amino acid substitution and has hyaluronidase activity.
[000270] [000270] Any of the modified PH20 polypeptides above provided here may have altered properties or activities, such as enhanced or enhanced, compared to the corresponding PH20 polypeptide not containing the amino acid modification (e.g., amino acid substitution). For example, the altered activities or properties can be increased catalytic activity and / or increased stability under denaturing conditions. The. Increased activity
[000271] [000271] Modified PH20 polypeptides or variants which contain one or more amino acid substitutions in a PH20 polypeptide and which have increased hyaluronidase activity compared to the corresponding PH20 polypeptide are not provided here, containing the amino acid substitution (s) by example, the polypeptide
[000272] [000272] The modified PH20 polypeptide may exhibit hyaluronidase activity that is at least or approximately at least or 120%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 200% , 250%, 300%, 350%, 400%, 500%, 1500%, 2000%, 3000%, 4000%, 5000% of the corresponding PH20 polypeptide hyaluronidase activity not containing the amino acid substitution (s) , for example, the PH20 polypeptide exposed in any of SEQ ID NOS: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof, under the same conditions. For example, hyaluronidase activity is increased at least or approximately at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times , 90 times, 100 times, 200 times, 300 times, 400 times or more.
[000273] [000273] In specific examples, the modified PH20 polypeptides contain an amino acid substitution at one or more amino acid positions identified as being associated with increased hyaluronidase activity. As described here, such positions have been identified using mutagenesis and selection or screening methods to identify those positions that result in increased hyaluronidase activity. PH20 polypeptide may also contain other modifications, such as other amino acid substitutions, which individually are not associated with increased activity since the resulting PH20 polypeptide exhibits increased hyaluronidase activity compared to PH20 not containing the modification (s) of amino acid, as amino acid substitution (s). The modified PH20 polypeptide provided here may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, or more amino acid substitutions. Additional modifications, such as insertions or deletions, can also be included. The amino acid substitution can be in a PH20 polypeptide as set out in any of SEQ ID NOS: 2,3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the same. For example, the substitution (s) can be in a human PH20 polypeptide, for example, any exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72 or a variant thereof .
[000274] [000274] For example, the modified PH20 polypeptides provided here contain an amino acid substitution at one or more amino acid positions corresponding to positions 1, 12, 15, 24, 26, 27, 29, 30,
[000275] [000275] Examples of amino acid substitutions in the modified PH20 polypeptides provided here include, but are not limited to, substitution: with histidine (H) in a position corresponding to position 1; Q in a position corresponding to position 1; And in a position corresponding to position 12; T in a position corresponding to position 12; V in a position corresponding to position 15; And in a position corresponding to position 24; H in a position corresponding to position 24; And in a position corresponding to position 26; K in a position corresponding to position 26; K in a position corresponding to position 27; R in a position corresponding to position 27; And in a position corresponding to position 29; I in a position corresponding to position 29; L in a position corresponding to position 29; M in a position corresponding to position 29; P in a position corresponding to position 29; S in a position corresponding to position 29; V in a position corresponding to position 29; G in a position corresponding to position 30; H in a position corresponding to position 30; K in a position corresponding to position 30; M in a position corresponding to position 30; R in a position corresponding to position 30; S in a position corresponding to position 30; A in a position corresponding to position 31; C in a position corresponding to position 31; H in a position corresponding to position 31; I in a position corresponding to position 31; K in a position corresponding to position 31; L in a position corresponding to position 31; P in a position corresponding to position 31; R in a position corresponding to position 31; S in a position corresponding to position 31; T in a position corresponding to position 31; V in a position corresponding to position 31; F in a position corresponding to position 32; G in a position corresponding to position 32; H in a position corresponding to position 32; W in a position corresponding to position 33; F in a position corresponding to position 37; N in a position corresponding to position 39; T in a position corresponding to position 39; R in a position corresponding to position 46; F in a position corresponding to position 48; H in a position corresponding to position 48; N in a position corresponding to position 48; Q in a position corresponding to position 52; K in a position corresponding to position 58; Q in a position corresponding to position 58; W in a position corresponding to position 63; V in a position corresponding to position 67; H in a position corresponding to position 68; Q in a position corresponding to position 68; A in a position corresponding to position 69; C in a position corresponding to position 69; F in a position corresponding to position 69; G in a position corresponding to position 69; I in a position corresponding to position 69; L in a position corresponding to position 69; M in a position corresponding to position 69; P in a position corresponding to position 69; R in a position corresponding to position 69; W in a position corresponding to position 69; Y in a position corresponding to position 69; A in a position corresponding to position 70; C in a position corresponding to position 70; F in a position corresponding to position 70; G in a position corresponding to position 70; H in a position corresponding to position 70; K in a position corresponding to position 70; L in a position corresponding to position 70; N in a position corresponding to position 70; P in a position corresponding to position 70; R in a position corresponding to position 70; S in a position corresponding to position 70; T in a position corresponding to position 70; V in a position corresponding to position 70; R in a position corresponding to position 71; S in a position corresponding to position 71; M in a position corresponding to position 72; Q in a position corresponding to position 72; H in a position corresponding to position 73; L in a position corresponding to position 73; W in a position corresponding to position 73; A in a position corresponding to position 74; C in a position corresponding to position 74; G in a position corresponding to position 74; N in a position corresponding to position 74; P in a position corresponding to position 74; R in a position corresponding to position 74; S in a position corresponding to position 74; V in a position corresponding to position 74; W in a position corresponding to position 74; F in a position corresponding to position 75; L in a position corresponding to position 75; R in a position corresponding to position 75; T in a position corresponding to position 75; G in a position corresponding to position 84; R in a position corresponding to position 84; A in a position corresponding to position 86; C in a position corresponding to position 87; T in a position corresponding to position 87; Y in a position corresponding to position 87; C in a position corresponding to position 92; I in a position corresponding to position 93; L in a position corresponding to position 93; R in a position corresponding to position 93; T in a position corresponding to position 93; R in a position corresponding to position 94; G in a position corresponding to position 97; Q in a position corresponding to position 118; F in a position corresponding to position 120; V in a position corresponding to position 120; Y in a position corresponding to position 120; H in a position corresponding to position 127; N in a position corresponding to position 127; G in a position corresponding to position 131; R in a position corresponding to position 131; V in a position corresponding to position 131; D in a position corresponding to position 135; G in a position corresponding to position 135; R in a position corresponding to position 135, with H in a position corresponding to position 141; Y in a position corresponding to position 141; R in a position corresponding to position 142; R in a position corresponding to position 147; V in a position corresponding to position 147; K in a position corresponding to position 148; G in a position corresponding to position 150; K in a position corresponding to position 151; L in a position corresponding to position 151; M in a position corresponding to position 151; Q in a position corresponding to position 151; R in a position corresponding to position 151; R in a position corresponding to position 152; G in a position corresponding to position 155; K in a position corresponding to position 155; D in a position corresponding to position 156; A in a position corresponding to position 163; And in a position corresponding to position 163; K in a position corresponding to position 163; R in a position corresponding to position 163; M in a position corresponding to position 164; D in a position corresponding to position 165; N in a position corresponding to position 165; A in a position corresponding to position 166; F in a position corresponding to position 166; H in a position corresponding to position 166; L in a position corresponding to position 166; Q in a position corresponding to position 166; R in a position corresponding to position 166; T in a position corresponding to position 166; Y in a position corresponding to position 166; L in a position corresponding to position 169; R in a position corresponding to position 170; K in a position corresponding to position 174; D in a position corresponding to position 198; K in a position corresponding to position 206; L in a position corresponding to position 206; N in a position corresponding to position 212; M in a position corresponding to position 213; N in a position corresponding to position 213; M in a position corresponding to position 215; S in a position corresponding to position 219; K in a position corresponding to position 233; R in a position corresponding to position 233; M in a position corresponding to position 234; R in a position corresponding to position 236; And in a position corresponding to position 237; S in a position corresponding to position 238; I in a position corresponding to position 247; T in a position corresponding to position 257; P in a position corresponding to position 259; Y in a position corresponding to position 260; K in a position corresponding to position 261; N in a position corresponding to position 261; K in a position corresponding to position 263; R in a position corresponding to position 263; A in a position corresponding to position 269; L in a position corresponding to position 271; M in a position corresponding to position 271; T in a position corresponding to position 272; D in a position corresponding to position 276; S in a position corresponding to position 276; Y in a position corresponding to position 276; K in a position corresponding to position 277; R in a position corresponding to position 277; T in a position corresponding to position 277; H in a position corresponding to position 278; K in a position corresponding to position 278; N in a position corresponding to position 278; R in a position corresponding to position 278; S in a position corresponding to position 278; T in a position corresponding to position 278; Y in a position corresponding to position 278; M in a position corresponding to position 282; V in a position corresponding to position 291; A in a position corresponding to position 293; C in a position corresponding to position 293; F in a position corresponding to position 293; M in a position corresponding to position 293; P in a position corresponding to position 293; Q in a position corresponding to position 293; V in a position corresponding to position 293; And in a position corresponding to position 305; G in a position corresponding to position 308; N in a position corresponding to position 308; And in a position corresponding to position 309; L in a position corresponding to position 309; N in a position corresponding to position 309; Q in a position corresponding to position 309; R in a position corresponding to position 309; T in a position corresponding to position 309; A in a position corresponding to position 310; G in a position corresponding to position 310; K in a position corresponding to position 313; R in a position corresponding to position 313; H in a position corresponding to position 315; I in a position corresponding to position 317; K in a position corresponding to position 317; R in a position corresponding to position 317; M in a position corresponding to position 318; H in a position corresponding to position 320; K in a position corresponding to position 320; R in a position corresponding to position 320; R in a position corresponding to position 324; A in a position corresponding to position 325; D in a position corresponding to position 325; And in a position corresponding to position 325; G in a position corresponding to position 325; H in a position corresponding to position 325; K in a position corresponding to position 325; M in a position corresponding to position 325; N in a position corresponding to position 325; Q in a position corresponding to position 325; S in a position corresponding to position 325; V in a position corresponding to position 326; I in a position corresponding to position 328; K in a position corresponding to position 328; L in a position corresponding to position 328; S in a position corresponding to position 328; Y in a position corresponding to position 328; G in a position corresponding to position 347; S in a position corresponding to position 347; V in a position corresponding to position 353; with T in a position corresponding to position 359; R in a position corresponding to position 371; P in a position corresponding to position 377; T in a position corresponding to position 377; W in a position corresponding to position 380; Y in a position corresponding to position 380; K in a position corresponding to position 389; M in a position corresponding to position 392; R in a position corresponding to position 395; M in a position corresponding to position 399; T in a position corresponding to position 399; W in a position corresponding to position 399; G in a position corresponding to position 405; D in a position corresponding to position 407; Q in a position corresponding to position 407; A in a position corresponding to position 409; Q in a position corresponding to position 409; T in a position corresponding to position 410; P in a position corresponding to position 418; F in a position corresponding to position 419; I in a position corresponding to position 419; K in a position corresponding to position 419; R in a position corresponding to position 419; S in a position corresponding to position 419; H in a position corresponding to position 421; K in a position corresponding to position 421; N in a position corresponding to position 421; Q in a position corresponding to position 421; R in a position corresponding to position 421; S in a position corresponding to position 421; K in a position corresponding to position 425; A in a position corresponding to position 431; H in a position corresponding to position 431; K in a position corresponding to position 431; Q in a position corresponding to position 431; R in a position corresponding to position 431; S in a position corresponding to position 431; V in a position corresponding to position 431; L in a position corresponding to position 433; R in a position corresponding to position 433; T in a position corresponding to position 433; V in a position corresponding to position 433; K in a position corresponding to position 436; I in a position corresponding to position 437; M in a position corresponding to position 437; T in a position corresponding to position 438; V in a position corresponding to position 439; H in a position corresponding to position 440; R in a position corresponding to position 440; F in a position corresponding to position 441; R in a position corresponding to position 442; A in a position corresponding to position 443; M in a position corresponding to position 443; M in a position corresponding to position 445; P in a position corresponding to position 445; A in a position corresponding to position 446; D in a position corresponding to position 447; N in a position corresponding to position 447; and / or with Q in a position corresponding to position 447, each with reference to amino acid positions exposed in SEQ ID NO: 3. The modified PH20 polypeptides may contain any one or more of the aforementioned amino acid substitutions, in any combination, with or without additional modifications, provided that the PH20 polypeptide has hyaluronidase activity, such as increased hyaluronidase activity compared to the PH20 polypeptide not containing the (s) modification (s), for example, at least 1.5-fold increased hyaluronidase activity.
[000276] [000276] In some examples, the modified PH20 polypeptides provided here contain one or more amino acid substitution (s) in a position (s) corresponding to position (s) 24, 29, 31, 48, 58, 69, 70, 75 , 84, 97, 165, 166, 271, 278, 317, 320, 325, and / or 326 with reference to positions set out in SEQ ID NO: 3. For example, exemplary amino acid substitutions include, but are not limited to, substitution with: E in a position corresponding to position 24; And in a position corresponding to position 29; V in a position corresponding to position 31; N in a position corresponding to position 48; K in a position corresponding to position 58; Q in a position corresponding to position 58; A in a position corresponding to position 69; F in a position corresponding to position 69; G in a position corresponding to position 69; P in a position corresponding to position 69; R in a position corresponding to position 69; A in a position corresponding to position 70; F in a position corresponding to position 70; G in a position corresponding to position 70; H in a position corresponding to position 70; H in a position corresponding to position 70; N in a position corresponding to position 70; R in a position corresponding to position 70; T in a position corresponding to position 70; V in a position corresponding to position 70; L in a position corresponding to position 75; T in a position corresponding to position 75; G in a position corresponding to position 84; G in a position corresponding to position 97; D in a position corresponding to position 165; L in a position corresponding to position 166; R in a position corresponding to position 166; T in a position corresponding to position 166; L in a position corresponding to position 271; H in a position corresponding to position 278; R in a position corresponding to position 278; K in a position corresponding to position 317; K in a position corresponding to position 320; And in a position corresponding to position 325, with G in a position corresponding to position 325; K in a position corresponding to position 325; N in a position corresponding to position 325; Q in a position corresponding to position 325; V in a position corresponding to position 326; each with reference to amino acid positions exposed in SEQ ID NO: 3. Modified PH20 polypeptides may contain any one or more of the aforementioned amino acid substitutions, in any combination, with or without additional modifications, provided that the PH20 polypeptide has hyaluronidase activity, such as increased hyaluronidase activity compared to PH20 polypeptide not containing ( s) modification (s), for example, at least 2.0 times of increased hyaluronidase activity.
[000277] [000277] Exemplary modified PH20 polypeptides that show increased activity compared to unmodified PH20 polypeptide (for example, set out in SEQ ID NO: 3) are any having the amino acid sequence exposed in any of SEQ ID NOS: 73, 78, 86, 89, 91, 95, 96, 99, 100, 105, 106, 108, 109, 111, 112, 113, 115, 117, 118, 119, 120, 123-126, 128-136, 139-141, 149, 154, 155, 159, 164, 165, 167, 173, 178, 181, 191-193, 195-197, 199-205, 207-221, 225, 226, 228, 229, 231, 233, 237- 239, 242, 247-254, 256, 257, 267, 269, 270, 277, 283, 293, 295, 296, 298, 300, 303, 308, 316, 318, 321, 322, 324, 325, 330, 334, 335, 338-340, 344, 348, 355, 367, 369, 371, 377, 384-388, 394, 398, 399, 401, 406-408, 410, 412, 414, 416, 419, 421- 426, 428, 430, 431, 435, 448, 455, 456, 459, 462, 463, 465, 469, 478-480, 482, 484, 490, 493, 497, 501, 503, 505, 506-508, 510-512, 514, 518, 522, 523, 527, 531, 533, 537-543, 545, 551, 558, 559, 561, 563-566, 569, 572, 574, 576, 579, 581-583, 585, 587, 588, 594, 596, 602, 605, 606, 609, 613, 618-620, 624-634, 637, 640-644, 647, 648, 652, 657, 675, 695, 698, 699, 700, 712, 717, 725, 731, 732, 734, 738, 742, 746, 748-750, 757, 760, 762-765, 768-773, 775, 779, 782, 783, 786-789, 794-797, 799-801, 807, 814, 816, 819, 822, 825, 826, 830, 836, 838, 844, 847, 851, 853 or having an amino acid sequence that is at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than sequence identity for any of SEQ ID NOS: 73, 78, 86, 89, 91, 95, 96, 99, 100, 105, 106, 108, 109, 111, 112, 113, 115, 117, 118, 119, 120, 123-126, 128-136, 139-141, 149, 154, 155, 159, 164, 165, 167, 173, 178, 181, 191-193, 195-197, 199-205, 207-221, 225, 226, 228, 229, 231, 233, 237- 239, 242, 247-254, 256, 257, 267, 269, 270, 277, 283, 293, 295, 296, 298, 300, 303, 308, 316, 318, 321, 322, 324, 325, 330, 334, 335, 338-340, 344, 348, 355, 367, 369, 371, 377, 384-388, 394, 398, 399, 401, 406- 408, 410, 412, 414, 416, 419, 421-426, 428, 430, 431, 435, 448, 455, 456, 459, 462, 463, 465, 469, 478-480, 482, 484, 490, 493, 497, 501, 503, 505, 506-508, 510-512, 514, 518, 522, 523, 527, 531, 533, 537-543, 545, 551, 558, 559, 561, 563-566, 569, 572, 574, 576, 579, 581-583, 585, 587, 588, 594, 596, 602, 605, 606, 609, 613, 618-620, 624-634, 637, 640-644, 647, 648, 652, 657, 675, 695, 698, 699, 700, 712, 717, 725, 731, 732, 734, 738, 742, 746, 748-750, 757, 760, 762-765, 768-773, 775, 779, 782, 783, 786-789, 794-797, 799-801, 807, 814, 816, 819, 822, 825, 826, 830, 836, 838, 844, 847, 851, 853 and contains the amino acid substitution and has increased hyaluronidase activity compared to the corresponding unmodified polypeptide. B. Increased stability
[000278] [000278] PH20 polypeptides that have increased stability are provided here. In particular, PH20 polypeptides show increased stability in vivo and / or in vitro. For example, PH20 polypeptides can exhibit increased stability under various storage conditions. The modified PH20 polypeptides provided here that exhibit increased stability exhibit, among other parameters, Increased resistance to denaturation conditions, including, but not limited to, denaturation conditions caused by temperature (e.g. high temperature such as heat), agitation, without salt or low salt content, and / or the presence of excipients. Exemplary excipients include, but are not limited to, nonstick, binders, coatings, fillers and thinners, flavors, colors, lubricants, glidants, preservatives, sorbents or sweeteners. For example, various excipients, such as condoms, can act as protein denaturing agents. Modified PH20 polypeptides provided here that exhibit increased protein stability exhibit reduced aggregation, reduced precipitation and / or increased activity when exposed to a denaturation condition compared to the corresponding PH20 not containing the amino acid substitution. For example, modified PH20 polypeptides provided here have at least or at least approximately or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120 %, 130%, 140%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500% or more of increased activity when exposed to a denaturation condition compared to the corresponding PH20 polypeptide not containing the amino acid substitution when exposed to the same denaturation condition.
[000279] [000279] The PH20 polypeptides provided here that have increased stability are variant or modified PH20 polypeptides that contain an amino acid substitution, deletion or insertion or other modification. Typically, PH20 polypeptides provided here that exhibit increased stability contain one or more amino acid substitutions in a PH20 polypeptide compared to the corresponding PH20 polypeptide not containing the amino acid substitution (s), for example, the PH20 polypeptide exposed in any of SEQ ID NOS: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of identity sequence with it. In particular, the variant or modified PH20 polypeptides provided here show increased stability compared to the corresponding PH20 polypeptide not containing the amino acid substitution, for example, the PH20 polypeptide exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72 and in particular the PH20 polypeptide set out in SEQ ID NO: 3.
[000280] [000280] In specific examples, the modified PH20 polypeptides contain an amino acid substitution at one or more amino acid positions identified as being associated with increased stability. As described here, such positions can be identified using mutagenesis and selection or screening methods to identify those positions that result in stability (e.g., increased activity) of the polypeptide compared to the corresponding PH20 not containing the modification after exposure to one or more denaturation conditions. PH20 polypeptide may also contain other modifications, such as other amino acid substitutions that individually are not associated with conferring stability, as long as the resulting modified PH20 polypeptide exhibits increased stability under one or more denaturation conditions compared to PH20 not containing the (s) ) amino acid modification (s), as amino acid substitution (s) and has hyaluronidase activity. The modified PH20 polypeptide provided here may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48.49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
[000281] [000281] Exemplary modified PH20 polypeptides provided here are PH20 polypeptides that exhibit increased stability after exposure to phenol compounds, elevated temperature (heat), and / or absence of NaCl. i. Phenophiles
[000282] [000282] Modified PH20 polypeptides which have increased stability in the presence of phenolic compounds are provided here. Multidose formulations must contain antimicrobial preservatives to protect them from microbial contamination. For parenteral drug products, including insulin and other therapeutic agents, the most common condoms are phenolic compounds, such as phenol, metacresol (m-cresol), benzyl alcohol, and parabens including methyl paraben and propyl paraben. Condoms must typically be present in sufficient concentrations to meet regulatory rules. For example, regulatory requirements state that the antimicrobial efficacy of the formulation must meet the requirements for testing the effectiveness of condoms (PET) in the target markets. Currently different regulatory bodies have different pharmacopoeia criteria for antimicrobial efficacy for pharmaceutical products designed for multiple dosing. The United States Pharmacopoeia (USP) and European Pharmacopoeia (EP) PET requirements differ considerably, imposing additional limitations on the development of multidose formulations. Table 4 shows the criteria for injecting drugs to meet the USP and EP criteria. Typically, formulations that meet EP antimicrobial requirements (EPA or EPB) contain more preservatives than those formulated only to meet USP antimicrobial requirements.
[000283] [000283] Antimicrobial condoms can interact with proteins resulting in aggregations and negative effects on stability. Thus, although a necessary component, condoms present a significant problem in the development of multidose, stable protein formulations because they typically induce protein aggregation in aqueous solution. In particular, increased or increased amounts of condoms can negatively impact protein stability, including effects on physical stability (aggregation or precipitation) that can impact protein activity. For example, to meet the requirements for EP condom effectiveness, relatively high amounts of phenolic compounds, such as phenol or m-cresol, may be required, which can influence the stability of the protein formulation. For example, condoms such as phenol, m-cresol and benzyl alcohol have been shown to induce aggregation of human growth hormone (Maa and Hsu (1996) Int. J. Pharm.140: 155–168), interleukin-1 receptor recombinant (Remmele (1998) Pharm. Res.15: 200–208), insulin-like growth factor I (Fransson (1997) Pharm. Res. 14: 606–612), rhIFN- (Lam (1997) Pharm. Res 14: 725–729) and cytochrome c (Singh et al (2011) J. Pharm Sci., 100: 1679-89). The destabilizing effect that condoms have on proteins in solution has been a limiting factor in the development of multi-dose formulations, and to date, most protein therapies have been formulated for individual use only.
[000284] [000284] Hyaluronidase PH20, like rHUPH20, rapidly loses activity in the presence of condoms, probably due to protein unvezesing and subsequent aggregate formation. For example, as shown in the Examples of the present invention, condoms reduce PH20 enzyme activity, particularly at elevated temperatures (see also US provisional application No. 61 / 520,962; and US applications No. 13 / 507,263 and 13 / 507,262). For example, after incubating with 0.4% m-cresol for 4 hours, PH20 (for example, rHuPH20) retains only approximately 10% of its activity (see, for example, example 5). When incubated in the presence of 0.1% phenol and 0.15% or 0.315% m-cresol for 6 days at 37ºC, PH20 (for example, rHuPH20) retains approximately 0% to 15% activity, depending on the presence of other excipients or amounts of other excipients in the formulation (see, for example, examples 9 and 10). For example, the presence of a higher concentration of salt generally increases the stability of PH20. In particular, the melting temperature of PH20, such as rHuPH20, is significantly reduced when phenolic preservatives, such as m-cresol, are added to the formulation. For example, the unfolding temperature of rHuPH20 is reduced from 44ºC to 24ºC. Lower PH20 split temperatures lead to increased PH20 aggregation, especially at elevated temperatures, and reduced enzyme activity. The destabilizing effect is probably due to the hydrophobic nature of phenolic condoms. The hydrophobicity of phenolic compounds can lead to interaction with rHuPH20 through non-specific binding to the protein, ultimately disrupting the structural integrity of rHuPH20. This translates into a significant loss of enzymatic activity for rHuPH20 in the presence of condoms.
[000285] [000285] The modified PH20 polypeptides provided here that have increased stability in the presence of phenolic condoms have more than 15% enzyme activity in the presence of at least one phenolic condom for at least 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks or more compared to the enzyme activity of the modified PH20 polypeptide in the absence of a condom for the same period of time and under the same conditions (except for the presence of a condom). In some instances, PH20 polypeptides modified in the absence of a condom for the same period of time and under the same conditions (except for the presence of a condom). In some instances, the modified PH20 polypeptides provided here have at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60 %, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of enzyme activity in the presence of a phenolic condom compared to the absence of a condom. For example, the phenolic preservative compound can be phenol, metacresol (m-cresol), benzyl alcohol, and / or parabens including methyl paraben or propylparaben.
[000286] [000286] In specific examples, increased stability in the presence of a condom is displayed under temperature conditions between or approximately between 0 ° C and 40 ° C, such as between or approximately between 2 ° C and 6 ° C, 24 ° C and 32 ° C or 35 ° C and 40 ° C, and generally at or approximately 4 ° C or 5 ° C, 30 ° C or 37 ° C. It is understood that since the elevated temperature can also have a destabilizing effect on PH20 activity (see below), the percentage of enzyme activity of a modified PH20 polypeptide provided here in the presence of a condom is higher at lower temperatures than at higher temperatures.
[000287] [000287] Generically, the modified PH20 polypeptides provided here have increased stability, and the enzyme activities observed, in the presence of an effective antimicrobial amount of condom that kills or inhibits the spread of microbial organisms in a sample composition.
[000288] [000288] For example, the modified PH20 polypeptides provided here have increased stability, and enzyme activity mentioned above, in the presence of a total amount of one or more phenolic preservative agents as a percentage (%) of mass concentration (weight / v) which is or is between 0.05% and 0.6%, 0.1% and 0.4%, 0.1% and 0.3%, 0.15% and 0.325%, 0.15% and 0, 25%, 0.1% and 0.2%, 0.2% and 0.3% or 0.3% and 0.4% inclusive.
[000289] [000289] Generally, modified PH20 polypeptides provided here have increased stability in the presence of m-cresol and / or phenol. For example,
[000290] [000290] In examples of the present invention, modified PH20 polypeptides have more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45 %, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzyme activity in the presence of at least approximately between or between 0.3 % and 0.4%, including m-cresol and / or phenol for at least 4 hours at 37 ° C compared to the enzyme activity of the modified PH20 polypeptide in the absence of the condom for the same period of time and under the same conditions ( except for the presence of a condom). For example, modified PH20 polypeptides have more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzyme activity in the presence of approximately or 0.4% m-cresol for at least 4 hours at 37 ° C compared to the enzyme activity of the modified PH20 polypeptide in the absence of a condom for the same period of time and under the same conditions (except for the presence of a condom). Modified PH20 polypeptides provided here also have more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% , 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzyme activity in the presence of at least approximately between or between 0.2% and 0.4%, including m-cresol and / or phenol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days at 37 ° C compared to the enzyme activity of the modified PH20 polypeptide in the absence of a condom for the same period time and under the same conditions (except for the presence of a condom). For example, modified PH20 polypeptides provided here have more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzyme activity in the presence of approximately or 0.10% phenol and approximately or 0.15% m-cresol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days at 37 ° C compared to the enzyme activity of the modified PH20 polypeptide in the absence of a condom for the same period of time and under the same conditions conditions (except for the presence of a condom). In other examples, modified PH20 polypeptides provided here have more than 15%, such as at least 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more enzyme activity in the presence of approximately or 0.315% m-cresol for at least 1 day, 2 days, 3 days, 4 days, 5 days or 6 days, generally for at least 6 days, at 37 ° C compared to the enzymatic activity of the modified PH20 polypeptide in the absence of a condom for the same period of time and under the same conditions (except for the presence of a condom).
[000291] [000291] For example, such modified PH20 polypeptides provided here that have increased stability to phenol compounds contain an amino acid substitution at one or more amino acid positions corresponding to positions 10, 12, 20, 22, 26, 34, 36, 46 , 50, 52, 58, 68, 70, 74, 82, 83, 84, 86, 97, 127, 131, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 204, 205 , 206, 213, 219, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401 , 407, 416, 419, 421, 431, 433, 439, 440, 443 or 445 with reference to amino acid positions exposed in SEQ ID NO: 3. For example, amino acid positions can be substitutions in one or more positions corresponding to the substitution of (P) in position 10 (P10), V12, A20, S22, L26, D34, S36, I46, G50, G52, V58, D68 , I70, T74, K82, I83, S84, Q86, T97, D127, N131, Q138, V142, Q143, L144, V166, I169, L174, H193, K195, K196, F204, N205, V206, D213, N219, Q234 , V237, A238, T240, E249, S261, A267, V277K279, G291, I309, M310, K314, S315, L317, Q347, P367, E375, K376, Y399, S401, S407, D416, A419, D421, D31 , E439, T440, P443 or I445 with reference to amino acid positions exposed in the
[000292] [000292] Exemplary amino acid substitutions in the modified PH20 polypeptides provided here include, but are not limited to, substitution with: glycine (G) in a position corresponding to position 10; K in a position corresponding to position 12; S in a position corresponding to position 20; T in a position corresponding to position 22; M in a position corresponding to position 26; W in a position corresponding to position 34; N in a position corresponding to position 36; L in a position corresponding to position 46; M in a position corresponding to position 50; T in a position corresponding to position 52; S in a position corresponding to position 52; C in a position corresponding to position 58; K in a position corresponding to position 58; R in a position corresponding to position 58; N in a position corresponding to position 58; Y in a position corresponding to position 58; P in a position corresponding to position 58; H in a position corresponding to position 58; P in a position corresponding to position 68; V in a position corresponding to position 70; And in a position corresponding to position 74; L in a position corresponding to position 82; N in a position corresponding to position 82; V in a position corresponding to position 83; Q in a position corresponding to position 83; S in a position corresponding to position 83; G in a position corresponding to position 83; N in a position corresponding to position 84; A in a position corresponding to position 86; K in a position corresponding to position 86; And in a position corresponding to position 97; L in a position corresponding to position 97; R in a position corresponding to position 127; R in a position corresponding to position 131; L in a position corresponding to position 138; K in a position corresponding to position 142; N in a position corresponding to position 142; P in a position corresponding to position 142; S in a position corresponding to position 142; T in a position corresponding to position 142; G in a position corresponding to position 143; K in a position corresponding to position 143; T in a position corresponding to position 144; Q in a position corresponding to position 166; T in a position corresponding to position 166; L in a position corresponding to position 169; G in a position corresponding to position 174; N in a position corresponding to position 174; Q in a position corresponding to position 193; T in a position corresponding to position 195; N in a position corresponding to position 195; And in a position corresponding to position 196; R in a position corresponding to position 196; P in a position corresponding to position 204; A in a position corresponding to position 205; And in a position corresponding to position 205; I in a position corresponding to position 206; A in a position corresponding to position 213; I in a position corresponding to position 219; M in a position corresponding to position 234; T in a position corresponding to position 237; H in a position corresponding to position 238; Q in a position corresponding to position 240; V in a position corresponding to position 249; A in a position corresponding to position 261; K in a position corresponding to position 261; T in a position corresponding to position 267; K in a position corresponding to position 277; H in a position corresponding to position 279; V in a position corresponding to position 279; V in a position corresponding to position 291; And in a position corresponding to position 309; Q in a position corresponding to position 310; Y in a position corresponding to position 314; Y in a position corresponding to position 315; N in a position corresponding to position 317; W in a position corresponding to position 317; D in a position corresponding to position 318; G in a position corresponding to position 347; A in a position corresponding to position 367; R in a position corresponding to position 375; R in a position corresponding to position 376; V in a position corresponding to position 399; And in a position corresponding to position 401; A in a position corresponding to position 407; L in a position corresponding to position 416; K in a position corresponding to position 419; H in a position corresponding to position 421; And in a position corresponding to position 431; T in a position corresponding to position 433; V in a position corresponding to position 433; C in a position corresponding to position 439; P in a position corresponding to position 440; G in a position corresponding to position 443; N in a position corresponding to position 445, each with reference to amino acid positions exposed in SEQ ID NO: 3.
[000293] [000293] The amino acid substitution (s) may be on a PH20 polypeptide as set forth in any of SEQ ID NOS: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity therewith. For example, the substitution (s) can be in a human PH20 polypeptide, for example, any exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72 or a variant of same.
[000294] [000294] Exemplary modified PH20 polypeptides that exhibit increased stability to phenol compounds compared to unmodified PH20 polypeptide (for example, set out in SEQ ID NO: 3) are any having the amino acid sequence exposed in any of SEQ ID NOS: 83 , 88, 93, 94, 101, 144, 148, 158, 171, 176, 175, 177, 178, 180, 182, 183, 184, 185, 194, 221, 240, 259, 260, 261, 262, 263 , 264, 268, 270, 272, 307, 309, 327, 334, 341, 351, 352, 353, 356, 357, 358, 359, 361, 424, 426, 430, 434, 436, 443, 444, 445 , 446, 447, 449, 450, 451, 454, 461, 467, 480, 487, 489, 492, 495, 504, 505, 509, 527, 544, 576, 589, 600, 603, 607, 612, 614 , 647, 658, 683, 687, 733, 736, 741, 754, 763, 768, 781, 796, 797, 809, 818, 829 or 837 or having an amino acid sequence that is at least 68%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of SEQ ID NOS: 83, 88, 93, 94, 101, 144, 148, 15 8, 171, 176, 175, 177, 178, 180, 182, 183, 184, 185, 194, 221, 240, 259, 260, 261, 262, 263, 264, 268, 270, 272, 307, 309, 327, 334, 341, 351, 352, 353, 356, 357, 358, 359, 361, 424, 426, 430, 434, 436, 443, 444, 445, 446, 447, 449, 450, 451, 454, 461, 467,
[000295] [000295] In particular, a modified PH20 polypeptide is provided here that contains an amino acid substitution with P in a position corresponding to amino acid residue 204 with reference to SEQ ID NO: 3. Typically, the modified PH20 polypeptide containing a substitution of amino acid F204P in an amino acid sequence exposed in any of SEQ ID NOS: 3, 7, 69, 72 or 32-66, or an amino acid sequence that has at least 75%, 80%, 85%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity for any of SEQ ID NOS: 3, 7, 69, 72 or 32-66 provided the modified polypeptide contains the amino acid substitution corresponding to F204P. In other cases, the modified PH20 polypeptide is a non-human polypeptide. For example, a modified PH20 polypeptide is provided here that contains an amino acid substitution F204P in an amino acid sequence set out in SEQ ID NO: 10, 12, 14, 857, 859, 861 or 870 or a sequence that has at least 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity for any of SEQ ID NOS: 10 , 12, 14, 857, 859, 861 or 870 provided that the modified polypeptide contains the amino acid substitution corresponding to F204P. In a further example, a modified PH20 polypeptide is provided here that contains an F205P amino acid substitution in an amino acid sequence exposed in SEQ ID NO: 24 or Y204P in SEQ ID NO: 31, or a sequence that has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity for SEQ ID NO: 24 or 31 An example of such a modified PH20 polypeptide is a polypeptide having the amino acid sequence shown in SEQ ID NO: 449, or having an amino acid sequence that has at least 68%, 70%, 75%, 80%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity for SEQ ID NO: 449 and contains the amino acid substitution F204P, has increased hyaluronidase activity and has increased stability to phenol compounds compared to the corresponding unmodified polypeptide (eg SEQ ID NO: 3). In any of the above examples, the modified PH20 polypeptide that contains an amino acid substitution with P in a position corresponding to amino acid residue 204 with reference to SEQ ID NO: 3 does not have the amino acid sequence exposed in SEQ ID NO: 15-22 , 28 or
[000296] [000296] In another example, a modified PH20 polypeptide is provided here that contains an amino acid substitution in a position corresponding to amino acid residue 58 with reference to SEQ ID NO: 3. Examples of amino acid substitutions are substitution with lysine (K) or arginine (R) in a position corresponding to amino acid residue 58 with reference to SEQ ID NO: 3. Typically, the modified PH20 polypeptide is a human polypeptide. For example, a modified PH20 polypeptide is provided here that contains an amino acid substitution V58K or V58R in an amino acid sequence exposed in any of SEQ ID NOS: 3, 7, 69, 72 or 32-66, or an amino acid sequence that at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
[000297] [000297] At high temperatures, PH20 hyaluronidases may lose activity.
[000298] [000298] PH20 polypeptides are provided here which have increased stability at elevated temperatures between or approximately between 30 ° C and 45 ° C, inclusive, as between or approximately between 35 ° C and 42 ° C, in particular at or approximately 37 ° C . For example, modified PH20 polypeptides are provided here that are stable at elevated temperatures greater than 32 ° C such as 35 ° C to 45 ° C, 37 ° C to 42 ° C and in particular at or approximately 37 ° C for at least 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, at least 6 days or at least 7 days. Modified PH20 polypeptides that exhibit stability at elevated temperatures can be used in applications where temperatures are high, may fluctuate or may increase. This can occur, for example, in administration methods using pumps or other continuous infusion devices.
[000299] [000299] In particular, modified PH20 polypeptides provided here that exhibit stability at elevated temperatures show increased hyaluronidase activity at elevated temperature compared to the corresponding PH20 polypeptide not containing the modification, for example, amino acid substitution. PH20 polypeptides may show increased hyaluronidase activity after incubation at elevated temperatures greater than 32 ° C such as 35 ° C to 45 ° C or 37 ° C to 42 ° C, in particular at or approximately 37 ° C for at least 4 hours , 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days 6 days or at least 7 days compared to the corresponding PH20 polypeptide not containing the modification incubated under the same conditions. For example, hyaluronidase activity can be increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400% , 500% or more compared to the corresponding PH20 polypeptide not containing the modification incubated under the same conditions. For example, hyaluronidase activity can be increased at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times , 3 times, 4 times, 5 times or more compared to the corresponding PH20 polypeptide not containing the modification incubated under the same conditions.
[000300] [000300] In other examples, modified PH20 polypeptides provided here that exhibit stability at elevated temperatures retain hyaluronidase activity at elevated temperatures compared to the activity of the modified PH20 polypeptide incubated at not elevated temperatures under the same conditions (except for differences in temperature) . For example, modified PH20 polypeptides have greater than or approximately 50%, as greater than or at least 55%, 60%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or 100% of activity at elevated temperatures greater than 32 ° C such as 35 ° C to 45 ° C or 37 ° C to 42 ° C, in particular in or approximately 37 ° C compared to PH20 activity at non-elevated temperatures between or approximately 2 ° C to 8 ° C. In some examples, modified PH20 polypeptides provided here that exhibit stability at elevated temperatures show increased activity at elevated temperatures compared to the activity of the modified PH20 polypeptide incubated at not elevated temperatures under the same conditions (except for the difference in temperature). For example, modified PH20 polypeptides have activity greater than or approximately 10% increased activity, such as greater than or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of activity at elevated temperatures greater than 32 ° C such as 35 ° C to 45 ° C or 37 ° C to 42 ° C, in particular at or approximately 37 ° C compared to PH20 activity at non-elevated temperatures between or approximately 2 ° C to 8 ° C. For example, modified PH20 polypeptides exhibit more than or at least approximately 1.1-fold hyaluronidase activity, such as greater than or at least 1.2-fold,
[000301] [000301] For example, such modified PH20 polypeptides provided here that have increased stability at elevated temperatures contain an amino acid substitution at one or more amino acid positions corresponding to positions 1, 11, 12, 14, 20, 26, 29, 34, 50, 58, 70, 82, 83, 84, 86, 87, 140, 142, 143, 147, 152, 166, 167, 172, 174, 178, 193, 195, 206, 212, 213, 219, 233, 237, 240, 267, 277, 291, 292, 309, 313, 314, 317, 318, 347, 367, 368, 371, 374, 389, 392, 395, 396, 406, 419, 421, 439 or 443 with reference to amino acid positions exposed in SEQ ID NO: 3. For example, amino acid positions can be substitutions in one or more positions corresponding to the substitution of (L) in position 1 (L1), N11, V12, F14, A20, L26, F29, D34, G50, V58, I70, K82 , I83, S84, Q86, D87, Q140, V142, Q143, T147, K152, V166, E167, G172, L174, N178, H193, K195, V206, D212, D213, N219, Q233, V237, T240, A267, V277 , G291, E292, I309, M313, K314, L317, L318, Q347, P367, D368, A371, L374, E389, E392, S395, E396, L406, A419, D421, E439 or P443, with reference to exposed amino acid positions in SEQ ID NO: 3. The resulting modified PH20 polypeptide exhibits increased stability at elevated temperatures greater than 32 ° C such as 35 ° C to 45 ° C, 37 ° C to 42 ° C and in particular at or approximately 37 ° C for at least 3 hours, 4 hours , 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, at least 5 days, at least 6 days, at least 7 days or more.
[000302] [000302] Exemplary amino acid substitutions in the modified PH20 polypeptides provided here include, but are not limited to, substitution with R in a position corresponding to position 1; S in a position corresponding to position 11; I in a position corresponding to position 12; V in a position corresponding to position 14; S in a position corresponding to position 20; M in a position corresponding to position 26; with R in a position corresponding to position 29; W in a position corresponding to position 34; M in a position corresponding to position 50; K in a position corresponding to position 58; Q in a position corresponding to position 58; Q in a position corresponding to position 58; V in a position corresponding to position 70; L in a position corresponding to position 82; Q in a position corresponding to position 83; R in a position corresponding to position 84; A in a position corresponding to position 86; S in a position corresponding to position 87; K in a position corresponding to position 140; S in a position corresponding to position 142; T in a position corresponding to position 142; K in a position corresponding to position 143; S in a position corresponding to position 147; T in a position corresponding to position 152; T in a position corresponding to position 166; D in a position corresponding to position 167; A in a position corresponding to position 172; G in a position corresponding to position 174; N in a position corresponding to position 174; R in a position corresponding to position 178; Q in a position corresponding to position 193; T in a position corresponding to position 195; I in a position corresponding to position 206; S in a position corresponding to position 212; A in a position corresponding to position 213; I in a position corresponding to position 219; G in a position corresponding to position 233; T in a position corresponding to position 237; A in a position corresponding to position 240; Q in a position corresponding to position 240; T in a position corresponding to position 267; And in a position corresponding to position 277; S in a position corresponding to position 291; H in a position corresponding to position 292; V in a position corresponding to position 292; S in a position corresponding to position 309; H in a position corresponding to position 313; S in a position corresponding to position 314; I in a position corresponding to position 317; T in a position corresponding to position 317; W in a position corresponding to position 317; R in a position corresponding to position 318; G in a position corresponding to position 347; A in a position corresponding to position 367; R in a position corresponding to position 368; S in a position corresponding to position 371; P in a position corresponding to position 374; A in a position corresponding to position 389; V in a position corresponding to position 392; A in a position corresponding to position 395; H in a position corresponding to position 396; N in a position corresponding to position 406; H in a position corresponding to position 419; K in a position corresponding to position 419; R in a position corresponding to position 421; S in a position corresponding to position 421; A in a position corresponding to position 439; C in a position corresponding to position 439; or G in a position corresponding to position 443, each with reference to amino acid residue positions exposed in SEQ ID NO: 3.
[000303] [000303] The amino acid substitution (s) may be in a PH20 polypeptide as set forth in any of SEQ ID NOS: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant the same having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity therewith. For example, the substitution (s) may be in a human PH20 polypeptide, for example, any exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72 or a variant thereof .
[000304] [000304] Exemplary modified PH20 polypeptides that have increased stability to phenol compounds compared to unmodified PH20 polypeptide (for example, set out in SEQ ID NO: 3) are any having the amino acid sequence exposed in any of the SEQ ID NOS: 79, 85, 87, 90, 93, 101, 114, 144, 171, 178, 181, 221, 259, 262, 269, 270, 282, 343, 356,
[000305] [000305] PH20 denatures in the presence of low salt or no salt. Thus, PH20 requires a high concentration of salt between or approximately between 140 mM to 200 mM to maintain stability. Other therapeutic agents, for example, insulin, show decreased solubility and increased crystallization / aggregation in the presence of high salt content. Thus, the high salt requirements of PH20 can affect the solubility and / or activity of co-formulated therapeutic agents, while the presence of low salt content can decrease the PH20 activity. This can create problems for generating PH20 co-formulations.
[000306] [000306] Modified PH20 polypeptides are provided here which have increased stability in the presence of low salt concentrations (eg NaCl) less than 100 mM, for example, less than 90 mM, 80mM, 70mM, 60 mM, 50 mM , 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. Generally, the modified PH20 polypeptides provided here exhibit stability in the presence of low salt concentrations, for example, low NaCl concentrations between or approximately between mM NaCl and 100 mM NaCl, such as between or approximately between 15 mM and 80 mM NaCl. The modified PH20 polypeptides provided here that have stability at low salt concentrations, such as low NaCl concentrations (i.e., less than 100 mM or less), have increased hyaluronidase activity compared to the corresponding PH20 not containing the (s) modification (s) (for example, amino acid substitutions). For example, modified PH20 polypeptides have activity greater than or approximately 10% increased activity, such as greater than or at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500% or more of activity at low salt concentrations, such as low NaCl concentrations (ie less than 100 mM), compared to the corresponding PH20 activity not containing the amino acid modification (s) (e.g., amino acid substitution (s)) under the same conditions. For example, modified PH20 polypeptides exhibit activity greater than or at least approximately 1.1 times the hyaluronidase activity, such as greater than or at least 1.2 times, 1.3 times, 1.4 times, 1.5 times,
[000307] [000307] Modified PH20 polypeptides containing one or more amino acid substitutions in a PH20 polypeptide and which are inactive are provided here, so the polypeptides do not have hyaluronidase activity or have low or decreased hyaluronidase activity. The modified PH20 polypeptides provided here that are generally inactive exhibit less than 20%, such as less than 105, of the hyaluronidase activity of a reference or wild-type PH20 polypeptide, such as the polypeptide exposed in SEQ ID NO: 3 or 7. For For example, modified PH20 polypeptides provided here that are inactive have 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0 less , 7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05% or less of the hyaluronidase activity of a reference PH20 polypeptide or wild type, as the corresponding polypeptide not containing the amino acid modification (for example, amino acid substitution), for example, a polypeptide exposed in SEQ ID NO: 3 or 7.
[000308] [000308] For example, PH20 polypeptides are provided here which are inactive and which are modified, for example, by amino acid substitution, compared to a reference PH20 polypeptide or wild type. For example, a modified PH20 polypeptide provided here that is inactive contains one or more amino acid substitutions in position (s) corresponding to position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 25, 27, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 94, 95, 96, 98, 99, 100,
[000309] [000309] Exemplary amino acid substitutions in any of the corresponding positions above are shown in table 5. Reference to the corresponding position in Table 5 is with reference to positions exposed in SEQ ID NO: 3. It is understood that substitutions can be made in the corresponding position in another PH20 polypeptide by aligning it with the sequence shown in SEQ ID NO: 3 (see, for example, figures 1 and 2), so the corresponding position is the aligned position. The amino acid substitution (s) may be in the corresponding position on a PH20 polypeptide as set forth in any of SEQ ID NOS: 2, 3, 6-66, 68-72, 856-861, 869 or 870 or a variant thereof having at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 86%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity thereto, as long as the resulting modified PH20 polypeptide is inactive. For example, the substitution (s) may be in a corresponding position in a human PH20 polypeptide, for example, any exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72, or a variant of it. In particular, any one or more of the substitutions in SEQ ID NO: 3, provided that the resulting modified PH20 polypeptide is inactive and has less than 20%, and generally less than 10%, of the PH20 polypeptide hyaluronidase activity exposed in SEQ ID NO: 3A TABLE 5: Inactive mutants Substitution position Substitution position Substitution position o o races races spond pondente teente 2 HKWY 3 AGKPTV 4 DEFGLPW
[000310] [000310] Modified PH20 polypeptides include those that contain chemical or post-translational modifications. In some instances, modified PH20 polypeptides provided here do not contain chemical or post-translational modifications. Chemical and post-translational modifications include, but are not limited to, PEGUylation, sialization, albumination, glycosylation, farnisylation, carboxylation, hydroxylation, phosphorylation and other polypeptide modifications known in the art.
[000311] [000311] Also, in addition to any one or more amino acid modifications, such as amino acid substitutions, provided here, modified PH20 polypeptides provided here can be conjugated or fused to any fraction using any method known in the art, including chemical and recombinant methods, with the condition that the resulting polypeptide retains hyaluronidase activity. For example, in addition to any one or more amino acid modifications, such as amino acid substitutions, provided here, modified PH20 polypeptides provided here may also contain other modifications that are or are not in the primary sequence of the polypeptide, including but not limited to, modification with a carbohydrate fraction, a polyethylene glycol (PEG) fraction, a sialic acid fraction, an Fc domain from G immunoglobulin, or any other domain or fraction. For example, such additional modifications can be made to increase the stability or serum half-life of the protein.
[000312] [000312] In some cases, the domain or other fraction is a target agent, including any agent that targets the conjugate to one or more cell types by selectively binding to a cell surface receptor or other cell surface fraction . For example, the domain or other fraction is a targeting agent that directs the conjugate to tumor cells. In such examples, a modified PH20 polypeptide, like any provided here, is linked directly or indirectly to a target agent. Such target agents include, but are not limited to, growth factors, cytosines, chymosins, antibodies, and hormones, or allelic variants, muteins, or fragments thereof as long as the target agent is internalized by a cell surface receptor. Additional, non-limiting, exemplary modifications are described below. A. decreased immunogenicity
[000313] [000313] The modified PH20 polypeptides provided here can be made to have decreased immunogenicity. Decreased immunogenicity can be effected by sequence changes that eliminate antigenic epitopes from the polypeptide or by altering post-translational modifications. A person skilled in the art is familiar with methods of identifying antigenic epitopes on a polypeptide (see, for example, Liang et al. (2009) BMC Bioinformatics, 10: 302; Yang et al. (2009) Rev. Med. Virol., 19: 77-96). In some examples, one or more amino acids can be modified to remove or alter an antigenic epitope.
[000314] [000314] In another example, altering the glycosylation of a protein can also affect immunogenicity. For example, alteration of the peptide glycosylation is considered, provided that the polypeptides minimally contain at least N-acetylglucosamine in amino acid residues corresponding to exposed amino acid residues such as N200, N333 and N358 of SEQ ID NO: 3 or 7.
[000315] [000315] For example, PH20 polypeptides can be modified in such a way that they do not have fucose, particularly bifucosylation. In particular, the PH20 polypeptides provided here are not bifucosylated. This can be achieved by expressing and producing the PH20 polypeptide in host cells that do not effect bifucosylation. Fucose is a deoxyxosis that is present in a wide variety of organisms, including mammals, insects and plants. Fucosylated glycans are synthesized in fucosyl transfereases, see for example, Ma et al., Glycobiology, 16 (12): 158R-184R, (2006); Nakayama et al., J. Biol. Chem., 276: 16100-16106 (2001); and Sturla et al., Glycobiology, 15 (10): 924-935 (2005). In humans, fucose often exists as a terminal modification for glycan structures, and the presence of α1,6- fucose linked to N-acetylglucosamine has been shown to be important in glycoprotein processing and recognition. In insects, N-glycan core structures have bifucosylation with α1,6- and α1,3-bonds. Insect cell nucleus fucosylation with α1,3-bonds generates a carbohydrate epitope that is immunogenic in humans (see, for example, US publication No. 20070067855). For example, PH20 polypeptides provided here can be generated in host cells that are unable to bifucosylate the polypeptide. Thus, although insect cells or other cells that bifucosylate can be used to express polypeptides, typically mammalian cells, such as CHO cells, are used.
[000316] [000316] In some examples, fucose-deficient or defucosylated PH20 polypeptides can be generated in insect cells with modified glycosylation targets,
[000317] [000317] In some examples, the modified PH20 polypeptides provided here are conjugated to polymers. Exemplary polymers that can be conjugated to PH20 polypeptides, include natural and synthetic homopolymers, such as polyols (ie, poly-OH), polyamines (ie, poly-NH2) and polycarboxylic acids (ie, poly-COOH), and additional heteropolymers, that is, polymers containing one or more different coupling groups, for example, hydroxyl groups and amine groups. Examples of suitable polymer molecules include polymer molecules selected from polyalkylene oxides (PAO), such as polyalkylene glycols (PAG), including polyethylene glycols (PEG), methoxy polyethylene glycols (mPEG), and polypropylene glycols, PEG-glycidyl ethers (epox-
[000318] [000318] Typically, polymers are polyalkylene oxides (PAO), such as polyethylene oxides, such as PEG, typically mPEG, which have few reactive groups capable of crosslinking. Typically, polymers are non-toxic polymeric molecules such as (methoxy) polyethylene glycol (mPEG) that can be coluvially conjugated to PH20 polypeptides (for example, with binding groups on the protein surface) using relatively simple chemistry.
[000319] [000319] Polymeric molecules suitable for attachment to PH20 polypeptides include, but are not limited to, polyethylene glycol (PEG) and PEG derivatives such as methoxy-polyethylene glycols (mPEG), PEG-glycidyl ethers (epox-PEG), PEG-oxycarbonylimidazole ( CDI-PEG), branched PEGs and polyethylene oxide (PEO) (see, for example, Roberts et al., Advanced Drug Delivery Review 2002, 54: 459-476; Harris and Zalipsky (eds.) “Poly (ethylene glycol), Chemistry and Biological Applications ”ACS Symposium Series 680, 1997; Mehvar et al., J. Pharm. Pharmaceut. Sci., 3 (1): 125-136, 2000; Harris and Chess (2003) Nat Rev
[000320] [000320] Various methods of modifying polypeptides by covalently bonding (conjugating) a PEG or PEG derivative (i.e., PEGUylation ”) are known in the art (see, for example, U.S. 2006/0104968; U.S.
[000321] [000321] Numerous reagents for PEGUilation have been described in the art. Such reagents include, but are not limited to, N-hydroxysuccinimidyl-activated PEG (NHS), mPEG succinimidyl, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinimidyl succinimidyl butaneate hydroxybutanoic acid, homobifunctional PEG-succinimidyl propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, PEG maleimide, PEG hydrazide, p-nitrophenyl carbonate PEG, mPEG-benzotriazol carbonate, mEGP, pEGP, butyldehyde, butyldehyde; mPEG methyl ketone, maleimide "without linker" mPEG, mPEG vinyl sulfone, mPEG thiol, mPEG orthopyridylthioester, mPEG orthopyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, vinylsulfone PEG-NHS, acrylate PEG-NHS, fluoresce , and biotin PEG-NHS (see for example, Monfardini et al., Bioconjugate Chem. 6: 62-69, 1995; Veronese et al., J. Bioactive Compatible Polymers 12: 197- 207, 1997; US 5. 672,662; U.S. 5,932,462; U.S. 6,495,659; U.S. 6,737,505; U.S. 4,002,531; U.S. 4,179,337; U.S.
[000322] [000322] Methods are provided here to identify a variant or modified hyaluronan degrading enzyme, such as a modified hyaluronidase or modified PH20 polypeptide, that exhibits an altered activity or property compared to an unmodified hyaluronan degrading enzyme. In particular, the methods provided here can be used to select one or more modified hyaluronan degrading enzymes, such as one or more modified hyaluronidase or PH20 polypeptide, which exhibits increased activity and / or increased stability in the presence of a denaturing condition or agent. For example, the methods can be used to identify a modified or variant hyaluronan degrading enzyme, such as a modified hyaluronidase or variant or modified PH20 variant or polypeptide, which exhibits increased stability due to increased resistance to denaturation conditions, including but not limited to a, denaturation conditions caused by temperature (e.g., high temperature such as heat), agitation, no salt or low salt content, presence of an excipient and / or a denaturing agent. Exemplary denaturing agents or excipients include, but are not limited to, non-stick, binders, coatings, fillers and thinners, flavors, colors, lubricants, glidants, preservatives, sorbents or sweeteners. For example, various excipients, such as condoms, can act as protein denaturing agents. In the method, the activity can also be compared with an unmodified hyaluronan degrading enzyme under the same denaturation condition, and an identified or selected modified hyaluronan degrading enzyme that has greater activity than the corresponding unmodified hyaluronan degrading enzyme.
[000323] [000323] In the method, one or more enzymes to degrade modified hyaluronan are provided. In some examples, a library of modified molecules is prepared. Methods of mutagenesis and generation of libraries or collections of variant molecules are described here and are known to a person skilled in the art using standard recombinant DNA techniques. In one example, the enzymes that are tested can be grouped and classified, so the method allows selection only of those enzymes that have a desired activity. In another example, the enzymes tested can be physically separated and classified individually, as by formatting in sets, as addressable sets.
[000324] [000324] In one aspect of the method, degraded modified hyaluronan enzymes are tested or classified for hyaluronidase activity in the presence and absence of one or more denaturing conditions or denaturing agent. After testing under the two sets of conditions, activities are evaluated to identify modified hyaluronan degrading enzymes that show activity in the presence of the denaturation condition. The desired level or amount of activity selected as cut in methods can be empirically determined by the user, and depends on factors such as the specific hyaluronan degrading enzyme, the desired application or use of the hyaluronan degrading enzyme, the specific denaturation condition or denaturation and other similar factors. Typically, a modified hyaluronan degrading enzyme is identified that exhibits at least 5% or 10% of the activity in the presence of a denaturing agent or condition compared to its absence, and generally at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, for example, at least 40% of the activity.
[000325] [000325] Additionally or alternatively, the activity of the modified hyaluronan degrading enzyme in the presence of one or more denaturing conditions or denaturing agents is compared with the corresponding unmodified hyaluronan degrading enzyme activity in the presence of the same (s) ) denaturation agent (s) or condition (s). In such examples, it is understood that the activity of the modified and unmodified enzyme is tested under the same conditions (for example, time, temperature, composition), except for the difference in the specific enzyme tested (unmodified versus modified). A modified hyaluronan-degrading enzyme is identified to have greater activity, such as at least 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300 %, 400%, 500% or more of the unmodified hyaluronan degrading enzyme activity.
[000326] [000326] The method can be executed a plurality of times, so the steps of the method are 1, 2, 3, 4, or 5 times. The method provided here is also iterative. In one example, after the method is run, any modified, identified hyaluronan degrading enzyme can be modified or additionally modified to increase or optimize activity.
[000327] [000327] A description of the method steps and method components is provided in the subsections that follow.
[000328] [000328] In the methods of the present invention, one or more modified hyaluronan degrading enzymes, such as a hyaluronidase or a PH20 polypeptide, are tested for a desired activity or property, such as increased stability (e.g., increased resistance to a denaturation condition) . The modified hyaluronan degrading enzyme can be modified compared to an unmodified hyaluronan degrading enzyme, like any hyaluronan degrading enzyme known in the art. Hyaluronan-degrading enzymes are a family of enzymes that degrade hyaluronic acid, which is an essential component of the extracellular matrix and a major constituent of the interstitial barrier. Hyaluronan-degrading enzymes act to degrade hyaluronan by cleaving hyaluronan polymers, which are composed of repeating disaccharide units: D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine (GlcNAc), linked together through glycosidic bonds - 1 → 4 and -1 → 3 alternating. By catalyzing the hydrolysis of hyaluronic acid, a major constituent of the interstitial barrier, hyaluronic degrading enzymes decrease the viscosity of hyaluronic acid, thereby increasing tissue permeability. Accordingly, enzymes that degrade hyaluronan for the uses and methods provided herein include any enzyme having the ability to catalyze the cleavage of a hyaluronan or polymer disaccharide chain. In some examples, the hyaluronan degrading enzyme cleaves the -1 → 4 glycosidic bond in the hyaluronan or polymer chain. In other examples, the hyaluronan-degrading enzyme catalyzes the cleavage of the -1 → 3 glycosidic bond in the hyaluronan or polymer chain.
[000329] [000329] Hyaluronan degrading enzymes include enzymes that are membrane bound or that are soluble forms that are secreted from cells. Thus, where hyaluronan degrading enzymes include a glycosylphosphatidylinositol (GPI) anchor signal sequence and / or are otherwise membrane-anchored or insoluble, such hyaluronan degrading enzymes can be supplied in soluble form by C-terminal truncation. or deleting all or a portion of the GPI anchor signal sequence to make the enzyme secreted and soluble. Thus, hyaluronan-degrading enzymes include truncated C-terminal variants, for example, truncated to remove all or a portion of a GPI anchor signal sequence. Examples of such soluble hyaluronidases are PH20 soluble hyaluronides, like any set out in US Patent No. 7,767,429; U.S. publication Nos. US 2004/0268425 and US 2010/0143457.
[000330] [000330] Exemplary hyaluronan degrading enzymes are human or non-human animal hyaluronidases, bacterial hyaluronidases, leeches or chondroitinases that have hyaluronan degrading activity, including soluble or truncated forms of which are activated. Hyaluronidases from exemplary non-human animals are any exposed in any of SEQ ID NOS: 8-31, 856-861, 869, 870, 871-886, or truncated, terminal C-shaped, mature variants that are soluble and active, or active forms of them. Exemplary human hyaluronidases are any exposed in any of SEQ ID NOS: 2, 3, 6, 7, 32-66, 68-72 or 887- 890, or mature C-terminal, truncated variants that are soluble and active, or active forms thereof, and in particular any of SEQ ID NOS: 3, 7, 32-66, 69 or
[000331] [000331] For example, one or more modified PH20 polypeptides are tested for a desired activity or property, such as increased stability (for example, increased resistance to a denaturation condition). The modified PH20 polypeptide can be modified compared to an unmodified PH20 polypeptide, such as any known native PH20 polypeptide, reference or wild-type polypeptide. For example, the modified PH20 polypeptide is modified in comparison to a soluble or active, full-length form of a PH20 polypeptide, like any exposed in any of SEQ ID NOS: 3, 7, 32-66, 69 or 72, or a polypeptide with at least 85%, such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity for any of SEQ ID NOS: 3, 7, 32-66, 69 or 72. In specific examples of the method of the present invention, the unmodified or starting PH20 polypeptide has the amino acid sequence exposed in SEQ ID NO: 3.
[000332] [000332] Libraries or collections of enzymes to degrade modified hyaluronan can be selected. Hyaluronan degrading enzymes can be modified by any process known to a person skilled in the art that can alter the structure of a protein. Examples of modifications include substitution, addition and deletion of one or more amino acids from the protein to form libraries or collections of enzymes to degrade modified hyaluronan. It is understood at the level of a person skilled in the art to generate variant or modified proteins for use in the methods of the present invention. Mutagenesis methods are well known in the art and include, for example, site-directed mutagenesis such as QuikChange (Stratagene) or saturation mutagenesis. Mutagenesis methods include, but are not limited to, site-mediated mutagenesis, PCR mutagenesis, cassette mutagenesis, site-directed mutagenesis, random point mutagenesis, mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, DNA mutagenesis modified by phosphorogen , mutagenesis using open duplex DNA, spot mismatch repair, mutagenesis using host strains deficient in repair, selection-restriction and purification-restriction, deletion mutagenesis, total gene synthesis mutagenesis, double-strand rupture repair, and many others known to knowledgeable people. In the methods of the present invention, mutagenesis can be carried out through the full length of a protein or in a region of a protein. Mutations can be made rationally or randomly.
[000333] [000333] In some examples, the methods provided here are performed in such a way that the identity of each mutant protein is known a priori before testing the protein. For example, the methods provided here can be useful for mutagenesis and test or classification methods that are addressable. This can allow for easy comparisons between the activities of tested proteins without the need for sequencing of identified proteins. For example, site-directed mutagenesis methods can be used to individually generate mutant proteins. Mutagenesis can be performed by replacing single amino acid residues at specific target positions one by one, in such a way that each individual mutant generated is the unique product of each single mutagenesis reaction. Mutant DNA molecules can be designed, generated by mutagenesis and cloned individually, as in addressable sets, in such a way that they are physically separated from each other and each is the unique product of an independent mutagenesis reaction. The amino acids selected to replace the target positions on the specific protein being optimized can be all of the remaining 19 amino acids, or a more restricted group containing only selected amino acids. In some methods provided here, each amino acid that is replaced is independently replaced by 19 of the remaining amino acids or less than 19 of the remaining amino acids, such as 10, 11, 12, 13, 14, 15, 16, 17 or 18 of the remaining amino acids .
[000334] [000334] Hyaluronidase activity or other activity of a composition containing a modified hyaluronan degrading enzyme is classified or tested under conditions that expose the hyaluronan degrading enzyme to a denaturation condition or a denaturing agent (presence of denaturation condition or denaturing agent). The denaturing condition or denaturing agent does not require a condition or agent that is completely deadly to the enzyme, but it is generally any condition or agent that destabilizes the enzyme activity over time. For example, the denaturation condition can be a condition caused by temperature (for example, elevated temperature as greater than or approximately or 30 ° C, for example, 30 ° C to 42 ° C as or approximately 37 ° C), agitation , no salt or low salt content (eg, NaCl), and / or caused by the presence of a denaturing agent such as the presence of excipients (eg, the presence of preservatives).
[000335] [000335] For the purpose of selecting or identifying a modified hyaluronan degrading enzyme that exhibits increased stability or stability under the condition of denaturation, activity can be compared with activity of the modified hyaluronan degrading enzyme in the absence of the condition of denaturation and / or activity of corresponding unmodified hyaluronan degrading enzyme in the presence of the denaturation condition. For example, the modified hyaluronan-degrading enzyme can also be classified or tested under the same conditions, except not including a denaturing condition or denaturing agent (no denaturing condition or denaturing agent). If desired, the corresponding unmodified hyaluronan degrading enzyme activity (for example, the hyaluronan degrading enzyme not containing the amino acid substitution (s) can also be tested under the same conditions as exposing the hyaluronan degrading enzyme to the same denaturation condition. or a denaturing agent.
[000336] [000336] For example, each element of a library or collection of modified hyaluronan degrading enzymes is incubated under or exposed to one or more denaturing conditions. Incubation or exposure can occur in vivo or in vitro. Typically, the assay is performed in vitro. The same modified enzyme is also exposed or incubated to a control or reference condition that does not contain the denaturation condition. The activities under the two conditions are compared to identify modified hyaluronan degrading enzymes that show stability after exposure to a denaturation condition or conditions. In addition, when classifying or identifying enzyme activity under the two different sets of conditions, generally the only conditions that vary in the assay refer to the presence or absence of one or more denaturation conditions. The other conditions of the assay, including but not limited to, time, temperature and / or other incubation conditions, may be the same for both sets of conditions.
[000337] [000337] For example, exposure can be obtained by incubating a modified hyaluronan degrading enzyme in an assay buffer or composition that has been modified or adjusted to contain a denaturing agent such as an excipient or low salt or no salt. Exemplary denaturing agents or excipients include, but are not limited to, non-stick, binders, coatings, fillers and thinners, flavors, colors, lubricants, glidants, preservatives, sorbents or sweeteners. The choice of buffer that is used can be empirically determined by a person skilled in the art depending on the specific parameter or parameters being modified. Exemplary assay buffers are Good buffers (see, for example, Good and others (1966) Biochemistry, 5: 467-477). Examples of such buffers include, but are not limited to, ACES, ADA, BES, Bicine, BIS-TRIS, CAPS, HEPES, MES, MOPS, PIPES, TRIS or Trizma® buffers. In addition, the amount or concentration of the excipient or salt can be empirically determined by a person skilled in the art depending on the choice of excipient or salt and the desired level or activity of the modified hyaluronan degrading enzyme.
[000338] [000338] In one example, the assay buffer or composition is modified by including an amount of a denaturing agent or denaturing excipient that is a preservative, for example, a phenolic preservative. The phenolic preservative can be phenol, metacresol (m-cresol), benzyl alcohol, and parabens including methyl paraben and propyl paraben. In particular, the phenolic condom is phenol and / or m-cresol. The total amount of one or more phenolic preservative agents as a percentage (%) of mass concentration (weight / v) can be between 0.05% and 0.6%, 0.1% and 0.4%, 0, 1% and 0.3%, 0.15% and 0.325%, 0.15% and 0.25%, 0.1% and 0.2%, 0.2% and 0.3% or 0.3% and 0.4% inclusive. In such an example, the activity of the modified hyaluronan degrading enzyme is tested or evaluated in the presence of such total amount (for example, between or approximately between 0.05% and 0.6%) of one or more condoms, for example, a or more phenolic condoms. In some instances, the modified hyaluronan degrading enzyme can also be tested or evaluated under control or reference condition in which the assay buffer or composition is not modified to contain a condom. In certain cases, as a control, the activity of modified hyaluronan degrading enzymes can also be compared with the corresponding unmodified hyaluronan degrading enzyme not containing the modification (s) under conditions that contain a preservative and / or under conditions that do not contain a preservative agent.
[000339] [000339] In another example, the test buffer is modified by the presence of a denaturation condition that is low in salt or without salt. As discussed elsewhere here, hyaluronan-degrading enzymes, such as PH20, generically require salt (for example, NaCl, Lys-Lys or MgCl2) for activity. Consequently, the absence of salt or low salt content is denaturing for the enzyme. In one example, the assay buffer is modified by adding an amount of salt that is less than 100 mM, for example, less than 90 mM, 80 mM, 70 mM, 60 mM, 50 mM, 40 mM, 30 mM, 25 mM, 20 mM, 15 mM, 10 mM, 5 mM or less. In such an example, the activity of the modified hyaluronan-degrading enzyme is tested in the absence of salt or in the presence of salt that is less than 100 mM. In some instances, the modified hyaluronan degrading enzyme can also be tested or evaluated under a control or reference condition in which the assay buffer contains a higher salt concentration, generally between or approximately between 140 mM and 200 mM. In certain cases, as a control, the activity of modified hyaluronan degrading enzymes can also be compared with the corresponding unmodified hyaluronan degrading enzyme not containing the modification (s) under conditions that contain low salt or no salt content, as less than 100 mM and / or under conditions that contain salt in an amount that is between or approximately between 140 mM and 200 mM.
[000340] [000340] Exposure of a hyaluronan-degrading enzyme to a denaturation condition can also be achieved by incubating a modified hyaluronan-degrading enzyme under conditions that are known to be denaturants, such as under high temperature conditions such as a temperature higher than or approximately or 30 ° C (e.g., 30 ° C to 42 ° C as or approximately 37 ° C) or stirring. For example, the activity of the modified hyaluronan degrading enzyme is tested at elevated temperatures greater than or approximately or 30 ºC to 42 ºC. in some instances, the modified hyaluronan degrading enzyme can also be tested or evaluated under a control or reference condition where the temperature is less than 30 ºC, such as between or approximately between 0 ºC and 25 ºC, for example, 0 ºC and 5 ºC or 18 ºC and 25 ºC. In certain cases, as a control, the activity of modified hyaluronan degrading enzymes can also be compared with the corresponding unmodified hyaluronan degrading enzyme not containing the modification (s) under elevated temperatures greater than or approximately or 42 ° C. ºC and / or temperatures below 30 ºC, such as between or approximately between 0 ºC and 25 ºC, for example, 0 ºC and 5 ºC or 18 ºC and 25 ºC.
[000341] [000341] The modified hyaluronan degrading enzyme can be exposed to one or more of the conditions. Exposure to one condition can occur simultaneously, subsequently, intermittently or periodically to exposure to one or more other conditions.
[000342] [000342] In one example, in the method of the present invention, the modified hyaluronan degrading enzyme is incubated or exposed to the condition of denaturation or denaturing agent before performing an assay for hyaluronidase activity. For example, the modified hyaluronan degrading enzyme is incubated in the presence of a denaturing agent or exposed to one or more denaturation conditions or control conditions, such as one or more of the denaturation conditions or control conditions as described above. Incubation or exposure can be for any desired period of time, and can be empirically determined by a person skilled in the art. For example, the modified hyaluronan degrading enzyme can be incubated or exposed to one or more denaturing conditions, denaturing agents or control conditions for or for approximately 1 minute to 1 month, such as 1 minute to 3 weeks, 1 minute to 2 weeks, 1 minute to 1 week, 1 minute to 24 hours, 1 minute to 12 hours, like 30 minutes to 6 hours or 1 hour to 4 hours, and generally or approximately at least 30 minutes, 1 hour, 2 hours, 3 hours , 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours or 12 hours. After the incubation or exposure time, the sample or composition containing the modified hyaluronan degrading enzyme (or unmodified control enzyme) is evaluated against the hyaluronidase assay. In another example, the modified hyaluronan degrading enzyme is exposed or incubated under one or more conditions of denaturation and is simultaneously or concurrently evaluated for hyaluronidase activity. In any examples where a modified hyaluronan degrading enzyme is evaluated, it is understood that an unmodified hyaluronan degrading enzyme not containing the modification (s) can also be evaluated under similar test conditions for comparison.
[000343] [000343] Assays for evaluating hyaluronidase activity are well known in the art. Examples of such assays are described in Section G. In one example, hyaluronidase activity can be assessed in a microturbity assay, in which the amount of undegraded HA is measured by the addition of an agent that precipitates HA (for example, cetyl pyridinium chloride (CPC) or acidified serum) after the enzyme is allowed to react with HA. In another example, hyaluronidase activity can be assessed using a microtiter assay in which residual biotinylated hyaluronic acid is measured after incubation with hyaluronidase (see, for example, Frost and Stern (1997) Anal. Biochem. 251: 263-269, US Pat. Publication No.
[000344] [000344] In the method after selecting modified hyaluronan degrading enzymes under one or more denaturation conditions, the hyaluronidase activities of the tested enzymes are compared. The method is put in place to identify a modified hyaluronan degrading enzyme that is more resistant to denaturation by a condition or a denaturing agent, so the activity of the enzyme is indicative of the stability of the enzyme as a measure of its resistance to denaturation. It is understood that some reduction in enzyme activity, as a result of denaturation, can be tolerated in various applications, and thus the method can be put in place to select a modified hyaluronan degrading enzyme that exhibits a required activity after exposure to a condition denaturation to allow its use or application (eg therapeutic activity). For example, a modified enzyme can be selected that loses activity more slowly than the corresponding reference or unmodified hyaluronan degrading enzyme, but whose retained activity is sufficient for a specific purpose or application.
[000345] [000345] In examples of the methods of the present invention, the activity of the modified hyaluronan degrading enzyme is evaluated after exposure to a first condition denaturation and also evaluated after exposure to a second condition which is a condition of non-denaturation or control, and the activities hyaluronidase results compared. For comparison, in some examples, activity can be represented as a ratio of activity or a percentage of activity under a condition of denaturation compared to a condition of non-denaturation or control. For example, where the parameter that differs between the first and second conditions in the presence of a condom (eg, phenolic condom), the activity can be represented as a ratio of activity or percentage of activity observed in the presence of a condom (eg, condom phenolic) versus activity in the absence of a condom (eg, phenolic condom). In another example, where the parameter that differs between the first and second condition is temperature, the activity can be represented as an activity ratio or percentage of activity observed in the presence of high temperature (for example, 30ºC to 42ºC) compared to activity in the presence of a lower temperature like 0ºC to 25ºC, for example 0ºC to 5ºC or 18ºC to 25ºC.
[000346] [000346] A modified hyaluronan degrading enzyme is selected or identified that retains or exhibits any desired activity in the presence of the denaturation condition compared to its absence. The specific cut of activity for enzyme selection here depends on the specific use and / or practice of the method and can be empirically determined depending on factors such as the specific denaturation condition or denaturing agent, the specific modified hyaluronan degrading enzyme, the desired application selected or identified hyaluronan degrading enzyme and other similar factors. Generally, a selected or identified modified hyaluronan degrading enzyme is stable if any detectable activity is measured or evaluated after exposure or incubation with a denaturing condition or denaturing agent. For example, an identified or selected modified hyaluronan degrading enzyme exhibits stability, or resistance to a denaturing condition or denaturing agent, if it has at least 5% or 10% of the activity of the same enzyme in the absence of the denaturing condition or denaturing agent. denaturation, and generally if the modified hyaluronan degrading enzyme has an activity that is at least 15% of the initial hyaluronidase activity before incubation in the presence of the denaturation condition. For example, a modified hyaluronan degrading enzyme is selected or identified that appears at least (or at least approximately) 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, 150% , 200%, 300%, 400%, 500% or more of the initial hyaluronidase activity of the modified hyaluronan degrading enzyme tested under a condition of non-denaturation or control.
[000347] [000347] In other examples of the methods of the present invention, the activity of the modified hyaluronan degrading enzyme is evaluated after exposure to a denaturation condition and the activity of the reference or unmodified hyaluronan degrading enzyme is also evaluated after exposure to the same conditions denaturation. In such examples, activities are compared when enzymes are exposed to the same conditions. For comparison, activity under a denaturation condition can be represented as an activity ratio or a percentage of activity of a modified hyaluronan degrading enzyme compared to a reference or unmodified hyaluronan degrading enzyme. In such examples, a modified hyaluronan-degrading enzyme is selected that has greater activity under a denaturing condition than the unmodified or reference hyaluronan-degrading enzyme. Thus, the modified hyaluronan degrading enzyme is one that is more resistant to the condition. For example, where the condition of denaturation is the presence of a condom (eg, phenolic condom) the activity observed in the presence of a condom (eg, phenolic condom) can be represented as an activity ratio or percentage of degrading enzyme activity modified hyaluronan compared to the unmodified or reference hyaluronan degrading enzyme. In another example, where the denaturation condition is high temperature, the activity observed in the presence of high temperature (for example, 30ºC to 42ºC) can be represented as an activity ratio or percentage of activity of the modified hyaluronan degrading enzyme compared to enzyme to degrade unmodified or reference hyaluronan.
[000348] [000348] In such examples, a modified hyaluronan-degrading enzyme, such as a modified PH20, is identified or selected that has an activity ratio that is greater than or at least 1.1, such that the enzyme has greater activity than the unmodified or reference hyaluronan degrading enzyme in the denaturation condition. For example, the ratio is at least or at least approximately 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
[000349] [000349] The method provided here is also iterative. In one example, after the method is performed, any modified hyaluronan degrading enzymes identified as having stability, such as increased stability, under a condition of denaturation, can be modified or further modified to increase or optimize stability. A secondary library can be created by introducing additional modifications to a first identified modified hyaluronan degrading enzyme. For example, modifications that have been identified as conferring stability, such as increasing stability, can be combined to generate a combinatorial library. The secondary library can be tested using the assays and methods described here.
[000350] [000350] In another example of an iterative aspect of the method, modified hyaluronan degrading enzymes that are identified as lacking stability as increased stability (for example, such that they are not active or have no increased activity under a condition of denaturation) , can be further modified and retested for stability under a denaturation condition. Additional modifications can be targeted near specific regions (for example, specific amino acid residues) associated with the activity and / or stability of the molecule. For example, residues that are generally associated with molecule activity and / or stability are critical residues that are involved in structural folding or other activities of the molecule. Consequently, such waste is required for activity, generally in any condition. Critical residues can be identified because, when changed, normal protein activity is removed or reduced. For example, critical residues can be identified that, when changed into a hyaluronan-degrading enzyme, have hyaluronidase activity removed or reduced in a control or normal test condition. An additional library of modified proteins can be generated with amino acid mutations targeted at or near the identified critical amino acid residues, as adjacent to the identified critical amino acid residues. In some examples, the mutations can be amino acid substitution in any other of up to 19 other amino acid residues. The secondary library can be tested using the assays and methods described here. E. PRODUCTION OF MODIFIED PH20 POLYPEPTIDES AND CODING NUCLEIC ACID MOLECULES
[000351] [000351] Polypeptides of a modified PH20 polypeptide set forth herein can be obtained by methods well known in the art for protein purification and recombinant protein expression. Polypeptides can also be synthesized chemically. Modified or variant forms, including truncated ones, can be constructed from a wild type polypeptide using standard recombinant DNA methods. For example, modified PH20 polypeptides can be constructed from a wild type polypeptide, as by site-directed mutagenesis.
[000352] [000352] Polypeptides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and classification of libraries, including classification of nucleic acid hybridization, classification based on antibody and classification based on activity.
[000353] [000353] For example, when polypeptides are produced by recombinant means, and a method known to those skilled in the art for identification of nucleic acids that encode desired genes can be used. Any method available in the art can be used to obtain a full or partial length cDNA (i.e., spanning the entire coding region) or genomic DNA clone encoding a PH20, such as from a tissue or cell source.
[000354] [000354] Methods for amplifying nucleic acids can be used to isolate nucleic acid molecules that encode a desired polypeptide, including, for example, polymerase chain reaction (PCR) methods. Examples of such methods include the use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). A nucleic acid-containing material can be used as a starting material from which a nucleic acid molecule encoding the desired polypeptide can be isolated. For example, mRNA and DNA preparations, cell extracts, tissue extracts, fluid samples (eg, blood, serum, saliva), samples from healthy and / or sick subjects can be used in amplification methods. The source may be of any eukaryotic species including, but not limited to, vertebrates, mammal, human, porcine, bovine, feline, avian, equine, canine and other primate sources. Nucleic acid libraries can also be used as a source of starting material. Primers can be designed to amplify a desired polypeptide. For example, primers can be designed based on expressed sequences from which a desired polypeptide is generated. Primers can be designed based on counter-translation of a polypeptide amino acid sequence. If desired, degeneration initiators can be used for amplification. Oligonucleotide primers that hybridize to sequences at the 3 'and 5' ends of the desired sequence can be used as primers to amplify by PCR sequences from a nucleic acid sample. Primers can be used to amplify the full-length PH20, or a truncated sequence thereof, as a nucleic acid that encodes any of the soluble PH20 polypeptides provided here. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.
[000355] [000355] Additional nucleotide sequences can be linked to a polypeptide encoding nucleic acid molecule, including linker sequences that contain restriction endonuclease sites for the purpose of cloning the synthetic gene into a vector, for example, an expression vector of protein or a vector designed for the amplification of the DNA sequences encoding nucleus protein. In addition, additional nucleotide sequences that specify functional DNA elements can be operably linked to a polypeptide-encoding nucleic acid molecule. Examples of such sequences include, but are not limited to, promoter sequences designed to facilitate expression of intracellular protein, and secretion sequences, for example,
[000356] [000356] In addition, tags or other fractions can be added, for example, to assist in the detection or purification of the affinity of the polypeptide. For example, additional nucleotide residue sequences such as base sequences specifying an epitope tag or other detectable marker can also be linked to enzyme-encoding nucleic acid molecules. Examples of such sequences include nucleic acid sequences encoding an HIs tag or Tag Flag.
[000357] [000357] Isolated and identified nucleic acids can then be inserted into an appropriate cloning vector. A large number of host-vector systems known in the art can be used. Possible vectors include, but are not limited to, plasmids or modified vectors, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as pCMV4, pBR322 or plasmid derivatives PUC or the Bluescript vector
[000358] [000358] If the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules can be enzymatically modified. Alternatively, any desired site can be produced by linking nucleotide sequences (linkers) on the ends of DNA; such linked ligands may contain specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and protein gene can be modified by homopolymeric tail formation.
[000359] [000359] Recombinant molecules can be introduced into host cells through, for example, transformation, transfection, infection, electroporation and sonoporation, so that many copies of the gene sequence are generated. In specific modalities, the transformation of host cells with recombinant DNA molecules that incorporate the isolated protein gene, cDNA, or synthesized DNA sequence allows the generation of multiple copies of the gene. In this way, the gene can be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, recovering the inserted gene from the isolated recombined DNA.
[000360] [000360] In addition to recombinant production,
[000361] [000361] The modifications provided here can be made by standard recombinant DNA techniques as is routine for a person skilled in the art. Any method known in the art to mutate any one or more amino acids in a target protein can be employed. Methods include mutagenesis directed to the standard site (using, for example, a kit like QuikChange available from Stratagene) of encoding nucleic acid molecules, or by solid phase polypeptide synthesis methods.
[000362] [000362] For recombinant expression of one or more of the desired proteins, such as any modified PH20 polypeptide described here, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, that is, a vector that contains the elements necessary for the transcription and translation of the inserted protein coding sequence. The necessary transcription and translation signals can also be provided by the native promoter for enzyme genes, and / or their flanking regions.
[000363] [000363] Vectors containing nucleic acid encoding the enzyme are also provided. Cells containing the vectors are also provided. The cells include eukaryotic and prokaryotic cells, and the vectors are any suitable for use in them. Generically, the cell is a cell that is capable of glycosylating the encoded protein.
[000364] [000364] Prokaryotic and eukaryotic cells containing the vectors are provided. Such cells include bacterial cells, yeast cells, fungus cells, Archea, plant cells, insect cells and animal cells. The cells are used to produce a protein of the same by growing the cells described above under conditions whereby the encoded protein is expressed by the cell, and recovering the expressed protein. For the purposes of the present invention, for example, the enzyme can be secreted into the medium.
[000365] [000365] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed protein in the desired mode. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing can impact the folding and / or function of the polypeptide. Different host cells, such as, but not limited to, CHO (DG44, DXB11, CHO-K1), HeLa, MCDK, 293 and WI38 have specific cell machinery and characteristic mechanisms for such post-translational activities and can be chosen to ensure modification and processing the introduced protein. Generally, the cell choice is one that is capable of introducing N-linked glycosylation into the expressed polypeptide. Consequently, eukaryotic cells containing the vectors are provided. Exemplary eukaryotic cells are Chinese mammalian hamster ovary (CHO) cells. For example, CHO cells deficient in dihydrofolate reductase (for example, DG44 cells) are used to produce polypeptides provided here. Note that bacterial expression of a PH20 polypeptide provided here will not result in a catalytically active polypeptide, however when combined with suitable glycosylation machinery, PH20 can be artificially glycosylated.
[000366] [000366] Vectors containing a nucleotide sequence encoding the modified PH20 polypeptide coupled to the native or heterologous signal sequence, as well as multiple copies thereof, are provided here. The vectors can be selected for expression of the enzyme protein in the cell or in such a way that the enzyme protein is expressed as a secreted protein.
[000367] [000367] A variety of vector-host systems can be used to express the protein coding sequence. These include, but are not limited to, mammalian cell systems infected with viruses (for example, vaccinia viruses, adenoviruses and other viruses); insect cell systems infected with viruses (for example, baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage; DNA, plasmid DNA or cosmid DNA. The elements of expression of vectors vary in their resistance and specificities. Depending on the host vector system used, any of a number of appropriate transcription and translation elements may be used.
[000368] [000368] Any methods known to those skilled in the art for inserting DNA fragments into a vector can be used to construct expression vectors containing a chimeric gene containing appropriate translation / transcription control signals and protein coding sequences. Such methods may include in vitro recombinant DNA and synthetic and in vivo recombinant techniques (genetic recombination). The expression of nucleic acid sequences encoding protein, or domains, derivatives, fragments or homologues thereof, can be regulated by a second nucleic acid sequence so that the genes or fragments thereof are expressed in a host transformed with (s) ) recombinant DNA molecule (s). For example, protein expression can be controlled by any promoter / enhancer known in the art. In a specific embodiment, the promoter is not native to the genes for a desired protein. Promoters that can be used include, but are not limited to, the SV40 initial promoter (Bernoist and Chambon, Nature 290: 304-310 (1981)), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto and others Cell 22: 787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78: 1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster and others, Nature 296: 39-42 (1982)); prokaryotic expression vector promoters, such as the -lactamase promoter (Jay et al., (1981) Proc. Natl. Acad. Sci. USA 78: 5543) or the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci USA 80: 21-25 (1983), see also Gilbert and Villa-Komaroff, “Useful Proteins from Recombinant Bacteria,” Scientific American 242: 74-94 (1980)); plant expression vector promoters, such as the nopaline synthase promoter (Herrera-Estrella et al., Nature 303: 209-213 (1984)) or the cauliflower mosaic virus RNA 35S promoter (Gardner et al., Nucleic Acids Res. 9: 2871 (1981)), and the ribulose bisphosphate carboxylase photosynthetic enzyme promoter (Herrera-Estrella et al., Nature 310: 115-120 (1984)); promoter elements of yeast and other fungi such as the Gal4 promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinase promoter, the alkaline phosphatase promoter, and the following animal transcription control regions that have tissue specificity and have been used in transgenic animals: elastase I gene control region that is active in pancreatic acinar cells (Swift et al., Cell 38: 639-646 (1984); Ornitz et al., Cold Spring Harbor Symp.
[000369] [000369] In a specific embodiment, a vector is used that contains a promoter operably linked to nucleic acids that encode a desired protein, or a domain, fragment, derivative or homologue thereof, one or more origins of replication, and optionally one or more more selectable markers (for example, an antibiotic resistance gene). Depending on the expression system, specific inhibition signals are also required for efficient translation of a PH20 sequence. These signals include the ATG initiation codon and adjacent sequences. In cases where the initiation codon and sequences upstream of PH20 or soluble forms thereof are inserted into the appropriate expression vector, no additional translation control signal is required. In cases where only a coding sequence, or a portion of it, is inserted, exogenous transcription control signals including the ATG initiation codon must be provided. In addition, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Elements of exogenous transcription and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by adding enhancers appropriate to the cell system in use (Scharf and others
[000370] [000370] Exemplary plasmid vectors for transformation of E.coli cells include, for example, pQE expression vectors (available from Qiagen, Valencia, CA; see also literature published by Qiagen describing the system). PQE vectors have a phage T5 promoter (recognized by E. coli RNA polymerase) and a double lac operator repression module to provide tightly regulated high-level expression of recombinant proteins in E. coli, a synthetic ribosomal binding site ( RBS II) for efficient translation, a 6xHis tag encoding sequence, t0 and T1 transcription terminators, Co1E1 origin of replication, and a beta-lactamase gene to confer resistance to ampicillin. The pQE vectors allow placement of a 6xHis tag at the N or C end of the recombinant protein. Such plasmids include pQE 32, pQE 30, and pQE 31 which provide multiple cloning sites for all three reading frames and provide the expression of N-terminal 6xHis labeled proteins, other exemplary plasmid vectors for transformation of E. coli cells include, for example, the expression vectors pET (see US patent 4,952,496; available from Novagen, Madison, WI; see also the literature published by Novagen describing the system). Such plasmids include pET 11a, which contains the T7lac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; pET 12a-c, which contains the T7 promoter, T7 terminator, and the E. coli ompT secretion signal; and pET 15b and pET19b (Novagen, Madison, WI), which contain a leading His-TagTM sequence for use in purification with a His column and a thrombin cleavage site that allows cleavage after purification on the column, the T7- lac and the T7 terminator.
[000371] [000371] Typically, vectors can be plasmids, stained glass vectors, or others known in the art, used for expression of the modified PH20 polypeptide in vivo or in vitro. For example, the modified PH20 polypeptide is expressed in mammalian cells, including, for example, Chinese Hamster Ovary (CHO) cells. An exemplary vector for mammalian cell expression is the HZ24 expression vector. The expression vector HZ24 was derived from the pCI vector structure (Promega). It contains DNA encoding the Beta-lactamase resistance gene (AmpR), an F1 origin of replication, a cytomegalovirus (CMV) initial-promoter / enhancer region, and an SV40 late polyadenylation signal (SV40). The expression vector also has an internal ribosome entry site (IRES) from the ECMV virus (Clontech) and the mouse dihydrofolate reductase (DHFR) gene.
[000372] [000372] Viral vectors, such as adenovirus, retrovirus or vaccinia virus vectors can be employed. In some examples, the vector is an attenuated or defective retroviral vector or another viral vector (see US patent No. 4,980,286). For example, a retroviral vector can be used (see Miller et al., Meth. Enzymol. 217: 581-599 (1993)). These retroviral vectors have been modified to delete retroviral sequences that are not necessary for packaging the viral genome and integration into host cell DNA.
[000373] [000373] In some examples, viruses armed with a nucleic acid encoding a modified PH20 polypeptide can facilitate its replication and spread to target tissue, for example. The target tissue can be a cancerous tissue so the virus is capable of selective replication in the tumor. The virus can also be a non-lytic virus in which the virus selectively replicates under a tissue-specific promoter. As viruses replicate, co-expression of the PH20 polypeptide with viral genes will facilitate the spread of the virus in vivo.
[000374] [000374] Modified PH20 polypeptides can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired proteins can be expressed in any appropriate organism to produce the required amounts and forms of proteins, such as those needed for administration and treatment. Expression hosts include prokaryotic and eukaryotic organisms such as E. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts may differ in their levels of protein production as well as the types of post-translational modifications that are present in the expressed proteins. The choice of expression host can be made based on these and other factors, such as safety and regulatory considerations, production costs and the need and methods for purification.
[000375] [000375] Many expression vectors are available and known to those skilled in the art and can be used for protein expression. The choice of expression vector will be influenced by the choice of the host expression system. In general, expression vectors can include transcription promoters and optionally enhancers, translation signals, and translation and transcription termination signals. Expression vectors that are used for stable transformation typically have a selectable marker that allows selection and maintenance of the transformed cells. In some cases, a source of replication can be used to amplify the vector's copy number.
[000376] [000376] Modified PH20 polypeptides can also be used or expressed as protein fusions. For example, an enzyme fusion can be generated to add additional functionality to an enzyme. Examples of enzyme fusion proteins include, but are not limited to, fusions of a signal sequence, a location-like tag, for example, a 6xHis or His6tag tag or a myc tag, or a purification tag, for example, a GST fusion; and a sequence to guide protein secretion and / or membrane association.
[000377] [000377] For high yield, long term production of recombinant proteins, stable expression is desired. For example, cell lines that stably express a modified PH20 polypeptide can be transformed using expression vectors that contain viral origins of replication or endogenous expression elements and a selectable marker gene. After the introduction of the vector, cells can be allowed to grow for 1-2 days in an enriched medium before being switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the sequences introduced. Resistant cells from stably transformed cells can be proliferated using tissue culture techniques appropriate to cell types.
[000378] [000378] Any number of selection systems can be used to recover transformed cell lines. These include, but are not limited to, herpes simplex virus thymidine kinase genes
[000379] [000379] The presence and expression of PH20 polypeptides can be monitored. For example, detection of a functional polypeptide can be determined by testing the conditioned medium for hyaluronidase enzyme activity under appropriate conditions. Exemplary assays to assess solubility and activity of expressed proteins are provided here. A. prokaryotic cells
[000380] [000380] Prokaryotes, especially E. coli,
[000381] [000381] Proteins, like any provided here, can be expressed in the cytoplasmic environment of E. coli. The cytoplasm is a reducing environment, and for some molecules, this can result in the formation of insoluble inclusion bodies. Reducing agents such as dithiothreotol and B-mercaptoethanol and denaturants such as guanidine-HCl and urea can be used to solubilize proteins again. An alternative approach performs protein expression in the periplasmic space of bacteria that provides an oxidation environment and disulfide and chaperonin-like isomerases, which can assist in the production of soluble protein. Typically, a leader sequence is fused to the protein to be expressed which orients the protein to the periplasm. The leader is then removed by signal peptidases within the periplasm. Examples of periplasmic targeting leader sequences include the pelB leader from the lyase pectate gene and the leader derived from the alkaline phosphatase gene. In some cases, periplasmic expression allows leakage of the expressed protein into the culture medium. Protein secretion allows quick and simple purification from the culture supernatant. Proteins that are not secreted can be obtained from the periplasm by osmotic lysis. Similar to cytoplasmic expression, in some cases proteins can become insoluble and denaturing and reducing agents can be used to facilitate solubilization and refolding. The temperature of induction and growth can also influence levels of expression and solubility, typically temperatures between 25ºC and 37ºC are used. Typically, bacteria produce glycosylated proteins. Thus, if proteins require glycosylation for function, glycosylation can be added in vitro after purification from host cells. B. Yeast cells
[000382] [000382] Yeasts such as Saccharomyces cerevisae, Schizosaccharomyces pombe, Yarrowia lipolytica, Kluyveromyces lactis and Pichia pastoris are well-known yeast expression hosts that can be used for protein production, as described here. Yeast can be transformed with episomal replication vectors or by stable chromosomal integration by homologous recombination. Typically, inducible promoters are used to regulate gene expression. Examples of such promoters include GAL1, GAL7 and GAL5 and metallothionein promoters, such as CUP1, AOX1 or other Pichia promoters or other yeast promoters. Expression vectors often include a selectable marker such as LEU2, TRP1, HIS3 and URA3 for selecting and maintaining transformed DNA. Proteins expressed in yeast are often soluble, co-expression with chaperonins such as Bip and protein disulfide isomerase can improve levels of expression and solubility. In addition, proteins expressed in yeast can be targeted for secretion using secretion signal peptide fusions as the yeast-like alpha-factor secretion signal from Saccharomyces cerevisae and fusions with yeast cell surface proteins as the receptor of married adhesion Aga2p or the glucoamylase Arxula adeninivorans. A protease cleavage site, as for the Kex-2 protease, can be constructed to remove the fused sequences from the expressed polypeptides as they exit the secretion pathway. Yeast is also capable of glycosylation in the Asn-X-Ser / Thr motifs. ç. Insects and insect cells
[000383] [000383] Insect cells, using baculovirus expression, are useful for expressing polypeptides such as PH20 polypeptides. Insect cells express high levels of protein and are capable of most of the post-translational modifications used by higher eukaryotes. Baculoviruses have a restrictive host range that improves safety and reduces regulatory concerns for eukaryotic expression. Typical expression vectors use a promoter for high level expression like the baculovirus polyhedrin promoter. Commonly used baculovirus systems include a baculovirus, such as the nuclear polyhedrosis virus Autographa californica (AcNPV) or the bombyx mori nuclear polyhedrosis virus (BmNPV), and an insect cell line, such as Sf9 derived from Spodoptera frugiperda, Pseudaletia unipuncta ( A7S) and Danaus plexippus (DpN1). For high-level expression, the nucleotide sequence of the molecule to be expressed is immediately fused downstream of the virus polyhedrin initiation codon. Mammalian secretion signals are precisely processed in insect cells and can be used to secrete the expressed protein into the culture medium. In addition, the cell lines Pseudaletia unipuncta (A7S) and Danaus plexippus
[000384] [000384] An alternative expression system in insect cells employs stably transformed cells. Cell lines such as Schnieder 2 (S2) and Kc (Drosophila melanogaster) cells and C7 cells (Aedes albopictus) can be used for expression. The metallothionein promoter Drosophila can be used to induce high levels of expression in the presence of induction of heavy metal with cadmium or copper. Expression vectors are typically maintained by the use of selectable markers such as neomycin and hygromycin. d. Mammalian expression
[000385] [000385] Mammalian expression systems can be used to express proteins including PH20 polypeptides. Expression constructs can be transferred to mammalian cells by viral infection such as adenovirus or by direct DNA transfer such as liposomes, calcium phosphate, DEA-dextran and by physical means such as electroporation and microinjection. Expression vectors for mammalian cells typically include an mRNA cover site, a TATA frame, a translation initiation sequence (Kozak consensus sequence) and polyadenylation elements. IRES elements can also be added to allow bicistronic expression with another gene, as a selectable marker. Such vectors often include transcription promoter enhancers for high level expression, for example, the SV40 enhancer-promoter, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter enhancers are active in many types of cells. Cell and tissue type promoters and enhancer regions can also be used for expression. Exemplary enhancer / promoter regions include, but are not limited to, those of genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, light chain myosin 2, and gonadotropic release hormone gene control. Selectable markers can be used to select by and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select only those cells that express the DHFR gene. Fusion with cell surface signaling molecules like TCR-ζ and FcεRI- can guide protein expression in an active state on the cell surface.
[000386] [000386] Many cell lines are available for mammalian expression including cells from mouse, rat, human, monkey, chicken and hamster. Exemplary cell lines include, but are not limited to CHO, Balb / 3T3, HeLa, MT2, mouse NS0 (non-secretion) and other myeloma cell lines, hybridoma and heteroibridoma cell lines, lymphocytes, fibroblasts, Sp2 / 0 , Sp2 / 0, COS, NIH3T3, HEK293, 293S, 2B8, and HKB cells. Cell lines are also available adapted to the serum-free medium that facilitates purification of secreted proteins from the cell culture medium. Examples include CHO-S cells (Invitrogen, Carlsbad, CA, cat # 11619-012) and the serum-free EBNA-1 cell line (Pham et al., (2003) Biotechnol. Bioeng. 84: 332-42.) . Cell lines are also available that are adapted to grow in special media optimized for maximum expression. For example, CHO DG44 cells are adapted to grow in suspension culture in a chemically defined, animal-free medium. and. Plants
[000387] [000387] Transgenic plant cells and plants can be used to express proteins like any one described here. Expression constructs are typically transferred to plants using direct DNA transfer such as microprojectile bombardment and PEG-mediated transfer in protoplasts, and with agrobacterium-mediated transformation. Expression vectors can include promoter and enhancer sequences, transcription termination elements and translation control elements. Expression vectors and transformation techniques are usually divided between dicot hosts, such as Arabidopsis and tobacco, and monocot hosts, such as corn and rice. Examples of plant promoters used for expression include the cauliflower mosaic virus promoter, the nopaline synthase promoter, the ribose bisphosphate carboxylase promoter and the ubiquitin and UBQ3 promoters. Selectable markers such as hygromycin, phosphomanosis isomerase and neomycin phosphotransferase are often used to facilitate selection and maintenance of transformed cells. Transformed plant cells can be maintained in culture as cells, aggregates (callus tissue) or regenerated in whole plants. Cells from transgenic plants can also include algae built to produce hyaluronidase polypeptides. Since plants have different glycosylation patterns than mammalian cells, this may influence the choice of protein produced in these hosts.
[000388] [000388] Host cells transformed with a nucleic acid sequence encoding a modified PH20 polypeptide can be cultured under conditions suitable for expression and recovery of the encoded protein from cell culture. The protein produced by a recombinant cell is generally secreted, but it can be contained intracellularly depending on the sequence and / or the vector used. As will be understood by those skilled in the art, expression vectors that contain nucleic acid encoding PH20 can be designed with signal sequences that facilitate direct secretion of PH20 through prokaryotic or eukaryotic cell membranes.
[000389] [000389] Thus, methods for purifying polypeptides from host cells will depend on the chosen host cells and expression systems. For secreted molecules, proteins are generally purified from the culture medium after removing the cells. For intracellular expression, cells can be lysed and proteins purified from the extract. When transgenic organisms such as transgenic plants and animals are used for expression, tissues or organs can be used as starting material to make a lysed cell extract. Additionally, the production of transgenic animals can include the production of polypeptides in milk or eggs, which can be collected, and if necessary, proteins can be extracted and further purified using standard methods in the art.
[000390] [000390] Proteins, such as modified PH20 polypeptides, can be purified using standard protein purification techniques known in the art including, but not limited to, SDS-PAGE, size fractionation and size exclusion chromatography, ammonium sulfate precipitation and ion exchange chromatography, such as anion exchange chromatography. Affinity purification techniques can also be used to improve the efficiency and purity of the preparations. For example, antibodies, receptors and other molecules that bind PH20 hyaluronidase enzymes can be used in affinity purification. for example, soluble PH20 can be purified from the conditioned medium.
[000391] [000391] Expression constructs can also be constructed to add an affinity tag to a protein such as a myc epitope, GST or His6 fusion and purified affinity with myc antibody, glutathione resin or Ni-resin, respectively. Such tags can be joined to the nucleotide sequence encoding a soluble PH20 as described elsewhere here, which can facilitate the purification of soluble proteins. For example, a modified PH20 polypeptide can be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification. Such domains of facilitating purification include, but are not limited to, metal chelation peptides as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin and the domain used in the purification system of FLAGS affinity / extension (Immunex Corp., Seattle Wash.). The inclusion of a cleavable linker sequence such as Factor XA or enterokinase (Invitrogen, San Diego, CA) between the purification domain and the expressed PH20 polypeptide is useful to facilitate purification. Such an expression vector provides expression of a fusion protein containing a PH20 polypeptide at and an enterokinase cleavage site. Histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography) while the enterokinase cleavage site provides a means to purify the polypeptide from the fusion protein.
[000392] [000392] Purity can be assessed by any method known in the art including gel electrophoresis, orthogonal HPLC methods, staining and spectrophotometric techniques. The expressed and purified protein can be analyzed using any assay or method known to a person skilled in the art, for example, any described in Section G. These include assays based on the physical and / or functional properties of the protein, including, but not limited to , analysis by gel electrophoresis, immunoassay and hyaluronidase activity assays.
[000393] [000393] Depending on the expression system and host cells used, the resulting polypeptide may be heterogeneous due to peptidases present in the culture medium after production and purification. For example, culture of PH20 soluble in CHO banknotes can result in a mixture of heterogeneous polypeptides.
[000394] [000394] Polyethylene glycol (PEG) has been widely used in biomaterials, biotechnology and medicine mainly because PEG is a water-soluble, non-toxic, biocompatible polymer that is typically non-immunogenic (Zhao and Harris, ACS Symposium Series 680: 458- 72 , 1997). In the field of drug delivery, PEG derivatives have been widely used in covalent bonding (ie, “PEGUIlation”) with proteins to reduce immunogenicity, proteolysis and kidney clearance and to increase solubility (Zalipsky, Adv. Drug Del. Rev 16: 157-82, 1995). Similarly, PEG has been linked to relatively hydrophobic drugs with low molecular weight to increase solubility, reduce toxicity and alter bi-distribution. Typically, PEGUIlated drugs are injected as solutions.
[000395] [000395] A closely related application is the synthesis of reticulated degradable PEG networks or formulations for use in drug supply since much of the same chemistry used in the design of soluble, degradable drug vehicles can also be used in the design of degradable gels ( Sawhney et al., Macromolecules 26: 581-87, 1993). It is also known that intermacromolecular complexes can be formed by mixing solutions of two complementary polymers. Such complexes are generally stabilized by electrostatic interactions (polyanion-polycation) and / or hydrogen bonds (polyacid-polybase) between the polymers involved, and / or by hydrophobic interactions between polymers in an aqueous environment (Krupers et al., Eur. Polym J. 32: 785-790, 1996). For example, mixing solutions of polyacrylic acid (PAAc) and polyethylene oxide (PEO) under the right conditions results in the formation of complexes based mostly on hydrogen bonding. Dissociation of these complexes under physiological conditions was used to supply free drugs (ie, not PEGUlated). In addition, complementary polymer complexes were formed from both homopolymers and copolymers.
[000396] [000396] Numerous reagents for PEGIlation have been described in the art. Such reagents include, but are not limited to, reacting the polypeptide with N-hydroxy succinimidyl-activated PEG (NJS), succinimidyl mPEG, mPEG2-N-hydroxysuccinimide, mPEG succinimidyl alpha-methylbutanoate, mPEG succinimidyl propionate, mPEG succinate succinimidyl mPEG carboxymethyl 3-hydroxybutanoic acid, homobifunctional PEG-succinimidil propionate, homobifunctional PEG propionaldehyde, homobifunctional PEG butyraldehyde, maleimide PEG, hydrazide, PEG p-nitrophenyl carbonate, mPEG-benzotriad, mPEG-benzotriazide, mPEG-benzotriad , mPEG acetyl, mPEG piperidone, mPEG methyl ketone, maleimide "no linker" mPEG, mPEG vinyl sulfone, mPEG thiol, mPEG ortho-pyridylthioester, mPEG ortho-pyridyl disulfide, Fmoc-PEG-NHS, Boc-PEG-NHS, acrilates -NHS, PEG-NHS fluorescein, and PEG-NHS biotin (see for example, Monfardini et al., Bioconjugate Chem. 6: 62-69, 1995; Veronese et al., J. Bioactive Compati ble Polymers 12: 197-207, 1997; U.S. 5,672,662; U.S. 5,932,462; U.S. 6,495,659; U.S. 6,737,505; U.S. 4,002,531; U.S. 4,179,337; U.S.
[000397] [000397] In one example, polyethylene glycol has a molecular weight ranging from approximately 3 kD to approximately 40 kD, and typically from approximately 5 kD to approximately 30 kD. Covalent binding of PEG to the drug (known as “PEGUIlation”) can be accomplished by known chemical synthesis techniques. For example, protein PEGIation can be accomplished by reacting NHS-activated PEG with the protein under appropriate reaction conditions.
[000398] [000398] Although numerous reactions have been described for PEGUIlation, those that are more generally applicable confer directionality, use mild reaction conditions, and do not require extensive downstream processing to remove toxic catalysts or biproducts. For example, monomethoxy PEG (mPEG) has only one reactive terminal hydroxyl, and thus its use somewhat limits the heterogeneity of the resulting protein-PEG product mixture. Activation of the hydroxyl group at the end of the polymer opposite the methoxy terminal group is generally necessary to perform efficient protein PEGIlation, with the aim of making derivatized PEG more susceptible to nucleophilic attack. The attacking nucleophile is usually the epsilon-amino group of a lysyl residue, but other amines can also react (for example, N-terminal alpha-amine or histidine ring amines) if local conditions are favorable. More targeted binding is possible in proteins containing a single lysine or cysteine. The residue mentioned last can be targeted by PEG-maleimide for specific thiol modification. Alternatively, PEG hydrazide can be reacted with a periodate oxidized hyaluronan degrading enzyme and reduced in the presence of NaCNBH3. More specifically, PEGUATED CMP sugars can be reacted with a hyaluronan degrading enzyme in the presence of appropriate glycosyl transferases. One technique is the "PEGUIlation" technique where a number of polymeric molecules are attached to the polypeptide in question. When using this technique, the immune system has difficulties in recognizing the epitopes on the surface of the polypeptide responsible for the formation of antibodies, thereby reducing the immune response. For polypeptides introduced directly into the circulatory system of the human body to provide a particular physiological effect (ie, pharmaceuticals) the typical potential immune response is an IgG and / or IgM response, while polypeptides that are inhaled through the respiratory system (ie , industrial polypeptide) can potentially cause an IgE response (that is, an allergic response). One of the theories that explain the reduced immune response is that the polymeric molecule (s) protects epitope (s) on the surface of the polypeptide responsible for the immune response that leads to antibody formation. Another theory or at least a partial factor is that the heavier the conjugate, the lower the resulting immune response.
[000399] [000399] Typically, to make the PH20 PEGUIlated polypeptide provided here, PEG fractions are conjugated, through covalent bonding, with the polypeptides. PEGUIlation techniques include, but are not limited to, specialized linkers and coupling chemicals (see, for example, Roberts, Adv. Drug Deliv.
[000400] [000400] As an exemplary illustrative method for making a PH20 PEGUIlated polypeptide, PEG aldehydes, succinimides and carbonates were individually applied to conjugate PEG fractions, typically succinimidyl PEGs, with rHuPH20. For example, rHuPH20 was conjugated to exemplary succinimidyl monoPEG reagents (mPEG) including succinimidyl-Mpeg propionates (mPEG-SPA), succinimil-mPEG butanoates (mpEG-SBA), and (to bind “branched” PEGs) mpEGβ-N- hydroxyl succinimide. These PEGUI succinimidyl esters contain carbon structures of different length between the PEG group and the activated crosslinker, and a single or branched PEG group. These differences can be used, for example, to provide different reaction kinetics and potentially limit sites available for PEG binding with rHuPH20 during the conjugation process.
[000401] [000401] Succinimidyl PEGs (as above) containing linear or branched PEGs can be conjugated to PH20. PEGs can be used to generate PH20s reproducibly containing molecules having, on average, between approximately three to six or three to six PEG molecules per hyaluronidase. Such rHuPH20 PEGUIlated compositions can be readily purified to provide compositions having specific activities of approximately 25,000 or
[000402] [000402] Using several PEG reagents, exemplary versions of a PH20 PEGUIlated polypeptide can be prepared, for example, using mPEG-SBA (30 kD), mPEG-SMB (30 kD), and branched versions based on mPEG2-NHS (40 kD ) and mPEG2-NHS (60 kD). PEGUIlated versions of PH20 can be generated using NHS chemicals, as well as carbonates and aldehydes, using each of the following reagents: branched mPEG2-NHS-40K, branched mPEG-NHS-10K, branched mPEG-NHS-20K, branched mPEG2-NHS-60K ; mPEG-SBA-5K, mPEG-SBA-20K, mPEG-SBA-30K; mPEG-SMB-20K, mPEG-SMB-30K; mPEG-butyraldehyde; mPEG-SPA-20K, mPEG-SPA-30K; and PEG-NHS-5K-biotin. PH20 PEGUkey can also be prepared using PEG reagents available from Dowpharma, a division of Dow Chemical Corporation; including PH20 polypeptides PEGUIlated with Dowpharma PEG-nitrophenylcarbonate (30 kDa) and with propionaldehyde PEG (30 kDa).
[000403] [000403] In one example, PEGUIlation includes conjugation of mPEG-SBA, for example, mPEG-SBA-30K (having a molecular weight of approximately 30 kDa) or another succinimidyl ester of a butaneic acid derivative PEG, with a polypeptide PH20. Succinimidyl esters of PEG butanoic acid derivatives such as mPEG-SBA-30K readily couple with amino groups of proteins. For example, covalent conjugation of m-PEG-SBA-30K and rHuPH20 (which is approximately 60 KDa in size) provides stable amide bonds between rHuPH20 and mPEG, as shown in scheme 1, below.
[000404] [000404] Typically, mPEG-SBA-30K or another PEG is added to the PH20 polypeptide in a 10: 1 molar ratio of PEG: polypeptide in an appropriate buffer, for example, 130 mM NaCl / 10 mM HEPES at pH 6.8 or 70 mM phosphate buffer, pH 7, followed by sterilization, for example, sterile filtration, and continued conjugation, for example, with stirring, overnight, at 4ºC in a cold room. In one example, the conjugated PEG-PH20 is concentrated and exchanged in buffer.
[000405] [000405] Other methods of coupling succinimidyl esters of PEG butanoic acid derivatives, such as mPEG-SBA-30K are known in the art (see, for example, U.S. 5,672,662; U.S. 6,737,505; and U.S. 2004/0235734). For example, a polypeptide, such as a PH20 polypeptide, can be coupled to an NHS-activated PEG derivative by reaction in a borate buffer (0.1 M, pH 8.0) for one hour at 4 ° C. The resulting pegylated protein can be purified by ultrafiltration. Another method reacts polypeptide with mPEG-SBA in deionized water to which triethyl amine is added to raise the pH to 7.2-9. The resulting mixture is stirred at room temperature for several hours to complete the PEGUIlation.
[000406] [000406] Methods for PEGUating PH20 polypeptides, including, for example, animal-derived hyaluronidases and bacterial hyaluronan degrading enzymes, are known to a person skilled in the art. See, for example, European patent no. EP 0400472, which describes the PEGIATION of chondroitin lyase ABC and bovine testis hyaluronidase. Also, US publication no. 2006014968 describes human hyaluronidase PEGIation derived from human PH20. For example, the PEGUIlated hyaluronan degrading enzyme generally contains at least 3 PEG fractions per molecule. In some examples, the PH20 polypeptide contains three to six PEG molecules. In other examples, the enzyme may have a molar ratio of PEG to protein between 5: 1 and 9: 1, for example, 7: 1. F. Pharmaceutical compositions and formulations, dosages and administration
[000407] [000407] Pharmaceutical compositions of any of the modified PH20 polypeptides are provided here for administration. Pharmaceutically acceptable compositions are prepared in view of approvals for a regulatory body or other organ prepared in accordance with a pharmacopoeia generally recognized for use in animals and humans. Typically, compounds are formulated in pharmaceutical compositions using techniques and procedures well known in the art (see, for example, Ansel Introduction to Pharmaceutical Dosage Forms, fourth edition, 1985, 126).
[000408] [000408] In particular, pharmaceutical compositions are provided here that are stable as a liquid formulation for extended periods of time for at least 1 month at temperatures from or approximately 2 ° C to 8 ° C, inclusive or for at least 2 days at a temperature of or approximately 30ºC to 42ºC, inclusive. Pharmaceutical compositions, in particular liquid formulations, may be limited by the stability of the active agent, which may be susceptible to the effects of storage conditions (storage time or period, temperature and / or agitation) and / or formulation components contained in the composition. Consequently, stable pharmaceutical compositions generally contain a modified PH20 polypeptide as described in section C.1.b which exhibits increased stability manifested as an increased resistance to one or more protein denaturation conditions. Such protein denaturation conditions may include, but are not limited to, an elevated temperature greater than or equal to or approximately 30 ° C, agitation, low salt or no salt, and the presence of excipients. Increased stability is characterized by improved shelf life, decreased fragmentation, and / or decreased aggregate formation, while still retaining the activity of the active agent (s), for example, hyaluronidase PH20. Such formulations can be supplied as "ready-to-use" liquid formulations without further reconstitution and / or without any requirement for further dilution. In some examples, formulations can also be prepared in a lyophilized or concentrated form.
[000409] [000409] Pharmaceutical compositions containing a modified PH20 polypeptide can be co-administered with another therapeutic agent. In such examples, the modified PH20 polypeptides can be formulated separately as a pharmaceutical composition and administered before, simultaneously with, intermittently with, or subsequent to a second composition containing an active therapeutic agent. In other examples, modified PH20 polypeptides can be co-formulated with pharmaceutical formulations of other therapeutic agents.
[000410] [000410] In particular, co-formulations containing a modified PH20 polypeptide as described herein and a therapeutic agent which is a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amebicidal agent, are provided herein trichomonic, an anti-parkinson agent, an antimalarial agent, an anticonvulsant agent, an antidepressant agent and an antiarthritic agent, an antifungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocking agent, an anesthetic agent, a bronchial swelling agent, a biocidal agent, a bactericidal agent, a bacteriostatic agent, a beta adrenergic blocking agent, a calcium channel blocking agent, a cardiovascular drug, contraceptive agent, decongestant agent, diuretic agent, depressant agent, diagnostic agent, electrolyte, hypnotic agent, hormone agent, hyperglycemic agent, muscle relaxant, muscle contraction agent, ophthalmic agent, parasympathomimetic agent, psychic energizing agent, sedative agent, sympathomimetic agent, tranquilizer , a urinary agent, a vaginal agent, a viricidal agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibiting agent, a polypeptide, a protein, a nucleic acid, a drug, a organic molecule or a sleep inducer. For example,
[000411] [000411] Formulations containing PH20 provided here, including separate formulations and co-formulations, are stable for extended periods of time, including at varying temperatures and under varying storage or conditions of use such as agitation. For example, the formulations provided here are stable and retain activity of active agent (s) (for example, hyaluronidase PH20), under “refrigerator” conditions, for example, at βºC to 8ºC, as at or approximately 4ºC, for at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months or 30 months or more. In another example, the formulations provided here are stable and retain activity of active agent (s), (for example, hyaluronidase PH20) at room temperature for example at 18 ºC to 32 ºC,
[000412] [000412] Compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and continuous release formulations. A composition can be formulated as a suppository, with traditional binders and vehicles such as triglycerides. The oral formulation can include standard vehicles such as pharmaceutical types of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate and other such agents. Topical formulations are also considered. The formulation must suit the mode of administration.
[000413] [000413] The formulation is generally made to suit the route of administration. Parenteral administration, generically characterized by injection or infusion, whether subcutaneously, intramuscularly, intravenously or intradermally is considered here. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent shortly before use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just before use and sterile emulsions. Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. For example, compositions containing a modified PH20 polypeptide, formulated separately or co-formulated with another therapeutic agent, can be supplied as a pharmaceutical preparation in liquid form as a solution, syrup or suspension. In liquid form, the pharmaceutical preparations can be supplied as a concentrated preparation to be diluted to a therapeutically effective concentration before use. Generically, the preparations are supplied in a dosage form that does not require dilution for use. In another example, pharmaceutical preparations can be presented in lyophilized form for reconstitution with water or another suitable vehicle before use.
[000414] [000414] Injectables are designed for local and systemic administration. For the purposes of the present invention, local administration is desirable for direct administration to the affected interstice. The solutions can be aqueous or non-aqueous. If administered intravenously, appropriate vehicles include physiological saline or phosphate buffered saline (PBS), and solutions containing solubilizing and thickening agents, such as glucose, polyethylene glycol and polypropylene glycol and mixtures thereof.
[000415] [000415] The concentration of the pharmaceutically active compound is adjusted so that an injection or infusion provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as known in the art. Parenteral unit dose preparations can be packaged, for example, in an ampoule, a cartridge, a vial or a syringe with a needle. The volume of reconstituted liquid solution or powder preparation, containing the pharmaceutically active compound, is a function of the disease to be treated and the specific industrial product chosen for packaging. All preparations for parenteral administration must be sterile, as known and practiced in the art. The percentage of active compound contained in such parenteral compositions is highly dependent on its specific nature, as well as the activity of the compound and the needs of the subject.
[000416] [000416] Pharmaceutical compositions may include vehicles or other excipients. For example, pharmaceutical compositions provided here may contain any one or more of a diluent (s), adjuvant (s), non-stick (s), binder (s), filler (s), flavor (s), color (s), lubricant (s), glidant (s), preservative (s), detergent (s), sorbent (s) or sweetener (s) and a combination thereof or vehicle with which a modified PH20 polypeptide is administered. For example, pharmaceutically acceptable vehicles or excipients used in parenteral preparations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, dispersing and suspending agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically substances acceptable. Formulations, including liquid preparations, can be prepared by conventional means with pharmaceutically acceptable additives or excipients.
[000417] [000417] Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, generally in purified form, together with an appropriate amount of vehicle in order to provide the form for proper administration to the patient. Such pharmaceutical vehicles can be sterile liquids, such as water or oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soy oil, mineral oil, and sesame oil. Water is a typical vehicle when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous glycerol and dextrose solutions can also be used as liquid carriers, particularly for injectable solutions. Examples of aqueous vehicles include sodium chloride injection, Ringer injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringer injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Dispersing and suspending agents include, but are not limited to, sorbitol syrup, hydrogenated cellulose derivatives or edible fats, sodium carboxymethyl cellulose, hydroxy propyl methyl cellulose and polyvinyl pyrrolidone. Emulsifying agents include, but are not limited to, lecithin or acacia. Detergents include, but are not limited to, Polysorbate 80 (Tween 80). Non-aqueous vehicles include, but are not limited to, almond oil, oily esters, or fractionated vegetable oils. Antimicrobial agents or preservatives include, but are not limited to, methyl or propyl-p-hydroxy benzoates or sorbic acid, m-cresol, phenol.
[000418] [000418] In particular, antimicrobial agents (eg, preservatives), in bacteriostatic or fungistatic concentrations (eg, an effective antimicrobial amount) can be added to parenteral preparations packaged in multiple dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxy benzoyl esters, thimerosal, benzalkonium chloride and benzethonium chloride.
[000419] [000419] The volume of the formulations including the formulations containing co-formulated or separately formulated pH provided here, can be any volume suitable for the container in which it is provided. In some examples, formulations are provided in a bottle, syringe, pen, reservoir for a pump or closed loop system, or any other appropriate container. For example, the formulations provided here are between or approximately between 0.1 ml and 500 ml, such as 0.1 ml and 100 ml, 1 ml and 100 ml, 0.1 ml and 50 ml, as at least or approximately at least or approximately or 0.1 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or more. The. Freeze dried powders
[000420] [000420] Of interest here are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They can also be reconstituted and formulated as solids or gels.
[000421] [000421] The freeze-dried, sterile powder is prepared by dissolving an enzyme compound in a buffer solution. The buffer solution may contain an excipient that improves the stability or other pharmaceutical component of the powder or reconstituted solution, prepared from the powder. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those skilled in the art provides the desired formulation. A liquid formulation as described above can be prepared. The resulting mixture is filtered sterile or treated to remove particulate materials and ensure sterility, and divided into vials for lyophilization. For example, lyophilized powder can be prepared by dissolving an excipient, such as dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other appropriate agent, in an appropriate buffer, such as citrate, potassium or sodium phosphate or another such buffer known to those skilled in the art. Then, a selected enzyme is added to the resulting mixture, and stirred until dissolved.
[000422] [000422] Each bottle is made to contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at approximately 4ºC until room temperature. Reconstitution of this lyophilized powder with an appropriate buffer solution provides a formulation for use in parenteral administration. B. Exemplary formulations
[000423] [000423] Single dose formulations of PH20 are known in the art. For example, recombinant Hylenex® (human hyaluronidase injection) contains, per mL, 8.5 mg NaCl (145 mM), 1.4 mg dibasic sodium phosphate (9.9 mM),
[000424] [000424] In addition to a therapeutically effective amount of a modified PH20 polypeptide and / or other therapeutic agent, exemplary pharmaceutical compositions provided here, including formulations containing separately formulated and co-formulated PH20, may contain a concentration of NaCl and are prepared at a pH required to maintain the stability of the active agent (s) (For example, hyaluronidase PH20 and / or other co-formulated therapeutic agent). For multi-dose formulations and other formulations stored for an extended period, the compositions generally also contain one or more preservatives. Additional stabilizing agents and other excipients can also be included. Exemplary components are described below. i. salt (for example, NaCl)
[000425] [000425] In the examples of the present invention, the pharmaceutical compositions provided here contain a concentration of salt, such as sodium chloride (NaCl), to maintain the stability of the active agent (s) (for example, hyaluronidase PH20 ). Salt, like NaCl, is generally required to retain PH20 stability and activity. Low salt concentrations generally less than 120 mM can have detrimental effects on PH20 activity over time and depending on temperature conditions. Consequently, the absence of salt (for example, NaCl) or a low concentration of salt (for example, NaCl) can result in instability of the protein. In some examples of the present invention, however, modified PH20 polypeptides that show increased stability in the absence of salt or low salt content, such as low or no NaCl (see, for example, section C.1.b.iii), do not are susceptible to denaturation. In addition, the presence of salt (for example, NaCl) may have different effects on other therapeutic agents. For example, insulin solubility and insulin analogues tend to increase with lower salt concentrations (eg <140 mM) and high salt concentrations can result in insulin crystallization / aggregation, especially at lower temperatures (see for for example, US provisional order No. 61 / 520,962; US Serial order No. 13 / 507,263 and 13 / 507,262; and international PCT order No. PCT / US2012 / 042816). Accordingly, pharmaceutical compositions provided here are prepared according to the requirements of the active agent (s). It is understood at the level of a person skilled in the art to assess the stability of the active agent (s) in the formulation and under various storage conditions (see, for example, section G). In specific examples from here, pharmaceutical compositions, including formulations containing separately formulated or co-formulated PH20 provided here, contain NaCl in a concentration between or approximately between 10 mM and 200 mM, such as 10 mM and 50 mM, 50 mM and 200 mM , 50 mM and 120 mM, 50 mM and 100 mM, 50 mM and 90 mM, 120 mM and 160 mM, 130 mM and 150 mM, 80 mM and 140 mM, 80 mM and 120 mM, 80 mM and 100 mM, 80 mM and 160 mM, 100 mM and 140 mM, 120 mM and 120 mM or 140 mM and 180 mM. ii. pH and buffer
[000426] [000426] In examples of the present invention, the pharmaceutical compositions provided here are prepared at a pH to maintain the stability of the active agent (s) (For example, hyaluronidase PH20). For example, the pharmaceutical compositions provided here are prepared at a pH between or approximately between 6.5 to 7.8 as between or approximately between 6.5 to 7.2, 7.0 to 7.8, 7.0 to 7 , 6 or 7.2 to 7.4. PH reference here is based on pH measurement at room temperature. It is understood that the pH may change during storage over time, but will typically remain between or between approximately pH 6.5 and or approximately 7.8. For example, the pH can vary by ± 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,
[000427] [000427] The compositions are generally prepared using a buffering agent that maintains the pH range. Any buffer can be used for formulations provided here as long as it does not adversely affect the stability of the active agent (s) (eg, hyaluronidase PH20) and support the required required pH range. Examples of particularly suitable buffers include Tris, succinate, acetate, phosphate buffers, citrate, aconite, malate and carbonate. Those skilled in the art, however, will recognize that formulations provided here are not limited to a specific buffer, as long as the buffer provides an acceptable degree of pH stability, or "buffer capacity" in the indicated range. Generally, a buffer has an adequate buffer capacity at approximately 1 pH unit of its pK (Lachman and others In: The Theory and Practice of Industrial Pharmacy 3rd edition (Lachman, L., Lieberman, HA. And Kanig, JL, Eds .), Lea and Febiger, Philadelphia, pp. 458-460, 1986). Buffer suitability can be estimated based on published pK tabs or can be determined empirically by methods well known in the art. The pH of the solution can be adjusted to the desired end point within the range as described above, for example, using any acceptable base or acid.
[000428] [000428] Buffers that can be included in the co-formulations provided here include, but are not limited to, Tris (tromethamine), histidine, phosphate buffers, such as dibasic sodium phosphate and citrate buffers. Such buffering agents can be present in co-formulations at concentrations between or approximately between 1 mM and 100 mM, such as 10 mM and 50 mM or mM and 40 mM, as at or approximately 30 mM. For example, such buffering agents can be present in co-formulations at a concentration of or approximately 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM , 60 mM, 65 mM, 70 mM, 75 mM, or more. iii. Condom (s)
[000429] [000429] In the examples of the present invention, multi-dose formulations or formulations stored for extended periods contain an antimicrobial effective amount of condom or mixture of condoms in an amount to have a bacteriostatic or fungistatic effect. In specific examples, condoms are present in a concentration sufficient to provide antimicrobial requirements for example, United States Pharmacopoeia (USP) and European Pharmacopoeia (EP), including EP antimicrobial requirements (EPA) and preferred EP antimicrobial requirements (EPB) (see table 4). Since the presence of condoms, and in particular phenolic condoms, can have detrimental effects on PH20 stability, such formulations typically contain a modified PH20 polypeptide that has increased stability in the presence of condoms, as described in Section C.1. bi of the present invention. Generally, the amount maintains the stability of the active agent (s) (for example, hyaluronidase PH20).
[000430] [000430] An effective antimicrobial condom amount is an amount that exhibits antimicrobial activity by killing or inhibiting the spread of microbial organisms in a sample of the composition as assessed in an antimicrobial condom effectiveness test (APET). A person skilled in the art is familiar with testing the effectiveness of an antimicrobial condom and standards to be met in accordance with USP and EPA or EPB to meet minimum requirements. In general, testing the effectiveness of an antimicrobial condom involves challenging a composition with prescribed inoculants of appropriate microorganisms, that is, bacteria, yeast and fungi, storing the inoculated preparation at a prescribed temperature, taking samples at specified intervals of time, and counting the organisms in the sample (see, Sutton and Porter, (2002) PDA Journal of Pharmaceutical Science and Technology 56 (4): 300-311; The United States Pharmacopeial Convention, Inc., (effective January 1, 2002), The United States Pharmacopeia 25th review, Rockville, MD,
[000431] [000431] Non-limiting examples of condoms that can be included in the co-formulations provided here include, but are not limited to, phenol, meta-cresol (m-cresol), methylparaben, benzyl alcohol, thimerosal, benzalkonium chloride, 4 -chloro-1-butanol, chlorhexidine dihydrochloride, chlorhexidine digluconate, L-phenyl alanine, EDTA, bronopol (2-bromo-2-nitropropane-1,3-diol), mercuric phenyl acetate, glycerol (glycerin), imidurea , chlorhexidine, sodium dehydroacetate, ortho-cresol (o-cresol), para-cresol (p-cresol), chlorocresol, cetrimide, benzethonium chloride, ethylparaben, propylparaben or butylparaben and any combination thereof. For example, formulations provided here may contain a single condom. In other examples the formulations contain at least two different condoms or at least three different condoms. For example, formulations provided here may contain two preservatives such as L-phenyl alanine and m-cresol, L-phenyl alanine and methylparaben, L-phenylalanine and phenol, m-cresol and methylparaben, phenol and methylparaben, m-cresol and phenol or others similar combinations. In one example, the condom in the formulation contains at least one phenolic condom. For example, the formulation contains phenol, m-cresol or phenol and m-cresol.
[000432] [000432] In the formulations provided here, the total amount of one or more preservative agents as a percentage (%) of mass concentration (weight / v) in the formulation can be, for example, between or between approximately 0.1% and 0.4%, such as 0.1% and 0.3%, 0.15% to 0.325%, 0.15% and 0.25%, 0.1% and 0.2%, 0.2% and 0.3%, or 0.3% and 0.4%. Generically, formulations contain less than 0.4% (weight / v) preservative. For example, the co-formulations provided here contain at least or approximately at least 0.1%, 0.12%, 0.125%, 0.13%, 0.14%, 0.15%, 0.16% 0, 17%, 0.175%, 0.18%, 0.19%, 0.2%, 0.25%, 0.3%, 0.325%, 0.35% but less than 0.4% of total condom.
[000433] [000433] In some examples, the formulations provided here contain between or between approximately 0.1% and 0.25% phenol and between or approximately between 0.05% and 0.2% m-cresol, such as between or approximately between 0 , 10% and 0.2% phenol and between or approximately between 0.06% and 0.18% m-cresol, or between or approximately between 0.1% and 0.15% phenol and between or approximately between 0.08 % and 0.15% m-cresol. For example, formulations provided here contain or contain approximately 0.1% phenol and 0.075% m-cresol; 0.1% phenol and 0.15% m-cresol; 0.125% phenol and 0.075% m-cresol; 0.13% phenol and 0.075% m-cresol; 0.13% phenol and 0.08% m-
[000434] [000434] In examples of the present invention, the pharmaceutical compositions provided herein may optionally contain one or more other stabilizing agent to maintain the stability of the active agent (s) (for example, hyaluronidase PH20). Included among the types of stabilizers that may be contained in the formulations provided here are amino acids, amino acid derivatives, amines, sugars, polyols, salts and buffers, surfactants and other agents. The formulations provided here contain at least one stabilizer. For example, the formulations provided here contain at least one, two, three, four, five, six or more stabilizers. Consequently, any one or more of an amino acid, derived from amino acid, amine, sugar, polyol, salt and buffer, surfactant and other agents can be included in the formulations of the present invention. Generally, the formulations of the present invention contain at least one surfactant and an appropriate buffer. Optionally, the formulations provided here may contain additional additional stabilizers. Other components include, for example, one or more tonicity modifiers, one or more antioxidating agents, or another stabilizer.
[000435] [000435] Exemplary amino acid stabilizers, amino acid derivatives or amines include, but are not limited to, L-arginine, glutamine, glycine, lysine, methionine, proline, Lys-Lys, Gly-Gly, trimethyl amine oxide ( TMAO) or betaine. Exemplary sugars and polyols include, but are not limited to, glycerol, sorbitol, mannitol, inositol, sucrose or trehalose. Exemplary salts and buffers include, but are not limited to,
[000436] [000436] In specific examples here, the formulations contain one or more detergents, as surfactants, to maintain the stability of the active agent (s), (for example, hyaluronidase PH20). For example, surfactants can inhibit PH20 aggregation and minimize absorptive loss. Surfactants are generally non-ionic surfactants. Surfactants that can be included in the formulations of the present invention include, but are not limited to, fatty and partial acid esters and ethers of polyhydric alcohols such as glycerol, or sorbitol, poloxamers and polysorbates. For example, exemplary surfactants in the formulations of the present invention include any one or more of poloxamer 188 (PLURONICS® as PLURONIC® F68), TETRONICS®, polysorbate 20, polysorbate 80, PEG 400, PEG 3000, Tween® (for example, Tween® 20 or Tween® 80), Triton® X-100, SPAN®, MYRJ®, BRIJ®, CREMOPHOR®, polypropylene glycols or polyethylene glycols. In some examples, the formulations of the present invention contain poloxamer 188, polysorbate 20, polysorbate 80, generically poloxamer 188 (pluronic F68). The formulations provided here generally contain at least one surfactant, such as 1, 2 or 3 surfactants.
[000437] [000437] In the formulations provided here, the total amount of one or more surfactants as a percentage (%) of mass concentration (weight / v) in the formulation can be, for example, between or between approximately 0.005% and 1.0% , such as between or between approximately 0.01% and 0.5%, such as 0.01% and 0.1% or 0.01% and 0.02%. Generically, formulations contain at least 0.01% surfactant and contain less than 1.0%, such as less than 0.5% or less than 0.1% surfactant. For example, the formulations provided here may contain at or approximately 0.001%, 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.08%, or 0.09% surfactant. In specific examples, the formulations provided here contain or contain approximately 0.01% to or even approximately 0.05% surfactant.
[000438] [000438] Tonicity modifiers can be included in the formulation provided here to produce a solution with the desired osmolality. The formulations provided here have an osmolality of between or approximately between 245 mOsm / kg and 305 mOsm / kg. For example, osmolality is or is approximately 245 mOsm / kg, 250 mOsm / kg, 255 mOsm / kg, 260 mOsm / kg, 265 mOsm / kg, 270 mOsm / kg, 275 mOsm / kg, 280 mOsm / kg, 285 mOsm / kg, 290 mOsm / kg, 295 mOsm / kg, 300 mOsm / kg or 305 mOsm / kg. In some examples, the formulations have an osmolality of or approximately 275 mOsm / kg. Tonicity modifiers include, but are not limited to, glycerin, NaCl, amino acids, polyalcohols, trehalose, and other salts and / or sugars. The specific amount can be empirically determined to retain enzyme activity and / or tonicity.
[000439] [000439] In other instances, glycerin (glycerol) is included in the formulations. For example, formulations provided here typically contain less than 60 mM glycerin, as less than 55 mM, less than
[000440] [000440] The formulations provided here may also contain antioxidants to reduce or prevent oxidation, in particular oxidation of the PH20 polypeptide. For example, oxidation can be carried out by high concentrations of surfactant or hyaluronan oligomers. Exemplary antioxidants include, but are not limited to, cysteine, tryptophan and methionine. In specific examples, the antioxidant is methionine. The formulations provided here can include an antioxidant at a concentration of between or approximately between 5 mM and or and approximately 50 mM, such as 5 mM to 40 mM, 5 mM and 20 mM or 10 mM and 20 mM. For example, methionine can be provided in formulations of the present invention in a concentration of between or approximately between 5 mM and or and approximately 50 mM, such as 5 mM and 40 mM, 5 mM and 20 mM or 10 mM and 20 mM. For example, an antioxidant, for example, methionine, can be included in a concentration that is or is approximately 5 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM,
[000441] [000441] The formulations provided here may also contain an amino acid stabilizer, which contributes to the stability of the preparation. The stabilizer can be a non-polar or basic amino acid. Exemplary non-polar and basic amino acids include, but are not limited to, alanine, histidine, arginine, lysine, ornithine, isoleucine, valine, methionine, glycine and proline. For example, the amino acid stabilizer is glycine or proline, typically glycine. The stabilizer can be a single amino acid or it can be a combination of 2 or more such amino acids. Amino acid stabilizers can be natural amino acids, amino acid analogs, modified amino acids or equivalent amino acids. Generically, the amino acid is an L-amino acid. For example, when proline is used as the stabilizer, it is generally L-proline. It is also possible to use amino acid equivalents, for example, proline analogs. The concentration of amino acid stabilizer, for example, glycine, included in the formulation ranges from 0.1 M to 1 M of amino acid, typically 0.1 M to 0.75 M, generally 0.2 M to 0.5 M, for example , at least or approximately 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M , 0.6 M, 0.7 M, 0.75 M or more amino acid. The amino acid, for example, glycine, can be used in a form of a pharmaceutically acceptable salt, such as hydrochloride, hydrobromide, sulfate, acetate, etc. The purity of the amino acid, for example, glycine, must be at least 98%, at least 99%, or at least 99.5% or more.
[000442] [000442] In the examples of the present invention, if necessary, hyaluronidase inhibitors are included in a formulation to stabilize PH20, in particular to reduce the effects of otherwise destabilizing agents and conditions, such as low salt, high pH, the presence of condoms and high temperatures, present in the formulation. Such a component is generally not necessary for pharmaceutical compositions containing a modified PH20 polypeptide as provided herein which exhibits increased stability under such conditions. When provided, the hyaluronidase inhibitor is provided at least in its equilibrium concentration. A person skilled in the art is familiar with several classes of hyaluronidase inhibitors (see, for example, Girish et al. (2009) Current Medicinal Chemistry, 16: 2261-2288, and references cited therein). A person skilled in the art knows or can determine by standard methods in the art the equilibrium concentration of a hyaluronidase inhibitor in a stable reaction or composition of the present invention.
[000443] [000443] An exemplary hyaluronidase inhibitor for use in the compositions of the present invention is hyaluronan (HA). Hyaluronic acid (HA, also known as hyaluronan and hyaluronate) is the natural substrate for PH20, HA is an unsulfated glycosaminoglycan that is widely distributed throughout all connective, epithelial and neural tissues. It is a polymer of up to 25,000 units of disaccharide, itself composed of D-glucuronic acid and D-N-acetylglucosamine. The molecular weight ranges from approximately 5 kDa to 200,000 kDa. HA of any size can be used in the compositions as a stabilizer. In some examples, HA is a disaccharide,
[000444] [000444] In some examples, a nicotinic compound is used as a stabilizing agent. Nicotinic compounds include, but are not limited to, nicotinamide, nicotinic acid, niacin, niacinamide,
[000445] [000445] Depending on the condition treated, other routes of administration, such as topical application, transdermal patches, oral and rectal administration are also considered here.
[000446] [000446] For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories include solid bodies for insertion into the rectum that melts or softens at body temperature releasing one or more pharmaceutically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or vehicles and agents for raising the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases can be used. Agents for raising the melting point of suppositories include spermaceti and wax. Rectal suppositories can be prepared by the compressed method or by molding. The typical weight of a rectal suppository is approximately 2 to 3 g. tablets and capsules for rectal administration are manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.
[000447] [000447] For oral administration, pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients as binding agents (for example, pregelatinized corn starch, polyvinyl pyrrolidone or hydroxy propyl methyl cellulose) ; fillers (for example, lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (for example, magnesium stearate, talc or silica); disintegrants (for example, potato starch or sodium starch glycolate); or wetting agents (for example, sodium lauryl sulfate). The tablets can be coated by methods well known in the art.
[000448] [000448] Formulations suitable for buccal (sublingual) administration include, for example, lozenges containing the active compound in a flavored base, usually sucrose and acacia or tragacanth; and tablets containing the compound in an inert base such as gelatin and glycerin or sucrose and acacia.
[000449] [000449] Typical mixtures are prepared as described for local and systemic administration. The resulting mixtures can be solutions, suspensions, emulsion or similar and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, skin adhesives or any other formulations suitable for topical administration.
[000450] [000450] The pharmaceutically acceptable compounds or derivatives thereof can be formulated as aerosols for topical application, such as by inhalation (see, for example, U. S. Nos.
[000451] [000451] The compounds can be formulated for local or topical application, as for topical application to the skin and mucous membranes, as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is considered for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound individually or in combination with other pharmaceutically acceptable excipients can also be administered.
[000452] [000452] Formulations suitable for transdermal administration are provided. They can be supplied in any suitable format, as discreet adhesives adapted to remain in close contact with the recipient's epidermis for an extended period of time. Such adhesives contain the active compound in an optionally buffered aqueous solution, for example, 0.1 to 0.2 M concentration with respect to the active compound. Formulations suitable for transdermal administration can also be provided by iontophoresis (see, for example, Tyle, P, Pharmaceutical Research 3 (6): 318-326 (1986)) and are typically in the form of an optionally buffered aqueous solution of the active compound .
[000453] [000453] Pharmaceutical compositions can also be administered by controlled release formulations and / or delivery devices (see, for example, in U.S. Patent Nos. 3,536,809; 3,598,123; 3,630,200;
[000454] [000454] The modified PH20 polypeptides provided here can be formulated as pharmaceutical compositions for single or multiple dosage administration. PH20 polypeptide is included in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects in the treated patient. The therapeutically effective concentration can be determined empirically by testing the polypeptides in in vitro and in vivo systems known as by using the assays provided here or known in the art (see for example, Taliani et al. (1996) Anal. Biochem., 240: 60 -67; Filocamo et al. (1997) J Virology, 71: 1417-1427; Sudo et al. (1996) Antiviral Res. 32: 9-18; Bouffard et al. (1995) Virology, 209: 52-59; Bianchi et al. (1996) Anal. Biochem., 237: 239-244; Hamatake et al. (1996) Intervirology 39: 249-258; Steinkuhler et al. (1998) Biochem., 37: 8899-8905; D'Souza et al. (1995) J Gen. Virol., 76: 1729-1736; Takeshita et al. (1997) Anal. Biochem., 247: 242-246; see also for example, Shimizu et al. (1994) J. Virol. 68: 8406-8408; Mizutani et al. (1996) J. Virol. 70: 7219-7223; Mizutani et al. (1996) Biochem. Biophys. Res. Commun., 227: 822-826; Lu et al. (1996) Proc. Natl. Acad. Sci (USA), 93: 1412-1417; Hahm et al., (1996) Virology, 226: 318-326; Ito and or others (1996) J. Gen. Virol., 77: 1043-1054; Mizutani et al. (1995) Biochem. Biophys. Res. Commun., 212: 906-911; Cho et al. (1997) J. Virol. Meth. 65: 201-207 and then extrapolated from them for dosages for humans.
[000455] [000455] The amount of a modified pH20 to be administered to treat a disease or condition can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be employed to help identify optimal dosage ranges. The precise dosage, which can be determined empirically, may depend on the specific enzyme, route of administration, type of disease to be treated and the severity of the disease.
[000456] [000456] Consequently, it is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known test protocols or by extrapolation from in vivo or in vitro test data. It should be noted that concentrations and dosage values can also vary with the severity of the condition being relieved. It should be further understood that for any specific subject, specific dosage regimens must be adjusted over time according to the individual need and the professional decision of the person who administers or supervises the administration of the compositions, and that the exposed concentration ranges here they are only exemplary and are not intended to limit the scope or use of compositions and combinations containing them. The compositions can be administered hourly,
[000457] [000457] Typically, a therapeutically effective dose of a modified PH20 enzyme is at or approximately 10 Units (U) at 500,000 Units, 100 Units at 100,000 Units, 500 Units at 50,000 Units, 1000 Units at 10,000 Units, 5000 Units at 7500 Units, 5000 Units to 50,000 Units, or 1,000 Units to 10,000 Units, generally 1,000 to 50,000 Units, in a stabilized solution or suspension or a lyophilized form. For example, a PH20 polypeptide can be administered in a dose of at least or approximately at least or 10 U, 20 U, 30 U, 40 U, 50 U, 100 U, 150 U, 200 U, 250 U, 300 U, 350 U, 400 U, 450 U, 500 U, 600 U, 700 U, 800 U, 900 U, 1000 U, 2,000 U, 3,000 U, 4,000 Units, 5,000 U or more. The formulations can be supplied in unit dose forms such as, but not limited to, ampoules, syringes and individually wrapped tablets or capsules.
[000458] [000458] The PH20 enzyme can be administered individually or with other pharmacologically effective agent (s) or therapeutic agent (s) in a total volume of 0.1 -100 ml, 1 -50 ml , 10-50 ml, 10-30 ml, 1-20 ml, or 1-10 ml, typically 10-50 ml. Typically, volumes of injections or infusions of a composition containing PH20 are at least or at least approximately 0.01 ml, 0.05 ml, 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0 , 5 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 20 ml, 30 ml, 40 ml, 50 ml or more. The formulations provided here contain a PH20 polypeptide modified in an amount between or approximately between 30 Units / mL and 3000 U / mL, 300 U / mL and 2000 U / mL or 600 U / mL and 2000 U / mL or 600 U / mL and 1000 U / mL, such as at least or approximately at least 30 U / mL, 35 U / mL, 40 U / mL, 50 U / mL, 100 U / mL, 200 U / mL, 300 U / mL, 400 U / ml, 500 U / ml, 600 U / ml, 700 U / ml, 800 U / ml, 900 U / ml, 1000 U / ml, 2000 U / ml or 3000 U / ml. For example, the formulations provided here contain a pH20 that is in an amount that is at least 100 U / ml to 1000 U / ml, for example at least or approximately at least or approximately or 600 U / ml.
[000459] [000459] PH20 polypeptide can be supplied as a solution in an amount that is at least or approximately or is 100 U / mL, 150 U / mL, 200 U / mL, 300 U / mL, 400 U / mL, 500 U / mL, 600 U / mL, 800 U / mL or 1000 U / mL, or can be supplied in a more concentrated form, for example in an amount that is at least or approximately or is 2000 U / mL, 3000 Units / mL , 4000 U / mL, 5000 U / mL, 8000 U / mL, 10,000 U / mL or 20,000 U / mL for use directly or for dilution to effective concentration before use. PH20 polypeptide compositions can be supplied as a liquid or lyophilized formulation.
[000460] [000460] When PH20 is co-formulated with a therapeutic agent, dosages can be provided as a ratio of the amount of a PH20 polypeptide to the amount of therapeutic agent administered. For example, a PH20 polypeptide can be administered in 1 hyaluronidase U / therapeutic agent U (1: 1) at 50: 1 or more,
[000461] [000461] The formulations provided here, including co-formulations and / or stable formulations, can be prepared for single dose administration, multiple dose administration or continuous infusion administrations. The implantation of a slow-release or continuous-release system, such that a constant dosage level is maintained (see, for example, U. S. Patent No. 3,710,795), is also considered here.
[000462] [000462] For example, formulations of pharmaceutically therapeutically active compounds and derivatives thereof are provided for administration to humans and animals in unit dosage forms or multiple dosage forms. For example, compounds can be formulated as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, or oil-water emulsions containing appropriate amounts of the compounds or pharmaceutically acceptable derivatives thereof. Each unit dose contains a predetermined amount of therapeutically active compound (s) sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, carrier or diluent. Examples of unit dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. A multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose forms. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of 560 ml or gallons. Consequently, a multiple dose form is a multiple of unit doses that are not segregated in the package. Generically, dosage forms or compositions containing active ingredient in the range of 0.005% to 100% with the remaining non-toxic carrier compound can be prepared.
[000463] [000463] The compositions provided here are typically formulated for subcutaneous administration, although other routes of administration are considered, such as any route known to those skilled in the art including, intramuscular, intraperitoneal, intravenous, intradermal, intralesional, intraperitoneal, epidural injection, vaginal, rectal, local, optic, transdermal administration or any route of administration. Formulations suitable for such routes are known to a person skilled in the art. Administration can be local, topical or systemic depending on the treatment site. Local administration in an area in need of treatment can be achieved by, for example, but not limited to, local infusion during surgery, topical application, for example, in combination with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant. The compositions can also be administered with other biologically active agents, sequentially, intermittently or in the same composition.
[000464] [000464] The most appropriate route in any given case depends on a variety of factors, such as the nature of the disease, the subject's tolerance for a specific route of administration, the severity of the disease, and the specific composition that is used. Typically, the compositions provided here are administered parenterally. In some examples, the modified PH20 polypeptide compositions are administered so that they reach the interstice of skin or tissues, thereby degrading the interstitial space for subsequent delivery of a therapeutic agent. Thus, in some examples, direct administration under the skin, as by subcutaneous administration methods, is considered. Thus, in one example, local administration can be achieved by injection, such as a syringe or other industrial product containing an injection device such as a needle. In another example, local administration can be achieved by infusion, which can be facilitated by the use of a pump or other similar device. Other modes of administration are also considered. For example, modified PH20 polypeptides, including conjugated forms with increased half-life such as PEGUIlated forms thereof, can be administered intravenously. Pharmaceutical compositions can be formulated in dosage forms appropriate for each route of administration.
[000465] [000465] Administration methods can be used to decrease the exposure of selected modified PH20 polypeptides to degradative processes, such as proteolytic degradation and immunological intervention through antigenic and immunogenic responses. Examples of such methods include local administration at the treatment site. Therapeutic PEGUlation increases resistance to proteolysis, increases plasma half-life, and decreases antigenicity and immunogenicity. Examples of PEGUIlation methodology are known in the art (see for example, Lu and Felix, Int. J. Peptide Protein Res., 43: 127-138, 1994; Lu and Felix, Peptide Res., 6: 140-6, 1993; Felix et al., Int. J. Peptide Res., 46: 253-64, 1995; Benhar et al., J. Biol. Chem., 269: 13398-404, 1994; Brumeanu et al., J Immunol., 154 : 3088-95, 1995; see also, Caliceti et al. (2003) Adv. Drug Deliv. Rev. 55 (10): 1261-77 and Molineux (2003) Pharmacotherapy 23 (8 Pt 2): 3S-8S). PEGUIlation can also be used to supply nucleic acid molecules in vivo. For example, adenovirus PEGIlation can increase gene transfer and stability (see, for example, Cheng et al. (2003) Pharm. Res. 20 (9): 1444-51).
[000466] [000466] Several other delivery systems are known and can be used to deliver selected PH20 polypeptides, but not limited to, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, and delivery of nucleic acid molecules encoding PH20 polypeptides selected as retrovirus delivery systems.
[000467] [000467] Consequently, in certain embodiments, liposomes and / or nanoparticles can also be used with administration of soluble PH20 polypeptides. Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously from multilamellar concentric bilayer vesicles (also called multilamellar vesicles (MLVs)). MLVs generally have diameters from 25 nm to 4 µm. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 angstroms containing an aqueous solution in the core.
[000468] [000468] Phospholipids can form a variety of different structures of liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low lipid to water ratios, liposomes are formed. Physical characteristics of liposomes depend on pH, ionic resistance and the presence of divalent cations. Liposomes can show low permeability to polar and ionic substances, however at high temperatures they undergo a phase transition that markedly changes their permeability. The phase transition involves a change in an orderly, closely packed structure, known as the gel state; to a less ordered structure, loosely packed, known as the fluid state. This occurs at a characteristic phase transition temperature and results in an increase in permeability to ions, sugars and drugs.
[000469] [000469] Liposomes interact with cells through different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system such as macrophages and neutrophils; adsorption on cell surfaces, either by weak non-specific hydrophobic or electrostatic forces, or by specific interactions with cell surface components; fusion with the plasma cell membrane by inserting the lipid bilayer of the liposome into the plasma membrane; with simultaneous release of liposomal content in the cytoplasm; and by transfer of liposomal lipids to cell or subcellular membranes, or vice versa, without any association of the liposome content. Variation in the liposome formulation can change which mechanism is operative, although more than one can operate at the same time. Nanocapsules can generally retain the compounds in a stable and reproducible way. To avoid side effects due to the intracellular polymeric overload, such ultrafine particles (with size around 0.1 m) must be designed using polymers capable of being degraded in vivo. Biodegradable cyanoacrylate-polyalkyl nanoparticles that meet these requirements are considered for use here, and such particles can be easily made.
[000470] [000470] Stable co-formulations of a fast-acting insulin, such as a fast-acting insulin analog and a modified PH20 polypeptide, are provided here. Any of the modified PH20 polypeptides provided here can be included in an insulin co-formulation, like any of the co-formulations described in U.S. Serial Nos. 13 / 507,263 or 13 / 507,262 or in international PCT application Serial No. PCT / US2012 / 042816.
[000471] [000471] In particular, the modified PH20 polypeptide is a modified PH20 polypeptide that has increased stability under denaturation conditions, like any exposed in sections C.1.b. In particular, the PH20 polypeptide is a modified PH20 polypeptide that has increased stability to one or more phenolic condoms, like any exposed in section C.1.b.i. For example, PH20 polypeptide is a modified PH20 polypeptide that contains an amino acid substitution with P in a position corresponding to position 204 with reference to amino acid positions exposed in SEQ ID NO: 3, as F204P with reference to any of SEQ ID NOS : 3, 7 or 32-
[000472] [000472] The fast-acting insulin can be a regular insulin or a fast-acting insulin analog. Insulin is a polypeptide that when processed is composed of 51 amino acids containing an A-and -B chain. generally, insulin contains an A-chain of approximately 21 amino acids and a B-chain of approximately 30 amino acids.
[000473] [000473] Co-formulations are stable as a liquid formulation for extended periods of time for at least 1 month at temperatures of or approximately 2 ° C to 8 ° C inclusive, or for at least 3 days at a temperature of or approximately 30 ° C to 42 ° C inclusive. For example, co-formulations are stable and retain PH20 hyaluronidase and insulin activity in “refrigerator” conditions, for example, at 2 ° C to 8 ° C, such as at or approximately 4 ° C, for at least 2 months, 3 months, 4 months, 5 months, 6 months, or 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months or 30 months or more. In another example, the formulations provided here are stable and retain PH20 hyaluronidase and insulin activity at room temperature for example at 18 ° C to 32 ° C, generally 20 ° C to 32 ° C, as 28 ° C to 32 ° C, for at least 2 weeks until 1 year, for example, at least 3 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, at least 7 months, at least 8 months, at least 9 months, or at least 1 year or more. In an additional example, the formulations provided here are stable and retain PH20 hyaluronidase and insulin activity at elevated temperatures of approximately or greater than 30 ° C, generally from or approximately 30 ° C to 42 ° C, such as 32 ° C to 37 ° C ° C or 35 ° C to 37 ° C or approximately or 37 ° C for at least 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days, 50 days, 60 days or more.
[000474] [000474] Assays to assess the stability of active agents are well known to a person skilled in the art. Section G provides exemplary assays to assess the stability of hyaluronidase PH20. Insulin stability can be assessed using similar methods well known to a person skilled in the art. For example, insulin stability and solubility can be assessed by visual assessment (for example, including changes in color, clarity, presence of aggregates or clod and material adhesion, or freezing), acid clarification, optical microscopy, liquid chromatography high-performance reverse phase (RP-HPLC), in vitro or in vivo bioassays and denaturation and non-denaturation (SEC) size exclusion chromatography. In vitro or in vivo bioassays for insulin activity include, but are not limited to, a competitive binding assay using cells that express insulin receptors (for example, human placental cell membranes) and radiolabeled insulin (see for example , Weiss et al., (2001) J. Biol. Chem. 276: 40018-40024; Duttaroy et al., (2005) Diabetes 54: 251- 258); insulin-stimulated glucose absorption (Louveau et al., (2004) J Endocrin. 181: 271-280, Duttaroy et al., (2005) Diabetes 54: 251-258); assays to assess glucose production in the presence of insulin (Wang et al., (2000) J. Biochem., 275: 14717-14721, Duttaroy et al., (2005) Diabetes 54: 251-258); and studies using models of diabetic and / or healthy animals (Atkinson et al., (1999) Nature Med. 5: 601-604; Nagoya-Shibata-Yasuda (NSY) mice, Zucker diabetic fatty (ZDF) rats and Gato-Katazaki ( GK) rats (Cefalu (2006) ILAR Journal 47: 186-198).
[000475] [000475] Examples of such formulations contain
[000476] [000476] The following stable formulations are exemplary only and provide a platform from which minor adjustments can be made. It is understood that very small changes in the concentrations of the various excipients and other components (for example ± 15% of the mentioned concentrations), or small changes in pH, can be made while retaining some if not all of the insulin solubility and stability and stability of PH20. Additional changes can also be made by adding or removing excipients. For example, the type of stabilization surfactant can be changed.
[000477] [000477] For example, exemplary co-formulations of the present invention contain 100 U / ml to 1000 U / ml of a modified PH20 polypeptide, and in particular at least or approximately at least or approximately 600 U / ml of a modified PH20 polypeptide ; 10 U / mL at 1000
[000478] [000478] In specific examples, fast-acting insulin is insulin aspart, insulin lispro or insulin glulisine. Exemplary co-formulations provided here that contain a modified PH20 polypeptide and insulin lispro are those that contain from or approximately 25 mM to or even approximately 35 mM Tris (for example, in or approximately 30 mM Tris); from or from approximately 70 mM to or up to approximately 100 mM NaCl (for example, at or approximately 80 mM or 100 mM NaCl); from or from approximately 10 mM to or up to approximately 30 mM methionine (e.g., at or approximately 10 mM or 20 mM methionine); from or from approximately 40 mM to or up to approximately 60 mM glycerin (for example, in or approximately 50 mM glycerin); from or from approximately 0.005% to or up to approximately 0.05% of poloxamer 188 (for example, in or approximately 0.01% of poloxamer 188); from or from approximately 0.017 mg zinc / 100 U insulin to or up to approximately 0.024 mg zinc / 100 U insulin (for example, 0.017 mg zinc / 100 U insulin, 0.018 mg / 100 U, 0.02 mg / 100 U, 0.022 mg / 100 U or 0.024 mg zinc / 100 U insulin); from or from approximately 0.08% to or up to approximately 0.17% phenol (for example, 0.1%, 0.125% or 0.13% phenol); and from or from approximately 0.07% to or up to approximately 0.17% m-cresol (for example, 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example, co-formulations can contain at or approximately 0.1% phenol and 0.015% m-cresol; in or approximately 0.125% phenol and 0.075% m-cresol; in or approximately 0.13% phenol and 0.075% m-cresol; in or approximately 0.13% phenol and 0.08% m-cresol; or at or approximately 0.17% phenol and 0.13% m-cresol. Such formulations of insulin lispro and a modified PH20 polypeptide are prepared at a pH of or approximately
[000479] [000479] Exemplary co-formulations provided here that contain a modified PH20 polypeptide and insulin aspart are those that contain from or from approximately 25 mM to or up to approximately 35 mM Tris (for example, in or approximately 30 mM Tris); from or from approximately 70 mM to or up to approximately 120 mM NaCl (for example, at or approximately 80 mM or 100 mM NaCl); from or approximately 2 mM to or up to approximately 30 mM methionine, such as 2 mM to 10 mM or 5 mM to 30 mM methionine (for example, at or approximately 5 mM, 10 mM or 20 mM methionine); from or from approximately 0.005% to or up to approximately 0.05% of poloxamer 188 (for example, in or approximately 0.01% of poloxamer 188); from or approximately 0.08% to or even approximately 0.17% phenol (for example, 0.1%, 0.125% or 0.13% phenol); and from or from approximately 0.07% to or up to approximately 0.17% m-cresol (for example, 0.075%, 0.08%, 0.13% or 0.15% m-cresol). For example, co-formulations can contain at or approximately 0.1% phenol and 0.015% m-cresol; in or approximately 0.125% phenol and 0.075% m-cresol; in or approximately 0.13% phenol and 0.075% m-cresol; in or approximately 0.13% phenol and 0.08% m-cresol; or at or approximately 0.17% phenol and 0.13% m-cresol. Such aspart insulin formulations and a modified PH20 polypeptide are prepared with a pH of or approximately
[000480] [000480] Additional exemplary formulations provided here that contain a modified PH20 polypeptide and insulin aspart are those that do not contain phenol. Such exemplary formulations contain from or from approximately 25 mM to or even approximately 35 mM Tris (for example, in or approximately 30 mM Tris); from or from approximately 70 mM to or up to approximately 120 mM NaCl (for example, at or approximately 80 mM or 100 mM NaCl); from or approximately 2 mM to or up to approximately 30 mM methionine, such as 2 mM to 10 mM or 5 mM to 30 mM methionine (for example, at or approximately 5 mM, 10 mM or 20 mM methionine); from or from approximately 0.005% to or up to approximately 0.05% of poloxamer 188 (for example, in or approximately 0.01% of poloxamer 188); and from or from approximately 0.07% to or up to approximately 0.4% m-cresol, as from or from approximately 0.2% to 0.4% m-cresol (e.g. 0.3%, 0.315%, 0.35%, 0.4% m-cresol). Such aspart insulin formulations and a modified PH20 polypeptide are prepared with a pH of or approximately
[000481] [000481] Exemplary co-formulations provided here that contain a modified PH20 polypeptide and insulin glulisine are those that contain from or from approximately mM to or up to approximately 35 mM Tris (for example, in or approximately 30 mM Tris); from or from approximately 100 mM to or up to approximately 150 mM NaCl (for example, at or approximately 100 mM or 140 mM NaCl); from or from approximately 10 mM to or up to approximately 30 mM methionine (e.g., at or approximately 10 mM or 20 mM methionine); from or from approximately 40 mM to or up to approximately 60 mM glycerin (for example, in or approximately 50 mM glycerin); from or from approximately 0.005% to or up to approximately 0.05% of poloxamer 188 (for example, in or approximately 0.01% of poloxamer 188); from or from approximately 0.08% to or up to approximately 0.17% phenol (for example, 0.1%, 0.125% or 0.13% phenol); and from or from approximately 0.07% to or up to approximately 0.17% m-cresol (for example, 0.075%, 0.08%, 0.13% or 0.15% m-
[000482] [000482] Pharmaceutical modified PH20 polypeptide compounds, or nucleic acids encoding such polypeptides, or derivatives or variants thereof, can be packaged as industrial products containing packaging material, a pharmaceutical composition that is effective for treating a disease or disorder, and a label indicating that the pharmaceutical composition or therapeutic molecule should be used to treat the disease or disorder. Combinations of a selected modified PH20 polypeptide, or a derivative or variant thereof and a therapeutic agent can also be packaged in an industrial product.
[000483] [000483] The industrial products provided here contain packaging materials. Packaging materials for use in the packaging of pharmaceutical products are well known to those skilled in the art. See, for example, US patents nos. 5,323,907, 5,052,558 and
[000484] [000484] Modified PH20 polypeptides, therapeutic agents and / or industrial products thereof can also be supplied as kits. Kits can include a pharmaceutical composition described here and an item for administration provided as an industrial product. For example, a PH20 polypeptide may be provided with a delivery device, such as a syringe, inhaler, measuring cup, drip, or applicator. The compositions can be contained in the item for administration or they can be provided separately to be added later. The kit can optionally include instructions for application including dosages, dosage regimens and instructions for modes of administration. The kits may also include a pharmaceutical composition described here and a diagnostic item. For example, such kits may include an item to measure the concentration, quantity or activity of the selected protease in a subject.
[000485] [000485] Assays can be used to assess the stability and activity of the PH20 polypeptides provided here. The assays can be used to evaluate the hyaluronidase activity of the PH20 polypeptide under specific conditions, temperature and / or over time. Such assays can be used, for example, to determine the stability of the PH20 polypeptide under specific denaturing conditions, including, but not limited to, elevated temperatures greater than or approximately or 30 ° C (for example, 30 ° C to 42 ° C such as or approximately 37 ° C), agitation, presence of excipients (eg, preservative) or low NaCl content or no NaCl (salt). For example, stability under specific conditions can be monitored by assessing activity, solubility, and stability (for example, formation of aggregates, etc.) in the absence of exposure to the denaturation condition and then at various points of time later in the presence of the condition . Consequently, stability can be assessed over time. Stability can also be assessed by comparing any one or more of activity, solubility or aggregation in the presence of one or more denaturation conditions compared to a native, wild-type or reference PH20 polypeptide. The assays can also be used to make minor adjustments to the formulations provided here while retaining the stability of the two active agents.
[000486] [000486] The activity of a modified PH20 polypeptide can be assessed using methods well known in the art. For example, the USP XXII hyaluronidase assay determines activity indirectly by measuring the amount of lower grade hyaluronic acid, or hyaluronan, (HA) substrate that remains after the enzyme is left to react with HA for 30 min. at 37 ° C (USP XXII-NF XVII (1990) 644-645 United States Pharmacopeia Convention, Inc, Rockville, MD). A Hyaluronidase Reference Standard (USP) or National Form (NC) standard Hyaluronidase solution can be used in an assay to determine the activity, in units, of any hyaluronidase. In one example, activity is measured using a micro turbidity assay. This is based on the formation of an insoluble precipitate when hyaluronic acid binds with a reagent that precipitates it, such as acidified serum or cetyl pyridinium chloride (CPC). Activity is measured by incubating hyaluronidase with sodium hyaluronate (hyaluronic acid) for a specified period of time (for example, 10 minutes) and then precipitating undigested sodium hyaluronate with the addition of acidified serum or CPC. The resulting sample turbidity is measured at 640 nm after a further development period. The decrease in turbidity resulting from hyaluronidase activity on the sodium hyaluronate substrate is a measure of hyaluronidase enzymatic activity.
[000487] [000487] In another example, hyaluronidase activity is measured using a microtiter assay in which residual biotinylated hyaluronic acid is measured after incubation with hyaluronidase (see, for example, Frost and Stern (1997) Anal. Biochem. 251: 263-269 , US patent publication No. 20050260186). The free carboxyl groups in the hyaluronic acid glucuronic acid residues are biotinylated, and the biotinylated hyaluronic acid substrate is covalently coupled to a microtiter plate. After incubation with hyaluronidase, the residual biotinylated hyaluronic acid substrate is detected using an avidin-peroxidase reaction, and compared with that obtained after reaction with hyaluronidase patterns of known activity.
[000488] [000488] Other assays for measuring hyaluronidase activity are also known in the art can be used in the methods of the present invention (see for example, Delpech et al., (1995) Anal. Biochem. 229: 35-41; Takahashi et al., ( 2003) Anal. Biochem. 322: 257-263).
[000489] [000489] Many hyaluronidase assays were based on measuring the generation of new groups of reducing N-acetylamino (Bonner and Cantey, Clin. Chim. Acta 13: 746-752, 1966), or loss of viscosity (De Salegui et al. , Arch. Biochem. Biophys.121: 548-554, 1967) or turbidity (Dorfman and Ott, J. Biol. Chem. 172: 367, 1948). With purified substrates, all of these methods are sufficient to determine the presence or absence of endoglycosidase activity.
[000490] [000490] Substantially purified glycosaminoglycan substrates can also be used in a Gel Change Assay. Glycosaminoglycans are mixed with a recombinant PH20, such as a soluble P20, to test for endoglycosidase activity that results in a change in substrate mobility in the gel. Examples of such substrates include, but are not limited to, chondroitin-4 and 6 sulfate, dermatan sulfate, heparan sulfate, which can be obtained from Sigma Chemical. Human umbilical cord hyaluronan can be obtained from ICN. For example, each test substrate can be diluted in or approximately 0.1 mg / mL in a buffer range of pH 3.5 - 7.5 in such an exemplary assay, in or approximately 10 µl samples of purified soluble PH20 or conditioned cell medium that express PH20 can be mixed with in or approximately 90 µl of test substrate in desired buffer and incubated for 3 hours at 37 ° C. After incubation, the samples are neutralized with sample buffer (Tris EDTA pH 8.0, Bromophenol blue and glycerol) followed by electrophoresis. Glycosaminoglycans can be detected using any method known in the art, for example, glycosaminoglycans can be detected by staining the gels using 0.5% Alcian blue in 3% glacial acetic acid overnight followed by discoloring in 7% glacial acetic acid. Degradation is determined by comparing substrate mobility in the presence and absence of enzyme.
[000491] [000491] Hyaluronidase activity can also be detected by substrate gel zymography (Guentenhoner et al. (1992) Matrix 12: 388-396). In this test, a sample is applied to an SDS-PAGE gel containing hyaluronic acid and the proteins in the sample separated by electrophoresis. The gel is then incubated in an enzyme assay buffer and subsequently colored to detect the hyaluronic acid in the gel. Hyaluronidase activity is visualized as a clean zone on the substrate gel.
[000492] [000492] The ability of a PH20 polypeptide, including a modified PH20 polypeptide provided here, to act as a dispersing or diffusing agent can also be assessed. For example, trypan blue dye can be injected subcutaneously with or without a PH20 polypeptide into the lateral skin on either side of the nude mice. The dye area is then measured, as with a microcaliber, to determine the PH20 polypeptide's ability to act as a dispersing agent (U.S. Pat. No. 20060104968).
[000493] [000493] The functional activity of a PH20 polypeptide can be compared and / or normalized to a reference standard using any of these assays. This can be done to determine what is the functionally equivalent amount of a PH20 polypeptide. For example, the ability of a PH20 polypeptide to act as a dispersing or diffusing agent can be assessed by injecting it into the side of the mice with trypan blue, and the amount required to obtain the same amount of diffusion as, for example, 100 units. of a Hyaluronidase Reference Standard, can be determined. The amount of PH20 polypeptide required is therefore functionally equivalent to 100 units of hyaluronidase.
[000494] [000494] The solubility of a PH20 polypeptide can be determined by any method known to a person skilled in the art. One method for determining solubility is detergent splitting. For example, a soluble PH20 polypeptide can be distinguished, for example, by dividing it in the aqueous phase of a solution of Triton® X-114 at 37 ° C (Bordier et al., (1981) J. Biol. Chem., 256: 1604 -1607). Membrane-fixed polypeptides, such as lipid-fixed hyaluronidases, including GPI-fixed hyaluronidases, will divide into the detergent-rich phase, but will divide into the aqueous or detergent-poor phase after treatment with phospholipase C. Phospholipase C is an enzyme that cleaves the phosphate bond -glycerol found in GPI-fixed proteins. PLC treatment will cause the release of GPI-bound proteins from the outer cell membrane.
[000495] [000495] The stability of a PH20 polypeptide provided here can also be assessed using other methods and assays known in the art. In addition to assessing stability based on hyaluronidase activity, stability can be assessed by visual inspection, percentage of recovery, protein purity and apparent melting temperature.
[000496] [000496] For example, protein purity can be measured by reverse phase high performance liquid chromatography (RP-HPLC). Protein purity, as determined by RP-HPLC, is the percentage of the main PH20 protein peak present, compared to all protein species present. In this way, RP-HPLC, and similar methods known to a person skilled in the art, can assess degradation of the enzyme. Protein purity can be assessed over time. Protein purity can also be assessed in the presence of one or more denaturation conditions and in varying amounts. The percentage of recovery can also be determined as the relative percentage of the polypeptide under various conditions (denaturation conditions, storage time, storage mode as container or container, or other similar parameters that can be changed) compared to a reference sample . The stability of PH20 polypeptide can also be determined by measuring the oxidation of hyaluronidase by RP-HPLC. The percentage of oxidation is a measure of the sum of peak areas of the largest (ox-1) and smallest (ox-2) peaks.
[000497] [000497] In one example, the melting temperature of a PH20 polypeptide, such as a modified PH20 polypeptide, can be determined by measuring the hydrodynamic radius of particles by dynamic light scattering under various conditions (for example, denaturation conditions or other conditions storage). An increase in particle size and a decrease in the melting temperature indicates denaturation and subsequent aggregation of hyaluronidase.
[000498] [000498] Other methods known to a person skilled in the art can be used to determine the stability of hyaluronidase in the co-formulations provided here, including polyacrylamide gel electrophoresis (PAGE), immunoblotting, nuclear magnetic resonance spectroscopy (NMR), spectrometry mass, circular dichroism (CD) and dye-based fluorescence assays.
[000499] [000499] The effect of administering a PH20 polypeptide, such as a modified PH20 polypeptide, individually or in combination with another therapeutic agent, on the pharmacokinetic and pharmacodynamic properties of any agent administered can also be assessed in vivo using animal models and / or human subjects, as in the setting of a clinical experiment. Pharmacokinetic or pharmacodynamic studies can be performed using animal models or can be performed during studies with patients administered with a PH20 polypeptide or modified PH20 polypeptide.
[000500] [000500] Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cinomolgus monkeys or rhesus monkeys. In some instances, pharmacokinetic or pharmacodynamic studies are performed using healthy animals. In other examples, studies are conducted using animal models of a disease for which hyaluronan therapy is considered, such as animal models of any disease or disorder associated with hyaluronan, for example, a tumor model.
[000501] [000501] The pharmacokinetic properties of a PH20 polypeptide, such as modified PH20 polypeptide, can be evaluated by measuring such parameters as the maximum concentration (peak), the peak time (that is, when maximum concentration occurs; Tmax), the minimum concentration ( that is, the minimum concentration between doses: Cmin), the elimination half-life (T1 / 2) and area under curve (ie, the area under the curve generated by plotting time versus concentration; AUC), after administration. The absolute bioavailability of hyaluronidase can be determined by comparing the area under the hyaluronidase curve after subcutaneous delivery (AUCsc) with the hyaluronidase AUC after intravenous delivery (AUCiv). Absolute bioavailability (F) can be calculated using the formula: F = ([AUC] sc × dosesc) / ([AUC] iv × doseiv). A different dose range and dosing frequency can be administered in pharmacokinetic studies to assess the effect of increasing or decreasing the concentration enzyme, such as modified PH20 polypeptide, in the dose. H. Methods of treatment and combination therapy
[000502] [000502] Methods and uses of any of the modified PH20 polypeptides provided here that exhibit hyaluronidase activity based on their ability to degrade glycosaminoglycan (s) as hyaluronan are provided here. Due to such activity, modified PH20 polypeptides can be used as a dispersion factor to increase the supply and / or bioavailability of therapeutic agents administered subcutaneously. The delivery of any therapeutic agent, including but not limited to, peptides, proteins, small molecule drugs, nucleic acids, or viruses can be facilitated or enhanced by coadministration with a modified PH20 polypeptide provided here. For example, modified PH20 polypeptides can be used to increase the supply of therapeutic agents such as antibodies (eg, monoclonal antibodies), cytosines, Immune globulin, an Insulin, or clotting factors, to a desired location, such as by increasing the penetration of chemotherapeutic agents in solid tumors. The modified PH20 polypeptides can also be used to treat a hyaluronan disease or disorder that is characterized by an excess or accumulation of hyaluronan. For example, modified PH20 polypeptides provided here can be used to treat a tumor, to treat accumulation of glycosaminoglycan in the brain; to treat a cardiovascular disorder; to treat an ophthalmic disorder; to treat lung disease; to treat cellulite; and / or to treat a proliferative disorder.
[000503] [000503] Other methods and uses of a PH20 polypeptide include any that are known to a person skilled in the art. For example, several forms of PH20 hyaluronidases have been prepared and approved for therapeutic use in humans. For example, animal derived hyaluronidase preparations include Vitrase® (ISTA Pharmaceuticals), a purified sheep testicular hyaluronidase, and Amphadase® (Amphastar Pharmaceuticals), a bovine testicular hyaluronidase. Hylenex® (Halozyme Therapeutics) is a recombinant human hyaluronidase produced by genetically formed Chinese hamster ovary (CHO) cells containing nucleic acid encoding soluble rHuPH20 (see for example, U.S. Patent No. 7,767,429). Approved therapeutic uses for hyaluronidases include use as an adjunct to increase the absorption and dispersion of other therapeutic agents for hypodermoclysis (administration of subcutaneous fluid), and as an adjunct in subcutaneous urography to improve resorption of radiopaque agents. In addition to these indications, hyaluronidases can be used as a therapeutic or cosmetic agent to treat additional diseases and conditions. For example, hyaluronidase is commonly used, for example, for peribulbar block in local anesthesia before ophthalmic surgery. The presence of the enzyme avoids the need for additional blockages and reduces the time for the onset of akinesia (loss of eye movement). Sub-Tenon block and peribulbar are the most common applications of hyaluronidase for ophthalmic procedures. Hyaluronidase can also promote akinesia in cosmetic surgery, such as blepharoplasty and facial lifts. It is understood that soluble PH20 hyaluronidases provided herein, including esPH20 hyaluronidases, can be used in any treatment method or combination therapy for which a PH20 hyaluronidase is used (see, for example, US Nos. US20040268425; US20050260186; US20060104968; and US Serial Nos. 12 / 381,844, published as US publication No. US20100074885; 12 / 386,249, published as US publication No. US20090311237; 12 / 387,225, published as US publication No. US20090304665; and 12 / 386,222, published as US publication No. US2010003238, each incorporated by reference in full).
[000504] [000504] Non-limiting, exemplary methods and uses are described in the following subsections.
[000505] [000505] As noted above, hyaluronidase is a dispersion or diffusion substance that modifies the permeability of connective tissue through the hydrolysis of hyaluronic acid, a polysaccharide found in the intercellular base substance of connective tissue, and of certain specialized tissues, such as humor citrus and umbilical cord. When no dispersion factors are present, materials injected subcutaneously, such as drugs, proteins, peptides and nucleic acid, spread very slowly. Co-injection with hyaluronidase, however, can cause rapid dispersion. The diffusion rate is proportional to the amount of enzymes, and the extent of diffusion is proportional to the volume of solution.
[000506] [000506] Modified PH20 polypeptides provided here can be used to promote or enhance delivery agents and molecules for any of a variety of mammalian tissues in vivo. It can be used to facilitate diffusion and therefore promote the supply of small molecule pharmacological agents as well as larger molecule pharmacological agents, such as proteins, nucleic acids and ribonucleic acids, and macromolecular compositions that may contain a combination of components including, but not limited to, nucleic acids, proteins, carbohydrates, lipids, molecules and drugs based on lipids (see, for example, US publications nos. US20040268425; US20050260186; and US20060104968). Modified PH20 polypeptides can be co-administered and / or co-formulated with a therapeutic agent to improve bioavailability as well as pharmacokinetic (PK) and / or pharmacodynamic (PD) characteristics of co-formulated or co-administered agents. PK / PD parameters that can be improved by the use of soluble PH20, such as esPH20, include such measures as Cmax (the maximum agent concentration obtained after absorption, for example, in the bloodstream), Tmax (the time required to obtain maximum concentration) , T1 / 2 (the time required for the concentration to halve), Cmin (the minimum concentration of agent after metabolism and excretion), AUC (area under the concentration versus time curve, a measure of the overall amount of bioavailability), concentrations in various tissues of interest (including, for example, the rate of obtaining desired concentrations, the general levels, and the duration of maintaining desired levels), and Emax (the maximum effect obtained).
[000507] [000507] The treatment methods provided here include combination therapies with a therapeutic agent for the treatment of a disease or disorder to which the therapeutic agent is a threat. Any therapeutic agent that improves and or otherwise decreases the severity of a disease or condition can be combined with a modified PH20 polypeptide provided here to increase the bioavailability of such a therapeutic agent. In particular, modified PH20 polypeptides provided here can be used in each and every combination described in orders, see, for example, US publications nos. US20040268425; US20050260186; US20060104968 and US Serial Nos. 12 / 381,844, published as US publication No. US20100074885; 12 / 386,249, published as US publication No. US20090311237; 12 / 387,225, published as US publication No. US20090304665; and 12 / 386,222, published as US publication No. US2010003238 in place of the disclosed hyaluronidase or hyaluronidase degrading enzyme.
[000508] [000508] Modified PH20 polypeptides can be administered before, subsequent to, intermittently with or simultaneously with the therapeutic agent preparation. Generally, the modified PH20 polypeptide is administered prior to or simultaneously with administration of the therapeutic agent preparation to allow PH20 to degrade hyaluronic acid in the interstitial space. PH20 can be administered at a site other than the site of administration of the therapeutic molecule, or soluble PH20 can be administered at a site equal to the site of administration of the therapeutic molecule.
[000509] [000509] Examples of pharmaceutical, therapeutic and cosmetic agents and molecules that can be administered with hyaluronidase include, but are not limited to, a chemotherapeutic or anticancer agent, an analgesic agent, an antibiotic agent, an anti-inflammatory agent, an antimicrobial agent, amebicidal agent, trichomonic agent, antiparkinson agent, antimalarial agent, anticonvulsant agent, antidepressant agent, antiarthritic agent, antifungal agent, antihypertensive agent, antipyretic agent, antipyretic agent parasitic, an antihistamine agent, an alpha-adrenergic agonist agent, an alpha blocking agent, an anesthetic agent, a bronchial dilating agent, a biocidal agent, a bactericidal agent, a bacteriostatic agent, a beta adrenergic blocking agent, a blocking agent calcium channel agent, a cardiovascular drug agent, a contraceptive agent, a cosmetic or aesthetic agent, a decongestant agent nant, a diuretic agent, a depressant agent, a diagnostic agent, an electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant, a muscle contraction agent, an ophthalmic agent, an agent parasympathomimetic, psychic energizing agent, sedative, sleep inducer, sympathomimetic agent, tranquilizer, urinary agent, vaginal agent, viricidal agent, vitamin agent, non-steroidal anti-inflammatory agent, or an angiotensin converting enzyme inhibiting agent,
[000510] [000510] For example, modified PH20 polypeptides provided here can be used to increase the supply of chemotherapeutic agents. Hyaluronidases have also been used to increase the activity of chemotherapy and / or tumor accessibility to chemotherapy (Schuller et al., 1991, Proc. Amer. Assoc. Cancer Res. 32: 173, abstract no. 1034; Czejka et al., 1990, Pharmazie 45: H.9; Baumgartner et al. (1988) Reg. Cancer Treat. 1: 55-58; Zanker et al. (1986) Proc. Amer. Assoc. Cancer Res. 27: 390). Combination chemotherapy with hyaluronidase is effective in treating a variety of cancers including urinary bladder cancer (Horn et al., 1985, J. Surg. Oncol. 28: 304-307), squamous cell carcinoma (Kohno et al., 94, J. Cancer Res. Oncol. 120: 293-297), breast cancer (Beckenlehner et al., 1992, J. Cancer Res. Oncol. 118: 591-596), and gastrointestinal cancer (Scheithauer et al., 1988, Anticancer Res 8: 391-396). In this example, the modified PH20 hyaluronidase increases the penetration of chemotherapeutic agents or other anticancer agents into solid tumors, thereby treating the disease.
[000511] [000511] Compositions containing soluble PH20 can be injected intratumorally with anticancer agents or intravenously for disseminated cancers or tumors difficult to reach. The anticancer agent can be a chemotherapeutic, an antibody, a peptide, or a gene, virus or DNA therapy vector. In addition, hyaluronidase can be used to recruit tumor cells in the cycling pool for sensitivity in previously chemorefractive tumors that have acquired resistance to multiple drugs (St Croix et al., (1998) Cancer Lett September 131 (1): 35-44).
[000512] [000512] Exemplary anticancer agents that can be administered after, coinciding with or before the administration of a soluble PH20, such as an esPH20, include, but are not limited to, Acivicina, Aclarubicina, Acodazois, Acroninas, Adozelesins, Aldesleucinas, alemtuzumabs; Alitretinoins (9-Cis retinoic acids), allopurinois, altretamines; Alvocidibs, Ambazonas, Ambomicinas, Ametantronas, amifostinas; Aminoglutetimides, Amsacrinas, anastrozois, Anaxironas, Ancitabinas, Antramicinas, Apaziquones, Argimesnas, arsenic trioxides; asparaginases; Asperlins, Atrimustins, Azacitidines, Azetepas, Azotomycins, Banoxanthrones, Batabulins, Batimastats, BCG live; Benaxibins, Bendamustines, Benzodepas, Bexarotenes; Bevacizumab; Bicalutamides; Bietaserpines; Biricodars; Bisantrenes; Bisantrenes; Bisnafide Dimesylates; Bizelesins; Bleomycins; Bortezomibs; Brequinars; Bropirimines; Budotitanes; Busulfans; Cactinomycins; Calusterones; Canertinibs; Capecitabines; Caracemides; Carbetimers; Carboplatins; Carboquones; Carmofurs; Carmustines with Polifeprosans; Carmustines; Carubicins; Carzelesins; Cedefingols; Celecoxibs; Cemadotins; Chlorambucils; Cioteronels; Ciplactin; Cirolemicina; Cisplatins; Cladribines; Clanfenurs; Clofarabines; Crisnatois; Cyclophosphamides; Liposomal cytarabine; Cytarabines; Dacarbazine; Dactinomycins; Darbepoetin Alfas; Liposomal Daunorubicin; Daunorubicinas / Daunomicinas; Daunorubicins; Decitabines; Denileucin Diftitoxes; Dexniguldipines; Dexones; Dexrazoxanes; Dezaguanines; Diaziquones; Dibrospidiums; Dienogests; Dinalins; Disermolides; Docetaxels;
[000513] [000513] Aldesleukins (for example, PROLEUKIN®); Alemtuzumabs (for example, CAMPATH®); Alitretinoins (for example, PANRETIN®); Alopurinois (for example, ZYLOPRIM®); Altretamines (for example, HEXALEN®); Amifostines (for example, ETHYOL®); Anastrozois (for example, ARIMIDEX®); arsenic trioxides (for example, TRISENOX®); Asparaginases (for example, ELSPAR®); Live BCG (for example, TICE® BCG); Bexarotenes (for example, TARGRETIN®); Bevacizumab (AVASTIN®); Bleomycins (for example, BLENOXANE®); Intravenous busulfan (for example, BUSULFEX®); Oral busulfan (for example, MYLERAN ™); Calusterones (for example, METHOSARB®); Capecitabines (for example, XELODA®); Carboplatins (for example,
[000514] [000514] For example, exemplary antibiotic agents include, but are not limited to, Aminoglycosides; Amphenols; Ansamycins; Carbacefems; Carbapenemos; Cephalosporins or Cefems; Cefamycins; Clavams; cyclic lipopeptides; Diaminopyrimidines; Ketolides; Lincosamides; Macrolides; Monobactams; Nitrofurans; Oxacefems; Oxazolidinones; Penems, thienamycins and several beta-lactams; Penicillins; polypeptide antibiotics; Quinolones; Sulphonamides; Sulphones; Tetracyclines; and other antibiotics (such as Clofoctols, Fusidic acids, Hexedines, Methenamines, Nitrofurantoins, Nitroxolines, Ritipenems, Taurolidines, Xibomols).
[000515] [000515] Also included among the exemplary therapeutic agents are coagulation factors or other blood modifiers such as antihemophilic factors, anti-inhibitor coagulant complexes, antithrombin III, coagulation factor V, coagulation factor VIII, coagulation factor IX, fractions plasma protein, von Willebrand factors; antiplatelet agents (including, for example, abciximabs, anagrelides, cilostazois, clopidogrel bisulfates, dipyridamoles, epoprostenools, eptifibatides, tirofibans; colony stimulating factors (CSFs) (including, for example, CSFs granulocytes and granulocytes
[000516] [000516] Exemplary antibodies or other therapeutic agents include, but are not limited to, Cetuximab (IMC-C225; Erbitux®); Trastuzumab (Herceptin®); Rituximab (Rituxan®; MabThera®); Bevacizumab (Avastin®); Alemtuzumab (Campath®; Campath-1H®; Mabcampath®); Panitumumab (ABX-EGF; Vectibix®); Ranibizumab (Lucentis®); Ibritumomab; Ibritumomab tiuxetan (Zevalin ®); Tositumomab; Iodine I 131 Tositumomab (BEXXAR®); Catumaxomab (Removab®); Gemtuzumab; Gemtuzumab ozogamycin (Mylotarg®); Abatacept (CTLA4-Ig; Orencia®); Belatacept (L104EA29YIg; LEA29Y; LEA); Ipilimumab (MDX-010; MDX-101); Tremelimumab (ticilimumab; CP-675.206); PRS-010 (see for example, US20090042785); PRS-050 (US7585940; US20090305982); Aflibercept (VEGF Trap, AVE005; Holash et al., (2002) PNAS 99: 11393-11398); Volociximab (M200); F200 (Fab fragment (human / murine) chimeric IgG4 from Volociximab (M200)); MORAb-009 chimeric human / mouse IgG1 (US20050054048); soluble fusion protein: Anti-mesothelin Fv bound to a truncated Pseudomonas exotoxin A (SS1P (CAT-5001); US20070189962); Cixutumumab (IMC-A12); Nimotuzumab (h-R3) (Spicer (2005) Curr Opin Mol Ther 7: 182-191); Zalutumumab (HuMax-EGFR; Lammerts van Bueren et al. (2008) PNAS 105: 6109-14);
[000517] [000517] In particular, therapeutic agents include, but are not limited to, immunoglobulins, Interferon beta, Interferon alfa-2as, Interferon alfa-1s, Interferon alfa-n3s, Interferon beta-1, Interferon beta-1as, Interferon beta-1as, Interferon gamma-lbs, Peg-interferon alfa-2 and Peginterferon alfa-2bs, insulin, bisphosphate (eg Pamidronates or Zoledronates), Docetaxels, Doxorubicins, Liposomal Doxorubicin and bevacizumabs.
[000518] [000518] Other exemplary therapeutic agents that can be combined with poor co-administration and / or co-formulation with a modified PH20 polypeptide provided here, include, but are not limited to, Adalimumabs, Agalsidase Betas, Alefacepts, Ampicillins, Anakinras, Polio vaccines, Anti -Tymocytes, Azithromycin, Becaplermins, Caspofungins, Cefazolins, Cefepimes, Cefotetans, Ceftazidimas, Ceftriaxones, Cetuximabs, Cilastatines, Clavulanic acids, Clindamycins, Darbepoetin Alfas, Daclizumaxines, Diphtheria, Etheria, Diphtheria, Diphtheria , Filgrastims, Fluconazois, follicle stimulating hormones, Folitropin Alfas, Folitropin Betas, Phosphenitoines, Gadodiamides, Gadopentetates, Gatifloxacins, Glatirameros, GM-CSF's, Goserelins, Goserelin acetates, Granisetrons, Haemophilus, Holstein hepatitis A, Hepatitis B vaccines, Ibritumomab Tiuxetans, Ibritum omabs, Tiuxetans, Immunoglobulins, Hemophilus influenza vaccines, influenza virus vaccines, Infliximabs, Insulins, Insulin Glargines, Interferons, Interferon alfa, Interferon Betas,
[000519] [000519] The methods provided here include methods of co-administering a modified PH20 polypeptide and an insulin to increase the subcutaneous supply of insulin, such as a fast-acting insulin (see for example, US Patent No. 7,767,429; US Patent No. 7,846,431; Publication US No. US20090304665; and US Order Serial Nos. 13 / 507,263; 13 / 507,262 and 13 / 507,261). Such methods include methods of direct administration, and pump and continuous infusion methods, including open and closed pump systems. For example, exemplary insulins that can be administered with a modified PH20 hyaluronidase provided here are fast-acting insulins or insulin analogs. For example, a coadministered insulin includes regular insulin, aspart insulin, lispro insulin, glulisine insulin or other similar analogue variants. Exemplary insulins are insulins that contain an A chain exposed to SEQ ID NO: 862 and a B chain exposed to SEQ ID NO: 863 or variants that contain one or more amino acid modifications compared to a human insulin exposed to SEQ ID NO: 862 and 863 (chains A and B). For example, exemplary insulin analogs are known to a person skilled in the art, and include, but are not limited to, those exposed in SEQ ID NOS: 862 (chain A) and having a B-chain exposed in any of SEQ ID NOS: 865 -867.
[000520] [000520] Co-formulations can be administered subcutaneously to treat any conditions that are favorable to treatment with insulin. Therapeutic uses include, but are not limited to, treatment for type 1 diabetes mellitus, type 2 diabetes mellitus, gestational diabetes, and for glycemic control in critically ill patients. For example, co-formulations of a fast-acting insulin and hyaluronan-degrading enzyme can be administered subcutaneously in separate doses, such as through a syringe or insulin pen, before a meal as insulin therapy in relation to dinner. in subjects with diabetes to obtain glycemic control. Co-formulations can also be administered subcutaneously or intraperitoneally using an insulin pump or in the context of a closed-loop system to continuously control blood glucose levels throughout the day and night and / or to control post-glycemic excursions.
[000521] [000521] For any disease or condition, including those all exemplified above, for which a fast-acting insulin is indicated or has been used and for which other agents and treatments are available, co-formulations can be used in combination with same. Depending on the disease or condition being treated, exemplary combinations include, but are not limited to, combinations with antidiabetic drugs, including, but not limited to, sulfonyl ureas, biguanides, meglitinides, thiazolidinediones, alpha-glucosidase inhibitors, peptide analogs, including glucagon-like peptide analogs (GLP) and gastric inhibitor peptide analogs (GIP) and DPP-4 inhibitors. In another example, the co-formulations of a fast-acting insulin and modified PH20 polypeptide described here can be administered in combination with, before, intermittently with or subsequent to, one or more other insulins, including fast-acting insulin, and insulins basal action.
[000522] [000522] In particular, hyaluronidase PH20 can be used to treat diseases or conditions associated with hyaluronan. Typically, diseases and conditions associated with hyaluronan are associated with elevated expression of hyaluronan in a tissue, cell, or body fluid (e.g., tumor tissue or tissue associated with tumor, blood, or interstitial space) compared to a control, for example. example, another tissue, cell or body fluid. The high expression of hyaluronan may be elevated in comparison to normal tissue, cell or body fluid, for example, a tissue, cell or body fluid that is analogous to the sample being tested, but isolated from a different subject, such as a subject who is normal (ie, does not have a disease or condition, or does not have the type of disease or condition that the subject being tested has), for example, a subject who does not have a condition or disease associated with hyaluronan. The elevated expression of hyaluronan can be elevated in comparison with an analogous tissue from another subject who has a similar condition or disease, but whose disease is not as severe and / or is not associated with hyaluronan or expressed relatively less hyaluronan and thus it is associated with hyaluronan to a lesser extent. For example, the subject being tested may be a subject with cancer associated with hyaluronan, where the amounts of HA in the tissue, cell or fluid are relatively high compared to a subject having a less severe cancer, such as an early, differentiated or another type of cancer. In another example, the cell, tissue or fluid contains high levels of hyaluronan compared to a control sample, such as a fluid, tissue, extract (eg, nuclear or cell extract), peptide or nucleic acid preparation, cell line , biopsy, standard or other sample, with a known amount or relative amount of HA, such as a sample, for example, a tumor cell line, known to express relatively low levels of HA, as exemplary tumor cell lines described here that express low levels of HA, for example, the HCT 116 cell line, the HT29 cell line, the NCl H460 cell line, the DU145 cell line, the Capan-1 cell line, and tumors from models tumor cells generated using such cell lines.
[000523] [000523] Conditions and diseases associated with hyaluronan include those associated with elevated interstitial fluid pressure, such as disc pressure, proliferative disorders such as cancer and benign prostatic hyperplasia and edema. Edema can result from or be manifested in, for example, organ transplantation, stroke or brain trauma. Proliferative disorders include, but are not limited to, cancer, smooth muscle cell proliferation, systemic sclerosis, liver cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy, for example, diabetic retinopathy or other retinopathies, cardiac hyperplasia, disorders associated with the reproductive system, such as benign prostatic hyperplasia (BPH) and ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hematomas, lymphangiomatosis, sarcoidosis, desmoid tumors. Cancers include blood / lymphatic and solid tumors and metastatic disease, and non-differentiated tumors. Treatment-sensitive tumors typically show cellular and / or stromal expression of a hyaluronan, compared to non-cancerous tissue of the same type of tissue or compared to a non-metastatic tumor of the same type of tumor. Cancers include any one or more of ovarian cancer, carcinoma in situ (ISC), squamous cell carcinoma (SCC), prostate cancer, pancreatic cancer, other gastric cancers, non-small cell lung cancer, breast cancer, cancer of the brain and colon cancer.
[000524] [000524] Modified pH20 polypeptides provided here, as PEGUIlated forms thereof, can be used to treat tumors. Thus, in addition to their indirect anticancer effects, hyaluronidases also have direct anticarcinogenic effects. Hyaluronidase prevents growth of transplanted tumors in mice
[000525] [000525] Cancer rich in hyaluronan can be a cancer in which cancer cells produce HALOs, cancers that have high expression of hyaluronan (as determined by immunostaining, for example, histological staining of sections from the tumor), cancers that have High HAS2 (Hialuronan synthase 2), cancers that do not produce hyaluronidase (HYAL1) in vitro. Hyaluronan-rich cancers can be identified by any method of evaluating hyaluronan expression, and other known methods for assaying mRNA / protein expression.
[000526] [000526] Several hyaluronan-rich cancers have been identified. In some cases, hyaluronan expression correlates with poor prognosis, for example, decreased survival rate and / or recurrence-free survival rate, metastasis, angiogenesis, invasion of cancer cells in other tissues / areas and other indicators of poor prognosis. Such correlation has been observed, for example, in hyaluronan-rich tumors including ovarian cancer, SCC, SSI, prostate cancer, lung cancer, including non-small cell lung cancer (NSCLC), breast cancer, colon cancer and pancreatic cancer (see, for example, Anttila et al., Cancer Research, 60: 150-155 (2000); Karvinen et al., British
[000527] [000527] Other diseases or conditions associated with hyaluronan that are associated with excess glycosaminoglycans and that can be treated with a modified PH20 polypeptide provided here include, but are not limited to, cardiovascular disease (for example, after reperfusion of ischemia; in arteriosclerosis; ); vitrectomy and ophthalmic disorders and conditions (for example, in methods to liquefy the vitreous mood of the eye; reduce postoperative pressure; other eye surgical procedures such as glaucoma, retinal and vitreous surgery and in corneal transplantation); in hypodermoclysis (that is, infusion of fluids and electrolytes in the hypodermis of the skin); cosmetic applications (for example, in the treatment of cellulite, “pig skin” edema or “orange peel” edema); organ transplantation (for example, associated with interstitial edema in relation to an organ graft); lung disease.
[000528] [000528] In additional examples of their therapeutic use, modified PH20 polypeptides provided here, can be used for such purposes as an antidote to local necrosis from paravenous injection of necrotic substances such as vinca alkaloids (Few et al.
[000529] [000529] Modified PH20 polypeptides can be used in the treatment of spinal cord injury by degrading chondroitin sulfate proteoglycans (CSPGs). After spinal cord injury, glial scars containing CSPGs are produced by astrocytes. CSPGs play a crucial role in inhibiting axon growth. In addition, CSPG expression has been shown to increase after injury to the central nervous system (CNS). Soluble PH20 can also be used to treat herniated discs in a process known as quiminucleolysis. ABC of chondroitinase, an enzyme cleaving similar substrates like hyaluronidase, can induce a reduction in intradiscal pressure in the lumbar spine. There are three types of disc injuries. A protruding disc is intact, but protruding. In the extruded disc, the fibrous wrapper tore and the NP drained out, but it is still connected to the disc. In a sequestered disc, a fragment of the NP has become detached from the disc and is free in the spinal canal. Quiminucleolysis is typically effective on protruding and extruded discs, but not on hijacked disc injuries.
[000530] [000530] Modified PH20 polypeptides provided here can be used as vaccines in contraceptive applications. PH20 is present in the male reproductive tract, and is expressed in both the testes and epididymis and is present in sperm. PH20 plays a role in fertilization by facilitating the entry of sperm through the cumulus layer that surrounds the unfertilized egg. PH20 is also able to bind to hyaluronic acid (HA) in the pellucid zone during early stages of fertilization. This connection also initiates intracellular signaling that assists in the acrosome reaction. PH20 immunization has been shown to be an effective contraceptive in male guinea pigs (Primakoff et al. (1988) Nature 335: 543- 546, Tung et al. (1997) Biol. Reprod. 56: 1133-1141). It has also been shown to be an effective contraceptive in female guinea pigs due to the generation of anti-PH20 antibodies that prevent sperm and egg binding. In the examples of the present invention, the modified PH20 polypeptides can be inactive enzymes, like any described in sections C.2. polypeptides can be administered directly or they can be administered as or recombinant virus to provide the antigen. I. EXAMPLES
[000531] [000531] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. EXAMPLE 1 Generation of recombinant human PH20 hyaluronidase (rHuPH20) A. Generation of a cell line that expresses rHuPH20
[000532] [000532] A recombinant human PH20 hyaluronidase rHuPH20 was generated as described in the US publication published no. US20110053247. In summary, plasmid pCI-PH20-IRES-DHFR-SV40pa (HZ24) (set out in SEQ ID NO: 5)
[000533] [000533] Non-transfected CHO DG44 cells growing in GIBCO-modified CD-CHO media for DHFR (-) cells, supplemented with 4 mM Glutamine and 18 mL / L Plurionic F68 / L (Gibco), were seeded in 0.5 x 106 cells / mL in a shaker flask in preparation for transfection. The cells were cultured at 37 ° C in 5% CO2 in a humidified incubator, shaking at 120 rpm. CHO DG44 cells not transfected in exponential growth were tested for viability before transfection.
[000534] [000534] Sixty million viable cells from the non-transfected CHO DG44 cell culture were pelleted and resuspended at a density of 2 x 107 cells in 0.7 mL of 2x transfection buffer (2x HeBS: 40 mM Hepes, pH 7.0, 274 mM NaCl, 10 mM KCl, 1.4 mM Na2HPO4, 12 mM dextrose). For each aliquot of cells suspended again, 0.09 mL (250 µg) of the linear HZ24 plasmid (linearized by digestion overnight with Cla I (New England Biolabs) was added, and the cell / DNA solutions were transferred to 0.4 cm gap BTX (Gentronics) electroporation at room temperature. A negative control electroporation was performed without plasmid DNA mixed with the cells. Plasmid / cell mixtures were electroporated with a 330 V capacitor discharge and 960 µF or at 350 V and 960 µF.
[000535] [000535] The cells were removed from the cuvettes after electroporation and transferred to 5 mL of CD-CHO medium modified for DHFR (-) cells, supplemented with 4 mM glutamine and 18 mL / L Plurionic F68 (L) (Gibco) and allowed to grow in one well of a 6 well tissue culture plate without selection for 2 days at 37ºC in 5% CO2 in a humidified incubator.
[000536] [000536] Two days after electroporation, 0.5 ml of tissue culture medium was removed from each well and tested for the presence of hyaluronidase activity, using the microturbity assay described in example 8. The results are shown in the table
[000537] [000537] Cells from transfection 2 (350V) were collected from the tissue culture well, counted and diluted to 1 × 104 to 2 × 104 viable cells per mL. A 0.1 ml aliquot of the cell suspension was transferred to each well of five 96-well round-bottom tissue culture plates. One hundred microliters of CD-CHO medium (GIBCO) containing 4 mM GlutaMAX ™ -1 supplement (GIBCO ™, Invitrogen Corporation) and without hypoxanthine and thymidine supplements were added to the wells containing cells (final volume 0.2 mL). Ten clones were identified from the 5 plates grown without methotrexate (table 7). Table 7. Hyaluronidase activity of identified clones Plate / cavity relative Hyaluronidase
[000538] [000538] Six HZ24 clones were expanded in culture and transferred to shaker flasks as single cell suspensions. Clones 3D3, 3E5, 2G8, 2D9, 1E11, and 4D10 were coated on 96-well Round-bottom tissue culture plates using an infinite two-dimensional dilution strategy in which cells were diluted 1: 2 below the plate, and 1: 3 across the plate, starting at 5000 cells in the upper left cavity. Diluted clones were cultured in a 500-well non-transfected CHO DG44 cells per well, to provide necessary growth factors for the initial days in culture. Ten plates were made per subclone, with 5 plates containing 50 nM methotrexate and 5 plates without methotrexate.
[000539] [000539] Clone 3D3 produced 24 visual subclones (13 from treatment without methotrexate, and 11 from treatment with 50 nM methotrexate). Significant hyaluronidase activity was measured in supernatants from 8 of the 24 subclones (> 50 units / mL), and these 8 subclones were expanded into T-25 tissue culture flasks. Clones isolated from the methotrexate treatment protocol were expanded in the presence of 50 nM methotrexate. The 3D35M clone was further expanded in 500 nM methotrexate giving rise to clones that produce hyaluronidase activity in excess of 1,000 units / mL in shaker flasks (3D35M clone; or Gen1 3D35M). a master cell bank (MCB) of 3D35M cells was then prepared. B. Production of Gen2 cells containing soluble human PH20 (rHuPH20)
[000540] [000540] The 3D35M Gen1 cell line described in example 1.A has been adapted to higher levels of methotrexate to produce generation 2 (Gen2) clones. 3D35M cells were seeded from cultures containing methotrexate established in CHO CD medium containing 4 mM GlutaMAX-1 ™ and 1.0 M methotrexate. The cells were adapted to a higher level of methotrexate by cultivating and passing the same 9 times over a period of 46 days in a humidified incubator at 7% CO2, 37ºC. the amplified cell population was cloned by limiting dilution in 96-well tissue culture plates containing medium with 2.0 M methotrexate. After approximately 4 weeks, clones were identified and clone 3E10B was selected for expansion. 3E10B cells were cultured in CD CHO medium containing 4 mM GlutaMAX-1 ™ and 2.0
[000541] [000541] Cell line amplification continued by culturing 3E10B cells in CD CHO medium containing 4 mM GlutaMAX-1 ™ and 4.0 eN methotrexate. After the 12th pass, the cells were frozen in flasks as a research cell bank (RCB). One RCB vial was thawed and cultured in a medium containing 8.0 M methotrexate. After 5 days, the methotrexate concentration in the medium was increased to 16.0 M, then 20.0 M 18 days later. The cells from the 8th passage in medium containing 20.0 M methotrexate were cloned by limiting dilution in 96 well tissue culture plates containing CD CHO medium containing 4 mM GlutaMAX-1 ™ and 20.0 M methotrexate. The clones were identified 5-6 weeks later and clone 2B2 was selected for expansion in medium containing 20.0 M methotrexate. After the 11th pass, 2B2 cells were frozen in flasks as a research cell bank (RCB).
[000543] [000543] The resulting 2B2 cells are CHO DG44 cells deficient in dihydrofolate reductase (dhfr-) that express soluble recombinant human PH20 (rHuPH20). Soluble PH20 is present in 2B2 cells at a copy number of approximately 206 copies / cell. Southern blot analysis of genomic 2B2 cell DNA digested into Spe I-, Xba I- and BamH I / Hind III- using a rHuPH20 specific probe revealed the following restriction digest profile: a ~ 7.7 kb main hybridization range and four smaller hybridization strips (~ 13.9, ~ 6.6, ~ 5.7 and ~ 4.6 kb) with Spe I digested DNA; a larger hybridization range of ~ 5.0 kb and two smaller hybridization ranges (~ 13.9 and ~ 6.5 kb) with DNA digested with Xba I; and a unique ~ 1.4 kb hybridization range observed using 2B2 DNA digested with BamH I / Hind III. C. Production of rHuPH20 soluble in Gen2 in 300 L of bioreactor cell culture
[000544] [000544] One vial of HZ24-2B2 was thawed and expanded from shaker flasks through 36 L rotating flasks in CD-CHO medium (Invitrogen, Carlsbad, CA) supplemented with 20 µM methotrexate and GlutaMAX-1 ™ (Invitrogen ). In summary, the cell flask was thawed in a 35 ° C water bath, the medium was added and the cells were centrifuged. The cells were resuspended in a 125 ml shaking flask with 20 ml of fresh medium and placed in a 7% CO2 incubator, 37 ° C. the cells were expanded to 40 ml in the 125 ml shake flask. When the cell density reached more than 1.5 x 10 6 cells / ml, the culture was expanded in a 125 ml spinner flask in a 100 ml culture volume. The flask was incubated at 37 ° C, 7% CO2. When the cell density reached more than 1.5 x 10 6 cells / ml, the culture was expanded in a 1 L spinner flask in 800 ml of culture volume and incubated at 37 ° C, 7% CO2. When the cell density reached more than 1.5 x 6 cells / mL, the culture was expanded in a 36 L spinner flask in 32 L culture volume and incubated at 37 ° C, 7% CO2.
[000545] [000545] A 400 L reactor was sterilized and 230 mL of CD-CHO medium was added. Before use, the reactor was checked for contamination. Approximately 30 L cells were transferred from the 36 L spinner flasks to the 400 L bioreactor (Braun) at an inoculation density of 4.0 x 105 viable cells per mL and a total volume of 260 L. Parameters were: temperature adjustment, 37ºC; impeller speed 40-55 RPM; container pressure: 3 psi, air spray 0.5 - 1.5 L / min; air coverage: 3 L / min. The reactor was sampled daily for cell counts, pH verification, media analysis, protein production and retention. Also, during the course nutrient feeds were added. In 120 h (day 5), 10.4 L of feed no. 1 / medium (4x CD-CHO + 33g / L glucose + 160 mL / L Glutamax-1 ™ + 83 mL / L Yeastolate + 33 mg / L rHuInsulin) was added. In 168 hours (day 7), 10.8 L of feed # 2 (2 × CD-CHO + 33 g / L Glucose + 80 mL / L Glutamax-1 ™ + 167 mL / L Yeastolate +
[000546] [000546] The culture was pumped by a peristaltic pump through four modules of the Milistak filtration system (Millipore) in parallel, each containing a layer of diatomaceous earth graded to 4-8 m and a layer of diatomaceous earth graded to 1.4 - 1.1 m, followed by a cellulose membrane, then through a second single Millistak filtration system (Millipore) containing a layer of diatomaceous earth graded to 0.4 - 0.11 µm and a layer of diatomaceous earth graduated to <0.1 µm, followed by a cellulose membrane, and then through a final 0.22 µm filter in a sterile, flexible single use bag with a capacity of 350 L. the harvested cell culture fluid was supplemented with 10 mM EDTA and 10 mM Tris at a pH of 7.5. The culture was concentrated 10 × with a tangential flow filtration apparatus (TFF) using four Sartoslice TFF 30 kDa molecular weight cutoffs (MWCO) polyether sulfone filter (PES) (Sartorious), followed by a 10 µm buffer exchange × with 10 mM Tris, 20 mM Na2SO4, pH 7.5 in a 0.22 µm final filter in a 50 L sterile storage bag.
[000547] [000547] The concentrated diafiltered crop was inactivated by viruses. Before viral inactivation, a 10% Triton® X-100, 3% tri (n-butyl) phosphate (TNBP) solution was prepared. The concentrated, diafiltered crop was exposed to 1% Triton® X-100, 0.3% TNBP for 1 hour in a 36 L glass reaction vessel immediately before purification on column Q. D. Purification of rHuPH20 soluble in Gen2
[000548] [000548] A Q Sepharose ion exchange column (Pharmacia) (9 L of resin, H = 29 cm, D = 20 cm) was prepared. Wash samples were collected to determine pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of mM Tris, 20 mM Na2SO4, pH 7.5. after viral inactivation, the diafiltered, concentrated crop was loaded onto the Q column at a flow rate of 100 cm / h. The column was washed with 5 column volumes of 10 mM Tris, 20 mM Na2SO4, pH 7.5 and 10 mM Hepes, 50 mM NaCl, pH7.0. The protein was eluted with 10 mM Hepes, 400 mM NaCl, pH 7.0 in a 0.22 µm final filter in a sterile bag. The eluate sample was tested for biocharge, protein concentration and hyaluronidase activity. A280 absorbance readings were taken at the beginning and end of the exchange.
[000549] [000549] Phenyl-Sepharose hydrophobic interaction chromatography (Pharmacia) was performed below. A phenyl - Sepharose (PS) column (19-21 L of resin, H = 29 cm, D = 30 cm) was prepared. The wash was collected and sampled in relation to pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate, 0.1 mM CaCl2, pH 7.0. The protein eluate from the Q sepharose column was supplemented with 2M ammonium sulfate, 1M potassium phosphate and 1M CaCl2 material solutions to provide final concentrations of 5 mM, 0.5 M and 0.1 mM, respectively. The protein was loaded onto the PS column at a flow rate of 100 cm / h and the column flow was collected through. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate and 0.1 mM CaCl2 pH 7.0 at 100 cm / h and the wash was added to the flow collected through. Combined with column washing, the flow through was passed through a 0.22 µm final filter into a sterile bag. The direct flow was sampled in relation to biocharge, protein concentration and enzyme activity.
[000550] [000550] An aminophenyl boronate column (Prometics) was prepared. The wash was collected and sampled in relation to pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 column volumes of 5 mM potassium phosphate, 0.5 M ammonium sulfate. The direct flow of OS containing purified protein was loaded onto the aminophenyl boronate column at a flow rate of 100 cm / h. The column was washed with 5 mM potassium phosphate, 0.5 M ammonium sulfate, pH
[000551] [000551] The hydroxyapatite (HAP) column (Biorad) was prepared. The wash was collected and tested for pH, conductivity and endotoxin (LAL assay). The column was equilibrated with 5 mM potassium phosphate, 100 mM NaCl, 0.1 mM CaCl2, pH 7.0. The protein purified with aminophenyl boronate was supplemented to final concentrations of 5 mM potassium phosphate and 0.1 mM CaCl2 and loaded onto the HAP column at a flow rate of 100 cm / h. The column was washed with 5 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl2. The column was then washed with 10 mM potassium phosphate, pH 7, 100 mM NaCl, 0.1 mM CaCl2. The protein was eluted with 70 mM potassium phosphate, pH 7.0 and passed through a sterile 0.22 µm filter into a sterile bag. The eluted sample was tested for biocharge, protein concentration and enzyme activity.
[000552] [000552] The HAP-purified protein was then passed through a virus removal filter. The sterile Viosart filter (Sartorius) was first prepared by washing with 2 L of 70 mM potassium phosphate, pH 7.0. prior to use, the filtered buffer was sampled for pH and conductivity. The protein purified by PAH was pumped through a peristaltic pump through the 20 nM virus removal filter. The protein filtered in 70 mM of potassium phosphate, pH 7.0 was passed through a final 0.22 µm filter into a sterile bag. The filtered sample was tested for protein concentration, enzyme activity, oligosaccharide tillering, monosaccharide and sialic acid. The sample was also tested for process-related impurities.
[000553] [000553] The protein in the filtrate was then concentrated to 10 mg / mL using a 10 kDa (MWCO) Sartocon Slice tangential flow filtration system (TFF) (Sartorius). The filter was first prepared by washing with 10 mM histidine, 130 mM NaCl, pH 6.0 and the permeate was sampled for pH and conductivity. After concentration, the concentrated protein was sampled and tested for protein concentration and enzyme activity. A 6 × buffer exchange was performed on the protein concentrated in the final buffer: 10 mM histidine, 130 mM NaCl, pH 6.0. After buffer exchange, the concentrated protein was passed through a 0.22 µm filter into a 20L sterile storage bag. The protein was sampled and tested for protein concentration, enzyme activity, free sulfhydryl groups , oligosaccharide modeling and osmolality. The batch number WRS2 was used as a standard in the tests described below, the results showed that the test description in relation to appearance was clear and colorless; the pH was 7.4; the endotoxin level was <0.01 EU / ml; osmolality was 308 mOsm / Kg; the density was 1.005 g / ml; the rHuPH20 content was
[000554] [000554] The sterile filtered bulk protein was then ascetically dispensed in 20 mL into 30 mL sterile Teflon bottles (Nalgene). The vials were then quickly frozen and stored at -20 ± 5 ° C. EXAMPLE 2 GENERATION OF PH20 MUTANT LIBRARY A. Cloning and mutagenesis
[000555] [000555] In this example, a human hyaluronidase PH20 library was created by cloning DNA encoding human PH20 into a plasmid followed by transfection and protein expression.
[000556] [000556] The library was created by mutagenesis of a PH20 template which is an optimized version of the PH20 codon with a leading IG Kappa sequence. Specifically, to generate the library of variants, the expression vector HZ24-PH20 (OHO) -IRES-SEAP (exposed in SEQ ID NO: 4) was used as a template, which contains the nucleotide sequence encoding PH20 exposed in SEQ ID NO : 1, which encodes a precursor PH20 exposed in SEQ ID NO: 2 or a mature PH20 exposed in SEQ ID NO: 3 that have no residues 1-22 corresponding to the sequence of IgK signals. The vector structure was derived from the original HZ24 vector containing the DHFR selection marker (see example 1 and SEQ ID NO: 5) with the addition of an IgK leader sequence and codon optimization. The expression vector was also modified to contain the gene for secreted alkaline phosphatase (SEAP). Consequently, in addition to the sequence coding PH, the HZ24-PH20 (OHO) -IRES-SEAP expression vector also contains an internal ribosome entry site (EMCV IRES) that is linked to the coding sequence for the alkaline phosphatase gene. secreted (SEAP), and a unique CMV promoter that triggers the expression of PH20 and SEAP in construction.
[000557] [000557] The first library was made to generate encoded variant proteins in which each of residues 23-469 of SEQ ID NO: 2 (corresponding to residues 1-447 of SEQ ID NO: 3 or residues 36-482 of SEQ ID NO: 6) was changed to one of approximately 15 amino acid residues, such that each element contained a single amino change. The resulting library contained 6753 variant elements, each containing a single amino acid mutation compared to residues 23-469 of SEQ ID NO: 2 (corresponding to residues 1-447 of SEQ ID NO: 3 or residues 36-482 of SEQ ID NO: : 6). Glycerol stocks from the resulting library were prepared and stored at -80 ° C. The amino acid substitutions (mut) in each element are listed in table 8 below, and correspond to amino acid substitutions with reference to the PH20 amino acid sequence exposed in SEQ ID NO: 3 (and SEQ ID NOS: 7 or 32-66, which are the mature sequence of PH20 or other C-terminally truncated fragments thereof). The corresponding shifted codons (cod) for each PH20 variant in the library are also listed in Table 8, and correspond to changes in nucleotide residue in the corresponding coding nucleotide for PH20 exposed as 1058-2464 of SEQ ID NO: 4. Each element was expressed and classified in relation to hyaluronidase activity as described below.
[000558] [000558] For expression of each mutant, plasmid DNA HZ24-PH20-IRES-SEAP containing cDNA encoding one of the PH20 variant or encoding wild-type PH20 was transferred to monolayer CHO-S cells (Invitrogen, Cat. No. 11619 -012) using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027) according to the protocol suggested by the manufacturer. CHO-S cells were seeded the night before transfection and cultured in DMEM with 10% FBS to be 80% confluent the next day. Next, the CHO-S cell medium was replaced with Optimem. A mixture of plasmid DNA and lipofectamine was made (0.2 µg DNA and 0.5 µL Lipofetamine). The Lipofectamine / DNA mixture was added to CHO-S cells and incubated overnight. The next day, the cells were supplemented with serum-free CD-CHO medium (Invitrogen, Cat. No. 10743-029). The supernatant from the transfected cells was collected at various time points after transfection, and generally 96 hours after transfection. The supernatant, containing the PH20 variant or PH20 wild type protein having an amino acid sequence set out in SEQ ID NO: 3, was stored at -20 ° C. The activities of the supernatants were classified as described in the following examples. EXAMPLE 3 CLASSIFICATION OF LIBRARY WITH A TEST OF HYALURONIDASE ACTIVITY TO IDENTIFY ACTIVITY CHANGES
[000559] [000559] In this example, supernatants of expressed PH20 variants generated in Example 2 were classified using a hyaluronidase activity assay to assess the activity of each mutant. In addition, the activity of secreted alkaline phosphatase (SEAP) was also measured to allow normalization of the PH20 activity of the mutants expressed for the wild type PH20. Active and inactive mutants have been identified.
[000560] [000560] An HA 1.2-MDa (Lifecore) has been biotinylated for use as a substrate in the hyaluronidase activity assay. First, 1.2 grams (g) of 1.2 MDa HA were dissolved at 4 ° C in 600 mL ddH20 for one week at a concentration of 2 mg / mL with stirring. Next, 645.71 mg of biotin hydrazide was dissolved in 100 ml DMSO at a concentration of 25 mM (6,458 mg / ml,
[000561] [000561] In four (4) sterile 1000-mL capped bottles, the following components were added at room temperature (RT) and in the following order with stirring: 1) 200 mL of 2 mg / mL of HA solution; 2) 80 mL of 0.5M MES, pH 5.0 with gentle mixing; and 3) 91.6 ml of ddH20 with gentle mixing. Then, 24 mL of 25 mM Biotin-hydrazide and 4 mL of 100X Sulfo-NHS solution were added sequentially immediately followed by the addition of 500 µL EDC. After adding each component, the solution was mixed by inverting three times and stirring. After the addition of the last component, the solution was mixed by stirring overnight at 4 ° C. then, guanidine hydrochloride was added to a final concentration of 4 M by adding 38.2 g per 100 ml and allowed to dissolve completely before adjusting the solution volume to 600 ml with ddH2O.
[000562] [000562] For dialysis, 200 mL of each batch of conjugated HA guanidine hydrochloride solution was transferred to dialysis membranes. During the course of three days, the solution was dialyzed against ddH2O with a change in ddH2O at least six times. The resulting volume of approximately 840 ml was adjusted to a final volume of 1000 ml with ddH2O. the final concentration of biotinylated hyaluronan (bHA) was 0.4 mg / mL.
[000563] [000563] The enzyme assay was a modification of the method described by et al. (1997) (A Microtiter-Based Assay for Hyaluronidase Activity Not Requiring Specialized Reagents. Analytical Biochemistry (1997) 251: 263-269) that provides a measurement of PH20 hyaluronidase activity.
[000564] [000564] First, the biotinylated HA substrate (bHA) was attached to microtiter plates to generate assay plates. Soon, 100 µl of 1 mg / mL b-HA in 0.5 M carbonate buffer (pH 9.6) was dispensed into each well of a high binding microplate (extra high binding Immunolon 4 HBX; Thermo Scientific). The plate was covered with a plate sealer and stored at 2-8 ° C for 24-48 hours.
[000565] [000565] Next, the assay plate was washed with 1 X phosphate buffered saline wash buffer (PBS) containing 0.05% (v / v) Tween 20 (PBST). PBST was generated from 1X PBS (generated from Catalog No. P5368, Sigma (10 mM phosphate buffer, 2.7 mM potassium chloride, 137 mM sodium chloride, pH 7.4) by placing the contents of a PBS package in a graduated cylinder 1 L with 800 mL of deionized water, dissolved by stirring or mixing and adding enough water to 1 L) by adding 500 µl Tween 20 (Catalog No. 6505; EMD Bioscience) to 900 mL of 1 X PBS and adding quantity enough water at 1 L. The wash was done using the BioTek ELx405 Select CW (BioTek) plate washer by washing five (5) times with 300 µl of PBST wash buffer per well for each wash. At the end of each wash, the plate was tapped on a paper towel to remove excess liquid from each well. Before incubation with samples, 200 µl of blocking buffer (1.0% w / v bovine serum albumin (BSA) in PBS) was added to each well and the assay plate was incubated at 37 ° C for approximately 1 hour before. The blocking buffer was generated by adding 2.5 g of BSA (Catalog No. 001-000-162; Jackson Immuno Research) to 200 ml 1 X PBS, stirring, adding a sufficient amount of 1 X PBS to 250 ml and filtration through a 0.2 µM PES filter unit.
[000566] [000566] The PH20 supernatants of the transfected wild-type or variant generated as described in example 1 were diluted 1:25 in duplicate assay diluent (pH 7.4 HEPES buffer; 10 mM HEPES, 50 mM NaCl, 1 mM CaCl2, 1 mg / mL BSA, pH 7.4, 0.05% Tween-20) on high coated 4XHB uncoated microplates. For the standard curve, 1: 3 serial dilutions of rHuPH20 (generated as described in example 1 with a specific activity of 145 U / mL) were made in duplicate assay diluent buffer starting at 3 U / mL for standards as follows: 3 U / ml, 1 U / ml, 1/3 U / ml, 1/9 U / ml, 1/27 U / ml, 1/81 U / ml, and 1/243 U / ml. One hundred microliters (100 µl) of each standard and sample were transferred to the assay plates and incubated for approximately 1.5 hours at 37 ° C.
[000567] [000567] After incubation, the plate was washed with PBST using the BioTek ELx405 Select CW plate washer by washing five (5) times with 300 µl of PBST washing buffer per well for each wash. At the end of each wash, the plate was tapped on a paper towel to remove excess liquid from each well. Then, 100 µl of diluted 1: 5000 streptavidin-HRP (SA-HRP) was added to each well of the plate and incubated at room temperature for approximately 1 hour. For dilution, a stock of 1 mg / mL streptavidin-HRP conjugate (Catalog No. 21126; Thermo Scientific) was diluted 1: 5000 in dilution buffer (1 mg / mL BSA, 0.025% Tween 20, 137 mM NaCl , 20 mM Tris pH 7.5). after incubation, the plate was washed with PBST using the BioTek ELx405 Select CW plate washer by washing five (5) times with 300 µl PBST of wash buffer per well for each wash. At the end of each wash, the plate was tapped on a paper towel to remove excess liquid from each well. Next, 100 µl of TMB solution (Catalog No. 52-00-03, KPL; room temperature and protected from light) was added to each well for approximately five (5) minutes at room temperature or until color development great was provided. To stop the reaction, 100 µl of 1.0 N sulfuric acid or TMB Stop solution (Catalog No. 50-85-06) was added to each well and the plates tapped to mix. Optical density was measured at 450 nm within 30 minutes of adding the stop solution. Since more PH20 in a standard or sample would lead to less bHA available to bind SA-HRP, the optical density value (450 nm) was inversely proportional to the concentration of hyaluronidase activity in each specimen.
[000568] [000568] The activity of secreted alkaline phosphatase (SEAP) in the cell culture supernatant was also measured using a placental alkaline phosphatase coloriostatic assay using pNPP as a phosphatase substrate (Anaspec SensoLyte pNPP SEAP kit; Catalog No. 72144, Anaspec) according to the manufacturer's instructions. The absorbance signal was measured at an optical density (OD) of 405 nm.
[000569] [000569] The criteria for the high yield classification (HTP) were that the transfected supernatant resulted in a SEAP signal of ≥ 0.1 and the signal for the wild-type control rHuPH20 produced a signal of ≥ 1 U / mL. Also, the criteria for each selection were that the standard curves had a signal-to-noise ratio (Y / N) for the standard of 0 U / mL versus the standard of 3 u / mL at OD405 of ≥ 5, had less than three (3) patterns with a coefficient of variation (CV) ≥ 10%, and at least four (4) of the patterns were in the linear range. EXAMPLE 4 PH20 VARIABLES SELECTED WITH ACTIVITY OF ALTERED HYALURONIDASE
[000570] [000570] Each variant generated was classified in relation to hyaluronidase activity as described in example 3. The expression SEAP was used to normalize the PH20 activity of each variant to the wild type PH20. Mutants were identified that exhibited altered hyaluronidase activity compared to the wild type.
[000571] [000571] Active mutants were selected so that at least one duplicate sample showed wild-type activity greater than 40% when normalized with SEAP activity. The active mutants identified are shown in table 9. The table shows the amino acid substitution compared to the exposed PH20 amino acid sequence SEQ ID NO: 3. The amino acid sequence of exemplary mutants is also exposed by reference to a SEQ ID NO. The table also shows the average hyaluronidase activity of tested duplicates normalized by SEAP values compared to the mean of wild-type PH20 activities on each plate, which were also normalized by their own SEAP values. For example, a value of 0.40 indicates that the variant has 40% of the hyaluronidase activity of the wild-type PH20, a value of 1 indicates that the variant has similar wild-type hyaluronidase activity, and a value of 3.00 indicates that the variant has 300% of PH20 hyaluronidase activity in the wild type or 3-fold increased activity compared to the wild type.
[000572] [000572] The results in Table 9 show that more than 600 mutants tested show activity that is increased compared to the wild type. For example, approximately 536 mutants exhibit 120% or more than 120% of wild-type hyaluronidase activity and approximately 75 of the mutants exhibit 300% or more than 300% of wild-type PH20 hyaluronidase activity. In particular, the results in table 9 show that the hyaluronidase activity compared to the wild type of mutant S69A is approximately 22 times; S69R mutant is approximately 14 times; I70A mutant is approximately 27 times; I70K mutant is approximately 14 times; I70R mutant is approximately 14 times; and I271L mutant is approximately 10 times. TABLE 9: ACTIVE MUTANTS SEQ AvgNorm mutant SEQ AvgNorm mutant SEQ AvgNorm mutant ID Act. ID Act. ID Act.
[000573] [000573] The other mutants that showed less than 20% wild-type PH20 hyaluronidase activity, at least in one of the duplicates, were re-classified to confirm that the dead mutants are inactive. To confirm the inactive mutants, the hyaluronidase activity assay described in example 3 was modified to incorporate a sample-substrate incubation step at 37 ° C overnight before measuring enzyme activity. The modified assay is designed to detect PH20 activities below 0.2 U / mL.
[000574] [000574] The preparation for the bHA coated plates and blocking of the plates prior to the addition of the transfected variant supernatants or wild-type PH20 was the same as described in example 3. The assay was modified as follows. First, transfected supernatants or wild-type PH20 containing no mutation generated as described in example 2 were diluted 1:25 in duplicate in assay diluent. For the standard curve, 1: 3 serial dilutions of rHuPH20 (generated as described in example 1) were made in duplicate assay diluent starting from 0.1 U / mL down to 0.00014 U / mL. An empty cavity was also included. Next, 100 µl of the diluted or standard samples were added to pre-designated wells of the blocked and blocked bHA plate and allowed to incubate at 37 ° C overnight. After incubation, the plates were washed and ligated with bHA detected as described above in example 3. The optical density was measured at 450 nm within 30 minutes of adding the stop solution.
[000575] [000575] The identified reconfirmed inactive mutants are shown in Table 10. The table shows the amino acid substitution compared to the PH20 amino acid sequence exposed in SEQ ID NO: 3. TABLE 10: inactive mutants N002H R060V R121W C189P P236I V287N L336W G377V N002K R060Y R121Y C189R P236L V287P L336Y G378D N002W L061A N122A C189S P236N V287Q A337C G378E N002Y L061E N122C C189T P236Q V287R A337F G378F F003A L061F N122E C189V P236T V287S A337G G378I F003G L061G N122F C189W P236Y Y288D A337I G378L F003K L061H N122I C189Y A238F Y288E A337K G378M F003P L061N N122K Y190C A238G Y288F A337L G378Q F003T L061P N122Q Y190E A238L Y288G A337M G378T F003V L061Q N122R Y190F A238P Y288H A337R G378W R004D L061R N122S Y190G A238V Y288I A337T G378Y R004E L061T N122T Y190H A238W Y288K A337W K379A R004F L061W N122V Y190K A238Y Y288P A338C K379C R004G L061Y W123A Y190L A239C Y288R A338D K379E R004L G062A W123C Y190N A239F Y288T A338E K379F R004P G062C W123D Y190Q A239G T289A A338F K379I R004W G062D W123E Y190R A239H T289C A338G K379L R004Y G062F W123H Y190S A239I T289E A338H K379M A005D G062I W123L Y190T A239L T289G A338I K379W A005G G062K W123M Y190V A239P T289H A338K F380C A005I G062L W123P Y190W A239R T289L A338L F380D A005L G062M W123Q N191A A239S T289P A338P F380E A005M G062P W123R N191E A239T T289Q A338R F380G A005N G062Q23819 G1962 A235
[000576] [000576] Supernatants of PH20 activity variants exposed in table 9, as identified in example 4, were tested for stability under thermophilic and / or phenophilic conditions. The assay for measuring hyaluronidase activity under phenophilic and temperature conditions using biotinylated HA (bHA) as a substrate for measuring hyaluronidase activity was modified from the original assay described in example 3 in which it incorporated a 4-hour incubation of 4 hours of samples with or without m-cresol before measuring enzyme activity. The assay was used to identify PH20 mutants with thermophilic properties (greater activity at 37ºC than at 4ºC) and / or phenophilic properties (greater activity in the presence of m-cresol than wild-type PH20).
[000577] [000577] Before incubating samples with bHA, samples of PH20 variant were diluted in designated wells of an uncoated 4xHB plate for pre-incubation at 37ºC for 4 hours under the following conditions: 1) pre-incubation at 37ºC with 0.4 % m-cresol; and 2) pre-incubation at 37ºC without 0.4% m-cresol. For pre-incubation at 37ºC with 0.4% m-cresol, an intermediate stock of 1% m-cresol was prepared from 50% (v / v) of m-cresol stock solution. Briefly, in a 2 mL Wheaton glass vial a stock of 50% m-cresol (Fluka, Catalog No. 65996; Spectrum, catalog No. C2773) was made in methanol based on density (D = 1,034 g / L ). the bottle was sealed and stored at -20 ° C with light protection in small aliquots. Then, the 1% intermediate material was generated by dilution in HEPES assay buffer (10 mM HEPES, 50 mM NaCl, 1 mM CaCl2, 1 mg / mL BSA, pH 7.4, 0.05% Tween-20) daily immediately before use in a vortex hood.
[000578] [000578] Next, duplicates of transfected variant supernatant samples exposed in table 9, generated as described above in example 2, were individually separately subjected to a 1: 2.5 dilution of 1% m-cresol in transfected supernatant / buffer HEPES test to obtain 0.4% final concentration of m-cresol. For pre-incubation at 37ºC m 0.4% m-cresol, transfected variant supernatant samples were subjected to a 1: 2.5 dilution in transfected supernatant / HEPES assay buffer. In addition, for each condition, an internal death control was also tested by boosting 3 U / mL of rHuPH20 in HEPES pH 7.4 buffer (
[000579] [000579] The preparation of the bHA coated plates and blocking of the plates prior to the addition of the transfected supernatants or wild-type PH20 was the same as described in example 3. The assay was further modified as follows. First, the samples were diluted 1:10 in duplicate in HEPES assay buffer on 4XHB plates. For each variant, the samples that were tested were 1) supernatant transfected variant not pre-incubated (without incubation; 4ºC); 2) pre-incubated transfected variant supernatants, pre-incubated at 37ºC for 4 hours with 0.4% m-cresol (Cresol); or 3) pre-incubated transfected variant supernatant, pre-incubated at 37ºC for 4 hours without 0.4% m-cresol (without cresol; 37 ° C). in addition, the reinforced samples were also tested. A standard curve using rHuPH20 was made as described in example 3 without m-cresol. One hundred microliters (100 µl) of each standard and sample were transferred to pre-designated wells of the blocked and bHA coated plate and incubated for approximately 1.5 hours at 37ºC. Thus, each sample of each variant was tested in quadruplicate due to the pre-incubation of duplicate samples of each variant supernatant transfected in the pre-incubation step and the additional duplicate of each sample in the bHA assay.
[000580] [000580] After incubation, the plates were washed and ligated with bHA detected as described above in example 3. The optical density was measured at 450 nm within 30 minutes of adding the stop solution.
[000581] [000581] U / mL activity was calculated from the standard curve and compared. The results were represented as the percentage (% 0 of activity remaining under each of the following parameters: activity ratio at 1) 37 ° C pre-incubation without m-cresol / 4 ° C; 2) 37 ° C after pre-incubation with m-cresol / 4 ° C; and 3) 37 ° C after pre-incubation with m-cresol / after pre-incubation at 37 ° C without m-cresol. Initial phenolic hits for reconfirmation were identified as those that in a duplicate assay had a percentage of activity remaining under condition 3) of ≥ β0% of the original activity at 37 ° C.
[000582] [000582] Initial hits were selected again using a new 6-well plate selection assay. For the new selection, plasmid DNA corresponding to the potential Hit was transformed into E. coli bacteria and plasmid DNA prepared and purified using MaxiPrep according to the manufacturer's instructions. The DNA sequence has been confirmed.
[000583] [000583] Plasmid DNA was transfected into monolayer CHO-S cells (Invitrogen, Cat. No. 11619-012) cultured in 6-well plates at a density of approximately 50-80% confluence using Lipofectamine 2000 (Invitrogen, Cat. No. 11668-027) according to the protocol suggested by the manufacturer. Transfections were performed in duplicate. The cells were incubated at 37ºC in a Co2 incubator for 96 hours post-transfection before collecting the supernatant for the assay. As controls, cells were also transfected with the expression vector HZ24-PH20 (OHO) -IRES-SEAP (SEQ ID NO: 4) which contains a codon-optimized wild type PH20 sequence (OHO). Simulated cells were also included as controls.
[000584] [000584] Ninety-six (96) hours after transfection, the supernatant was collected from each sample,
[000585] [000585] The results were evaluated as described above. Absolute hyaluronidase activity (U / mL) was generated from the standard curve. In addition, the percentage of activity was determined as an activity ratio at 37 ° C / 4 ° C, 37 ° C plus m-cresol / 4 ° C, and 37 ° C plus m-cresol / 37 ° C. The results are shown in tables 11 and 12 below. TABLE 11: Absolute hyaluronidase activity 37 ° C with m-cresol Without incubation 37 ° C without cresol Mutant (37 ° C plus m- (4 ° C) (37 ° C) cresol) L001A 2,993 2,511 3,529 3,214 0.287 0.295 L001E 2,669 2,539 2,862 3,179 0.376 0.341 L001G 0.348 0.583 0.596 0.676 0.055 0.031 L001Q 5,135 6,443 6,133 5,719 0.621 0.636 L001R 5,603 4,390 6,576 7,042 0.458 0.396 P006A 2.965 3.208 4.088 3.495 0.404 0.435 V0.08 1.47 0.46 0.47 0.47 0.47 0.47 0.820 0.688 0.123 0.114 P010H 0.473 0.485 0.624 0.548 0.000 0.000 N011S 0.862 0.962 1,313 1,263 0.094 0.064 V012E 11,019 5,519 5,312 5.528 0.753 0.934 V012I 2.804 3.844 3.610 6.566 0.106 0.090 V012K 1.24 1.914 2.63 0.901 0.017 0.017 A020S 1,494 2,205 2,822 2,620 0.413 0.397 S022T 3,035 3,788 3,375 3,273 0.684 0.748 L026M 1,482 1,226 2,027 1,704 0.224 0.178 K028R 0.944 0.845 1,043 0.925 0.112 0.095
[000586] [000586] For F204P mutant, the above results from supernatant tested from transient transfection of CHO-S cells incubated in the presence of m-cresol in a bHA enzyme activity assay show that the mutant protein F204P was highly resistant to the treatment of 0 , 4% m-cresol. The results showed that the activity that remained after 4 hours of incubation yields 0.4% of m-cresol at 37ºC was approximately equal to the activity observed when the enzyme was incubated at 4ºC or at 37ºC in the absence of m-cresol. The positive control (WT PH20 - OHO) showed a reduction in activity of 75% and 83% on the day of the assay (as tested from two different OHO transfections). This demonstrated that the F204P phenophile was able to retain 60% to 90% or more of its activity above the residual activity of the wild-type PH20 control enzyme.
[000587] [000587] To confirm the stability of F204P after treatment with m-cresol or exposure to increased temperature, a second transfection of F204P was performed in duplicate using CHO-S cells, and clarified supernatant was tested again for its stability at 4ºC, 37ºC for 4 hours with 0.4% m-cresol and at 37ºC for 4 hours without 0.4% m-cresol. The results confirmed that the mutant enzyme F204P retained a high amount of hyaluronidase activity after a 4-hour incubation in m-cresol at 37ºC. the results were similar to the results seen in the first selection of the mutant, with F204P retaining 57% more than 90% of its activity above the residual PH20 control enzyme residual activity after the 4-hour incubation.
[000588] [000588] A summary of the F204P enzyme activity compared to the wild type control is set out in Table 13.
[000589] [000589] cDNA containing HZ24-PH20-IRES-SEAP plasmid DNA encoding one of the PH20 variant was transfected into monolayer CHO-S cells as generically described in example 2. CHO-S cells were cultured in shake flasks using CD-CHO medium supplemented with GlutaMAX (8 mM). On the day of transfection, flasks were prepared of approximately 300 mL in volume containing CHO-S cells at a density of approximately 1.0 x 10 6 cells / mL. each 300 ml vial was transfected using 375 µg of plasmid DNA encoding the F204P mutant combined with 375 µL of Freestyle MAX transfection reagent. The transfected plasmid DNA had a nucleotide sequence exposed in SEQ ID NO: 4 containing a codon change from TTC to CCT at nucleotide positions 1733-1735, thereby encoding the F204P mutant. the transfected cells were then allowed to remain in culture for 96 hours, after which the cells and medium were harvested and pooled. The cells were pelleted by centrifugation (4000 x g, β0 '), and the supernatant retained for purification of the F204P protein (approximately 4.5 liters).
[000590] [000590] The crude supernatant was concentrated 20x using a 30 kDa tangential flow filter system (TFF) (Millipore Pellicon XL, Bimax 30, 200 mL empty volume; 50 cm2 surface filter area) until the volume was approximately 450 mL . The permeate was saved for testing to detect flow through the F204P protein. An exchange of free flow buffer for the retentate was then performed using 4 liters of buffer (10 mM NaPO4; 25 mM NaCl, pH 7.2). The volume of the retentate was reduced again to approximately 200 ml, and then the remaining permeate in the system was purged (empty volume ~ 200 ml) washed using approximately 50 ml of buffer to provide a final concentrated product of approximately 450 ml.
[000591] [000591] An affinity column was prepared by coupling Rabbit anti-rHuPH20 IgG purified by CNBr activated Sepharose 4 Fast Flow antigen affinity (GEHealth catalog No. 17-0981-01). Briefly, 0.7 g of pre-activated Sepharose 4 powder was suspended in 1 mM HCl in a 10 mL glass column for 30 minutes to allow the powder to swell. The solution was drained from the column and washed with 15 volumes of gel (approximately 30 mL) of 1 mM cold gravity HCl. The column was washed with 5 volumes of coupling buffer gel (0.1M NaHCO3, 0.5M NaCl at pH 8.3). then, 5 mg of rabbit anti-rHuPH20 Ig> 1.0 mg / ml in coupling buffer was added to the column in a protein / gel ratio of 2-3 mg / ml of gel. The column was rotated head to head at 4 ° C overnight. The direct flow was collected to determine coupling efficiency. The gel was washed with 2 volumes of coupling buffer gel, and then washed and resuspended in 1 M ethanol amine pH 9.5 for 2 hours at room temperature to block unused activated sites. The gel was sided 6 times with 5 volumes of gel per wash alternating coupling buffer and 0.1 NaAc, 0.5M NaCl, pH 4.5. The gel was then washed with 10 volumes of TBS gel (20 mM Tris-HCl, 0.15 M NaCl, pH
[000592] [000592] The concentrated supernatant product was subsequently loaded onto an anti-rHuPH20 affinity column at an approximate rate of 5 mL / min. Elution was carried out according to standard procedure using a GE ™ AKTA FPLC purification system (GE Healthcare, Product No. 18-1900-26), whereby the protein was eluted by washing with low pH glycine (0.1 M glycine -HCl, pH 2.5) in 1 mL fractions. Each fraction was immediately neutralized by adding 100 µL of 1M Tris, pH 7.5.
[000593] [000593] The eluted protein was assayed by decomposing protein strips on a 4-20% SDS-PAGE gradient Tris-glycine gel. SeeBlue®Plus2 pre-colored MW standards (Life Technologies; Catalog No. LC5925) were used as molecular weight standards, and 50 ng rHuPH20 (as described in example 1) was used as a positive control. The polyacrylamide gel was stained with Instant Blue to sample total protein from each fraction. To confirm that the bands on the gel are PH20, the gel was transferred to a PVDF membrane (Invitrogen), which was subjected to Western Blot using primary rabbit anti-PH20 antibody generated by immunizing rabbits with rHuPH20 and a secondary anti-rabbit HRP antibody - goat (Calbiochem, Cat. No. DC03L).
[000594] [000594] Next, the direct flow from the initial charge of the affinity column was reloaded on the column twice due to the low capacity of the affinity column. All fractions containing the protein were then combined resulting in a total volume that was approximately 13 ml. This product was then dialyzed overnight versus four liters of buffer (10 mM NaPO4, 140 mM NaCl, pH 7.2) using a Slide-A-Lyzer G2 dialysis cassette (20,000 MWCO) with a capacity of 15 mL. The buffer was then changed and the product dialyzed against a second new amount of four liters of the same buffer. The F204P protein was then concentrated using an Amicon Ultra Centrifuge column (Millipore; 10,000 MWCO) to a final volume of approximately 450 µL (10 minutes at 4000 xg).
[000595] [000595] The purified protein was characterized in relation to its protein concentration, activity and purity.
[000596] [000596] To determine the protein concentration of the purified protein, a quantification ELISA was performed as described in example 7. Also, the hyaluronidase activity was determined as described in example 3. The protein concentration after centrifugation was estimated to be approximately 400 µg / mL. The purified protein was also decomposed on a 4-20% SDS-PAGE gradient Tris-glycine gel which was then stained with Instant Blue. The staining results demonstrated that the protein was essentially a single molecular weight protein of approximately 63 kDa, similar to the rHuPH20 control. No appreciable degradative product was detected by this method. Approximate yields of the protein at various time points and activity during purification are described in Table 14. TABLE 14: characterization of purification steps ELISA Assay activity Quant Conc. Active Activity Active Volume Phase From Protein and Purification and Total Total Protein (mL) (U / mL) to (µg) ca (U) (µg / mL) (U / µg) Supernatural 4500 2.66 11.700 0.046 207 56.5 te Conc. after TFF & 450 42 18,900 0.4 178 105.9 buffer exchange Grouped fractions 5-7 after AC, 0.45 11.741 5283 396 180 35.3 Dialysis & Conc. - A280
[000597] [000597] The purity of the purified protein was determined by reverse phase HPLC (RP-HPLC). The elution time from the reverse phase column was essentially identical to that observed with recombinant human hyaluronidase (HUB), and provides a basis for roughly estimating the purity of the sample by approximately 80-90%. EXAMPLE 7 QUANTIFICATION USING ELISA
[000598] [000598] Quantification of PH20 or variants was performed using ELISA that captures the protein using a monoclonal anti-rHuPH20 capture antibody. Specifically, the day before ELISA, 96-well 4HBX plates were coated with capture antibody (polyclonal rabbit anti-PH20 antibody purified with Protein G generated by immunizing rabbits with rHuPH20; 1 mg / mL of material) at 1 µg / mL in 100 mM phosphate (pH 7.2) in a total volume of 100 µL per well. The plates were stored at 4 ° C overnight. The next day, the plates were washed 5x with 1xPBS in 300 µL / well with a plate washer. After each wash, the plates were dried on a paper towel. Then, the plates were blocked with 200 µL PBS containing Tween 20 (1xPBST) per well at room temperature for 1 hour.
[000599] [000599] Standards and samples have been added to the plate. For generation of the standard, a material of 1 mg / mL of rHuPH20 (example 1) was recently diluted in 50 µg / mL in HEPES pH 7.4 assay buffer as an intermediate material. Then, for the standards, the 50 µg / mL material was diluted in duplicates in 360 µL of 0.5xPBST at 300 ng / mL for the first standard (first line). For the other standard lines, 240 µL 0.5xPBST were added to each well, and 1: 3 serial dilutions were made. For the transfected supernatant samples, 360 µL per well was added in duplicate on the first line, and each was also serially diluted as described above in
[000600] [000600] An anti-PH20 antibody conjugated to HRP was prepared for detection using an HRP conjugation kit (Pierce, Thermo-Fisher; catalog No. 31489). 1 mg of a rabbit polyclonal antibody purified with protein G generated by immunizing rabbits with rHuPH20 was diluted in 1 ml PBS and 1 ml 2 x carbonate kit buffer. Next, 100 µL of peroxidase was added to 1 mL of the above antibody solution and incubated at room temperature for 1 hour. Then, 10 µL of NaBH4 material were added in an exhaust fan, and the sample incubated at room temperature for 20 minutes. To cool the reaction, 20 µL of ethanolamine was added and incubated at room temperature for 15 minutes. To this, 1/25 volume 5% human serum albumin (0.1 mL syringe) was added to provide a 2 mg / mL albumin stock reaction. the pH was adjusted to approximately 7.9 by adding 250 µL of 1 M Tris pH
[000601] [000601] Antibodies were detected using the anti-PH20 antibody conjugated to HRP which was diluted 1000x in 0.5x PBST. 100 µL of the diluted antibody was added to all wells of the plate and the plate incubated for an additional 2 hours at room temperature. After incubation, the plates were washed 5x with 1xPBST at 300 µL / well using a plate washer.
[000602] [000602] The enzymatic activity of PH20 in samples such as cell cultures, purification fractions and purified solutions was determined using a turbidimetric assay, which is based on the formation of an insoluble precipitate when hyaluronic acid binds with cetylpyridinium chloride (CPC). Activity is measured by incubating PH20 with hyaluronan for a defined period of time (30 minutes) and then precipitating undigested hyaluronan with the addition of CDC. The turbidity of the resulting sample is measured at 640 nm. The decrease in turbidity resulting from enzyme activity in the hyaluronan substrate is a measure of the enzymatic activity of PH20. The method is done using a calibration curve generated with dilutions of a PH20 test work reference standard (rHuPH20 standard generated as described in example 1) and measurements of sample activity are made against that calibration curve.
[000603] [000603] Sample dilutions and standards were prepared in the Enzyme Diluent Solution (70 mM NaCl, 0.1% human serum albumin [HSA], 0.67 g / L gelatin hydrolyzate in 25 mM PIPES buffer, pH 5.5). the samples were diluted to an appropriate concentration. Hyaluronic acid (HA, average MW 20 - 50 kDa) from Lifecore Biomedical (Chaska, MN) was also prepared in 1 mg / mL in a substrate solution containing 25 mM PIPES, 70 mM NaCl at pH 5.5. equal amounts of the two solutions above were mixed to prepare a 1 ml reaction mixture and incubated at 37 ° C for 30 min. The reaction was stopped by adding 4 ml of cetylpyridinium chloride solution (CPC, 5.0 mg / ml). After a brief vortex formation, the turbidity of the sample mixture was read at 640 nm and the activity was determined by fitting against a standard curve. Specific activity (Units / mg) was calculated by dividing the enzyme activity (U / mL) by the protein concentration (mg / mL). EXAMPLE 9 VARIANT STABILITY F204P-PH20 IN CONDOM
[000604] [000604] To confirm the selection results, an amount estimated to be approximately 450 U / ml of the purified F204P protein as described in example 6 was formulated in 10 mM sodium phosphate, pH
[000605] [000605] Physicians' results of enzyme activity are shown in Table 15. As can be seen, wild-type control rHuPH20 showed a rapid decrease in activity when incubated at 37 ° C in the presence of phenolic condoms. In contrast, the F204P mutant did not show significant loss in activity throughout the study. The results also show that PH20 activity is retained after incubation for up to 4 weeks at 5 ° C and 30 ° C compared to rHuPH20 wild-type control activity not containing the mutation. These results confirm that F204P tolerates EPB level of condom (0.1% phenol and 0.15% m-cresol) and is stable at 37 ° C for at least 6 days at 5 ° C and 30 ° C for more month.
[000606] [000606] The PH20 F204P variant was tested for stability in a co-formulation containing an insulin analogue (insulin aspart or insulin lispro).
[000607] [000607] In the co-formulations tested, insulin lispro was a commercial product (Insulin Lispro: Eli Lilly Humalog® (insulin Lispro) 100 U / mL, Lot A572364).
[000608] [000608] In the tested co-formulations, the aspart insulin analog was a reprocessed aspart prepared by grouping 12 vials (10 mL each) of a commercial product (Aspart Insulin: Novo Nordisk, NovoRapid® (Aspart insulin), Lot XS60195), which was then concentrated using an Amicon Ultracel-10 K column concentrator until the final concentration was approximately 5 times the original concentration. The insulin analogue was precipitated by adding 1 M sodium acetate, pH 5.3 and mM zinc chloride (ZnCl2, EMD, Cat No. ZX0065-1) to 1/10 of the volume of protein solution. The solution was placed on ice for 30 minutes followed by centrifugation at 5600 rpm for 20 minutes in an Avanti J-E centrifuge with JS-5.3 oscillating bucket rotor (Beckman Coulter). The supernatant was decanted and the pellet was suspended again and washed with a solution of 20 mM sodium acetate, 2 mM zinc chloride, pH 5.5. the suspense solution was again centrifuged as described above. The washing step was repeated a total of 5 times. A final wash was carried out with 20 mM sodium acetate, pH 5.5 to remove all traces of zinc chloride. The resulting protein slurry was dissolved with water containing 20 mM HCl. After total dissolution, 250 mM Tris, pH 10.7 was added to a final Tris concentration of 20 mM. The pH of the resulting solution was adjusted such that the insulin analog was formulated as described below and the protein concentration was adjusted to approximately 15-20 mg / ml. An insulin analogue prepared in this way typically had a yield of approximately 90%, with a residual preservative concentration less than 100 times the starting material.
[000609] [000609] Briefly, three (3) formulations were generated each containing 600 units (U) of wild type PH20-F204P or rHuPH20 (generated as described in example 1) for a total of 6 formulations as set out in Table 16: TABLE 16 : Summary of Modified Insulin Formulations Anti- Tensoat Preservative Metal Buffer API Ox ivo os tonic ID pH age PH20 NaPO4 Tris / NaCl Metion Glice Zn F68 ha m- (U / mL HCl ina l Cresol analogue (mg / mL) rina) F1.Humal
[000610] [000610] Each formulation solution was dispensed in 0.5 ml aliquots in 2 ml type I borosilicate USP glass bottles with a chlorobutyl rubber stopper and an aluminum seal. The flasks were incubated at 5 ° C, 30 ° C and 37 ° C. The samples were taken from the incubator at scheduled time points for measurements of enzyme activity as described in example 8.
[000611] [000611] The results of the enzyme activity doctors for samples incubated at 37 ° C, 30 ° C and 5 ° C are shown in Tables 17-19, respectively. at 37 ° C, the enzymatic activity of samples containing wild-type rHuPH20 (F2, F4 and F6) was almost completely lost in two days of incubation. In contrast, after 6 days of incubation at 37 ° C, formulation F3 and F5, which contains PH20-F204P, lost only approximately 10% and
[000612] [000612] A similar trend towards enzymatic activities from ampoules incubated at 30 ° C was observed between PH20-F204P and rHuPH20. For formulations that contain an EPA condom level, the differences between wild type and F204P were dramatic (Table 17; F1 and F5 vs. F2 and F6). When the condom concentration was reduced to an EPB level (F3 and F4), the F204P still exceeded the performance of wild-type rHuPH20, although there was slightly higher stability of rHuPH20 compared to EPA conditions. At both EPA and EPB preservative levels, PH20-F204P was able to maintain its activity for up to 14 days at 30 ° C when 100 mM NaCl was included in the formulation. Table 17. Enzymatic activity of wild-type and mutant F204P rHuPH20 incubated at 37 ° C PH20 U / mL activity, (% of activity remaining) Activity
[000613] [000613] The PH20 V58R variant was expressed in CHO-S cells as described in example 2 or example 6. The transfected plasmid DNA had a nucleotide sequence exposed in SEQ ID NO: 4 containing a codon change from GTC to CGG at nucleotide positions 1295-1297, thereby encoding the V58R mutant. The V58R mutant was tested for stability in a coformulation containing aspart insulin (aspart insulin analogue prepared as described in example 10) and under EPA or EPB preservative levels. Briefly, four (4) formulations were generated each containing 600 units (U) of wild-type PH20-V58R or rHuPH20 (generated as described in example 1) as shown in Table 20. Formulations F1 and F2 represent the condom levels of EPB while formulations F3 and F4 represent EPA condom levels.
[000614] [000614] Each formulation solution was dispensed in 0.5 mL aliquots in 2 mL USP type 1 borosilicate glass bottles with a chlorobutyl rubber stopper and an aluminum seal. The flasks were incubated at 30 ° C and 37 ° C. The samples were removed from the incubator at programmed time points for measurements of enzyme activity as described in example 8.
[000615] [000615] The results of enzyme activity measurements for samples incubated at 37 ° C and 30 ° C are shown in Table 21 and Table 22. At 37 ° C, the enzymatic activity of samples containing wild-type rHuPH20 (F2 and F4 ) was almost completely lost in two days of incubation. In contrast, after 6 days of incubation at 37 ° C, formulations F1 (EPB) and F3 (EPA), containing V58R-PH20, lost only approximately 25% and 40% of activity, respectively. At 30 ° C, the enzymatic activity of samples containing wild-type rHuPH20 was also dramatically reduced in the presence of EPA or EPB condom levels in one month of incubation, although there was a slightly less dramatic loss in activity in the presence of condom levels. EPB. In contrast, for V58R-PH20, there was no loss of enzyme activity for any formulation tested up to 1 month.
[000616] [000616] The PH20 variant V58R-PH20 was compared with F204P in relation to its stability in a co-formulation containing insulin aspart (insulin analogue aspart prepared as described in example 10) and under preservative levels of EPA or EPB. Briefly, eight (8) formulations were generated as shown in table 23. Formulations F1-F4 represent EPB condom levels while formulations F5-F8 represent EPA condom levels. Formulations F3 and F4 and formulations F7 and F8 were identical and represent the wild type control formulations used for EPB or EPA studies, respectively.
[000617] [000617] Each formulation solution was dispensed in 0.5 mL aliquots in 2 mL type I borosilicate glass vials with a chlorobutyl rubber stopper and an aluminum seal. The flasks were incubated at 30 ° C and 37 ° C. the samples were removed from the incubator at programmed time points for measurements of enzyme activity as described in example 8.
[000618] [000618] The results show that the percentage of hyaluronidase activity in the formulations tested after pre-incubation at 37 ºC was slightly higher for the two PH20 mutants when formulated in EPB and non-EPA preservative levels. Although the percentage of remaining activity was greater than 80% for the two mutants tested after 6 days of incubation in formulations containing EPB condom levels, it was lower in the presence of EPA condom levels. For example, the 6-day remaining activity at EPA condom levels was slightly less than 80% after 6 days for F204P-PH20, while it was only approximately 40% for V58R-PH20. Consequently, the results also show that at 37 ºC, V58R-PH20 is somewhat less stable than F204P-PH20, in particular, in a formulation with EPA preservative levels. After incubation at 30 ºC for at least one week, the F204P-PH20 and V58R-PH20 were stable and showed almost 100% of initial activity in the presence of both EPA and EPB condom levels. In contrast, rHuPH20 showed only approximately 40% of its initial activity after 4 weeks at 30 ºC in the presence of EPB condom levels, while it did not show detectable activity after 4 weeks at 30ºC in the presence of EPA condom levels. EXAMPLE 12 EXPRESSION OF F204P-PH20 USING A VECTOR OF EXPRESSION OF LENTIVIRUS
[000619] [000619] A lentivirus expression vector, pLV-EF1a-PH20 (F204P) -IRES-GFP-Bsd was generated containing a codon-optimized mutant hyaluronidase cDNA encoding F204P-PH20. The sequence of pLV-EF1a- PH20 (F204P) -IRES-GFP-Bsd is set out in SEQ ID NO: 925. The pLV-EF1a-PH20 (F204P) -IRES-GFP-Bsd vector contains an Ampicillin Resistance (AmpR) gene located in nucleotides 8611-9471, an EF1a promoter in residues 1933 to 2327, an IRES in residues 4786-5370, a GFP-Bsd at residues 5394-6527 and nucleotides encoding F204P-PH20 at residues 3369-4781.
[000620] [000620] Lentivirus was produced as described in Bandaranayake et al. ((2011) Nucleic Acids Research, 39: and 143). Briefly, 293T cells (ATCC) were coated in 6 x 106 cells on 10 cm tissue culture plates. After 24 hours, 6 µg of psPAX2 (SEQ ID NO: 926; plasmid Addgene No. 12260), 3 µg of PMD2.G (SEQ ID NO: 927; plasmid Addgene # 12259) and 9 µg of pLV lentiviral vector plasmid -EF1a-PH20 (F204P) -IRES- GFP-Bsd were mixed in 1.5 mL Opti-MEM (Life Technologies). 45 μL of Lipofectamine 2000 (LF2000; Life Technologies) was diluted in 1.5 mL Opti-MEM (Life Technologies). DNA and LF2000 were mixed gently, and incubated at room temperature for 20 minutes to allow DNA and lipids to form complexes. Meanwhile, the overnight culture medium was replaced with
[000621] [000621] CHO-S cells (Invitrogen) were cultured in CHO-S medium (Invitrogen) with agitation at 120 rpm at 37 ºC and 5% CO2 in bleed 125-mL shake flasks (Nalgene). For transduction, CHO-S cells were added to wells of a six-well plate in 2 × 106 cells per well in 2 ml of CHO-S medium containing 4 µg / ml hexadimethrin bromide in a final concentration of 4 µg / mL (Polybrene; SIGMA). Virus was added to each well in a multiplicity of infection (MOI) of 10 and the cells were incubated with shaking (120 rpm) at 37 ºC and 5% CO2 for 6 hours. The cells were then harvested and pelleted by low speed centrifugation (500 x g, 5 min). The transduction medium was removed and replaced with 10 mL of new CHO-S medium (Invitrogen) supplemented with GlutaMax (50 mL / liter) and transferred to a T-25 flask. Three days after infection, blasticidin (Invitrogen) was added to the growth medium at a concentration of 1 µg / mL. Hi medium was changed regularly at 3-4 day intervals, and the cells were transferred to a T75 flask for expansion. Two weeks after the initial infection, the cells were expanded into shaker flasks and maintained in culture using medium containing 1 µg / mL of blasticidine. F204P-PH20 protein secreted in CHO-S medium was collected and purified by affinity chromatography using an anti-rHuPH20 affinity column as described in the example
[000622] [000622] The secondary structure and melting temperature of the PH20 F204P variant were tested and compared with wild-type rHuPH20 (generated as described in example 1) to further assess the stability of the variant. The secondary structure was tested by circular dichroism. A Jasco J-810-150S equipped with PTC-424S was used for the CD spectral measurement and the CD spectra were collected by Spectra Manager (Version 1.5, Jasco). The procedures for instrumental assembly and data collection are described in Table 24. Table 24: Operating conditions for CD spectroscopy Parameters conditions 3 Nitrogen flow rate 25 feet / h Sample temperature 30-75 ºC Sample concentration Approx. 0.1 mg / mL 1 mm cell path length Wavelength 220 nm Data pitch 1 ºC Delay time 60 seconds Temperature slope 1 ºC / min standard sensitivity Response 4 seconds Bandwidth 1 nm
[000623] [000623] Two hundred (200) µL of a 0.1 mg.mL protein sample diluted in Mcllvaine’s buffer (McIlvaine (1921) JBC 49: 183) adjusted to pH 6.5 were prepared. A series of samples of the F204P variant was also generated which varied in pH by adjustment using Mcllvaine's buffer to a pH range of 5.0 to 7.5 as shown in Table 25. In addition, the samples were also generated by adjusting the NaCl concentration to 17.5 mM to 140 mM as shown in Table 26. The samples were filtered using a 0.2 µm syringe filter prior to measurement. Similar samples were generated for rHuPH20.
[000624] [000624] The results show that the secondary structure of F204P is similar to rHuPH20. As a function of temperature, circular dichroism showed that a change in absorption was measured with increasing temperatures. As a function of pH, the distribution of Tm was closely comparable for F204P and rHuPH20 and the highest Tm for each was obtained between pH 5.5 and pH 6.0. The results, however, showed that Tm of the F204P variant was approximately 9 ºC higher in all tested ranges than wild-type rHuPH20. This result indicated that the F204P mutant is more stable against thermal stress conditions. As a function of salt, the results show that F204P and rHuPH20 of the wild type showed an increasing Tm with a higher concentration of salt, showing that both have a proportional slope towards the concentration of salt. Example 14 Evaluation of enzymatic activity in an intradermal trypan blue dispersion assay
[000625] [000625] The dispersion activity of the PH20 F204P variant was evaluated using an in vivo dye dispersion assay. In summary, purified PH20 variant F204P (prepared as described in example 12) and wild-type rHuPH20 (prepared as described in example 1) were both formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5)
[000626] [000626] Forty (40) µL of samples were administered by a single intradermal injection. The dye dispersion area was measured at 2.5, 5, 10, 15 and 20 minutes post-injection and was recorded by photographic imaging by photograph of the injection site with a Nikon D90 digital camera with 60 mm main lens. A laser distance meter (Leica D3) was used to precisely position the camera at a predetermined distance from the trypan Blue dye area in the animal. the dye area was determined using Image-Pro Analyzer 7.0 (MediaCybernetics, Inc). The calculated areas were expressed as mm2.
[000627] [000627] The results are shown in Table 28. The results showed that the dispersion activity of the PH20 F204P variant was substantially identical to the dispersion activity of rHuPH20. The ability to increase the dye dispersion area was dose dependent, with the two proteins having an activity greater than 500 U / mL. The results also showed that the dye dispersion area increased with time post-intradermal injection. The dye dispersion areas of rHuPH20 and F204P-PH20 were significantly larger than the dye dispersion areas for the controls (p <0.05) at all time points when formulated at all concentrations (5, 50 and 500 U / mL) with the exception of rHuPH20 at the lowest concentration (5 U / mL). When compared to each other, rHuPH20 and F204P-PH20 showed similar dispersion effects, although there was a significant difference in dispersion between the two groups at 5 U / mL and 500 U / mL but not at 50 U / mL. In summary, the results show that rHuPH20 and F204P-PH20 provided a statistically significant increase in the dye dispersion area compared to vehicle control. TABLE 28: dispersion of trypan blue group average area (mm2). 2.5 min 5 min 10 min 15 min 20 min (n = 6) 1: 37.44 ± 38.16 ± 43.71 ± 45.70 ± 48.77 ± Control 2.81 3.33 2.12 2.38 2.14 2:
[000628] [000628] The activity of F204P-PH20 was evaluated and compared with rHuPH20 to measure the amount of time required for the dermal barrier to reconstitute after intradermal administration of hyaluronidase. Dermal reconstitution was assessed by comparing the duration of hyaluronidase spreading activity as assessed by monitoring the diffusion area of 0.4% trypan blue over time. The proteins used in the study were purified PH20 F204P variant (prepared as described in example 12) and wild type rHuPH20 (prepared as described in example 1) that were both formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH
[000629] [000629] All mice received two intradermal doses of vehicle control or rHuPH20 or F204P-PH20 at 100 U / mL in 0.04 mL study time 0. The same control or test article was injected into the opposite side sides of each animal (right, R; left, L). Injection sites were marked with a permanent marker. Trypan blue color (0.4% liquid solution; 15250, Invitrogen) was administered in a volume of 0.04 mL by intradermal injection at the same injection site a
[000630] [000630] The results are shown in Table 30. The results show that when the dye dispersion area was measured at various points of time after administration of the test or control article, there was a statistically significant increase in the dye dispersion area in 30 min And 1 hour after injection of rHuPH20 or F204P-PH20. At 4 hours after administration of the enzymes, however, there was no statistically significant increase in the dye dispersion area compared to control. In addition, no statistically significant difference in the dye dispersion area was observed between the rHuPH20 and F204P-PH20 treatment groups. Therefore, the duration of dispersion activity of rHuPH20 and F204P were similar and show that rHuPH20 and F204P-PH20 have comparable in vivo performance. TABLE 30: Dermal reconstitution Ponto Min. Vehicle rHuPH20 F204P-PH20 after injection time 30 5 49.96 ± 2.05 80.84 ± 8.03 80.76 ± 4.46 20 64.42 ± 2.49 94.55 ± 7.09 95.75 ± 5.18 1 hour 5 58.01 ± 3.21 82.56 ± 6.40 77.11 ± 3.18 20 65.19 ± 6.21 96.19 ± 6.39 91.45 ± 1.73 4 5 52.10 ± 3.47 67.19 ± 2.39 67.33 ± 3.93 hours 20 57.69 ± 3.92 81.15 ± 4.45 82.21 ± 4.14 24 5 49.87 ± 3.25 59.01 ± 2.15 54.91 ± 3.54 hours 20 57.15 ± 3.47 67.65 ± 2.27 62.91 ± 3.30 48 5 53.64 ± 2.99 53.53 ± 4.88 55.64 ± 7.19 hours 20 61.57 ± 4.02 66.33 ± 4.12 63.11 ± 5.97 Example 16
[000631] [000631] The pharmacokinetics (PK) of rHuPH20 and F204P-PH20 were compared after administration into the tail vein, intravenously, by measuring plasma hyaluronidase levels over time after administration. The proteins used in the study were purified PH20 variant F204P (prepared as described in example 12; batch concentration 1.02 mg / mL) and wild-type rHuPH20 (prepared as described in example 1; batch concentration 0.95 mg / mL) formulated in API buffer (10 mM Histidine, 130 mM NaCl, pH 6.5). The proteins were prepared at a concentration of 0.087 mg / ml in API buffer for a dose volume of approximately 5 ml. An animal that was not administered with protein was used as a control (pre-dose control). Forty-two (42) male CD-1 mice (~ 20- grams) were used in the study with six animals per treatment group as set out in Table 31. TABLE 31: Pharmacokinetics of Single Intravenous Dose of rHuPH20 or F204P-PH20 group Number of Dose Article Euthanasia Volume test animals (mg / kg) dose (No.) (mL / kg) 1 6 (Nos. 1- Without N / AN / A pre-dose 6) treatment 2 6 (Nos. 7- rHuPH20 0.433 5 1 min 12) 3 6 (Nos. rHuPH20 0.433 5 5 ± 1 min 13-18) 4 6 (Nos. rHuPH20 0.433 5 10 ± 2 19-24) min 5 6 (Nos. F204P-PH20 0.433 5 1 min 25-30)
[000632] [000632] The mice were administered intravenously 0.433 mg / kg rHuPH20 or F204P-PH20 by injection into the tail vein. Blood samples were obtained from animals 1 minute, 5 minutes and 10 minutes after administration. Blood samples were obtained by terminal bleeding (cardiac puncture) and collected in blood collection tubes containing anticoagulant EDTA for the preparation of plasma. Blood samples were centrifuged at 500 g for 10 minutes and the plasma removed and frozen at -80º C until evaluation of hyaluronidase activity using the microturbity assay described in example 8.
[000633] [000633] The results are shown in Table 32. The results show that hyaluronidase activity is not detected in plasma before treatment with hyaluronidase. Within 1 minute of treatment with rHuPH20 or F204P-PH20 hyaluronidase, there is a detectably high amount of hyaluronidase activity present in the plasma, which is similar between the two treatment groups. Over time, hyaluronidase activity rapidly decreases for both treatment groups, although hyaluronidase activity is detectably present in the plasma 10 minutes after administration. At the time points of 5 minutes and 10 minutes post administration, plasma activity in animals treated with F204P-PH20 is greater than in animals treated with rHuPH20. This shows that F204P-PH20 has somewhat greater activity over an extended period of time, and therefore has a longer half-life in vivo than rHuPH20. Table 32: rHuPH20 and F204P-PH20 activity (U / mL) in K2EDTA mouse plasma Time point (min) Pre-dose 1 minute 5 minutes 10 minutes Anima U protein / mL Anima U / mL Anima U / mL Anima U / mL l No. l No. l No. l No. a rHuPH20 1 BQL 7 235 13 18.3 19 3.76 2 BQL 8 13.5 14 7.70 20 3.70 3 BQL 9 278 15 8.85 21 2.64 4 BQL 10 328 16 10.5 22 2.70 5 BQL 11 356 17 12.8 23 2.36 6 BQL 12 287 18 18.0 24 2.80 F204P- 1 BQL 25 249 31 48.0 37 11.5 PH20 2 BQL 26 223 32 21.6 38 11.4 3 BQL 27 246 33 38.4 39 10.1 4 BQL 28 246 34 38.6 40 12.2 5 BQL 20 0.696 35 38.2 41 10.8 6 BQL 30 257 36 28.5 42 10.2 BQL - below the quantifiable limit <0.625 U / mL with minimum dilution required a - Hemolysate
[000634] [000634] Since modifications will be evident to those skilled in the art, it is intended that this invention be limited only by the scope of the attached claims.

权利要求:
Claims (20)
[1]
1. Modified PH20 polypeptide characterized by the fact that it comprises an amino acid substitution in an unmodified PH20 polypeptide, in which: 5 the unmodified PH20 polypeptide comprises the amino acid sequence exposed in one of SEQ ID NOS: 3, 7 and 32- 66; the amino acid substitution is in an amino acid position corresponding to a position 10, 12, 20, 22, 26, 34, 36, 46, 50, 52, 58, 68, 70, 74, 82, 83, 84, 86, 97, 127, 138, 142, 143, 144, 166, 169, 174, 193, 195, 196, 204, 205, 206, 213, 234, 237, 238, 240, 249, 261, 267, 277, 279, 291, 309, 310, 314, 315, 317, 318, 347, 367, 375, 376, 399, 401, 407, 416, 419, 421, 431, 433, 439, 440, 443 or 445 with reference to the positions of amino acid exposed in SEQ ID NO: 3, with the proviso that if the modified PH20 polypeptide includes only a single amino acid substitution, the substitution does not correspond to amino acid substitutions V12A or E249Q with reference to the amino acid positions exposed in SEQ ID NO: 3; the corresponding amino acid positions are identified by aligning the PH20 polypeptide with the polypeptide set out in SEQ ID NO: 3; the modified PH20 polypeptide has increased stability in the presence of a phenolic condom (s) compared to the unmodified PH20 polypeptide not containing the amino acid substitution; the increased stability is manifested as increased hyaluronidase activity in the presence of the phenolic condom (s) compared to the unmodified PH20 polypeptide hyaluronidase activity without containing the amino acid substitution in the presence of the same (s) condom (s), and activity is compared under the same conditions.
[2]
2. Modified PH20 polypeptide according to claim 1, characterized by the fact that the condom is a phenolic condom that is m-cresol, 5 phenol, or m-cresol and phenol.
[3]
3. Modified PH20 polypeptide according to claim 1 or 2, characterized by the fact that at least one amino acid substitution is selected from substitutions with: T in a position corresponding to position 52; K in a position corresponding to position 58; R in a position corresponding to position 58; V in a position corresponding to position 83; P in a position corresponding to position 204; M in a position corresponding to position 234; A in a position corresponding to position 261; Q in a position corresponding to position 310; and H in a position corresponding to position 421, with reference to the amino acid residue positions exposed in SEQ ID NO: 3.
[4]
Modified PH20 polypeptide according to any of claims 1, 2 or 3, characterized in that it comprises substitution with P in a position corresponding to position 204 in a PH20 polypeptide with reference to the amino acid residue positions exposed in SEQ ID NO: 3.
[5]
5. Modified PH20 polypeptide according to any one of claims 1, 2, 3 or 4, characterized in that up to 5 amino acid substitutions are compared to an unmodified PH20 polypeptide of any of SEQ ID NOS: 3, 7 and 32-66.
[6]
Modified polypeptide according to any one of claims 2, 3, 4 or 5, characterized in that it comprises substitution with P in a position corresponding to position 204 in a PH20 polypeptide and up to 5 additional amino acid substitutions, in which the amino acid substitution (s) is / are in an unmodified PH20 polypeptide consisting of the 5 amino acid sequence shown in SEQ ID NO: 3.
[7]
7. Modified PH20 polypeptide according to any one of claims 1, 2, 3, 4, 5 or 6, characterized in that the modified PH20 polypeptide is glycosylated.
[8]
Modified PH20 polypeptide according to any one of claims 1, 2, 3, 4, 5, 6 or 7, characterized in that it is modified by conjugation with a polymer, for example dextran or PEG, and / or is conjugated to a fraction selected from a multimerization domain, toxin, detectable label and drug.
[9]
9. Nucleic acid molecule, characterized by the fact that it encodes a modified PH20 polypeptide according to any one of claims 1 to 8.
[10]
10. Vector, characterized by the fact that it contains the nucleic acid molecule according to claim 9 or that it encodes a polypeptide according to any one of claims 1 to 8.
[11]
11. Isolated cell or cell culture, characterized by the fact that it contains the vector according to claim 10.
[12]
12. Method for producing a modified PH20 polypeptide, characterized in that it comprises: introducing the nucleic acid according to claim 9 or the vector according to claim 10 into a cell capable of incorporating N-linked sugar fractions into the polypeptide; culturing the cell under conditions whereby an encoded modified PH20 polypeptide is produced and secreted by the cell; and optionally, recovering the expressed polypeptide.
[13]
Pharmaceutical composition characterized in that it is a PH20 polypeptide modified according to any one of claims 1 to 8 in a pharmaceutically acceptable excipient.
[14]
14. Pharmaceutical composition according to claim 13, characterized by the fact that the composition contains one or more phenolic preservative (s).
[15]
15. Pharmaceutical composition according to claim 13 or 14, characterized in that it also contains a therapeutically active agent.
[16]
16. Pharmaceutical composition according to claim 15, characterized by the fact that the therapeutic agent is an insulin.
[17]
17. Pharmaceutical composition, according to claim 15, characterized by the fact that the therapeutic agent is a fast-acting insulin.
[18]
18. Pharmaceutical composition according to claim 15, characterized by the fact that the therapeutic agent is a fast-acting insulin that is a regular insulin or is an insulin analogue.
[19]
19. Pharmaceutical composition according to any one of claims 13, 14, 15, 16, 17 or 18, characterized in that it is for use in the treatment of a condition or disease associated with hyaluronan, or for use in the treatment of diabetes, or for use in providing a therapeutic agent to a subject.
[20]
Pharmaceutical composition according to any one of claims 13, 14, 15, 16, 17 or 18, characterized in that it is for use in the treatment of a tumor.
FIGURE
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法律状态:
2020-11-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2021-01-19| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-03-16| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-04-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-12-07| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201161631313P| true| 2011-12-30|2011-12-30|

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US61/631,313|2011-12-30|

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US201261796208P| true| 2012-11-01|2012-11-01|

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US61/796,208|2012-11-01|

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PCT/US2012/072182|WO2013102144A2|2011-12-30|2012-12-28|Ph20 polypeptede variants, formulations and uses thereof|

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