VARIANT HETEROMULTIMERIC PROTEIN OR MODIFIED IGG ANTIBODY, ISOLATED NUCLEIC ACID, EXPRESSION VECTOR,

专利摘要:
variant heteromulimeric protein or modified igg antibody, isolated nucleic acid, expression vector, host cell, method of producing the variant heteromulimeric protein and composition are described in the present application fc variants and methods for the efficient production of antibodies and other complexes of multimeric proteins (collectively referred to in this application as proteins - heteromultimeric). heteromultimeric proteins may be able to specifically bind to more than one target. the targets can be, for example, different epitopes on a single molecule or located on different molecules. the methods combine efficiency, high level of gene expression, proper assembly and ease of purification for heteromultimeric proteins. the invention also provides methods of using these heteromultimeric proteins, and compositions, kits and articles of manufacture that comprise these antibodies.
公开号:BR112013019499A2
申请号:R112013019499-5
申请日:2012-02-03
公开日:2020-03-10
发明作者:Michael Elliott J.；Scheer Justin
申请人:Genentech, Inc.；
IPC主号:

专利说明:

"PROTEIN HETEROMULTIMÉRICAS variants or IgG MODIFIED NUCLEIC ACID EXPRESSION VECTOR, HOST CELLS, METHODS FOR PRODUCING PROTEIN heteromultimeric VARIANT or antibody IgG modified COMPOSITION, METHOD FOR PREPARING A protein heteromultimeric AND PROTEINS heteromultimeric VARIANT" [001] This order claims priority to the provisional US patent application for serial ^Q 61 / 439,750, entitled “Variants Of Fc And Methods For Its Production”, filed on February 4, 2011.
Field of the Invention [002] The invention relates to Fc variants, methods for their generation and antibodies and Fc fusions that comprise Fc variants.
Background of the Invention [003] Monoclonal antibodies of the IgG type contain two identical antigen binding arms and a constant domain (Fc). Antibodies with a different specificity in their binding arms generally do not occur in nature and therefore have to be produced with the help of chemical engineering (eg, chemical crosslinking, etc.), recombinant DNA and / or cell fusion technology .
[004] Bispecific antibodies can simultaneously bind to two different antigens. This property allows the development of therapeutic strategies that are not possible with conventional monoclonal antibodies. The large panel of imaginative bispecific antibody formats that was developed reflects the strong interest in these molecules. See Berg J, Lotscher E, Steimer KS, et al., ‘Bispecific antibodies that mediate killing of cells infected with human immunodeficiency virus of any strain,” Proc Natl Acad Sci USA (1991) 88 (11):
2/160
4723-4727 and Fischer N and Leger 0., "Biospecific Antibodies: Molecules That Enable Novel Therapeutic Strategies", Pathobiology (2007) 74: 3-14.
[005] Another class of multispecific molecules is that of recombinant fusion proteins. Recombinant fusion proteins consisting of the extracellular domain of immunoregulatory proteins and the constant domain (Fc) of immunoglobulin (Ig) represent a developing class of human therapeutics. Immunoadhesins combine the binding region of a protein sequence, with a desired specificity, with the effector domain of an antibody. Immunoadhesins have two important properties that are significant for their potential as therapeutic agents: target specificity and pharmacokinetic stability (in vivo half-life, which is comparable to that of antibodies). Immunoadhesins can be used as antagonists to inhibit or block harmful interactions, or as an agonist to mimic or improve physiological responses. See Chamow SM, Zhang DZ, Tan XY, et al., “A humanized, bispecific immunoadhesin-antibody that retargets CD3 + effectors to kill HIV-1infected cells”, J Hematother 1995; 4 (5): 439-446.
[006] Other multispecific molecules have been discussed elsewhere. Examples include, but are not limited to: Fisher et al., Pathobiology (2007) 74: 3-14 (review of various bispecific formats); US patent 6,660,843, issued December 9, 2003 to Feige et al. (peptibodies); US patent publication 2002-004587 published January 10, 2002 (multispecific antibodies); US patent 7612181 issued November 3, 2009 to Wu et al. (double variable domain format); US patent 6,534,628, Nord K et al., Prot Eng (1995) 8: 601-608, Nord K et al., Nat Biotech (1997) 15: 772-777, and Gronwall et al., Biotechnol Appl Biochem. (2008) Jun; 50 (Pt 2): 97-112 (Affibodies); Martens et al., Ciin Cancer Res (2006), 12: 6144-6152 and Jin et al., Cancer Res (2008) 68 (11): 4360-4368 (antibodies to
3/160 one arm); Bostrom et al., Science (2009) 323: 1610-1614 (Dual Action Fab, antibodies with a mixture of valence aka). Other formats are known to those skilled in the art.
[007] The manufacture of quality clinical material remains a challenge for antibodies in general and especially for the multispecific molecules described above. As mentioned above, there are several routes for the production of molecules with mixed connecting arms, i.e., connecting arms that are not identical to each other. Each of these methods has its disadvantages.
[008] Chemical cross-linking is labor intensive, since the relevant species may still need purification from homodimers and other by-products. In addition, chemical modification steps can alter the integrity of proteins, thus leading to poor stability. Thus, this method is often inefficient and can lead to loss of antibody activity.
[009] Cell fusion technology (eg hybrid hybridomas) expresses two heavy and two light chains that assemble at random leading to the generation of 10 antibody combinations. The desired heteromultimeric antibodies are only a small fraction of the antibodies thus produced. Purifying the desired heteromultimeric proteins dramatically reduces production yields and increases manufacturing costs.
[010] Recombinant DNA techniques have been used to generate several heteromultimeric formats, for example, single-stranded Fv, diabody, etc., which do not comprise an Fc domain. A major disadvantage of this type of antibody molecule is the lack of the Fc domain and thus the ability of the antibody to trigger an effector function (for example, complement activation, binding to the Fc receptor, etc.). Of that
4/160 form, a bispecific antibody comprising a functional Fc domain is desirable.
[011] Recombinant DNA techniques have also been used to generate “knob-into-hole” of bispecific antibodies. See US patent application 20030078385 (Arathoon et al. Genentech). A limitation of this strategy is that the light chains of the two parental antibodies must be identical to avoid mismatches and the formation of undesirable and / or inactive molecules to be expressed in the same cell.
[012] In addition, the limiting event during annealing and purification is redox efficiency. The oxidized heterodimer typically only produces 70 to 80% of the protein after this step (BioAnalyzer and MS-TOF). The remainder of 20 to 30% of the antibody is dimeric and devoid of a covalent bond (SEC-LLS). This can be removed, but it significantly impacts the total yield. Thus, there remains a need to improve the total yield in the production of antibodies, especially heterodimers. Described in the present application are Fc variants that can improve the total yield of antibodies, heterodimers and the like, as well as methods for their generation. These and other aspects and advantages of the invention will be apparent from the description of the invention provided in the present application.
Brief Description of the Invention [013] The production of heteromultimeric proteins, for example, multispecific antibodies, using current techniques has disadvantages, including the production of a mixture of products, reduced yield and reduced / eliminated effector function, among others. Thus, it is desirable to produce heteromultimeric proteins efficiently and at high levels.
[014] The production of antibody molecules, by various means, is generally well understood. US patent 6331415 (Cabilly et al.), By
5/160 example, describes a method for recombinant immunoglobulin production, where the heavy and light chains are expressed simultaneously from a single vector or from two separate vectors in a single cell. Wibbenmeyer et al., (1999, Biochim Biophys Acta 1430 (2): 191-202) and Lee and Kwak (2003, J. Biotechnology 101: 189-198) describe the production of monoclonal antibodies from heavy and light chains produced separately, with the use of plasmids expressed in separate cultures of E. coll. Several other techniques relevant to antibody production are described, for example, in Harlow, et al, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988) and WO 2006028936. Despite this, each of them has disadvantages, such as low yield and use of chemicals, etc.
[015] Disclosed in the present application are Fc variants that comprise at least two mutations in amino acid residues that provide a basis for improving the total yield in the production of heteromultimeric proteins.
[016] This invention provides an easy and efficient production process / method that allows economical production of heteromultimeric proteins, for example, multispecific antibodies, which comprise an Fc variant polypeptide.
[017] In a first embodiment, a variant heteromultimeric protein is provided which comprises an Fc variant of a wild type Fc polypeptide. In various embodiments, the Fc variant comprises at least one, two, three, four, five, six, seven, eight, nine or ten amino acid modifications in the Fc region of said wild-type Fc polypeptide resulting in a variant protein that exhibits decreased spacing (for example, bulge / bulge pairing), formation
6/160 decreased head-tail or increased total yield compared to wild type Fc polypeptide. In some embodiments, it is preferred that the Fc variant comprises two, three or four amino acid modifications capable of disrupting the formation of homodimers as described in the present application. In some embodiments, the Fc variant comprises a substitution at residues 241 and 243, in at least one heavy chain with an amino acid that is different from that present in a wild type Fc polypeptide. In some embodiments, mutations in at least one heavy chain are selected from F241R / F243S and F241S / F243R. In some embodiments, the Fc variant, moreover, comprises modification (s) of protrusion in orifice.
[018] In a second embodiment, an isolated nucleic acid encoding the variant heteromultimeric protein or the modified IgG antibody as described in the present application is provided.
[019] In a third embodiment, an expression vector is provided that encodes the variant heteromultimer protein or the modified IgG antibody as described in the present application.
[020] In a fourth embodiment, a host cell is provided that comprises a nucleic acid molecule or an expression vector that comprises the nucleic acid molecule as described in the present application. In some embodiments, the host cell is a CHO cell. In some embodiments, the host cell is an E. coli cell.
[021] In one embodiment, a method of producing the variant heteromultimer protein or the modified IgG antibody described in the present application comprises:
(a) cultivation of a host cell; and (b) recovering the variant heteromultimer protein or the modified IgG antibody from the cell culture.
7/160 [022] In some embodiments, recovery involves lysing the cells.
[023] In some embodiments, the method of preparing a heteromultimeric protein comprising the heteromultimeric protein comprising a first Fc-containing polypeptide that has a first heterodimerization domain and a second Fc-containing polypeptide that has a second heterodimerization domain, wherein the second domain of heterodimerization interacts with the polypeptide comprising a cavity comprising the replacement of two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine, and in which said original amino acids are replaced by selected import residues from the group consisting of alanine, serine, threonine and valine. In one embodiment, the Fc polypeptide comprising a cavity comprises replacing threonine at position 366 with serine, amino acid numbering according to the EU numbering scheme of Kabat et al., Above. In one embodiment, the Fc polypeptide comprising a cavity comprises replacing leucine at position 368 with alanine, amino acid numbering according to the EU numbering scheme of Kabat et al., Above. In one embodiment, the Fc polypeptide comprising a cavity comprises the substitution of tyrosine at position 407 by alanine, numbering amino acids according to the EU numbering scheme of Kabat et al., Above. In one embodiment, the Fc polypeptide comprising a cavity comprises two or more substitutions of amino acids selected from the group consisting of T366S, L368A and Y407V, numbering amino acids according to the EU numbering scheme of Kabat et al., Above . In some embodiments of these antibody fragments, the Fc polypeptide comprising the lump comprises replacing threonine at position 366 with tryptophan,
8/160 amino acids according to the EU numbering scheme of Kabat et al., Above.
[024] In one embodiment, heteromultimeric proteins produced by the methods described in the present application are provided.
[025] It is understood that the methods of the invention may include other steps that are generally routine steps, evident for starting and / or completing the process encompassed by methods of the invention as described in the present application. For example, in one embodiment, step (a) of a method of the invention is preceded by a step in which a nucleic acid encoding an Fc variant polypeptide is introduced into a first host cell, and a nucleic acid encoding a second polypeptide containing hinge is introduced into a second host cell. In one embodiment, methods of the invention further comprise a step of purifying heteromultimeric proteins that have specific binding to at least two distinct targets. In one embodiment, no more than 10%, 15%, 20% or 30% of isolated polypeptides are present as monomers and / or dimers of the heavy-light chain prior to the heteromultimeric protein purification step. In one embodiment, monomers can have less than about 30% of unwanted polypeptide contaminants that need to be removed before purifying heteromultimeric proteins.
[026] In one embodiment, the first and / or second polypeptide containing Fc is an antibody heavy chain. In a further embodiment, the antibody heavy chain is paired with an antibody light chain to provide a heavy-light chain pair. In some embodiments, the heavy-light chain pair is covalently linked. In another embodiment, the heavy-light chain pair defines a target link arm. In some achievements,
9/160 the target connecting arms are identical. In some embodiments, each target liaison arm recognizes two distinct targets.
[027] In another embodiment, the first and / or second Fc-containing polypeptide comprises a variable heavy chain domain. In another embodiment, the first and / or second Fc-containing polypeptide comprises a receptor binding domain. In some embodiments, the first and / or second Fc-containing polypeptide are substantially identical (i.e., the heterodimerization domain may not be identical to regions outside the heterodimerization domain that are identical). In some embodiments, the first and / or second polypeptide containing Fc are not identical.
[028] In some embodiments, the heteromultimeric protein is selected from the group consisting of an antibody, a bispecific antibody, a multispecific antibody, an arm antibody, a monospecific monovalent antibody, a multispecific monovalent antibody, a bispecific maxibody, an immunoadhesin, a peptibody, a bispecific peptibody, a monovalent peptibody and an affibody and a receptor fusion protein.
[029] In some embodiments, said heteromultimeric proteins comprise a hinge region that has at least one, at least two, at least three, at least four, or any integer up to all the cysteine residues that are normally capable of forming a heavy interchain disulfide bond. In some embodiments, additional cysteines have been introduced in the hinge region.
[030] A heteromultimeric protein of the invention can also be an antibody fragment such as, for example, an Fc or Fc fusion polypeptide, as long as it comprises the Fc region of an immunoglobulin. An Fc fusion polypeptide generally comprises an Fc polypeptide (or a fragment thereof) fused to a heterologous polypeptide sequence
10/160 (as an antigen binding domain), as an extracellular receptor domain (ECD) fused to an immunoglobulin Fc polypeptide (e.g., ECD Fit receptor fused to an IgG2 Fc). For example, in one embodiment, an Fc fusion polypeptide comprises a VEGF binding domain, which can be a VEGF receptor, which includes fit, flk, etc. A heteromultimeric protein of the invention generally comprises a heavy chain constant domain and a light chain constant domain. In one embodiment, a heteromultimeric protein of the invention comprises a modification (for example, but not limited to, insertion of one or more amino acids, for example, to form a dimerization sequence, such as leucine zipper), to form dimerization or interchanging multimerization. In some of these embodiments, the heteromultimeric protein comprises a dimerization domain (such as a leucine zipper sequence), for example, fused to the C-terminus of the heavy chain fragment. In some of these embodiments, the heteromultimer protein comprises a dimerization domain that comprises mutations to provide a "protrusion in orifice" dimerization domain (as defined below below).
[031] In some embodiments of the methods and heteromultimeric proteins of the invention, the Fc-containing polypeptides comprise at least one feature that promotes proper orientation of the Fc-containing polypeptides relative to each other, while improving the total yield of the first and second polypeptides containing Fc. This characteristic (s) improves the yield, purity and / or homogeneity of heteromultimeric protein populations obtained by methods of the invention, as described in the present application. In one embodiment, the Fc polypeptides of a first Fc-containing polypeptide and a second Fc-containing polypeptide meet / interact at an interface. In
11/160 some embodiments, in which the Fc polypeptides of the first and second Fc-containing polypeptides are at an interface, the interface of the second Fc polypeptide comprises a protrusion that can be positioned in a cavity at the interface of the first Fc polypeptide. In one embodiment, the first Fc polypeptide was changed from a model / original polypeptide to encode the cavity, or the second Fc polypeptide was changed from a model / original polypeptide to encode the lump, or both. In one embodiment, the first Fc polypeptide was changed from a template / original polypeptide to encode the cavity, and the second Fc polypeptide was changed from a template / original polypeptide to encode the lump, or both. In one embodiment, the interface of the second Fc polypeptide comprises a protrusion that can be positioned in a cavity at the interface of the first Fc polypeptide, wherein the cavity or protuberance, or both, has been introduced at the interface of the first and second Fc polypeptides, respectively. In some embodiments in which the first and second Fc polypeptides meet at an interface, the interface of the first Fc polypeptide comprises a protrusion that can be positioned in a cavity at the interface of the second Fc polypeptide. In one embodiment, the second Fc polypeptide was changed from a model / original polypeptide to encode the cavity, or the first Fc polypeptide was changed from a model / original polypeptide to encode the lump, or both. In one embodiment, the second Fc polypeptide was changed from a model / original polypeptide to encode the cavity, and the first Fc polypeptide was changed from a model / original polypeptide to encode the lump, or both. In one embodiment, the interface of the first Fc polypeptide comprises a protrusion that can be positioned in a cavity at the interface of the second Fc polypeptide, in
12/160 that the protuberance or cavity, or both, were introduced at the interface of the first and the second Fc polypeptides, respectively.
[032] In one embodiment, each lump and cavity comprises a naturally occurring amino acid residue. In one embodiment, the Fc polypeptide comprising the lump is generated by replacing an original residue from the interface of a model / original polypeptide with an imported residue that has a side chain volume greater than the volume of the original residue. In one embodiment, the Fc polypeptide comprising the lump is generated by a method comprising a step in which the nucleic acid encoding an original residue from the interface of said polypeptide is replaced by a nucleic acid encoding an imported residue that has a side chain volume larger than the original volume. In one embodiment, the original residue is threonine. In one embodiment, the imported residue is arginine (R). In one embodiment, the imported residue is phenylalanine (F). In one embodiment, the imported residue is tyrosine (Y). In one embodiment, the imported waste is tryptophan (W). In one embodiment, the imported residue is R, F, Y or W. In one embodiment, the bulge is generated by replacing two or more residues in a template / original polypeptide. In one embodiment, the Fc polypeptide comprising a lump comprises replacing threonine at position 366 with tryptophan, amino acid numbering according to the EU numbering scheme of Kabat et al. (pages 688-696 in Sequences of Proteins of Immunological Interest, 5th Ed, Vol 1 (1991,.. NIH, Bethesda, MD)).
[033] In some embodiments, the Fc polypeptide comprising a cavity is generated by replacing an original residue at the interface of a template / original polypeptide with an imported residue that has a side chain volume greater than the volume of the original residue. For example, the Fc polypeptide that comprises the cavity can be generated by
13/160 a method comprising a step in which the nucleic acid encoding a source residue from the interface of said polypeptide is replaced by a nucleic acid encoding an imported residue that has a side chain volume less than the volume of the original. In one embodiment, the original residue is threonine. In one embodiment, the original residue is leucine. In one embodiment, the original residue is tyrosine. In one embodiment, the imported waste is not cysteine (C). In one embodiment, the imported waste is alanine (A). In one embodiment, the imported waste is serine (S). In one embodiment, the imported waste is threonine (T). In one embodiment, the imported waste is valine (V). A cavity can be generated by replacing one or more original residues of a model / original polypeptide. For example, in one embodiment, the Fc polypeptide comprising a cavity comprises replacing two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine. In one embodiment, the Fc polypeptide comprising a cavity comprises two or more imported residues selected from the group consisting of alanine, serine, threonine and valine. In some embodiments, the Fc polypeptide comprising a cavity comprises the replacement of two or more original amino acids selected from the group consisting of threonine, leucine and tyrosine, and in which said original amino acids are replaced by imported residues selected from the group consisting of alanine, serine, threonine and valine. In one embodiment, the Fc polypeptide comprising a cavity comprises replacing threonine at position 366 with serine, amino acid numbering according to the EU numbering scheme of Kabat et al., Above. In one embodiment, the Fc polypeptide comprising a cavity comprises replacing leucine at position 368 with alanine, amino acid numbering according to the EU numbering scheme of Kabat et al., Above. In one realization, the
14/160 Fc polypeptide comprising a cavity comprises replacing tyrosine at position 407 with alanine, numbering amino acids according to the EU numbering scheme of Kabat et al., Above. In one embodiment, the Fc polypeptide comprising a cavity comprises two or more substitutions of amino acids selected from the group consisting of T366S, L368A and Y407V, numbering amino acids according to the EU numbering scheme of Kabat et al., Above . In some embodiments of these antibody fragments, the Fc polypeptide comprising the lump comprises the replacement of threonine at position 366 by tryptophan, amino acid numbering according to the EU numbering scheme of Kabat etal., Above.
[034] In one embodiment, the interface of the first Fc polypeptide and / or the second Fc polypeptide has been mutated to comprise substitutions at residues 241 and 243, in at least one heavy chain with an amino acid that is different from that present in a type Fc polypeptide wild. In some embodiments, mutations in at least one Fc polypeptide, for example, of the heavy chain, are selected from F241R / F243S and F241S / F243R. In some embodiments, the Fc variant, moreover, comprises modification (s) of protrusion in orifice.
[035] In various embodiments, the Fc polypeptide of the first and second polypeptides of the heavy chain may or may not be identical, as long as they are capable of dimerization, to form an Fc region (as defined in the present application). A first Fc polypeptide is generally linked adjacent to one or more domains of an immunoglobulin heavy chain on a single polypeptide, for example, with hinge, constant and / or variable domain sequences. In one embodiment, the first Fc polypeptide comprises at least a portion (including all) of a hinge sequence, at least a portion (including all) of
15/160 a Ch2 domain and / or at least a portion (including all) of a Ch3 domain. In one embodiment, the first Fc polypeptide comprises the hinge sequence and the Ch2 and Ch3 domains of an immunoglobulin. In one embodiment, the second Fc polypeptide comprises at least a portion (including all) of a hinge sequence, at least a portion (including all) of a Ch2 domain and / or at least a portion (including all) of a Ch3 domain . In one embodiment, the first Fc polypeptide comprises the hinge sequence and the Ch2 and Ch3 domains of an immunoglobulin. In one embodiment, the second Fc polypeptide comprises at least a portion (including all) of a hinge sequence, at least a portion (including all) of a Ch2 domain and / or at least a portion (including all) of a Ch3 domain . In one embodiment, the second Fc polypeptide comprises the hinge sequence and the Ch2 and Ch3 domains of an immunoglobulin. In one embodiment, an antibody of the invention comprises the first and the second Fc polypeptides, each of which comprises at least a portion of at least one antibody constant domain. In one embodiment, the antibody constant domain is a Ch2 and / or Ch3 domain. In any of the embodiments of an antibody of the invention comprising a constant domain, the antibody constant domain can be of any class of immunoglobulin, for example, an IgG. The source of immunoglobulin can be of any species of suitable origin (for example, an IgG can be human IgG1), or synthetically.
[036] In one embodiment, a first light chain polypeptide and a second light chain polypeptide in a first and second target molecule binding arm, respectively, of an antibody of the invention comprise different / distinct antigen binding determinants ( for example, different / distinct variable domain strings). In one embodiment, a first polypeptide in the chain
16/160 light and a second light chain polypeptide in a first and second target molecule binding arm, respectively, of an antibody of the invention comprise the same (i.e., common) antigen binding determinant, for example, the same variable domain string.
[037] The methods of the invention are capable of generating heteromultimeric molecules with high homogeneity. Consequently, the invention provides methods in which at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96% , 97%, 98%, 99% of the polypeptides are in a complex comprising a first pair of heavy and light chain polypeptides and a second pair of heavy and light chain polypeptides. In one embodiment, the invention provides methods in which between about 60% and 99%, 70% and 98%, 75% and 97%, 80% and 96%, 85% and 96% or 90 and 95% of the polypeptides are in a complex comprising a first pair of heavy and light chain polypeptides and a second pair of heavy and light chain polypeptides.
[038] In one embodiment, an antibody of the invention is selected from the group consisting of IgG, IgE, IgA, IgM and IgD. In some embodiments, the hinge region of an antibody of the present invention is preferably an immunoglobulin selected from the group consisting of IgG, IgA and IgD. For example, in some embodiments, an antibody or hinge region of an antibody is IgG, which in some embodiments is either lgG1 or lgG2 (for example, lgG2a or lgG2b). In some embodiments, an antibody of the invention is selected from the group consisting of IgG, IgA and IgD. In one embodiment, the antibody is of human, humanized, chimeric or non-human origin (for example, murine).
[039] The heteromultimeric proteins of the invention are generally capable of binding, preferably specifically, to antigens. These antigens include, for example, tumor antigens, regulatory factors for
17/160 cell survival, factors regulating cell proliferation, molecules associated with the development and differentiation of tissues (for example, known or suspected of contributing to them in a functional way), cell surface molecules, lymphokines, cytokines, molecules involved in regulation of the cell cycle, molecules involved in vasculogenesis and molecules associated with angiogenesis (for example, known or suspected of contributing in a functional way to it). An antigen to which a heteromultimeric protein of the invention is able to bind can be a member of a subgroup of one of the categories mentioned above, in which the other subgroups of said category comprise other molecules / antigens that have a distinctive feature (with respect to antigen of interest). An antigen of interest can also be considered to belong to two or more categories. In one embodiment, the invention provides a heteromultimeric protein that binds, preferably specifically, to a tumor antigen that is not a cell surface molecule. In one embodiment, a tumor antigen is a cell surface molecule, such as a receptor polypeptide. In another example, in some embodiments, a heteromultimeric protein of the invention binds, preferably specifically, to a tumor antigen that is not a cluster differentiating factor. In another example, a heteromultimeric protein of the invention is capable of binding, preferably specifically, to a cluster differentiation factor, which in some embodiments is not, for example, CD3 or CD4. In some embodiments, a heteromultimeric protein of the invention is an anti-VEGF antibody. In some embodiments, a heteromultimeric protein of the invention is a bispecific antibody selected from the group consisting of IL-lalfa / IL-lbeta, IL-12 / IL-18; IL-13 / IL-9; IL-13 / IL-4; IL-13 / IL-5; IL5 / IL-4; IL-13 / IL-lbeta; IL-13 / IL-25; IL-13 / TARC; IL-13 / MDC; IL-13 / MEF; ILIS / TGF-β; IL-13 / LHR agonist; IL-12 / TWEAK, IL-13 / CL25; IL-13 / SPRR2a;
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IL-13 / SPRR2b; IL-13 / ADAM8, IL-13 / PED2, IL17A / IL17F, CD3 / CD19, CD138 / CD20; CD138 / CD40; CD19 / CD20; CD20 / CD3; CD38 / CD138;
CD38 / CD20; CD38 / CD40; CD40 / CD20; CD-8 / IL-6; CD20 / BR3, TNFalfa / TGFbeta, TNFalfa / IL-lbeta; TNFalpha / IL-2, TNF alpha / IL-3, TNFv / IL-4, TNFalpha / IL-5,
TNFalfa / IL6, TNFalfa / IL8, TNFalpha / IL-9, TNFalpha / IL-10, TNFalfa / IL-11,
TNFalpha / IL-12, TNFv / IL-13, TNFalpha / IL-14, TNFalpha / IL-15, TNFalpha / IL-16,
TNFalfa / IL-17, TNFv / IL-18, TNFalfa / IL-19, TNFalfa / IL-20, TNFval / IL-23, TNFalfa / IFNalpha, TNFalpha / CD4, TNFalpha / VEGF, TNFalpha / MIF, TNFalfa / ICAM- 1, TNFalpha / PGE4, TNFalpha / PEG2, TNFalpha / RANK ligand. TNFalfa / Te38; TNFalfa / BAFF; TNFalfa / CD22; TNFalfa / CTLA-4; TNFalfa / GP130; TNFa / IL
12p40; VEGF / HER2, VEGF-A / HER2, VEGF-A / PDGF, HER1 / HER2, VEGFA / VEGF-C, VEGF-C / VEGF-D, HER2 / DR5, VEGF / IL-8, VEGF / MET, receiver
VEGFR / MET, VEGFR / EGFR, HER2 / CD64, HER2 / CD3, HER2 / CD16,
HER2 / HER3; EGFR / HER2, EGFR / HER3, EGFR / HER4, IL-13 / CD40L, IL4 / CD40L, TNFR1 / IL-1R, TNFR1 / IL-6R, TNFR1 / IL-18R, EpCAM / CD3, MAPG / CD28, EGFR / CD64, CSPGs / RGM A; CTLA-4 / BTNO2; IGF1 / IGF2; IGF1 / 2 / Erb2B; MAG / RGM A; NgR / RGM A; NogoA / RGM A; OMGp / RGM A; PDL-1 / CTLA-4; and RGM A / RGM B, ΙΙ1β / ΙΙ18, NRP1 / VEGFA, VEGFA / NRP2, cMET / EGFR, ALK1 / BMP9, VEGFA / a531, HER1 / HER3-BU and CMV. In some embodiments, a heteromultimeric protein of the invention binds to at least two target molecules selected from the group consisting of: α5β1, ALK1, BMP9, IL-lalfa, IL-lbeta, TARC, MDC, MEF, TGF-β, LHR agonist, TWEAK, CL25, SPRR2a, SPRR2b, ADAM8, PED2, CD3, CD4, CD16,
CD19, CD20, CD22, CD28, CD40, CD38, CD64, CD138, CD-8, BR3, TNFalfa,
TGF-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL- 14,
IL-15, IL-16, IL-17, IL-17A, IL-17F, IL-18, IL-19, IL-20, IL-23, IL-25, IFNalfa, MIF,
ICAM-1, PGE4, PEG2, RANK linker, Te38, BAFF, CTLA-4, GP130, IL-12p40,
VEGF, VEGF-A, PDGF, HER1, HER2, HER3, HER3-BU, HER4, VEGF-C,
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VEGF-D, DR5, cMET, MET, MET receptor, VEGFR, EGFR, CD40L, TNFR1, IL-1 R, IL-6R, IL-18R, EpCAM, MAPG, CSPGs, BTNO2, IGF1, IGF2, IGF1 / 2 , Erb2B, MAG, NgR, NogoA, NRP1, NRP2, OMGp, PDL-I, RGM A and RGM B. In some embodiments, a heteromultimeric protein of this invention binds to CD3 and at least one additional target molecule selected from BLR1 , BR3, CD19, CD20, CD22, CD72, CD79A, CD79B, CD180 (RP105), CR2, FcRH1, FcRH2, FcRH5, FCER2, FCRL4, HLA-DOB and NAG14.
[040] The first and second host cells in the methods of the invention can be grown in any environment that allows expression and isolation of the polypeptides of interest. For example, in one embodiment, the first and second host cells in a method of the invention are grown as separate cell cultures. In another embodiment, the first and second host cells in a method of the invention are grown as mixed cultures that comprise both cells.
[041] Heteromultimeric proteins can be modified to increase and / or add additional desired characteristics. These characteristics include biological functions, such as immune effector functions, desirable half-life / in vivo release, bioavailability, biodistribution or other pharmacokinetic characteristics. Such modifications are well known in the art and can also be determined empirically, and may include modifications by components that may or may not be peptide-based. For example, antibodies can be glycosylated or non-glycosylated, generally depending, at least in part, on the nature of the host cell. Preferably, the antibodies of the invention are non-glycosylated. A non-glycosylated antibody produced by a method of the invention can subsequently be glycosylated, for example, using in vitro glycosylation methods, well known in the art. As described
20/160 above and in the present application, heteromultimeric proteins of the invention can be produced in a prokaryotic cell such as, for example, E. coli. Heteromultimeric proteins produced in E. coli are generally non-glycosylated and lack the biological functions normally associated with glycosylation profiles found in heteromultimeric proteins produced in a mammalian host cell (eg, CHO).
[042] The invention also provides immunoconjugates that comprise a heteromultimeric protein of the invention conjugated to a heterologous component. Any heterologous component would be suitable, as long as its conjugation with the antibody does not substantially reduce the desired function and / or characteristic of the antibody. For example, in some embodiments, an immunoconjugate comprises a heterologous component that is a cytotoxic agent. In some embodiments, said cytotoxic agent is selected from the group consisting of a radioactive isotope, a chemotherapeutic agent and a toxin. In some embodiments, said toxin is selected from the group consisting of calicheamicin, maytansine and trichotene. In some embodiments, an immunoconjugate comprises a heterologous component that is a detectable marker. In some embodiments, said detectable marker is selected from the group consisting of a radioactive isotope, a member of a ligand-receptor pair, a member of an enzyme-substrate pair and a member of an energy transfer pair fluorescence resonance.
[043] In one embodiment, the invention provides compositions that comprise a heteromultimeric protein of the invention and a carrier, which in some embodiments is pharmaceutically acceptable.
[044] In another embodiment, the invention provides compositions that comprise an immunoconjugate as described in the present application and a vehicle, which in some embodiments is pharmaceutically acceptable.
21/160 [045] In one embodiment, the invention provides a composition comprising a population of multispecific heteromultimeric proteins of the invention. As would be evident to a technician in the subject, in general this composition would not be completely homogeneous (that is, 100%). However, as described in the present application, the methods of the invention are capable of producing a substantially homogeneous population of multispecific heteromultimeric proteins. For example, the invention provides a composition comprising heteromultimeric proteins, in which at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 85%, 90%, 95% , 96%, 97%, 98%, 99% of said heteromultimeric proteins are a multispecific antibody (for example, a bispecific antibody, etc.) of the invention, as described in the present application.
[046] In another embodiment, the invention provides articles of manufacture comprising a container and a composition contained therein, wherein the composition comprises a heteromultimeric protein (for example, an antibody) of the invention. In another embodiment, the invention provides articles of manufacture comprising a container and a composition contained therein, wherein the composition comprises an immunoconjugate, as described in the present application. In some embodiments, these articles of manufacture also include instructions for use of said composition.
[047] In yet another embodiment, the invention provides polypeptides that encode a heteromultimeric protein of the invention. In yet another embodiment, the invention provides polynucleotides that encode an immunoconjugate, as described in the present application. In some embodiments, nucleic acids (that is, polynucleotides) are isolated.
22/160 [048] In one embodiment, the invention provides recombinant vectors to express a molecule (for example, an antibody) of the invention. In another embodiment, the invention provides recombinant vectors to express an immunoconjugate of the invention.
[049] Any of a series of host cells can be used in the methods of the invention. Such cells are known in the art (some of which are described in the present application), or can be determined empirically with respect to compatibility for use in the methods of the present invention with the use of known routine techniques. In one embodiment, a host cell is a prokaryote. In some embodiments, a host cell is a gram-negative bacterial cell. In one embodiment, a host cell is E. coli. In some embodiments, E. coli is from a lipoprotein-deficient strain (ΔΙρρ). In some embodiments, the genotype of an E. coli host cell is devoid of degP genes and pre and anchors a mutant spr gene. In one embodiment, a host cell is a mammal, for example, a Chinese Hamster Ovary (CHO) cell.
[050] In one embodiment, the invention provides host cells that comprise a polynucleotide or recombinant vector of the invention. In one embodiment, a host cell is a mammalian cell, for example, a Chinese Hamster Ovary (CHO) cell. In one embodiment, a host cell is a prokaryotic cell. In some embodiments, a host cell is a gram-negative bacterial cell, which in some embodiments is E. coli. The host cells of the invention can further comprise a polynucleotide or recombinant vector that encodes a molecule whose expression thereof in a host cell increases the yield of a heteromultimeric protein in a method of the invention. For example, this molecule can be a chaperone protein. In
23/160 an embodiment, said molecule is a prokaryote polypeptide selected from the group consisting of DsbA, DsbC, DsbG and FkpA. In some embodiments, said polynucleotide or recombinant vector encodes both DsbA and DsbC. In some embodiments, an E. coli host cell is from a strain deficient in endogenous protease activity. In some embodiments, the genotype of an E. coli host cell is that of an E. coli strain that is devoid of degP genes and pre and anchors a mutant spr gene. In some embodiments, the genotype of an E. coli host cell is ΔΙρρ.
[051] Heteromultimeric proteins of the invention find a variety of uses in a variety of configurations. In one example, a heteromultimeric protein of the invention is a therapeutic antibody. In another example, a heteromultimeric protein of the invention is an agonist antibody. In another example, a heteromultimeric protein of the invention is an antagonist antibody. A heteromultimeric protein of the invention can also be a diagnostic antibody. In yet another example, a heteromultimeric protein of the invention is a blocking antibody. In another example, a heteromultimeric protein of the invention is a neutralizing antibody.
[052] In one embodiment, the invention provides methods of treating or postponing a disease in a subject, said methods which comprise administering a heteromultimeric protein of the invention to said subject. In one embodiment, the disease is cancer. In another embodiment, the disease is associated with deregulation of angiogenesis. In another embodiment, the disease is an immune dysfunction, such as rheumatoid arthritis, immune thrombocytopenic purpura, systemic lupus erythematosus, etc.
[053] In one embodiment, the invention provides the use of a heteromultimeric protein (for example, an antibody) of the invention in the preparation of a medicament for therapeutic and / or prophylactic treatment
24/160 of a disease, such as cancer, tumor, cell proliferative dysfunction, immune dysfunction (such as autoimmune) and / or angiogenesis-related dysfunction.
[054] In one embodiment, the invention provides the use of a nucleic acid of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as a cancer, a tumor, a cell proliferative dysfunction, an immune disease (such as autoimmune) and / or angiogenesis-related dysfunction.
[055] In one embodiment, the invention provides the use of an expression vector of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as a cancer, a tumor, a cell proliferative disorder, a dysfunction immune (such as autoimmune) and / or angiogenesis-related dysfunction.
[056] In one embodiment, the invention provides the use of a host cell of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as a cancer, a tumor, a cell proliferative dysfunction, an immune dysfunction (such as autoimmune) and / or angiogenesis-related dysfunction.
[057] In one embodiment, the invention provides the use of an article of manufacture of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as a cancer, a tumor, a cell proliferative disorder, a dysfunction immune (such as autoimmune) and / or angiogenesis-related dysfunction.
[058] In one embodiment, the invention provides the use of a kit of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease, such as a cancer, a tumor, a cell proliferative dysfunction, an immune dysfunction ( as autoimmune) and / or angiogenesis-related dysfunction.
25/160 [059] Other objects, characteristics and advantages of the present invention will be evident from the detailed description below. It should be understood that, however, the detailed description and the specific examples, while indicating preferred embodiments of the invention, are provided by way of illustration only, since various changes and modifications within the scope and spirit of the invention will be evident to a technician in the subject from this detailed description.
Brief Description of the Figures [060] Figure 1 illustrates a completely oxidized half-antibody. No “bulge” or “orifice” or other domains of heterodimerization are shown. The half-antibody represented in this figure is a lgG1 isotype. One skilled in the art will appreciate that the other immunoglobulin isotypes can be visualized as half-antibodies with the corresponding inter- and intrachain bonds. In an intact Ab the hinge cysteines will form interchain disulfide bridges.
[061] Figure 1B illustrates an entire bispecific antibody. The heavy-chain disulfide bridges are not shown in the hinge region.
[062] Figures 2A and B illustrate plasmids that encode the protuberances and the half-antibody holes, respectively.
[063] Figure 3 illustrates the production of heteromultimeric proteins, for example, bispecific antibodies, using separately expressed and genetically engineered half-antibodies. The BsAb produced typically has two different heavy chains with each being paired with a light chain of the same origin. In this method, each light chain is not necessarily the same for each half-antibody.
[064] Figure 4A is a flow diagram for the production of bispecific antibodies using elaborated half-antibodies
26/160 genetically and expressed separately. In this method, the chemical redox is used.
[065] Figure 4B shows a Coomassie-stained gel. The two half-antibodies were analyzed under conditions of reduction and non-reduction by SDS-PAGE. The predominant fraction is the 75kD light chain-heavy chain pair for each half-antibody under non-reducing conditions. Under reduction conditions (eg treatment with DTT) each chain is visible as a separate band.
[066] Figure 4C shows the results of ESI-TOF mass spectrometry of a half-antibody with and without a 1mM Netylmaleimide (NEM) treatment. No change in the mass of the antibody medium is observed under treatment with NEM, indicating that all cysteines are fully oxidized. The oxidized hinge cysteines are represented as a cyclic disulfide of the represented amino acid sequence. The expected mass for the antibody medium is 72,548 Daltons, which is observed by mass spectrometry, indicating no covalent adduct.
[067] Figure 4D shows the carboxymethyl chromatogram (CM), the photograph of an SDS-PAGE gel and the deconvoluted mass for the production of a bispecific anti-EGFR / anti-c-met antibody. CM chromatography produces a single peak which is subsequently analyzed by SDS-PAGE. The main band in the gel is that of the entire bispecific antibody (i.e., intact). A smaller band can also be seen in the 75kD range. The main band was subsequently analyzed by mass spectrometry and indicated that only the detectable intact antibody product was in accordance with the theoretical MW of a bispecific anti-EGFR / anti-c-met antibody.
[068] Figure 5 is a flow diagram for the large-scale production of bispecific antibodies using genetically engineered half-antibodies and expressed separately.
27/160 [069] Figure 6 is a graphical representation of the protrusions in holes of the Fc heterodimer. The heterodimer was crystallized in the presence of a mini-Z domain peptide (not shown). The mini-Z peptide has been linked to the Ch2-Ch3 interface and, presumably, helps to stabilize the F2 Ch2 regions. The protrusion and orifice structure makes contact with the Ch3 domains and is not significantly different from the agglutinated wild type IgGj Fc's.
[070] Figure 7 is a graphical representation of head-tail pairs suggested by analyzing the crystalline structure of Fc. The pairings may be partially responsible for the inefficiency of the redox. The head-tail pairing of the Fc’s causes a portion of the antibodies to escape the disulfide pairing of the hinge. Although dimeric, the uncertainty of his true identity requires its removal. This impacts the total efficiency / efficiency of the annealing process and remains a target for improving the platform.
[071] Figures 8A to D show the content of the dimer of different Fc variants. The Fc's gel filtration analysis shows different extensions of dimerization. The mutant protuberances demonstrate decreased non-covalent homodimerization, when compared to wild type versions of both protuberance and orifice.
[072] Figure 9 shows the contact regions between the protrusion-protrusion of FCs. Panel A shows the hydrophobic contacts. The protrusions-protuberances of Fc homodimers are rendered as drawings with light shading for chain A and dark shading for chain B. The hydrophobic residues Y349, L368, K370, F405 and Y407 in the CH2 domain A chains associated with residues F241, F243, V262, and V264 in the CH3 domain B's chains. P395 and P396 create a hydrophobic pocket between the two CH2 domains. Panel B shows the hydrophilic contacts that are shown on that panel. The lumps-lumps
28/160 of Fc homodimers are rendered as drawings with light shading for chain A and dark shading for chain B. Residues T350, K370 and D399 in A's chains of CH2 domain associated with residues S239, V240, R301 and K334 in B's chains domain name CH3. N389, Y391 and K392 form interactions between the two CH2 domains.
[073] Figure 10 is a bar graph showing the rate of disulfide formation to produce the correct protrusion in the heterodimer orifice. When the half-antibodies are initially mixed together, a low level of homodimers is seen at time zero. As reduced glutathione begins to reduce disulfide on the hinge, homodimers begin to disappear. As time progresses, glutathione begins the oxidation process and an increase in the formation of intact IgG is seen.
[074] Figure 11 is a sequence of curves of the surface plasmon resonance (SPR) spectrum from experiments using an immobilized ligand. The analyte was passed over the ligand and homodimerization was monitored. Analyte concentrations were tested at 6.25 nM, 12.5 nM, 25 nM, 50 nM, 100 nM and 200 nM. The ligands were: (A) The wild type protuberance, (B) wild type orifice, (C) F241R / F243S protuberance, (D) F241S / F243R protuberance and (E) F241S / F243R orifice. The y-axis shows the response units and the x-axis represents time (seconds).
Abbreviations
ADCC = antibody-dependent cell-mediated cytotoxicity
API = Anti-pathogen immunoadhesins
BPI = bactericidal / increasing protein permeability
C1 q = Complement factor 1 q
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CD = Differentiation cluster
CDC = Complement-dependent cytotoxicity
CH1 or Ch1 = constant domain of the first heavy chain CH2 or Ch2 = constant domain of the second heavy chain CH3 or Ch3 = constant domain of the third heavy chain CH4 or Ch4 = constant domain of the fourth heavy chain CL or Cl = constant domain of the light chain
CTLA = Molecule associated with cytotoxic T lymphocyte
Fc = Crystallizable fragment
FcyR = Gamma receptor for the Fc portion of IgG
HIV = Human immunodeficiency virus
ICAM = intercellular adhesion molecule
BsAb = Bispecific Antibody
BsDb = Bispecific bspecific dsFv = Bisulfide stabilized Fv
Fc = Constant fragment of an antibody
Fd = Vh + Ch1 of an antibody
FcR = Fc receptor
Fv = Variable fragment of an antibody
IgG = Immunoglobulin G mAb = Monoclonal antibody
PBL = peripheral blood lymphocyte scDb = single chain scFv = single chain Fv (scFv) ₂ = tandem scFv-scFv Tandab = tandem diabody
VH or Vh = Variable domain of an antibody heavy chain VL or Vl = Variable domain of an antibody light chain
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Detailed Description of the Invention [075] The invention will now be described in detail for reference only using the following definitions and examples. All patents and publications, including all sequences disclosed in those patents and publications, cited in this application are expressly incorporated by reference.
[076] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. Singleton, et al., Dictionary Of Microbiology And Molecular Biology, 2Ed., John Wiley and Sons, New York (1994), and Hale & Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide a general dictionary for a person skilled in the art for many of the terms used in that invention. Although any of the methods and materials similar or equivalent to those described in the present application can be used in the practice or testing of the present invention, the preferred methods and materials are described. Numeric ranges are inclusive of the numbers that define the range. Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' orientation; amino acid sequences are written from left to right in amino orientation for carboxy, respectively. Physicians are particularly directed to Sambrook et al., 1989, and Ausubel FM et al., 1993, for definitions and terms of the technique. It is understood that this invention is not limited to the particular methodology, protocols and reagents described, as these may vary.
[077] Numeric ranges are inclusive of the numbers that define the range.
[078] Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' orientation; the strings
31/160 of amino acids are written from left to right in amino orientation for carboxy, respectively.
[079] The titles provided in the present application are not limitations on the various aspects or embodiments of the invention, which can be obtained by reference to the specification as a whole. Consequently, the terms defined immediately below are more fully defined by reference to the specification as a whole.
I. Definitions [080] A "heteromultimer", "heteromultimeric complex" or "heteromultimeric protein" refers to a molecule that comprises at least one first Fc-containing polypeptide and a second Fc-containing polypeptide, where the second Fc-containing polypeptide is differs in the amino acid sequence from the first Fc-containing polypeptide by at least one amino acid residue. The heteromultimer can comprise a "heterodimer" formed by the first and second polypeptides containing Fc or they can form tertiary structures of higher order where polypeptides are present in addition to the first and second polypeptides containing Fc. The heteromultimer polypeptides can interact with each other by a non-peptide, covalent bond (eg, disulfide bridge) and / or a non-covalent interaction (eg, hydrogen bonds, ionic bonds, van der Waals forces and / or hydrophobic interactions).
[081] As used in the present application, "heteromultimerization domain" refers to changes or additions to a biological molecule, in order to promote formation of heteromultimer and prevent the formation of homomultimer. Any heterodimerization domain that has a strong preference for the formation of heterodimers over homodimers is within the scope of the invention. Illustrative examples include, but are not limited to, for example, US patent application 20030078385 (Arathoon et al. 32/160
Genentech; which describes protrusions in holes); WO 2007147901 (Kjaergaard et al. - Novo Nordisk: which describes ionic interactions); WO 2009089004 (Kannan et al. - Amgen: which describes electrostatic targeting effects); provisional US patent application 61 / 243,105 (Christensen et al. - Genentech; which describes coiled spirals). See also, for example, Pack, P. & Plueckthun, A., Biochemistry 31, 1579-1584 (1992) which describes leucine zipper or Pack et al., Bio / Technology 11, 1271-1277 (1993) which describes propeller-turn-propeller motif. The term "heteromultimerization domain" and "heterodimerization domain" are used interchangeably in the present application.
[082] The term "antibody" in the present application is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivative thereof , as long as they exhibit the desired biological activity (for example, epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments as described in the present application. An antibody can be human, humanized and / or mature in affinity.
[083] As a reference framework, as used in the present application an antibody will refer to the structure of an immunoglobulin G (IgG). However, one skilled in the art would understand / recognize that an antibody of any class of immunoglobulin can be used in the inventive method described in the present application. For clarity, an IgG molecule contains a pair of identical heavy chains (HCs) and a pair of identical light chains (LCs). Each LC has a variable domain (Vl) and a constant domain (Cl), while each HC has a variable domain (Vh) and three constant domains (Ch1, Ch2 and Ch3). The Ch1 θ Ch2 domains are connected by a
33/160 hinge region. This structure is well known in the art. Reference is made to Figure 1B.
[084] As used in the present application, "antibody medium" refers to an immunoglobulin heavy chain associated with an immunoglobulin light chain. An exemplary half-antibody is described in Figure 1A. One skilled in the art will immediately understand that a half-antibody can also have an antigen-binding domain that consists of a single variable domain.
[085] The term "maxibody" refers to a fusion protein that comprises a scFv fused to an Fc polypeptide. Reference is made to Figure 8a of WO 2009089004. Reference is made to Figure 2 of WO 2009089004 for a bispecific maxibody.
[086] The term "Ch2 domain" of a human IgG Fc region generally spans from about 231 residues to about 340 IgG according to the EU numbering system. The Ch2 domain is the only one in which it is not closely paired with another domain. Preferably, two N-linked branched carbohydrate chains are interposed between the two Ch2 domains of an intact native IgG molecule. It has been speculated that carbohydrate may provide a substitute for domain-domain matching and help stabilize the Ch2 domain. Burton, Molec. Immunol. 22: 161-206 (1985).
[087] The term "Ch3 domain" encompasses the extension of C-terminal residues to a Ch2 domain in an Fc region (that is, from about amino acid residue 341 to about amino acid residue 447 of an IgG according to with the EU numbering system).
[088] The term "Fc region", as used in the present application, generally refers to a complex of dimers comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein the C-terminal polypeptide sequence is the one in
34/160 which is obtained by papain digestion of an intact antibody. The Fc region can comprise native or variant Fc sequences. Although the limits of the Fc sequence of an immunoglobulin heavy chain may vary, the Fc sequence of the human IgG heavy chain is generally defined to extend from an amino acid residue, around the Cys226 position, or from about the Pro230 position, until the carboxyl termination of the Fc sequence. Unless otherwise specified in the present application, the numbering of amino acid residues in the Fc region or constant region is in accordance with the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ^to Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc sequence of an immunoglobulin generally comprises two constant domains, a Ch2 domain and a Ch3 domain, and optionally comprises a Ch4 domain. By "Fc polypeptide" in the present application is meant one of the polypeptides that make up an Fc region, for example, a monomeric Fc. An Fc polypeptide can be obtained from any suitable immunoglobulin, such as IgGi, IgG2, IgGg or IgG4, IgA, IgE, IgD or IgM subtypes. The Fc region comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, whose region is also the part recognized by Fc receptors (FcR) found in certain types of cells. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (usually at its N termination). In some embodiments, an Fc polypeptide does not comprise a functional or wild-type hinge sequence.
[089] A "functional Fc region" has an "effector function" of a native sequence Fc region. Exemplary "effector functions" include C1q binding, CDC, Fc receptor binding, ADCC, phagocytosis, infra-regulation
35/160 cell surface receptors (e.g., B cell receptor, BCR), etc. These effector functions generally require the Fc region to be combined with a binding domain (for example, a variable domain of the antibody) and can be evaluated using various assays, as disclosed, for example, in the definitions of the present application.
[090] An "native sequence Fc region" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Native sequence human Fc regions include a native sequence human lgG1 Fc region (allotypes A and non-A), native sequence human lgG2 Fc region, native sequence human IgGg Fc region, native sequence human lgG4 Fc region , as well as naturally occurring variants.
[091] A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution (s). Preferably, the variant Fc region has at least one amino acid substitution as compared to a native sequence Fc region or the Fc region of a parent polypeptide, for example, from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the parental polypeptide Fc region. The variant Fc region in the present application will preferably have at least about 80% homology with a native sequence Fc region and / or with a Fc region of a parent polypeptide and more preferably at least about 90% homology with that, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to that.
36/160 [092] "Component Fc" as used in the present application refers to a hinge region, a Ch2 domain or a Ch3 domain of an Fc region.
[093] In certain embodiments, the Fc-containing polypeptide comprises an IgG Fc region, preferably derived from a wild-type human IgG Fc region. By "wild-type" human IgG Fc is meant a sequence of naturally occurring amino acids within the human population. Of course, just as the Fc sequence can vary slightly between individuals, one or more changes can be made to a wild type sequence and still remain within the scope of the invention. For example, the Fc region may contain additional changes that are not related to the present invention, such as a mutation at a glycosylation site or inclusion of an unnatural amino acid. When used in conjunction with protrusion, orifice or protrusion / orifice, “wild type” is intended to refer to the protein sequence that has only the protuberance, orifice or protuberance / orifice mutations introduced, but is usually to understand the sequence that occurs naturally within the human population.
[094] The term "variable region" or "variable domain" refers to the domain of an heavy or light chain of an antibody that is involved in binding the antibody to the antigen. The heavy chain and light chain variable domains (Vh and Vl, respectively) of a native antibody, generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al. , Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single Vh or Vl domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be isolated using a Vh domain or
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Vl of an antibody that binds to the antigen to select a library of complementary V1 or Vh domains, respectively. See, for example, Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).
[095] The term "Fab" as used in the present application refers to an antigen-binding fragment of an antibody. As noted above, papain can be used to digest an intact antibody. Papain digestion of antibodies produces two identical antigen binding fragments, that is, "Fab" fragments and a residual "Fc" fragment (i.e., the Fc region, above). The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (V _H ), and the first constant domain of a heavy chain (C _H 1).
[096] The phrase "antigen binding arm", "target molecule binding arm", "target binding arm" and variations thereof, as used in the present application, refer to a component part of a heteromultimeric protein of the invention that has the ability to specifically link to a target of interest. Generally, and preferably, the antigen binding arm is a complex of immunoglobulin polypeptide sequences, for example, CDR and / or variable domain sequences of an immunoglobulin light or heavy chain.
[097] A "target" or "target molecule" refers to a component recognized by a heteromultimeric protein binding arm. For example, if the heteromultimeric protein is an antibody, then the target may be epitopes on a single molecule or on different molecules, or a pathogen or a tumor cell, depending on the context. Similarly, if the heteromultimer protein is an Fc receptor fusion protein, the target would be the cognate binding partner for the receptor. A person skilled in the art will appreciate that the target is determined by the specificity of
38/160 target link arm connection and that different target link arms can recognize different targets. A target preferably binds to a heteromultimeric protein of that invention with an affinity higher than 1 µm Kd (according to Scatchard analysis). Examples of target molecules include, but are not limited to, soluble whey proteins and / or their receptors, such as cytokines and / or cytokine receptors, adhesins, growth factors and / or their receptors, hormones, viral particles (for example, RSV protein, CMV, StaphA, influenza, hepatitis C virus), microorganisms (eg, bacterial cell proteins, fungal cells), adhesins, CD proteins and their receptors.
[098] An example of an "intact" or "whole" antibody is one that comprises an antigen binding arm, as well as a Cl and at least heavy chain constant domains, Ch1, Ch2 and Ch3. The constant domains can be native sequence constant domains (for example, human native sequence constant domains) or amino acid sequence variants thereof.
[099] The term "coupling" as used in the present application refers to the steps necessary to join the first and second polypeptides containing Fc to each other, for example, formation of a covalent bond. These steps comprise the reduction, annealing and / or oxidation of cysteine residues in the hinge region of the first and second polypeptides containing Fc to form an interchain disulfide bridge. Coupling can be achieved by chemical crosslinking or using a redox system. See, for example, Humphreys et al., J. Immunol. Methods (1998) 217: 1-10 and Zhu et al., Cancer Lett., (1994) 86: 127-134.
[0100] The term "multispecific antibody" is used in the broadest sense and specifically covers an antibody that has polyepitopic specificity. These multispecific antibodies include, but are not limited to,
39/160 an antibody comprising a heavy chain variable domain (Vh) and a light chain variable domain (Vl), in which the VhVl unit has polyepitopic specificity, whose antibodies have two or more Vl and Vh domains with each unit VhVl binding to a different epitope, whose antibodies have two or more unique variable domains with each unique variable domain binding to a different epitope, whole antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabody, bispecific and tribodies , antibody fragments that have been covalently or non-covalently linked. “Polyepitopic specificity” refers to the ability to specifically bind to two or more different epitopes, on the same target (s) or different target (s). “Monospecific” refers to the ability to attach to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody, which binds to each epitope with an affinity of 5 μΜ to 0.001 pM, 3 μΜ to 0.001 pM, 1 μΜ to 0.001 pM, 0.5 μΜ to 0.001 pM or 0.1 μΜ to 0.001 pM. An illustrative drawing of a bispecific is provided in Figure 1B.
[0101] "Antibody fragments" comprise a portion of an intact antibody, preferably antigen binding or a variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies (Db); tandem bodies (taDb), linear antibodies (for example, US patent 5,641,870; Zapata et al., Protein Eng. 8 (10): 1057-1062 (1995)); single arm antibodies, single variable domain antibodies, minibodies, single chain antibody molecules; and multispecific antibodies formed from antibody fragments (for example, which include, but are not limited to, Db-Fc, taDb-Fc, taDb-Cn3 and (scFV) 4-Fc).
[0102] The term “single domain antibodies” (sdAbs) or “variable single domain antibodies (SVD)” generally refers to antibodies
40/160 in which a single variable domain (Vh or Vl) can confer binding to the antigen. In other words, the single variable domain does not need to interact with another variable domain in order to recognize the target antigen. Single domain antibodies consist of a single monomeric variable antibody domain (Vh or Vl) in each antigen binding arm. Examples of single domain antibodies include those derived from camelids (llamas and camels) and cartilaginous fish (eg, sharks) and those derived from recombinant methods from humans and mouse antibodies (Ward et al., Nature (1989) 341 : 544-546; Dooley and Flajnik, Dev Comp Immunol (2006) 30: 43-56; Muyldermans et al., Trend Biochem Sei (2001) 26: 230-235; Holt et al., Trends Biotechnol (2003): 21 : 484-490; WO 2005/035572; WO 03/035694; Davies and Riechmann, Febs Lett (1994) 339: 285290; WO 00/29004; WO 02/051870). A single variable domain antibody may be present in an antigen binding arm (for example, homo or heteromultimer) with other variable regions or variable domains, the case of which is not a single domain antibody.
[0103] The term "orifice protuberance" or "KnH" as mentioned in this application refers to the technology that directs the pairing of two polypeptides together in vitro or in vivo by introducing a knob into a polypeptide and a cavity (hole) in the other polypeptide at an interface where they interact. For example, KnHs were introduced at the Fc: Fc binding interfaces, Cl: Ch1 interfaces or antibody Vh / Vl interfaces (for example, US patent 20007/0178552, WO 96/027011, WO 98/050431 and Zhu et al (1997) Protein Science 6: 781-788). This is especially useful in directing the pairing of two different heavy chains together during the production of multispecific antibodies. For example, multispecific antibodies that have KNH in their Fc regions may further comprise
41/160 unique variable domains linked to each Fc region, or comprise different heavy chain variable domains that pair with different light chain or similar variable domains. KnH technology can also be used to pair two extracellular domains of the receptor together or any other polypeptide sequence that comprises different target recognition sequences (for example, including aff / 'bodies, peptibodies and other Fc fusions).
[0104] "Fv" consists of a dimer from a variable region domain of the heavy chain and the light chain in close non-covalent association. From the folding of these two domains, six hypervariable loops originate (3 loops of each of the H and L chains) that contribute to the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of a Fv that comprises only three antigen-specific CDRs) has the ability to recognize and bind to the antigen, although often with lower affinity than that of the entire binding site .
[0105] "Single chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the Vh and VL antibody domains connected in a single polypeptide chain. Preferably, the sFv polypeptide additionally comprises a polypeptide linker between the Vh and V1 domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore eds., Springer-Verlag, New York, pages 269-315 (1994); Malmborg et al., J. Immunol. Methods 183: 7-13, 1995.
[0106] The term “diabody” refers to small antibody fragments, prepared by the construction of sFv fragments (see
42/160 the previous paragraph) with short ligands (about 5 to 10 residues) between the Vh and Vl domains, so that the interchain, but not the V domains pairing interchain, is achieved, resulting in a bivalent fragment, that is, a fragment that has two antigen binding sites. Bispecific diabody bodies are heterodimers with two crossover sFv fragments, in which the Vh and Vl domains of the two antibodies are present in the different polypeptide chains. Diabodies are more fully described, for example, in EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Know. USA, 90: 6444-6448 (1993).
[0107] The term "arm antibody" or "arm antibodies" refers to an antibody comprising (1) a variable domain joined by a peptide bridge to the polypeptide comprising a Ch2 domain, a Ch3 domain or a domain Ch2-Ch3 and (2) a second domain Ch2, Ch3 or Ch2-Ch3, in which a variable domain is not joined by a peptide bridge to a polypeptide comprising the second domain Ch2, Ch3 or Ch2-Ch3. In one embodiment, the one-arm antibody comprises 3 polypeptides (1) a first polypeptide comprising a variable domain (for example, Vh), Ch1, Ch2 and Ch3, (2) a second polypeptide comprising a variable domain (for example , Vl) and a Cl domain, and (3) a third polypeptide comprising a Ch2 and Ch3 domain. In another embodiment, the antibody in an arm has a partial hinge region containing the two cysteine residues that form disulfide bridges that bind to constant heavy chains. In one embodiment, the antibody variable domains of an arm form an antigen-binding region. In another embodiment, the antibody variable domains of an arm are unique variable domains, where each unique variable domain is an antigen-binding region. In one embodiment, the arm antibody is a single variable domain antibody.
43/160 [0108] The antibodies of the invention can be "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a class or subclass of particular antibody, while the rest of the chain (s) is identical or homologous to the corresponding sequences in antibodies derived from other species or belonging to another class or subclass of antibodies, as well as fragments of those antibodies, as long as they exhibit the desired biological activity (US patent 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)). Chimeric antibodies of interest in the present application include primatized antibodies that comprise variable domain antigen binding sequences derived from a non-human primate (e.g., Old World monkey, Simian, etc.) and human constant region sequences.
[0109] "Humanized" forms of non-human antibodies (eg, rodent) are chimeric antibodies that contain minimal sequence derived from the non-human antibody. Most humanized antibodies are human immunoglobulins (receptor antibody), in which residues from a hypervariable region of the receptor are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit or non-primate. human, having the desired antibody specificity, affinity and capacity. In some examples, residues from the human immunoglobulin framework (FR) region are replaced by corresponding non-human residues. In addition, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications are still made to refine the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one and typically two
44/160 variable domains, where all or substantially all hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For additional details, see Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2: 593-596 (1992).
[0110] "Pepticibody" or "pepibodies" refers to a fusion of randomly generated peptides with an Fc domain. See US patent 6,660,843, issued December 9, 2003 to Feige et al. (incorporated by reference in its entirety). They include one or more peptides linked to the N-termination, C-termination, amino acid side chains or to more than one of these sites. The peptibody technology allows the design of therapeutic agents that incorporate peptides that target one or more ligands or receptors, tumor-homing peptides, membrane-carrying peptides and the like. The peptibody technology has proven to be useful in the design of a series of these molecules, including linear peptides and constricted disulfide, “tandem peptide multimers” (that is, more than one peptide in a single chain of an Fc domain). See, for example, US patent 6,660,843; US patent application 2003/0195156, published on October 16, 2003 (corresponding to WO 02/092620, published on November 21, 2002); US patent application 2003/0176352, published on September 18, 2003 (corresponding to WO 03/031589, published on April 17, 2003); US serial number ^Q 09 / 422,838, filed on October 22, 1999 (corresponding to WO 00/24770, published on May 4, 2000); US patent application 2003/0229023, published on 11
45/160 December 2003; WO 03/057134, published on July 17, 2003; US patent application 2003/0236193, published on December 25, 2003 (corresponding to PCT / US04 / 010989, filed on April 8, 2004); US serial number ^Q 10 / 666,480, filed on September 18, 2003 (corresponding to WO 04/026329, published on April 1, 2004), each of which is incorporated into this application as a reference in its entirety.
[0111] "Affibodies" or "Affibody" refer to the use of a peptide-bridged protein to an Fc region, where the protein is used as a framework to provide a binding surface for a target molecule. Protein is often a naturally occurring protein such as Staphylococcal Protein A or IgG-binding domain B, or protein Z derived therefrom (see Nilsson et al. (1987), Prot Eng 1, 107-133, and patent US 5,143,844) or a fragment or derivative thereof. For example, affibodies can be created from variants of Z proteins that have altered binding affinity for the target molecule (s), in which a segment of the Z protein has been mutated by random mutagenesis to create a library variants capable of binding to a target molecule. Examples of affibodies include US patent 6,534,628, Nord K. et al., Prot Eng 8: 601-608 (1995) and Nord K et al, Nat Biotech 15: 772-777 (1997). Biotechnol Appl Biochem. June 2008; 50 (Pt 2): 97-112.
[0112] As used in the present application, the term "immunoadhesin" designates molecules that combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, immunoadhesins comprise a fusion of an amino acid sequence with a desired binding specificity, in which the amino acid sequence is different from antigen recognition and an antibody binding site (that is, it is "heterologous" in
46/160 compared to an antibody constant region), and an immunoglobulin constant domain sequence (for example, Ch2 and / or Ch3 sequence of an IgG). Exemplary adhesin sequences include contiguous amino acid sequences that comprise a portion of a receptor or ligand that binds to a protein of interest. Adhesin sequences can also be sequences that bind to a protein of interest, but are not receptor or ligand sequences (e.g., peptide adhesin sequences). These polypeptide sequences can be selected or identified by various methods, include phage display techniques and high-capacity classification methods. The immunoglobulin constant domain sequence in the immunoadhesin can be obtained from any immunoglobulin, such as subtypes lgG1, lgG2, lgG3 or lgG4, IgA (including lgA1 and lgA2), IgE, IgD or IgM.
[0113] "Complex" or "complexed", as used in the present application, refers to the association of two or more molecules that interact with each other through bonds and / or forces (for example, van der Waals forces, hydrophobic, hydrophilic) that are not peptide bridges. In one embodiment, the complex is heteromultimeric. It is to be understood that the term "protein complex" or "polypeptide complex" as used in the present application, includes complexes that have a non-protein entity conjugated to a protein in the protein complex (for example, including, but not limited to, molecules chemicals, such as a toxin or a detection agent).
[0114] A heteromultimeric protein of that invention "that binds" to an antigen of interest is one that binds to the target with sufficient affinity, so that the heteromultimeric protein is useful as a diagnostic and / or therapeutic agent to target a protein , a cell or tissue that expresses the target and does not interact significantly with other proteins. In these embodiments, the extent of binding of the heteromultimeric protein to
47/160 a “non-target” protein will be less than about 10% of the antibody binding to its specific target protein, as determined, for example, by fluorescence activated cell separation analysis (FACS), radioimmunoprecipitation (RIA) or ELISA. Regarding the binding of a heteromultimeric protein to a target molecule, the term "specific binding" or "specifically binds to" or is "specific to" a particular target or an epitope on a particular polypeptide target, means binding that is different from measurable form from a non-specific interaction (for example, a non-specific interaction may be linked to bovine serum albumin or casein). Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of unmarked target. In that case, specific binding indicates whether binding of the labeled target to a probe is competitively inhibited by the excess of unmarked target. The term "specific binding" or "specifically binds to" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide target, as used in the present application, can be displayed, for example, by a molecule that has a Kd for the target of at least about 200 nM, alternatively of at least about 150 nM, alternatively of at least about 100 nM, alternatively of at least about 60 nM, alternatively of at least about 30 nM, alternatively of at least about 8 nM, alternatively at least alternatively at least about 40 nM, alternatively at least about 10 nM, alternatively at least about 4 nM, less than about 50 nM, alternatively at least about 20 nM, alternatively at least about 6 nM, alternatively at least about 2 nM, alternatively at least about 1 nM, or more.
48/160
In one embodiment, the term "specific binding" refers to the binding where a heteromultimeric protein binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope.
[0115] "Binding affinity" generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (for example, an antibody) and its binding partner (for example, an antigen). Unless otherwise stated, as used in the present application, "binding affinity" refers to the intrinsic binding affinity that reflects a 1: 1 interaction between members of a binding pair (for example, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). For example, Kd may be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or stronger. Affinity can be measured by common methods known in the art, including those described in the present application. Antibodies with low affinity generally bind slowly to antigens and tend to dissociate easily, whereas antibodies with high affinity generally bind faster to antigens and tend to stay on longer. A variety of methods for measuring binding affinity are known in the art and any of these can be used for the purposes of the present invention.
[0116] In one embodiment, the “Kd” or “Kd value” according to this invention is measured using surface plasmon resonance assays using a BIAcore ™ -2000 or a BIAcore ™ -3000 (BIAcore, Inc., Piscataway, NJ) at 25 ^Q C CM5 chip with immobilized target (e.g., antigen) to approximately 10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated
49/160 with N-ethyl-N - (3-dimethylaminopropyl) -carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), according to the supplier's instructions.
The antigen is diluted with 10 mM sodium acetate, pH 4.8, in 5 pg / mL (approximately 0.2 μΜ) before injection, at a flow rate of 5 pL / minute to achieve approximately 10 response units ( RU) of coupled protein.
[0117] After the injection of antigen, 1M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (for example, 0.78 nM to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25 ^Q C at a flow rate of approximately 25 pL / min. Association rates (k _on ) and dissociation rates (k _O ff) are calculated using a Langmuir one-to-one connection model (BIAcore Evaluation Software version 3.2) by simultaneously adjusting the association and dissociation sensorgram. The dissociation equilibrium constant (Kd) is calculated as the ratio k ₀ ff / k _0n . See, for example, Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the association rate exceeds 10 ⁶ M ' ¹ S' ¹ by the surface plasmon resonance assay above, then the association rate can be determined using a fluorescent suppression technique that measures the increase or decrease in the emission intensity fluorescence (excitation = 295 nm; emission = 340 nm, 16 nm band-pass) at 25 ^Q C in a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations antigen as measured on a spectrometer, such as a flow stop equipped spectrophotometer (Aviv Instruments) or an SLMAminco 8000 series spectrophotometer (ThermoSpectronic) with a mixing crucible.
[0118] "Biologically active" and "biological activity" and "biological characteristics", in relation to a heteromultimeric protein of this invention, such as an antibody, fragment or derivative thereof, means to have the
50/160 ability to bind to a biological molecule, unless otherwise specified.
[0119] "Isolated" when used to describe the various polypeptide heteromultimers, means a heteromultimer that has been separated and / or recovered from a cell or cell culture from which it was expressed. Contaminating components of its natural environment are materials that could interfere in the diagnosis or therapeutic use for the heteromultimer and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the heteromultimer will be purified (1) to more than 95% by weight of protein, as determined by the Lowry method and with the highest preference to more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, using a rotating cup sequencer or (3) until homogeneous, by SDS-PAGE under conditions of reduction or non-reduction, with the use of Coomassie blue or preferably, silver stain. Generally, however, the isolated polypeptide will be prepared by at least one purification step.
[0120] The heteromultimers of the present invention are generally purified to substantial homogeneity. The terms "substantially homogeneous", "substantially homogeneous form" and "substantial homogeneity" are used to indicate that the product is substantially free of by-products originating from combinations of undesirable polypeptides (for example, homomultimers).
[0121] Expressed in terms of purity, substantial homogeneity means that the amount of by-products does not exceed 10%, 9%, 8%, 7%, 6%, 4%, 3%, 2% or 1% by weight, or is less than 1% by weight. In one embodiment, the by-product is less than 5%.
51/160 [0122] "Biological molecule" refers to a nucleic acid, a protein, a carbohydrate, a lipid and combinations thereof. In one embodiment, the biological molecule exists in nature.
[0123] By "linked" or "bonds" as used in the present application is meant either a direct peptide bridge bond between the first and the second amino acid sequence or a bond that involves a third amino acid sequence that is linked by peptide and between the first and the second amino acid sequence. For example, a linker peptide attached to the C-terminal end of one amino acid sequence and the N-terminal end of the other amino acid sequence.
[0124] By "linker" as used in the present application is meant an amino acid sequence of two or more amino acids in length. The linker can consist of neutral polar or non-polar amino acids. A linker can be, for example, from 2 to 100 amino acids in length, such as between 2 and 50 amino acids in length, for example, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids of lenght. A binder can be "cleavable", for example, by autocleaving, or enzymatic or chemical dividing. Divide sites of amino acid sequences and enzymes and chemicals that cleave at these sites are well known in the art and are also described in the present application.
[0125] By a "rope" as used in the present application is meant an amino acid ligand that joins two other amino acid sequences. A string, as described in the present application, can link the N-terminus of an immunoglobulin heavy chain variable domain with the C-terminus of an immunoglobulin light chain constant domain. In specific embodiments, a string is between about 15 and 50 amino acids in length, for example, between 20 and 26 amino acids in length (for example, 20, 21, 22, 23, 24, 25 or 26 amino acids in length
52/160 length). The rope can be “cleavable”, for example, by self-cleavage, or enzymatic or chemical dividing, using standard methods and reagents in the art.
[0126] Enzymatic divination of a "ligand" or "rope" may involve the use of an endopeptidase such as, for example, Lys-C, Asp-N, Arg-C, V8, Glu-C, chymotrypsin, trypsin , pepsin, papain, thrombin, Genenase, Factor Xa, TEV (cysteine protease from tobacco etch virus), enterokinase, HRV C3 (human rhinovirus protease C3), quininogenase, as well as subtilisin-like pro-protein convertase (for example , Furin (PC1), PC2 or PC3) or dibasic N-arginine convertase. Chemical divination may involve the use of, for example, hydroxylamine, N-chlorosuccinimide, Nbromo-succinimide or cyanogen bromide.
[0127] A "Lys-C endopeptidase dividing site" as used in the present application is a lysine residue in an amino acid sequence that can be cleaved on the C-terminal side by Lys-C endopeptidase. Lys-C endopeptidase divides on the C-terminal side of a lysine residue.
[0128] A “chaotropic agent” means a water-soluble substance that disrupts the three-dimensional structure of a protein (for example, an antibody) by interfering with the stabilization of intramolecular interactions (for example, hydrogen bonds, van der Waals forces or hydrophobic effects). Exemplary chaotropic agents include, but are not limited to, urea, guanidine-HCI, lithium perchlorate, histidine and arginine.
[0129] A “mild detergent” means a water-soluble substance that disrupts the three-dimensional structure of a protein (for example, an antibody) by interfering with the stabilization of intramolecular interactions (for example, hydrogen bonds, van der Waals forces or hydrophobic effects), but that do not permanently disrupt the protein structure as it causes a loss of biological activity (that is, no
53/160 denatures the protein). Exemplary mild detergents include, but are not limited to, Tween (eg, Tween-20), Triton (eg, Triton X-100), NP-40 (nonyl phenoxypolyethoxyethanol), Nonidet P-40 (octyl phenoxylpolyethoxyethanol) and sodium dodecyl sulfate (SDS).
[0130] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or a variant amino acid sequence Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytoxicity, Fc receptor binding, antibody-dependent cell-mediated cytoxicity (ADCC); phagocytosis, infrarregulation of cell surface receptors (for example, B cell receptor) and B cell activation.
[0131] "Antibody dependent cell mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to the Fc receptors (FcRs) present in certain cytotoxic cells (for example, Natural Killer (NK) cells , neutrophils, and macrophages) allow these cytotoxic effector cells to specifically bind to a target cell that produces antigen and subsequently eliminate the target cell with cytotoxic agents. Antibodies "embrace" cytotoxic cells and are absolutely necessary for this destruction. Primary cells for ADCC mediation, NK cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, as described in US patents 5,500,362 or 5,821,337. Effector cells useful for these assays include peripheral blood mononuclear cells (PBMC) and Natural Killer cells (NK). Alternatively, or
54/160 in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model as disclosed in Clynes et al., Proc. Natl. Acad. Know. USA 95: 652-656 (1998).
[0132] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is a human FcR. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor) and includes receptors of the subclasses FcyRI, FcyRII and FcyRIII, including allelic variants and alternatively joined forms of these receptors. FcyRII receptors include FcyRIIA (an "activating receptor") and FcyRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ mainly in their cytoplasmic domains. The FcyRIIA activation receptor contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain. The FcyRIIB inhibitory receptor contains a tyrosine-based immunoreceptor inhibition motif (ITIM) in its cytoplasmic domain (see revision M. Daêron, Annu. Rev. Immunol. 15: 203-234 (1997)). The FcRs were reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-492 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Ciin. Med. 126: 330-41 (1995). Other FcRs, including those to be identified in the future, are encompassed by the term “FcR” in this application. The term also includes the neonatal receptor, FcRn, which is responsible for transferring maternal IgGs to fetuses (Guyer et al., J. Immunol. 117: 587 (1976) and Kim etal., J. Immunol. 24: 249 ( 1994)).
[0133] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer cells (NK), monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK cells being
55/160 preferred. Effector cells can be isolated from a native source, for example, from the blood.
[0134] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by binding the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that are linked to its cognate antigens. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro etal., J. Immunol. Methods 202: 163 (1996).
[0135] The term "therapeutically effective amount" refers to an amount of an antibody, antibody fragment or derivative to treat a disease or dysfunction in a subject. In the case of a tumor (for example, cancerous tumor), the therapeutically effective amount of the antibody or antibody fragment (for example, a multispecific antibody or antibody fragment) can reduce the number of cancer cells, reduce the size of the main tumor, inhibit (i.e., decrease to a certain extent and preferably interrupt) the infiltration of cancer cells into peripheral organs, inhibit (that is, decrease to a certain extent and preferably interrupt) tumor metastasis, inhibit, to some extent, tumor growth, and / or relieve to some extent one or more of the symptoms associated with the dysfunction. To some extent, the antibody, or antibody fragment, can prevent the growth and / or destroy existing cancer cells, which can be cytostatic and / or cytotoxic. For cancer therapy, in vivo efficacy can be measured, for example, by assessing duration of survival, time to disease progression (TTP), response rates (RR), response duration and / or quality of life.
[0136] "Reduce or inhibit" means the ability to cause a general decrease, preferably 20% or more, more
56/160 preferably 50% or more, and more preferably 75%, 85%, 90%, 95% or more. Reducing or inhibiting may refer to the symptoms of the dysfunction being treated, the presence or size of metastasis, the size of the primary tumor or the size or number of blood vessels in angiogenic disorders.
[0137] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by irregular cell growth / proliferation. This definition includes benign and malignant cancer. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of these cancers include squamous cell cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous lung carcinoma, peritoneum cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer , pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary glandular carcinoma, kidney cancer (for example , renal cell carcinoma), liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma and various types of head and neck cancer. "Early stage cancer" means cancer that is not invasive or metastatic or is classified as a Stage 0, I or II cancer. The term "pre-cancerous" refers to a condition or growth that typically precedes or develops in cancer. “Non-metastatic” means cancer that is benign or that remains in the initial site and that has not penetrated the lymphatic system, blood vessels or tissues except the initial site. Generally, cancer does not
57/160 metastatic is any cancer that is in Stage 0, I or II and occasionally a cancer in Stage III.
[0138] An "allergic or inflammatory disorder" in the present application is a disease or disorder that results from an overactivation of an individual's immune system. Exemplary allergic or inflammatory disorders include, but are not limited to, asthma, psoriasis, rheumatoid arthritis, atopic dermatitis, multiple sclerosis, systemic lupus, erythematosus, eczema, organ transplantation, age-related macular degeneration, Crohn's disease, ulcerative colitis, eosinophilic esophagitis and autoimmune diseases associated with inflammation.
[0139] An "autoimmune disease" in the present application is a disease or dysfunction that arises and is directed against the individual's own tissues, a cosegregate or manifestation of the same, or resulting from conditions from this. Examples of autoimmune diseases or disorders include, but are not limited to, arthritis (rheumatoid arthritis, such as acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, and juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, deforming arthritis, primary chronic polyarthritis, reactive arthritis and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as psoriasis, psoriasis, psoriasis, psoriasis and nail psoriasis, dermatitis including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis and atopic dermatitis, x-linked hyper-lgM syndrome, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including autoimmune urticaria cr onica, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis as sclerosis
58/160 systemic, multiple sclerosis (MS) as optic-spinal MS, progressive pimary MS (PPMS) and remitting-recurrent MS (RRMS), progressive systemic sclerosis, arteriosclerosis, disseminated sclerosis and ataxic sclerosis, inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune-mediated gastrointestinal diseases, colitis such as ulcerative colitis, ulcerative colitis, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis and transmural colitis, and autoimmune bowel disease), gangrenous pyoderma, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological dysfunction, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases like anaphylaxis and allergic and atopic rhinitis, encephalitis like encephalitis Rasmussen's alitis, brain stem encephalitis, uveitis as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phatoantigenic uveitis, posterior uveitis or autoimmune uveitis, glomerulonephritis (glomerulonephritis) with or without nephrotic syndrome as or without nephrotic syndrome Primary GN, immune-mediated GN, membranous GN (nephropathic membranous), idiopathic membranous GN or idiopathic membranous neuropathy, proliferative membrane or membranous GN (MPGN), including Type I and Type II, and rapid progression GN, allergic conditions and responses, allergic reactions, eczema including allergic or atopic eczema, asthma such as bronchial asthma and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) as Cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), diabetes mellitus (Type I) of
59/160 juvenile principle, including pediatric insulin dependent diabetes mellitus (IDDM), adult principle diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with cytokine and T lymphocyte-mediated acute and delayed hypersensitivity, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis (including large vessel vasculitis (including polymyalgia rheumatica and Giant cell arteritis (from Takayasu)), medium vessel vasculitis (including Kawasaki disease and nodular polyarteritis) , microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous or hypersensitivity vasculitis, systemic necrotizing vasculitis and ANCA-associated vasculitis, such as vasculitis or Churg-Strauss syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, positive anemia of positive Coombs, Diamond Blackfan anemia, hemolytic anemia o u immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis , CNS inflammatory disorders, multiple organ injury syndrome such as those resulting from septicemia, trauma or hemorrhage, diseases mediated by antigen-antibody complex, antiglomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet's disease, Castleman, Goodpasture's syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, pemphigoid such as vesicular pemphigoid and skin pemphigoid, pemphigus (including common pemphigus, pemphigus foliaceus, pemphigus pemphigoid, muco-membranous and pituitary erythematosopathy), poliend , Reiter's disease or syndrome, immune complex nephritis , antibody-mediated nephritis, optic neuromyelitis, polyneuropathy, chronic neuropathy such as IgM polyneuropathies or neuropathy
60/160 IgM-mediated, thrombocytopenia (as developed by patients with myocardial infarction, for example), including thrombotic thrombocytopenic purpura (TTP) and autoimmune or immune thrombocytopenia mediated as idiopathic purpura (ITP) including chronic or acute ITP, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis) or subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism polyglandular syndrome such as autoimmune polyglandular syndrome (or polyglandular endocrinopathic syndrome), paraneoplastic syndrome, including neurological pareneoplastic syndrome such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, rigid man or rigid person syndrome, encephalomyelitis such as allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE), myasthenia gravis as myasthenia gravis associated with thymoma, cerebellar degeneration, neuromyotonia, opsoclone or opsoclone myoclonus syndrome (WHO) and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or chronic autoimmune active hepatitis, lymphoid interstitial pneumonitis, bronchiolitis obliterans (without transplantation) against NSIP, GuillainBarré syndrome, Berger disease (IgA nephropathy), idiopathic IgA nephropathy, dermatosis by Linear IgA, primary biliary cirrhosis, pneumocirrhosis, autoimmune enteropathy syndrome, celiac disease, celiac psilosis (gluten enteropathy), refractory psilosis, idiopathic psilosis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclone myoclonus syndrome (WHO), polychondritis such as refractory or relapsing polyondritis, pulmonary alveolar proteinosis
61/160 amyloidosis, scleritis, non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (for example, benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraines, migraines , arrhythmia, muscle dysfunction, deafness, blindness, periodic paralysis, and CNS channelopathy, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological dysfunction, fibromyalgia, endemic syndrome, fibromyalgia , adrenalitis, gastric atrophy, pre-senile dementia, demyelinating diseases such as autoimmune demyelinating diseases, diabetic neuropathy, Dressier syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmobilitis, sclerodactyly, and telangiectasia), male autoimmune infertility female disease mixed connective tissue disease, Chagas disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird breeding lung, allergic granulomatous angitis, benign lifocytic angitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, leprosis, malaria, leishmaniasis, kipanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis , interstitial pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, elevated and reduced erythema, fetal erytoblastosis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, flariase, cyclitis as chronic cyclitis, heterochronic cyclitis, iridocyclitis or Fuch's cyclitis Henoch-Schonlein, immunodeficiency virus infection INSTANCE human (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection,
62/160 Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis obliterans, thyrotoxicosis, dorsalis tabs, chorioiditis, giant cell polymyalgia, endocrine ophthalmology, pneumonitis, endocrine pneumonia chronic, dry keratoconjunctivitis, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal exchange neuropathy, hereditary benign lesion and reperfusion and ischemia, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, thrush, aphthous stomatitis, arteriosclerotic diseases, aspermiogenesis, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, phaco-anaphylactic endophthalmia, allergic entente, leprous nodular erythema, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, sensory loss from Hamman-Rich, hearing loss , hemoglobinuria paroxism hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, gangrenous pyoderma, acquired antisepticism, atrophy of acquired disease, atrophy non-malignant thymoma, vitiligo, SCID and diseases associated with the Epstein-Barr virus, acquired immunodeficiency syndrome (AIDS), parasitic diseases such as Leishmania, toxic shock syndrome, poisoned food, conditions involving T cell infiltration, leukocyte adhesion deficiency, immune responses associated with acute or delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, diseases mediated by the antigen-antibody complex, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oof oritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, peripheral neuropathy, syndrome
63/160 polyglandular type I autoimmune, idiopathic adult hypoparathyroidism (AOIH), total alopecia, dilated cardiomyopathy, bullous epidermolysis acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, purulent sinusitis, and purulent sinusitis, not purulent sinusitis and purulent sinusitis, purulent or non-purulent sinusitis. chronic, ethmoid, frontal, maxillary, or sphenoid sinusitis, a dysfunction related to eosinophils, eosinophilic pulmonary infiltration, eosinophilic myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, and bronchopathic bronchosomal pneumonia, anaphylaxis, seronegative spondyloarthritis, polyiendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis, Bruton syndrome, transient childhood hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia telangiectasia, associated autoimmune dysfunctions collagen deficiency, rheumatism, neurological disease, ischemia-reperfusion dysfunction, reduced blood pressure response, vascular dysfunction, antiectasis, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease accompanying vascularization, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, reperfusion injury to myocardium or other tissues, dermatosis with acute inflammatory components, acute purulent meningitis or other inflammatory disorders of the central nervous system, ocular and orbital inflammatory disorders, syndromes associated with granulocyte transfusion, cytokine-induced toxicity , acute serious inflammation, chronic incurable inflammation, pyelitis, pneumonocirrhosis, diabetic retinopathy, major diabetic artery dysfunction, endoarterial hyperplasia, peptic ulcer, valvulitis and endometriosis.
[0140] The term "cytotoxic agent" as used in this application refers to a substance that inhibits or prevents the function of a cell and / or causes destruction of a cell. The term is intended to include isotopes
64/160 radioactive (eg At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , Ra ²²³ , P ³² and Lu radioactive isotopes), chemotherapeutic agents, for example, methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof, such as nucleolytic enzymes, antibiotics and toxins, such as small molecule toxins or toxins enzymatically active bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, and the various antitumor, anticancer and chemotherapeutic agents disclosed in the present application. Other cytotoxic agents are described in the present application. A tumoricidal agent causes destruction of tumor cells.
[0141] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; sulfonated alkyls such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethyleneethiophosphoramide and trimethylolomelamine; acetogenins (especially bulatacin and bulatacinone); delta-9tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacone; lapacol;
colchicines; betulinic acid; a camptothecin (including the synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin and 9-aminocamptothecin); briostatin;
calistatin; CC-1065 (including adozelesin, carzelesin and bizelesin synthetic analogs); podophyllotoxin; podophyllinic acid; teniposide; cryptoficina (particularly cryptoficina 1 and criptoficina 8); dolastatin; duocarmycin (including synthetic analogs of KW-2189 and CB1-TM1); eleuterobin; pancratistatin; a sarcodictine; spongistatin; nitrogen mustards
65/160 such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mecloretamine, meclorethamine hydrochloride, melphalan, novembicin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enedin antibiotics (eg, calicheamicin, especially calicheamicin gamma 1 (see, for example, Agnew, Chem Inti. Ed. Engl., 33: 183-186 (1994)); dinemicin, including dinamycin A; a speramycin; as well as neocarzinostatin chromophores and antiobiotic chromophores (enedin related chromoproteins), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophylline, cromomycin, dacorin-5-duborinomycin; norleucine, doxorubicin ADRIAMYCIN® (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinodoxorubicin and deoxidoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycomycin, potassium chloride, mycophenolic acid, mycotomycin, in mycomycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6mercaptopurine, tiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacytidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutetimide, mitotane, trilostane; restorative folic acid like frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; ansacrine; bestrabucila; bisanthrene; edatraxate; defofamine; demecolcine; diaziquone; elfornitine; ellipinium acetate; an epothilone;
66/160 etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; fenamet; pirarubicin; losoxantrone; 2-ethyl hydrazide; procarbazine; PSK® polysaccharide complexes (JHS Natural Products, Eugene, OR); razoxan; rhizoxin; sizofirana; spirogermanio; tenuazonic acid; triaziquone; 2.2 ’, 2” -trichloro triethyl amine; trichothecenes (especially T2 toxin, verracurin A, roridin A and anguidine); urethane; vindesina (ELDISINE®, FILDESIN®); dacarbazine; manomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids, for example, TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremofor-free ABRAXANE ™, modified albumin nanoparticle paclitaxel formulation (American Pharmaceutical Partners, Schaumberg, IL) and TAXOTERE® doxetaxel (Rhône- Poulenc Rorer, Antony, France); chloranbucyl; gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN®); oxaliplatin; leukovovine; vinorelbine (NAVELBINE®); new chair; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase RFS 2000 inhibitor; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine (XELODA®); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone, and FOLFOX, an abbreviation for an oxaliplatin (ELOXATIN ™) treatment regimen combined with 5-FU and leukovovine .
[0142] Also included in this definition are anti-hormonal agents that act to regulate, reduce, block or inhibit the effects of hormones that can promote cancer growth, and are many
67/160 times in the form of systemic or whole body treatment. They can be the hormones themselves. Examples include antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including tamoxifen NOLVADEX®), raloxifene EVISTA®, droloxifene, 4-hydroxy tamoxifen, trioxiphen, queoxifene, LY117018, onapristone and toremifene; antiprogesterones; estrogen receptor infraregulators (ERDs); ovarian suppression or blocking function agents, for example, hormone agonists that release luteinizing hormone (LHRH) like leuprolide acetate LUPRON® and ELIGARD®, goserelin acetate, buserelin acetate and tripterelin; other antiandrogens such as flutamide, nilutamide and bicalutamide; and aromatase inhibitors that inhibit the aromatase enzyme, which regulates the production of estrogen in the adrenal glands such as, for example, 4 (5) -imidazoles, aminoglutetimide, megestrol acetate MEGASE®, exemestane (AROMASIN®), formestane, fadrozola, vorozola RIVISOR ®, letrozola FEMARA® and anastrozola ARIMIDEX®. In addition, this definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, zoledronic acid / zoledronate ZOMETA®, alendronate FOSAMAX®, pamidronate AREDIAEL®, tiludron® or tiludon® ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, specifically those that inhibit expression of genes in the signaling pathways involved in abnormal cell proliferation such as, for example, PKC-alpha, Raf, H-Ras and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine and VAXID® vaccine; topoisomerase 1 inhibitor LURTOTECAN®; rmRH ABARELIX®; lapatinib ditosylate (a double small molecule tyrosine kinase inhibitor
68/160
EGFR and ErbB2 (also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[0143] A "growth inhibitory agent" when used in the present application refers to a component or composition that inhibits the growth of a cell, both in vitro and in vivo. Thus, the growth inhibitory agent may be one that significantly reduces the percentage of cells in the S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (in one location, except in the S phase), as agents that induce G1 disruption and M-phase disruption. Classical M-phase blockers include creases (eg, vincristine and vinblastine), taxanes and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide and bleomycin. G1-disrupting agents also affect S-phase disruption, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, meclorethamine, cisplatin, methotrexate, 5-fluorouracil and ara-C. Additional information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation, oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders: Philadelphia, 1995), especially page 13. Taxanes (paclitaxel and docetaxel) are anticancer drugs, both derived from the yew. Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semi-synthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules of tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
[0144] "Anticancer therapy" as used in the present application refers to a treatment that reduces or inhibits cancer in a subject. Examples of anticancer therapy include cytotoxic radiation therapy, as well as
69/160 administration of a therapeutically effective amount of a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, a cancer vaccine, an angiogenesis inhibitor, a prodrug, a cytokine, a cytokine antagonist, a corticosteroid, an immunosuppressive agent, an antiemetic, an antibody or antibody fragment, or an analgesic for the subject.
[0145] The term "prodrug", as used in this application, refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted to the most active parental form. See, for example, Wilman, “Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pages 375-382, 615 ^Q Meeting Belfast (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery”, Directed Drug Delivery, Borchardt etal., (Ed.), Pages 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide prodrugs or optionally substituted phenylacetamide prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted to more active free cytotoxic drugs. Examples of cytotoxic drugs that can be derived into a pro-drug form for use in this invention include, but are not limited to, the chemotherapeutic agents described above.
[0146] The term "cytokine" is a generic term for proteins released by a population of cells that act in another cell as intercellular mediators. Examples of such cytokines are lymphokines, monocytes and traditional polypeptide hormones. Included among cytokines
70/160 are growth hormones such as human growth hormone, human growth hormone (HGH), N-methionyl, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH); epidermal growth factor (EGF); liver growth factor; fibroblast growth factor (FGF); prolactin; placental lactogen; alpha and beta factor of tumor necrosis; Mullerian inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors like NGF-alpha; platelet growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and -gamma; colony-stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL -10, IL11, IL-12; IL-18 is a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors including LIF and ligand kit (KL). As used in the present application, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalent of native sequence cytokines.
[0147] By "cytokine antagonist" is meant a molecule that partially or completely blocks, inhibits or neutralizes the biological activity of at least one cytokine. For example, cytokine antagonists can inhibit cytokine activity by inhibiting cytokine expression and / or secretion, or by binding to a cytokine or cytokine receptor. Cytokine antagonists include antibodies, synthetic peptides or
71/160 native sequence, immunoadhesins, and small molecule antagonists that bind to a cytokine or cytokine receptor. The cytokine antagonist is optionally conjugated or fused to a cytotoxic agent. Exemplary TNF antagonists are etanercept (ENBREL®), infliximab (REMICADE®) and adalimumab (HUMIRA ™).
[0148] The term "immunosuppressive agent", as used in the present application, refers to substances that act to suppress or mask the immune system of the subject being treated. These include substances that suppress cytokine production, downregulate or suppress self-antigen expression, or mask MHC antigens. Examples of immunosuppressive agents include 2-amino-6-aryl-5-substituted pyrimidines (see US patent 4,665,077); mycophenolate mofetil such as CELLCEPT®, azathioprine (IMURAN®, AZASAN® / 6-mercaptopurine; bromocriptine; danazol; dapsone; glutaraldehyde (which masks MHC antigens, as described in US patent 4,120,649); anti-idiotypic antibodies to antigen antigens MHC and MHC fragments; cyclosporine A; steroids, such as corticosteroids and glucocorticosteroids, for example, prednisone, prednisolone, such as PEDIAPRED® (prednisolone sodium phosphate) or ORAPRED® (prednisolone sodium phosphate solution), methylprednisolone and dexametas ; methotrexate (oral or subcutaneous) (RHEUMATREX®, TREXALL ™); hydroxychloroquine / chloroquine; sulfasalazine; leflunomide; cytokines or cytokine receptor antagonists, including anti-interferon -γ, β or -α antibodies, necrosis factor a antibodies anti-tumor (infliximab or adalimumab), anti-TNFα immunoadhesin (ENBREL®, etanercept), anti-tumor necrosis factor-β antibodies, anti-interleukin-2 antibodies and receptor antibodies anti-IL-2 antibodies; anti-LFA-1 antibodies, including anti-CD11a and anti-CD18 antibodies; anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; polyclonal or pan-T antibodies, or monoclonal anti-CD3 or anti-CD4 / CD4a antibodies;
72/160 soluble peptide containing an LFA-3 binding domain (WO 90/08187); streptokinase; TGF-β; streptodornase; RNA or DNA from the host; FK506; RS-61443; deoxiespergualin; rapamycin; T cell receptor (Cohen etal., US patent 5,114,721); T cell receptor fragments (Offner et al. Science 251: 430-432 (1991); WO 90/11294; laneway, Nature 341: 482 (1989); and WO 91/01133); T cell receptor antibodies (EP patent 340,109) such as T10B9; cyclophosphamide (CYTOXAN®); dapsone; penicillamine (CUPRIMINE®); plasma exchange; or intravenous immunoglobulin (IVIG). These can be used alone or in combination with each other, in particular combinations of steroid and another immunosuppressive agent or as combinations followed by a maintenance dose with a non-steroidal agent to reduce the need for steroids.
[0149] An "analgesic" refers to a drug that acts to inhibit or suppress pain in a subject. Exemplary painkillers include non-steroidal anti-inflammatory drugs (NSAIDs), including ibuprofen (MOTRIN®), naproxen (NAPROSYN®), acetylsalicylic acid, indomethacin, sulindac and tolmetin, including salts and derivatives thereof, as well as several other drugs used for reduce acute pain that may occur, including anticonvulsants (gabapentin, phenylamine, carbamazepine) or tricyclic antidepressants. Specific examples include acetaminophen, aspirin, amitriptyline (ELAVIL®), carbamazepine (TEGRETOL®), phenyltoin (DILANTIN®), gabapentin (NEURONTIN®), (E) -N-vanylyl-8-methyl-6-noneamide (CAPSAICINA®) or a nerve block.
[0150] “Corticosteroids” refer to any one of several synthetic or naturally occurring substances, with the general chemical structure of steroids that mimic or enhance the effects of naturally occurring corticosteroids. Examples of synthetic corticosteroids include
73/160 prednisone, prednisolone (including methylprednisolone), dexamethasone, triamcinolone and betamethasone.
[0151] A "cancer vaccine" as used in the present application is a composition that stimulates an immune response in an individual against cancer. Cancer vaccines typically consist of a source of cancer-associated material or cells (antigen) that may be autologous (from it) or allogeneic (from others) to the subject, along with other components (for example, adjuvants) ) to further stimulate and increase the immune response against the antigen. Cancer vaccines can result in stimulation of the subject's immune system to produce antibodies to one or more specific antigens, and / or to produce killer T cells to attack cancer cells that have those antigens.
[0152] "Cytotoxic radiation therapy" as used in the present application refers to radiation therapy that inhibits or prevents cell function and / or causes cell destruction. Radiotherapy therapy may include, for example, irradiation of an external beam of light or therapy with a labeled radioactive agent, such as an antibody. The term is intended to include the use of radioactive isotopes (for example, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , Ra ²²³ , P ³² and Lu radioactive isotopes) .
[0153] A "subject" is a vertebrate, like a mammal, for example, a human. Mammals include, but are not limited to, farm animals (such as cows), sport animals, domestic animals (such as cats, dogs and horses), primates, mice and rats.
[0154] Except where otherwise indicated by context, the terms "first" polypeptide and "second" polypeptide, and variants thereof, are simply generic identifiers, and are not to be taken as a specific identification or a specific polypeptide or a antibody component of the invention.
74/160 [0155] Commercially available reagents cited in the Examples were used according to the manufacturer's instructions, unless otherwise indicated. The origin of these cells identified in the Examples below, and throughout the specification, by the ATCC accession numbers is the American Culture Type Collection, Manassas, VA. Unless otherwise noted, the present invention uses standard recombinant DNA technology procedures, such as those described above and in the following books: Sambrook et al., Above. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, NY, 1989); Innis et al., PCR Protocols ·. A Guide to Methods and Applications (Academic Press, Inc., NY, 1990); Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Press, Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (IRL Press, Oxford, 1984); Freshney, Animal Cell Culture, 1987; Coligan et al., Current Protocols in Immunology, 1991.
[0156] Throughout this specification and the claims, the word "understands" or variations such as "understand" or "that understands" should be understood to imply the inclusion of a declared whole number or a group of whole numbers, but not the exclusion of any other whole number or group of whole numbers.
II. Construction of Heteromultimeric Proteins [0157] Typically, the heteromultimeric proteins described in the present application will comprise a significant portion of an antibody Fc region.
Heteromultimerization domains [0158] Heteromultimeric proteins comprise a heteromultimerization domain. To generate a substantially homogeneous population of heterodimeric molecules, the heterodimerization domain must have a strong preference for the formation of heterodimers over homodimers.
75/160
Although the heteromultimeric proteins exemplified in the present application use orifice protrusion technology to facilitate heteromultimerization, those skilled in the art will appreciate other heteromultimerization domains useful in the present invention.
Orifice Lumps [0159] The use of orifice lumps as a method of producing multispecific antibodies is well known in the art. See US patent 5,731,168 issued March 24, 1998 assigned to Genentech, PCT publication WO 2009089004 published July 16, 2009 and granted to Amgen, and US patent publication 20090182127 published July 16, 2009 and granted for Novo Nordisk A / S. See also Marvin and Zhu, Acta Pharmacologica Sincia (2005) 26 (6): 649658 and Kontermann (2005) Acta Pharacol. Sin., 26: 1-9. A brief discussion is provided in this application.
[0160] The "bulge" refers to at least one side chain of amino acid, which protrudes from the interface of a first polypeptide and is therefore positionable in a compensatory cavity at the adjacent interface (that is, the interface of a second polypeptide) in order to stabilize the heteromultimer, and thereby favor the formation of heteromultimer over homomultimer formation, for example. The bulge can exist at the original interface or can be introduced synthetically (for example, by changing the nucleic acid that encodes the interface). Normally, the nucleic acid encoding the interface of the first polypeptide is altered to encode the bulge. To achieve this, the nucleic acid that encodes at least one "original" amino acid residue at the interface of the first polypeptide is replaced by nucleic acid that encodes at least one "imported" amino acid residue that has a greater side chain volume than the original amino acid residue. It will be appreciated that there may be more than one
76/160 original and corresponding imported waste. The upper limit for the number of original residues that are replaced is the total number of residues at the interface of the first polypeptide. The side chain volumes of the various amine residues are shown in the table below.
Table 2
Properties of Amino Acid Residues
Amino Acid One Letter Abbreviation PASTA ^a (daltons) VOLUME ^b (Angstrom ³ ) Accessible Surface Area ⁰ (Angstrom ² ) Alanine (Ward) THE 71.08 88.6 115 Arginine (Arg) R 156.20 173.4 225 Asparagine (Asn) N 114.11 117.7 160 AcidAspartic (Asp) D 115.09 111.1 150 Cysteine (Cys) Ç 103.14 108.5 135 Glutamine (Gin) Q 128.14 143.9 180 Glutamic Acid (Glu) AND 129.12 138.4 190 Glycine (Gly) G 57.06 60.1 75 Histidine (His) H 137.15 153.2 195 Isoleucine (lie) I 113.17 166.7 175 Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18 168.6 200 Methionine (Met) M 131.21 162.9 185 Phenylalinine (Phe) F 147.18 189.9 210 Proline (Pro) P 97.12 122.7 145 Serina (Ser) s 87.08 89.0 115 Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) w 186.21 227.8 255 Tyrosine (Tyr) Y 163.18 193.6 230 Valina (Go) V 99.14 140.0 155
^a Molecular weight of amino acids minus that of water. Values of
Handbook of Chemistry and Physics, 43 ^â ed. Cleveland, Chemical Rubber
Publishing Co., 1961.
^b AA values Zamyatnin, Prog. Biophys. Mol. Biol. 24: 107123, 1972.
77/160 ^c Values of C. Chothia, J. Mol. Biol. 105: 1-14, 1975. The accessible surface area is defined in Figures 6 to 20 of that reference.
[0161] Preferred imported residues for the formation of a bulge are usually naturally occurring amino acid residues and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W). The most preferred are tryptophan and tyrosine. In one embodiment, the original residue for the formation of the bulge has a small side chain volume, such as alanine, asparagine, aspartic acid, glycine, serine, threonine or valine.
[0162] A "cavity" refers to at least one amino acid side chain that is embedded from the interface of a second polypeptide and therefore accommodates a corresponding protuberance at the adjacent interface of a first polypeptide. The cavity can exist at the original interface or can be introduced synthetically (for example, by changing the nucleic acid that encodes the interface). Normally, the nucleic acid encoding the interface of the second polypeptide is altered to encode the well. To achieve this, the nucleic acid that encodes at least one “original” amino acid residue at the interface of the second polypeptide is replaced by DNA that encodes at least one “imported” amino acid residue that has a smaller volume of the side chain than the residue of original amino acid. It will be appreciated that there may be more than one original and corresponding imported waste. The upper limit for the number of original residues that are replaced is the total number of residues at the interface of the second polypeptide. The side chain volumes of the various amino acid residues are shown in Table 2 above. Preferred imported residues for the formation of a cavity are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T) and valine (V). The
78/160 most preferred are serine, alanine or threonine. In one embodiment, the original waste for the formation of the cavity has a large side chain volume, such as tyrosine, arginine, phenylalanine or tryptophan.
[0163] An "original" amino acid residue is one that is replaced by an "imported" residue that may have a side chain volume less or greater than the original residue. The imported amino acid residue may be a naturally occurring or non-naturally occurring amino acid residue, but it is preferably the first. The naturally occurring amino acid residues are those residues encoded by the genetic code and listed in Table 2 above. By "non-naturally occurring" amino acid residue is meant a residue that is not encoded by the genetic code, but is capable of covalently attaching adjacent to the amino acid residue (s) in the polypeptide chain . Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine and other analogues of amino acid residues such as, for example, those described in Ellman et al. Meth. Enzym. 202: 301336 (1991). To generate this naturally occurring amino acid residue, the procedures by Noren et al. Science 244: 182 (1989) and Ellman et al., Above can be used. In short, this involves chemical activation of a tRNA suppressor with a non-naturally occurring amino acid residue followed by transcription and translation of RNA in vitro. The method of the present invention involves replacing at least one original amino acid residue, but more than one original residue can be replaced. Typically, no more than the total residues at the interface of the first or second polypeptide will comprise residues of original amino acids that are replaced. Typically, the original waste for replacement is "rooted". "Rooted" means that the residue is essentially inaccessible to the solvent. Generally, imported waste does not
79/160 is cysteine to prevent possible oxidation or mismatch of disulfide bridges.
[0164] The protrusion is “positionable” in the cavity, which means that the spatial location of the protuberance and the cavity at the interface of a first polypeptide and a second polypeptide, respectively, and the sizes of the protuberance and cavity are such that the bulge can be located in the cavity without significantly disturbing the normal association of the first and second polypeptides at the interface. Since protuberances such as Tyr, Phe and Trp do not typically extend perpendicularly from the interface axis and have preferential conformations, the alignment of a protuberance with a corresponding cavity is based on the projection of the protuberance / pairing of the cavity based on a three-dimensional structure like that obtained by X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved with the use of techniques widely accepted in the art.
[0165] By "original nucleic acid or template" is meant the nucleic acid that encodes a polypeptide of interest that can be "altered" (that is, genetically engineered or mutated) to encode a protuberance or cavity. The original or starting nucleic acid can be a naturally occurring nucleic acid, or it can comprise a nucleic acid that has undergone the previous change (for example, a humanized antibody fragment). By "alteration" of the nucleic acid is meant that the original nucleic acid is mutated by insertion, deletion or substitution of at least one codon encoding an amino acid residue of interest. Typically, a codon that encodes an original waste is replaced by a codon that encodes an imported waste. Techniques for genetically modifying DNA in this way have been reviewed in Mutagenesis: _a Practical Approach, M. J. McPherson, Ed., (IRL Press, Oxford, UK. (1991), and
80/160 includes site-directed mutagenesis, cassette mutagenesis and polymerase chain reaction (PCR) mutagenesis, for example ,. By mutation an original nucleic acid / template, original polypeptide / template encoded by the original nucleic acid / template is thereby altered accordingly.
[0166] The protuberance or cavity can be "introduced" at the interface of a first or a second polypeptide by synthetic means, for example, by recombinant techniques, peptide synthesis in vitro, those techniques for introducing amino acid residues that do not occur previously described naturally, by enzymatic or chemical coupling of peptides or some combination of these techniques. Consequently, the bulge or cavity that is "introduced" is "unnaturally occurring" or "non-native", which means that it does not exist in nature or in the original polypeptide (for example, a humanized monoclonal antibody).
[0167] Generally, the imported amino acid residue to form the bulge has a relatively small number of "rotamers" (for example, about 3 to 6). A "rotamer" is an energetically favorable conformation of an amino acid side chain. Rotamer numbers for the various amino acid residues are reviewed in Ponders and Richards, J. Mol. Biol. Biol. 193: 775-791 (1987).
Other Mutations [0168] The Fc polypeptides described in the present application may have mutations that confer decreased detachment, decreased headstock formation or increased total yield compared to the wild type Fc polypeptide or protuberance in Fc polypeptide orifice. The Fc variant comprises at least one, two, three, four, five, six, seven, eight, nine or ten substitutions in residues selected from S239, V240, F241, F243, V264, R301, K334, Y349, T350, L368, K370, N389, Y391, K392, P395, P396, D399, F405, Y407 on at least one heavy chain with
81/160 an amino acid that is different from that present in a wild type Fc polypeptide. It may be desirable to alter the effector function and it is contemplated that some of the mutations may increase or decrease the effector function. It is preferred that the mutations do not significantly alter other functional characteristics of the antibody, for example, the effector function.
III. Vectors, Host Cells and Recombinant Methods [0169] For recombinant production of a heteromultimeric protein (for example, an antibody) of the invention, the encoding nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of DNA) or for expression . The DNA encoding the antibody is immediately isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that are able to specifically bind to the genes encoding the antibody's heavy and light chains). Many vectors are available. The choice of the vector depends, in part, on the host cell to be used. Generally, the preferred host cells are both prokaryotic and eukaryotic in origin (usually from mammals, but also including fungi (for example, yeast), insects, plants and nucleated cells from other multicellular organisms). It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IGA, IgD and IgE constant regions, and that these constant regions can be obtained from any human or animal species.
The. Generation of Heteromultimeric Proteins Using Prokaryotic Host Cells
i. Vector Construction [0170] Polynucleotide sequences encoding polypeptide components of the heteromultimeric proteins (for example, an antibody) of the invention can be obtained using techniques of
82/160 standard recombination. The desired polynucleotide sequences can be isolated and sequenced from, for example, cells that produce antibody, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the sequences that encode the polypeptides are inserted into a recombinant vector, capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. The selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains several components, depending on its function (amplification or expression of heterologous polynucleotide, or both) and its compatibility with the specific host cell in which it resides. The components of the vector generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, a heterologous nucleic acid insert and a sequence transcription termination.
[0171] In general, plasmid vectors that contain replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector usually carries a replication site, as well as marker sequences that are capable of providing phenotypic selection in the transformed cells. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species. pBR322 contains genes that code for resistance to ampicillin (Amp) and tetracycline (Tet) and thus provides easy means for identifying transformed cells. pBR322, its derivatives or other microbial plasmids or bacteriophages also
83/160 can contain, or be modified to contain, promoters that can be used by the microbial organism to express endogenous proteins. Examples of pBR322 derivatives used for expression of specific antibodies are described in detail in Carter etal., US patent 5,648,237.
[0172] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transformation vectors along with these hosts. For example, bacteriophage such as ÀGEM.TM.-11 can be used in the manufacture of a recombinant vector that can be used to transform susceptible host cells, such as E. coli LE392.
[0173] The expression vector of the invention can comprise two or more pairs of cistron promoter, which encode each of the components of the polypeptide. A promoter is an untranslated regulatory sequence located upstream (5 ') in relation to a cistron that modulates its expression. Prokaryotic promoters can typically be divided into two classes, inducible and constitutive. An inducible promoter is a promoter that initiates increased levels of cistron transcription under its control, in response to changes in the condition of the culture, for example, the presence or absence of a nutrient or a change in temperature.
[0174] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be functionally linked to the DNA cistron that encodes, for example, the light or heavy chain, by removing the promoter from the DNA source, through restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the invention. Both, the native promoter sequence and many heterologous promoters, can be used for amplification and / or direct expression of the target genes. In some embodiments, heterologous promoters are used, as they generally allow for greater
84/160 higher transcription and yield of the expressed target gene, compared to the native target polypeptide promoter.
[0175] Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the β-galactamase and lactose promoter systems, a tryptophan (trp) promoter system and hybrid promoters such as the tac or trc promoter. However, other promoters that are functional in bacteria (such as other known bacterial promoters or phages) are also suitable. Their nucleotide sequences were published, thereby allowing a person skilled in the art to functionally link them to the cistrons that encode the heteromultimeric protein genes, for example, the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269), using ligands or adapters to provide any necessary restriction sites.
[0176] In one embodiment of the invention, each cistron in the recombinant vector comprises a secretion signal sequence component that directs the translocation of the expressed polypeptides across a membrane. In general, the signal sequence can be a component of the vector, or it can be a part of the DNA of the target polypeptide that is inserted into the vector. The signal sequence selected for the purpose of this invention must be that recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequences for heterologous polypeptides, the signal sequence is replaced by a prokaryote signal sequence selected, for example, from the group consisting of alkaline phosphatase, penicillinase, Ipp or enterotoxin II leaders (STII ) heat stable, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.
85/160 [0177] In another embodiment, the production of immunoglobulins, according to the invention, can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences in each cistron. In this regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins in the cytoplasm. Certain host strains (for example, E. coli trxB 'strains) provide conditions in the cytoplasm that are favorable for disulfide bridge formation, thereby allowing for folding and appropriate assembly of subunits of the expressed protein. See Proba and Pluckthun Gene, 159: 203 (1995).
[0178] Prokaryote host cells suitable for expression of heteromultimeric proteins (e.g., antibodies) of the invention include Archaebacteria and Eubacteria, as Gram-negative or Gram-positive organisms. Examples of useful bacteria include Escherichia (for example, E. coll), Bacilli (for example, B. subtilis), Enterobacteria, Pseudomonas species (for example, P. aeruginosa), Salmonella typhimurium, Serratia marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla or Paracoccus. In one embodiment, gram-negative cells are used. In one embodiment, E. coli cells are used as hosts for the invention. Examples of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular Biology, volume 2 (Washington, DC: American Society for Microbiology, 1987), pages 1190-1219; ATCC deposit N ^Q 27,325) and derivatives thereof, including lineage 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA (nmpc-fepE) degP41 kan ^R (US patent 5,639,635). Other strains and derivatives thereof, such as E. coli 294 (ATCC 31.446), E. coll B, E. colix 1776 (ATCC 31.537) and E. coli RV308 (ATCC 31.608) are also suitable. In one embodiment, E. coli ΔΙρρ finds specific use. These examples are illustrative rather than limiting. Methods
86/160 for constructing derivatives of any of the aforementioned bacteria that have defined genotypes are known in the art and described, for example, in Bass et al., Proteins, 8: 309-314 (1990). Generally, it is necessary to select the appropriate bacteria taking into account the replicon replicability in the cells of a bacterium. For example, E. coll, Serratia or Salmonella species may be suitable for use as hosts when well-known plasmids such as pBR322, pBR325, pACYC177 or pKN410 are used to provide the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors can be desirably incorporated into the cell culture.
ii. Polypeptide Production [0179] Host cells are transformed with the expression vectors described above and cultured in a conventional modified nutrient medium, appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences.
[0180] Transformation means introducing DNA into the prokaryote host, so that the DNA is replicable, both as an extrachromosomal element and as a chromosome member. Depending on the host cell used, the transformation is done using standard techniques, appropriate for these cells. Calcium treatment, which employs calcium chloride, is generally used for bacterial cells that contain substantial barriers in the cell wall. Another method for transformation employs polyethylene glycol / DMSO. Yet another technique used is electroporation.
[0181] Prokaryote cells used to produce the polypeptides of the invention are cultured in a medium known in the art and suitable for culture of the selected host cells. Examples of suitable media include Luria broth (LB) plus nutrient supplements
87/160 required. In some embodiments, the medium also contains a selection agent, chosen based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added in the medium for the growth of cells that express the ampicillin resistance gene.
[0182] Any necessary supplement, in addition to sources of carbon, nitrogen and inorganic phosphate, can also be included in appropriate concentrations, introduced alone or as a mixture with another supplement or medium, as a source of complex nitrogen. Optionally, the culture medium can contain one or more reducing agents, selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol.
[0183] Prokaryotic host cells are grown at appropriate temperatures. For E. coli growth, for example, the preferred temperature is in the range of about 20 ^Q C to about 39 ^Q C, more preferably about 25 ^Q C to about 37 ^Q C, even more preferably about of 30 ^Q C. The pH of the medium can be any pH in the range of about 5 to about 9, depending mainly on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, and more preferably about 7.0.
[0184] If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for activation of the promoter. In one embodiment of the invention, PhoA promoters are used to control transcription of the polypeptides. Consequently, the transformed host cells are cultured in a phosphate-limiting medium for induction. Preferably, the phosphate limiting medium is C.R.A.P medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263: 133-147). A variety of other inductors
88/160 can be used, according to the construct of the vector employed, as is known in the art.
[0185] In one embodiment, the first and second host cells containing Fc are cultured separately and the expressed polypeptides of the present invention are secreted and recovered from the host cell periplasm, separately. In a second embodiment, the first and second Fc-containing host cells are cultured separately and before isolation of the Fc-containing polypeptides, the two host cell cultures are mixed together, and the cells are pelleted. In a third embodiment, the first and second host cells containing Fc are grown separately, centrifuged and resuspended separately and then mixed together before isolation of the Fc-containing polypeptides. In a fourth embodiment, the first and second host cells containing Fc are cultured together in the same culture vessel. Protein recovery typically involves disruption of the microorganism's cell membrane, usually by means such as osmotic shock, sonication or lysis. Once cells are disrupted, cell fragments or whole cells can be removed by centrifugation or filtration. Proteins can also be purified, for example, by resin affinity chromatography. Alternatively, proteins can be transported to and isolated from the culture medium. The cells can be removed from the culture and the culture supernatant filtered and concentrated for further purification of the produced proteins. The expressed polypeptides, moreover, can be isolated and identified, using commonly known methods, such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay. The isolated polypeptides will be used to produce the heteromultimeric proteins.
89/160 [0186] In one embodiment of the invention, the production of heteromultimeric protein (e.g., antibody) is conducted in large quantities by a fermentation process. Several batch-fed batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, preferably around 1,000 to 100,000 liters of capacity. These fermenters use impulse stirrers to distribute oxygen and nutrients, especially glucose (the preferred source of carbon / energy). Small-scale fermentation generally refers to fermentation in a fermenter that has no more than approximately 100 liters of volumetric capacity, and can vary from about 1 liter to about 100 liters.
[0187] In a fermentation process, the induction of protein expression is typically initiated after the cells have grown under conditions suitable for a desired density, for example, an OD ₅₅₀ of about 180 to 220, in which the cell stages are primary stationary phase. Several inductors can be used, according to the construct of the vector employed, as is known in the art and described above. The cells can be cultured for shorter periods before induction. The cells are usually induced for about 12 to 50 hours, although longer or shorter induction periods can be used.
[0188] To improve the yield and production quality of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the assembly and appropriate folding of secreted heteromultimer proteins (eg antibodies), additional vectors that overexpress chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and / or DsbG) or FkpA (a peptidilprolil cis , transisomerase with chaperone activity) can be
90/160 used to co-transform host prokaryotic cells. Chaperone proteins have been shown to facilitate folding and the appropriate solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274: 19601-19605; Georgiou et al., US patent 6,083,715; Georgiou et al., US patent 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275: 17100-17105; Ramm and Pluckthun (2000) J. Biol. Chem. 275: 17106-17113; Arie et al. (2001) Mol. Microbiol. 39: 199-210.
[0189] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient in proteolytic enzymes can be used for the present invention. For example, host cell lines can be modified to effect genetic mutation (s) in genes encoding known bacterial proteases, such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and described, for example, in Joly et al. (1998), Proc. Natl. Acad. Know. USA 95: 2773-2777; Georgiou et al., US patent 5,264,365; Georgiou etal., US patent 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).
[0190] In one embodiment, strains of E. coli deficient in proteolytic enzymes and transformed with plasmids that overexpress one or more chaperone proteins are used as host cells in the expression system of the invention. In a second embodiment, the E. coli strain is deficient for an outer membrane lipoprotein (ΔΙρρ).
wm. Purification of Heteromultimeric Protein [0191] In one embodiment, the heteromultimeric protein produced in the present application is further purified to obtain preparations that are substantially homogeneous for further testing and use. Can be
91/160 standard protein purification methods known in the art are employed. The following procedures are exemplary suitable purification procedures: fractionation in immunoaffinity or ion exchange columns, precipitation in ethanol, reverse phase HPLC, chromatography on silica or cation exchange resin such as DEAE, chromato-focusing, SDS-PAGE, precipitation with ammonium sulfate and gel filtration using, for example, Sephadex G-75.
[0192] In one embodiment, Protein A immobilized on a solid phase is used for immunoaffinity purification, for example, of entire antibody products of the invention. Protein A is a 41 kD cell wall protein from Staphylococcus aureas that binds with a high affinity to the antibody Fc region. Lindmark et al. (1983) J. Immunol. Meth. 62: 1-13. The solid phase in which Protein A is immobilized is preferably a column comprising a glass or silica surface, more preferably a controlled porous glass column or a column of silicic acid. In some applications, the column was coated with a reagent, such as glycerol, in an attempt to prevent non-specific adherence of contaminants.
[0193] According to the first purification step, the preparation derived from cell culture, as described above, is applied to the solid phase of immobilized Protein A to allow specific binding of the antibody of interest to Protein A. The solid phase is then washed to remove contaminants not specifically bound to the solid phase. The heteromultimeric protein (e.g., antibody) is recovered from the solid phase by elution.
B. Generation of Heteromultimeric Proteins Using Eukaryotic Host Cells [0194] The components of the vector generally include, but are not limited to, one or more of the following: a signal sequence, a source of
92/160 replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence.
i. Signal Sequence Component [0195] A vector for use in a eukaryotic host cell can also contain a signal sequence or other polypeptide that has a specific dividing site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably one that is recognized and processed (ie, cleaved by a signal peptidase) by the host cell. In the expression mammalian cells, the mammalian signal sequence, as well as viral secretory leaders, for example, the herpes simplex gD signal, are available. The DNA for this precursor region is linked in the reading frame to the DNA encoding the heteromultimeric protein (s) (for example, antibodies).
ii. Origin of Replication [0196] Generally, an origin of replication component is not required for expression vectors in mammals. For example, the SV40 origin can typically be used only because it contains the initial promoter.
wm. Selection Gene Component [0197] Expression and cloning vectors can contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, neomycin, methotrexate or tetracycline, (b) auxotrophic complement deficiencies, where relevant, or (c) non-critical nutrient supply available from complex media.
[0198] An example of a selection scheme uses a drug to stop the growth of a host cell. These cells, which are successfully transformed with a heterologous gene, produce a protein
93/160 which confers resistance to the drug and, thus, survive the selection regime. Examples of these dominant selections use the drugs neomycin, mycophenolic acid and hygromycin.
[0199] Another example of selectable markers suitable for mammalian cells are those capable of identifying cells competent to occupy the antibody's nucleic acid, such as DHFR, thymidine kinase, metallothionein-1 and II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.
[0200] For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive DHFR antagonist. An appropriate host cell when wild-type DHFR is employed is the Chinese hamster ovarian cell line (CHO) deficient in DHFR activity (eg ATCC CRL-9096).
[0201] Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or cotransformed with DNA sequences encoding an antibody, wild-type DHFR protein and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), they can be selected by cell growth in a medium that contains a selection agent for the selectable marker, such as an aminoglycoside antibiotic, for example, kanamycin, neomycin or G418. See, for example, US patent 4,965,199.
iv. Promoter Component [0202] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is functionally linked to the nucleic acid of the desired Fc-containing polypeptide (s) (eg, antibody). Promoter sequences are known for eukaryotes. In practice, all eukaryotic genes have a region rich in
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AT, located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence of 70 to 80 bases found upstream from the beginning of the transcription of many genes is a CNCAAT region where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence, which can be the signal for adding the poly A tail to the 3' end of the coding sequence. All of these sequences are properly inserted into eukaryotic expression vectors.
[0203] Transcription of the desired Fc-containing polypeptide (s) (eg, antibody) from vectors in mammalian host cells is controlled, for example, by promoters obtained from virus genomes, such as polyoma viruses , fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian 40 virus (SV40), from heterologous mammalian promoters, for example , the actin promoter or an immunoglobulin promoter, or from heat shock promoters, provided as promoters that are compatible with the host cell systems.
[0204] The initial and final promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment, which also contains the SV40 viral origin of replication. The immediate initial promoter of human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expression of DNA in mammalian hosts that uses bovine papilloma virus as a vector is disclosed in US patent 4,419,446. A modification of this system is described in US patent 4,601,978. See also Reyes et al., Nature 297: 598-601 (1982) on human β-interferon cDNA expression in mouse cells by controlling a thymidine kinase promoter from the herpes virus
95/160 simple. Alternatively, the long terminal repeat of the Rous Sarcoma Virus can be used as a promoter.
v. Enhancer Element Component [0205] DNA transcription encoding the desired Fc-containing polypeptide (s) (eg, antibody) by more evolved eukaryotes can be increased by inserting an enhancer sequence into the vector. Many enhancer sequences from mammalian genes are now known (for example, globin, elastase, albumin, α-fetoprotein and insulin genes). In addition, an enhancer can be used from a eukaryotic cell virus. Examples include the SV40 enhancer on the end side of the origin of replication (100270 bp), the enhancer of the cytomegalovirus initial promoter, the polyoma enhancer on the end side of the origin of replication and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) for a description of elements for increasing activation of eukaryotic promoters. The enhancer can be joined to the vector in a 5 'or 3' position for the antibody polypeptide coding sequence, as long as the increase is achieved, but is generally located at a 5 'site from the promoter.
saw. Transcription Termination Component [0206] Expression vectors used in eukaryotic host cells will typically also contain sequences necessary for transcription termination and mRNA stabilization. These sequences are commonly available from the 5 'and, occasionally 3', untranslated regions, from eukaryotes or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA that encodes an antibody. A component of the useful transcription termination is the polyadenylation region of bovine growth hormone. See document WO 94/11026 and the expression vector disclosed therein.
96/160 vii. Host Cell Selection and Transformation [0207] Host cells suitable for cloning or expressing DNA in the vectors in the present application include the more evolved eukaryotic cells described in the present application, including vertebrate host cells. The propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 lines transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney lineage (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); kidney cells from hamster cubs (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; and a strain of human hepatoma (Hep G2).
[0208] Host cells are transformed with the expression or cloning vectors described above, to produce polypeptide (s) containing the desired Fc (eg antibody) and cultured in conventional modified nutrient medium as appropriate to induce promoters, select transformers or amplify the genes encoding the desired sequences.
97/160 saw. Cultivation of Host Cells [0209] Host cells used to produce a desired Fc-containing polypeptide (s) (e.g., antibody) of this invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma) and Dulbecco's Eagle Modified Medium ((DMEM), Sigma) are suitable for culturing host cells In addition, any of the means described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. BiochemA02-.255 (1980), US patents 4,767,704; 4,657,866; ; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or US patent Re. 30,985 can be used as a culture medium for host cells. Any of these media can be supplemented, as needed with hormones and / or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (as a drug GENTAMYCIN ™), trace elements (defined as inorganic components commonly present in final concentration in the micromolar range) and glucose or an equivalent energy source. Any other necessary supplement can also be included in appropriate concentrations that would be known to those skilled in the art. Cultivation conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and will be evident to a person skilled in the art.
ix. Purification of Heteromultimeric Proteins [0210] When using recombinant techniques, polypeptides containing Fc can be produced intracellularly or secreted directly into the medium. If the Fc-containing polypeptide is produced
98/160 intracellularly, as a first step, the particulate fragment, from both host cells and lysed fragments, is removed, for example, by centrifugation or ultrafiltration. When the Fc-containing polypeptide is secreted into the medium, supernatants from these expression systems are usually first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor like PMSF can be included in any of the previous steps to inhibit proteolysis and antibiotics can be included to prevent the growth of unforeseen contaminants.
[0211] The heteromultimer composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity linker depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix in which the affinity binder is attached is most often agarose, but other matrices are available. Mechanically stable matrices, such as controlled porous glass or poly (styrene-divinyl) benzene, allow for faster flow rates and shorter processing times than those that can be achieved with agarose. Where the antibody comprises a Ch3 domain, Bakerbond ABX ™ resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation in an ion exchange column, precipitation in
99/160 ethanol, Reverse Phase HPLC, silica chromatography, SEPHAROSE ™ heparin chromatography, chromatography on an anionic or cationic exchange resin (such as a polyaspartic acid column), chromatofocusing, SDSPAGE and ammonium sulfate precipitation are also available , depending on the antibody to be recovered.
[0212] After any preliminary purification step (s), the mixture comprising the antibody of interest and the contaminants can be subjected to hydrophobic interaction chromatography at low pH, with the use of an elution buffer at a pH between about 2.5 to 4.5, preferably carried out at low salt concentrations (for example, from about 0 to 0.25 M of salt). The production of heteromultimeric proteins may alternatively or additionally (for any of the foregoing specific methods) comprise dialysis of a solution comprising a mixture of the polypeptides.
x. Production of Antibodies Using Baculovirus [0213] Recombinant baculoviruses can be generated by cotransfection of a plasmid encoding an antibody or antibody fragment and BaculoGold ™ DNA virus (Pharmingen) in an insect cell such as a Spodoptera frugiperda cell (for example, Sf9 cells; ATCC CRL 1711) or an S2 cell of Drosophila melanogaster using, for example, lipofectin (commercially available from GIBCO-BRL). In a particular example, an antibody sequence is fused upstream of a marker epitope contained within a baculovirus expression vector. As a marker epitope, it includes the poly-His marker. A variety of plasmids can be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen) or pAcGP67B (Pharmingen). Briefly, the sequence encoding an antibody or a fragment of it can be amplified by PCR with
100/160 primers complementary to the 5 'and 3' regions. The 5 'primer may incorporate restriction enzyme site flanking (selected). The product can then be digested with those restriction enzymes selected and subcloned into the expression vector.
[0214] After transfection with the expression vector, host cells (e.g., Sf9 cells) are incubated for 4 to 5 days at 28 ^Q C and the released virus is collected and used for further amplifications. Viral infection and protein expression can be performed as described, for example, by O'Reilley et al. (Baculovirus expression vectors: A Laboratory Manual. Oxford: Oxford University Press (1994)).
[0215] The expressed poly-His labeled antibody can then be purified, for example, by Ni ²⁺ -chelated affinity chromatography as follows. The extracts can be prepared from Sf9 cells infected with recombinant virus as described by Rupert et al. (Nature 362: 175-179 (1993)). Briefly, the Sf9 cells are washed, resuspended in sonication buffer (25 ml HEPES, pH 7.9; 12.5 mM MgCh; 0.1 mM EDTA; 10% glycerol; 0.1 NP-40 %; 0.4 M KCI), and twice sonicated for 20 seconds on ice. The sonicates are cleared by centrifugation and the supernatant is diluted 50 times in loading buffer (50 mM phosphate, 300 mM NaCI, 10% glycerol, pH 7.8) and filtered through a 0.45 μιτι filter. An NTA Ni ²⁺ agarose column (commercially available from Qiagen) is prepared with a 5 ml bottom volume, washed with 25 ml of water and equilibrated with 25 ml of loading buffer. The filtered cell extract is loaded onto a column at 0.5 ml per minute. The column is washed to the A280 baseline with loading buffer, where the collection of the dot fraction is initiated. Then, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCI, 10% glycerol, pH 6.0), which elutes non-specifically bound proteins.
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After reaching the A280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and silver staining or Western blot with NTA-Ni ²⁺ conjugate for alkaline phosphatase (Qiagen). The fractions containing the eluted His10-labeled antibody are pooled and dialyzed against loading buffer.
[0216] Alternatively, antibody purification can be performed using known chromatography techniques, including, for example, Protein A or Protein G column chromatography. In one embodiment, the antibody of interest can be recovered at from the solid phase of the column by elution in a solution containing a chaotropic agent or mild detergent. Exemplary chaotropic agents and mild detergents include, but are not limited to, Guanidine-HCI, urea, lithium perchlorate, Arginine, Histidine, SDS (sodium dodecyl sulfate), Tween, Triton and NP-40, all of which are commercially available available.
IV. Formation / Assembly of Heteromultimeric Proteins [0217] The formation of the complete heteromultimeric protein involves reassembly of the first and second polypeptides containing Fc by disulfide bridge formation, which in the present invention is referred to as refolding. Refolding includes the association of the first Fc-containing polypeptide with the second Fc-containing polypeptide and the formation of interchain disulfide bridges. Refolding, also called renaturation, in the present invention, is done in vitro.
[0218] Host cells can be cultured using the methods described above either as separate cultures or as a single culture. In one method, the first host cells and second host cells are grown in the same culture vessels (sometimes referred to as co-culture or a mixed culture). In another method, the first and
102/160 second host cells are grown in separate culture vessels. In one method, the separate cultures are processed separately, then mixed / combined before the cell membrane ruptures. In another method, the separate cultures are mixed, then processed before the cell membrane ruptures. In one method, the separate cultures are mixed without further processing before the cell membrane is disrupted. In one method, the single culture comprising the first and second host cells is processed before the cell membrane ruptures. In another method, the cultured cells are not processed before the cell membrane ruptures. The processing of the cells comprises centrifugation and resuspension in an appropriate buffer (for example, extraction buffer).
[0219] Extraction buffers are known in the art and the skilled person will be able to determine which buffer to use without undue experimentation.
[0220] Host cell membranes are disrupted using methods known in the art. These methods include cell membrane permeabilization and cell membrane disintegration. Permeabilizing the cell membrane refers to making the membrane “permeable”, for example, by inserting holes, without destroying the overall integrity of the membrane so that the cell remains viable. In other words, permeabilization provides macromolecular movement across the cell membrane and preserves the cell structure sufficiently to allow for continuous cell viability. In contrast, the disintegration of the cell membrane results in cell contents that are released into the extracellular medium and cell death.
[0221] Methods for disrupting cell membranes include, but are not limited to, enzymatic lysis, sonication, osmotic shock,
103/160 passage through a microfluidizer, addition of EDTA, use of various detergents, solvents (such as toluene, dimethyl sulfoxide, etc.), surfactants (eg, Triton-X 100, Tween 20, etc.), hypotonic buffers, use freezing / thawing, electroporation, and passing through a stainless steel ball homogenizer.
[0222] Once the Fc-containing polypeptides are released from the cell (both by permeabilization and by disintegration) the heteromultimerization domains will lead to the association of heteromultimeric proteins. The inter-chain disulfide formation of the associated Fc-containing polypeptides can proceed with or without the addition of reducing agents. The resulting disulfide bound to the heteromultimeric protein is then purified. Optionally, it can be formulated for research, diagnosis, therapeutic purposes or other purposes.
V. Target Molecules [0223] Examples of molecules that can be targeted by a heteromultimeric protein of that invention include, but are not limited to, soluble whey proteins and their receptors and other membrane-bound proteins (eg adhesins).
[0224] In another embodiment the heteromultimeric protein of the invention is able to bind to one, two or more cytokines, cytokine-related proteins, and cytokine receptors selected from the group consisting of BMPI, BMP2, BMP3B (GDFIO), BMP4, BMP6, BMP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), EPO, FGFI (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST) , FGF5, FGF6 (HST-2), FGF7 (KGF), FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNAI, IFNA2, IFNA4, IFNA5 , IFNA6, IFNA7, IFNBI, IFNG, IFNWI, FELI, FELI (EPSELON), FELI (ZETA), IL1A, IL1B, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B , IL13, IL14, IL15, IL16, IL17, IL17B, IL18,
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IL19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29, IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFB3, LTA (TNF-b), LTB, TNF (TNF-a ), TNFSF4 (0X40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL), TNFSF7 (CD27 ligand), TNFSF8 (CD30 ligand), TNFSF9 (4-1BB ligand), TNFSFIO (TRAIL), TNFSF1I (TRANCE), TNFSF12 (AP03L), TNFSF13 (April), TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF, VEGFB, VEGFC, ILIR1, IL1R2, IL1RL1, LL1 RL2, IL2RA, ILRG2 , IL3RA, IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, ILIORA, ILIORB, IL1IRA, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA, IL21R, IL22R, IL1, IL1, IL1R RAPL2, IL1RN, IL6ST, IL18BP, IL18RAP, IL22RA2, AIFI, HGF, LEP (leptin), PTN and THPO.
[0225] In another embodiment, a target molecule is a chemokine, chemokine receptor or a chemokine-related protein selected from the group consisting of CCLI (I-309), CCL2 (MCP -1 / MCAF), CCL3 (MIP -la), CCL4 (MIP-lb), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCLH (eotaxin), CCL13 (MCP-4), CCL15 (MIP-ld), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19 (MDP-3b), CCL20 (MIP-3a), CCL21 (SLC / exodus-2), CCL22 (MDC / STC-I), CCL23 ( MPIF-I), CCL24 (MPIF-2 / eotaxin2), CCL25 (TECK), CCL26 (eotaxin-3), CCL27 (CTACK / ILC), CCL28, CXCLI (GROI), CXCL2 (GRO2), CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL10 (IP 10), CXCL11 (l-TAC), CXCL12 (SDFI), CXCL13, CXCL14, CXCL16, PF4 (CXCL4), PPBP ( CXCL7), CX3CL1 (SCYDI), SCYEI, XCLI (lymphotactin), XCL2 (SCM-lb), BLRI (MDR15), CCBP2 (D6 / JAB61), CCRI (CKRI / HM145), CCR2 (mcp-IRB / RA), CCR3 (CKR3 / CMKBR3), CCR4, CCR5 (CMKBR5 / ChemR13), CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6), CCR7 (CKR7 / EBII), CC R8 (CMKBR8 / TERI / CKR-LI), CCR9 (GPR-9-6), CCRLI (VSHKI), CCRL2 (L-CCR), XCRI (GPR5 / CCXCRI), CMKLRI, CMKORI (RDCI), CX3CR1 (V28) , CXCR4, GPR2 (CCRIO), GPR31, GPR81 (FKSG80),
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CXCR3 (GPR9 / CKR-L2), CXCR6 (TYMSTR / STRL33 / Bonzo), HM74, IL8RA (IL8Ra), IL8RB (IL8Rb), LTB4R (GPR16), TCPIO, CKLFSF2, CKLFSF3, CKLFSF5, CKLFSF5, CKLFSF5 , BDNF, C5R1, CSF3, GRCCIO (CIO), EPO, FY (DARC), GDF5, HDFIA, DL8, PRL, RGS3, RGS13, SDF2, SLIT2, TLR2, TLR4, TREMI, TREM2 and VHL.
[0226] In another embodiment, the heteromultimeric proteins of the invention are able to bind to one or more targets selected from the group consisting of ABCFI; ACVRI; ACVRIB; ACVR2; ACVR2B; ACVRLI; AD0RA2A; Aggrecan; AGR2; AICDA; AIFI; AIGI; AKAPI; AKAP2; AMH; AMHR2; ANGPTI; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOCI; AIR; AZGPI (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF (BLys); BAGI; BAII; BCL2; BCL6; BDNF; BLNK; BLRI (MDR15); BMPI; BMP2; BMP3B (GDFIO); BMP4; BMP6; BMP8; BMPRIA; BMPRIB; BMPR2; BPAGI (plectin); BRCAI; C19orflO (IL27w); C3; C4A; C5; C5R1; CANTI; CASP1; CASP4; CAVI; CCBP2 (D6 / JAB61); CCLI (1-309); CCLII (eotaxin); CCL13 (MCP-4); CCL15 (MIP-ld); CCL16 (HCC-4); CCL17 (TARC); CCL18 (PARC); CCL19 (MIP-3b); CCL2 (MCP -1); MCAF; CCL20 (MIP-3a); CCL21 (MTP-2); SLC; exodus-2; CCL22 (MDC / STC-I); CCL23 (MPIF-1); CCL24 (MPIF-2 / eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK / ILC); CCL28; CCL3 (MTP-la); CCL4 (MDP-lb); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (mcp-2); CCNAI; CCNA2; CCNDI; CCNEI; CCNE2; CCRI (CKRI / HM145); CCR2 (mcp-IRB / RA); CCR3 (CKR3 / CMKBR3); CCR4; CCR5 (CMKBR5 / ChemR13); CCR6 (CMKBR6 / CKR-L3 / STRL22 / DRY6); CCR7 (CKR7 / EBII); CCR8 (CMKBR8 / TERI / CKR-LI); CCR9 (GPR-9-6); CCRLI (VSHKI); CCRL2 (L-CCR); CD164; CD19; CDIC; CD20; CD200; CD22; CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44; CD45RB; CD52; CD69; CD72; CD74; CD79A; CD79B; CD8; CD80; CD81; CD83; CD86; CDHI (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7; CDH8;
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CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKNIA (p21 Wapl / Cipl); CDKNIB (p27Kipl); CDKNIC; CDKN2A (P16INK4a); CDKN2B; CDKN2C; CDKN3; CEBPB; CERI; CHGA; CHGB; Chitinase; CHST10; CKLFSF2; CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7 (claudin-7); CLN3; CLU (clusterin); CMKLRI; CMKORI (RDCI); CNRI; COL18A1; COLIAI; COL4A3; COL6A1; CR2; CRP; CSFI (MCSF); CSF2 (GM-CSF); CSF3 (GCSF); CTLA4; CTNNBI (b-catenin); CTSB (cathepsin B); CX3CL1 (SCYDI); CX3CR1 (V28); CXCLI (GROI); CXCL10 (IP10); CXCLII (1-TAC / IP-9); CXCL12 (SDFI); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3 (GRO3); CXCL5 (ENA-78 I LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3 (GPR9 / CKR-L2); CXCR4; CXCR6 (TYMSTR / STRL33 I Bonzo); CYB5; CYCI; CYSLTRI; DAB2IP; DES; DKFZp451 J0118; DNCLI; DPP4; E2F1; ECGFI; EDGI; EFNAI; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENO1; ENO2; ENO3; EPHB4; EPO; ERBB2 (Her-2); EREG; ERK8; ESRI; ESR2; F3 (TF); FADD; FasL; FASN; FCERIA; FCER2; FCGR3A; FGF; FGFI (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14; FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23; FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9; FGFR3; FIGF (VEGFD); FELI (EPSILON); FILI (ZETA); FLJ12584; FLJ25530; FLRTI (fibronectin); FLTI; FOS; FOSLI (FRA-I); FY (DARC); GABRP (GABAa); GAGEBI; GAGECI; GALNAC4S-6ST; GAT A3; GDF5; GFI1; GGT1; GM-CSF; GNASI; GNRHI; GPR2 (CCRIO); GPR31; GPR44; GPR81 (FKSG80); GRCCIO (CIO); GRP; GSN (Gelsolin); GSTPI; HAVCR2; HDAC4; HDAC5; HDAC7A; HDAC9; HGF; HIFIA; HDPI; histamine and histamine receptors; HLA-A; HLADRA; HM74; HMOXI; HUMCYT2A; ICEBERG; ICOSL; ID2; IFN-a; IFNAI; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgama; DFNWI; IGBPI; IGFI; IGFIR; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-I; IL10; IL10RA; IL10RB; IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1; IL13RA2;
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IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; IL17R; IL18; IL18BP; IL18R1; IL18RAP; IL19; IL1 A; IL1B; ILIF10; IL1F5; IL1F6; IL1F7; IL1F8; IL1F9; IL1HYI; IL1RI; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1; IL1 RL2, ILIRN; IL2; IL20; IL20RA; IL21R; IL22; IL22R; IL22RA2; IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG; IL3; IL30; IL3RA; IL4; IL4R; IL5; IL5RA; IL6; IL6R; IL6ST (glycoprotein 130); EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB; DL9; DL9R; DLK; INHA; INHBA; INSL3; INSL4; IRAKI; ERAK2; ITGAI; ITGA2; ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b 4 integrin); JAGI; JAKI; JAK3; JUN; K6HF; KAII; KDR; KITLG; KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3; KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6 (hair specific type H keratin); LAMAS; LEP (leptin); Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2; LTBR; MACMARCKS; MAG or Omgp; MAP2K7 (c-Jun); MDK; MIBI; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2; MMP9; MS4A1; MSMB; MT3 (metallothionectin-ll); MTSSI; MUCI (mucin); MYC; MYD88; NCK2; neurocane; NFKBI; NFKB2; NGFB (NGF); NGFR; NgR-Lingo; NgRNogo66 (Nogo); NgR-p75; NgR-Troy; NMEI (NM23A); N0X5; NPPB; NROBI; NR0B2; NRIDI; NR1D2; NR1H2; NR1H3; NR1H4; NR1I2; NR1I3; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1; NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRPI; NRP2; NT5E; NTN4; ODZI; OPRDI; P2RX7; PAP; PARTI; PATE; PAWR; PCA3; PCNA; PDGFA; PDGFB; PECAMI; PF4 (CXCL4); PGF; PGR; phosphatane; PIAS2; PIK3CG; PLAU (uPA); PLG; PLXDCI; PPBP (CXCL7); PPID; PRI; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP; PSCA; PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21Rac2); RARB; RGSI; RGS13; RGS3; RNFIIO (ZNF144); ROBO2; S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mamaglobin2); SCGB2A2 (mamaglobin 1); SCYEI (endothelial monocyte activation cytokine); SDF2; SERPINAI; SERPINA3; SERP1NB5 (maspin); SERPINEI (FATHER-I); SERPDMF1; SHBG;
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SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2; SPPI; SPRRIB (Sprl); ST6GAL1; STABI; STAT6; STEAP; STEAP2; TB4R2; TBX21; TCPIO; TDGFI; TEK; TGFA; TGFBI; TGFBIII; TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; THIL; THBSI (thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissue factor; TLRIO; TLR2; TLR3; TLR4; TLR5; TLR6; TLR7; TLR8; TLR9; TNF; TNF-a; TNFAEP2 (B94); TNFAIP3; TNFRSFIIA; TNFRSFIA; TNFRSFIB; TNFRSF21; TNFRSF5; TNFRSF6 (Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSFIO (TRAIL); TNFSFI 1 (TRANCE); TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15 (VEGI); TNFSF18; TNFSF4 (0X40 linker); TNFSF5 (CD40 linker); TNFSF6 (FasL); TNFSF7 (CD27 linker); TNFSF8 (CD30 linker); TNFSF9 (4-1BB linker); TOLLIP; Toll-like receivers; TOP2A (topoisomerase Ea); TP53; TPMI; TPM2; TRADD; TRAFI; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREMI; TREM2; TRPC6; TSLP; TWEAK; VEGF; VEGFB; VEGFC; versican; VHL C5; VLA-4; XCLI (lymphotactin); XCL2 (SCM-lb); XCRI (GPR5 / CCXCRI); YYI; and ZFPM2.
[0227] Preferred molecular targets for antibodies encompassed by the present invention include CD proteins such as CD3, CD4, CD8, CD16, CD19, CD20, CD34; CD64, CD200 members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mad, p150.95, VLA-4, ICAM-1, VCAM, alpha4 / beta7 integrin, and alpha / beta3 integrin including both alpha and beta subunits thereof (for example, anti- CD11a, anti-CD18 or anti-CD11b); growth factors like VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (□ alfalFN); TNFalpha, an interleukin, such as IL-1beta, IL-3, IL-4, IL-5, IL-8, IL-9, IL-13, IL17A / F, IL-18, IL-13Ralfa1, IL13Ralfa2, IL- 4R, IL-5R, IL-9R, IgE; blood group antigens; flk2 / flt3 receptor; obesity receptor (OB); mpl receiver; CTLA-4; protein RANKL, RANK, RSV F, protein C, etc.
109/160 [0228] In one embodiment, the heteromultimeric proteins of this invention bind to the protein related to the low density lipoprotein receptor (LRP) -1, LRP-8 or transferrin receptor, and at least one target selected from of the group consisting of 1) beta-secretase (BACE1 or BACE2), 2) alpha-secretase, 3) gamma-secretase, 4) tau-secretase, 5) amyloid precursor protein (APP), 6) death receptor 6 ( DR6), 7) beta amyloid peptide, 8) alpha-synuclein, 9) Parquina, 10) Huntingtin, 11) p75 NTR, and 12) caspase-6.
[0229] In one embodiment, the heteromultimeric proteins of this invention bind to at least two target molecules selected from the group consisting of: IL-lalfa and IL-lbeta, IL-12 and IL-18; IL-13 and IL-9; IL-13 and IL4; IL-13 and IL-5; IL-5 and IL-4; IL-13 and IL-lbeta; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC; IL-13 and MEF; IL-13 and TGF-β; IL-13 and LHR agonist; IL-12 and TWEAK, IL13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 and ADAM8, IL-13 and PED2, IL17A and IL17F, CD3 and CD19, CD138 and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD38 and CD138; CD38 and CD20; CD38 and CD40; CD40 and CD20; CD-8 and IL-6; CD20 and BR3, TNFalpha and TGF-beta, TNFalfa and IL-lbeta; TNFalpha and IL-2, TNFalpha and IL-3, TNFalpha and IL-4, TNFalpha and IL-5, TNFalpha and IL6, TNFalpha and IL8, TNFalpha and IL-9, TNFalpha and IL-10, TNFalpha and IL-11 , TNFalfa and IL-12, TNFalfa and IL-13, TNFalfa and IL-14, TNFalfa and IL-15, TNFalfa and IL-16, TNFalpha and IL-17, TNFalfa and IL-18, TNFalfa and IL-19, TNFalfa and IL-20, TNFalfa and IL-23, TNFalfa and IFNalfa, TNFalfa and CD4, TNFalfa and VEGF, TNFalfa and MIF, TNFalfa and ICAM-1, TNFalfa and PGE4, TNFalfa and PEG2, TNFalfa and RANK ligand, TNFalfa and Te38; TNFalfa and BAFF; TNFalfa and CD22; TNFalpha and CTLA-4; TNFalfa and GP130; TNFa and IL-12p40; VEGF and HER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5, VEGF and IL-8, VEGF and MET, VEGFR and MET receptor, VEGFR and EGFR, HER2 and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR (HERI) and HER2, EGFR and HER3, EGFR and
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HER4, IL-13 and CD40L, IL4 and CD40L, TNFR1 and IL-1 R, TNFR1 and IL-6R and TNFR1 and IL-18R, EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 and BTNO2; IGF1 and IGF2; IGF1 / 2 and Erb2B; MAG and RGM A; NgR and RGM A; NogoA and RGM A; OMGp and RGM A; PDL-I and CTLA-4; and RGM A and RGM B.
[0230] Soluble antigens or fragments thereof, optionally conjugated to other molecules can be used as immunogens to generate antibodies. For transmembrane molecules, such as receptors, fragments of these (for example, the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells that express the transmembrane molecule can be used as the immunogen. These cells can be derived from a natural source (for example, cancer cell lines) or they can be cells that have been transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for the preparation of antibodies will be apparent to those skilled in the art.
SAW. Activity Assays [0231] The heteromultimeric proteins of the present invention can be characterized by their physical / chemical properties and biological functions by various assays known in the art.
[0232] Heteromultimer proteins can furthermore be characterized by a series of assays that include, but are not limited to, N-terminal sequencing, amino acid analysis, high performance liquid chromatography (HPLC) by size exclusion without denaturation , mass spectrometry, ion exchange chromatography and papain digestion.
[0233] In certain embodiments of the invention, the immunoglobulins produced in the present application are analyzed for their biological activities. In some embodiments, the immunoglobulins of the present invention
111/160 are tested for their antigen binding activities. Antigen binding assays that are known in the art and can be used in the present application include, without limitation, any direct or competitive binding assay using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), sandwich immunoassays, immunoprecipitation assays, fluorescent immunoassays and protein A immunoassays. An illustrative antigen binding assay is provided below in the Examples section.
[0234] In one embodiment, the present invention contemplates an altered antibody that has some, but not all, effector functions, which make it a desired candidate for many applications in which the half-life of the antibody in vivo is still important for certain effector functions (as an add-on and ADCC) are unnecessary or harmful. In certain embodiments, the Fc activities of the heteromultimeric protein produced are measured to ensure that only the desired properties are maintained. In vitro and / or in vivo cytotoxicity assays can be conducted to confirm the reduction / depletion of CDC and / or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the heteromultimeric protein is devoid of FcyR binding (then likely to be devoid of ADCC activity), but maintains FcRn binding capacity. The main cells for ADCC mediation, NK cells, express only FcyRIII, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression in hematopoietic cells is summarized in Table 3, on page 464 by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). An example of an in vitro assay to assess ADCC activity of a molecule of interest is described in US patents 5,500,362 or 5,821,337. Effector cells useful for these assays include peripheral blood mononuclear cells (PBMC) and natural killer cells (NK).
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Alternatively, or in addition, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model as disclosed in Clynes et al. PNAS (USA) 95: 652-656 (1998). C1q binding assays can also be performed to confirm that the antibody is unable to bind to C1q and, therefore, devoid of CDC activity. To assess complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). FcRn binding and in vivo release / half-life determinations can also be performed using methods known in the art.
VII. Conjugated Proteins [0235] The invention also provides conjugated proteins, such as conjugated or immunoconjugated antibodies (for example, "antibody-drug conjugates" or "ADC"), comprising any of the heteromultimeric proteins described in the present application (for example, an antibody produced according to the methods described in the present application), where one of the light or heavy chain constant regions is conjugated to a chemical molecule such as a dye or cytotoxic agent, such as a chemotherapeutic agent, a drug, a growth inhibitory agent , a toxin (for example, an enzymatically active bacterial, fungal, plant or animal toxin or fragments thereof), or a radioactive isotope (i.e., a radioconjugate). In particular, as described in the present application, the use of heteromultimerization domains allows the construction of antibodies that contain two different heavy chains (HC1 and HC2), as well as two different light chains (LC1 and LC2). An immunoconjugate constructed using the methods described in the present application can contain the cytotoxic agent conjugated to a constant region of only one of the heavy chains (HC1 or HC2) or only one of the light chains (LC1 or LC2). In addition, due to the immunoconjugate, the cytotoxic agent can be attached to only a single
In the heavy or light chain, the amount of cytotoxic agent to be administered to an individual is reduced in relation to administration of an antibody that has the cytotoxic agent attached to both heavy and light chains. Reducing the amount of cytotoxic agent to be administered to a subject limits the adverse side effects associated with the cytotoxic agent.
[0236] The use of antibody-drug conjugates for the delivery site of cytotoxic or cytostatic agents, that is, drugs to destroy or inhibit tumor cells in the treatment of cancer (Syrigos and Epenetos, Anticancer Research 19: 605-614 (1999 Niculescu-Duvaz and Springer, Adv. Drg. Del. Rev. 26: 151-172 (1997); US patent 4,975,278) allows targeted distribution of the drug component to tumors, and intracellular accumulation in the same, where systemic administration these unconjugated drug agents can result in unacceptable levels of toxicity to normal cells, as well as tumor cells sought to be eliminated (Baldwin et al., Lancet (March 15, 1986): 603-605 (1986); Thorpe, (1985 ) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, A. Pinchera et al. (Ed.s), pages 475-506). Through this, maximum efficiency is sought with minimal toxicity. Both polyclonal antibodies and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., Cancer Immunol. Immunother. 21: 183-187 (1986)). The drugs used in these methods include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al., (1986) above). Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, small molecule toxins such as geldanamycin (Mandler et al., Jour, of the Nat. Cancer Inst. 92 (19): 1573-1581 ( 2000); Mandler et al., Bioorganic & Med. Chem. Letters 10: 1025-1028 (2000); Mandler et al., Bioconjugate Chem. 13: 786-791 (2002)), maytansinoids (EP patent 1391213; Liu et al., Proc. Natl. Acad. Sci.
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USA 93: 8618-8623 (1996)), and calicheamicin (Lode etal., Cancer Res. 58: 2928 (1998); Hinman et al., Cancer Res. 53: 3336-3342 (1993)). Toxins can carry out their cytotoxic and cytostatic effects through mechanisms that include binding to tubulin, binding to DNA or inhibition of topoisomerase. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.
[0237] Chemotherapeutic agents useful in the generation of immunoconjugates are described in the present application (for example, above). Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, active diphtheria toxin non-binding fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain , alpha-sarcin, Aleurites fordii protein, diantin proteins, American Phytolaca proteins (PAPI, PAPII and PAP-S), carantia momordica inhibitor, curcine, crotin, saponaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes. See, for example, WO 93/21232 published on October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ 1, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re. Antibody conjugates and cytotoxic agents are produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCI), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azide compounds (such as bis (p-azidobenzoyl) hexanediamine), bisdiazonium derivatives (such as bis- (p-diazoniumbenzyl) -ethylenediamine) , diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Triamine acid
115/160 pentaacetic 1-isothiocyanatobenzyl-3-methyldiethylene with carbon 14 (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotide to the antibody. See, for example, WO 94/11026.
[0238] Conjugates of an antibody and one or more small molecule toxins, such as calicheamicins, maytansinoids, dolastatins, aurostatins, trichothecenes and CC1065, and derivatives of these toxins that have active toxin, are also contemplated in the present application.
i. Maytansine and Maytansinoids [0239] In some embodiments, the immunoconjugate comprises an antibody of the invention (complete or fragments), conjugated to one or more maytansinoid molecules.
[0240] Maytansinoids are mitotic inhibitors that act by inhibiting the polymerization of tubulin. Maytansine was first isolated from the East African bush, Maytenus serrata (US patent 3,896,111). Subsequently, it has been discovered that certain microbes also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (US patent 4,151,042). Synthetic maytansinol and derivatives and analogues thereof are disclosed, for example, in US patents 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308 .269, 4,309,428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663 and 4,371,533 .
[0241] Maytansinoid drug components are attractive drug components in antibody-drug conjugates because they are: (i) relatively accessible to be prepared by fermentation or chemical modification, derivation of fermentation products, (ii) receptive to derivatization with functional groups suitable for conjugation to antibodies, through non-disulfide ligands, (iii) stable in plasma, and (iv) effective against a variety of tumor cell lines.
116/160 [0242] Immunoconjugates containing maytansinoids, methods for producing them and their therapeutic uses are disclosed, for example, in US patents 5,208,020, 5,416,064 and European patent EP 0 425 235 B1, whose disclosures are expressly incorporated into this application as a reference. Liu et al., Proc. Natl. Acad. Know. USA 93: 8618-8623 (1996) described immunoconjugates that comprise a maytansinoid called DM1, linked to the monoclonal antibody C242, directed against human colorectal cancer. The conjugate has been shown to be highly cytotoxic towards cultured colon cancer cells and has shown antitumor activity in in vivo tumor growth assays. Chari et al., Cancer Research 52: 127-131 (1992) describe immunoconjugates in which a maytansinoid was conjugated, through a disulfide linker, to a murine A7 antibody, which binds to an antigen of the human colon cancer cell line , or another TA.1 murine monoclonal antibody that binds to the HER-2 / neu oncogene. The cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro on the human breast cancer cell line SK-BR-3, which expresses 3 x 10 ⁵ HER-2 surface antigens per cell. The conjugated drug reached a degree of cytotoxicity similar to the maytansinoid-free drug, which could be increased by increasing the number of maytansinoid molecules per antibody molecule. The A7-maytansinoid conjugate showed low systemic cytotoxicity in mice.
[0243] Antibody-maytansinoid conjugates are prepared by chemical bonds in an antibody and a maytansinoid molecule without significantly decreasing the biological activity of both the antibody and the maytansinoid molecule. See, for example, US patent 5,208,020 (its disclosure is expressly incorporated into this application as a reference). An average conjugation of 3 to 4 maytansinoid molecules per antibody molecule showed efficacy in increasing the cytotoxicity of
117/160 target cells, without negatively affecting the function or solubility of the antibody, however, it would be expected that even a toxin / antibody molecule would increase the cytotoxicity above that of the naked antibody. Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in US patent 5,208,020 and in other patents and non-patent publications referred to above. Preferred maytansinoids are maytansinol and maytansinol analogues, modified in the aromatic ring or in other positions of the maytansinol molecule, such as various maytansinol esters.
[0244] There are many linking groups known in the art to make antibody-maytansinoid conjugations, including, for example, those disclosed in US patent 5,208,020 or EP 0 425 235 B1, Chari et al., Cancer Research 52: 127 -131 (1992), and publication of US patent application 2005/0169933, the disclosures of which are expressly incorporated into the present application as a reference. Antibodymaino-transinoid conjugates comprising the SMCC binding component can be prepared as disclosed in US patent application publication 2005/0169933. Linking groups include disulfide groups, thioether groups, labile acid groups, photolabile groups, peptidase labile groups or labile esterases groups, as disclosed in the patents identified above, with disulfide and thioether groups being preferred. Additional linking groups are described and exemplified in the present application.
[0245] Antibody and maytansinoid conjugates can be produced using a variety of bifunctional protein coupling agents such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), derivatives of bifunctional imidoesters (such as dimethyl adipimidate HCI), esters
118/160 active (like disuccinimidyl suberate), aldehydes (like glutaraldehyde), bis-azido compounds (like bis- (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (like bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5difluoro-2,4-dinitrobenzene). Particularly preferred coupling agents for providing a disulfide bond include, N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) (Carlsson et al., Biochem. J. 173: 723-737 [1978]) and N-succinimidyl-4 - (2-pyridylthio) pentanoate (SPP).
[0246] The ligand can be attached to the maytansinoid molecule in various positions, depending on the type of bond. For example, an ester bond can be formed by reacting with a hydroxyl group, using conventional coupling techniques. The reaction can occur in position C-3 which has a hydroxyl group, in position C-14 modified with hydroxymethyl, in position C-15 modified with a hydroxyl group and in position C-20 which has a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.
II. Auristatins and Dolastatins [0247] In some embodiments, the immunoconjugate comprises an antibody of the invention conjugated to dolastatins or peptide analogs of dolostatins and derivatives, auristatins (US patents 5,635,483 and 5,780,588). Dolastatins and auristatins have been shown to not interfere with microtubule dynamics, GTP hydrolysis and nuclear and cellular division (Woyke et al., Antimicrob. Agents and Chemother. 45 (12): 3580-3584 (2001) and has anticancer activity (US patent 5,663 .149) and antifungal (Pettit et al., Antimicrob. Agents Chemother. 42: 2961-2965 (1998)). The drug component, dolastatin or auristatin, can be attached to the antibody via the N (amine) or C ( carboxyl) of the peptide drug component (WO 02/088172).
119/160 [0248] Exemplary auristatin realizations include the drug components monomethyluristatin DE and DF linked at the N-termination, disclosed in “Monomethylvaline Compounds Capable of Conjugation to Ligands”, US patent application publication 2005/0238649, the disclosure of which is expressly incorporated into this application as a reference.
[0249] Typically, peptide-based drug components can be prepared by forming a peptide bridge between two or more amino acids and / or peptide fragments. These peptide bridges can be prepared, for example, according to the liquid phase synthesis method (see, E. Schroder and K. Lübke, “The Peptides”, volume 1, pages 76-136, 1965, Academic Press) is well known in the field of peptide chemistry. The drug components auristatin / dolastatin can be prepared according to the methods of: US patents 5,635,483 and 5,780,588; Pettit et al., J. Nat. Prod. 44: 482-485 (1981); Pettit et al., AntiCancer Drug Design 13: 47-66 (1998); Poncet, Curr. Pharm. Des. 5: 139-162 (1999); and Pettit, Fortschr. Chem. Org. Naturst. 70: 1-79 (1997). See also Doronina, Nat Biotechnol. 21 (7): 778-784 (2003); and “Monomethylvaline Compounds Capable of Conjugation to Ligands”, US patent application publication 2005/0238649, which is fully incorporated into this application as a reference (which discloses, for example, binders and methods of preparing monomethylvaline compounds, such as MMAE and MMAF conjugated to binders).
III. Calicheamicin [0250] In other embodiments, the immunoconjugate comprises an antibody of the invention conjugated to one or more molecules of calicheamicin. The calicheamicin family of antibiotics is capable of producing double strand DNA breaks at subpicomolar concentrations. For the preparation of conjugates of the calicheamicin family, see US patents 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,5,770,710, 5,773,001 and 5,877,296
120/160 (all granted to the American Cyanamid Company). Analog structures of calicheamicin that can be used include, but are not limited to, γ · /, ₂ ', α ₃ ', N-acetyl-γι ¹ , PSAG and θ'ι (Hinman et al., Cancer Research 53: 3336-3342 (1993), Lode et al., Cancer Research 58: 2925-2928 (1998) and the aforementioned US patents to American Cyanamid). Another anti-tumor drug to which the antibody can be conjugated is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not immediately cross the plasma membrane. Therefore, the cellular perception of these agents, through antibody-mediated internalization, greatly increases their cytotoxic effects.
iv. Other Cytotoxic Agents [0251] Other antitumor agents that can be conjugated to the antibodies of the invention or produced according to the methods described in the present application include BCNU, streptozoicin, vincristine and 5-fluorouracil, the family of agents collectively known as the LL-E33288 complex , described in US patents 5,053,394 and 5,770,710, as well as speramicins (US patent 5,877,296).
[0252] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, active non-binding fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, chain Modecin, alpha-sarcin, Aleurites fordii proteins, diantin proteins, American Phytolaca proteins (PAPI, PAPII, and PAP-S), carantia momordica inhibitor, curcine, crotine, saponary officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin and trichothecenes (see, for example, WO 93/21232, published on October 28, 1993).
[0253] The present invention, moreover, contemplates an immunoconjugate formed between an antibody and a compound with activity
121/160 nucleolytic (for example, a ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).
[0254] For the selective destruction of a tumor, the antibody may comprise a highly radioactive atom. A variety of radioactive isotopes are available for the production of radioconjugate antibodies. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and Lu radioactive isotopes. When the conjugate is used for detection, it can comprise a radioactive atom for scintigraphic studies, for example, ^99m tc or I ¹²³ , or a rotating marker for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri ), such as again iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0255] The radio or other markers can be incorporated into the conjugate in a known manner. For example, the peptide can be biosynthesized or it can be synthesized by chemical amino acid synthesis with the use of suitable precursor amino acids involving, for example, fluorine19 in place of hydrogen. Markers such as ^99m tc or I ¹²³ , Re ¹⁸⁶ , Re ¹⁸⁸ and In ¹¹¹ can be attached via a cysteine residue on the peptide. Yttrium-90 can be attached via a lysine residue. The IODOGEN method (Fraker et al., Biochem. Biophys. Res. Commun. 80: 49-57 (1978)) can be used to incorporate iodine-123. Monoclonal Antibodies in Immunoscintigraphy (Chatal, CRC Press 1989) describes other methods in detail.
[0256] Antibody and cytotoxic agent conjugates can be produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), succinimidil-4- (N- maleimidomethyl) cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional imidoester derivatives (like dimethyl adipimidate HCI), active esters (like disuccinimidyl suberate), aldehydes (like glutaraldehyde),
122/160 bis-azido compounds (like bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (like bis- (p-diazoniumbenzoyl) -ethylenediamine), diisocyanates (like toluene 2,6-diisocyanate) and compounds of bis-active fluorine (such as 1,5difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Triamino pentaacetic acid 1-isothiocyanatobenzyl-3-methyldiethylene with carbon 14 (MX-DTPA) is an exemplary chelating agent for the conjugation of radionucleotide to the antibody. See, for example, WO 94/11026. The ligand can be a "cleavable ligand" that facilitates the release of the cytotoxic drug into the cell. For example, a labile acid linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or bisulfide containing linker (Chari et al., Cancer Research 52: 127-131 (1992); US patent 5,208,020).
[0257] The compounds of the invention expressly contemplate, but are not limited to, ADC prepared with crosslinking reagents: BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl- (4-vinylsulfone) benzoate) that are commercially available (for example, together to Pierce Biotechnology, Inc., Rockford, IL., USA). See pages 467-498, 2003-2004 Applications Handbook and Catalog.
v. Preparation of Conjugated Antibodies [0258] In the conjugated antibodies of the invention, an antibody is conjugated to one or more components (for example, drug components), for example, about 1 to about 20 components per antibody, optionally via a linker. Conjugated antibodies can be prepared by several routes, using organic chemistry reactions, conditions and reagents known to those skilled in the art, including: (1) reaction of a
123/160 nucleophilic group of an antibody with a divalent binding reagent through a covalent bond, followed by reaction with a component of interest; and (2) reacting a nucleophilic group of a component with a divalent binding reagent through a covalent bond, followed by reaction with the nucleophilic group of an antibody. Additional methods for preparing conjugated antibodies are described in the present application.
[0259] The binding reagent can be composed of one or more binding components. Exemplary binding components include 6maleimidocaproil (“MC”), maleimidopropanoil (“MP”), valine-citrulline (“val-cit”), alanine-phenylalanine (“ala-phe”), p-aminobenzyloxycarbonyl (“PAB”), NSuccinimidil 4- (2-pyridylthio) pentanoate (“SPP”), N-Succinimidyl 4- (Nmaleimidomethyl) cyclohexane-1 carboxylate (“SMCC '), and N-Succinimidil (4-iodoacetyl) aminobenzoate (“ SIAB ”). Additional binding components are known in the art and some are described in the present application. See also “Monomethylvaline Compounds Capable of Conjugation to Ligands”, US patent application publication 2005/0238649, the content of which is incorporated into this application as a reference in its entirety.
[0260] In some embodiments, the linker may comprise amino acid residues. Exemplary amino acid linking components include a dipeptide, a tripeptide, a tetrapeptide or a pentapeptide. Exemplary dipeptides include: valine-citrulline (vc or val-cit), alaninephenylalanine (af or ala-phe). Exemplary tripeptides include: glycine-valinacitrulin (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues that comprise an amino acid binding component include naturally occurring ones, as well as secondary amino acids and analogues of non-naturally occurring amino acids, such as citrulline. The binding components of amino acids can be created and optimized in their selectivity for enzymatic divation by specific enzymes, for example,
124/160 a tumor associated protease, cathepsin B, C and D or a plasmin protease.
[0261] Nucleophilic groups in antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, for example, lysine, (iii) side chain thiol groups, for example , cysteine, and (iv) hydroxyl groups of sugar or amine, where the antibody is glycosylated. The amine, thiol and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with the electrophilic groups in the binding components and binding reagents including: (i) active esters, such as NHS esters, HOBt esters, haloformates and acid haloids; (ii) alkyl and benzyl haloids, such as haloacetamides; (iii) aldehyde groups, ketones, carboxyl and maleimide. Certain antibodies have reducible interchain disulfides, that is, cysteine bridges. Antibodies can be produced by reaction to conjugate with binding reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus theoretically form two nucleophile reactive thiols. Additional nucleophilic groups can be introduced into the antibodies by reacting lysines with 2-iminothiolane (Traut's reagent), resulting in the conversion of an amine to a thiol. Reactive thiol groups can be introduced into the antibody (or fragment thereof) by introducing one, two, three, four or more cysteine residues (for example, preparing mutant antibodies that comprise one or more non-native cysteine amino acid residues).
[0262] Conjugated antibodies of the invention can also be produced by modifying the antibody to introduce electrophilic components, which can react with the nucleophilic substituent on the binding reagent, drug or other component. Glycosylated antibody sugars can be oxidized, for example, with periodate oxidizing reagents, to form aldehyde or ketone groups, which can react with the amine group of
125/160 binding reagents, drugs or other components. The resulting Schiff imine base groups can form a stable bond or can be reduced, for example, by borohydride reagents to form stable amine bonds. In one embodiment, the reaction of the carbohydrate portion of a glycosylated antibody, with both galactose oxidase and sodium metaperiodate, can produce carbonyl groups (aldehyde and ketone) in the protein that can react with appropriate groups in the drug or another component (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing serine or N-terminal threonine residues can react with sodium metaperiodate, resulting in the production of an aldehyde in place of the first amino acid (Geoghegan and Stroh, Bioconjugate Chem.: 138-146 (1992); US patent 5,362,852). That aldehyde can react with a drug component or nucleophile linker.
[0263] Likewise, nucleophilic groups in a component (such as a drug component) include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thioseminarbazone, hydrazine carboxylate and arylhydrazide groups capable of reacting to forming covalent bonds with electrophilic groups in the binding components and binding reagents, including: (i) active esters, such as NHS esters, HOBt esters, haloformates and acid haloids; (ii) alkyl and benzyl haloids, such as haloacetamides; (iii) aldehyde groups, ketones, carboxyl and maleimide.
[0264] Alternatively, a fusion protein comprising the antibody and the cytotoxic agent can be produced, for example, by recombinant techniques or peptide synthesis. The length of the DNA can comprise respective regions that encode the two portions of the conjugate, either adjacent to each other or separated by a region that encodes a linker peptide that does not destroy the desired properties of the conjugate. In yet another embodiment, the antibody can be conjugated to a "receptor" (such as streptavidin) for use in the pre-targeted tumor, where the conjugate
126/160 antibody-receptor is administered to the individual, followed by removal of unbound conjugate from the circulation with the use of a cleaning agent and then administration of a “binder” (eg avidin) that is conjugated to a cytotoxic agent ( for example, a radionucleotide).
VIII. Utility [0265] The present Fc-variant polypeptides described in the present application find industrial applicability in the production of heteromultimeric proteins.
[0266] The heteromultimeric proteins described in the present application are useful in, for example, therapeutic methods in vitro, ex vivo and in vivo. The invention provides several methods based on the use of one or more of these molecules. In certain pathological conditions, it is necessary and / or desirable to use heteromultimeric proteins, for example, multispecific antibodies. The invention provides these heteromultimeric proteins, which can be used for a variety of purposes, for example, as therapeutic, prophylactic and diagnostic. For example, the invention provides methods of treating a disease, said methods comprising administering to a subject in need of treatment of a heteromultimeric protein of the invention, in which the disease is treated. Any of the heteromultimeric proteins of the invention described in the present application can be used in the therapeutic (or prophylactic or diagnostic) methods described in the present application.
[0267] For example, when the heteromultimeric protein is multivalent, a valuable benefit is the enhanced greediness it represents for its antigens. In addition to having high intrinsic affinity in an antigen-based binding unit (ie, a Fab), normal IgG antibodies also exploit the avidity effect to increase their association with antigens as a result of their bivalent binding towards targets .
127/160 [0268] A heteromultimeric protein directed against two separate epitopes on the same antigen molecule can not only provide the benefit of enhanced binding avidity (due to bivalent binding), but can also acquire new properties that are not associated with either of parental antibodies. Thus, the heteromultimeric proteins of the invention find use, for example, in blocking receptor-ligand interactions.
[0269] The heteromultimeric proteins described in the present application also find use in the application of simultaneously blocking the signaling pathways of two targets with one molecule.
IX. Therapeutic Uses [0270] The heteromultimeric proteins as antibodies and antibody fragments described in the present application (for example, an antibody and / or fragment thereof, produced according to the methods described in the present application) can be used for therapeutic applications. For example, these heteromultimeric proteins can be used for the treatment of tumors, including precancerous, non-metastatic, metastatic and cancerous tumors (for example, early-stage cancer), for the treatment of allergic or inflammatory disorders, or for the treatment autoimmune disease, or for the treatment of a subject at risk of developing cancer (for example, breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma, or ovarian cancer), an allergic or inflammatory disorder , or an autoimmune disease.
[0271] The term cancer encompasses a set of proliferative disorders, including, but not limited to, pre-cancerous growths, benign tumors and malignant tumors. Benign tumors remain localized and do not have the ability to infiltrate, invade or metastasize in distant locations. Malignant tumors will invade and
128/160 will damage other tissues around them. They can also acquire the ability to disconnect from where they started and spread to other parts of the body (metastasize), usually through the bloodstream or through the lymphatic system, where the lymph nodes are located. Primary tumors are classified by the type of tissue from which they arise, metastatic tumors are classified by the type of tissue from which cancer cells are derived. Over time, the cells of a malignant tumor become abnormal and look less and less like normal cells. This change in the appearance of cancer cells is called the tumor grade, and cancer cells are described as being well differentiated, moderately differentiated, weakly differentiated or undifferentiated. Well-differentiated cells are almost normal in appearance and resemble the normal cells from which they originated. Undifferentiated cells are cells that have become so abnormal that it is no longer possible to determine the origin of the cells.
[0272] The tumor can be a solid tumor, a non-solid tumor or a soft tissue tumor. Examples of soft tissue tumors include leukemia (eg, chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenic leukemia, mature mature B-cell lymphoblastic leukemia, chronic lymphocytic leukemia, polyphocytic leukemia, or hairy cell leukemia, ) or lymphoma (for example, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma or Hodgkin's disease). A solid tumor includes any cancer of the body's tissues except blood, bone marrow or the lymphatic system. Solid tumors, moreover, can be separated into those of epithelial cell origin and those of non-epithelial cell origin. Examples of solid epithelial cell tumors include tumors of the gastrointestinal tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach,
129/160 duodenum, small intestine, large intestine, anus, gallbladder, lip, nasopharynx, skin, uterus, male genital organ, urinary organs, bladder and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and bone tumors.
[0273] Epithelial cancers generally progress from a benign tumor to a preinvasive stage (eg, carcinoma in situ), to a malignant cancer, which has penetrated the basement membrane and invaded the subpithelial stroma.
[0274] Multispecific protein complexes can also be used in these therapeutic applications and antibodies that bind to HER2, in particular can be used to treat breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma or ovarian cancer.
[0275] Other subjects who are candidates for receiving the compositions of this invention have or are at risk of developing, abnormal proliferation of fibrovascular tissue, acne rosacea, acquired immunodeficiency syndrome, artery acclusion, atopic keratitis, bacterial ulcer, Bechets disease, blood born tumors, obstructive carotid disease, choroidal neovascularization, chronic inflammation, chronic retinal detachment, chronic uveitis, chronic vitritis, excessive use of contact lenses, corneal graft rejection, corneal neovascularization, corneal graft neovascularization, Crohn's disease, Eales' disease, epidemic keratoconjunctivitis, fungal ulcer, Herpes simplex infections, Herpes zoster infections, hyperviscosity syndrome, Kaposi's sarcoma, leukemia, lipid degeneration, Lyme disease, marginal keratolysis, Mooren ulcer, infections by Mooren Mycobacterium except leprosy, myopia, disease ocular neovascular, optical excavation, Osler-Weber syndrome (Osler-WeberRendu), osteoarthritis, Paget's disease, pars planitis, pemphigoid, filectenulose,
130/160 polyarteritis, post-laser complications, protozoan infections, elastic pseudoxanthoma, sciatic pterigium keratitis, radial keratotomy, retinal neovascularization, prematurity retinopathy, retrolental fibroplasia, sarcoid, scleritis, sickle cell anemia, Sogrens syndrome, solid tumors, solid tumors Stargart's disease, Steven Johnson's disease, upper limbic keratitis, syphilis, systemic lupus, Terrien's marginal degeneration, toxoplasmosis, trauma, Ewing's sarcoma tumors, neuroblastoma tumors, osteosarcoma tumors, retinoblastoma tumors, rhabdomyosomatic tumors, colitis , occlusive venus, vitamin A deficiency and Wegener's sarcoidosis, unwanted angiogenesis associated with diabetes, parasitic diseases, abnormal wound healing, post-surgery hypertrophy, injury or trauma (eg, acute lung injury / ARDS), hair growth inhibition , inhibition of ovulation and formation of corpus luteum, inhibition of and implantation and inhibition of embryonic development in the uterus.
[0276] Examples of allergic or inflammatory disorders or autoimmune diseases or disorders that can be treated with the use of an antibody produced according to the methods described in this application include, but are not limited to, arthritis, such as acute arthritis, chronic rheumatoid arthritis , gout arthritis, acute gout arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, vertebral arthritis, juvenile rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, chronic arthritis, deformities primary, reactive arthritis and ankylosing spondylitis, inflammatory hyperproliferative skin disease, psoriasis such as plaque psoriasis, guttate psoriasis, postular psoriasis and nail psoriasis, atopic diseases including atopy, such as hay fever, Job's syndrome, dermatitis including contact dermatitis , chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, herpetiform dermatitis and atopic dermatitis, x-linked hyper-lgM syndrome,
131/160 urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including autoimmune urticaria, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), MS as systemic sclerosis, MS as MS progressive pimara (PPMS) and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, disseminated sclerosis and ataxic sclerosis, inflammatory bowel disease (IBD) (eg Crohn's disease, mediated autoimmune gastrointestinal diseases, colitis such as ulcerative colitis , ulcerative colitis, microscopic colitis, collagenous colitis, polyposis colitis, necrotizing enterocolitis and transmural colitis, and autoimmune intestinal inflammatory disease, gangrenous pyoderma, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, including respiratory distress syndrome (respiratory distress syndrome) ARDS), meningitis, inflammation of all parts of the uvea, iritis, choroiditis, and autoimmune hematological dysfunction, rheumatoid spondylitis, sudden hearing loss, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen encephalitis, brain stem encephalitis , uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis, posterior uveitis or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome such as chronic or acute GN, as a primary, GN-like glomerulonephritis, primary, GN Membranous GN (membranous neupathic), idiopathic membranous GN or idiopathic membranous neuropathy, membranous proliferative GN (MPGN), including Type I and Type II, and fast-progressing GN, allergic conditions and responses, allergic reactions, eczema including allergic or atopic eczema, asthma such as bronchial asthma and autoimmune asthma, conditions involving infiltration of T cells and chronic inflammatory responses, immune reactions against foreign antigens, such as fetal blood groups A-B-0 during pregnancy, inflammatory disease
132/160 chronic pulmonary, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) as cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome, disseminated lupus erythematosus, lupus (including nephritis, cerebritis, pediatric, non-renal , discoid, alopecia), juvenile diabetes mellitus (Type I), including pediatric insulin dependent diabetes mellitus (IDDM), adult principle diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with hypersensitivity acute and delayed cytokine and T lymphocyte-mediated, tuberculosis, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitis (including large vessel vasculitis (including rheumatic polymyalgia and Giant cell arteritis) from vasayasu), vasculitis medium (including Kawasaki disease and polyarteritis nodosa), polyarte microscopic ritis, CNS vasculitis, necrotizing vasculitis or hypersensitivity vasculitis, systemic necrotizing vasculitis, such as vasculitis or Churg-Strauss syndrome (CSS)), temporal arteritis, aplastic anemia, aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those resulting from septicemia, trauma or hemorrhage, diseases mediated by antigen-antibody complex, anti-glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Bechet's disease, Castleman's syndrome, Goodpasture, Reynaud's syndrome, Sjorgen's syndrome, St's syndrome evens-Johnson, pemphigoid as vesicular pemphigoid and skin pemphigoid, pemphigus (including common pemphigus, pemphigus foliaceus, pemphigus
133/160 mucous membranous pemphigoid and erythematous pemphigus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, optic neuromyelitis, polyneuropathy, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy (thrombocytopathy) developed by patients with myocardial infarction, for example), including thrombotic thrombocytopenic purpura (TTP), post-transfusion purpura (PTP), heparin-induced thrombocytopenia and autoimmune or immune thrombocytopenic mediated as idiopathic purpura (ITP) including chronic or acute ITP autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), or sub-acute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism Severe, polyglandular syndrome with the autoimmune polyglandular syndrome (or polyglandular endocrinopathic syndrome), paraneoplastic syndrome, including neurological pareneiplastic syndrome such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, rigid man or rigid person syndrome, encephalomyelitis such as allergic encephalomyelitis and experimental allergic encephalomyelitis (EAE) myasthenia gravis such as myasthenia gravis associated with thymoma, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (WHO), sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupid hepatitis, chronic active hepatitis, interstitial pneumonitis lymphoid (LIP), bronchiolitis obliterans (without transplantation) against NSIP, Guillain-Barré syndrome, Berger disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pneumocirrosis, autoimmune enteropathy syndrome, celiac disease, ps celiac ilosis (gluten enteropathy), refractory psilosis, idiopathic psilosis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; disease
134/160 by Lou Gehrig), coronary artery disease, autoimmune inner ear disease (AIED), or autoimmune hearing loss, opsoclone myoclonus syndrome (WHO), polychondritis such as refractory or relapsing polychondritis, pulmonary alveolar proteinosis, amyloidosis, scleritis , non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B-cell lymphocytosis (for example, benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, muscular dysfunction, arrhythmia, muscular dysfunction , deafness, blindness, periodic paralysis, and CNS canalopathy, autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveoretinitis, autoimmune hepatological dysfunction, fibromyalgia, multiple endocrine failure, Schmidt syndrome, gastric atrophy senile, demyelinating diseases like aut demyelinating diseases oimmune and chronic inflammatory demyelinating polyneuropathy, diabetic neuropathy, Dressier syndrome, alopecia areata, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmobilitis, sclerodactyly, and telangiectasia), male and female autoimmune infertility, ankylosing spondylitis, connective tissue disease, connective tissue disease Chagas disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird breeder's lung, allergic granulomatous angitis, benign lifocytic angitis, Alport's syndrome, alveolitis such as allergic alveolitis and alveolitis fibrosing, interstitial lung disease, transfusion reaction, leprosis, malaria, leishmaniasis, kipanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, Caplan's syndrome, dengue, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial fibrosis pulmonary, fibrosis idiopathic pulmonary disease, cystic fibrosis, endophthalmitis, elevated and reduced erythema, fetal erythroblastosis, eosinophilic faciitis,
135/160
Shulman, Felty's syndrome, flariase, cyclitis like chronic cyclitis, heterochronic cyclitis, iridocyclitis or Fuch's cyclitis, Henoch-Schonlein purpura, human immunodeficiency virus (HIV) infection, ecovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection , rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, obliterating thromboangitis, thyrotoxicosis, dorsalisis , chorioiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, dry keratoconjunctivitis, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal exchange neuropathy, hereditary benign lesion and reperfusion and ischemia, retinal autoimmunity, bronchial inflammation, joint inflammation chronic obstructive airway disease itself lycosis, canker sore, aphthous stomatitis, atherosclerotic diseases, aspermiogenesis, autoimmune hemolysis, Boeck's disease, cryoglobulinemia, Dupuytren's contracture, phaco-anaphylactic endophthalmos, allergic entente, leprous nodular erythema, idiopathic facial paralysis, chronic fatigue syndrome, chronic fatigue syndrome -Rich, sensory hearing loss, paroxysmal hemoglobinuria, hypogonadism, regional ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic aphthelmia, granulomatous orchitis, pancreatitis, acute gangrene, thyroiditis, thyroiditis, pyridoderma acquired spleen, infertility due to antisperm antibiotics, non-malignant thymoma, vitiligo, SCID and diseases associated with the Epstein-Barr virus, acquired immunodeficiency syndrome (AIDS), parasitic diseases such as Lishmania, toxic shock syndrome, poisoned food, conditions involving infiltration of cell T, leukocyte adhesion deficiency, immune responses associated with acute or delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte diapedesis, injury syndrome
136/160 multiple organs, diseases mediated by the antigen-antibody complex, antiglomerular-based membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, syndrome nephrotic, insulitis, polyendocrine failure, peripheral neuropathy, autoimmune type I polyglandular syndrome, adult-onset idiopathic hypoparathyroidism (AOIH), total alopecia, dilated cardiomyopathy, acquired bullous epidermolysis (EBA), hemochromatosis, myocarditis, nephrotic syndrome, cholangitis purulent and non-purulent, acute and chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis and eosinophil-related dysfunction such as eosinophilia, pulmonary eosinophilic infiltration, eosinophilic myalgia syndrome, Loffler syndrome, chronic eosinophilic pneumonia, tropical eosinophilia, and pulmonary eosinophilia bronchopneumonic pergylosis, aspergilloma or granuloma containing eosinophils, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, chronic mucocutaneous candidiasis; with collagen disease, rheumatism, neurological disease, reperfusion ischemic dysfunction, reduced blood pressure response, vascular dysfunction, antiectasia, tissue damage, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and disease that accompanies vascularization, allergic hypersensitivity disorders, glomerulonephritis , reperfusion injury, reperfusion injury of the myocardium or other tissues, dermatosis with acute inflammatory components, acute purulent meningitis, or other inflammatory disorders of the central nervous system, inflammatory disorders ocular and orbital, syndromes associated with granulocyte transfusion, cytokine-induced toxicity, serious acute inflammation, chronic intractable inflammation, pyelitis, pneumonocirrhosis, retinopathy
137/160 diabetic, dysfunction of large diabetic artery, endoarterial hyperplasia, peptic ulcer, valvulitis and endometriosis.
[0277] In addition to therapeutic uses, the antibiotics of the invention can be used for other purposes, including diagnostic methods, such as diagnostic methods for the diseases and conditions described in the present application.
X. Dosages, Formulations and Duration [0278] The proteins of the invention will be formulated, dosed and administered in a manner consistent with good medical practice. Factors for consideration in this context include the specific dysfunction to be treated, the specific mammal to be treated, the clinical condition of each subject, the cause of the dysfunction, the location of distribution of the agent, the method of administration, the administration schedule and other factors known to medical specialists. The "therapeutically effective amount" of the proteins to be administered will be guided by these considerations and is the minimum amount needed to prevent, improve or treat a particular disorder (for example, a cancer, allergic or inflammatory disorder, or autoimmune disorder). Proteins need not be, but are optionally formulated with one or more agents currently used to prevent or treat dysfunction. The effective amount of these other agents depends on the amount of proteins present in the formulation, the type of dysfunction or treatment and the other factors discussed above. These are generally used in the same dosages and with routes of administration as used above or about 1 to 99% of the dosages used to date. Generally, cancer relief or treatment involves a decrease in one or more symptoms or medical problems associated with the cancer. The therapeutically effective amount of the drug may accompany one or more of the following combinations: reduce (at least 10%, 20%, 30%, 40%, 50%, 60%,
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70%, 80%, 90%, 100% or more) the number of cancer cells, reduce or inhibit the size of the tumor or tumor mass, inhibit (that is, decrease, to some extent and / or stop) cell infiltration cancerous in peripheral organs, reduce hormonal secretion in the case of adenomas; reduce the density of the blood vessel; inhibit tumor metastasis, reduce or inhibit tumor growth; and / or relieve in one way, one or more of the symptoms associated with cancer. In some embodiments, proteins are used to prevent the occurrence or recurrence of cancer or an autoimmune disorder in the subject.
[0279] In one embodiment, the present invention can be used to increase the duration of survival of a human subject susceptible to cancer or diagnosed with cancer or autoimmune dysfunction. Survival duration is defined as the time from the first administration of the drug to death. Survival duration can also be measured by the stratified risk rate (HR) of the treatment group versus the control group, which represents the risk of death for a patient during treatment.
[0280] In yet another embodiment, the treatment of the present invention significantly increases the response rate in a group of human subjects susceptible or diagnosed with a cancer who are treated with various anticancer therapies. The response rate is defined as the percentage of treated individuals who responded to treatment. In one embodiment, the combination treatment of the invention with the use of proteins of that invention and surgery, radiation therapy, or one or more chemotherapy agents significantly increases the response rate in the group of subjects treated compared to the group treated only with surgery, radiation or chemotherapy, the increase that has a chi-square p value of less than 0.005. Additional measures of therapeutic effectiveness in the treatment of cancer are described in US patent application publication 20050186208.
139/160 [0281] Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional carriers, excipients , or physiologically acceptable stabilizers (Flemington's Pharmaceutical Sciences (20th edition) , ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA). Acceptable vehicles include saline buffers or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers like polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins; chelating agents such as EDTA; sugar alcohols like mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or PEG.
[0282] Optionally, but preferably, the formulation contains a pharmaceutically acceptable salt, preferably sodium chloride and preferably in concentrations close to physiological. Optionally, the formulations of the invention can contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration ranges from 0.1 to 2.0%, typically v / v. Suitable preservatives include those known in the pharmaceutical art. Benzyl alcohol, phenol, m-cresol, methylparaben and propylparaben are preferred preservatives. Optionally, the formulations of the invention can include a pharmaceutically acceptable surfactant at a concentration of 0.005 to 0.02%.
[0283] The formulation in the present application may also contain more than one active component, as necessary for the specific indication to be treated, preferably those with activities
140/160 complementary that do not adversely affect each other. These molecules are suitably present in combination, in amounts that are effective for the intended purpose.
[0284] The active ingredients can also be retained in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsule and poly (methylmethacylate) microcapsule, respectively, in distribution systems. colloidal drug (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These techniques are disclosed in Flemington’s Pharmaceutical Sciences, above.
[0285] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing heteromultimeric protein, whose matrices are in the form of molded articles, for example, films or microcapsules. Examples of sustained release matrices include polyester, hydrogels (for example, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides (US patent 3,773,919), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid and leuprolide acetate) and poly-D - (-) - 3-hydroxybutyric acid. While polymers such as ethylene vinyl acetate and glycolic lactic acid allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated heteromultimeric protein (s) remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37 ^Q C, which results in a loss of biological activity
141/160 and possible changes in immunogenicity. Rational strategies can be developed for stabilization, depending on the mechanism involved. For example, if the aggregation mechanism has been shown to have an intermolecular SS bond formation through thio-disulfide interchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilization from acid solutions, content moisture control, appropriate use of additives and development of compositions of specific polymeric matrices.
[0286] The proteins described in the present application (for example, a heteromultimeric protein, such as an antibody produced according to the methods in the present application) are administered to a human subject according to known methods, such as intravenous administration, for example, as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes. Local administration may be particularly desirable if extensive side effects or toxicity are associated with antagonism to the target molecule recognized by the proteins. An ex vivo strategy can also be used for therapeutic applications. Ex vivo strategies involve transfection or transduction cells obtained from the subject with a polynucleotide that encodes a protein of that invention. The transfected or transduced cells are then returned to the subject. The cells can be of any type of a wide variety including, without limitation, hematopoietic cells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells, T cells or B cells), fibroblasts, epithelial cells, endothelial cells, keratinocytes or muscle cells.
[0287] In one example, the protein complex (for example, a heteromultimeric protein such as a multispecific antibody produced according to the methods described in the present application) is administered
142/160 locally, for example, by direct injections, when the dysfunction or location of the tumor allows and the injections can be repeated periodically. The protein complex can also be delivered systemically to the subject or directly to tumor cells, for example, to a tumor or to a tumor bed followed by surgical excision of the tumor, in order to prevent or reduce local recurrence or metastasis.
XI. Articles of Manufacture [0288] Another embodiment of the invention is an article of manufacture containing one or more protein complexes described in the present application and materials useful for the treatment or diagnosis of a dysfunction (for example, an autoimmune disease or cancer). The article of manufacture comprises a container and a label or package insert in or associated therewith. Suitable containers include, for example, bottles, vials, syringes, etc. Containers can be manufactured from a variety of materials, such as glass or plastic. The container contains a composition that is effective in treating the condition and may have a sterile access port (for example, the container may be a bag of intravenous solution or a bottle that has a cap that can be pierced by a hypodermic injection needle). At least one active agent in the composition is a heteromultimeric protein (for example, an antibody or antibody fragment) of the invention. The label or package insert indicates that the composition is used to treat the particular condition. The label or package insert will also further comprise instructions for administering the heteromultimeric protein composition to the subject. Manufacturing articles and kits comprising combinatorial therapies described in this application are also contemplated.
[0289] Package insert refers to the instructions included, as usual, in commercial packaging of therapeutic products that contains information about the indications, use, dosage, administration, contraindications and / or
143/160 warnings regarding the use of these therapeutic products. In certain embodiments, the package insert indicates that the composition is used to treat breast cancer, colorectal cancer, lung cancer, renal cell carcinoma, glioma or ovarian cancer.
[0290] Additionally, the article of manufacture may also comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), buffered saline, Ringer's solution and dextrose solution. In addition, it may include other materials considered from a commercial and user point of view, including other buffers, thinners, filters, needles and syringes.
[0291] Kits are also provided that are useful for various purposes, for example, for purifying or immunoprecipating an antigen (for example, HER2 or EGFR) from cells. For isolation and purification of an antigen (for example, HER2 or EGFR), the kit may contain a heteromultimeric protein (for example, an EGFR / HER2 antibody) coupled to beads (for example, sepharose beads). Kits containing the heteromultimeric protein (s) for detection and quantification of the antigen in vitro, can be supplied, for example, in an ELISA or Western blot. Like the article of manufacture, the kit comprises a container and a label or label on the container or associated therewith. The container contains a composition that comprises at least one heteromultimeric protein (for example, a multispecific antibody or an antibody fragment) of the invention. Additional containers that contain, for example, diluents and buffers or control antibodies may be included. The label or package insert can provide a description of the composition, as well as instructions for the intended in vitro or diagnostic use.
144/160 [0292] The foregoing description is considered to be sufficient to allow a person skilled in the art to practice the invention. The following Examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the invention, in addition to those presented and described in the present application will become apparent to those skilled in the art from the preceding descriptions and are included in the scope of the appended claims.
[0293] In the following experimental disclosure, the following abbreviations apply: eq (equivalent); M (Molar); μΜ (micromolar); N (Normal); mol (moles); mmol (millimoles); pmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); pg (micrograms); L (liters); mL (milliliters); μΙ_ (microliters); cm (centimeters); mm (millimeters); gm (micrometers); nm (nanometers); ^Q C. (degrees Centigrade); h (hours); min (minutes); s (seconds); msec (milliseconds); ADCC (antibody dependent cell cytotoxicity)); BsAb (bispecific antibody); Cl (constant domain of the light chain); Ch (heavy chain constant domain); CMC (complement-mediated cytotoxicity); Fab (antigen binding fragment); Fc (crystallized fragment); Fv (variable fragment (Vl + Vh)); EGFR (epidermal growth factor receptor); HC (heavy chain); IGFR (insulin-like growth factor receptor); LC (light chain); scFv (single chain variable fragment (V1 and Vh attached by an amino acid ligand)); VEGF (vascular endothelial growth factor); VEGFR2 (vascular endothelial growth factor receptor 2); Vh (heavy variable domain); Vl (light variable domain).
Examples [0294] The present invention is described in more detail in the following examples which are in no way intended to limit the scope of the
145/160 invention as claimed. The attached Figures are intended to be considered an integral part of the specification and description of the invention. All references cited are specifically incorporated into this application as a reference, for everything described in this regard. The following examples are offered to illustrate, but not to limit the claimed invention.
Example 1
Construction of Expression Vectors [0295] This example illustrates the nucleic acid construct used to transform host cells.
[0296] In general, both heavy and light chain DNA coding sequences were cloned into an expression plasmid that contained separate promoter elements for each of the sequences and antibiotic resistance for the selection of bacterial cells that contained the plasmid of expression. The vector constructs also encode the heat-stable enterotoxin II (STII) secretion signal (Picken et al., 1983, Infect. Immun. 42: 269-275 and Lee et al., 1983, Infect. Immun. 42: 264 -268) for the export of antibody polypeptides in the periplasmic space of bacterial cells. The transcription of each chain is controlled by the phoA promoter (Kikuchi et al., 1981, Nucleic Acids Res., 9: 5671-5678) and the translational control is provided by the STII signal sequence variants, previously described, of translational strength known relative, which contain silent codon changes in the translation initiation region (TIR) (Simmons and Yansura, 1996, Nature Biotechnol. 14: 629-634 and Simmons et al., 2002, J. Immunol Methods, 263: 133- 147). A schematic drawing of the protuberance and orifice of plasmids is shown in Figures 2A and 2B, respectively.
146/160 [0297] While the present invention is not based on specific antibody binding sequences and is applicable to any half-antibody combinations, the Examples of the present application are directed to heteromultimeric antibodies directed to c-met, EGFR, IL- 4 and IL-3. Examples of anti-c-met antibodies are provided in US patents 7,472,724 and 7,498,420. Examples of anti-EGFR antibodies are provided in US Provisional Patent Application 61 / 210,562 (filed March 20, 2009), US patent application publication 20080274114 (published on November 6, 2008) and US patent 5,844,093 (granted on December 1, 1998). Examples of anti-IL-13 antibodies are described in US patent 7,501,121 (issued March 10, 2009), US patent 7,615,213 (issued November 10, 2009), WO 2006/085938 (published in August 17, 2006), US patent application publication 20090214523 (published August 27, 2009) and US patent 7,674,459 (granted March 9, 2010). Examples of anti-IL-4 antibodies are described in US patent application publication 20080241160 (published October 2, 2008) and US patent 6,358,509 (granted March 19, 2002).
[0298] Each half-antibody had both a lump and a genetically engineered orifice (cavity) in the heavy chain as described in US patent 7,642,228. Briefly, a Ch3 lump mutant was generated first. A library of Ch3 orifice mutants was then created by randomizing residues 366, 368 and 407, in proximity to the protrusion in the partner's Ch3 domain. In the following examples, the bulge mutation is T366W and the orifice has T366S, L368A and Y407V mutations in an IgG1 skeleton. Equivalent mutations in other immunoglobulin isotypes are easily determined by a person skilled in the art. In addition, a technician will readily appreciate the fact that
147/160 the use of two half-antibodies of the same isotype for the elaboration of the bispecific. Half-antibodies of different isotypes can be used, but may need additional mutations.
[0299] In some examples, each half-antibody had additional mutations introduced into residues F241 and F243 in the Ch2 domains. Spot mutations were introduced using techniques known in the art to change wild-type phenylalanine to both serine and arginine, so the combination was both F241S / F243R and F241 R / F243S.
[0300] Although the vector described in this Example is for both anti-c-Met and anti-EGFR medium, a person skilled in the art will readily appreciate the fact that any antibody can be encoded in the plasmid. The starting plasmid for all constructs used in the present application is the previously described separate anti-tissue factor cistron plasmid, paTF50, with relative TIs of 1 for heavy chain and 1 for light chain (Simmons et al., 2002, J Immunol Methods, 263: 133-147 and US patent 6,979,556). An increase in the relative TIR forces was used to increase the expression titers of these half-antibodies.
Example 2
Production of Heteromultimeric Protein Using Separate Cell Cultures [0301] The following example shows the production of heteromultimeric proteins when cells expressing monomeric components (for example, a half-antibody) are grown in separate cultures. In this method, the cells are cultured and induced to express the antibody medium in separate cultures. In these methods, the components can be purified first and then combined to form the heteromultimeric protein.
148/160 [0302] In this method, a nucleic acid encoding the polypeptide containing the first Fc (for example, a half-antibody (protuberance)) is introduced into a first host cell and a nucleic acid encoding the polypeptide containing the second Fc ( for example, a half-antibody (orifice) is introduced into a second host cell. Although this example illustrates the formation of a BsAb, a person skilled in the art will readily appreciate the fact that the methods described are applicable to any heteromultimeric protein that comprises a hinge region, for example, affibodies, etc.
Independent Production of Bulk and Orifice of Half-Antibodies in Separate Cultures, Separate Purification of Half-Antibodies, Mixture and Redox Reaction for Formation of Intact BsAb [0303] Half-antibodies containing both bulge and orifice mutations (with or without mutations F241 and F243) were generated in cultures separated by the expression of heavy and light chains using the constructs described in Example 1 in a bacterial host cell, for example, E. coli. See Figures 3 and 4A. In this method, the protuberance of the half-antibody was an anti-EGFR and the orifice of the half-antibody was an anti-c-met. The expression plasmids of Example 1 were introduced into the host strains of E. coli 33D3 (Ridgway et al. (1999) 59 (11): 2718) or 64B4 (W3110 AfhuA AphoA HvG + Aprc spr43H1 AdegP AmanA lacl ^q AompT) and transformants were selected on LB plates containing carbenicillin. The transformants were then used to inoculate a start culture LB containing carbenicillin and was grown from this overnight with shaking at 30 ^Q C. The culture was diluted 100X beginner in a limiting means CRAP phosphate (Simmons et al., 2002 , J. Immunol Methods, 263: 133-147) containing carbenicillin, and this was cultured for 24 hours with stirring at 30 ^Q C. cultures were spun, and cell pellets were
149/160 frozen until antibody purification begins. The pellets were thawed and resuspended in an extraction buffer containing 25 mM Tris base with pH 7.5 adjusted with hydrochloric acid, 125 mM NaCI and 5 mM EDTA (TEB or Tris Extraction Buffer) with a volume: weight ratio of 100 mL of TBS to 5 g of cell pellet, and extracted by disturbing the cells with the use of microfluids by passing the resuspended mixture through a microfluidizer from Microfluidics Corporation, model 11 OF (Newton, MA), three times. The bacterial cell extract was then clarified by centrifugation for 20 min at 15,000 x g, and the supernatant collected and filtered through a 0.22 micron acetate filter before purification.
[0304] Each half-antibody was purified separately by Protein A capture, followed by cation exchange chromatography. Clarified cell extracts from the orifice half-antibody were loaded onto a 1 mL HiTrap MabSelect ™ SuRe column purchased from GE Healthcare (Pistcataway, NJ) at 2 mL / min. After loading, the column was washed with 10 column volumes (CV) of 40 mM sodium citrate, pH 6, 125 mM sodium chloride and 5 mM EDTA, followed by 5 column volumes of 20 mM sodium citrate at pH 6 to facilitate capture by the cation exchange column. Affinity-captured half-antibodies were eluted with 10 column volumes (CV) of 0.1 mM acetic acid (pH 2 to 3) and directly captured on a strong 1 ml HiTrap SP-HP cation exchange column acquired from the GE Healthcare. The column was washed with 10 CV of buffer A containing 25 mM 2- (A / -morpholino) ethanesulfonic acid (MES), pH 5.8. The half-antibodies were eluted with a linear gradient of 0 to 50% buffer B (25 mM MES, pH 5.8 and 1 M sodium chloride (NaCI)). Both proteins eluted between 20 and 40% B and the eluting peak was determined by UV absorbance at 290 nm and by non-reducing SDS-PAGE analysis of the collected fractions combined separately as the protrusion or media orifice
150/160 antibody. Both proteins generally exhibited a major elution peak and all fractions that contained the heavy and light chain species oxidized with each other were included in the mixture. The analysis of purified half-antibodies by reducing and non-reducing SDS-PAGE is shown in Figure 4B. The results indicate that the majority of the expressed and captured protein is 75 kD in size. This data was confirmed by ESI-TOF mass spectrometry as shown in Figure 4C. The mass of the half-antibodies were the expected masses, indicating that there was no disulfide adduct in any cysteine, including the two cysteine residues in the hinge region. To determine whether hinge cysteines were reduced to exhibit a free reactive thiol, proteins were reacted at neutral pH with 1 mM Netylmaleimide (NEM) for one hour before analysis by mass spectrometry. The protein mass was unchanged indicating that the hinge cysteines were most likely oxidized to one another in an intrachain disulfide, for example, a cyclic disulfide. To form a completely intact and bispecific antibody using these two half-antibodies (orifice and protuberance), it was first necessary to reduce the intrachain disulfides in the hinge region to release the cysteine-free thiols so that they could later be oxidized to the other heavy chain to form the 150 kD bispecific antibody.
[0305] To perform the annealing, reduction and reoxidation of complementary half-antibodies for the formation of intact bispecific molecules, the following procedure was developed. After independent isolation, the purified proteins were combined to equal masses in the Combination step of the procedure (shown in Figure 5), the pH of the mixture was adjusted to 8 by adding one tenth of the volume of 1 M Tris, pH 8, and the proteins were reduced with 2 mM dithioltreitol (DTT) at room temperature. After reduction for 2 h, the combined proteins were
151/160 subjected to a buffer exchange for 25 mM Tris, pH 8, and 125 mM NaCI using 5 ml Zeba Desalt centrifuge columns (Pierce, Rockford, IL), resulting in a volume of about 4 ml with a protein concentration of 1 mg / mL. The proteins were then annealed by heating the mixture at 37 ^Q C for 3 h, followed by cooling at room temperature, about 24 ^Q C. The annealed antibodies were concentrated using spin concentrators cutting with MW 10 kD to a volume of 0.5 ml with a protein concentration of about 10 mg / ml, and oxidized by air while dialyzed in 50 mM Tris, pH 8, and 150 mM NaCI with 10 kD membranes (SpectrumLabs, Rancho Dominguez, CA ). After oxidation overnight at room temperature, the oxidized material was run on an S-200 filtration gel column (22 mL S-200 Tricorn purchased from GE Healthcare) in a buffer containing 25 mM MES, pH 6, and 300 mM NaCI. The intact antibody was combined and diluted 10 times in water.
[0306] The BsAb protein was then purified by cation exchange chromatography using carboxymethyl (CM) resin (1 mL HiTrap CM-FF, GE Healthcare) and pH gradient for elution from 4.5 to 9.2 . The composition of buffers A and B consisted of 20 mM sodium citrate, 30 mM MES, 20 mM HEPES, 20 mM imidazole, 20 mM Tris, 20 mM CAPS and 25 mM NaCI, where buffer A is adjusted to pH 4 , 2 with HCI and buffer B is adjusted to pH 9.2 (or 10.4) using NaOH. The purified material obtained after CM chromatography was analyzed by mass spectrometry to determine the exact molecular composition (Figure 4D). The mass spectrometry analysis indicated that the only detectable intact antibody product had a MW of 146,051.89, which corresponds almost identically to the heterodimeric species of anti-EGFR / anti-met orifice with a theoretical MW of 145,051.75 . The performance of this procedure,
152/160 starting with about 2 mg of lump and 2 mg of orifice, was about 0.5 to 1 mg.
[0307] The same procedure was performed for half antibodies containing the F241 and F243 mutations.
Example 3
Crystallization of Fc [0308] During the formation process, certain losses are associated with malformed disulfide bridges, as demonstrated by the low yields in Example 2. This example is an analysis of the different structural forms of the Fc protrusion and orifice observed by crystallography by X-ray. The crystalline structure of Fc was obtained by several heterodimeric proteins, with only the bulge and orifice mutations described above.
Lump and Fc Hole for Crystallography [0309] An antibody hole lump in an arm consisting of a lgG1 heavy chain (hole), a light chain and a truncated heavy chain Fc (lump) has been purified from E coli using standard antibody purification methods. See, for example, WO 2005/063816. The purified antibody was digested arm with a ratio w / w 1/1000 with lysine endopeptidase C for 15 min at 37 ^Q C. The digestion was stopped with 5 μΜ protease inhibitor NALFA-tosyl-L-lysinyl -chloromethyl ketone (TLCK). Antibody from an intact arm and FAb were removed from the protrusion in an Fc orifice using kappa selection resin, which does not bind to free Fc. The Fc hole protrusion was then purified on an S75 column (GE Biosciences) before crystallization. The resulting bulge and orifice of Fc fragments have the following sequences (the number of initial residues based on the entire IgG 1 heavy chain is provided):
153/160
Chain 1 (hole) (SEQ ID NO: 1)
223 THTCPPCPAP ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSREEMTKNQ VSLSCAVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFFLVSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGK
Chain 2 (bulge) (SEQ ID NO: 2)
221 DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK [0310] The crystallization conditions required the addition of a mini-Z domain of protein A (reference Starovasnik below) the in order to stabilize the protein. The protein was subjected to a buffer exchange for 0.15 M NaCI, 50 mM Tris, pH 8, and was concentrated to 15 mg / ml. The equimolar concentration of the Mini-Z peptide was added and incubated overnight. Proteins were crystallized by the suspended drop method (see, for example, Experimental and theoretical analysis of the rate of solvent equilibration in the hanging drop method of protein crystal growth. Journal of Crystal Growth, Vol. 90, Issues 1-3, 2 July 1988, pp. 117 129) at 18 ^Q C with a reservoir buffer containing 20% w / v PEG2000MME, 0.1 M MES pH 6.5, isopropanol, 10% v / v. Protein slides were formed after 1 week. Data collected on the ALS 5.0.2 beamline.
[0311] The minimized version of Protein A, domínio-domain (“mini-Z”) was prepared as previously described (Starovasnik MA, Braisted AC, Wells JA (1997) Proc. Natl. Acad. Sci. USA Vol. 94, pages 10080-10085).
154/160 [0312] The orifice protuberance of the Fc heterodimer was crystallized in the presence of a mini-Z domain peptide (as described above). See Figure 6. The mini-Z peptide has been linked to the CH2CH3 interface and probably helps to stabilize the F2 CH2 regions. The structure makes contact with the two CH3 domains and is not significantly different from the non-glycosylated wild-type IgG1 Fc's.
PROTUBERANCE-Fc PROTUBERANCE FOR CRYSTALLOGRAPHY [0313] The protuberance-protuberance protein was produced by expression in E. cell, as described above, except for the Fc variants that were expressed as individual Fc chains with hinge cysteines removed by serine mutation in order to prevent covalent dimerization (see, for example, WO 2006028936), and the cation exchange step has been omitted. The Fc lump-lump was present as a non-covalent dimer and isolated by a combination of Protein A affinity chromatography and gel filtration using an S200 column (GE Biosciences). The purified protein was used for crystalline selection. For crystalline growth screens, the protein was subjected to a buffer exchange for PBS and concentrated at 10 mg / mL. Protein crystallized in 20% w / v PEG2000-monomethyl ether (MME), 0.2 M ammonium sulfate, 0.1 M sodium cacodylate, pH 6.5, with 2 μΙ of protein in 2 μΙ of reservoir by the drop method suspended at 18 ^Q C. Thick slides appeared after five days and data were collected on the beamline ALS 5.0.1. using a 25% w / v cryoprotectant PEG2000-MME.
[0314] The sequence for the lump sequence was (SEQ ID NO: 3):
221 DKTHTSPPSP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTKQPREQTREK
155/160
NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK [0315] The orifice-orifice homodimer was prepared in the same way as the variant protuberance.
[0316] The sequence for the orifice sequence was (SEQ ID NO: 4):
221 DKTHTSPPSP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK [0317] The Fc's homodimeric, however, formed a forming head to tail having any of the Fab's of the bispecific 180 ^Q from each other, as described in Figure 7. Not only would this be a new conformation for an Fc, it would also prevent normal oxidation of the hinge since the disulfide pairs are at opposite ends of the Fc. Without a corresponding Fc, hinge cysteines tend to cyclise and then become non-reactive, unless reoxidized. Thus, these structures suggest alternative conformations that can contribute to the inefficiency of the redox reaction.
[0318] Table 1 shows the Data Collection and Refinement for homodimers - protrusion / protrusion and orifice / orifice - and the heterodimer - protuberance / orifice. Based on the analysis of the crystalline structure, Table 3, below, of the contact residues was developed.
Table 3
Chain A Chain B S239 K370 V240 K370 F241 L368
156/160
Chain A Chain B F241 K370 F243 F405 F243 Y407 P244 V397 V264 Y349 R301 T350 K334 D399 Y349 D265 L368 V262 K392 N389 K392 Y391 P395 P396
[0319] For clarity of understanding, both Chains A and B have contact sets. The table above represents one of the two sets. Therefore, a complete table would have twice as many contacts listed above. For example, S239 in A contacts K370 in B, and S239 in B makes contact with K370 in A.
Example 4
Orientation Stabilization [0320] The limiting step during annealing and purification is the redox reaction step. The oxidized heterodimer typically comprises only 70 to 80% of the protein after this step (BioAnalyzer and MS-TOF). The remaining 20 to 30% of the antibody is dimeric and is devoid of a covalent bond (SEC-LLS). This can be removed, but it significantly affects the total yield. Thus, to test whether it is possible to compromise head-to-tail associations, and thus improve bispecific recoveries, a protrusion of Fc's was generated with the F241S / F243R or F241 R / F243S mutations.
157/160 [0321] The Fc's protrusion and orifice have approximately the same amount of homodimers by size exclusion chromatography. Fc bulge mutations reduced the amount of homodimer present compared to the wild type (ie, bulge mutations only) by up to 83.5% (see Figure 8). See Table 4 below.
Table 4
Lump F241S / F243R Lump F241R / F243S Reduction in homodimer content 83.5% 64.5%
[0322] The annealing of the lump and orifice of the bispecific antibody is enhanced with the Fc mutants compared to the wild type lump. The percentage of intact antibody quantified by the bioanalyser is 27.6% for the wild type pair, 46.4% for the F241S / F243R mutant protuberance and 45.5% for the F241R / F243S mutant protuberance. Both the F241S / F243R and F241R / F243S mutations compromise the hydrophobic centers of the head-tail pairing. The incorporation of the mutations in the protuberance chain reduced the amount of homodimer in relation to the wild type protuberance and seems to be beneficial for the improvement of the yield. It is believed that similar mutations in just one wild-type orifice or in the protrusion in wild-type Fc orifice would result in similar improvements.
Temporal Evolution of Bispecific Glutathione [0323] F241S / F243R mutations increased the rate of correct disulfide formation among bispecific antibodies. Half antibodies were mixed 1: 1 in 200 mM arginine succinate, pH 8.5, with an excess of 200 M of reduced glutathione at room temperature. During the reaction, samples were collected at the following points: 0, 3 h, 6
158/160 h, 12 h, 24 h, 36 h, 48 h and 72 h. Upon collection, an equivalent volume of 0.15 M acetic acid was added to stop the formation reaction. The samples were then analyzed in an Agilent BioAnalyzer 2100 with a Protein 230 kit. Using F241S / F243R mutations in the protuberance and / or half-antibody hole, the oxidation rate was increased under these conditions over time from 12 to 48 h (Figure 10). This demonstrates the advantage of these changes in increasing training rates and overall training efficiency.
Bulk and Orifice Associations through Plasmonic Surface Resonance (SPR) [0324] Using a BioRad ProteOn ™ system, wild-type bulge binders, wild-type hole, F241R / F243S protuberance, F241S / F243R protuberance and F241S / protuberance F243R were immobilized on an NHS activated sensor. The analyte, protuberance F241S / F243R, was passed through the sensor in 25 mM Tris, 150 mM NaCI and 0.05% Tween-20, pH 8.0. Analyte concentrations were 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM and 6.25 nM. The ProteOn ™ chip was regenerated between each analyte concentration using 10 mM Glycine, pH 3. The data demonstrates that F241 / F243 mutations do not interfere with CH3 heterodimerization and neither is there any measurable homodimerization with or without these changes at these concentrations (Figures 11A-E). The Kd for heterodimers is in the single-digit nanomolar range in both cases.
[0325] It is understood that the examples and achievements described in this application are for illustrative purposes only and that various modifications or changes in consideration of these will be suggested to those skilled in the art and should be included within the spirit and limit of this request and scope of the attached claims. All publications, patents and
159/160 patent applications cited in this application are incorporated in full into this application as a reference for all purposes.
Table 1 Data Collection and Refinement for Bulge / Bulge, Bulge / Hole / MiniZ and Hole / Hole
Bulge /Bulge (T366W) Lump / Protuberance / MiniZT366W +T366S / L368A / Y407V + MiniZ Hole / Hole T366S / L368 A / Y407V Data collect ALS 5.0.1 ALS 5.0.2 CLS CMCFID space.group P3i21 P2i C2 cellular upity(A, °) a = b = 44.50, c = 205.6 a = 79.24, b = 66.08, c = 102.9, β = 95.2 a = 75.47, b = 44.48, c = 68.90, β = 106.9 V _M (Â ³ / Dalton) 2.3 2.3 2.5 Resolution (Â) 50-2.5 (2.592.50) 50 - 2.7 (2.80 2.70) 50-2.1(2.18-2.10) Rsym ^{a, b} 0.090 (0.427) 0.109 (0.486) 0.090 (0.566) Number of observations 54606 10730 24715 Unique reflections 9058 30484 13006 Redundancy 5.8 (4.2) 3.5 (3.3) 1.9 (1.8) Integrity (%) ^b 96.2 (82.3) 99.5 (98.7) 99.4 (99.7) l / al ^b 15 (2.3) 11 (2.3) 13 (2.7) Wilson B (Â ² ) 45 56 34 Refinement Resolution (Â) 50 - 2.5 50 - 2.7 50 - 2.1 Number of reflections (F> 0o (F)) 8853 28356 12850 R ^c Final, Rlivre 0.222, 0.287 0.229, 0.294 0.197, 0.256 Molecules / unit and asymmetric 0.5 2 0.5 protein waste 208 968 208
160/160
Bulge /Bulge (T366W) Lump / Protuberance / MiniZT366W +T366S / L368A / Y407V + MiniZ Hole / Hole T366S / L368 A / Y407V solvent molecules 41 0 106 ^d atoms 1717 (29) 7811 (0) 1763 (23) Average B-factor ( ^A T 44/43/44 28/18/33/18: 29A, B / D, E /G.H / l, J: everything 39/38/39 Lj ^ Rmsd actions 0.009 0.016 0.009 Angulos Rmsd (°) 1.1 1.6 1.1 Bs connected, Rmsd (A ² ) 2.8 / 2.8 4.0 / 4.5 4.5 / 4/3 Number of TLS groups 4 12 2 Ramachandran(%) 91.8 / 7.1 / 0 / 1.1 88.6 / 10.9 / 0.5 / 0 95.1 / 4.4 / 0.5 /0
^a Rsym = Σ | | I | - | <l> | | / Σ | <l> |, where I is the intensity of a single observation and <l> the average intensity for equivalent symmetry observations.
^b in parentheses, for the highest wrap resolution.
^c R = Σ | Εο-Εο | / Σ | Εο |, where Fo and Fc are observed and the amplitudes of structural factors are calculated, respectively. Free is calculated as R for hijacked reflections of refinement.
^d in parentheses, the number of atoms assigned is less than the occupancy unit.
^and protein / solvent / all atoms or A, B / D, E / G, H / i, J: everything.

权利要求:
Claims (26)
[1]
Claims
1. VARIANT HETEROMULTIMERIC PROTEIN OR MODIFIED IgG ANTIBODY, characterized by comprising an Fc variant of a wild-type Fc polypeptide, said Fc variant comprising at least two amino acid modifications in the Fc region of said wild-type Fc polypeptide, in which said variant protein exhibits separation decreased, decreased head-tail formation or increased total yield compared to the wild type Fc polypeptide.
[2]
2. PROTEIN OR ANTIBODY according to claim 1, characterized in that they additionally comprise knob-into-hole modifications.
[3]
3. VARIANT HETEROMULTIMERIC PROTEIN OR MODIFIED IgG ANTIBODY, characterized by comprising an Fc variant of a wild type Fc polypeptide, in which said Fc variant comprises substitutions in residues 241 and 243 (EU numbering) in at least one heavy chain with an amino acid which is different from that present in a wild-type Fc polypeptide, thereby causing decreased unbundling, decreased head-tail formation or increased total yield compared to IgG antibody without substitutions at residues 241 and 243 (EU number).
[4]
4. PROTEIN OR ANTIBODY according to claim 3, characterized by comprising mutations in at least one heavy chain selected from F241R / F243S and F241S / F243R.
[5]
PROTEIN OR ANTIBODY according to one of claims 3 to 4, characterized in that it additionally comprises orifice bulge modification (s).
[6]
6. PROTEIN OR ANTIBODY, according to claim 5, characterized by the modification of protuberance
2/5 understand replacing an original amino acid residue from the Fc polypeptide interface with an amino acid residue with a side chain greater than the original amino acid residue, in which the substitute amino acid residue is selected from the group consisting of tryptophan, phenylalanine , tyrosine and arginine.
[7]
PROTEIN OR ANTIBODY according to one of claims 5 to 6, characterized in that the protuberance modification comprises T366W substitution (EU numbering).
[8]
PROTEIN OR ANTIBODY according to one of Claims 5 to 7, characterized in that the orifice modification comprises replacing an original amino acid residue at the Fc polypeptide interface with an amino acid residue with a side chain smaller than the original amino acid residue, in that the substitute amino acid residue is selected from the group consisting of serine, threonine, valine and alanine.
[9]
9. PROTEIN OR ANTIBODY according to claim 8, characterized in that the orifice modification comprises two or more amino acid substitutions selected from the group consisting of T366S, L368A and Y407V (EU numbering).
[10]
PROTEIN OR ANTIBODY according to one of claims 5 to 9, characterized by the heavy chain comprising the F241R / F243S or F241S / F243R substitution further comprising a bulge modification.
[11]
PROTEIN OR ANTIBODY according to one of claims 5 to 10, characterized by the heavy chain comprising the F241R / F243S or F241S / F243R substitution additionally comprising an orifice modification.
3/5
[12]
PROTEIN OR ANTIBODY according to one of Claims 5 to 11, characterized in that both heavy chains comprise the F241R / F243S or F241S / F243R substitution.
[13]
PROTEIN OR ANTIBODY according to one of claims 1 to 12, characterized in that the protein or antibody is a multispecific antibody.
[14]
14. NUCLEIC ACID isolated, characterized by encoding the heteromultimeric variant protein or the modified IgG antibody, as defined in one of claims 1 to 13.
[15]
15. EXPRESSION VECTOR, characterized in that it comprises a nucleic acid, as defined in claim 14.
[16]
16. HOSTING CELL, characterized by comprising the nucleic acid, as defined in claim 14 or the expression vector, as defined in claim 15.
[17]
17. HOSTING CELL, characterized by producing the variant heteromultimeric protein or the modified IgG antibody, as defined in one of claims 1 to 13.
[18]
18. CELL according to one of claims 16 to 17, characterized in that it is a CHO cell or an E. coli cell.
[19]
19. METHOD TO PRODUCE A VARIANT HETEROMULTIMERIC PROTEIN OR MODIFIED IgG ANTIBODY, characterized in that it comprises culturing the cell, as defined in one of claims 16 to 17, and recovering the variant heteromultimer protein or the modified IgG antibody from the cell culture.
[20]
20. COMPOSITION, characterized in that it comprises a variant heteromultimeric protein or the modified IgG antibody, as defined in one of claims 1 to 13.
4/5
[21]
21. METHOD FOR PREPARING A HETEROMULTIMERIC PROTEIN comprising a first Fc-containing polypeptide having a first heterodimerization domain and a second Fc-containing polypeptide having a second heterodimerization domain, wherein the first and / or second Fc-containing polypeptide comprises substitutions in the residues 241 and 243 (EU numbering), the method characterized by comprising the steps of:
(a) providing a first polypeptide that contains purified Fc, having a first heterodimerization domain;
(b) providing a second polypeptide containing purified Fc, having a second heterodimerization domain;
(c) combining the first and second polypeptides that contain Fc; and (d) refolding the first Fc-containing polypeptide with the second Fc-containing polypeptide to form a heteromultimeric protein.
[22]
22. The method of claim 21, further comprising the step of purifying the heteromultimeric protein.
[23]
23. METHOD according to one of claims 21 to 22, characterized in that the first and / or second Fc-containing polypeptide comprises substitutions F241 R / F243S or F241S / F243R.
[24]
24. METHOD according to claim 23, characterized by the first and / or second polypeptide containing Fc additionally comprising orifice bulge modification (s).
[25]
25. VARIANT HETEROMULTIMERIC PROTEIN, characterized by being produced by the method, as defined in claim 19.
5/5
[26]
26. VARIANT HETEROMULTIMERIC PROTEIN, characterized by being produced by the method, as defined in claim 21.

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KR20140043722A|2014-04-10|

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CN103649117A|2014-03-19|

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EP2670776B1|2018-11-21|

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法律状态:
2020-03-24| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2020-11-17| 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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2020-12-29| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-03-30| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-10-13| 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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US201161439750P| true| 2011-02-04|2011-02-04|

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US61/439,750|2011-02-04|

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PCT/US2012/023749|WO2012106587A1|2011-02-04|2012-02-03|Fc VARIANTS AND METHODS FOR THEIR PRODUCTION|

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