Compositions and methods for the therapy and diagnosis of breast cancer

专利摘要:
The present invention relates to compositions and methods for the treatment and diagnosis of cancer, in particular breast cancer. Exemplary compositions include a breast cancer polypeptide, an immunogenic portion thereof, a polynucleotide encoding the polypeptide, an antigen presenting cell expressing the polypeptide and one or more T cells specific for the cell expressing the polypeptide. The compositions described above are useful, for example, in the diagnosis, prevention and / or treatment of diseases, in particular breast cancer.
公开号:KR20030028474A
申请号:KR1020027015801
申请日:2001-05-22
公开日:2003-04-08
发明作者:프루다키스토니엔；리드스티븐지；스미스존엠；미셔린다이；딜론데이빈씨；레터마크더블유；왕아이준；스케이키야시르에이더블유；할록커수잔엘；데이크레이그에이치
申请人:코릭사 코포레이션；
IPC主号:

专利说明:

Compositions and methods for the treatment and diagnosis of breast cancer {Compositions and methods for the therapy and diagnosis of breast cancer}
[2] Breast cancer is an important health problem for women in the United States and around the world. While considerable research has been done regarding the detection and treatment of the disease, breast cancer is the second leading cause of cancer-related deaths in women, with more than 180,000 women in the United States suffering from this disease each year. For women in North America, the chance of developing breast cancer in their lifetime is now 1 / 8th of a second.
[3] At present, there are no vaccines or other universally successful methods useful for the prevention or treatment of breast cancer. The disease is currently being treated according to a combination of early diagnosis (via normal breast diagnostic procedures) and aggressive treatment, and aggressive treatment may include one or more treatments such as surgery, radiotherapy, chemotherapy and hormone therapy. have. The course of treatment for certain breast cancers is often selected based on a variety of prognostic variables, including analysis of specific tumor markers (eg, Porter-Jordan and Lippman, Breast Cancer 8: 73-100 (1994)). However, the use of established markers often results in difficult interpretations, and the high mortality observed in breast cancer patients suggests that improvements are needed for the treatment, diagnosis and prevention of the disease.
[4] Therefore, there is a need in the art for improved methods for the treatment and diagnosis of breast cancer. The present invention fulfills this need and provides other advantages associated with it.
[5] Summary of the Invention
[6] In one embodiment, the present invention provides a kit comprising (a) the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (b) the complement of the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (c) a sequence consisting of 20 or more consecutive residues of the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (d) sequences that hybridize under moderately stringent conditions to the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (e) a sequence having at least 75% identity with the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325, and 327 to 330; (f) a sequence having at least 90% identity with the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325, and 327 to 330; And (g) a degenerate variant of the sequence provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 It provides a polynucleotide composition.
[7] In one preferred specific embodiment, the polynucleotide composition of the present invention is at a level of at least about 2 times, preferably at least about 5 times, and most preferably at least about 10 times higher than normal tissue, about 20 of the breast cancer samples tested. It is expressed in at least%, preferably at least about 30% and most preferably at least about 50%.
[8] In another aspect, the present invention provides a polypeptide composition comprising an amino acid sequence encoded by a polynucleotide sequence described above.
[9] The invention further provides a polypeptide composition comprising an amino acid sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 299, 300, 304-306, 308-312, 314, 326 and 331-334.
[10] In certain preferred embodiments, the polypeptides and / or polynucleotides of the invention are immunogenic, ie they can elicit an immune response, in particular a humoral and / or cellular immune response, as described further herein.
[11] The invention further relates to the polypeptide sequences set forth in SEQ ID NOs: 299, 300, 304 to 306, 308 to 312, 314,326 and 331 to 334 or SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313 Preferably at least about 50%, preferably at least about 70% and more preferably at least about 50% of the immunogenic activity of the polypeptide sequences encoded by the polynucleotide sequences shown in 314, 316, 317, 325 and 327-330 Provided are fragments, variants, and / or derivatives of the polypeptide and / or polynucleotide sequences described herein having a level of at least 90%.
[12] The present invention further provides a polynucleotide encoding the polypeptide described above, an expression vector comprising the polynucleotide, and a host cell transformed or transfected with the expression vector.
[13] In another aspect, the present invention provides a pharmaceutical composition comprising a polypeptide or polynucleotide as described above and a physiologically acceptable carrier.
[14] In a related aspect of the invention, pharmaceutical compositions, eg vaccine compositions, are provided for prophylactic or therapeutic use. Such compositions generally comprise an immunogenic polypeptide or polynucleotide of the present invention and an immunostimulant such as an adjuvant.
[15] The invention further comprises (a) an antibody or antigen-binding fragment which specifically binds to a polypeptide of the invention or a fragment thereof; And (b) provides a pharmaceutical composition comprising a physiologically acceptable carrier.
[16] In a further aspect, the present invention provides a pharmaceutical composition comprising (a) an antigen presenting cell expressing a polypeptide as described above and (b) a pharmaceutically acceptable carrier or excipient. Exemplary antigen presenting cells include dendritic cells, macrophages, monocytes, fibroblasts and B cells.
[17] In a related aspect, there is provided a pharmaceutical composition comprising (a) an antigen presenting cell expressing a polypeptide as described above and (b) an immunostimulant.
[18] The invention further provides, in another embodiment, a fusion protein comprising one or more polypeptides as described above, as well as a polynucleotide encoding said fusion protein, typically a physiologically acceptable carrier and / or immunostimulant. It is provided in the form of a pharmaceutical composition comprising, for example, in the form of a vaccine composition. The fusion protein may comprise multiple immunogenic polypeptides or portions / variants thereof as described herein, further comprising one or more polypeptide fragments to facilitate expression, purification and / or immunogenicity of the polypeptide (s). It may include.
[19] In a further aspect, the present invention provides a method of stimulating an immune response in a patient, preferably a T cell response in a human patient, comprising administering the pharmaceutical composition described herein. The patient may have breast cancer, in which case the method provides a method of treating the disease, or a patient at risk for the disease may be treated prophylactically.
[20] In a further aspect, the present invention provides a method of inhibiting the occurrence of cancer in a patient, comprising administering to the patient a pharmaceutical composition as mentioned above. The patient may have breast cancer, in which case the method provides a method of treating the disease, or a patient at risk for the disease may be prophylactically treated.
[21] The invention further includes in another embodiment, contacting the biological sample with a T cell that specifically reacts with the polypeptide of the invention under conditions and for a sufficient time to remove cells expressing the protein from the biological sample. Thus, a method of removing tumor cells from a biological sample is provided.
[22] In a related aspect, a method of inhibiting the development of cancer in a patient is provided, comprising administering to the patient a biological sample treated as described above.
[23] In other embodiments, the T cells are subjected to (i) a polypeptide as described above, (ii) a polynucleotide encoding the polypeptide, and / or under conditions and for a time sufficient to stimulate and / or propagate the T cells. iii) A method of stimulating and / or proliferating a T cell specific for a polypeptide of the invention, including contacting at least one of the antigen presenting cells expressing said polypeptide. Also provided are isolated T cell populations comprising the T cells prepared as above.
[24] In a further aspect, the present invention provides a method of inhibiting the occurrence of cancer in a patient, comprising administering to the patient an effective amount of a T cell population as described above.
[25] The present invention further provides CD4 ⁺ and / or CD8 ⁺ T cells from a patient comprising (a) (i) a polypeptide comprising at least an immunogenic portion of the polypeptide described herein; (ii) a polynucleotide encoding said polypeptide; And (iii) incubating with at least one of the antigen presenting cells expressing the polypeptide; (b) providing a method of inhibiting the occurrence of cancer in a patient, by administering an effective amount of proliferated T cells to the patient, thereby inhibiting the occurrence of cancer in the patient. Proliferated cells may be cloned, but not necessarily, prior to administration to a patient.
[26] In a further aspect, the present invention provides a method of contacting a subject, comprising (a) contacting a biological sample obtained from a patient with a binder that binds to a polypeptide as mentioned above; (b) determining the amount of polypeptide binding to the binder in the sample; (c) comparing the amount of polypeptide to a predetermined cut-off value to determine the presence or absence of cancer in the patient, thereby determining the presence or absence of cancer, preferably breast cancer in the patient. In a preferred specific embodiment, the binder is an antibody, more preferably a monoclonal antibody.
[27] The invention also provides, in another aspect, a method of monitoring cancer progression in a patient. The method comprises the steps of (a) contacting a biological sample obtained from a patient within a suitable time at the first time point with a binder that binds to the aforementioned polypeptide; (b) detecting the amount of polypeptide binding to the binder in the sample; (c) repeating steps (a) and (b) with a biological sample obtained from the patient within a suitable time at subsequent time points; (d) monitoring the progression of the cancer in the patient by comparing the amount of polypeptide detected in step (c) with the amount detected in step (b).
[28] The invention further provides, in another embodiment, (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide encoding a polypeptide of the invention; (b) detecting the level of polynucleotide, preferably mRNA, that hybridizes to said oligonucleotide in the sample; (c) comparing the levels of polynucleotides that hybridize to oligonucleotides to a predetermined cut-off value to determine the presence or absence of cancer in the patient, wherein the method comprises determining the presence or absence of cancer in the patient. to provide. In certain specific embodiments, the amount of mRNA can be, for example, via a polymerase chain reaction using a polynucleotide encoding a polypeptide as described above, or one or more oligonucleotides hybridizing to one or more complements of the polynucleotide. Is detected. In another specific embodiment, the amount of mRNA is detected using hybridization techniques, using polynucleotides encoding the aforementioned polypeptides, or oligonucleotide probes that hybridize to the complement of the polynucleotides.
[29] In a related aspect, (a) contacting a biological sample obtained from a patient with an oligonucleotide that hybridizes to a polynucleotide encoding a polypeptide of the invention; (b) detecting the amount of polynucleotide hybridizing to said oligonucleotide in the sample; (c) repeating steps (a) and (b) at subsequent time points using biological samples obtained from the patient at suitable times; (d) comparing the amount of polynucleotides detected in step (c) with the amount detected in step (b) to monitor the progress of the cancer in the patient, the method comprising monitoring the progress of the cancer in the patient do.
[30] In a further aspect, the present invention provides antibodies, such as monoclonal antibodies, as well as diagnostic kits comprising said antibodies that bind to the polypeptides described above. Also provided is a diagnostic kit comprising one or more oligonucleotide probes or primers as described above.
[31] These and other aspects of the invention will be apparent with reference to the following detailed description and the accompanying drawings. All references described herein are incorporated herein by reference in their entirety, as if each were individually cited.
[1] The present invention relates generally to the treatment and diagnosis of cancer, such as breast cancer. The present invention more specifically relates to a polypeptide comprising at least a portion of a breast cancer protein, and to a polynucleotide encoding said polypeptide. Such polypeptides and polynucleotides are useful in pharmaceutical compositions, such as vaccines, and other compositions for the diagnosis and treatment of breast cancer.
[32] 1 shows differential display PCR products isolated by gel electrophoresis, obtained from cDNA prepared from normal breast tissues (lanes 1 and 2) and cDNA prepared from breast cancer tissues of the same patient (lanes 3 and 4). Arrows indicate bands corresponding to B18Ag1.
[33] FIG. 2 is a northern blot comparing the levels of B18Ag1 mRNA in breast cancer tissues (lane 1) to levels in normal breast tissues.
[34] 3 shows the levels of B18Ag1 mRNA in breast cancer tissues compared to levels in various normal and non-breast cancer tissues as measured by RNase protection assays.
[35] 4 is a genomic clone map showing the location of additional retroviral sequences obtained from the ends of the XbaI restriction digests associated with B18Ag1 (provided in SEQ ID NOs: 3-10).
[36] 5A and 5B show sequencing strategy, genomic organization, and predicted open reading frame for retroviral elements containing B18Ag1.
[37] 6 shows the nucleotide sequence of a representative breast cancer-specific cDNA B18Ag1.
[38] 7 shows the nucleotide sequence of a representative breast cancer-specific cDNA B17Ag1.
[39] 8 shows the nucleotide sequence of a representative breast cancer-specific cDNA B17Ag2.
[40] 9 shows the nucleotide sequence of a representative breast cancer-specific cDNA B13Ag2a.
[41] 10 shows the nucleotide sequence of a representative breast cancer-specific cDNA B13Ag1b.
[42] 11 shows the nucleotide sequence of a representative breast cancer-specific cDNA B13Ag1a.
[43] 12 shows the nucleotide sequence of a representative breast cancer-specific cDNA B11Ag1.
[44] 13 shows the nucleotide sequence of a representative breast cancer-specific cDNA B3CA3c.
[45] 14 shows the nucleotide sequence of a representative breast cancer-specific cDNA B9CG1.
[46] 15 shows the nucleotide sequence of a representative breast cancer-specific cDNA B9CG3.
[47] 16 shows the nucleotide sequence of a representative breast cancer-specific cDNA B2CA2.
[48] 17 shows the nucleotide sequence of a representative breast cancer-specific cDNA B3CA1.
[49] 18 shows the nucleotide sequence of a representative breast cancer-specific cDNA B3CA2.
[50] 19 shows the nucleotide sequence of a representative breast cancer-specific cDNA B3CA3.
[51] 20 shows the nucleotide sequence of a representative breast cancer-specific cDNA B4CA1.
[52] 21A shows RT-PCR analysis of breast cancer genes in breast cancer tissues (lanes 1 to 8) and normal breast tissues (lanes 9 to 13) and H ₂ O (lane 14).
[53] Figure 21B shows prostate cancer (lanes 1 and 2), colon cancer (lane 3), lung cancer (lane 4), normal prostate (lane 5), normal colon (lane 6), normal kidney (lane 7), normal liver (lane 8) ), Normal lung (lane 9), normal ovary (lanes 10 and 18), normal pancreas (lanes 11 and 12), normal skeletal muscle (lane 13), normal skin (lane 14), normal stomach (lane 15), normal testes (Lane 16), normal small intestine (lane 17), HBL-100 (lane 19), MCF-12A (lane 20), breast cancer (lanes 21 to 23), H ₂ O (lane 24), and colon cancer (lane 25) RT-PCR analysis of breast cancer genes is shown.
[54] 22 shows recognition of B11Ag1 peptide (referred to as B11-8) by anti-B11-8 CTL strains.
[55] FIG. 23 shows recognition of cell lines transformed with antigen B11Ag1 by anti-B11-8 specific clone A1.
[56] 24 shows recognition of lung adenocarcinoma strain (LT-140-22) and breast adenocarcinoma strain (CAMA-1) by B1-8 specific clone A1.
[57] The present invention relates generally to compositions and their use in the treatment and diagnosis of cancer, in particular breast cancer. As described further below, exemplary compositions of the invention include polypeptides, in particular immunogenic polypeptides, polynucleotides encoding such polypeptides, antibodies and other binding agents, antigen presenting cells (APCs) and immune system cells (eg, T cells), for example, but not limited to.
[58] The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, immunology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for illustrative purposes. Such techniques are described in detail in the literature. See, eg, Sambrook, et al. Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al. Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & Higgins, eds., 1985); Transcription and Translation (B. Hames & Higgins, eds., 1984); Animal Cell Culture (R. Freshney, ed., 1986); Perbal, A Practical Guide to Molecular Cloning (1984).
[59] All publications, patents, and patent applications cited herein above or below are hereby incorporated by reference in their entirety.
[60] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural marks unless the content clearly dictates otherwise.
[61] Polypeptide Composition
[62] As used herein, the term “polypeptide” is used in its conventional meaning, ie as a sequence of amino acids. Polypeptides are not limited to products of a particular length, ie, peptides, oligopeptides and proteins are included within the definition of a polypeptide, and the term may be used interchangeably herein unless stated otherwise. The term also does not cite or exclude modifications after expression of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, which occur naturally and non-naturally. . The polypeptide may be an entire protein, or a subsequence thereof. Particular polypeptides of interest in the context of the present invention are epitopes, ie amino acid subsequences comprising antigenic determinants that are substantially involved in the immunogenic properties of the polypeptide and can elicit an immune response.
[63] Certain exemplary polypeptides of the invention comprise a polynucleotide sequence of any one of SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330, moderately Or, alternatively, to a polynucleotide sequence represented by any one of SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 under high stringent conditions. And those encoded by the hybridizing sequence. Certain other exemplary polypeptides of the invention comprise the amino acid sequence represented by any one of SEQ ID NOs: 299, 300, 304-306, 308-312, 314, 326, and 331-334.
[64] Polypeptides of the invention are often cited herein as breast cancer proteins or breast cancer polypeptides, indicating that their identification is based at least in part on their increased expression levels in breast cancer samples. Thus, "breast cancer polypeptide" or "breast cancer protein" generally refers to a polypeptide sequence of the invention, or a polynucleotide sequence that encodes the polypeptide, as determined using representative assays provided herein, At least two times, and preferably at least five times more, levels of expression in normal tissues, a substantial proportion of breast cancer samples, such as at least about 20% of tested breast cancer samples, more preferably about 30 At least%, and most preferably at least about 50%. Based on the increase in expression levels in tumor cells, the breast cancer polypeptide sequences of the present invention have specific uses, both as diagnostic markers as well as therapeutic targets, as further described below.
[65] In certain preferred embodiments, the polypeptides of the invention are immunogenic, ie they detectably respond in immunoassays (such as ELISA or T-cell stimulation assays) using antisera and / or T-cells from patients with breast cancer. . Screening of immunogenic activity can be performed using techniques known to those skilled in the art. For example, the screening can be performed using a method as described in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In one illustrative example, the polypeptide can be immobilized on a solid support to contact the patient serum to allow the antibody in the serum to bind to the immobilized polypeptide. Unbound serum can then be removed and bound antibody can be detected using, for example, ¹²⁵ I-labeled Protein A.
[66] As will be appreciated by those skilled in the art, immunogenic portions of the polypeptides described herein are also included in the present invention. As used herein, an “immunogenic moiety” is, in itself, an immunologically reactive (ie specifically binding to) B-cell and / or T-cell surface antigen receptor that recognizes a polypeptide. Fragments of the immunogenic polypeptides of the invention. Immunogenic moieties can generally be identified using known techniques as summarized in Paul, Fundamental Immunology, 3rd ed., 243-247 (Raven Press, 1993) and references cited therein. Such techniques include screening polypeptides for their ability to react with antibodies, antisera and / or T cell lines or clones. As used herein, antisera and antibodies, when they specifically bind to an antigen (ie, they react with proteins in ELISA or other immunoassays, but not detectably with unrelated proteins), "Antigen-specific". The antisera and antibodies can be prepared as described herein and using known techniques.
[67] In one preferred specific embodiment, the immunogenic portion of a polypeptide of the invention is antisera and / or T at a level substantially less than the reactivity of the full length polypeptide (eg, in ELSIA and / or T cell reactivity assays). It is the part that reacts with the cell. Preferably, the immunogenic activity level of the immunogenic portion is at least about 50%, preferably at least about 70%, and most preferably at least about 90% of the immunogenicity of the full length polypeptide. In certain instances, the preferred immunogenic portion will be identified as having an immunogenic activity level that is greater than the immunogenic activity of the corresponding full length polypeptide, eg, at least about 100% or at least 150% of the immunogenic activity. Can be.
[68] In certain other specific embodiments, exemplary immunogenic moieties can include peptides that lack an N-terminal leader sequence and / or a transmembrane domain. Other exemplary immunogenic moieties will contain small N- and / or C-terminal deletions (eg, 1-30 amino acids, preferably 5-15 amino acid deletions) relative to the mature protein.
[69] In another specific embodiment, a polypeptide composition of the invention also comprises a T cell and / or antibody produced against a polypeptide of the invention, in particular a polypeptide having an amino acid sequence described herein or an immunogenic fragment or variant thereof. It may include one or more immunologically reactive polypeptides.
[70] In another specific embodiment of the invention, one or more of the polypeptides described herein, or a continuous nucleic acid sequence contained in the polynucleotides described herein, or hybridized with one or more of these sequences under conditions of moderate to high stringency A polypeptide is provided that comprises one or more polypeptides capable of inducing T cells and / or antibodies that are immunologically reactive with one or more polypeptides encoded by one or more nucleic acid sequences.
[71] In another aspect, the invention provides a polypeptide fragment comprising at least about 5, 10, 15, 20, 25, 50, or 100 contiguous amino acids, or any intermediate length thereof, of a polypeptide composition as disclosed herein, for example Or those shown in SEQ ID NOs: 299, 300, 304 to 306, 308 to 312, 314, 326 and 331 to 334 or SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, Provided are those encoded by the polynucleotide sequences shown in the sequences of 317, 325 and 327-330.
[72] In another aspect, the present invention provides variants of the polypeptide compositions described herein. Polypeptide variants generally encompassed by the present invention are typically about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% along the polypeptide sequences set forth herein and their lengths. At least 94%, 95%, 96%, 97%, 98% or 99% identity (measured as described below).
[73] In one preferred specific embodiment, polypeptide fragments and variants provided by the present invention are immunologically reactive with antibodies and / or T cells that are reactive with the full length polypeptides specifically set forth herein.
[74] In another preferred specific embodiment, the polypeptide fragments and variants provided by the present invention are at least about 50%, preferably at least about 70%, and most preferred of the immunogenic activity exhibited by the full length polypeptide sequence specifically presented herein. Preferably at least about 90% immunogenic activity level.
[75] As used herein, polypeptide “variants” are polypeptides that are typically different from the polypeptides specifically described herein with one or more substitutions, deletions, additions and / or insertions. Such variants may be naturally occurring, or may, for example, modify one or more polypeptide sequences of the invention and assess their immunogenic activity as described herein and use one of a number of techniques known in the art. This can be produced synthetically.
[76] For example, certain exemplary variants of the polypeptides of the invention include those in which one or more portions, such as an N-terminal leader sequence or transmembrane domain, have been removed. Other exemplary variants include variants in which a small portion (eg, 1-30 amino acids, preferably 5-15 amino acids) is removed from the N- and / or C-terminus of the mature protein.
[77] In many instances, variants will contain conservative substitutions. A “conservative substitution” is a substitution where an amino acid is substituted with another amino acid having similar properties such that one skilled in the art of peptide chemistry predicts that the secondary structure and degree of hydration of the polypeptide will not substantially change. As described above, modifications can be made in the structure of the polynucleotides and polypeptides of the present invention and still yield functional molecules encoding variant or derivative polypeptides having desirable properties, such as immunogenic properties. Can be. If it is desired to change the amino acid sequence of a polypeptide to produce an equivalent, or more improved immunogenic variant or portion of the invention, one of ordinary skill in the art will typically employ one or more codons encoding the DNA sequence according to Table 1. Will change.
[78] For example, certain amino acids can be substituted with other amino acids in the protein structure without significant loss of ability to bind interactively with structures such as, for example, antigen-binding regions or binding sites on the substrate molecule of the antibody. Because the ability to interact and the properties of a protein are defined as the biological functional activity of the protein, certain amino acid sequence substitutions can be performed within the protein sequence, and of course the DNA coding sequence underlying it, and nevertheless have similar properties. Proteins can be obtained. Accordingly, it is understood that various changes may be made in the peptide sequence of the described composition or in the corresponding DNA sequence encoding the peptide, without significant loss of its biological utility or activity.
[79]
[80] In carrying out such changes, the hydropathic index of amino acids can be taken into account. The importance of the degree of hydration amino acid index in contributing interactive biological functions on proteins is generally understood in the art [Kyte and Doolittle, 1982, incorporated herein by reference]. It is recognized that the relative degree of hydration of amino acids affects the secondary structure of the protein, thereby defining interactions with other molecules of the protein, such as enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is becoming. Each amino acid was assigned a hydration index based on its hydrophobicity and charge properties (Kyte and Doolittle, 1982). This value is isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
[81] It is known that certain amino acids can be substituted with other amino acids having similar hydration indices or scores to yield proteins with similar biological activity, i.e., biologically functional equivalent proteins. In carrying out such changes, substitution of amino acids having a hydration index within the range of ± 2 is preferred, particularly preferably within ± 1, even more preferably within ± 0.5. It is understood in the art that substitution of similar amino acids can be performed effectively based on hydrophilicity. See US Pat. No. 4,554,101; Which is hereby incorporated by reference in its entirety] states that since the largest local mean hydrophilicity of a protein is affected by the hydrophilicity of its adjacent amino acids, it correlates with the biological properties of the protein.
[82] As detailed in US Pat. No. 4,554,101, the following hydrophilicity values were assigned to amino acid residues: arginine (+3.0); Lysine (+3.0); Aspartate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+3.0); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). It is understood that amino acids can be substituted with other amino acids having similar hydrophilicity values to still obtain biological equivalents, particularly immunologically equivalent proteins. In this variation, substitutions of amino acids having hydrophilicity within ± 2 are preferred, with preference being within ± 1, even more particularly preferably within ± 0.5.
[83] Thus, as summarized above, amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into account various aforementioned properties are known to those skilled in the art and include arginine and lysine; Glutamate and aspartate; Serine and threonine; Glutamine and asparagine; And valine, leucine and isoleucine.
[84] In addition, any polynucleotide may be modified to further increase stability in vivo. Possible modifications include the addition of flanking sequences at the 5 'and / or 3' ends; The use of phosphorothioate or 2'O-methyl instead of phosphodiesterase bonds in the backbone; And / or including but not limited to inosine, quaosine and wibutosin as well as unusual bases such as acetyl-, methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine and uridine It doesn't work.
[85] Amino acid substitutions may be further performed based on similarities in the polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphoteric properties of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid, and positively charged amino acids include lysine and arginine; Amino acids leucine, isoleucine, and valine with uncharged polar head groups having similar hydrophilicity values; Glycine and alanine; Asparagine and glutamine; And serine, threonine, phenylalanine and tyrosine. Other amino acid groups that may exhibit conservative changes include (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; And (5) phe, tyr, trp, his. Variants may also contain, or optionally non-conservative changes. In a preferred specific embodiment, variant polypeptides differ from the native sequence by substitution, deletion or addition of up to 5 amino acids. Variants may also (or optionally) be modified by, for example, deletions or additions of amino acids that minimally affect the immunogenicity, secondary structure and hydrophobic properties of the polypeptide.
[86] As noted above, the polypeptide may comprise a signal (or leader) sequence at the N terminus of the protein, which directs the transport of the protein simultaneously with or after translation. The polypeptide may also be conjugated to a linker or other sequence (eg, poly-His) for ease of synthesis, purification or identification of the polypeptide, or to enhance binding of the polypeptide to a solid support. For example, the polypeptide can be conjugated to an immunoglobulin Fc region.
[87] When comparing polypeptide sequences, the two sequences are said to be “identical” if the amino acid sequences in the two sequences are identical when arranged for maximum correspondence, as described below. Comparison between two sequences is typically performed by comparing sequences on a comparison window to identify and compare local regions of sequence similarity. A “comparative window” as used herein refers to at least about 20 contiguous portions, generally 30 to about 75, which allows for optimal alignment of the two sequences and then comparing the sequences to the same number of contiguous portions of the reference sequence. , From 40 to about 50 consecutive sections.
[88] Optimal alignment of sequences for comparison can be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) In the Lasergene suite of bioinformatics software using default variables. The program embodies several arrangements described in the following documents [Dayhoff, M.O. (1978) A model of evolutionary chagne in proteins-Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5: 1151-153; Myers, E.W. and Muller W. (1988) CABIOS 4: 11-17; Robinson, 425; Sneath, P.H.A. and Sokal, R. R. (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Fransisco, CA; Wilbur, W.J. and Lipman, D. J. (1983) Proc. Natl. Acad. Sci. USA 80: 726-730.
[89] Alternatively, the optimal alignment of sequences for comparison can be found in Smith and Waterman (1981) Add. APL. By a local confirmation algorithm of Math 2: 482, Meedleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the confirming arrangement algorithm of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444], the computerized means of the algorithm [GAP, BESTFIT, BLAST, FASTA and TFASTA, in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, , WI], or by scrutiny.
[90] One preferred example of a suitable algorithm for determining percent sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, respectively. BLAST and BLAST 2.0 can be used, for example, in conjunction with the variables described herein to determine the percent sequence identity of the polynucleotides and polypeptides of the invention. Software for performing BLAST analysis is officially available through the National Center for Biotechnology Information (NCBI). For amino acid sequences, a scoring matrix can be used to calculate cumulative scoring. The elongation of the word hit in each direction is reduced when the cumulative array score decreases by an amount of X from its maximum achievement; Due to accumulation of one or more negative-scoring residue sequences, when the cumulative score reaches zero or less; Or when one of the two ends of the sequence is reached. The BLAST algorithm variables W, T, and X determine the sensitivity and speed of the array.
[91] In one preferred approach, “percentage of sequence identity” is determined by comparing two optimally arranged sequences on a comparison window of 20 or more positions, wherein the position of the polypeptide in the comparison window is determined by two sequences. Contains up to 20%, typically 5 to 15%, or 10 to 12% of additions or deletions (eg gaps) compared to the reference sequence (which does not include additions or deletions) for can do. The percentage determines the number of positions occurring in both sequences to yield the number of positions where identical amino acid residues matched, dividing the matched positions by the number of total positions in the reference sequence (ie window size) and sequence It is calculated by multiplying by 100 to calculate the identity (%).
[92] In another exemplary specific embodiment, the polypeptide comprises multiple polypeptides as described herein, or comprises polypeptides as described herein and unrelated sequences, such as one or more known tumor proteins. Fusion polypeptides. The fusion partner may, for example, assist in providing a T helper epitope (immunological fusion partner), preferably a T helper recognized by humans, or expressing (expressing) the protein at a higher yield than the native recombinant protein. Enhancer). Certain preferred fusion partners are both immunological and expression enhancing fusion partners. Other fusion partners can be selected to increase the solubility of the polypeptide or to target the polypeptide into the desired intracellular compartment. Further fusion partners also include an affinity tag that facilitates purification of the polypeptide.
[93] Fusion polypeptides can generally be prepared using standard techniques, including chemical conjugation. Preferably, the fusion polypeptide is expressed as a recombinant polypeptide, allowing for increased levels of production compared to non-fused polypeptides in the expression system. In brief, each DNA sequence encoding a polypeptide component can be combined and linked into a suitable expression vector. The 3 'end of the DNA sequence encoding one polypeptide component, with or without a peptide linker, is linked to the 5' end of the DNA sequence encoding the second polypeptide component so that the reading frames of the sequences are on the same Let's do it. This translates into a single fusion polypeptide that retains the biological activity of the polypeptides of both components.
[94] Peptide linker sequences can be used to sequester the first and second polypeptide components at a position sufficiently apart to ensure that each polypeptide is folded into its secondary and tertiary structure. The peptide linker sequence is introduced into the fusion polypeptide using standard techniques known in the art. Suitable peptide linker sequences can be selected based on the following variables: (1) the ability to allow flexible stretched forms; (2) the ability to not allow secondary structures to interact with functional epitopes on the first and second polypeptides; And (3) absence of hydrophobic or charged residues capable of reacting with the polypeptide functional epitope. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other contiguous neutral amino acids such as Thr and Ala can also be used as the linker sequence. Amino acid sequences that may be usefully used as linkers are described in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., Proc. Natl. Acad. Sci. USA 83: 8258-8262, 1986; US Pat. No. 4,935,233 and US Pat. No. 4,751,180. The linker sequence may generally be from 1 to about 50 amino acids in length. Linker sequences are unnecessary if the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent spatial interference.
[95] The linked DNA sequence is operably linked to a suitable transcriptional or translational regulatory element. The regulatory element involved in DNA expression is located just before 5 'of the DNA sequence encoding the first polypeptide. Similarly, stop codons and transcription termination signals required to terminate translation are located just after 3 'of the DNA sequence encoding the second polypeptide.
[96] Fusion polypeptides can include polypeptides as described herein with unrelated immunogenic proteins such as immunogenic proteins capable of inducing a recall response. Examples of such proteins include tetanus, tuberculosis and hepatitis proteins. See, eg, Stoute et al. New Engl. J. Med., 336: 86-91, 1997].
[97] In one preferred specific embodiment, the immunological fusion partner is derived from a mycobacterium species, eg, mycobacterium tuberculosis-derived Ra12 fragment. Methods of expression of Ra12 compositions and heterologous polynucleotide / polypeptide sequences and / or use thereof in immunogenicity are described in US Patent No. 60 / 158,585, the contents of which are incorporated herein by reference in their entirety. do. Briefly, Ra12 represents a polynucleotide region that is a subsequence of Mycobacterium tuberculosis MTB32A nucleic acid. MTB32A is M. It is a serine protease of 32 KD molecular weight encoded by a gene in toxic and non-toxic strains of tuberculosis. Nucleotide and amino acid sequences of MTB32A have been described [eg, US Patent Application 60 / 158,585; See also: Skeiky et al., Infection and Immun. (1999) 67: 3998-4007, incorporated herein by reference. C-terminal fragments of the MTB32A coding sequence are expressed at high levels and remain as soluble polypeptides during the purification process. Ra12 can also enhance the immunogenicity of heterologous immunogenic polypeptides fused thereto. One preferred Ra12 fusion polypeptide comprises a 14KD C-terminal fragment corresponding to amino acid residues 192 to 323 of MTB32A. Other preferred Ra12 polynucleotides generally include at least about 15 contiguous nucleotides encoding at least a portion of the Ra12 polypeptide, at least about 30, at least about 60, at least about 100, at least about 200, or at least about 300 nucleotides. It includes. Ra12 polynucleotides may comprise a native sequence (ie, an endogenous sequence encoding a Ra12 polypeptide or portion thereof) or may comprise a variant of the sequence. Ra12 polynucleotide variants may contain one or more substitutions, additions, deletions and / or insertions in which the biological activity of the encoded fusion polypeptide is substantially reduced compared to a fusion polypeptide comprising a native Ra12 polypeptide. . The variant preferably exhibits at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity with the polynucleotide sequence encoding the native Ra12 polypeptide or portion thereof.
[98] In another preferred specific embodiment, the immunological fusion partner is derived from protein D, which is the surface protein of the Gram-negative bacterium haemophilus influenza B (WO 91/18926). Preferably, the protein D derivative comprises approximately the first 1/3 of the protein (eg, the first N-terminal 100 to 110 amino acids) and the protein D derivative may be lipidated. In certain preferred specific embodiments, the first 109 residues of the lipoprotein D fusion partner are included on the N-terminus to provide a polypeptide having additional exogenous T cell epitopes. Increase expression in E. coli (resulting in function as an expression enhancer). Lipid tails ensure optimal presentation of antigen to antigen presenting cells. Other fusion partners include NS1 (hemagglutinin), a non-structural protein of influenza viruses. Typically, 81 N-terminal amino acids are used, but different fragments including T helper epitopes may be used.
[99] In another specific embodiment, the immunogenic fusion partner is a protein known as LYTA or a portion thereof (preferably C-terminal portion). LYTA is derived from Streptococcus pneumoniae, which strain synthesizes N-acetyl-L-alanine amidase known as amidase LYTA [encoded from LytA gene; Gene 43: 265-292, 1986. LYTA is an autolytic enzyme that specifically degrades certain bonds in the peptidoglycan backbone. The C-terminal domain of the LYTA protein is involved in affinity for certain choline analogs such as choline or DEAE. This property is useful for the expression of fusion proteins. It has been used in the development of E. coli C-LYTA expressing plasmids. Purification of hybrid proteins containing C-LYTA fragments at the amino terminus has been described (Biotechnology 10: 795-798, 1992). In a preferred specific embodiment, repeating portions of LYTA can be introduced into the fusion polypeptide. Repetitive portions are found in the C-terminal region starting at residue 178. Certain preferred repeating portions introduce residues 188-305.
[100] Another exemplary specific embodiment relates to a fusion polypeptide and a polynucleotide encoding it, wherein the fusion partner is directed to the endosome / lysosomal compartment, as described in US Pat. No. 5,633,234. And a targeting signal that can be directed. When fused to this targeting signal, the immunogenic polypeptides of the invention will bind more effectively with type II MHC molecules and thus provide CD4 ⁺ T cell specific enhanced in vivo stimulation for the polypeptide.
[101] Polypeptides of the invention are prepared using one of a variety of known synthetic and / or recombinant techniques, the latter being further described below. Polypeptides, portions of which are generally less than about 150 amino acids, or other variants thereof, may be prepared synthetically, generally using techniques known to those skilled in the art. In one illustrative example, the polypeptide is synthesized using one of commercially available solid phase techniques, such as Merrifield solid phase synthesis, in which amino acids are added to a continuously growing amino acid chain. See Merrifield, J. Am. Chem. Soc. 85: 2149-2146, 1963. Equipment for autosynthesis of polypeptides is commercially available from the source (Perkin Elmer / Applied BioSystems Division, Foster City, Calif.) And can be operated according to the manufacturer's instructions.
[102] In general, polypeptide compositions of the present invention (including fusion polypeptides) are isolated. An "isolated" polypeptide has been removed from its natural environment. For example, a natural protein or polypeptide is isolated when isolated from some or all of the coexisting materials in nature. Preferably, the polypeptide is also purified, for example at least about 90% pure, more preferably at least about 95% pure, and most preferably at least about 99% pure.
[103] Polynucleotide composition
[104] In another aspect, the present invention provides a polynucleotide composition. The terms "DNA" and "polynucleotide" are used herein interchangeably to mean discrete DNA molecules that do not contain the entire genomic DNA of a particular species. As used herein, “isolated” means that the polynucleotide is substantially removed from another coding sequence, and the DNA molecule is a large chromosomal fragment or other functional gene or polypeptide encoding region, such as a large portion of unrelated coding DNA. It does not contain a part. Of course, this means a DNA molecule as naturally isolated and does not exclude genes or coding regions that are subsequently added to the fragment artificially.
[105] As will be appreciated by those of skill in the art, the polynucleotide compositions of the present invention are genomic sequences, extracellular and plasmid-encoded sequences, and smaller genetic engineering adapted to express or express proteins, polypeptides, peptides, and the like. May comprise an engineered gene fragment. The fragments can be naturally isolated or artificially modified synthetically.
[106] In addition, as will be appreciated by those skilled in the art, the polynucleotides of the present invention may be single stranded (coding or antisense) or double stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules may comprise mRNAs that contain introns and that do not contain HnRNA molecules or introns that correspond to DNA molecules in a one-to-one manner. Additional coding or non-coding sequences may be present in the polynucleotides of the present invention, but are not required, and the polynucleotides may be linked to other molecules and / or support materials, but are not necessary.
[107] The polynucleotide may comprise a native sequence (ie, an endogenous sequence encoding a polypeptide / protein or portion thereof), or a variant or derivative of said sequence, and preferably a sequence encoding an immunological variant or derivative It may include.
[108] Thus, in another aspect of the invention, according to some of the polynucleotide sequences set forth in one of SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 Or all; The complement of the polynucleotide sequence set forth in one of SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; And a degenerate variant of the polynucleotide sequence shown in any one of SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 Is provided. In certain preferred specific embodiments, the polynucleotide sequences shown herein encode an immunogenic polypeptide, as described above.
[109] In other related specific embodiments, the invention is substantially identical to the sequences described herein in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 At least about 70% identity, as compared to the polynucleotide sequence of the present invention using, for example, the methods described herein (eg, BLAST analysis using standard variables, as described below), Preferably, polynucleotide variants having at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity are provided. Those skilled in the art will recognize that these values can be appropriately adjusted to determine the corresponding identity of the protein encoded by the two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, read frame localization, and the like. will be.
[110] Typically, polynucleotide variants preferably have one or more substitutions such that the immunogenicity of the polypeptides encoded by the variant polynucleotides is not substantially reduced compared to the polypeptides encoded by the polynucleotide sequences specifically set forth herein. , Additions, deletions and / or insertions. The term “variant” should also be understood to include homologous genes of heterologous origin.
[111] In additional specific embodiments, the present invention provides polynucleotide fragments comprising consecutive stretches of varying lengths of sequences that are the same or complementary to one or more of the sequences described herein. For example, at least about 10, 15, 20, 30, 40, 50, 75, 100, 150, 200, 300, 400, 500, or 1000 consecutive nucleotides of one or more of the sequences described herein, as well as But polynucleotides are provided that include all intermediate lengths of that length. In the above, the "intermediate length" is 16, 17 18, 19 and the like; 21, 22, 23, and the like; 30, 31, 32, and the like; 50, 51, 52, 53, and the like; 100, 101, 102, 103, and the like; Quoted values such as 150, 151, 152, 153 and the like and from 200 to 500; It means any length between values including all integers between 500 and 1000.
[112] In another specific embodiment of the present invention, a polynucleotide composition is provided that is capable of hybridizing under moderate to high stringent conditions to a polynucleotide sequence or fragment thereof provided herein, or a complementary sequence thereof. Hybridization techniques are known in the field of molecular biology. For illustrative purposes, suitable moderately stringent conditions for testing hybridization of polynucleotides of the present invention with other polynucleotides include pre-washing in a solution of 5 × SSC, 0.5% SDS, 1.0 mM EDTA, pH 8.0; Hybridization overnight at 5 × SSC at 50 ° C. to 60 ° C .; Then wash twice for 20 minutes using 2 X, 0.5 X and 0.2 X SSC, each containing 0.1% SDS at 65 ° C. Those skilled in the art will appreciate that the stringency of hybridization can be easily controlled by methods such as varying the salt content of the hybridization solution and / or the hybridization performance temperature. For example, in other specific embodiments, suitable high stringency hybridization conditions include the conditions described above, except for raising the hybridization temperature, for example, raising the temperature to 60-65 ° C. or 65-70 ° C. do.
[113] In certain preferred embodiments, the polynucleotides described above, such as polynucleotide variants, fragments and hybridization sequences, encode polypeptides that are immunologically cross-reactive with the polypeptide sequences specifically set forth herein. In another preferred specific embodiment, the polynucleotide is a polypeptide having an immunogenic activity level of at least about 50%, preferably at least about 70%, and more preferably at least about 90%, relative to the polypeptide sequences specifically presented herein. Encrypt it.
[114] The polynucleotides, or fragments thereof, of the present invention may be mixed with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding fragments, and the like, regardless of the length of the coding sequence itself, Their overall length can vary considerably. Accordingly, it is understood that nucleic acid fragments of any length may be used and the overall length is preferably limited by the ease of manufacture and use in the intended recombinant DNA protocol. For example, exemplary polynucleotide fragments having a total length (including all intermediate lengths) of about 10,000, about 5,000, about 3,000, about 2,000, about 1,000, about 500, about 200, about 100, about 50 base pairs in length It is understood that it is useful in many implementations of the invention.
[115] When comparing polynucleotide sequences, the two sequences are referred to as "identical" if the nucleotide sequences in the two sequences are identical when arranged for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences on a comparison window to identify and compare local regions of sequence similarity. As used herein, “comparative window” means a segment of at least about 20 contiguous positions, generally 30 to about 75, 40 to about 50 contiguous positions, wherein two sequences are optimally aligned. The sequence can then be compared to the reference sequence at the same number of consecutive positions.
[116] Optimal alignment of sequences for comparison can be performed using the Megalign program (DNASTAR, Inc., Madison, Wis.) In the Lasergene suite of bioinformatics software, using default variables. The program embodies several arrangements described in the following references [Dayhoff, M.O. (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J. (1990) Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M. (1989) CABIOS 5: 151-153; Myers, E.W. and Muller W. (1988) CABIOS 4: 11-17; Robinson, E.D. (1971) Comb. Theor 11: 105; Santou, N. Nes, M. (1987) Mol. Biol. Evol. 4: 406-425; Sneath, P.H.A. and Sokal, R. R. (1973) Numerical Taxonomy-the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D. J. (1983) Proc. Natl. Acad. Sci. USA 80: 726-730.
[117] In addition, the optimal arrangement of sequences for comparison is described in Smith and Waterman (1981) Add. APL. By the local identity algorithm of Math 2: 482, Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, by Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444], the computerized means of the algorithm (GAP, BESTFIT, BLAST, FASTA and TFASTA, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI in the Wisconsin Genetics Software Package). ) Or by scrutiny.
[118] One preferred example of an algorithm that is suitable for determining percent sequence identity and percent sequence similarity is described in Altschul et al. (1977) Nucl. Acids Res. 25: 3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215: 403-410, the BLAST and BLAST 2.0 algorithms described, respectively. BLAST and BLAST 2.0 can determine the percent sequence identity to the polynucleotides of the invention, for example, using the variables described herein. Software for performing BLAST analysis is commercially available through the National Center for Biotechnology Information (NCBI). In one illustrative example, cumulative scoring is calculated using the variables M (compensation score for matching residue pairs; always> 0) and N (penalty score for mismatching residues; always <0) for nucleotides. can do. The elongation of the word hit in each direction is reduced when the cumulative array score decreases by an amount of X from its maximum achievement; Due to accumulation of one or more negative-scoring residue sequences, when the cumulative score reaches zero or less; Or when one of the two ends of the sequence is reached. The BLAST algorithm variables W, T, and X determine the sensitivity and speed of the array. The BLASTIN program (for nucleotide sequences) defaults to a word length of 11 (W), an expectation of 10 (E) and a BLOSUM62 scoring matrix (Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915] array, 50 (B), 10 expected (E), M = 5, N = -4 and comparison of the two chains.
[119] Preferably, “% sequence identity” is determined by comparing two optimally arranged sequences on a comparison window of 20 or more positions, wherein the polynucleotide sequence portion within the comparison window is for optimal alignment of the two sequences. Compared to a reference sequence (which does not include additions or deletions), it may comprise up to 20%, generally 5-15%, or 10-12% additions or deletions (ie, gaps). The percentage (%) measures the number of sites where identical nucleic acid bases occur in two sequences to obtain the number of matched positions, and the number of matched positions as the number of total positions in the reference sequence (ie, window size). Dividing and multiplying the result by 100 yields a percentage of sequence identity.
[120] Those skilled in the art will understand that due to the degeneracy of the genetic code, there are numerous nucleic acid sequences encoding the polypeptides described herein. Some of these polynucleotides have minimal homology with the nucleotide sequences of any native gene. Nevertheless, various polynucleotides are expressly included in the present invention due to their differences in codon usage. In addition, alleles of genes comprising the polynucleotide sequences provided herein are included within the scope of the present invention. An allele is an endogenous gene that is modified as a result of one or more mutations such as deletions, additions, and / or substitutions of nucleotides. The mRNA and protein obtained may have a modified structure or function, but is not necessarily so. Alleles can be identified using standard techniques (hybridization, amplification and / or database sequence comparison).
[121] Thus, in another specific embodiment of the present invention, mutagenesis methods such as site-specific mutagenesis are used to prepare immunogenic variants and / or derivatives of the polypeptides described herein. In this way, certain modifications in the polypeptide sequence can be carried out through mutagenesis of the underlying polynucleotides encoding it. Such techniques provide a direct method for preparing and testing sequence variants, for example by introducing one or more nucleotide sequence changes into a polynucleotide, reflecting one or more of the above considerations.
[122] Site-specific mutagenesis may include specific oligonucleotides encoding the DNA sequence of the desired mutation, as well as a sufficient number of contiguous primers to provide sufficient size and sequence complexity of primer sequences to form stable dimers that extend on both sides of the deletion junction. It is possible to prepare mutants through the use of nucleotides. Mutations can be used in selected polynucleotide sequences to improve, change, reduce, modify or otherwise change the properties of the polynucleotide itself and / or to change the properties, activity, composition, stability or primary structure of the encoded polypeptide. have.
[123] In certain specific embodiments of the invention, we intend for mutagenesis of the described polynucleotide sequences to change one or more properties of the encoded polypeptide, such as the immunogenicity of the polypeptide vaccine. Site-specific mutagenesis techniques are known in the art and are widely used to prepare variants of both polypeptides and polynucleotides. For example, site-specific mutagenesis is often used to alter specific portions of DNA molecules. In this specific embodiment, primers are typically used that include a length of about 14 to about 25 nucleotides, causing about 5 to about 10 residues to change on both sides of the junction of the sequence.
[124] As will be appreciated by those skilled in the art, site-specific mutagenesis techniques often employ phage vectors that exist in both single and double strand forms. Typical vectors useful for site-specific mutagenesis include vectors such as M13 phage. Such phages are readily available and their use is generally known to those skilled in the art. Double-stranded plasmids are also commonly used in site directed mutagenesis which eliminates the step of delivering the gene of interest to the phage from the plasmid.
[125] Generally, site-directed mutagenesis is thus performed by first obtaining a single chain vector or by separately melting the two chains of a double stranded vector comprising within the sequence a DNA sequence encoding the desired peptide. Oligonucleotide primers with the desired mutated sequence are generally prepared synthetically. This primer is then annealed with a single chain vector and e. Contact with a DNA polymerase enzyme such as E. coli polymerase I Klenow fragment completes the synthesis of the chain with the mutation. Thus, heterodimers are formed such that the first chain encodes a non-mutated sequence and the second chain has the desired mutation. Then, using these heterodimeric vectors, E. Suitable clones, such as E. coli cells, are transformed and clones comprising the recombinant vector with the mutated sequence arrangement are selected.
[126] The preparation of sequence variants of selected peptide-encoded DNA fragments using site-directed mutagenesis provides a means for producing potentially useful species, but sequence variants of the peptides and other methods by which DNA sequences encoding them can be obtained. It is not intended to be limited because it exists. For example, a recombinant vector encoding the desired peptide sequence can be treated with a mutagenesis agent such as hydroxyamine to obtain sequence variants. Specific details related to these methods and protocols are described in Maloy et al., 1994; Segal, 1976; Prokop and Bajpai, 1991; Kuby, 1994; and Maniatis et al., 1982, each of which is incorporated herein by reference.
[127] As used herein, the term “oligonucleotide directed mutagenesis method” refers to template-dependent methods and vectors that result in an increase in the detectable signal concentration such as amplification or amplification of a specific nucleic acid molecule relative to the initial concentration. It is associated with mediated proliferation. As used herein, the term "oligonucleotide directed mutagenesis method" is intended to mean a method associated with template-dependent elongation of a primer molecule. The term template dependent method relates to nucleic acid synthesis of RNA or DNA molecules in which the sequence of the newly synthesized nucleic acid chain is determined by known laws of complementary base pairs (see, eg, Watson, 1987). Typically, vector mediated methods involve the introduction of a nucleic acid fragment into a DNA or RNA vector, cloning amplification of the vector, and recovery of the amplified nucleic acid fragment. Examples of such methods are provided in US Pat. No. 4,237,224, which is specifically incorporated herein by reference in its entirety.
[128] In another method for the preparation of polypeptide variants of the invention, repetitive sequence recombination as described in US Pat. No. 5,837,458 can be used. In this method, repetitive cycles and selection of recombination and screening are performed to "evolve" individual polynucleotide variants of the invention, for example, with enhanced immunogenic activity.
[129] In another specific embodiment of the invention, the polynucleotide sequences provided herein can be advantageously used as probes or primers for nucleic acid hybridization. As such, it is meant that nucleic acid fragments comprising the sequence region of a contiguous sequence of at least about 15 nucleotides in length that have the same sequence or are complementary to a contiguous sequence of 15 nucleotides in length described herein will be particularly useful. Longer contiguous identical or complementary sequences such as about 20, 30, 40, 50, 100, 200, 500, 1000 (including all intermediate lengths thereof) and even sequences up to full length sequences may also be used in certain specific embodiments. Will be used.
[130] Due to the ability of the nucleic acid probe to hybridize specifically to the sequence of interest, the nucleic acid probe can be used to detect the presence of complementary sequences in a given sample. However, other uses are also contemplated, such as the use of sequence information for the production of mutant species primers, or the primers used in the production of other genetic constructs.
[131] Examples of polynucleotide molecules having a sequence region consisting of about 10 to 14, 15 to 20, 30, 50, or 100 to 200 nucleotides (also including intermediate lengths) that are the same or complementary to the polynucleotide sequences described herein For example, it is particularly considered as a hybridization probe for use in Southern and Northern blotting. This enables the gene product or fragment thereof to be analyzed in both various cell types and various bacterial cells. The overall size of the fragment as well as the size of the complementary stretch (es) will ultimately depend on the intended use or application of the particular nucleic acid fragment. Smaller fragments generally have utility in hybridization embodiments, where the length of continuous complementarity regions may vary from about 15 to about 100 nucleotides, although larger successive complementary stretches of the complementary sequence are intended to be detected. It can be used depending on the length.
[132] The use of hybridization probes of about 15 to 25 nucleotides in length makes it possible to form stable and selective dimeric molecules. Molecules having consecutive complementarity sequences of at least 15 bases in length of elongate or greater are generally preferred for increasing the stability and selectivity of hybrids, thereby improving the quality and level of the particular hybrid molecule obtained. In general, it would be desirable to design nucleic acid molecules having 15-25 consecutive nucleotides, or, if desired, longer gene-complementary sequences.
[133] The hybridization probe can be selected from any portion of one of the sequences described herein. What is needed is to review any contiguous sequence of a sequence presented herein, or a sequence of length that includes from about 15 to 25 nucleotides to a full length sequence, to be used as a probe or primer. The choice of probe and primer sequences can be determined by various variables. For example, primers from the ends of the entire sequence can be used.
[134] Small polynucleotide fragments or fragments can be readily prepared by direct synthesis of the fragments by chemical methods, for example, as commonly practiced using automated oligonucleotide synthesizers. In addition, fragments can be applied to nucleic acid regeneration techniques such as the PCR ^TM technique of US Pat. No. 4,683,202, incorporated herein by reference, to introduce selected sequences into recombinant vectors for recombinant production, and to those skilled in the field of molecular biology. It can be obtained by recombinant DNA techniques generally known to the.
[135] The nucleotide sequences of the present invention can be used due to their ability to form dimeric molecules selectively with complementary stretches of the entire gene or gene fragment of interest. Depending on the intended application, various hybridization conditions will typically be used to achieve varying selectivity of the probe for the target sequence. For applications requiring high selectivity, relatively stringent conditions that typically form hybrids will be used, for example provided at salt concentrations of from about 0.02 M to about 0.15 M salt at temperatures of from about 50 ° C. to about 70 ° C. As will be appreciated, relatively low salt and / or high temperature conditions will be selected. The selection conditions rarely allow for inconsistencies between the probe and the template or target chain and will be particularly suitable for separating related sequences.
[136] Of course, for certain applications, for example, when one wants to make a mutant using a mutant primer chain hybridized to a basic template, less stringent (reduced stringency) hybridization conditions allow the formation of heterodimers. Typically you will need it. In such circumstances, it may be desirable to use salt conditions, such as from about 0.15 M to about 0.9 M salt, at temperatures between about 20 ° C and about 55 ° C. Cross-hybridized species can thus be readily identified as positive hybridization signals for control hybridization. In any case, in general, conditions can be made more stringent by adding an increase in formamide, which is understood to destabilize the hybrid dimer in the same manner as the temperature rise. Thus, the hybridization conditions can be easily adjusted, and thus can generally be a method of selection depending on the desired result.
[137] According to another specific aspect of the present invention, there is provided a polynucleotide composition comprising an antisense oligonucleotide. Antisense oligonucleotides have proven to be effectively targeted inhibitors of protein synthesis and consequently provide a therapeutic method for diseases that can be treated by inhibiting the synthesis of proteins involved in the disease. The efficacy of antisense oligonucleotides to inhibit protein synthesis is well established. For example, the synthesis of polygalactauronase and muscarin type 2 acetylcholine receptors is inhibited by antisense oligonucleotides directed against their respective mRNA sequences (US Pat. No. 5,739,119 and US Pat. No. 5,759,829). . In addition, examples of antisense inhibition have been demonstrated in nuclear protein cyclins, multiple drug resistance genes (MDG1), ICAM-1, E-selectin, STK-1, striatum GABA _A receptors and human EGF [Jaskulski et al., Science . 1988 Jun 10; 240 (4858): 1544-6; Vasantahkumar and Ahmed, Cancer Commun. 1989; 1 (4): 225-32; Peris et al., Brain Res Mol Brain Res. 1998 Jun 15; 57 (2): 310-20; US Patent No. 5,801,154; US Patent No. 5,789,573; U.S. Patent 5,178,709 and U.S. Patent 5,610,288]. Antisense constructs have also been described that can be used to inhibit and treat various abnormal cell proliferations, such as cancer [US Pat. No. 5,747,470; U.S. Patent 5,591,317 and U.S. Patent 5,783,683].
[138] Thus, in certain embodiments, the present invention provides oligonucleotide sequences comprising all or a portion of any sequence capable of specifically binding to the polynucleotide sequence described herein or its complement. In one specific embodiment, the antisense oligonucleotides comprise DNA or derivatives thereof. In another specific embodiment, the oligonucleotides comprise RNA or derivatives thereof. In a third specific embodiment, oligonucleotides comprise modified DNA comprising a phosphorothioated modified backbone. In a fourth specific embodiment, the oligonucleotide sequence comprises a peptide nucleic acid or derivative thereof. In each case, preferred compositions comprise sequence regions that are complementary to, and more preferably substantially complementary to, and even more preferably completely complementary to one or more portions of the polynucleotides described herein. The selection of antisense compositions specific for a given gene sequence is based on analysis of the selected target sequence and measurements on secondary structure, T _m , binding energy and relative stability. Antisense compositions can be selected based on their ability not to form dimers, hairpins or other secondary structures that reduce or inhibit specific binding to target mRNAs in a host cell. More preferred target regions of the mRNA are sequence regions located at or adjacent to the translational initiation codon AUG and substantially complementary to the 5 'region of the mRNA. Such secondary structure analysis and target site selection considerations are described, for example, in OLIGO Primer Analysis Software v.4 and / or BLASTIN 2.0.5 Algorithm Software [Altschul et al., Nucleic Acids Res. 1997, 25 (17): 3389-402.
[139] It is also contemplated to use an antisense delivery method using a short peptide vector named MPG (27 residues). MPG peptides contain a hydrophobic domain derived from the fusion sequence of HIV gp41 and a hydrophilic domain from the nuclear localization sequence of the SV40 T-antigen [Morris et al., Nucleic Acids Res. 1997 Jul 15; 25 (14): 2730-6]. Several molecules of MPG peptides have been demonstrated to coat antisense oligonucleotides and to be delivered into mammalian cells cultured within 1 hour with relatively high efficiency (90%). In addition, interaction with MPG strongly increases both the stability of the oligonucleotides to nucleases and the ability to cross cell membranes.
[140] According to another specific aspect of the invention, the polynucleotide compositions described herein are used in the design and preparation of ribozyme molecules for inhibiting expression of tumor polypeptides and proteins of the invention in tumor cells. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific manner. Ribozymes have specific catalytic domains with endonuclease activity [Kim and Cech, Proc. Natl. Acad. Sci. USA. 1987 Dec; 84 (24): 8788-92; Forster and Symons, Cell. 1987 Apr 24; 49 (2): 211-20]. For example, numerous ribozymes often speed up phosphoester transfer reactions with high specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate [Cech et al., Cell. 1981 Dec; 27 (30 Pt 2): 487-96; Michel and Westhof, J. Mol. Biol. 1990 Dec 5; 216 (3): 585-610; Reinhold-Hurek and Shub, Nature 1992 May 14; 357 (6374): 173-6. This specificity is due to the requirement that the substrate must bind through specific base pair interactions to the ribozyme's internal guide sequence (“IGS”) prior to the chemical reaction.
[141] Six basic kinds of natural enzymatic RNA are currently known. Each can catalyze the hydrolysis of RNA phosphodiester under physiological conditions and consequently cleave other RNA molecules. In general, enzymatic nucleic acids act by first binding to target RNA. The binding occurs through the target binding portion of the enzymatic nucleic acid, which is present in close proximity to the enzyme portion of the enzymatic nucleic acid molecule that acts to cleave the target RNA. Thus, enzymatic nucleic acids first recognize and bind to target RNA through complementary base pairs and, once bound to the correct binding site, enzymatically cleave the target RNA. Strategic cleavage of the target RNA will destroy the ability of direct synthesis of the encoded protein of the RNA. After the enzymatic nucleic acid binds to and cleaves its RNA target, it can be isolated from the RNA to find another target and repeatedly bind to the new target and cleave it.
[142] The enzymatic properties of ribozymes are numerous techniques such as antisense techniques, in which the nucleic acid molecule simply binds to the nucleic acid target and blocks its translation, since the concentration of ribozyme required for therapeutic treatment is lower than that of the antisense oligonucleotide. Advantageous over these. This advantage reflects the ability of ribozymes to act enzymatically. That is, a single ribozyme molecule can cleave numerous target RNA molecules. In addition, ribozymes are highly specific inhibitors that have inhibitory specificities depending on the target RNA cleavage mechanism as well as the base pairing mechanism that binds to the target RNA. A single mismatch or base substitution at a position adjacent to the cleavage site can completely eliminate the catalytic activity of the ribozyme. Similar inconsistencies in antisense molecules do not interfere with its action [Woolf et al., Proc. Natl. Acad. Sci. USA. 1992 Aug 15; 89 (16): 7305-9]. Thus, the specificity of action of ribozymes is greater than the specificity of action of antisense oligonucleotides that bind to the same RNA site.
[143] Enzymatic nucleic acid molecules can be formed in hammerheads, hairpins, hepatitis δ virus, group I introns or RNaseP RNAs (in combination with RNA guide sequences) or neurospora VS RNA motifs. Examples of hammerhead motifs are described in Rossi et al. Nucleic Acids Res. 1992 Sep 11; 20 (17): 4559-65. Examples of hairpin motifs are described in Hamel et al. Eur. Pat. Appl. No. EP 0360257; Hampel and Tritz, Biochemistry 1989 Jun 13; 28 (12): 4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan 25; 18 (2): 299-304 and U.S. Patent 5,631,359. Examples of hepatitis δ viral motifs are described in Perrotta and Been, Biochemistry. 1992 Dec 1; 31 (47): 11843-52. Examples of RNaseP motifs are described in Guerrier-Takada et al., Cell. 1983 Dec; 35 (3 Pt 2): 849-57. Neurospora VS RNA ribozyme motifs are described in Saville and Collins, Cell. 1990 May 18; 61 (4): 685-96; Saville and Collins, Proc. Natl. Acad. Sci. USA 1991 Oct 1; 88 (19): 8826-30; Collins and Olive, Biochemstry. 1993 Mar 23; 32 (11): 2795-9. Examples of group I introns are described in US Pat. No. 4,987,071. What is important in the enzymatic nucleic acid molecule of the invention is that it has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has a nucleic acid sequence that confers RNA cleavage activity to or within the substrate binding site. will be. Thus, ribozyme constructs are not limited to the specific motifs mentioned herein.
[144] Ribozymes are synthesized as described and designed in vitro and in vitro and as described in International Patent Publications WO 93/23569 and WO 94/02595, each of which is specifically incorporated herein by reference. Can be tested in vivo. The ribozyme can also be optimized for delivery. While specific examples are provided, one of ordinary skill in the art will recognize that equivalent RNA targets in other species can be used as needed.
[145] Ribozyme activity can be modified to modify the length of ribozyme binding arms or to prevent their degradation by serum ribonucleases (see, eg, WO 92/07065; WO 93/15187; WO 91/03162; European Patent Publication No. 92110298.4; US Patent No. 5,334,711 and WO 94/13688, which describe various chemical modifications that can be carried out on sugar residues of enzymatic RNA molecules], modifications that enhance their efficiency in cells, And by chemically synthesizing ribozymes with removal of stem II bases to shorten RNA synthesis time and reduce chemical requirements.
[146] Sullivan et al. WO 94/02595 describes a general method of delivery of enzymatic RNA molecules. Ribozymes can be administered to cells by a variety of methods known to those of skill in the art, which may be entrapped in liposomes, by iontophoresis, or by other vehicles such as hydrogels, cyclodextrins, biodegradable nanocapsules and But not limited to, by introduction into bioadhesive microspheres. For certain indications, ribozymes may be delivered directly into cells or tissues ex vivo in the presence or absence of the vehicle described above. Alternatively, the RNA / vehicle mixture may be delivered locally by direct inhalation, by direct infusion or by use of a catheter, inhalation pump or stent. Other routes of delivery include, but are not limited to, intravenous, intramuscular, subcutaneous or joint injection, aerosol inhalation, oral (tablet or pill form), local, systemic, intraocular, intraperitoneal and / or intrathecal delivery. It is not limited. More detailed descriptions of ribozyme delivery and administration are provided in International Patent Publications WO 94/02595 and WO 93/23569, each of which is specifically incorporated herein by reference.
[147] Another way to accumulate ribozyme (s) in cells at high concentrations is to introduce ribozyme-encoding sequences into the DNA expression vector. Transcription of ribozyme sequences is driven by promoters of eukaryotic RNA polymerase I (pol I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcription from the pol II or pol III promoter will be expressed at high levels in all cells, and the level of pol II promoter in a given cell type will depend on the nature of the gene regulatory sequences (enhancers, silencers, etc.) that are present adjacently. Prokaryotic RNA polymerase promoters may also be used, provided prokaryotic RNA polymerase enzymes are expressed in suitable cells. Ribozymes expressed from these promoters also functioned in mammalian cells. Such transcription units include, but are not limited to, plasmid DNA vectors, viral DNA vectors (e.g., adenovirus or adeno-associated vectors), or viral RNA vectors (e.g., retrovirus Semiki Forest virus, Sindbis virus vector). It can be introduced into a variety of vectors for introduction into mammalian cells, including but not limited to.
[148] In another specific embodiment of the present invention, a peptide nucleic acid (PNA) composition is provided. PNA is a DNA mimetic with a nucleobase attached to the pseudopeptide backbone [Good and Nielsen, Antisense Nucleic Acid Drug Dev. 1997 7 (4) 431-37. PNA can be used in many methods that have traditionally used RNA or DNA. Often PNA sequences perform better in techniques than corresponding RNA or DNA sequences and have utility that RNA or DNA does not have. An overview of PNA, including methods of making PNA, properties of use and methods of use, is disclosed in Corey, Trends Biotechnol. 1997 Jun; 15 (6): 224-9. As such, in certain specific embodiments, a PNA sequence complementary to one or more portions of an ACE mRNA sequence can be prepared, and the PNA composition can be used to modulate, alter, attenuate or reduce ACE-specific mRNA. This can change the level of ACE activity in the host cell to which the PNA composition is administered.
[149] PNA has a 2-aminoethyl-glycine bond that replaces the normal phosphodiester backbone of DNA [Nielsen et al., Science 1991 Dec 6; 254 (5037): 1497-500; Hanvey et al., Science. 1992 Nov. 27; 258 (5087): 1481-5; Hyrup and Nielsen, Bioorg Med Chem. 1996 Jan; 4 (1): 5-23]. This chemistry has three important consequences: first, in contrast to DNA or phosphorothioate oligonucleotides, PNA is a neutral molecule; Second, PNA is achiral and there is no need to develop stereoselective synthesis; Third, other methods have been used, including the modified Merifield method, but PNA synthesis uses standard Boc or Fmoc protocols for solid phase peptide synthesis.
[150] PNA monomers or ready-made oligomers are commercially available from PerSeptive Biosystems, Framingham, Mass. PNA synthesis by the Boc or Fmoc protocol is facilitated using manual or automated protocols [Norton et al., Bioorg Med Chem. 1995 Apr; 3 (4): 437-45]. Manual protocols are used for the preparation of chemically modified PNAs or for the simultaneous synthesis of a family of tightly packed PNAs.
[151] As with peptide synthesis, the success of a particular PNA synthesis will depend on the nature of the selected sequence. For example, in theory, the PNA can introduce any mixture of nucleotide bases, but the presence of adjacent furins can result in one or more residue deletions in the product. These challenges are anticipated and it is proposed to repeat the coupling of residues which are likely to be added inefficiently in the preparation of PNA using adjacent furins. The PNA is then purified by reverse phase high pressure liquid chromatography to provide yields and purity of products similar to those observed during peptide synthesis.
[152] Modifications of PNA for a given application can be achieved by coupling amino acids during solid phase synthesis or by attaching a compound containing a carboxylic acid group to the exposed N-terminal amine. Alternatively, the PNA can be modified after synthesis by coupling to the introduced lysine or cysteine. The ease of modification of PNAs facilitates optimization for better solubility or for specific functional requirements. Once synthesized, the identification of PNA and its derivatives can be confirmed by mass spectrometry. Several studies have performed and used modifications of PNA [eg, Norton et al., Bioorg Med Chem. 1995 Apr; 3 (4): 437-45; Petersen et al., J. Pept Sci. 1995 May-Jun; 1 (3): 175-83l Orum et al., Biotechniques. 1995 Sep; 19 (3): 472-80; Footer et al., Biochemistry. 1996 Aug 20; 35 (33): 10673-9; Griffith et al., Nucleic Acids Res. 1995 Aug 11; 23 (15): 3003-8; Pardrige et al., Proc. Natl. Acad. Sci. USA. 1995 Jun 6; 92 (12): 5592-6; Boffa et al., Proc. Natl. Acad. Sci. USA. 1995 Mar 14; 92 (6): 1901-5; Gambacorti-Passerini et al., Blood. 1996 Aug 15; 88 (4): 1411-7; Armitage et al., Proc. Natl. Acad. Sci. USA. 1997 Nov. 11; 94 (23): 12320-5; Seeger et al., Biotechniques. 1997 Sep; 23 (3): 512-7]. US Pat. No. 5,700,922 discusses PNA-DNA-PNA chimeric molecules and their use in diagnostics, protein regulation in organisms, and acceptable therapeutic conditions for therapeutic agents.
[153] Methods for characterizing antisense binding properties of PNAs are described in Rose, Anal Chem. 1993 Dec 15; 65 (24): 3545-9; and Jensen et al., Biochemistry. 1997 Apr. 22; 36 (16): 5072-7. Rose used capillary gel electrophoresis to measure the binding of PNA to complementary oligonucleotides by measuring relative binding kinetics and stoichiometry. Similar types of measurement methods were performed by Jensen et al. Using BIAcore ^™ technology.
[154] Other applications of PNA described and apparent to those skilled in the art include DNA chain invasion, antisense inhibition, mutation analysis, transcriptional enhancers, nucleic acid purification, isolation of transcriptionally active genes, blocking transcription factor binding, genome cleavage, biosensors , In situ hybridization, and the like.
[155] Polynucleotide Identification, Characterization, and Expression
[156] Polynucleotide compositions of the present invention can be identified, prepared and / or manipulated using one of a variety of established techniques (e.g., generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring). Harbor Laboratories, Cold Spring Harbor, NY, 1989, and other similar references. For example, polynucleotides can be identified by screening microarrays of cDNA for tumor-associated expression (ie, two or more times more expression in normal tissues, as measured using the representative assays provided herein). . Such screening is described, for example, by manufacturer's instructions (and basically in Schena et al., Proc. Natl. Acad. Sci. USA 93: 10641-10619, 1996 and Heller et al., Proc. Natl. Acad. Sci. USA 94: 1250-2155, 1997), using a microarray technique from Affymetrix, Inc., Santa Clara, Calif. Alternatively, polynucleotides can be amplified from cDNA prepared from cells expressing the proteins described herein, such as tumor cells.
[157] Numerous template dependency methods are used to amplify target sequences of interest present in a sample. One of the most known amplification methods is polymerase chain reaction (PCR ^™ ), which is described in detail in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated herein by reference in its entirety. It is described. Briefly, in PCR ^™ , two primer sequences are prepared that are complementary to regions on opposite complementary chains of a target sequence. Excess deoxynucleoside triphosphate is added to the reaction mixture with DNA polymerase (eg, Taq polymerase). If the target sequence is present in the sample, the primer will bind to the target and the polymerase will be stretched by adding nucleotides along the target sequence. By raising and lowering the temperature of the reaction mixture, the elongated primer will dissociate from the target to form the reaction product, and excess primer will bind to the target and reaction product and the process will be repeated. Preferably, reverse transcription and PCR ^TM amplification procedures can be performed to quantify the amount of mRNA amplified. Polymerase chain reaction methods are known in the art.
[158] One of a number of template dependency procedures, many of which are variants of PCR ^TM amplification techniques, are readily known and useful in the art. Illustratively, certain such methods include, for example, ligase chain reactions (referred to as LCR) described in European Patent Publication Nos. 320,308 and 4,883,750; Qbeta replicaases described in PCT International Publication No. PCT / US87 / 00880; Chain transfer amplification (SDA) and recovery chain reaction (RCP). Other amplification methods are also described in British Patent Application No. 2 202 328 and PCT Publication No. PCT / US89 / 01025. Other nucleic acid amplification procedures include transcription based amplification systems (TAS) (PCT WO 88/10315), including nucleic acid sequence based amplification and 3SR. European Patent Publication No. 329 822 describes nucleic acid amplification processes involving cyclically synthesized single stranded RNA (“ssRNA”) and double stranded DNA (dsDNA). PCT WO 89/06700 discloses a nucleic acid sequence amplification scheme based on the transcription of numerous RNA copies of a sequence after hybridization to a target single-stranded DNA (“ssDNA”) of a promoter / primer sequence. Describe. Other amplification methods, such as "RACE" [Frohman, 1990], and other amplification methods, such as "unidirectional PCR" [Ohara, 1989], are known to those skilled in the art.
[159] The amplified portions of the polynucleotides of the invention can be used to separate full length genes from suitable libraries (eg, tumor cDNA libraries) using known techniques. Within this technique, libraries (cDNA or genomic) are screened using one or more polynucleotides or primers suitable for amplification. Preferably, the library is size-selected to include larger molecules. Random primed libraries are also preferred for identifying the 5 'and upstream regions of the gene. Genomic libraries are preferred for obtaining introns and elongating 5 'sequences.
[160] For hybridization techniques, partial sequences can be labeled using known techniques (eg nick-detox or end-labels using ³² P). The bacterial or bacteriophage libraries are then screened by hybridizing filters containing generally modified bacterial colonies (or lawns containing phage plaques) with labeled probes. Sambrook et al., Molecular. Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989]. Colonies or plaques to hybridize are selected and stretched, and DNA is isolated for further analysis. cDNA clones can be analyzed to determine the amount of additional sequences, for example, by PCR using primers from partial sequences and primers from vectors. Restriction maps and partial sequences can be generated to identify one or more overlapping clones. The complete sequence can then be measured using standard techniques, which may involve generating a series of deletion clones. The overlapping sequences obtained can then be duplicated into one contiguous sequence. Full length cDNA molecules can be generated by linking suitable fragments using known techniques.
[161] Alternatively, amplification techniques as described above may be useful for obtaining full length coding sequences from partial cDNA sequences. One such amplification technique is reverse PCR [Triglia et al., Nucl. Acids Res. 16: 8186, 1988, which uses restriction enzymes to generate fragments in known regions of the gene. The fragment is then closed with intramolecular binding and used as a template for PCR with various primers derived from known regions. In another method, a sequence adjacent to a partial sequence can be recovered as a primer specific for the linker sequence and known region by amplification with a primer. The amplified sequence typically undergoes a second amplification process with the same linker primer and a second primer specific for a known region. Modifications in this process, using two primers that initiate stretching in opposite directions from known sequences, are described in WO 96/38591. Another such technique is known as "fast amplification of the cDNA terminus" or "RACE". The technique involves identifying the 5 'and 3' sequences of known sequences using internal primers and external primers that hybridize to a polyA region or vector sequence. Additional techniques include capture PCR [Lagerstrom et al., PCR Methods Applic. 1: 111-19, 1991 and working PCR [Parker et al., Nucl. Acids Res. 19: 3055-60, 1991. Other methods using amplification can also be used to obtain full length cDNA sequences.
[162] In certain instances, as available from GenBank, it is possible to obtain full length cDNA sequences by sequence analysis provided in an expressed sequence tag (EST) database. The search for redundant ESTs can generally be performed using a known program (eg, NCBI BLAST survey), which can be used to generate continuous full length sequences. Full length DNA sequences can also be obtained by analysis of genomic fragments.
[163] In another specific embodiment of the invention, polynucleotide sequences or fragments thereof, or fusion proteins or functional equivalents thereof, encoding polypeptides of the invention can be used in recombinant DNA molecules to direct expression of the polypeptide in a suitable host cell. have. Due to the inherent degeneracy of the genetic code, it is possible to generate other DNA sequences that encode identical or functionally equivalent amino acid sequences, which can be used to clone and express a given polypeptide.
[164] As will be appreciated by those skilled in the art, in certain instances it may be advantageous to prepare polypeptide-encoded nucleotide sequences having non-natural codons. For example, a codon preferred by a particular prokaryotic or eukaryotic host can be selected to produce a recombinant RNA transcript with desirable properties such as increased protein expression or longer half-lives than transcript half-lives generated from native sequences. .
[165] In addition, the polynucleotide sequences of the present invention can be genetically modified using methods generally known in the art to modify the polypeptide coding sequence for a variety of reasons, which modifications alter the cloning, processing and / or expression of the gene product. But not limited thereto. For example, DNA sequencing by random fragmentation and PCR combinations of gene fragments and synthetic oligonucleotides can be used to genetically engineer nucleotide sequences. In addition, site-directed mutagenesis can be used to insert new restriction sites, modify glycosylation patterns, change codon preferences, prepare splice variants, introduce mutations, and the like.
[166] In another specific embodiment of the invention, a fusion protein can be encoded by linking a native, modified or recombinant nucleic acid sequence to a heterologous sequence. For example, to screen peptide libraries for inhibitors of polypeptide activity, it may be useful to encode chimeric proteins that can be recognized by commercial antibodies. Fusion proteins can also be genetically engineered to contain a cleavage site located between the polypeptide-encoding sequence and the heterologous protein sequence so that the polypeptide can be cleaved and purified separately from the heterologous residues.
[167] Sequences encoding the polypeptide of interest can be synthesized in whole or in part using chemical methods known in the art. See Caruthers, M.H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232]. Alternatively, the protein itself may be prepared using chemical methods to synthesize an amino acid sequence, or portion thereof, of a polypeptide. For example, peptide synthesis can be performed using various solid phase techniques [Roberge, J. Y. et al. (1995) Science 269: 202-204], automated synthesis can be synthesized using, for example, an ABI 431A peptide synthesizer [Perkin Elmer, Palo Alto, CA].
[168] Newly synthesized peptides can be prepared using preparative high performance liquid chromatography (e.g., Creighton, T. (1983) Proteins, Structures and Molecular Principles, WH Freeman and Co., New York, NY) or other comparable techniques available in the art. Can be substantially purified. The composition of the synthetic peptides can be confirmed by amino acid analysis or sequencing (eg, Edman degradation method). In addition, the amino acid sequence of the polypeptide, or a portion thereof, may be modified during direct synthesis and / or a variant polypeptide may be prepared using chemical methods using sequences from other proteins or any portion thereof.
[169] For expression of a preferred polypeptide, the nucleotide sequence encoding the polypeptide, or functional equivalent thereof, can be inserted into a suitable expression vector, ie, a vector containing elements necessary for the transcription and translation of the inserted coding sequence. Methods known to those skilled in the art can be used to construct expression vectors containing sequences encoding polypeptides of interest and suitable transcriptional and translational regulatory elements. Such methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.
[170] Various expression vectors / host systems can be used to contain and express polynucleotide sequences. These include microorganisms such as bacteria transformed using recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; Yeast transformed using yeast expression vectors; Insect cell systems infected using virus expression vectors (eg, baculovirus); Plant cell systems transformed with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (eg, Ti or pBR322 plasmid); Or animal cell systems, but is not limited thereto.
[171] The “regulatory element” or “regulatory sequence” present in an expression vector is the non-toxic region (enhancer, promoter, 5 ′ and 3 ′ non-toxic region) of the vector that interacts with the host cell protein to perform transcription and translation. The element can vary in its strength and specificity. Depending on the vector system and host used, any number of suitable transcriptional and translational elements can be used, including constitutive and inducible promoters. For example, in cloning in bacterial systems, inducible promoters such as PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) Or PSORT1 plasmid (Gibco BRL, Gaithersburg, MD) can be used. have. In mammalian cell systems, promoters from mammalian genes or mammalian viruses are generally preferred. If it is necessary to generate a cell line containing multiple copies of the sequence encoding the polypeptide, vectors based on SV40 or EBV can be advantageously used with suitable selectable markers.
[172] In bacterial systems, any one of a number of expression vectors can be selected depending on the intended use for the expressed polypeptide. For example, where a large amount is needed, for example, in the case of antibody induction, a vector may be used that directs high levels of expression of the easily purified fusion protein. The vector is versatile. The sequence encoding the coli cloning and expression vector, eg, the polypeptide of interest, is linked into the vector in the same frame with the sequences for the amino terminal Met of β-galactosidase and the following seven residues, BLUESCRIPT (Stratagene) from which a hybrid protein can be produced; pNI vectors [Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem. 264: 5503-5509, etc., but is not limited thereto. pGEX vectors (Promega, Madison, Wis.) can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, the fusion protein is soluble and can be readily purified from lysed cells by adsorption to glutathione-agarose beads and then eluting in the presence of free glutathione. The protein produced in this system is designed to include heparin, thrombin, or factor XA protease cleavage sites, so that the cloned polypeptide of interest can be freely released from the GST residue.
[173] In yeast, Saccharomyces cerevisiae, numerous vectors containing constitutive or inducible promoters can be used, such as alpha factor, alcohol oxidase, and PGH. For an overview, see Ausubel et al. (Above) and Grant et al. (1987) Methods Enzymol. 153: 516-544.
[174] When plant expression vectors are used, expression of polypeptide coding sequences can be driven by any one of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with an omega leader sequence from TMV [Takamatsu, N. (1987) EMBO J. 6: 307-311]. Alternatively, plant promoters such as small subunits of RUBISCO or heat shock promoters may be used [Coruzzi, G. et al. (1984) EMBO J. 3: 1671-1680; Broglie, R. et al. (1984) Science 224: 838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17: 85-105. Such constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. The technique is described in a number of overviews generally available [see, eg, Hobbs, S. or Murry, L.E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw Hill, New York, N.Y.,; pp. 191-196].
[175] Insect systems can also be used to express insect polypeptides. For example, in one such system, Spodoptera frugiperda cells or Tricoplusia larbae (Autographa californica) nuclear polyhedron virus (AcNPV) as a vector. Trichoplusia larvae) can express foreign genes. Sequences encoding polypeptides can be cloned into non-essential regions of the virus, such as the polyhedrin gene and placed under the control of the polyhedrin promoter. Successful insertion of the polypeptide-encoding sequence inactivates the polyhedrin gene and produces a recombinant viral defect coating protein. Recombinant virus is, for example, S. a. It can be used to infect either Pruiferpera cells or Tricoflucia lar embryo, wherein the polypeptide of interest can be expressed [Engelhard, E.K. et al., (1994) Proc. Natl. Acad. Sci. 91: 3224-3227.
[176] In mammalian host cells, numerous viral based expression systems are generally available. For example, when adenoviruses are used as expression vectors, sequences encoding polypeptides of interest can be linked into adenovirus transcription / detoxification complexes consisting of late promoter and trimer leader sequences. Insertion of the viral genome into the non-essential El or E3 regions can be used to obtain live viruses capable of expressing polypeptides in infected host cells [Logan, J. and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81: 3655-3659. In addition, transcriptional enhancers, such as the Raus sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells.
[177] Certain initiation signals can also be used to achieve more effective transcription of sequences encoding polypeptides of interest. The signal comprises an ATG start codon and contiguous sequences. If the sequence encoding the polypeptide, its start codon, and upstream sequences are inserted into a suitable expression vector, no additional transcriptional or translational control signal may be required. However, if only the coding sequence, or part thereof, is inserted, an exogenous translational control signal comprising the ATG start codon must be provided. In addition, the start codon must be present in the correct reading frame to ensure readout of the entire insert. Exogenous detoxification elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression is described by Scharf, D. et al. (1994) Results Probl. Cell Differ. 20: 125-162, enhancers can be included by incorporating enhancers suitable for the particular cellular system used.
[178] In addition, host cell lines may be chosen for their ability to modulate the expression of the inserted sequences or to process the expressed protein in a preferred manner. Modifications of such polypeptides include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing, which cleaves the “prepro” form of a protein, may also be used to facilitate correct insertion, folding, and / or function. CHO, COS, Different host cells, such as Hela, MDCK, HEK293, and WI38, can be selected to ensure accurate modification and processing of foreign proteins.
[179] For long term, high yield production of recombinant proteins, stable expression is generally preferred. For example, a cell line stably expressing a polynucleotide of interest can be transformed using an expression vector that can contain the replication origin and / or endogenous expression elements of the virus and selectable marker genes in the same or separate vectors. have. After introduction of the vector, cells can be grown on rich media for 1-2 days and then transferred to selection media. The purpose of the selectable marker is to confer select resistance, which allows the growth and recovery of cells that successfully express the introduced sequence. Resistant clones of stably transformed cells can be propagated using tissue culture techniques appropriate for the cell type.
[180] Any number of selection systems can be used to recover the transformed cell line. It is known that the herpes simplex virus thymidine kinase [Wigler, M. et al. (1977) Cell 11: 223-32 and adenine phosphoribosyltransferase [Lowy, I. et al. (1990) Cell 22: 817-23] genes, including, but not limited to. In addition, anti-metabolite, antibiotic or herbicide tolerance can be used as a basis for selection, for example, dhfr [Wigler, M. et al., Which confers resistance to methotrexate, respectively. (1980) Proc. Natl. Acad. Sci. 77: 3567-70; Npt confers resistance to aminoglycosides, neomycin and G-418 [Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150: 1-14; And pat which confers resistance to closulfuron and phosphinothricin acetyltransferase [Murry, supra]. Additional selectable genes have been described, for example trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine [Hartman, S.C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85: 8047-51. The use of visible markers such as anthocyanins, beta-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin, have gained popularity and are either transient or can be attributed to specific vector systems as well as identification of transformants. Widely used to quantify stable protein expression levels [Rhodes, CA] et al. (1995) Methods Mol. Biol. 55: 121-131.
[181] Although the presence / absence of marker gene expression suggests that the gene of interest is also present, its presence and expression may need to be identified. For example, when a polypeptide coding sequence is inserted into a marker gene sequence, recombinant cells containing the sequence will be identified as lack of marker gene function. Alternatively, the marker gene can be located in series with the polypeptide coding sequence under the control of a single promoter. Expression of marker genes in response to induction or selection generally also indicates expression of the tandem genes.
[182] Alternatively, host cells containing and expressing the desired polynucleotide sequences can be identified by a variety of methods known in the art. Such methods include, for example, DNA-DNA, DNA-RNA hybridization and protein bioassays or immunoassay techniques, including membrane, solution, or chip-based techniques for detection and / or quantification of nucleic acids or proteins. However, it is not limited thereto.
[183] Various protocols for detecting and measuring expression of polynucleotide encoded products using polyclonal or monoclonal antibodies specific for the product are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS). Two site, monoclonal-based immunoassays using monoclonal antibodies for two non-intrusive epitopes on a given polypeptide may be preferred for certain applications, but competitive binding assays may also be used. These and other assays are described in particular in hampton, R. et al. 1990; Serological Methods, a Laboratory Manual, APS Press, St Paul. Minn .; and Maddox, D.E. et al. 1983; J. Exp. Med. 158: 1211-1216.
[184] A wide variety of labeling and conjugation techniques are known to those skilled in the art and can be used in a variety of nucleic acid and amino acid assays. Methods for labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include oligolabeling, nick translation, terminal labeling or PCR amplification using labeled nucleotides. Alternatively, the sequence or any portion thereof can be cloned into a vector for the preparation of mRNA probes. Such vectors are known in the art and are commercially available and can be used to synthesize RNA probes in vitro by adding suitable RNA polymerases such as T7, T3 or SP6 and labeled nucleotides. These procedures can be performed using a variety of commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent agents, chemiluminescent agents, or colorants as well as substrates, cofactors, inhibitors, magnetic particles, and the like.
[185] Host cells transformed with the polynucleotide sequence of interest can be cultured under conditions suitable for expression of the cell culture and recovery from the cell culture. Proteins produced by recombinant cells may be secreted or contained within cells, depending on the sequence and / or vector used. In addition, as will be appreciated by those skilled in the art, expression vectors containing polynucleotides of the present invention may be designed to contain signal sequences that direct the secretion of a polypeptide encoded through a prokaryotic or eukaryotic cell membrane. have. Other recombinant constructions can be used to bind a sequence encoding a polypeptide of interest to a nucleotide sequence encoding a polypeptide domain that can facilitate purification of soluble proteins. The purification promotion domain includes a metal chelating peptide such as a histidine-tryptophan module that enables purification on immobilized metals, a Protein A domain that allows purification on immobilized immunoglobulins, and a FLAGS elongation / affinity purification system from Immunex Corp., Seattle, Wash.), But is not limited to such. Cleavable linker sequences (Invitrogen, San Diego, Calif.) Such as those specific for factor XA or enterokinase between the purification domain and the encoded polypeptide can be included to facilitate purification. One such expression vector is provided for the expression of a fusion protein containing a polypeptide of interest and a nucleic acid encoding six histidine residues prior to a thioredoxin or enterokinase cleavage site. Histidine residues are described in Porath, J. et al., 1992, Prot. Exp. Prif. 3: 263-281, while promoting purification on IMIAC (fixed metal ion affinity chromatography), the enterokinase cleavage site provides a method of purifying the desired polypeptide from the fusion protein. A discussion of vectors containing fusion proteins is described in Kroll, D.J. et al., 1993; DNA Cell Biol. 12: 441-453.
[186] In addition to recombinant production methods, polypeptides or fragments thereof of the present invention may be prepared using solid phase techniques such as those described by Merrifield J. (1963) J. Am. Chem. Soc. 85: 2149-2154] for direct peptide synthesis. Protein synthesis can be performed by manual techniques or by automation. Automated synthesis can be accomplished using, for example, an apparatus (Applied Biosystems 431A Peptide Synthesizer, manufactured by Perkin Elmer). Alternatively, the various fragments can be chemically synthesized individually and combined by chemical methods to make whole molecules.
[187] Antibody Compositions, Fragments thereof and Other Binders
[188] According to another aspect, the invention further provides binding agents, such as antibodies and antigen-binding fragments thereof, which exhibit immunological binding to a tumor polypeptide or portion thereof, variant or derivative thereof described herein. An antibody, or antigen-binding fragment thereof, reacts with a polypeptide of the invention at a detectable level (e.g., in an ELISA assay) and does not detectably react with an unrelated polypeptide under similar conditions. And "specifically bind", "immunologically bind" and / or "immunologically reactive".
[189] As used in this context, immunological binding generally refers to the type of noncovalent interactions that occur between an immunoglobulin molecule and an antigen that is specific for the immunoglobulin. The intensity or affinity of an immunological binding interaction can be expressed as the dissociation constant (K _d ) of the interaction, where smaller K _d represents greater affinity. The immunological binding properties of the selected polypeptides can be quantified using methods known in the art. This method involves the determination of antigen binding site / antigen complex formation and dissociation rate, wherein the ratio depends on the concentration of the complex partner, the affinity of the interaction and geometric variables that affect the ratio equally in both directions. Follow. Thus, both the "on rate constant" (K _on ) and the "off rate constant" (K _off ) can be determined by the calculation of the concentration and the substantial rate of binding and dissociation. The ratio of K _off / K _on allows the deletion of all variables not related to the affinity, and therefore equals the dissociation constant K _d . See literature [Generally, Davies et al. (1990) Annual Rev. Biochem. 59: 439-473.
[190] An “antigen binding site” or “binding portion” of an antibody refers to the portion of an immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") regions. Three very different stretches of the V region of the heavy and light chains are referred to as "hypervariable regions" between more conservative flanking stretches known as "skeletal regions" or "FRs". Thus, "FR" refers to an amino acid sequence found naturally between or adjacent to hypervariable regions in immunoglobulins. In antibody molecules, the three hypervariable regions in the light chain and the hypervariable regions in the heavy chain are appropriately positioned relative to each other in three-dimensional space to form an antigen binding surface. The antigen binding surface is complementary to the three dimensional surface of the bound antigen and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity determining regions" or "CDRs".
[191] The binder may further be used to identify patients with or without cancer, such as breast cancer, using the representative assays provided herein. For example, other binding agents that bind antibodies or tumor proteins will preferably generate signals indicative of the presence of cancer in at least about 20% of the disease patients, and more preferably at least about 30% of patients. Alternatively or in addition, the antibody will generate a negative signal indicative of the absence of the disease in at least about 90% of individuals who do not have cancer. To determine whether a binder meets these requirements, biological samples (eg, blood, serum, sputum, urine and / or from patients with or without cancer (as determined using standard clinical trials) Tumor biopsies) can be analyzed as described herein for the presence of a polypeptide that binds to the binder. Preferably, a statistically significant number of samples with or without the disease will be analyzed. While each binder must meet the above criteria, one of ordinary skill in the art will recognize that binders can be mixed to improve sensitivity.
[192] Any formulation that meets the above requirements may be a binder. For example, the binding agent can be a ribosome, RNA molecule or polypeptide with or without peptide component. In a preferred specific embodiment, the binding agent is an antibody or antigen binding fragment thereof. Antibodies can be prepared by one of a variety of techniques known to those of ordinary skill in the art. See, eg, Harlow and lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, antibodies are prepared by cell culture techniques, such as through the production of monoclonal antibodies as described herein, or through transfection of antibody genes into a suitable bacterial or mammalian cell host, thereby enabling the production of recombinant antibodies. can do. In one technique, an immunogen comprising a polypeptide is first injected into one of a wide variety of mammals (eg, mouse, rat, rabbit, sheep or goat). In this step, the polypeptide of the present invention can act as an immunogen without modification. Alternatively, especially for relatively short polypeptides, a better immune response can be elicited when the polypeptide is bound to a carrier protein such as bovine serum albumin or keyhole limpet hemocyanin. The immunogen is preferably injected into an animal host according to a predetermined schedule, in combination with one or more booster immunizations, and the animal is periodically drawn. Polyclonal antibodies specific for the polypeptide can then be purified from the antisera by, for example, affinity chromatography using the polypeptide bound to a suitable solid support.
[193] Monoclonal antibodies specific for the antigenic polypeptide of interest are described, for example, in Kohler and Milstein, Eur. J. Immunol. 6: 511-519, 1976 and its improvement methods. Briefly, these methods relate to the preparation of immortal cell lines that can produce antibodies with the desired specificity (ie, reactivity with the polypeptide of interest). Such cell lines can be prepared, for example, from spleen cells obtained from an immunized animal as described above. The splenocytes are then immortalized, for example, by fusion with a myeloma cell fusion partner, preferably a cell that is syngeneic with the immunized animal. Various fusion techniques can be used. For example, splenocytes and myeloma cells can be mixed in a nonionic detergent for several minutes and then plated at low density on a selection medium that supports the growth of hybrid cells but inhibits the growth of myeloma cells. Preferred selection techniques use HAT (hypoxanthine, aminopterin, thymidine). After sufficient time, usually about 1 to 2 weeks, colonies of hybrids are observed. A single colony is selected to test its culture supernatant for binding activity to the polypeptide. Hybridomas with high reactivity and specificity are preferred.
[194] Monoclonal antibodies can be isolated from the supernatants of growing hybridoma colonies. In addition, various techniques can be used to increase yield, such as infusion of hybridoma cells into the peritoneal cavity of a suitable vertebrate host, such as a mouse. The monoclonal antibodies can then be recovered from ascites or blood. Contaminants can be removed from the antibody using conventional methods such as chromatography, gel filtration, precipitation, and extraction methods. Polypeptides of the invention can be used in purification methods, for example in affinity chromatography steps.
[195] Numerous therapeutically useful molecules are known in the art, including antigen-binding sites capable of exhibiting the immunological binding properties of antibody molecules. Proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which ("F (ab)" fragments) each contain a covalent heterodimer containing an intact antigen-binding site do. The enzyme pepsin cleaves the IgG molecule to provide several fragments comprising an "F (ab ') ₂ " fragment containing all of the antigen binding sites. "Fv" fragments are produced by preferential proteolytic cleavage of IgM, rarely cleaving IgG or IgA immunoglobulin molecules. However, Fv fragments are more generally derived using recombinant techniques known in the art. Fv fragments include non-covalent V _H :: V _L heterodimers comprising antigen binding sites that contain a significant portion of the antigen recognition and binding capacity of native antibody molecules. See Inbar et al. (1972) Proc. Natl. Acad. Sci. USA 69: 2659-2662; Hochman et al. (1976) Biochem 15: 2706-2710; and Ehrlich et al. (1980) Biochem 19: 4091-4096.
[196] Single-chain Fv ("sFv") polypeptides are covalently linked V _H :: V _L heterodimers expressed from gene fusions comprising V _H -V _L -coding genes bound by peptide encoding linkers. See Houston et al. (1988) Proc. Natl. Acad. Sci. USA 85 (16): 5879-5883]. Numerous methods identify chemical structures for converting light and heavy chain polypeptide chains that are naturally aggregated from the antibody V region but are chemically separated into sFv molecules that will be folded into a three-dimensional structure substantially similar to that of the antigen binding site. It has been described in order to. See, eg, US Pat. Nos. 5,091,513 and 5,132,405, Huston et al .; And US Pat. No. 4,946,778, Lander et al.].
[197] Each of the molecules described above comprises a set of heavy and light chain CDRs each dispersed between a set of heavy and light chain FRs that provide support to the CDRS and define the spatial correlation of the CDRs with respect to each other. As used herein, the term "CDR set" refers to three hypervariable regions of the heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, the three regions are named "CDR1", "CDR2" and "CDR3", respectively. Thus, the antigen binding site comprises six CDRs, including a set of CDRs from each of the heavy and light chain V regions. Polypeptides comprising a single CDR (eg, CDR1, CDR2 and CDR3) are referred to herein as "molecular recognition units". Crystallographic analysis of numerous antigen-antibody complexes demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contacts being formed with heavy chain CDR3. Thus, molecular recognition units are primarily concerned with the specificity for the antigen binding site.
[198] As used herein, the term “FR set” refers to the four flanking amino acid sequences that make up the CDRs of the CDR sets of the heavy or light chain V region. While certain FR residues may contact the antigen to which they bind, FR is involved in folding the V region to the antigen binding site, in particular bringing the FR residues immediately adjacent to the CDRs. Within the FR, certain amino residues and certain structural properties are very conservative. In this regard, all V region sequences contain an internal disulfide loop of about 90 amino acid residues. When the V region is folded to a binding site, the CDRs appear as protruding loop motifs that form the antigen binding surface. Regardless of the exact CDR amino acid sequence, it is generally recognized that there is a conserved structural region of the FR that affects the folded form of the CDR loop into a particular “normative” structure. In addition, certain FR residues are known to participate in non-covalent domain contact that stabilizes the interaction of antibody heavy and light chains.
[199] Numerous “humanized” antibody molecules have been described that contain antigen binding sites derived from non-human immunoglobulins and include chimeric antibodies having rodent V regions fused to human constant domains and their associated CDRs [Winter et al. (1991) Nature 349: 293-299; Lobuglio et al. (1989) Proc. Natl. Acad. Sci. USA 86: 4220-4224; Shaw et al. (1987) J Immunol. 138: 4534-4538; and Brown et al. (1987) Cancer Res. 47: 3577-3583], chimeric antibodies with rodent CDRs grafted to human support FRs prior to fusion to suitable human antibody constant regions [Riechmann et al. (1988) Nature 332: 323-327; Verhoeyen et al. (1988) Science 239; 1534-1536; and Jones et al. (1986) Nature 321: 522-525, and chimeric antibodies with rodent CDRs supported by a recombinantly veneered rodent FR (European Patent Application No. 519,596, published Dec. 23, 1992). . Such “humanized” molecules are designed to minimize undesirable immunological responses to rodent antihuman antibody molecules that limit the persistence and effect of therapeutic application of these residues on human receptors.
[200] As used herein, the terms "treated FR" and "recombinant FR" refer to heterologous molecules that include antigen binding sites that retain substantially all of the native FR polypeptide folding structure, for example, By selective replacement of FR residues from human rodent heavy or light chain V regions with human FR residues. The veneering technique is based on the understanding that the ligand binding properties of the antigen binding site are largely determined by the structure and relative placement of the heavy and light chain CDR sets within the antigen binding surface. See Davies et a. (1990) Ann. Rev. Biochem. 59: 439-473. Thus, antigen binding specificity can be conserved in human antibodies only if the CDR structure, interaction with each other and its interaction with the rest of the V region domain are carefully maintained. By using treatment techniques, it is possible to selectively replace external (eg, solvent-accessible) FR residues that are easily contacted by the immune system with human residues to include weakly immunogenic, or substantially non-immunogenic, treated surfaces. It provides a hydride molecule.
[201] The method of treatment is useful for human antibody variable domains accumulated in Kabat el al., Sequences of Proteins of Immunological Interest, 4th ed., (US Dept. of Health and Human Services, US Government Printing Office, 1987). Sequence data, updates to the Kabat database, and other available US and foreign databases (both nucleic acids and proteins) are used. Solvent accessibility of the V region amino acids can be inferred from known three-dimensional structures for human and murine antibody fragments. There are two general steps to processing murine antigen binding sites. First, the FR of the variable region of the antibody molecule of interest is compared with the corresponding FR sequence of the human variable domain obtained from the above identified source. The most homologous human V region is then compared residue to residue relative to the corresponding murine amino acid. The residues in the murine FR that differ from the human counterparts are replaced with residues indicated as human residues using recombinant techniques known in the art. Residue conversion is performed with residues that are at least partially exposed (easily solvent accessible) and care must be taken in the replacement of amino acid residues that may have a significant effect on the tertiary structure of the V region domains such as proline, glycine and charged amino acids.
[202] In this way, the non-covalent linkage between the obtained “treated” murine antigen binding site, murine CDR residues, residues substantially adjacent to the CDRs, residues found to be covered or nearly covered (solvent inaccessible), heavy and light chain domains To retain residues that are believed to participate in sexual (eg, electrostatic and hydrophobic) contacts, and those from the conserved structural regions of the FRs that are thought to affect the "normative" tertiary structure of the CDR loops. Is designed. This design criterion is then used to prepare a recombinant nucleotide sequence that binds the CDRs of both the heavy and light chains of the murine antigen binding site into the human FR, which is a mammal for expression of a recombinant human antibody that exhibits the antigenic specificity of the murine antibody molecule. It can be used to transfect cells.
[203] In another specific embodiment of the invention, the monoclonal antibodies of the invention can be coupled to one or more therapeutic agents. Suitable agents in this regard include radionuclides, differentiation inducing agents, agents, toxins and derivatives thereof. Preferred radionuclides include ⁹⁰ Y, ¹²³ I, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹¹ At and ²¹² Bi. Preferred agents include methotrexate, and pyrimidine and purine analogs. Preferred differentiating derivatives include phorbol esters and butyric acid. Preferred toxins include lysine, abrin, diphtheria toxin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigella toxin, and the Capricorn antiviral protein.
[204] The therapeutic agent can be coupled (eg covalently) to a suitable monoclonal antibody either directly or indirectly (eg, via a linker group). Direct reaction between the agent and the antibody is possible when the agent and the antibody each have a substituent that can react with each other. For example, a nucleophilic group, such as an amino or sulfhydryl group, on one side may be a carbonyl-containing group, such as an anhydride or an acid halide, or a good leaving group (eg, a halide) on the other. It may react with an alkyl group containing.
[205] Alternatively, it may be desirable to bind the therapeutic agent and antibody through a linker group. The linker group can act as a spacer to keep the antibody at regular intervals from the agent so as not to interfere with the binding capacity. The linker group may also act to increase the chemical reactivity of the substituents on the agent or antibody and consequently increase the coupling efficacy. Increasing chemical reactivity can also promote the use of agents, or otherwise the use of functional groups on agents that are otherwise impossible.
[206] It will be appreciated by those skilled in the art that bi- or multi-functional preparations of both the same or different functions (eg, as described in the catalog of Pierce Chemical Co., Rockford, IL) can be used as the linker group. Will be obvious. Coupling can be carried out, for example, via amino groups, carboxyl groups, sulfhydryl groups or oxidized carbohydrate moieties. There are numerous references (e.g., US Pat. No. 4,671,958, Rodwell et al.) Describing the methods.
[207] If the therapeutic agent is more potent when released from the antibody portion of the immunoconjugate of the invention, it would be desirable to use a cleavable linker group during or during localization into the cell. Numerous different cleavable linker groups have been described. Mechanisms for intracellular release of agents from this group of linkers include reduction of disulfide bonds (eg, US Pat. No. 4,489,710, Spitler), light irradiation of photolabile bonds (eg, US Pat. No. 4,625,014, Senter et. al.], hydrolysis of derivatized amino acid side chains [eg, US Pat. No. 4,638,045, Kohn et al.], serum complement-mediated hydrolysis [eg, US Pat. No. 4,671,958, Rodwell et al. .], And cleavage by acid-catalyzed hydrolysis (eg, US Pat. No. 4,569,789, Blattler et al.).
[208] It may be desirable to couple one or more agents to the antibody. In one specific embodiment, multiple molecules of the agent are bound to one antibody molecule. In another embodiment, more than one type of agent can be bound to one antibody. Regardless of certain specific embodiments, immunoconjugates with one or more agents can be prepared in a variety of ways. For example, linkers may be used that couple one or more agents directly to the antibody molecule or provide multiple sites for attachment. Alternatively, carriers can be used.
[209] The carrier can be attached to the formulation in a variety of ways, including covalent bonds, either directly or through a linker group. Suitable carriers include proteins such as albumin (eg, US Pat. No. 4,507,234, Kato et al.), Polysaccharides such as peptides and aminodextrans (US Pat. No. 4,699,784, Shih et al.). The carrier is also bound to the formulation by noncovalent bonds or by entrapment such as in liposome vesicles (eg, US Pat. Nos. 4,429,008 and 4,873,088). Carriers specific for radionuclide preparations include radiohalogenated small molecules and chelating agents. For example, US Pat. No. 4,735,792 describes representative radiohalogenated small molecules and their synthesis. Radionuclide chelating agents can be formed from chelating compounds, including metals or metal oxides, those containing nitrogen and sulfur atoms as donor atoms for binding radionuclides. For example, US Pat. No. 4,673,562, Davison et al., Describes representative chelated compounds and their synthesis.
[210] T cell composition
[211] In another embodiment, the present invention provides T cells specific for the tumor polypeptides, variants or derivatives thereof described herein. The cells can generally be prepared in vitro or ex vivo, using standard methods. For example, T cells may be obtained from a patient's bone marrow, peripheral blood or a fraction of bone marrow or peripheral blood, such as a commercially available cell separation system such as the Isolex ^™ system manufactured by Nexell Therapeutics, Inc., Irvine, CA; See also US Pat. No. 5,240,856; US Patent No. 5,215,926; WO 89/06280; WO 91/16116 and WO 92/07243. Alternatively, T cells can be derived from related or unrelated human, non-human mammals, cell lines or cultures.
[212] T cells can be stimulated with a polypeptide, a polynucleotide encoding a polypeptide and / or an antigen presenting cell (APC) expressing the polypeptide. The stimulation is performed for conditions and time sufficient to allow production of T cells specific for the polypeptide of interest. Preferably, the tumor polypeptides or polynucleotides of the present invention are present in a variety of delivery vehicles, such as microspheres, to facilitate the production of specific T cells.
[213] When T cells specifically proliferate, secrete, or kill cytokines, which are coated with the polypeptide of the invention or express genes encoding the polypeptide, the T cells are specific for the polypeptide of the invention. I think. T cell specificity can be assessed using any one of a variety of standard techniques. For example, in chromium release assays or proliferation assays, the stimulation index of at least two-fold increase in fusion and / or proliferation relative to the negative control indicates the specificity of T cells. Such assays are described, for example, in Chen et al., Cancer Res. 54: 1065-1070, 1994. Alternatively, detection of T cell proliferation can be accomplished by various known techniques. For example, T cell proliferation can be detected by measuring the rate of increase in DNA synthesis (eg, by pulse labeling T cell cultures with tritiated thymidine and measuring the amount of tritiated thymidine introduced into the DNA). have. Contact with the tumor polypeptide (100 ng / ml-100 µg / ml, preferably 200 ng / ml-25 µg / ml) for 3-7 days typically results in a two-fold increase in the proliferation of T cells. Such contact for 2 to 3 hours results in activation of T cells as measured using standard cytokine assays, which, in cytokine assays, doubled at cytokine (eg TNF or IFN-γ) release levels. Is an indicator of T cell activation. Coligan et al., Current Protocols in Immunology, vol. 1, Wiley Interscience (Greene 1998). T cells activated in response to tumor polypeptide, polynucleotide or polypeptide expressing APCs may be CD4 ⁺ and / or CD8 ⁺ . Tumor polypeptide-specific T cells can be propagated using standard techniques. In a preferred specific embodiment, the T cells are derived from a patient who is an associated or unrelated donor and is administered to the patient after stimulation and proliferation.
[214] For therapeutic purposes, CD4 ⁺ or CD8 ⁺ T cells that proliferate in response to tumor polypeptides, polynucleotides or APCs can be proliferated numerically in vitro or in vivo. Proliferation of the T cells in vitro can be accomplished in a variety of ways. For example, a T cell corresponds to a tumor polypeptide, or an immunogenic portion of said polypeptide, in the presence or absence of a stimulatory cell that synthesizes a T cell growth factor, such as interleukin-2 and / or a tumor polypeptide. Can be reexposed to short peptides. Alternatively, one or more T cells proliferating in the presence of a tumor polypeptide can be propagated numerically by cloning. Cell cloning methods are known in the art and include limiting dilution.
[215] Pharmaceutical composition
[216] In a further specific embodiment, the present invention provides a polynucleotide, polypeptide, T-cell and / or described herein in a pharmaceutically acceptable carrier for administration to a cell or animal, alone or in combination with one or more therapeutic embodiments. Antibody composition relates to one or more formulations.
[217] If desired, the compositions described herein can be administered in combination with other agents, such as, for example, other proteins or polypeptides or various pharmaceutically active agents. In fact, as long as the additional agent does not cause significant adverse effects upon contact with the target cell or host tissue, there are virtually no restrictions on the other ingredients that may also be included. Thus, the composition may be delivered with various other agents as needed in certain instances. The composition may be purified from a host cell or other biological source, or may be chemically synthesized as described herein. Likewise, the composition may further comprise a substituted or derivatized RNA or DNA composition.
[218] Accordingly, in another aspect of the invention, there is provided a pharmaceutical composition comprising one or more of the polynucleotides, polypeptides, antibodies, and / or T-cell compositions described herein in combination with a physiologically acceptable carrier. In certain preferred specific embodiments, the pharmaceutical compositions of the invention comprise the immunogenic polynucleotide and / or polypeptide compositions of the invention for use in prophylactic and therapeutic vaccine applications. Vaccine formulations are generally described, for example, in M.F. Powell and M.J. Newman, eds., "Vaccine Design (the subunit and adjuvant approach)", Plenum Press (NY, 1995). In general, the composition will comprise one or more polynucleotide and / or polypeptide compositions of the present invention in parallel with one or more immunostimulating agents.
[219] Any one of the pharmaceutical compositions described herein may contain pharmaceutically acceptable salts of the polynucleotides and polypeptides of the invention. The salts include, for example, organic bases (eg primary, secondary and tertiary amines and basic amino acids) and inorganic bases (eg sodium, potassium, lithium, ammonium, calcium and magnesium salts). And can be prepared from pharmaceutically acceptable non-toxic bases.
[220] In another specific embodiment, an exemplary immunogenic composition of the invention, eg, a vaccine composition, comprises DNA encoding one or more polypeptides as described above, resulting in the polypeptide being produced in situ. As noted above, polynucleotides may be administered in a variety of delivery systems known to those of ordinary skill in the art. Indeed, numerous gene delivery techniques are known in the art, for example, see Rolland, Crit. Rev. Therap. Drug Carrier Systems 15: 143-198, 1998 and references cited therein. Suitable polynucleotide expression systems will of course contain the necessary regulatory DNA regulatory sequences (eg, suitable promoters and termination signals) for expression in the patient. Alternatively, the bacterial delivery system may involve the administration of bacteria (eg, Bacillus-Calmete-Guerin) that express an immunogenic portion of the polypeptide on its cell surface and secrete the epitope.
[221] Thus, in certain specific embodiments, polynucleotides encoding the immunogenic polypeptides described herein are introduced into suitable mammalian host cells using one of a number of known viral based systems. In one exemplary specific embodiment, retroviruses provide a convenient and effective platform for gene delivery systems. Selected nucleotide sequences encoding polypeptides of the invention may be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the patient. Many exemplary retrovirus systems have been described (eg, US Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, A.D. (1990) Human Gene Therapy 1: 5-14; Scarpa et al. (1991) Virology 180: 849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109.
[222] In addition, numerous exemplary adeno-based systems have also been described. Unlike retroviruses that integrate into the host genome, adenoviruses exist outside the chromosome, minimizing the risks associated with insertional mutagenesis [Haj-Ahmad and Graham (1986) J. Virol. 57: 267-274; Bett et al. (1993) J. Virol. 67: 5911-5921; Mittereder et al. (1994) Human Gene Therapy 5: 717-729; Seth et al. (1994) J. Virol. 68: 933-940; Barr et al. (1994) Gene Therapy 1: 51-58; Berkner, K.L. (1988) BioTechniques 6: 616-629; and Rich et al. (1993) Human Gene Therapy 4: 461-476.
[223] Various adeno-associated virus (AAV) vector systems have also been developed for polynucleotide delivery. AAV vectors can be readily constructed using techniques known in the art. See literature [eg, US Pat. Nos. 5,173,414 and 5,139,941; International Patent Publications WO 92/01070 and WO 93/03769; Lebkowski et al. (1988) Molec. Cell Biol. 8: 3988-3996; Vincent et al (1990) Vaccine 90 (Cold Spring Harbor Laboratory Press); Carter, B.J. (1992) Current Opinion in Biotechnology 3: 533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158: 97-129; Therapy 1: 165-169; and Zhou et al. (1994) J. Exp. Med. 179: 1867-1875.
[224] Additional viral vectors useful for delivering polynucleotides encoding polypeptides of the present invention by gene transfer include those derived from the Fox family of viruses such as vaccinia virus and avian poxvirus. For example, vaccinia virus recombinants expressing new molecules can be constructed as follows. The DNA encoding the polypeptide is first inserted into a suitable vector so as to be adjacent to vaccinia promoter and flanking vaccinia DNA sequences such as sequences encoding thymidine kinase (TK). This vector can then be used to transfect cells simultaneously infected with vaccinia. Homologous recombination introduces the gene encoding the vaccinia promoter and the polypeptide of interest into the viral genome. The resulting TK.sup. (-) Recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and collecting viral plaques resistant to it.
[225] Vaccinia-based infection / transfection systems can be readily used to provide inducible transient expression or co-expression of one or more polypeptides described herein in host cells of an organism. In this particular system, cells are first infected in vitro with vaccinia virus recombinants encoding bacteriophage T7 RNA polymerase. Such polymerases exhibit a sophisticated specificity that only transfers the template with the T7 promoter. After infection, cells are transfected with polynucleotides or polynucleotides of interest driven by the T7 promoter. The polymerase expressed in the cytoplasm from vaccinia virus recombinants transcribs the transfected DNA into RNA, which is then translated into polypeptides by a host translational organ. The method provides high levels of transient intracellular production of RNA and its translation products. See, eg, Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87: 6743-6747; Fuerst et al. Proc. Natl. Acad. Sci. USA (1986) 83: 8122-8126.
[226] Alternatively, avian pox viruses such as chicken pox and canary pox viruses can be used to deliver the coding sequence of interest. Recombinant avian poxviruses that express immunogens from mammalian hospitals are known to provide protective immunity when administered to non-algal species. The use of the Avipox promoter may be particularly desirable in humans and other mammalian species, since members of the genus Abifox can be abundantly replicated in susceptible avian species and are therefore non-infectious in mammalian cells. Methods for producing recombinant Abipox virus are known in the art and use genetic recombination, as described above for the production of vaccinia virus. See literature [eg, WO 91/12882; WO 89/03429 and WO 92/03545.
[227] Any one of a number of alphavirus vectors may also be used to deliver the polynucleotide compositions of the present invention, see, for example, US Pat. No. 5,843,723; 6,015,686; 6,008,035 and 6,015,694. Certain vectors based on Venezuelan equine encephalitis (VEE) can also be used, and illustrative examples can be found in US Pat. Nos. 5,505,947 and 5,643,576.
[228] In addition, Michael et al. J. Biol. Chem. (1993) 268: 6866-6869 and Wagner et al. Proc. Natl. Acad. Sci. Molecular conjugate vectors, such as the adenovirus chimeric vectors described in USA (1992) 89: 6099-6103, can also be used to deliver genes under the present invention.
[229] Additional exemplary information for these and other known virus-based delivery systems can be found, for example, in Fisher-Hoch et al., Proc. Natl. Acad. Sci. USA 86: 317-321, 1989; Flexner et al., Ann. N.Y. Acad. Sci. 569: 86-103, 1989; Flexner et al., Vaccine 8: 17-21, 1990; U.S. Patents 4,603,112, 4,769,330 and 5,017,487; WO 89/01973; U.S. Patent 4,777,127, UK Patent 2,200,651; European Patent No. 0,345,242; WO 91/02805; Berkner, Biotechniques 6: 616-627, 1988; Rosenfeld et al., Science 252: 431-434, 1991; Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219, 1994; kass-Eisler et al., Proc. Natl. Acad. Sci. USA 90: 11498-11502, 1993; Guzman et al., Circulation 88: 2838-2848, 1993; and Guzman et al., Cir. Res. 73: 1202-1207, 1993.
[230] In certain specific embodiments, the polynucleotides may be integrated into the genome of the target cell. Such integration may be performed at specific positions and orientations through homologous recombination (gene replacement) or may be integrated at random nonspecific positions (gene growth). In a further specific embodiment, the polynucleotides can remain stable intracellularly as individual episomal fragments of DNA. The polynucleotide fragment or "episome" encodes sufficient sequence to be maintained and replicated independently of or in conjunction with the host cell cycle. The manner in which the expression construct is delivered intracellularly and the polynucleotides maintained in the cell depends on the type of expression construct used.
[231] In another specific embodiment of the invention, polynucleotides are described, for example, in Ulmer et al., Science 259: 1745-1749, 1993 and as disclosed in Cohen, Science 259: 1691-1692, 1993 As such, it is administered / delivered as “extracted” DNA. Uptake of the naked DNA can be increased by coating the DNA with biodegradable beads, which are effectively delivered intracellularly.
[232] In another specific embodiment, the compositions of the present invention can be delivered via particle bombardment methods, many of which have been described. In one illustrative example, gas-driven particle acceleration is achieved using devices such as those manufactured by Manufacturer (Powderject Pharmaceuticals PLC, Oxford, UK) and Manufacturer (Powderject Vaccines Inc., Madison, WI), Specific examples thereof include US Pat. No. 5,846,796; 6,010,478; 6,010,478; No. 5,865,796; 5,584,807 and European Patent No. 0500799. This method provides a needle-free delivery method wherein anhydrous powder formulations of fine particles, such as polynucleotides or polypeptide particles, are accelerated at high speed in a helium gas jet produced by a manual device, thereby bringing the particles of interest. Promote into target tissue.
[233] In related specific embodiments, other devices and methods that may be useful for gas-driven needle-free injection of a composition of the present invention include those provided by the manufacturer (Bioject, Inc., Portland, OR), including Specific examples are described in US Pat. Nos. 4,790,824, 5,064,413, 5,312,335, 5,383,851, 5,399,163, 5,520,639 and 5,993,412.
[234] In another specific embodiment, the pharmaceutical compositions described herein will comprise one or more immunostimulatory agents in addition to the immunogenic polynucleotides, polypeptides, antibodies, T-cells and / or APC compositions of the invention. Immunostimulatory agent means essentially any substance that enhances or enhances an immune response (antibody and / or cell-mediated) to an exogenous antigen. One preferred type of immunostimulant is an adjuvant. Many adjuvants are materials designed to protect antigens from rapid catabolism, such as aluminum hydroxide or mineral oil, and immune response stimulants such as lipid A, Bortadella pertussis. ) Or Mycobacterium tuberculosis induced protein. Certain adjuvants are commercially available and include, for example, Freund's incomplete and complete adjuvants (Difco Laboratories, Detroit, MI); Merck adjuvant 65 (manufactured by Merck and Company, Inc., Rahway, NJ), AS-2 (SmithKline Beecham, Philadelphia, PA), aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate, calcium, iron or Salts of zinc, insoluble suspensions of acylated tyrosine, acylated sugars, cationic or anionic derivatized polysaccharides, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A and quill A. Cytokines such as GM-CSF, Interleukin-2, -7, -12 and other growth factors can also be used as an adjuvant.
[235] In certain specific embodiments of the invention, the adjuvant composition is preferably one that induces an excellent Th1 type immune response. High levels of Th1-type cytokines (eg, IFN-γ, TNF-α, IL-2 and IL-12) tend to be beneficial for induction of cell mediated immune responses to administered antigens. In contrast, high levels of Th2-type cytokines (eg, IL-4, IL-5, IL-6, and IL-10) tend to be beneficial for inducing a humoral immune response. After application of the vaccine as provided herein, the patient will support an immune response including Th-1 and Th-2 type responses. In a preferred specific embodiment, the response is that in which Th-1 type is preferred, the level of Th1-type cytokines will further increase above the level of Th2-type cytokines. Levels of these cytokines can be readily assessed using standard assays. For an overview of the cytokine family, see Mosmann and Coffman, Ann. Rev. Immunol. 7: 145-173, 1989.
[236] Certain preferred adjuvants for preferentially inducing a Th1-type response include, for example, mixtures of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A with aluminum salts. do. MPL Adjuvant is commercially available from Corixa Corporation, Seattle, WA (see, eg, US Pat. Nos. 4,436,727, 4,877,611, 4,866,034, and 4,912,094). CpG-containing oligonucleotides, wherein the CpG dinucleotides are not methylated, also preferentially induce a Th1 response. Such oligonucleotides are known in the art and are described, for example, in WO 96/02555, WO 99/33488 and US Pat. Nos. 6,008,200 and 5,856,462. Immunostimulatory DNA sequences are also described, for example, in Sato et al., Science 273: 352, 1996. Still other preferred adjuvants include saponins such as quill A, or derivatives thereof including QS21 and QS7 (manufactured by Aquila Biopharmaceuticals Inc., Framingham, MA); Escin, Digitonin, or Gipposophila or Chenopodium quinoa saponins. Other preferred formulations include a mixture of two or more of the group comprising one or more saponins of the adjuvant mixture of the invention, eg, QS21, QS7, Quill A, β-escin or digitonin.
[237] Alternatively, the saponin formulation may be a chitosan or other polycationic polymer, polaractide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, polysaccharides or chemically modified polysaccharides. And particles consisting of liposomes and lipid-based particles, particles consisting of glycerol monoesters, and the like. Saponins may also form particle structures such as liposomes or ISCOMs in the presence of cholesterol. In addition, saponins can be formulated in non-particulate solutions or suspensions with polyoxyethylene ethers or esters, or in particulate structures such as pacilamellar liposomes or ISCOMs. Saponins are also Carbopol It may be formulated with such excipients to increase viscosity, or may be formulated in anhydrous powder form with powder excipients such as lactose.
[238] In one preferred specific embodiment, the adjuvant system is a mixture of monophosphoryl lipid A and saponin derivatives, for example QS21 and 3D-MPL as described in WO 94/00153. Mixtures of adjuvant, or QS21 as described in WO 96/33739, include less reactive compositions quenched with cholesterol. Other preferred formulations include oil-in-water emulsions and tocopherols. QS21, 3D-MPL in oil-in-water emulsion Another particular preferred adjuvant formulation using adjuvants and tocopherols is described in WO 95/17210.
[239] Another enhanced adjuvant system relates to mixtures of CpG-containing oligonucleotides and saponin derivatives, in particular mixtures of CpG and QS21 and is described in WO 00/09159. Preferably, the formulation additionally comprises an oil-in-water emulsion and tocopherol.
[240] Another exemplary adjuvant for use in the pharmaceutical compositions of the present invention is described in pending US patent applications 08 / 853,826 and 09 / 074,720, the contents of which are incorporated herein by reference in their entirety. Montanaide ISA 720 (manufactured by Seppic, France), SAF (manufactured by Chiron, California, United States), ISCOM (manufactured by CSL), MF-19 (manufactured by Chiron), and adjuvant's SBAS series (e.g. For example, SBAS-2 or SBAS-4, SmithKline Beecham, Rixensart, Belgium, Detox (Enhanzyn) ) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosamide 4-phosphates (AGP); And polyoxyethylene ether adjuvants such as those described in WO 99 / 52549A1.
[241] Other preferred adjuvants include adjuvant molecules of Formula (I).
[242] HO (CH ₂ CH ₂ O) _n -AR
[243] In the above formula,
[244] n is 1 to 50,
[245] A is a bond or -C (O)-,
[246] R is C _1-50 alkyl or _{phenylC 1-50} alkyl.
[247] One specific embodiment of the invention is that n is 1 to 50, preferably 4 to 24, most preferably 9, R component is C _1-50 , preferably C ₄ -C ₂₀ alkyl and most preferably It consists of a vaccine formulation comprising a polyoxyethylene ether of formula I wherein C ₁₂ alkyl and A is a bond. The concentration of polyoxyethylene ether may range from 0.1 to 20%, preferably from 0.1 to 10%, and most preferably from 0.1 to 1%. Preferred polyoxyethylene ethers are polyoxyethylene-9-lauryl ether, polyoxyethylene-9-stearyl ether, polyoxyethylene-8-stearyl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene- 35-lauryl ether and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12 ^th edition: entry 7717). Such adjuvant molecules are described in WO 99/52549.
[248] The polyoxyethylene ether according to formula (I) may optionally be mixed with another adjuvant. For example, the preferred adjuvant composition is preferably a mixture with CpG as described in the publication GB 9820956.2.
[249] According to another specific embodiment of the invention, the immunogenic compositions described herein are introduced into the host via APCs such as dendritic cells, macrophages, B cells, monocytes and other cells that can be engineered to be effective antigen presenting cells (APCs). Delivered. The cells are genetically engineered to increase antigen presentation ability, to improve activation and / or maintenance for T cell responses, to have anti-cancer effects on their own and / or to be immunologically compatible with the recipient (ie, matched). HLA haploid), but is not necessary. APCs can generally be isolated from one of a variety of biological fluids and organs, including tumors and surrounding tumor tissues, and can be autologous, pseudo, syngeneic or heterologous cells.
[250] Certain preferred specific embodiments of the invention use dendritic cells or progenitor cells thereof as antigen-presenting cells. Dendritic cells are very potent APCs (Banchereau and Steinman, Nature 392: 245-251, 1998) and have been shown to be effective as physiological adjuvants exhibiting prophylactic or therapeutic anticancer immunity (eg, Timmerman and Levy, Ann. Rev. Med. 50: 507-529, 1999]. In general, dendritic cells (in situ radial, with visible cytoplasmic processes visible in vitro), the ability to absorb, process and present antigens with high efficiency, and to activate natural T cell responses Can be identified based on his ability to Dendritic cells, of course, can be genetically engineered to express specific cell-surface receptors or ligands not normally found on dendritic cells in vivo or ex vivo, and such modified dendritic cells are included in the present invention. As an alternative to dendritic cells, secreted vesicle antigen-filled dendritic cells (so-called exosomes) can be used in the vaccine. See Zitvogelet al., Nature Med. 4: 594-600, 1998.
[251] Dendritic cells and progenitor cells can be obtained from peripheral blood, bone marrow, tumor penetrating cells, peritumoral tissue penetrating cells, lymph nodes, spleen, skin, umbilical cord blood or any other suitable tissue or fluid. For example, dendritic cells can be differentiated ex vivo by adding a mixture of cytokines such as GM-CSF, Il-4, IL-13 and / or TNFα to monocytes harvested from peripheral blood. Alternatively, CD34 positive cells harvested from peripheral blood, umbilical cord blood, or bone marrow can be used to induce differentiation, maturation and proliferation of GM-CSF, IL-3, TNFα, CD40 ligand, LPS, flt3 ligand and / or dendritic cells. In addition to the culture medium mixture of (s) can be differentiated into dendritic cells.
[252] Dendritic cells are easily classified as "mature" and "mature" cells, allowing a simple way to distinguish two characterized phenotypes. However, this nomenclature is not intended to exclude all intermediate stages of possible differentiation. Immature dendritic cells are characterized as APCs with high capacity for antigen uptake and processing, which is associated with high expression of Fcγ receptors and mannose receptors. Mature phenotypes typically include lower expression of these markers, and type I and II MHCs, adhesion molecules (eg, CD54 and CD11) and costimulatory molecules (eg, CD40, CD80, CD86 and 4- 1BB) is characterized by high expression of cell surface molecules involved in T cell activation.
[253] APCs are generally transfected with the polynucleotides (or portions thereof or other variants thereof) of the invention to express the encoded polypeptide or immunogenic portion thereof at the cell surface. The transfection may be performed ex vivo, and therefore, a pharmaceutical composition comprising the transfected cells may be used for therapeutic purposes, as described herein. Alternatively, a gene delivery vehicle that targets dendritic cells or other antigen presenting cells can be administered to the patient, resulting in transfection that occurs in vivo. In vivo and ex vivo transfection of dendritic cells is generally carried out by any method known in the art as described in WO 97/24447, or by Mahvi et al., Immunology and cell Biology 75: 456. -460, 1997]. Antigen filling of dendritic cells allows the dendritic cells or progenitor cells to be combined with tumor polypeptide DNA (extracted or in plasmid vectors) or RNA, or antigen-expressing recombinant bacteria or viruses (eg vaccinia, chicken fox, adenovirus). Or lentiviral). Prior to filling, the polypeptide may be covalently conjugated to an immunogenic partner (eg, carrier molecule) that provides T cell help. Alternatively, dendritic cells can be pulsed with an immunological counterpart that is separate from the polypeptide or in the presence of the polypeptide.
[254] Suitable carriers known to those of ordinary skill in the art can be used in the pharmaceutical compositions of the present invention, while the type of carrier will typically vary depending on the dosage form. The compositions of the present invention may be formulated in any suitable mode of administration, including, for example, topical, oral, nasal, intramucosal, intravenous, intracranial, intraperitoneal, injury and intramuscular administration.
[255] Carriers for use in the pharmaceutical compositions may be biocompatible and biodegradable. In certain specific embodiments, the formulation preferably provides a relative constant level of active ingredient release. However, in other specific embodiments, an immediate rapid release rate upon administration may be desirable. The formulation of the composition is within the level of ordinary skill in the art using known techniques. Exemplary carriers useful in this regard include microparticles such as poly (lactide-co-glycolide), polyacrylates, latex, starch, cellulose, dextran and the like. Other exemplary sustained release carriers include outer layers comprising non-liquid hydrophilic nuclei (eg, crosslinked polysaccharides or oligosaccharides), and optionally amphoteric compounds such as phospholipids. See, eg, US patents. 5,151,254 and PCT applications WO 94/20078, WO 94/23701 and WO 96/06638. The amount of active compound contained in a sustained release formulation is determined by the site of implantation, the rate of release and the expected duration, the nature of the treated or prevented condition.
[256] In another exemplary specific embodiment, biodegradable microspheres (eg polylactate polyglycolate) are used as carriers of the compositions of the present invention. Suitable biodegradable microspheres are described, for example, in US Pat. Nos. 4,897,268, 5,075,109, 5,928,647, 5,811,128, 5,820,883, 5,853,763, 5,814,344, 5,407,609 and 5,942,252. It is described. Modified hepatitis B nuclear protein carrier systems as described in WO 99/40934 and the references cited therein will also be useful in numerous applications. Another exemplary carrier / delivery system uses a carrier comprising a microparticle-protein complex capable of inducing a type I restricted cytotoxic T lymphocyte response in a host, as described in US Pat. No. 5,928,647.
[257] Pharmaceutical compositions of the invention often contain buffers (eg, neutral buffered saline or phosphate buffered saline), carbohydrates (eg, glucose, mannose, sucrose or dextran), mannitol, proteins, polypeptides, Or solutes, suspensions that give isotonic, hypotonic or mild hypertonicity to formulations with amino acids such as glycine, antioxidants, antibacterial agents, chelating agents such as EDTA or glutathione, adjuvant (e.g. aluminum hydroxide), recipient's blood Agent, thickener and / or preservative. Alternatively, the composition of the present invention may be formulated as a lyophilisate.
[258] The pharmaceutical compositions described herein may be present in unit dosage or multi-dose containers, such as sealed ampoules or vials. The container is typically sealed in this manner to maintain the sterility and stability of the formulation until use. In general, formulations may be stored as suspensions, solvents or emulsions in oily or aqueous vehicles. Alternatively, the pharmaceutical composition may be stored in a lyophilized state, requiring only the addition of a sterile liquid carrier immediately before use.
[259] The development of suitable dosage and therapeutic regimens for use with certain compositions described herein among various therapeutic regimens, including, for example, oral, parenteral, intravenous, intranasal and intramuscular administration and formulations are known in the art and Some of which are briefly discussed below for the general purpose of illustration.
[260] In certain applications, the pharmaceutical compositions described herein can be delivered to the animal via oral administration. As such, such compositions may be formulated with inert diluents or assimitable edible carriers, which may be enclosed in hard or soft gelatin capsules, which may be compressed into tablets, or may be incorporated directly into edible foods. .
[261] The active compound may be used in the form of tablets, buccal tablets, oral tablets, capsules, elixirs, suspensions, syrups, wafers, etc., ingested in combination with excipients. See, e.g., Mathiowitz et al., Nature 1997 Mar 27; 386 (6623): 410-4, Hwang et al., Crit. Rev. Ther. Drug Carrier Syst 1998; 15 (3): 243-84; U.S. Patent 5,641,515, U.S. Patent 5,580,579 and U.S. Patent 5,792,451]. Tablets, tablets, pills, capsules, and the like may also contain any of a variety of additional ingredients, including, for example, binders such as gum tragacanth, acacia, corn starch, or gelatin; Excipients such as dicalcium phosphate; Disintegrants such as corn starch, potato starch, alginic acid and the like; Lubricants such as magnesium stearate; And sweetening agents such as sucrose, lactose or saccharose, or flavoring agents such as peppermint, camellia oil, or cherry flavoring. When the dosage unit form is a capsule, it may contain a liquid carrier in addition to the substances of this type. Various other substances may be present as the skin agent or may be present to modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with sherlock, sugar, and the like. Of course, any material used to prepare any unit form should be pharmaceutically pure and substantially nontoxic in the amount used. In addition, the active compounds may be incorporated into sustained release preparations and formulations.
[262] Typically, such formulations contain at least about 0.1% of the active ingredient, but the percentage (%) of the active ingredient may, of course, conveniently be about 1 or 2% and about 60% or 70% or more of the weight or volume of the total formulation. have. Naturally, the amount of active compound (s) in each therapeutically useful composition can be prepared in this manner such that a suitable dosage form is obtained in a given unit dosage form of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product storage half-life, as well as pharmaceutical considerations, will be foreseen by those skilled in the manufacture of the pharmaceutical formulations. As such, various dosage forms and treatment regimens are desirable. can do.
[263] For oral administration, the compositions of the present invention may alternatively be incorporated with one or more excipients in the form of gargles, cream dentifrices, buccal tablets, oral sprays, or sublingual orally administered formulations. Alternatively, the active ingredients may be incorporated into oral solvents such as sodium borate, glycerin and potassium bicarbonate or dispersed in cream dentifrice, or added in a therapeutically effective amount in a composition that may include water, binders, abrasives, flavors, foaming agents, and wetting agents. Can be. Alternatively, the composition may be prepared in tablets or solvents that can be placed sublingually or otherwise dissolved in the premises.
[264] Under certain circumstances, it would be desirable to deliver the pharmaceutical compositions described herein parenterally, intravenously, intramuscularly, or intraperitoneally. Such approaches are known in the art, some of which are further described, for example, in US Pat. No. 5,543,158, US Pat. No. 5,641,515 and US Pat. No. 5,399,363. In certain specific embodiments, a solution of the active compound as a free base or pharmaceutically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersants can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations will generally contain a preservative to inhibit the growth of microorganisms.
[265] Exemplary pharmaceutical forms suitable for injectable use include sterile aqueous solvents or dispersants and sterile powders for the immediate preparation of sterile injectable solutions or dispersions (see, eg, US Pat. No. 5,466,468). In all cases, the formulation must be sterile and liquefied to an easily injectable degree. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, polyols (eg, glycerol, propylene glycol and liquid polyethylene glycols, etc.), suitable mixtures thereof, and / or vegetable oils. Suitable fluidity can be maintained, for example, by using a skin agent such as lecithin, by maintaining the required particle size in the case of dispersants and / or by using surfactants. Prevention of microbial action can be facilitated with various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars and sodium chloride. Prolonged absorption of the injectable compositions can be achieved by use in compositions of agents that delay absorption, such as aluminum monostearate and gelatin.
[266] In one specific embodiment, for parenteral administration in an aqueous solution, the solution must be suitably buffered as appropriate and the liquid diluent must first be given isotonicity with sufficient saline or glucose. Such specific aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used will be known to those skilled in the art in light of the present disclosure. For example, one dosage form may be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of bulk subcutaneous injection or injected at the indicated site of infusion. See, eg, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580. Certain changes in dosage form will necessarily occur depending on the condition of the patient being treated. In addition, for human administration, the formulation will of course meet sterile, pyrogenic and general safety and purity standards, preferably in accordance with the provisions of the FDA Secretariat's Biologics standard.
[267] In another specific embodiment of the present invention, the compositions described herein may be formulated in neutral or salt form. Exemplary pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of the protein), which are formed, for example, with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid and mandelic acid. . Salts formed with free carboxyl groups can also be derived from, for example, inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides, or organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. When formulated, the solution will be administered in a manner that is compatible with the dosage form and is therapeutically effective in this amount.
[268] Carriers may further include any and all solvents, dispersion media, vehicles, skins, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. Such media and formulations for pharmaceutically active substances are known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is envisaged. Additional active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically acceptable” means molecular entities and compositions that, when administered to humans, do not cause allergic or similar adverse reactions.
[269] In certain specific embodiments, the pharmaceutical composition may be delivered by nasal spray, inhalation, and / or other aerosol delivery vehicles.
[270] Methods of delivering genes, nucleic acids and peptide compositions directly to the lungs via nasal aerosol sprays have been described, for example, in US Pat. No. 5,756,353 and US Pat. No. 5,804,212. Likewise, intranasal microparticle resins [Takenaga et al., J Controlled Release 1988 Mar 2; 52 (1-2): 81-7] and delivery of medicaments using lysophosphatidyl-glycerol compounds [US Pat. No. 5,725,871] are also known in the pharmaceutical art. Likewise, drug delivery through exemplary mucosa in the form of a polytetrafluoroethylene support matrix is described in US Pat. No. 5,780,045.
[271] In certain specific embodiments, liposomes, nanocapsules, microparticles, lipid particles, vehicles and the like are used to introduce the compositions of the invention into suitable host cells / organisms. In particular, the compositions of the invention can be encapsulated in lipid particles, liposomes, vehicles, nanospheres or nanoparticles and formulated for delivery. Alternatively, the compositions of the present invention can be covalently or noncovalently bound to the surface of the carrier vehicle.
[272] Formulation and use of liposomes and liposome type preparations as potential pharmaceutical carriers are generally known to those skilled in the art. See, eg, Lasic, Trends Biotechnol 1998 Jul; 16 (7): 307-21; Takakura, Nippon Rinsho 1998 Mar; 56 (3) 691-5; Chandran et al., Indian J Exp Biol. 1997 Aug; 35 (8): 801-9; Nargalit, Crit Rev Ther Drug Carrier Syst. 1995; 12 (2-3): 233-61; US Patent No. 5,567,434; US Patent No. 5,552,157; US 5,565,213; US Pat. No. 5,738,868 and US Pat. No. 5,795,587, each of which is specifically incorporated herein by reference in its entirety.
[273] Liposomes have been used successfully with numerous cell types that are difficult to normally transfect by other methods, including T cell suspensions, primary hepatocyte cultures and PC12 cells [Renneisen et al., J Biol Chem . 1990 Sep 25; 265 (27): 16337-42; Muller et al., DNA Cell Biol. 1990 Apr; 9 (3): 221-9]. In addition, liposomes are free from DNA length limitations typical of viral based delivery systems. Liposomes have been used to effectively introduce genes, various agents, radiopharmaceuticals, enzymes, viruses, transcription factors, allosteric effectors and the like into a variety of cultured cell lines and animals. In addition, the use of liposomes does not appear to be associated with an autoimmune response or unacceptable toxicity after systemic delivery.
[274] In certain specific embodiments, liposomes are formed from phospholipids dispersed in an aqueous medium and simultaneously form a multiplate concentric bilayer vesicle (or multiplate vesicle (MLV)).
[275] Alternatively, in another specific embodiment, the present invention provides pharmaceutically acceptable nanocapsule formulations of the compositions of the present invention. Nanocapsules can generally capture compounds in a stable and renewable manner. See, eg, Quintanar-Guerrero et al., Drug Dev Ind Pharm. 1998 Dec; 24 (12): 1113-28. In order to avoid side effects due to intracellular polymeric overload, the ultrafine particles (about 0.1 μm in size) can be designed using polymers that can degrade in vivo. Such particles are described, for example, in Couvreur et al., Crit Rev Ther Drug Carrier Syst. 1988; 5 (1): 1-20; zur Muhlen et al., Eur J Pharm Biopharm. 1998 Mar; 45 (2): 149-55; Zambaux et al., J Controlled Release. 1998 Jan 2; 50 (1-3): 31-40; and US Pat. No. 5,145,684.
[276] Cancer treatment method
[277] In a further aspect of the invention, the pharmaceutical compositions described herein can be used for the treatment of cancer, in particular for immunotherapy of breast cancer. In this method, the pharmaceutical compositions described herein are administered to a patient, typically a warm blooded animal, preferably a human. The patient may or may not have cancer. Thus, the pharmaceutical composition can be used to prevent progression to cancer or to treat patients with cancer. Pharmaceutical compositions and vaccines may be administered before or after surgical removal of the primary tumor and / or before or after treatment such as administration of radiotherapy or conventional chemotherapeutic agents. As discussed above, administration of the pharmaceutical composition is carried out in any suitable manner, including by intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, transdermal, intranasal, intravaginal, local and oral routes. Can be.
[278] In certain specific embodiments, the immunotherapy may be active immunotherapy wherein the treatment is administered to an endogenous host immune system against the tumor by administering an immune response-modifying agent (eg, polypeptides and polynucleotides as provided herein). It is associated with in vivo stimulation of.
[279] In another specific embodiment, the immunotherapy may be passive immunotherapy, wherein the treatment directly or indirectly mediates anticancer effects and does not establish established tumor-immune responsiveness (eg, effectors) that are not necessarily dependent on an intact host immune system. Cell or antibody). Examples of effector cells include T cells, T lymphocytes (eg, CD8 ⁺ cytotoxic T lymphocytes and CD4 ⁺ T-helper tumor invasive lymphocytes) discussed above expressing a polypeptide provided herein, killer cells (eg, Natural killer cells and lymphokine-activated killer cells), B cells and antigen presenting cells (eg, dendritic cells and macrophages). T cell receptors and antibody receptors specific for the polypeptides cited herein can be cloned, expressed and delivered into other vectors or effector cells for adopted immunotherapy. The polypeptides provided herein can also be used to generate antibodies or anti-idiotype antibodies (as described above and in US Pat. No. 4,918,164) for passive immunotherapy.
[280] Effector cells may generally be obtained in sufficient amounts for immunotherapy adopted by in vitro growth, as described herein. Culture conditions for propagating single antigen specific effector cells into billions for maintenance of antigen recognition in vivo are known in the art. Such in vitro culture conditions typically use intermittent stimulation with antigen, often in the presence of cytokines (eg, IL-12) and non-differentiating feeder cells. As noted above, immunoreactive polypeptides as provided herein can be used to rapidly propagate antigen specific T cell cultures to produce a sufficient number of cells for immunotherapy. In particular, antigen presenting cells such as dendritic cells, macrophages, monocytes, fibroblasts and / or B cells can be pulsed with immunoreactive polypeptides or transfected with one or more polynucleotides using standard techniques known in the art. . For example, antigen presenting cells can be transfected with polynucleotides having promoters suitable for increasing expression in recombinant viruses or other expression systems. Cultured effector cells for use in therapy should be able to grow, distribute widely and survive long term in vivo. Studies have shown that cultured effector cells can be induced to grow in vivo by repeated stimulation with antigens supplemented with IL-12 and can survive in significant numbers for long periods. See, eg, Cheever et al. 157: 177, 1997].
[281] Alternatively, vectors expressing the polypeptides cited herein can be introduced into antigen presenting cells from a patient and cloned in vitro and transplanted back into the same patient. The transfected cells can be reintroduced to the patient using any manner known in the art, preferably by intravenous, intracavity, intraperitoneal or intratumoral administration in sterile form.
[282] Routes and frequencies of administration of therapeutic compositions as described herein, as well as dosages, will vary from individual to individual and can be readily established using standard techniques. In general, pharmaceutical compositions and vaccines may be administered by injection (eg, transdermally, intramuscularly, intravenously or subcutaneously), intranasally (eg, aspiration) or orally. Preferably, from about 1 to about 10 doses may be administered for a 52 week period. Preferably, 6 doses are administered at 1 month intervals and booster vaccination may then be provided periodically. Other protocols may be appropriate for individual patients. Suitable dosages are amounts of compounds which, when administered as described above, can promote an anticancer immune response and are at least 10-50% or more above the basal (ie, untreated) level. The response can be monitored by measuring anticancer antibodies in the patient or by vaccine-dependent production of cytolytic effector cells capable of killing the cancer cells of the patient in vitro. The vaccine also results in an immune response that results in improved clinical outcomes (eg, more frequent alleviation, complete or partial also longer disease-free survival) in vaccinated patients compared to non-vaccinated patients. Generally, in pharmaceutical compositions and vaccines comprising one or more polypeptides, the amount of each polypeptide present in the dosage form ranges from about 25 μg to 5 mg per kg host. Suitable dosage sizes will vary depending on the size of the patient, but typically range from about 0.1 ml to about 5 ml.
[283] In general, suitable dosages and therapeutic regimens provide the active compound (s) in an amount sufficient to provide therapeutic and / or prophylactic benefits. The response can be monitored by establishing improved clinical outcome (eg, more frequent alleviation, complete or partial and also longer disease-free survival) in treated patients compared to untreated patients. Increases in pre-existing immune responses to tumor proteins are generally correlated with improved clinical outcomes. The immune response can generally be assessed using standard proliferation, cytotoxicity or cytokine assays, which can be performed using samples obtained from patients before and after treatment.
[284] Cancer Detection and Diagnostic Compositions, Methods, and Kits
[285] Generally, the cancer is based on the presence of one or more breast cancer proteins in a biological sample (eg, blood, serum, sputum, urine, and / or tumor biopsy) obtained from the patient and / or polynucleotides encoding the protein. Can be detected from. That is, the protein can be used as a marker indicating the presence or absence of cancer such as breast cancer. The protein may also be useful for the detection of other cancers. The binding agents provided herein generally make it possible to detect the level of antigen that binds to the agent in a biological sample. Polynucleotide primers and probes can be used to detect levels of mRNA encoding tumor proteins, which is indicative of the presence or absence of cancer. In general, breast cancer sequences are present at levels three or more times higher in tumor tissue than normal tissue.
[286] There are a variety of assay formats known to those skilled in the art for using binders to detect polypeptide markers in a sample. See, eg, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. In general, the presence or absence of cancer in a patient can include (a) contacting a biological sample obtained from the patient with a binder, (b) detecting the level of polypeptide bound to the binder in the sample, and (c) a predetermined cut-off value. It can be measured by comparing polypeptide levels.
[287] In a preferred specific embodiment, the assay involves the use of a binder bound on a solid support to bind or remove the polypeptide from the rest of the sample. The bound polypeptide can then be detected using a detector that contains a reporter group and that binds specifically to the binder / polypeptide complex. Such detection agents include, for example, binding agents that specifically bind to polypeptides or antibodies or other agents that specifically bind to binding agents such as anti-immunoglobulins, protein G, protein A or lectins. Alternatively, competitive assays can be used, in which the polypeptide is labeled with a reporter group, followed by incubation of the sample and binder followed by binding to the immobilized binder. The extent to which the components of the sample inhibit the binding of the labeled polypeptide to the binder is an indication of the reactivity of the sample with the fixed binder. Suitable polypeptides for use within the assay include the full length breast cancer protein and polypeptide portion thereof to which the binder binds, as described above.
[288] The solid support can be any substance known to those skilled in the art to which the tumor protein can be attached. For example, the solid support may be a test well or nitrocellulose or other suitable membrane on a microtiter plate. Alternatively, the support may be a bead or disk, for example glass, fiberglass, latex, or a plastic material such as polystyrene or polyvinylchloride. The support may also be magnetic particles or a fiber optical sensor such as described, for example, in US Pat. No. 5,359,681. The binder can be immobilized on a solid support using various techniques known in the art, which are fully described in the patent and scientific literature. In the context of the present invention, the term "immobilization" refers to both non-covalent and covalent bonds, such as adsorption, which may be direct bonds between the functional groups on the agent and the support or through crosslinkers. Immobilization to microtiter plates or membranes by adsorption is preferred. In this case, adsorption can be achieved by contacting the binder with the solid support in a suitable buffer for a suitable time. The contact time will vary depending on the temperature, but is typically about 1 hour to about 1 day. Generally, contacting the wells of the plastic microtiter plate with a binder amount of about 10 ng to about 10 μg, and preferably about 100 ng to about 1 μg, is sufficient to fix the appropriate amount of binder.
[289] Covalent attachment of the binder to the solid support can generally be achieved by reacting the support with a bifunctional agent that first reacts with both the support and a functional group such as a hydroxy or amino group on the binder. For example, the binder may be covalently attached to the support with a suitable polymer coating by using benzokinone or by condensing an aldehyde group on the support with an amine and active hydrogen on the binding partner. See, for example, Pierce Immunotechnology Catalog and Handbook, 1991, at A12-A13].
[290] In certain specific embodiments, the assay is two antibody sandwich assays. This assay first contacts an antibody immobilized on a well of a solid support, often a microtiter plate, to allow the polypeptide in the sample to bind to the immobilized antibody. The unbound sample is then removed from the immobilized polypeptide-antibody complex and a detector containing a reporter group (preferably, a secondary antibody capable of binding to different sites of the polypeptide) is added. Thereafter, the amount of detection agent that remains bound to the solid support is detected using a method suitable for the particular reporter group.
[291] More specifically, once the antibody is immobilized on a support as described above, the protein binding site remaining on the support is typically blocked. Any suitable blocking agent known to those skilled in the art is used, such as bovine serum albumin or Tween 20 ^™ (Sigma Chemical Co., St. Louis, Mo.). The immobilized antibody is then incubated with the sample and the polypeptide is allowed to bind to the antibody. Samples may be diluted with a suitable diluent such as phosphate-buffered saline (PBS) prior to incubation. In general, a suitable contact time (ie, incubation time) is a time sufficient to detect the presence of the polypeptide in a sample obtained from an individual with breast cancer. Preferably, the contact time is a time for the level of binder to be at least about 95% achieved between the bound polypeptide and the unbound polypeptide. Those skilled in the art will appreciate that the time required to achieve equilibrium can be readily determined by analyzing the level of binding occurring over a period of time. At room temperature, an incubation time of about 30 minutes is generally sufficient.
[292] Unbound sample can then be removed by washing the solid support with a suitable buffer such as PBS containing 0.1% Tween20 ^™ . Secondary antibodies containing a reporter group can be added to the solid support. Preferred reporter groups include the groups cited above.
[293] The detector is then incubated with the immobilized antibody-polypeptide complex for a time sufficient to detect the bound polypeptide. Suitable amounts of time can generally be determined by analyzing the level of binding that occurs over a time period. The unbound detector is then removed and the bound detector is detected using a reporter group. The method used to detect the reporter group is determined by the characteristics of the reporter group. In the case of radioactive groups, scintillation coefficients or radiographs are generally suitable. Spectrophotometry can be used to detect dyes, luminescent groups and fluorescent groups. Biotin can be detected using avidin bound to different reporter groups (generally radioactive or fluorescent groups or enzymes). Enzyme reporter groups can generally be detected by adding a substrate (usually for a certain time) and then by spectrophotometric or other analysis of the reaction product.
[294] To determine the presence or absence of a cancer, such as breast cancer, a signal detected from a group of reporters bound to and remaining on a solid support is generally compared with a signal corresponding to a predetermined cut-off value. In one preferred specific embodiment, the cut-off value for cancer detection is the average signal obtained when the immobilized antibody is incubated with a sample from a patient who does not have cancer. In general, a sample that generates a signal with three standard deviations above a predetermined cut-off value is considered to be positive for the cancer. In optional preferred specific embodiments, the cut-off value is determined by Sackett et al., Clinical Epidemiology: A Basic Science for Clinical Medicine, Little Brown and Co., 1985, p. 106-7] to determine using the receiver operator curve (ROC). Briefly, in this specific embodiment, the cut-off value is derived from a plot of true positive ratio (ie susceptibility) and false positive ratio (100% -specificity) pairs corresponding to possible cut-off values, respectively, for the diagnostic test results. You can decide. The cut-off value closest to the upper left corner (ie, the value containing the maximum area) is the most accurate cut-off value, and a sample that generates a signal higher than the cut-off value measured in this way is considered positive. Can be. In addition, the cut-off value can be shifted left along the plot to minimize the positive ratio or to the right to minimize the negative ratio. In general, a sample that produces a signal larger than the cut-off value measured in this way is considered positive for the cancer.
[295] In a related specific embodiment, the assay is carried out in a flow-through or strip test format, wherein the binder is immobilized on a membrane such as nitrocellulose. In the flow-through test, the polypeptide in the sample binds to the fixed binder while the sample passes through the membrane. The secondary labeled binder then binds to the binder-polypeptide complex while the solution containing the secondary binder flows through the membrane. Thereafter, detection of bound secondary binder is performed as described above. In the strip test format, one end of the membrane to which the binder is bound is immersed in the solution containing the sample. The sample travels along the membrane through the region containing the secondary binder to the immobilized binder region. The concentration of secondary binder in the immobilized antibody region indicates the presence of cancer. Typically, the concentration of secondary binder at the site produces a line-like pattern that is visually readable. Absence of the pattern indicates negative results. In general, the amount of binder bound on the membrane will provide a visually identifiable pattern if the biological sample contains a level of polypeptide sufficient to generate a positive signal in two antibody sandwich assays, in the format discussed above. Selected to generate. Preferred binding agents for use in the assays are antibodies and antigen-binding fragments thereof. Preferably, the amount of antibody immobilized on the membrane ranges from about 25 ng to about 1 μg, and more preferably from about 50 ng to about 500 ng. The tests can typically be performed using very small amounts of biological samples.
[296] Of course, there are many other assay protocols suitable for use with the tumor proteins or binders of the invention. The above techniques are intended to be illustrative only. For example, it will be apparent to those skilled in the art that the protocols can be readily modified to use a tumor polypeptide to detect antibodies that bind the polypeptide in a biological sample. Detection of such tumor protein specific antibodies may be correlated with the presence of cancer.
[297] Cancer may also or alternatively be detected based on the presence of T cells that specifically react with tumor proteins in a biological sample. In certain methods, a biological sample comprising CD4 ⁺ and / or CD8 ⁺ T cells isolated from a patient is used to express a tumor polypeptide, a polynucleotide encoding said polypeptide and / or an APC expressing at least an immunogenic portion of said polypeptide. Incubated with and detect the presence or absence of specific activation of T cells. Suitable biological samples include, but are not limited to, isolated T cells. For example, T cells can be isolated from patients by conventional techniques (eg Ficoll / Hypaque density gradient centrifugation of peripheral blood lymphocytes). T cells can be incubated with polypeptides in vitro for 2-9 days (typically 4 days) at 37 ° C. It would be desirable to incubate another aliquot of T cell sample in the absence of tumor polypeptide for use as a control. In CD4 ⁺ T cells, activation is preferably detected by assessing the proliferation of T cells. In CD8 ⁺ cells, activation is preferably detected by assessing cytotoxic activity. At least two-fold greater proliferation level and at least 20% greater cytotoxic activity level than in disease-free patients indicate the presence of cancer in the patient.
[298] As noted above, cancer may also or alternatively be detected based on the level of mRNA encoding a tumor protein in a biological sample. For example, two or more oligonucleotide primers can be used in a polymerase chain reaction (PCR) based assay to amplify a portion of tumor cDNA derived from a biological sample, wherein one or more of the oligonucleotide primers encode a tumor protein. Is specific for (ie, hybridizes to) polynucleotides. The amplified cDNA is then separated and detected using techniques known in the art such as gel electrophoresis. Similarly, oligonucleotide probes that specifically hybridize to polynucleotides encoding tumor proteins can be used in hybridization assays to detect the presence of polynucleotides encoding tumor proteins in biological samples.
[299] In order to perform hybridization under assay conditions, the oligonucleotide primers and probes are at least about 60%, preferably at least 10 parts of the polynucleotides encoding tumor proteins of the invention that are at least 10 nucleotides and preferably at least 20 nucleotides in length Comprises at least about 75%, and more preferably at least about 90% identical oligonucleotide sequences. Preferably, the oligonucleotide primers and / or probes hybridize to polynucleotides encoding polypeptides described herein under moderately stringent conditions, as defined above. Oligonucleotide primers and / or probes that may be usefully used in the diagnostic methods described herein are 10 to 40 or more nucleotides in length. In a preferred specific embodiment, the oligonucleotide primers comprise at least 10 contiguous nucleotides, more preferably at least 15 contiguous nucleotides of a DNA molecule having a sequence as described herein. Techniques for both PCR based assays and hybridization assays are known in the art. See, for example, Cold Spring Harbor Symp. Quant. Biol., 51: 263, 1987; Erlich et al., PCR Technology, Stockton Press, NY, 1989].
[300] One preferred assay uses RT-PCR, where PCR is applied with respect to reverse transcription. Typically, RNA is extracted from biological samples such as biopsy tissue and reverse transcribed to produce cDNA molecules. PCR amplification with one or more specific primers produces cDNA molecules, which can be isolated and visualized using, for example, gel electrophoresis. Amplification is performed on biological samples obtained from test patients and individuals who do not have cancer. The amplification reaction can be carried out on several dilutions of doubled cDNA. More than two-fold increase in expression in several dilutions of the test patient sample is typically considered positive compared to the same dilution of the sample that did not develop cancer.
[301] In another specific embodiment, the compositions described herein can be used as markers for cancer progression. In this specific embodiment, assays as described above for the diagnosis of cancer can be performed over time, assessing the level change of reactive polypeptide (s) or polynucleotide (s). For example, assays can be performed every 24 to 72 hours for 6 months to 1 year, after which time as necessary. In general, cancer is ongoing in patients with increasing polypeptide or polynucleotide levels over time. In contrast, cancer is not in progress if the level of reactive polypeptide or polynucleotide remains constant or decreases over time.
[302] Certain in vivo diagnostic assays can be performed directly on tumors. The assay involves contacting tumor cells with a binder. The bound binder can then be detected either directly or indirectly through a reporter group. Such binders may also be used in histological applications. Alternatively, polynucleotide probes can be used in such applications.
[303] As noted above, to improve sensitivity, multiple tumor protein markers can be analyzed in a given sample. Binders specific for the different proteins provided herein can be mixed in a single assay. In addition, multiple primers or probes may be used simultaneously. The selection of tumor protein markers can be based on routine experimentation to determine mixtures that result in optimal sensitivity. In addition, or alternatively, assays for tumor proteins provided herein may be combined with assays for other known tumor antigens.
[304] The invention further provides a kit for use in one of the above diagnostic methods. The kit typically contains two or more components necessary to perform a diagnostic assay. The components may be compounds, reagents, containers and / or devices. For example, one container in a kit may contain a monoclonal antibody or fragment thereof that specifically binds a tumor protein. The antibody or fragment may be provided attached to a support material as described above. One or more additional containers may include elements such as reagents or buffers used in the assay. The kit may also or optionally contain a detection reagent as described above containing a reporter group suitable for direct or indirect detection of antibody binding.
[305] Alternatively, the kit can be designed to detect the level of mRNA encoding tumor protein in a biological sample. The kit generally comprises one or more oligonucleotide probes or primers as described above that hybridize to a polynucleotide encoding a tumor protein. Additional components that may be present in the kit include secondary oligonucleotides and / or diagnostic reagents or containers to facilitate detection of polynucleotides encoding tumor proteins.
[306] The following examples are provided for illustrative purposes and are not intended to be limiting.
[307] Example 1
[308] Preparation of breast cancer specific cDNA using differential display RT-PCR
[309] This example illustrates the preparation of cDNA molecules encoding breast tumor specific polypeptides using differential display screens.
[310] A. Preparation of B18Ag1 cDNA and Characterization of mRNA Expression
[311] Tissue samples are removed from the patient and then prepared from breast cancer and normal tissue of a patient with breast cancer identified by etiology. Normal RNA and tumor RNA were extracted from the sample and mRNA was isolated (dT) converted to cDNA using ₁₂ AG (SEQ ID NO: 130) bound 3 ′ primers. Thereafter, differential display PCR was performed using a randomly selected primer (CTTCAACCTC) (SEQ ID NO: 103). Amplification conditions are standard buffer containing 1.5 mM MgCl ₂ , 20 pmol primer, 500 pmol dNTP, and 1 unit of Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ). 40 cycles of amplification were performed using 94 ° C denaturation 30 seconds, 42 ° C annealing 1 minute, and 72 ° C extension 30 minutes. RNA fingerprints containing 76 amplified products were obtained. The RNA fingerprint of breast cancer tissue was at least 98% identical to that of normal breast tissue, but one band was repeatedly observed to be specific for the RNA fingerprint pattern of the tumor. These bands were cut from silver stained gels and sequenced by subcloning into a T-vector (Novagen, Madison, Wis.).
[312] The sequence of cDNA cited as B18Ag1 is provided in SEQ ID NO: 1. Database searches of GENBANK and EMBL revealed that the first cloned B18Ag1 fragment was 77% identical to the endogenous human retroviral element S71, a truncated retroviral element homologous to the apes sarcoma virus (SSV). S71 contains an incomplete gag gene, part of the pol gene and an LTR-like structure at the 3 'end (Werner et al., Virology 174: 225-238 (1990)). B18Ag1 is also 64% identical to SSV in the region corresponding to the P30 (gag) locus. B18Ag1 contains three individual incomplete reading frames containing regions that share significantly homology with the various gag proteins of retroviruses that infect mammals. In addition, homology to S71 includes thousands of base sequences, including LTRs, but not limited to gag genes.
[313] B18Ag1-specific PCR primers were synthesized using computer analysis instructions. RT-PCR amplification (40 cycles of 30 seconds at 94 ° C., 42 ° C. 30 seconds at 60 ° C., 30 seconds at 72 ° C.) confirmed that B18Ag1 exhibited substantial mRNA sequences present at relatively high levels in the breast cancer tissue of the patient. It was. Primers used for amplification were B18Ag1-1 (CTG CCT GAG CCA CAA ATG) (SEQ ID NO: 128) and B18Ag1-4 (CCG GAG GAG GAA GCT AGA GGA ATA) at pH 8.5 and 3.5 mM magnesium concentrations, And B18Ag1-2 (ATG GCT ATT TTC GGG GCC TGA CA) (SEQ ID NO: 126) and B18Ag1-3 (CCG GTA TCT CCT CGT GGG TAT T) (SEQ ID NO: 127) at pH 9.5 and 2 mM magnesium concentrations. The same experiment showed excessively low to no levels of expression in normal breast tissue of the patient (see FIG. 1). Later, using RT-PCR experiments, B18Ag1 mRNA was present in nine different breast cancer samples (from Brazil and US patients), but absent or excessively low levels in non-mammary tissue corresponding to each cancer patient. Indicated. RT-PCR analysis also indicates that B18Ag1 transcript is absent in various normal tissues (lymph nodes, myocardium and liver) and at relatively low levels in PBMCs and lung tissues. The presence of B18Ag1 mRNA in breast cancer samples, and its absence in normal tissues, was confirmed by Northern blot analysis, as shown in FIG. 2.
[314] Different expressions of B18Ag1 in breast cancer tissues have also been confirmed by RNase protection assays. 3 shows the levels of B18Ag1 mRNA in various tissue types as measured in four different RNase protection assays. Lanes 1 to 12 represent various normal breast tissue samples, lanes 13 to 25 represent various breast cancer samples, lanes 26 and 27 represent normal prostate samples, lanes 28 and 29 represent prostate cancer samples, and lanes 30 to 32 Represents colon cancer tissue, lane 33 represents normal artery, lane 34 represents normal small intestine, lane 35 represents normal skin, lane 36 represents normal lymph node, lane 37 represents normal ovary, and lane 38 is normal Liver, lane 39 represents normal skeletal muscle, lane 40 represents first normal gastric sample, lane 41 represents second normal gastric sample, lane 42 represents normal lung, lane 43 represents normal kidney, Lane 44 represents normal pancreas. Interlaboratory comparisons were facilitated by normalizing different assay results for this positive control, including the positive control RNA of known β-actin message enriched in each assay.
[315] RT-PCR and Southern blot analysis revealed the B18Ag1 locus present in human genomic DNA as an endogenous retroviral element of a single copy. Approximately 12-18 kb of genomic clones were isolated using the first B18Ag1 sequence as a probe. Four additional subclones were separated by XbaI cleavage. Additional retroviral sequences obtained from the ends of the XbaI digests of these clones (located as shown in FIG. 4) are shown in SEQ ID NOs: 3 to 10, wherein SEQ ID NO: 3 is a 10 labeled sequence in FIG. SEQ ID NO: 4 indicates the position of the 11 to 29 labeled sequence, SEQ ID NO: 5 indicates the position of the 3 labeled sequence, SEQ ID NO: 6 indicates the position of the 6 labeled sequence, and SEQ ID NO: 7 12 indicates the position of the labeled sequence, SEQ ID NO 8 indicates the position of 13 labeled sequences, SEQ ID NO 9 indicates the position of the 14 labeled sequences and SEQ ID NO 10 indicates the positions of the 11 to 22 labeled sequences.
[316] Subsequent studies demonstrated that 12-18 kb genomic clones contained about 7.75 kb of retroviral elements, as shown in FIGS. 5A and 5B. The sequence of this retroviral element is shown in SEQ ID NO: 141. Numbered lines at the top of FIG. 5A represent the sense chain sequences of retroviral genomic clones. The box below this line indicates the location of the selected restriction site. Arrows show the different redundancy clones used to sequence retroviral elements. The direction of the arrow indicates whether the single-pass subclonal sequence corresponds to the sense chain or the antisense chain. 5B is a schematic of the retroviral element containing B18Ag1 depicting the organization of the viral gene in the element. The white box corresponds to the predicted reading frame starting from methionine, found through the element. Each of the six similar read frames appear as indicated on the left side of the box, with frames 1 to 3 corresponding to those identified in the sense chain.
[317] Using the cDNA of SEQ ID NO: 1 as a probe, a longer cDNA containing minimum nucleotide difference (less than 1%) is obtained compared to the genomic sequence shown in SEQ ID NO: 141 (SEQ ID NO: 227).
[318] B. Preparation of cDNA Molecules Encoding Different Breast Cancer-Specific Polypeptides
[319] Normal RNA and tumor RNA were prepared and mRNA isolated and then converted to cDNA using (dT) ₁₂ AG attached 3 ′ primers as described above. Thereafter, differential display PCR was performed using randomly selected primers of SEQ ID NOs: 87-125. The amplification conditions are as noted above and the bands observed to be specific for the RNA fingerprint pattern of the tumor were cut from silver stained gels to obtain a T-vector (Novagen, Madison, WI) or a pCRII vector (Invitrogen). , San Diego, CA) and subcloned and sequenced. The sequence is shown in SEQ ID NOs: 11-86. Of the 79 sequences isolated, 67 were identified as novel (SEQ ID NOS: 11-26 and 28-77) (see Figures 6-20).
[320] Extended DNA sequence (SEQ ID NO: 290) for antigen B15Ag1 (first identified partial sequence provided in SEQ ID NO: 27) was obtained in further studies. Comparison of SEQ ID NO: 290 with those in the GenBank as described above confirmed homology with the known human β-A activin gene. Further studies separated the full length cDNA sequence for antigen B21GT2 (also referred to as B311D; originally identified partial cDNA sequence provided in SEQ ID NO: 56). The full length sequence is shown in SEQ ID NO: 307 and the corresponding amino acid sequence is shown in SEQ ID NO: 308. Further studies isolated splice variants of B311D. The B311D clone of SEQ ID NO: 316 was sequenced and XhoI / NotI fragments from this clone were gel purified and randomly primed for use as probes in further screening to label 32P-cDTP to obtain additional B311D gene sequences. Two fractions of the human breast cancer cDNA bacterial library were screened using standard techniques. One of the clones isolated in this manner produced an additional sequence comprising the poly A + tail. The determined cDNA sequence of this clone (quoted as B311D_BT_1A) is shown in SEQ ID NO: 317. The sequences of SEQ ID NOs: 316 and 317 were found to share identity above the 464 bp region, and the sequences diverge to the vicinity of the poly A + sequence of SEQ ID NO: 317.
[321] Subsequent studies identified additional 146 sequences (SEQ ID NOs 142-289), of which 115 were new (SEQ ID NOs 142, 143, 146-152, 154-166, 168-176, 178-192, 194 to 198, 200 to 204, 206, 207, 209 to 214, 216, 218, 219, 221 to 240, 243 to 245, 247, 250, 251, 253, 255, 257 to 266, 268, 269, 271 to 273, 275, 276, 278, 280, 281, 284, 288 and 291). To the extent of the inventors' knowledge, none of the above identified sequences have ever been found to be expressed at higher levels in human breast cancer tissues than normal breast tissues.
[322] In further studies, several different splice forms of antigen B11Ag1 (also referred to as B305D) are isolated, each of the various splice forms comprising slightly different forms of the B11Ag1 coding frame. Splice concatenation sequences define individual exons, which produce a variety of splice forms in various patterns and arrangements. Primers were designed and tested for expression patterns of each of the exons using RT-PCR as described below. Each exon was found to exhibit the same expression pattern as the original B11Ag1 clone and the expression is breast cancer-, normal prostate- and normal testis-specific. The determined cDNA sequences for the isolated protein coding exons are provided in SEQ ID NOs: 292-298, respectively. The foreseen amino acid sequences corresponding to the sequences of SEQ ID NOs: 292 and 298 are shown in SEQ ID NOs: 299 and 300. Further studies using rapid amplification of the cDNA termini (RACE), 5 'specific primers for one of the splice forms of B11Ag1 provided above, and breast adenocarcinoma were identified as isoforms B11C-15, B11C-8 and B11C-9,16. This resulted in the separation of the three additional associated splice forms cited. Scheduled cDNA sequences for such isoforms are provided in SEQ ID NOs: 301-303, and the corresponding predicted amino acid sequences are provided in SEQ ID NOs: 304-306.
[323] The protein coding region of B11C-15 (SEQ ID NO: 301, also cited as B305D isoform C) was used as the query sequence in the BLASTN survey of the Genbank DNA database. It was found to match the genomic clone from chromosome 21 (approval number AP001465). The pairwise arrangement provided in the BLASTN results was used to identify potential exons, ie coding sequences, of chromosome 21 corresponding to B305D sequences. Based on the BLASTN pairwise arrangement, the following fragments of GenBank recording AP001465 are placed together: base pairs 67978-68499, 72870-72987, 73144-73335, 76085-76206, 77905-78085, 80520-80624, 87602-87633. This sequence was deleted using the DNA Star Seqman program in a manner such that it was aligned with the B305D isoform C sequence and the excess sequence retained the sequence most similar to B305D. The final edited form of chromosome 21 sequence showed 96.5% identity with B305D. This obtained edited sequence from chromosome 21 was then deciphered and found to contain no stop codons other than the final stop codon at the same position as for B305D. Like B305D, chromosome 21 sequence (as provided in SEQ ID NO: 325) encodes a protein with 384 amino acids (SEQ ID NO: 326). The arrangement of the B305D isoform C protein (SEQ ID NO: 304) and the protein showed 90% amino acid identity.
[324] Homologs were identified by examining the High Throughput Genome Sequencing (HTGS) database (NCBI, National Institutes for Health, Bethesda, MD) using the cDNA sequence of B305D isoform C (SEQ ID NO: 301). Homologs were identified on chromosome 2 (clone ID 983818), chromosome 10 (clone ID 10933022), and chromosome 15 (clone ID 11560284). This homologue shared at least 90% identity with B305D isoform C at the nucleic acid level. All three of these homologues encode a 384 amino acid ORF that shares at least 90% identity with the amino acid sequence of SEQ ID 304. Further investigation of the GenBank database with the sequence of SEQ ID NO: 301 yielded partial sequence homologues on chromosome 22 (clone ID 5931507). The cDNA sequences of chromosomes 2, 10, 15, and 22 homologues were constructed based on homology with conservative sequences at the B305D isoform C and intron-exon junctions. The cDNA sequences for chromosomes 22, 2, 15 and 10 homologues are shown in SEQ ID NOs: 327 to 330, respectively, and the corresponding amino acid sequences are shown in SEQ ID NOs: 331, 334, 333 and 332, respectively.
[325] In subsequent studies on B305D isoform A (cDNA sequence provided in SEQ ID NO: 292), the cDNA sequence (provided in SEQ ID NO: 313) was found to contain additional guanine residues at position 884, thereby framing in an open reading frame Resulted in migration. The determined DNA sequence of the ORF is provided in SEQ ID NO: 314. This frame shift produces a protein sequence of 293 amino acids (provided in SEQ ID NO: 315) which contains the C-terminal domain common for other isoforms of B305D but differs in the N-terminal region.
[326] Example 2
[327] Preparation of B18Ag1 DNA from Human Genomic DNA
[328] This example illustrates the preparation of B18Ag1 DNA by amplifying human genomic DNA.
[329] B18Ag1 DNA can be prepared from 250 ng of human genomic DNA using the following amplification parameters using 20 pmol of B18Ag1 specific primer, 500 pmol of dNTP, and 1 unit of Taq DNA polymerase (Perkin Elmer, Branchburg, NJ): 94 30 seconds denaturation at &lt; RTI ID = 0.0 &gt; C, &lt; / RTI &gt; The final increase (42 ° C. anneal temperature) is 25 cycles. Primers were selected using computer analysis. The primers synthesized were B18Ag1-1, B18Ag1-2, B18Ag1-3, and B18Ag1-4. Primer pairs that can be used are 1 + 3, 1 + 4, 2 + 3 and 2 + 4.
[330] After gel electrophoresis, the band corresponding to B18Ag1 DNA can be excised and cloned into a suitable vector.
[331] Example 3
[332] Preparation of B18Ag1 DNA from Breast Cancer cDNA
[333] This example illustrates the preparation of B18Ag1 DNA by amplification from human breast cancer cDNA.
[334] The first chain cDNA was 500ng of poly A + RNA in a final volume of 30 μl, 200 pmol of primer (T) ₁₂ AG (ie, TTT TTT TTT TTT AG) (SEQ ID NO: 130), 1 × first chain reverse transcriptase buffer, DTT 6.7 mM , dNTP 500 mmol, and RNA prepared from human breast cancer tissue in a reaction mixture containing 1 unit of AMV or MMLV reverse transcriptase (from all sources, eg Gibco-BRL, Grand Island, NY). After the first chain synthesis, cDNA is diluted about 25-fold and 1 μl is used for amplification as described in Example 2. Certain primer pairs can generate heterologous transcript populations, but primers B18Ag1-2 (5'ATG GCT ATT TTC GGG GGC TGA CA) (SEQ ID NO: 126) and B18Ag1-3 (5'CCG GTA TCT CCT CGT GGG TAT T (SEQ ID NO: 127) yields a single 151 bp amplification product.
[335] Example 4
[336] Identification of B and T Cell Epitopes of B18Ag1
[337] This example illustrates the identification of B18Ag1 epitopes.
[338] The B18Ag1 sequence can be screened using a variety of computer algorithms. In order to determine B cell epitopes, sequences were determined by Hopp, Prog. Clin. Biol. Res. 172B: 367-77 (1985) or by Cease et al., J. Exp. Med. 164: 1779-84 (1986) or Spouge et al., J. Immunol. 138: 204-12 (1987)] can be screened for hydrophobicity and hydrophilicity values. Additional Type II MHC (antibody or B cell) epitopes are described in AMPHI [eg, Margalit et al., J. Immunol. 138: 2213 (1987)] or the same program as Rothbard and Taylor, EMBO J. 7: 93 (1988).
[339] Once peptides (15-20 amino acids in length) have been identified using this technique, individual peptides can be identified using automated peptide synthesis apparatus (e.g., available from manufacturers such as Perkin Elmer / Applied Biosystems Division, Foster City, CA) and Mary. Synthesis can be accomplished using techniques such as field synthesis. After synthesis, peptides can be used to screen serum collected from normal or breast cancer patients to determine if a patient with breast cancer has an antibody that is reactive to the peptide. The presence of the antibody in breast cancer patients will confirm the immunogenicity of the specific B cell epitope of interest. Peptides may also be tested for their ability to produce serological or humoral immunity in animals (mouse, rats, rabbits, chimpanzees, etc.) following immunization in vivo. The generation of peptide specific antiserum following said immunization further confirms the immunogenicity of that specific B cell epipot.
[340] To identify T cell epitopes, the B18Ag1 sequence can be screened using different computer algorithms useful for identifying 8-10 amino acid motifs in the B18Ag1 sequence capable of binding to type I HLA MHC molecules. Rammensee et al., Immunogenetics 41: 178-228 (1995). After synthesis, the peptides were subjected to standard binding assays [eg, Sette et al., J. Immunol. 153: 5586-92 (1994)] can be used to test their ability to bind Type I MHC, and more importantly, for example, in Bakker et al., Cancer Res. 55: 5303-34 (1995); Visseren et al., J. Immunol. 154: 3991-98 (1995); Kawakami et al., J. Immunol. 154: 3961-68 (1995); and Kast et al., J. Immunol. 152: 3904-12 (1994), can be used to test their ability to produce antigen-reactive cytotoxic T cells following in vitro stimulation of a patient or normal peripheral monocyte cells. Successful in vitro production of T cells capable of killing autologous (with the same type I MHC molecule) tumor cells following in vitro peptide stimulation confirms the immunogenicity of the B18Ag1 antigen. In addition, the peptides can be used to generate murine peptides and B18Ag1 reactive cytotoxic T cells following in vivo immunization in transgenic mice for expression of certain human type I MHC haploids [Vitiello et al., J. Exp. . Med. 173: 1007-15 (1991).
[341] A representative list of predicted B18Ag1 B cells and T cell epitopes digested according to the predicted Type I HLA MHC binding antigens is shown below:
[342] Foreseen Th Motif (B Cell Epitope) (SEQ ID NOs: 131-133)
[343] SSGGRTFDDFHRYLLVGI
[344] QGAAQKPINLSKXIEVVQGHDE
[345] SPGVFLEHLQEAYRIYTPFDLSA
[346] Foreseen HLA A2.1 Motif (T Cell Motif) (SEQ ID NOS: 134-140)
[347] YLLVGIQGA
[348] GAAQKPINL
[349] NLSKXIEVV
[350] EVVQGHDES
[351] HLQEAYRIY
[352] NLAFVAQAA
[353] FVAQAAPDS
[354] Example 5
[355] Identification of T Cell Epitopes of B11Ag1
[356] This example illustrates the identification of a B11Ag1 (or cited as B305D) epitope. Four peptides cited as B11-8, B11-1, B11-5 and B11-12 (SEQ ID NOs: 309 to 312, respectively) were derived from the B11Ag1 gene.
[357] Human CD8 T cells were primed in vitro for peptide B11-8 using dendritic cells according to the protocol of Van Tsai et al., Critical Reviews in Immunology 18: 65-75, 1998. The resulting CD8 T cell cultures were tested for their ability to recognize the B11-8 peptide or B-LCL strain, the negative control peptide shown as JY. Briefly, T cells were incubated with autologous monocytes in the presence of 10 μg / ml peptide, 10 ng / ml IL-7 and 10 μg / ml IL-2 and specifically lysed the target cells in a standard 51-Cr release assay. Analyze their ability to As shown in FIG. 22, bulk culturers demonstrated strong recognition of B11-8 peptide but weaker of peptide B11-1.
[358] Clones from these CTL strains were rapidly stretched and isolated using monoclonal antibody PKT3 and human IL-2. As shown in FIG. 23, in addition to being able to recognize specific peptides, these clones (referred to as A1) recognized JY LCL transduced with B11Ag1 gene. These data demonstrate that B11-8 is a naturally processed epitope of the B11Ag1 gene. In addition, these T cells were further recognized to recognize and lyse established tumor cell lines that naturally express B11Ag1 in a HLA-A2 restricted manner (FIG. 24). T cells strongly recognize A2 + breast carcinoma (CAMA-1) transduced with B11Ag1, as well as lung adenocarcinoma (LT-140-22) that naturally expresses B11Ag1 transduced with HLA-A2, but have non-morphology Residual or other negative tumor lines (SW620) are not recognized.
[359] These data clearly demonstrate that such human T cells recognize B11-specific peptides as well as transduced cells and naturally expressing tumor cell lines.
[360] Using the procedure described above, the CTL lines generated for antigens B11-5 and B11-12 were identified to recognize the corresponding peptide coated targets.
[361] Example 6
[362] Characterization of Breast Cancer Genes Identified by Differential Display PCR
[363] The specificity and susceptibility of breast cancer genes found by differential display PCR were measured using RT-PCR. This process allows for rapid evaluation of breast cancer gene mRNA expression semiquantitatively without the use of large amounts of RNA. Using gene specific primers, mRNA expression levels in various tissues were tested and 8 breast cancers, 5 normal breasts, 2 prostate cancers, 2 colon cancers, 1 lung cancer and 14 other normal adult tissues, namely normal Prostate, colon, kidney, liver, lung, ovary, pancreas, skeletal muscle, skin, stomach and testes.
[364] To ensure the semiquantitative properties of RT-PCR, β-actin was used as an internal control of each of the tested tissues. Serial dilutions of the first chain cDNA were prepared and RT-PCR analysis was performed using β-actin specific primers. The dilutions were then chosen that were susceptible enough to allow linear amplification of the β-actin template and reflect the difference in the initial copy number. Under these conditions, β-actin levels were measured for each reverse transcriptional response from each tissue. DNA contamination was minimized by treating with DNase and ensuring negative results when using single-chain cDNA prepared without the addition of reverse transcriptase.
[365] Using gene specific primers, mRNA expression levels were measured in various tissues. To date, 38 genes have been successfully tested by RT-PCR, of which five have shown excellent specificity and sensitivity to breast cancer (B15AG-1, B31GA1b, B38GA2a, B11A1a and B18AG1a). 21A and 21B show the results of three of these genes: B15AG-1 (SEQ ID NO: 27), B31GA1b (SEQ ID NO: 148) and B38GA2a (SEQ ID NO: 157). Table I summarizes the expression levels of all genes tested in normal breast tissue and breast cancer, and other tissues.
[366] Percentage of breast cancer antigens expressed in various tissues Breast tissueOverexpressed in Breast Cancer84% Equally expressed in normal and breast cancer16% Other organizationsOverexpressed in breast cancer but not expressed in all normal tissues9% Overexpressed in breast cancer but also expressed in certain normal tissues30% Overexpressed in breast cancer but equally expressed in all other tissues61%
[367] Example 7
[368] Preparation and Characterization of Antibodies Against Breast Cancer Polypeptides
[369] Polyclonal antibodies against breast cancer antigen B305D were prepared as follows.
[370] this. Breast cancer antigens expressed in E. coli recombinant expression system were grown overnight in LB broth with suitable antibiotics at 37 ° C. in shake incubators. The next morning, 10 ml of the culture was added overnight to a 2 × YT + 500 ml of suitable antibiotic in a 2 L-baffle Erlenmeyer flask. When the optical density (560 nm) of the culture reached 0.4 to 0.6, the cells were induced with IPTG (1 mM). Four hours after induction with IPTG, cells were harvested by centrifugation. The cells were then washed with phosphate buffered saline and centrifuged again. The supernatant was discarded and cells were frozen or immediately processed for future use. 20 ml of lysis buffer was added to the cell pellet and vortexed. Since, this. To disrupt the E. coli cells, the mixture was passed through a French press at a pressure of 16,000 psi. The cells were then centrifuged again and the supernatants and pellets examined by SDS-PAGE for fractionation of recombinant proteins. For proteins local to the cell pellet, the pellet was resuspended in 10 mM Tris pH 8.0, 1% CHAPS and the inclusion body pellets were washed and centrifuged again. The process was repeated two more times. Washed inclusion pellets were dissolved with 8M urea or 6M guanidine HCl containing 10 mM Tris pH 8.0 + 10 mM imidazole. The dissolved protein was added to 5 ml of nickel-chelate resin (Qiagen) and incubated for 45 minutes to 1 hour with constant shaking at room temperature. After incubation, the resin and protein mixture was poured into a disposable column and the flowthrough was collected. The column was then washed with 10-20 column volumes of solubilization buffer. The antigen was then eluted from the column using 8M urea, 10 mM Tris pH 8.0 and 300 mM imidazole and collected in 3 ml fractions. For further purification, the collected fractions were passed through an SDS-PAGE gel.
[371] As a final purification step, a strong anion exchange resin such as HiPrepQ (Biorad) was equilibrated with a suitable buffer and the fractions collected from above were charged to the column. Antigen was eluted from the column with increasing salt gradient. Fractions were collected through a column and fractions collected from the column by another SDS-PAGE gel. The collected fractions were dialyzed against 10 mM Tris pH 8.0. The protein was then dialyzed through a 0.22 micron filter and the antigen was frozen until needed for immunization.
[372] 400 μg of B305D antigen was mixed with 100 μg of muramydipeptide (MDP). Every 4 weeks, rabbits were boosted to 100 μg mixed with the same volume of incomplete Freund's adjuvant (IFA). Seven days after each boost, animals were bled. Blood was incubated at 4 ° C. for 12-24 hours and then centrifuged to generate serum.
[373] 96 well plates were incubated with 50 ml (typically 1 μg) of recombinant protein for 20 hours at 4 ° C. to coat the B305D antigen. 250 ml of BSA blocking buffer was added to the wells and incubated for 2 hours at room temperature. Plates were washed six times with PBS / 0.01% Tween. Rabbit serum was diluted in PBS. 5 ml of diluted serum was added to each well and incubated for 30 minutes at room temperature. The plates were washed as described above and then 50 ml of goat anti-horseradish peroxidase (HRP) was added at 1: 10000 dilution and incubated for 30 minutes at room temperature. The plate was again washed as described above and 100 ml of TMB microwell peroxidase substrate was added to each well. After 15 minutes of incubation in the dark at room temperature, the colorimetric reaction was terminated with 100 mL of 1N H ₂ SO ₄ and immediately read at 450 nm. Polyclonal antibodies showed immunoreactivity against B305D.
[374] Immunohistochemical (IHC) analysis of B305D expression in breast cancer and normal tissue samples was performed as follows. Paraffin-impregnated formal fixed tissue was sliced into 8 micron sections. Steam heat induced epitope recovery (SHIER) in 0.1 M sodium citrate buffer (pH 6.0) was used as optimal staining conditions. Sections were incubated with 10% serum / PBS for 5 minutes. Primary antibody was added to each section at the indicated concentrations for 25 minutes and incubated with anti-rabbit or anti-mouse biotinylated antibody for 25 minutes. Endogenous peroxidase activity was blocked with three 1.5 minute incubations with hydrogen peroxide. An avidin biotin complex / horseradish peroxidase (ABC / HRP) system was used with the DAB chromagen to visualize antigen expression. Slides were largely stained with hematoxylin. B305D expression was detected in both breast cancer and normal breast tissue. However, staining intensity was much lower in normal samples than in breast samples and surface expression of B305D was only observed in breast cancer tissues.
[375] A summary of real-time PCR and immunohistochemical analysis of B305D expression in a wide range of normal tissues is shown in Table II below. These results demonstrate that B305D is minimally expressed in the testis, indeterminate in the gallbladder, and not detected at all in all other tissues tested.
[376] mRNAIHC stainingOrganization typesummary Medium positivePositiveTesticleSmall minority of testicles are nuclear stained; Sperm negative; Siminoma negative NegativeNegativeThymusNo manifestation N / ANegativearteryNo manifestation NegativeNegativeSkeletal muscleNo manifestation NegativePositive (weak dye)IntestineNo manifestation NegativePositive (weak dye)ovaryNo manifestation Negative pituitaryNo manifestation NegativePositive (weak dye)CamouflageNo manifestation NegativeNegativespinal cordNo manifestation NegativeNegativespleenNo manifestation NegativeNegativeUreterNo manifestation N / ANegativeGallbladderNo manifestation N / ANegativeplacentaNo manifestation NegativeNegativethyroidNo manifestation NegativeNegativeHeartNo manifestation NegativeNegativekidneyNo manifestation NegativeNegativeliverNo manifestation NegativeNegativeBrain-cerebellumNo manifestation NegativeNegativecolonNo manifestation NegativeNegativeskinNo manifestation NegativeNegativemarrowNo manifestation N / ANegativeParathyroid glandNo manifestation NegativeNegativelungsNo manifestation NegativeNegativeesophagusNo manifestation NegativePositive (weak dye)WombNo manifestation NegativeNegativesuprarenal bodyNo manifestation NegativeNegativePancreasNo manifestation N / ANegativeLymph nodesNo manifestation NegativeNegativeBrain-cortexNo manifestation N / ANegativeFallopian tubesNo manifestation NegativePositive (weak dye)bladderNo manifestation NegativeN / AboneNo manifestation NegativeN / ASalivary glandsNo manifestation NegativeN / AActivation PBMCNo manifestation NegativeN / APause PBMCNo manifestation NegativeN / AAgencyNo manifestation NegativeN / AVena cavaNo manifestation NegativeN / AretinaNo manifestation NegativeN / AcartilageNo manifestation
[377] Example 8
[378] Protein Expression of Breast Cancer Antigens
[379] In this embodiment, Expression and purification of breast cancer antigen B305D in E. coli and mammalian cells is described.
[380] this. The expression of B305D isoform C-15 (SEQ ID NO: 301; translated into 384 amino acids) in E. coli was found in M. pET17b. This was achieved by cloning the open reading frame of B305D isoform C-15 downstream of the first 30 amino acids of the tuberculosis antigen Ra12 (SEQ ID NO: 318). First, the gene can be cloned into Ra12 at the EcoRI site by mutating the internal EcoRI site in the B305D ORF without changing the protein sequence. PCR primers used for site-directed mutagenesis are shown in SEQ ID NO: 319 (cited by AW012) and SEQ ID NO: 320 (cited by AW013). The ORF of EcoRI site-modified B305D was then amplified by PCR using primers AW014 (SEQ ID NO: 321) and AW015 (SEQ ID NO: 322). PCR products were cleaved with EcoRI and linked into Ra12 / pET17b at the EcoRI site. The sequence of the resulting fusion construct (Ra12mB11C) was confirmed by DNA sequencing. The DNA sequence determined for the fusion construct is provided in SEQ ID NO: 323 and the amino acid sequence is provided in SEQ ID NO: 324.
[381] The fusion construct was BL21 (DE3) CodonPlus-RIL. Transformed into E. coli (Stratagene) and grown overnight in LB with kanamycin. The obtained culture was induced with IPTG. Proteins were transferred to PVDF membranes, blocked with 5% skim milk (in PBS-Twin buffer), washed three times and incubated with mouse anti-His tag antibody (Clontech) for 1 hour. The membrane was washed three times and probed with HRP-protein A (Zymed) for 30 minutes. Finally, the membranes were washed three times and developed with ECL (Amersham). Expression was detected by Western blot.
[382] For recombinant expression in mammals, B305D isoform C-15 (SEQ ID NO: 301; translated into 384 amino acids) was subcloned into mammalian expression vectors pCEP4 and pcDNA31 (Invitrogen). This construct was transfected into HEK293 cells using Fugene 6 reagent (Roche). Briefly, HEK cells were plated and grown overnight at a density of 100,000 cells / ml in DMEM (Gibco) containing 10% FBS (Hyclone). The following day, 2 μl of Fugin 6 reagent was added to 100 μl of FBS without FBS and incubated for 15 minutes at room temperature. The Fusine 6 / DMEM mixture was added to 1 μg of B305D / pCEP4 or B305D / pcDNA plasmid DNA and then incubated for 15 minutes at room temperature. The purine / DNA mixture was then added into HEK293 cells and incubated with 7% CO ₂ at 37 ° C. for 48-72 hours. Cells were washed with PBS, collected by centrifugation and pelleted.
[383] For Western blot analysis, whole cell lysates were generated by incubating the cells in Triton-X100 containing lysis buffer for 30 minutes on ice. The lysate was then clarified by centrifugation at 10,000 rpm for 5 minutes at 4 ° C. Samples were diluted with SDS-PAGE fill buffer containing beta-mercaptoethanol, boiled for 10 minutes and then loaded onto SDS-PAGE gels. Proteins were transferred to nitrocellulose and probed using Protein A purified anti-B305D rabbit polyclonal serum (prepared as described above) at a concentration of 1 μg / ml. The blot was shown with goat anti-rabbit Ig coupled to HRP and incubated on ECL substrate. Expression of B305D was detected in HEK293 lysates transfected with B305D, but not in control HEK293 cells transfected with the vector alone.
[384] For FACS analysis, cells were further washed with ice cold staining buffer and then incubated with 1: 100 dilution of goat anti-rabbit Ig (H + L) -FITC reagent (Southern Biotechnology) for 30 minutes on ice. . After three washes, the cells were resuspended in staining buffer containing propidium iodide (PI), an important dye that allows identification of permeable cells and analyzed by FACS. FACS analysis showed surface expression of B305D protein.
[385] From the foregoing, although specific specific aspects of the invention have been described herein for purposes of illustration, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims.

权利要求:
Claims (17)
[1" claim-type="Currently amended] (a) the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (b) the complement of the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (c) a sequence consisting of 20 or more consecutive residues of the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (d) sequences that hybridize under moderately stringent conditions to the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330; (e) a sequence having at least 75% identity with the sequences provided in SEQ ID NOs: 1, 3-86, 142-298, 301-303, 307, 313, 314, 316, 317, 325, and 327-330; (f) a sequence having at least 90% identity with the sequences provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325, and 327 to 330; And (g) a degenerate variant of the sequence provided in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 Isolated polynucleotides.
[2" claim-type="Currently amended] (a) SEQ ID NOs: 299, 300, 304 to 306, 308 to 312, 314, 326 and 331 to 334; (b) a sequence encoded by the polynucleotide of claim 1; (c) a sequence having at least 70% identity with the sequence encoded by the polynucleotide of claim 1; And (d) an amino acid sequence selected from the group consisting of a sequence having at least 90% identity with a sequence encoded by the polynucleotide of claim 1.
[3" claim-type="Currently amended] An expression vector comprising the polynucleotide of claim 1 operably linked to an expression control sequence.
[4" claim-type="Currently amended] A host cell transformed or transfected with the expression vector according to claim 3.
[5" claim-type="Currently amended] An isolated antibody, or antigen-binding fragment thereof, that specifically binds to the polypeptide of claim 2.
[6" claim-type="Currently amended] (a) obtaining a biological sample from the patient; (b) contacting the biological sample with a binder that binds to the polypeptide of claim 2; (c) detecting the amount of polypeptide binding to the binder in the sample; And (d) comparing the amount of polypeptide to a predetermined cut-off value to determine the presence of cancer in the patient.
[7" claim-type="Currently amended] A fusion protein comprising at least one polypeptide according to claim 2.
[8" claim-type="Currently amended] Oligonucleotides that hybridize with the sequences shown in SEQ ID NOs: 1, 3 to 86, 142 to 298, 301 to 303, 307, 313, 314, 316, 317, 325 and 327 to 330 under moderately stringent conditions.
[9" claim-type="Currently amended] Under conditions and for a time sufficient to allow stimulation and / or proliferation of the T cells, the T cells are treated with (a) the polypeptide according to claim 2; (b) a polynucleotide according to claim 1; And (c) contacting at least one component selected from the group consisting of antigen presenting cells expressing a polypeptide according to claim 2, thereby stimulating and / or proliferating T cells specific for the tumor protein.
[10" claim-type="Currently amended] An isolated T cell population comprising T cells prepared according to the method of claim 9.
[11" claim-type="Currently amended] A first component selected from the group consisting of a physiologically acceptable carrier and an immunostimulant, and (a) the polypeptide according to claim 2; (b) a polynucleotide according to claim 1; (c) an antibody according to claim 5; (d) a fusion protein according to claim 7; (e) the T cell population according to claim 10; And (f) a second component selected from the group consisting of antigen presenting cells expressing the polypeptide according to claim 2.
[12" claim-type="Currently amended] A method of stimulating an immune response in a patient, comprising administering the composition of claim 11 to the patient.
[13" claim-type="Currently amended] A method of treating cancer in a patient, comprising administering the composition of claim 11 to the patient.
[14" claim-type="Currently amended] (a) obtaining a biological sample from the patient; (b) contacting the biological sample with an oligonucleotide according to claim 8; (c) detecting the amount of polynucleotide hybridizing with the oligonucleotide in the sample; And (d) measuring the presence of cancer in the patient by comparing the amount of polynucleotide hybridizing to the oligonucleotide to a predetermined cut-off value.
[15" claim-type="Currently amended] A diagnostic kit comprising at least one oligonucleotide according to claim 8.
[16" claim-type="Currently amended] A diagnostic kit comprising a detection reagent comprising at least one antibody according to claim 5 and a reporter group.
[17" claim-type="Currently amended] (a) CD4 + and / or CD8 + cells isolated from a patient, comprising: (i) the polypeptide according to claim 2; (ii) a polynucleotide according to claim 1; And (iii) incubating with at least one component selected from the group consisting of antigen presenting cells expressing the polypeptide of claim 2 to propagate T cells; And (b) administering to the patient an effective amount of proliferated T cells to inhibit the progression of the cancer in the patient.

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EP1988097A1|2008-11-05|Compositions and methods for the therapy and diagnosis of prostate cancer

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US6630574B1|2003-10-07|Compositions and methods for the therapy and diagnosis of lung cancer

---

JP2002532093A|2002-10-02|Compositions and methods for treatment and diagnosis of ovarian cancer

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US20040101899A1|2004-05-27|Compositions and methods for the therapy and diagnosis of breast cancer

同族专利:

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公开号 | 公开日

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AU6489301A|2001-12-03|

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WO2001090152A3|2002-06-20|

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JP2004508812A|2004-03-25|

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NO20025594L|2003-01-21|

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EP1696028A3|2006-12-27|

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US20020068285A1|2002-06-06|

---

NO20025594D0|2002-11-21|

---

IL152847D0|2003-06-24|

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EP1696028A2|2006-08-30|

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EP1283885A2|2003-02-19|

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CN1443239A|2003-09-17|

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CA2409299A1|2001-11-29|

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CZ20024213A3|2003-09-17|

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HU0302566A2|2003-10-28|

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WO2001090152A2|2001-11-29|

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BR0111024A|2004-06-22|

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MXPA02011563A|2003-06-06|

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PL366066A1|2005-01-24|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2000-05-24|Priority to US57750500A

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2000-05-24|Priority to US09/577,505

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2000-06-08|Priority to US59058300A

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2000-06-08|Priority to US09/590,583

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2000-10-26|Priority to US09/699,295

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2000-10-26|Priority to US09/699,295

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2001-03-16|Priority to US09/810,936

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2001-03-16|Priority to US09/810,936

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2001-05-22|Application filed by 코릭사 코포레이션

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2003-04-08|Publication of KR20030028474A

优先权:

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申请号 | 申请日 | 专利标题

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US57750500A| true| 2000-05-24|2000-05-24|

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US09/577,505|2000-05-24|

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US59058300A| true| 2000-06-08|2000-06-08|

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US09/590,583|2000-06-08|

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US09/699,295|US6828431B1|1999-04-09|2000-10-26|Compositions and methods for the therapy and diagnosis of breast cancer|

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US09/699,295|2000-10-26|

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US09/810,936|US20020068285A1|1996-01-11|2001-03-16|Compositions and methods for the therapy and diagnosis of breast cancer|

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US09/810,936|2001-03-16|