Angiopoietin-2 specific binding agents

专利摘要:
The present invention relates to specific binding agents such as fully human antibodies that bind to angiopoietin-2. The invention also relates to heavy chain fragments, light chain fragments and CDRs of the antibody as well as methods of making and using such antibodies.
公开号:KR20040051606A
申请号:KR10-2004-7005381
申请日:2002-10-11
公开日:2004-06-18
发明作者:올라이너조나단다니엘
申请人:암젠 인코포레이티드；
IPC主号:

专利说明:

ANGIOPOIETIN-2 SPECIFIC BINDING AGENTS}
[1] This application claims the advantages of US Provisional Serial No. 60 / 328,624, filed October 11, 2001, which is incorporated herein by reference.
[3] Angiogenesis, in which new blood vessels form from existing vessels, is essential for many physiological and pathological processes. In general, angiogenesis is tightly regulated by pro- and anti-angiogenic factors only in the case of cancers, diseases of the angiogenesis of the eye, arthritis and psoriasis, the process may fail. Folkman, J., Nat. See Med., 1: 27-31 (1995).
[4] It is known that many diseases are associated with uncontrolled or undesirable angiogenesis. Such diseases include, but are not limited to: ocular neovascularization, such as retinopathy (including diabetic retinopathy); Age-related macular degeneration; Psoriasis; Hemangioblastoma; Hemangioma; Arteriosclerosis; Inflammatory diseases such as rheumatoid or rheumatic inflammatory diseases (particularly arthritis including rheumatoid arthritis) or other chronic inflammatory disorders such as chronic asthma; Atherosclerosis or atherosclerosis after transplantation; Endometriosis; And neoplastic diseases (eg, leukemias and lymphomas) such as solid tumors and liquid or hematopoietic tumors. Other diseases associated with undesirable angiogenesis will be apparent to those skilled in the art.
[5] Although most signaling systems are involved in angiogenesis regulation, they exhibit optimal properties and one of the most endothelial-selective systems is also called Tie-2 receptor tyrosine kinase ("Tie-2" or "Tie-2R"), Also referred to as "ORK"; mouse Tie-2 also named "tek") and its ligand, angiopoietin (Gale, NW and Yancopoulos, GD, Genes Dev. 13: 1055-1066 (1999). Four known angiopoietins are angiopoietin-1 ("Ang-1") to angiopoietin-4 ("Ang-4"). Such angiopoietins are also termed "Tie-2 ligands" (Davis, S., et al. Cell, 87: 1161-1169 [1996]; Cytogenet Cell Genet, Grosios, K., et al. 84: 118- 120 [1999]; Investigative Ophthalmology & Visual Science, 42: 1617-1625 [1999]; Current Biology, Koblizek, TI, et al. 8: 529-532 [1998]; Lin, P., et al. Proc Natl Acad Sci USA, 95: 8829-8834 [1998], Maisonpierre, PC, et al. Science, 277: 55-60 [1997]; Papapetropoulos, A., et al. Lab Invest, 79: 213-223 [ 1999; Sato, TN, et al. Nature, 375: 70-74 [1998], Shyu, KG, et al. Circulation, 98: 2081-2087 [1998]; Suri, C., et al., Cell, 87: 1171- 1180 [1996]; Suri, C., et al. Science, 282: 468-471 [1998]; Valenzuela, DM, et al. Proceedings of the National Academy of Sciences of the USA, 96: 1904-1909 [1999]; Witzenbichler , B., et al., J Biol Chem, 273: 18514-18521 [1998]). While binding of Tie-2 and Ang-1 stimulates receptor phosphorylation in cultured endothelial cells, Ang-2 has been observed to both agonize and agonize Tie-2 receptor phosphorylation (Davis , S., et al. [1996], supra; Maisonpierre, PC, et al. [1997], supra; Kim, I., JH Kim, et al. Oncogene 19 (39): 4549-4552 (2000); Teichert Cardiovascular Research 49 (3): 659-70 (2001) by Kuliszewska, K., PC Maisonpierre, et al.
[6] The phenotypes of mouse Tie-2 and Ang-1 knockout are similar, and Ang-1-stimulated Tie-2 phosphorylation is responsible for reconstructing angiogenesis in the uterus through maintenance of endothelial cell-support cell adhesion. Mediate formation and stabilization (Dumont, DJ, et al., Genes & Development, 8: 1897-1909 [1994]; Sato, TN, et al. Nature, 376: 70-74 [1995]; Suri, C. et al. , Et al. [1996], supra. The role of Ang-1 in vascular stabilization is believed to be in maintaining Ang-1 expression broadly and structurally in adults (Hanahan, D., Science, 277: 48-50 [1997]; Zagzag, D., et al. Experimental Neurology, 159: 391-400 [1999]. In contrast, Ang-2 expression, when it is believed that Ang-2 blocks Ang-1 function, primarily limits the site of vascular remodeling and thereby causes vascular plasticity that promotes angiogenesis. Induce states (Hanahan, D., [1997], supra; Holash, J., et al., Science, 284: 1994-1998 [1999]; Maisonpierre, PC, et al. [1997], supra).
[7] Numerous published studies have demonstrated blood vessel-selective Ang-2 expression in disease states associated with angiogenesis. Such pathological symptoms include, for example, psoriasis, macular degeneration and cancer (American Journal of Pathology, Bunn, G., et al., 155: 1967-1976 [1999]; Cancer Research, et al., Etoh, T., et al. 61: 2145-2153 [2001]; Hangai, M., et al. Investigative Ophthalmology & Visual Science, 42: 1617-1625 [2001], Holash, J., et al. [1999], supra; Kuroda, K., et al. Journal of Investigative Dermatology, 116: 713-720 [2001]; Otani, A., et al. Investigative Ophthalmology & Visual Science, 40: 1912-1920 [1999]; Stratmann, A., et al. American Journal of Pathology, 153 : 1459-1466 [1998]; Tanaka, S., et al. J Clin Invest, 103: 34-345 [1999]; Yoshida, Y., et al., International Journal of Oncology, 15: 1221-1225 [1999]; Yuan , K., et al., Journal of Periodontal Research, 35: 165-171 [2000]; Zagzag, D., et al. (1999), supra). Most of these studies focus on cancer, among which various types of tumors show Ang-2 expression in blood vessels. In contrast to Ang-2 expression in pathological angiogenesis, Ang-2 expression in normal tissues is extremely limited (Maisonpierre, PC, et al. [1997], supra; Mezquita, J., et al. Biophysical Research Communications, 260: 492-498 [1999]. In normal adults, the three main sites of angiogenesis are the ovary, placenta and uterus; This site detects Ang-2 mRNA as primary tissue in normal (ie, non-cancerous) tissue.
[8] Certain functional studies suggest that Ang-2 may be involved in tumor angiogenesis. Ahmad et al. (Cancer Res., 61: 1255-1259 [2001]) describe Ang-2 over-expression and this Ang-2 over-expression is associated with increased tumor growth in xenograft models of mice. It is shown. See also Etoh et al. And Tanaka et al., And the data in these documents indicate that Ang-2 overexpression is associated with tumor vascular hyperplasia. In contrast, however, Yu et al. (Am. J. Path., 158: 563-570 [2001]) found that transfectants were identified by Ang-2 overexpression in Lewis lung carcinoma and TA3 breast carcinoma cells. Data is shown indicating prolonged survival in injected mice.
[9] Over the years, various publications have suggested Ang-1, Ang-2 and / or Tie-2 as possible targets for anticancer treatment. For example, US Pat. Nos. 6,166,185, 5,650,490, and 5,814,464 each describe the concept of anti-Tie-2 ligand antibodies and receptor bodies. Lin et al. (Proc. Natl. Acad. Sci USA, 95: 8829-8834 [1998]) disclose that mice are injected with adenoviruses expressing soluble Tie-2; This soluble Tie-2 has been described to reduce the number and size of tumors seen in mice. In a related study, Lin et al. (J. Clin. Invest., 100: 2072-2078 [1997]) describe the injection of soluble forms of Tie-2 into rats; Compounds are described to reduce tumor size in rats. Siemeister et al. (Cancer Res., 59: 3185-3189 [1999]) form human melanoma cell lines expressing the extracellular domain of Tie-2, which are injected into nude mice to inject soluble Tie-2. Has been shown to exhibit “significant inhibition” of tumor growth and tumor angiogenesis. In view of this fact, it is certain that both Ang-1 and Ang-2 bind to Tie-2, but whether Ang-1, Ang-2 or Tie-2 is a definite target for anti-cancer treatment from these studies. Is not obvious.
[10] Fusion of certain peptides to stable plasma proteins such as Ig constant regions to enhance the half-life of these molecules is described, for example, in PCT Publication No. WO 00/24782 (published May 4, 2000). .
[11] Fusion of proteins or fragments thereof into stable plasma proteins such as Ig constant regions to enhance the half-life of such molecules is described in the following literature (see, eg, US Pat. No. 5,480,981; Zheng et al. Immunol., 154: 5590-5600, (1995); Fisher et al., N. Engl. J. Med., 334: 1697-1702, (1996); Van Zee, K. et al. J. Immunol., 156: 2221. -2230, (1996); U.S. Patent No. 5,808,029, issued September 15, 1998; Nature of Capon et al., 337: 525-531 (1989); Immunotech., 1: 95-105, ( 1995); WO 97/23614, published July 3, 1997; PCT / US97 / 23183, filed December 11, 1997; Linsley, J. Exp. Med., 174: 561-569. (1991), see WO 95/21258, published August 10, 1995).
[12] Effective anti-Ang-2 treatment is effective in a large population of cancer patients. This is because most solid tumors require neovascularization, which grows beyond the size of 1-2 millimeters in diameter. The treatment is broadly applied, for example, in diseases related to retinopathy, arthritis and psoriasis as well as other angiogenesis.
[13] There is a need to demonstrate new agents that specifically recognize and bind Ang-2. Such agents are useful for diagnostic screening and therapeutic intervention in disease states associated with Ang-2 activity.
[14] It is therefore an object of the present invention to provide specific binders of Ang-2 that modulate Ang-2 activity.
[15] Summary of the Invention
[16] The present invention provides an antibody comprising a heavy chain and a light chain, the heavy chain comprising a variable region of a heavy chain selected from the following:
[17]
[18] The light chain comprises the variable region of the light chain selected from:
[19]
[20] The present invention also provides a specific binder comprising at least one peptide selected from the following and fragments thereof:
[21]
[22] It will be appreciated that the specific binding agent can be, for example, an antibody such as polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody or fully human antibody. The antibody will also be a single chain antibody. The invention also relates to hybridomas producing the monoclonal antibodies of the invention.
[23] It will also be appreciated that the present invention relates to the conjugates described herein. The conjugate can be, for example, a specific binding agent (such as an antibody) of the invention.
[24] The invention also relates to a nucleic acid molecule encoding a specific binding agent (antibody) of the invention, a vector comprising said nucleic acid molecule as well as a host cell comprising said vector.
[25] The present invention also provides a method of preparing a specific binder comprising the following steps:
[26] (a) transforming a host cell with at least one nucleic acid molecule encoding the specific binding agent of claim 1;
[27] (b) expressing a nucleic acid molecule in the host cell; And
[28] (c) separating the specific binder. The invention also provides a method of making an antibody comprising the following steps:
[29] (a) transforming a host cell with at least one nucleic acid molecule encoding an antibody of the invention;
[30] (b) expressing a nucleic acid molecule in the host cell; And
[31] (c) separating the specific binder.
[32] The invention also relates to a method of inhibiting undesirable angiogenesis in a mammal by administering a therapeutically effective amount of a specific binding agent of the invention. The invention also provides a method of treating cancer in a mammal by administering a therapeutically effective amount of a specific binding agent of the invention.
[33] The invention also relates to a method of inhibiting undesirable angiogenesis in a mammal by administering a therapeutically effective amount of an antibody of the invention. Additionally the present invention provides a method for treating cancer in a mammal comprising administering a therapeutically effective amount of an antibody of the invention.
[34] The invention also relates to a pharmaceutical composition comprising the specific binding agent of the invention and a pharmaceutically acceptable formulation agent. The pharmaceutical composition will comprise the antibody of the invention and a pharmaceutically acceptable agent.
[35] The present invention provides a method of modulating or inhibiting angiopoietin-2 activity by administering one or more specific binding agents of the present invention. The invention also provides a method of modulating or inhibiting angiopoietin-2 activity by administering an antibody of the invention.
[36] The invention also relates to a method of controlling at least one vascular permeability or plasma leakage in a mammal by administering a therapeutically effective amount of a specific binding agent of the invention. The present invention also administers a therapeutically effective amount of the specific binding agent of the present invention in mammals with the following diseases: ocular neovascular disease, obesity, hemangioblastoma, hemangioma, atherosclerosis, inflammatory disease, inflammatory disorders, atherosclerosis, A method of treating at least one of endometriosis, neoplastic disease, bone-related diseases or psoriasis.
[37] The invention also provides a method of controlling at least one vascular permeability or plasma leakage in a mammal by administering a therapeutically effective amount of an antibody of the invention. The present invention also administers a therapeutically effective amount of the antibody of the present invention in mammals with ocular neovascular disease, obesity, hemangioblastoma, hemangioma, atherosclerosis, inflammatory diseases, inflammatory disorders, atherosclerosis, uterus A method of treating at least one of endometriosis, neoplastic disease, bone-related disease or psoriasis.
[38] Moreover, the present invention relates to a method for treating cancer in a mammal by administering a therapeutically effective amount of a specific binding agent of the invention and a chemotherapeutic agent. It will be appreciated by those skilled in the art that there is no need for simultaneous administration of the specific binder and chemotherapeutic agent.
[39] The invention also relates to a method for treating cancer in a mammal by administering a therapeutically effective amount of an antibody of the invention and a chemotherapeutic agent. Specific binders and chemotherapeutic agents do not need to be administered simultaneously.
[40] The present invention also provides specific binding agents comprising Complementarity determining region 1 (CDR 1) of one of the following:
[41]
[42] The invention also relates to specific binding agents comprising complementary determinant 2 (CDR 2) of one of the following:
[43]
[44] The invention also relates to specific binding agents comprising complementary determinant 3 (CDR 3) of one of the following:
[45]
[46] The present invention also provides nucleic acid molecules encoding specific binders of the invention.
[47] Moreover, the present invention provides angiopoietin-2 in the biological sample by (a) contacting the biological sample with the specific binder of the present invention and (b) measuring the binding degree of the specific binder to the sample. It relates to a method for measuring the concentration of. In addition, the present invention provides a concentration of angiopoietin-2 in the biological sample by (a) contacting the antibody of the present invention with a biological sample and (b) measuring the degree of binding of the antibody to the sample. It relates to how to measure.
[48] The invention also relates to a method of inhibiting undesirable angiogenesis in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. The invention also relates to a method of modulating angiogenesis in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. The invention also relates to a method of inhibiting tumor growth characterized by undesirable angiogenesis in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition as described herein. Additionally, the present invention relates to a method of treating cancer in a mammal comprising administering a therapeutically effective amount of a polypeptide or composition and a chemotherapeutic agent as described herein. In a preferred embodiment, the chemotherapeutic agent is at least one of 5-FU, CPT-11 and Taxotere. However, other suitable chemotherapeutic agents and other cancer therapies may also be used.
[49] Specific binding agents of the invention can be used to treat a number of diseases associated with unregulated or undesirable angiogenesis. These diseases include angiogenesis, psoriasis, hemangioblastoma, hemangiomas, arteriosclerosis, rheumatoid or rheumatoid inflammatory diseases of the eye, such as retinopathy (including diabetic retinopathy and age-related macular degeneration) and arthritis including rheumatoid arthritis. Same inflammatory diseases or chronic asthma, other chronic inflammatory diseases such as arterial or post-trans atherosclerosis, endometriosis, and neoplastic diseases such as, for example, solid tumors and liquid tumors (e.g., leukemia) It is not limited to this. Additional diseases that can be treated by administering specific binding agents will also be apparent to those skilled in the art. Such additional diseases include, but are not limited to, bone-related disorders including obesity, vascular permeability, plasma leakage and osteoporosis. Accordingly, the present invention also relates to methods of treating such diseases associated with uncontrolled or undesirable angiogenesis.
[50] Other embodiments of the invention will become apparent from the detailed description herein.
[2] The present invention relates to specific binders that recognize and bind to angiopoietin-2 (hereinafter referred to as Ang-2). In particular, the present invention relates to the production, diagnostic and therapeutic uses of monoclonal and polyclonal antibodies and fragments thereof that specifically bind to Ang-2.
[51] 1 shows tumor size (y-axis) versus time (x-) in tumorigenic mice treated with an anti-Ang-2 antibody (clone 533, 537 or 544), control antibody or phosphate buffered saline (PBS) of the present invention. Axis). Examples are shown in more detail.
[52] 2A, 2B and 2C show the control peptibody, Tie2-Fc, C2B8 or 5B12 as well as the present invention, which binds to the full-length human Ang-2 (hAng-2), the N-terminus and C-terminus of hAng-2 Epitope mapping data (OD 370) for peptibody TN8-Con4-C, L1-7-N and 12-9-3-C are shown. Examples are shown in more detail.
[53] The headings in the paragraphs are used herein for structural purposes only and are not intended to limit the subject matter described.
[54] Standard techniques can be used to produce recombinant DNA molecules, proteins and antibodies as well as tissue culture and cell transformation. Enzymatic reactions and purification techniques are typically performed according to the manufacturer's instructions or as described herein by Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY [1989]) or as described herein. It may generally be carried out in the art using conventional methods. Unless a specific definition is required, the terms and laboratory methods and techniques used in connection with analytical chemistry, synthetic organic chemistry, medicinal chemistry and chemistry are well known in the art and commonly used. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and transport, and treatment of patients.
[55] Justice
[56] The following terms, as used herein, are understood by the following definitions, except where indicated otherwise.
[57] The term "Ang-2" refers to polypeptides described in Figure 6 of US Pat. No. 6,166,185 ("Tie-2 ligand-2") or fragments thereof as well as allelic variants, splice variants, derivatives, substitutions, deletions and / or Insert variants, fusion peptides and polypeptides, and related polypeptides, including interspecies homologs. Ang-2 polypeptides may or may not include additional terminal residues, such as, for example, leader sequences, target sequences, amino terminal methionine, amino terminal methionine and lysine residues and / or markers or fusion protein sequences, depending on the method of preparation. have.
[58] The term “biologically active” when used in connection with an Ang-2 or Ang-2 specific binder, refers to a peptide or polypeptide having at least one activity characteristic of an Ang-2 or Ang-2 specific binder. Specific binding agents of Ang-2 have an agonist, antagonist or neutralizing or blocking activity against at least one biological activity of Ang-2.
[59] The term “specific binder” denotes a molecule, preferably a proteinaceous molecule, that binds to Ang-2 (and variants and derivatives thereof as described herein) having affinity better than other angiopoietins. Specific binding agents are preferably proteins, peptides, nucleic acids, carbohydrates, lipids or low molecular weight compounds which bind to Ang-2. In a preferred embodiment, the specific binding agents of the present invention are polyclonal antibodies, monoclonal antibodies (mAb), chimeric antibodies, CDR-graft antibodies, multi-specific antibodies, bispecific antibodies, catalytic antibodies, humanized antibodies, Antibodies such as human antibodies, anti-genotype (anti-Id) antibodies and antibodies that can be labeled in water-soluble or bound forms, as well as fragments, variants or derivatives thereof, which are known by known techniques to It can be used in combination. Such techniques include, but are not limited to, enzymatic cleavage, chemical cleavage, peptide synthesis or recombinant techniques. Anti-Ang-2 specific binding agents of the invention can bind to portions of Ang-2 that modulate (eg, inhibit or promote) Ang-2 and / or other Ang-2-associated activity.
[60] The term "polyclonal antibody" refers to a heterologous mixed antibody that recognizes and binds different epitopes on the same antigen surface. Polyclonal antibodies can be obtained from crude serum preparations or can be purified using, for example, antigen affinity chromatography or A protein / G protein affinity chromatography.
[61] The term "monoclonal antibody" refers to a collection of antibodies encoded by the same nucleic acid molecule, optionally produced by a single hybridoma or other cell line, or transgenic animal, wherein each monoclonal antibody is Typically the same epitope on the antigen surface will be recognized. The term "monoclonal" is not limited to any particular method of making an antibody, nor is it limited to antibodies produced from specific species, such as mice, rats, and the like.
[62] The term “chimeric antibody” refers to an antibody that has a portion of a heavy and / or light chain that is identical or homologous to the sequence in an antibody belonging to a class or subclass of a particular antibody or derived from a particular species, whereas The remainder of the light chain is identical or homologous to the sequence in antibodies belonging to the class or subclass of other antibodies or derived from other species. Also included are fragments of the antibody that exhibit the desired biological activity (ie, the ability to specifically bind Ang-2). See US Pat. No. 4,816,567 and Morrison et al. Proc Natl Acad Sci (USA), 81: 6851-6855 [1985].
[63] The term "CDR graft antibody" refers to an antibody that has been recombinantly inserted into the outline of another antibody of the same or different species or isotype from a particular antibody of a particular species or isotype.
[64] The term "multi-specific antibody" refers to an antibody having a variable region that recognizes one or more epitopes on one or more antigen surfaces. A subclass of this type of antibody means "bi-specific antibody", which recognizes two different epitopes on the same or different antigen surface.
[65] A “catalyst” antibody refers to an antibody that binds a component having one or more cytotoxic or, more generally, one or more biological activities to a targeting binder.
[66] The term “humanized antibody” indicates that the antibody outline region is derived from the human body but each CDR is a specific type of CDR-grafted antibody substituted with a CDR derived from another species, such as a mouse CDR. The term "CDR" is defined below.
[67] The term “complete human” antibody refers to an antibody in which both the CDRs and the outer structure are derived from one or more human DNA molecules.
[68] The term “anti-genotype” antibody refers to any antibody that specifically binds to another antibody that recognizes the antigen. Anti-genotypic antibodies are, for example, Ang-2-specific, except for antibodies that arise by immunizing an animal with an Ang-2-specific antibody or Ang-2 binding fragment thereof rather than the Ang-2 polypeptide itself or a fragment thereof. It can be produced by the methods described herein for the production of antibodies.
[69] As used herein, the term “variant” includes polypeptides in which amino acid residues are inserted, deleted and / or substituted into the naturally occurring (or at least known) amino acid sequence of a binder. Variants of the invention include fusion proteins as described below.
[70] “Derivatives” include binders that have been chemically modified in some way other than insertional, deletional or substitutional variants.
[71] “Specifically binds to Ang-2” refers to a specific binding agent of the invention (eg, an antibody or fragment thereof) that recognizes and binds to a mature full-length or part-length human Ang-2 polypeptide or ortholog thereof. ) Or its affinity (eg, measured by an affinity ELISA or Biacore assay as described herein) or neutralizing ability (eg, by a neutralizing ELISA assay or similar assay as described herein). Measurement) is at least 10 times, up to 50 times, up to 100, 250 or 500 times or even up to at least 1000 times the affinity or neutralization capacity of another angiopoietin or other peptide or polypeptide.
[72] The term “antigen binding domain” or “antigen binding region” refers to a specific binder (eg, an antibody molecule) that contains a specific binder amino acid residue (or other component) that is a binder that interacts with the antigen and has specificity and affinity for the antigen. It means part of. In antibodies, antigen-binding domains are generally referred to as "complementarity-determinants or CDRs".
[73] The term “epitope” refers to the portion of any molecule that can be recognized and bound to a specific binding agent, such as an antibody, in one or more antigen binding regions of the binding agent. Epitopes generally consist of chemically active surface groups of molecules such as, for example, amino acids or carbohydrate side chains, and have specific charge properties as well as specific third-order structural properties. As used herein, epitopes may or may not be contiguous. In addition, the epitope contains the same tertiary structure as the epitope used to generate the antibody, but would similarly contain little or no amino acid residues found in Ang-2 used to stimulate the antibody immune response.
[74] The term “inhibitory and / or neutralizing epitope”, when combined with a specific binding agent such as an antibody, results in the loss (or at least a decrease) in vivo, ex vivo or in situ of the biological activity of a molecule, cell or organism comprising such an epitope. Epitopes are shown. In the literature of the present invention, neutralizing epitopes are located or bound on the biologically active region of Ang-2. Optionally, the term "active epitope" refers to Ang-2 when bound by a specific binding agent of the present invention, such as an antibody. Epitopes that activate or at least maintain a biologically active conformation of a.
[75] The term “antibody fragment” refers to a peptide or polypeptide comprising a more intact antibody. A complete antibody comprises two functionally independent parts or fragments: an antigen binding fragment known as "Fab" and a carboxy terminal crystallizable fragment known as "Fc". Fab fragments comprise the first constant region from both heavy and light chains (CH1 and CL1) with variable regions from both heavy and light chains that bind to specific antigens. Each heavy and light chain variable region comprises three complementarity determining regions (CDRs) and an outline amino acid residue separated from each CDR. The Fc region includes the second and third heavy chain constant regions (CH2 and CH3) and includes effector functions such as complement activity and attack by phagocytes. In some antibodies, the Fc and Fab regions are separated by antibody “hinge regions” and the hinge region will bind the Fab or Fc fragment depending on how proteolytically cleaved the full length of the antibody is. For example, the resulting Fc fragment and hinge region bind due to cleavage of the antibody by protease papain, but the fragment is provided by cleavage by protease papain, which hinge region also binds to both Fab fragments at the same time. In fact, since the two Fab fragments are covalently linked after papain cleavage, the resulting fragment is named F (ab ') 2 fragment.
[76] While the Fab has a short serum half life, the Fc domain will have a relatively long serum half life. See Nature, 337: 525-31 (1989) by Capon et al. When expressed as part of a fusion protein, the Fc domain may provide a long half-life or provide functions such as Fc receptor binding, A protein binding, complement binding and even transplacental delivery to the protein to be fused. The Fc region may be a naturally occurring Fc region or may be modified to enhance certain properties such as therapeutic properties or cycle time.
[77] The term “variable region” or “variable domain” refers to the portion of the light and / or heavy chain of an antibody that generally comprises approximately 120 to 130 amino acids in the heavy chain and about 100 to 110 amino-terminal amino acids in the light chain. it means. Variable regions generally differ in amino acid sequence among antibodies of the same species. The variable region of an antibody generally determines the binding and specificity of each specific antibody for a particular antigen. Variability in the sequence is concentrated in this region, referred to as complementarity-determinants (CDRs), and the more highly conserved regions in the variable domains are termed the border regions (FR). The CDRs of the light and heavy chains comprise amino acids that are highly reactive to the direct interaction of the antigen with the antibody, but the amino acids of the FRs can have a significant effect on the antigen binding / recognition described below.
[78] The term "light chain" used in reference to an antibody is collectively referred to as two distinct types, termed kappa (κ) or lambda (λ), based on the amino acid sequences of the constant domains.
[79] The term "heavy chain" as used in connection with an antibody is referred to collectively as five distinct types, named alpha, delta, epsilon, gamma and mu, based on the amino acids of the heavy chain constant domains. Combinations of light and heavy chains are given in five known antibody classes: IgA, IgD, IgE, each comprising four known subclasses of known IgG named IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ IgG and IgM.
[80] The term "naturally occurring", when used in association with a biological material such as a nucleic acid molecule, polypeptide, host cell, or the like, indicates that it is not found in nature or modified by humans.
[81] The term “isolated” when used in connection with Ang-2 or a specific binding agent of Ang-2, refers to a compound isolated from at least one hybrid polypeptide or compound found in the natural environment, preferably for therapeutic or diagnostic use. Compounds that are substantially isolated from other hybrid polypeptides of mammals in conflict.
[82] The term “mature” when used in connection with an Ang-2, anti-Ang-2 antibody or other protein specific specific binding agent of Ang-2, refers to a peptide or polypeptide lacking a leader or signal sequence. For example, when a binder of the present invention is expressed in prokaryotic host cells, a "mature" peptide or polypeptide lacks additional amino acid residues (leading sequences, such as amino terminal methionine or one or more methionine and lysine residues). ) May be included. Peptides or polypeptides prepared in this form can be used with or without additional amino acid residues removed.
[83] The terms "effective amount" and "therapeutically effective amount", when used in connection with a specific binding agent of Ang-2, are amounts of specific binding agents that are useful or necessary for significantly changing the degree of one or more biological activities of Ang-2. Indicates. The degree of Ang-2 activity may be increased or decreased. Preferably, Ang-2 activity is reduced.
[84] Specific Binders and Antibodies
[85] As used herein, the term “specific binder” refers to a molecule that specifically recognizes and binds Ang-2 as described herein. Suitable specific binding agents include but are not limited to antibodies and derivatives, polypeptides and small molecules thereof. Suitable specific binders were prepared by methods known in the art. Exemplary Ang-2 polypeptide specific binding agents of the present invention may bind to specific portions of the Ang-2 polypeptide, and may preferably modulate the activity or function of the Ang-2 polypeptide.
[86] Specific binding agents such as antibodies and antibody fragments that specifically bind Ang-2 polypeptides are within the scope of the present invention. Antibodies include monoclonal polyclonal, monoclonal (mAbs), recombinant, chimeric, CDR-grafted humans, such as human, single chain, catalytic, multispecific and / or bispecific, as well as fragments thereof. , Variants and / or derivatives.
[87] Generally, polyclonal antibodies directed to Ang-2 polypeptides are those wherein the Ang-2 polypeptide or fragment thereof is present in an animal (eg, rabbit, hamster, goat, sheep, horse, pig, rat, ger) with or without an adjuvant. To bil, guinea pig, mouse or a suitable mammal as well as other non-mammalian species). The adjuvant includes, but is not limited to: Freund's complete and incomplete adjuvant, inorganic gels such as ammonium hydroxide and lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyholes Surface-active substrates such as limpet hemocyanin and dinitrophenol. BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are potentially useful human adjuvants. This is useful for conjugating antigen polypeptides to carrier proteins such as keyhole limpet hemocyanin, serum, albumin, bovine tyroglobulin or soy trypsin inhibitors that confer immunogenicity to the immunized species. In addition, flocculants such as alum were used to enhance the immune response. After immunization, blood of the animals was drawn and the serum was assayed for anti-Ang-2 polypeptide antibody titers that can be measured by the assays described herein below in "Examples". Polyclonal antibodies were purified from serum using polyclonal antibodies in serum from which polyclonal antibodies are detected, or using, for example, antigen affinity chromatography or A protein or G protein affinity chromatography.
[88] Monoclonal antibodies directed against Ang-2 polypeptides are produced, for example, but not limited to, using traditional "hybridoma" methods or newer "phage display" techniques. For example, hybridoma methods as described in Nature 256: 495 (1975) to Kohler et al., Which are incorporated herein by reference; Immunol Today 4: 72 (1983) by Kosbor et al., Proc Natl Acad Sci (US) 80: 2026-2030 (1983), incorporated herein by reference, from Marcel Decker Inc., New York, USA. Obtained Monoclonal Antibody Production Techniques and Applications, pp. 51-63, (1987) human B-cell hybridoma technology; And EBV-Hybridoma technology described by Monoclonal Antibodies and Cancer Therapy of Cole et al., Obtained from Alan Alice Inc., New York, USA. The present invention also provides hybridoma cell lines that produce monoclonal antibodies that react with Ang-2 polypeptides.
[89] If hybridoma technology is used, myeloma cell lines can be used. Preferably, the cell line suitable for use in the hybridoma-producing fusion process produces non-antibodies with high fusion efficiency and is capable of growing the cell line in a specific medium that only grows the desired fusion cells (hybridoma). Have enzyme deficiency. For example, cell lines used for mouse fusion include Sp-20, P3-X63 / Ag8, P3-X63-Ag8.653, NS1 / 1.Ag4 1, Sp210-Ag14, FO, NSO / U, MPC-11 , MPC11-X45-GTG1.7 and S194 / 5XXO Bul; Cell lines used for rat fusion include R210.RCY3, Y3-Ag1.2.3, IR983F and 4B210. Other cell lines useful for cell fusion include U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6. Hybridomas and other cell lines producing monoclonal antibodies are expected to be novel compositions of the invention.
[90] Phage display technology was also used to generate monoclonal antibodies from certain species. Preferably, the technique was used to produce fully human monoclonal antibodies that are polynucleotides encoding a single Fab or Fv antibody fragment expressed on the surface of phage particles. J Mol Biol 227: 381 (1991) by Hoogenboom et al., Incorporated herein by reference; J Mol Biol 222: 581 (1991) by Marks et al .; Or US Pat. No. 5,885,793. Each phage can be “screened” using the binding assays described herein to identify antibody fragments having affinity for Ang-2. Therefore, the process is similar to immune screening through the display of antibody fragment repertory on the surface of fibrous bacteriophages. This procedure is described in PCT Application No. PCT / US98 / 17364, filed by Applicant Adams et al., Which describes the approach of high-affinity and functional functional antibody fragments for MPL- and msk-receptors with this approach. Describes separation. In this approach, a complete repertoire of the human antibody gene was described previously (see Mullinax et al. Proc Natl Acad Sci (US) 87: 8095-8099 (1990), incorporated herein by reference). Can be made by cloning human genes rearranged naturally.
[91] Once the polypeptide sequences encoding the respective chains of the full-length monoclonal antibodies or Fab or Fv fragments (class) of the invention are identified, then host cells such as eukaryotic and prokaryotic cells are routinely tested and well known in the art. Was used to express monoclonal antibody polypeptide using recombinant techniques. Optionally, a polynucleotide encoding a preferred specific binding agent by a method of expressing a polynucleotide molecule encoding a monoclonal antibody or other specific binding agent in the transformation of an animal, for example, mouse, rabbit, goat or cow Inserted into the genome of the recipient animal. In one aspect, polynucleotides encoding monoclonal antibodies or other specific binding agents can be linked to breast-specific regulatory sequences, and chimeric polynucleotides can be inserted into the germline of the target animal. The resulting transgenic animal then produces milk containing the desired antibody [J Immunol Meth 231: 147-157 (1999); See Immunol Today 8: 364-370 (2000) by Little et al.]. Moreover, plants are used to express and produce Ang-2 specific binding agents, such as monoclonal antibodies, by transfecting into a suitable plant a polynucleotide encoding a monoclonal antibody or other specific binding agent.
[92] In another embodiment of the present invention, monoclonal antibodies, polyclonal antibodies or fragments thereof derived from species other than human can be "humanized" or "chimerized". Methods of humanizing non-human antibodies are well known in the art (see US Pat. Nos. 5,859,205, 5,585,089 and 5,693,762, which are incorporated herein by reference). Humanization, for example, the methods described in this field [Nature 321: 522-525 (1986) to Jones et al., Incorporated herein by reference; Riechmann et al. Nature, 332: 323-327 (1988); Science 239: 1534-1536 (1988) by Verhoeyen et al., For example, to at least a portion of a complementarity-determining region (CDRs) of rodents, for a corresponding region of human antibodies. It was performed by replacing. The present invention also provides variants and derivatives of the human antibodies as are well known in the art and described herein.
[93] In addition, the present invention includes fully human antibodies that bind Ang-2 polypeptides, as well as fragments, variants, and / or derivatives thereof. Such antibodies can be produced using the phage display technology described above. Alternatively, a transgenic animal (eg, a mouse) can be used to produce the human antibody repertoire when no endogenous immunoglobulin is produced in producing the antibody. This can be achieved by immunizing an animal with an Ang-2 antibody or fragment thereof in which the Ang-2 fragment has a unique amino acid sequence for Ang-2. See, eg, Proc Natl Acad Sci (US), 90: 2551-2555 (1993) by Jakobovits et al .; Nature 362: 255-258 (1993) by Jakobovits et al .; See Bruggermann et al. Year in Immuno, 7:33 (1993). In certain methods, the transgenic animal can be transformed by disabling endogenous loci encoding the heavy and light chains of immunoglobulins and inserting the loci encoding human heavy and light chain proteins into its genome. It was then hybridized with partially modified animals with fewer than the full complement of those modifications to obtain animals with all of the desired immune system modifications. When administered with an immunogen, the transgenic animal can produce an antibody having a human variable region comprising a human (eg, rather than a mouse) amino acid sequence that is immuno-specific to the desired antigen. See PCT Application Nos. PCT / US96 / 05928 and PCT / US93 / 06926, which are incorporated herein by reference. Human antibodies can also be produced by expressing recombinant DNA in hybridoma cells or in host cells as described herein.
[94] It can be transformed in many different ways. See, eg, Immunol Today 17: 391-7 (1996), Bruggeman et al., Incorporated herein by reference. In a particular approach, minigene loci are constructed so that the gene segments of the germline conformation are artificially densified from each other. Due to size limitations (ie, typically 30 kb or less), the resulting minigenic locus may contain a limited number of different gene segments, but can still produce large repertoires of antibodies. Minigenic loci containing only human DNA sequences, including promoters and enhancers, are sufficiently functional in transgenic mice.
[95] When a large number of gene segments were required in transgenic animals, yeast artificial chromosomes (YACs) were used. YACs range from hundreds of kb to 1 Mb and can be inserted into the mouse (or other suitable animal) genome via direct microinjection into the egg or by introducing YAC into an embryonic stem-ES line. . In general, YACs can be inserted into ES cells by lipofection of purified DNA or by yeast spheroplasm fusion, where the purified DNA is transported into micelles and the fusion is fused in a similar manner to the hybridoma fusion protocol. . DNA insertion following selection of the preferred ES cells can be achieved by including selectable markers known in the art on YAC.
[96] As another alternative, a bacteriophage P1 vector amplified in bacterial E. coli hosts was used. Generally, the vector carries less inserted DNA than YAC and easily grows clones with a yield high enough to microinject directly into mouse eggs. The use of reaction mixtures of different P1 vectors appears to cause high levels of homologous recombination.
[97] Once suitable transgenic mice (or other suitable animals) are identified using techniques known in the art for detecting serum levels of circulating antibodies (e.g., ELISA), the transgenic animals are then disrupted with endogenous Ig loci. Hybridized with mice. The result is a progeny, wherein essentially all B cells express human antibodies.
[98] As another alternative, the entire animal Ig locus was replaced with the human Ig locus and the resulting animals expressed only human antibodies. In another approach, a portion of an animal's locus was replaced with a region specific and corresponding to the human locus. In certain cases, the resulting animal expresses chimeric antibodies according to the substitution characteristics of the mouse Ig locus as opposed to fully human antibodies.
[99] It can also be produced by exposing human splenocytes (B cells or T cells) to an antigen ex vivo and then reconstituting the cells exposed in immunocompromised mice such as, for example, SCID or nod / SCID. J Immunol, 160: 2051-2058 [1998] to Brams et al., Incorporated herein by reference; See Nat Med, 6: 103-106 [2000] by Carballido et al. Alternatively, transplantation of human fetal tissue into SCID mice (SCID-hu) has resulted in the development of organ hematopoiesis and human T-cells [McCune et al. Science 241: 1532-1639 (1988); See Sem Immunol 8: 243-248 (1996) by Ifversen et al.]. The humoral immune response in the chimeric mice is entirely dependent on the co-development of animal T-cells (see Immunol 83: 1271-179 (1994), Martensson et al., Incorporated herein by reference). As an alternative, human peripheral blood lymphocytes were implanted intraperitoneally (or alternatively) in SCID mice (see Nature 335: 256-259 (1988) by Mosier et al., Incorporated herein by reference). Transplanted cells were first antigen-stimulated or anti-human CD40 monocles such as Staphylococcal Enterotoxin A (SEA) (see Immunol 84: 224-230 (1995) by Martensson et al., Incorporated herein by reference). High levels of B-cells were produced when treated with ronal antibodies (see Blood 86: 1946-1953 (1995) to Murphy et al., Incorporated herein by reference).
[100] Alternatively, a fully synthetic human heavy chain repertoire can be created from unrearranged V gene segments by constructing each human VH segment with a random nucleotide D segment along with a human J segment [Hoogenboom, herein incorporated by reference]. Et al., J Mol Biol 227: 381-388 (1992). The light chain repertoire is also constructed by combining the respective V and J fragments (see EMBO J 13: 3245-3260 (1994) by Griffiths et al., Which is incorporated herein by reference). The nucleotide encoding the complete antibody (ie both heavy and light chains) was linked with a single chain Fv fragment and the polynucleotide was conjugated to a nucleotide encoding the fibrous phage minor envelope protein. When expressing the fusion protein on the surface of the phage, polynucleotides encoding specific antibodies were identified by selection using immobilized antigen.
[101] In another approach, antibody fragments are constructed from two Fab fragments, one of which is fused to phage protein and the rest secreted into the bacterial periplasm [Nucl Acids Res 19 by Hoogenboom, incorporated herein by reference]. 4133-4137 [1991]; See Barbas et al., Proc Natl Acad Sci (US) 88: 7978-7982 (1991).
[102] Large-scale production of chimeric, humanized, CDR-grafts and fully human antibodies or fragments thereof generally consists of recombinant methods. Polynucleotide molecules (classes) encoding the heavy and light chains of each antibody or fragment thereof were inserted into and expressed in a host cell using the materials and procedures described herein. In a preferred embodiment, the antibodies are produced in mammalian host cells such as CHO cells. Details of the production are described below.
[103] Fusion Partners of Specific Binders
[104] In a further embodiment of the invention, the amino acid sequence of the variable domain of an Ang-2 antibody, such as a heavy chain variable region having an amino acid sequence as described herein or a light chain variable region having an amino acid sequence as described herein, is provided. The comprising polypeptides were fused to one or more domains of the Fc region of human IgG at the N-terminus or C-terminus. When the Fc domain is constructed with therapeutic proteins, such as Fabs of Ang-2-specific antibodies, they provide longer half-lives or provide functions such as Fc receptor binding, protein A binding, complement binding and even transplacental delivery. [See Nature 337: 525-531 (1989) by Capon et al., Incorporated herein by reference].
[105] In Examples, the antibody hinges, CH2 and CH3 regions, are specific binding polypeptides such as anti-Ang-2 Fab or Fv fragments (eg, obtained from phage display libraries) in a manner known to those skilled in the art. Fused at the N-terminus or C-terminus of. The resulting fusion protein was purified using A protein or G protein affinity column. Peptides and proteins fused to the Fc region have been found to exhibit substantially greater half-life than their counterparts that are not fused in vivo. Fusion to the Fc region also contemplates dimerization / multimerization of the fusion polypeptide. The Fc region may be a naturally occurring Fc region or may be altered to enhance certain properties such as therapeutic properties, cycle time, reduced aggregation, and the like. In other embodiments known in the art, Fc regions of human or other species or synthesized Fc regions are fused to the N-terminus of CD30L to treat Hodgkin's disease, degenerative lymphoma and T-cell leukemia (US Patent No. 5,480,981), the Fc region is fused to a TNF receptor to treat pulmonary pneumoniae (see Fisher et al., N Engl J Med, 334: 1697-1702 (1996), incorporated herein by reference); and The Fc region is fused to the Cd4 receptor to treat AIDS (see Nature, 337: 525-31 (1989) by Capon et al., Incorporated herein by reference).
[106] Catalytic antibodies are another form of fusion molecule and include antibodies that bind one or more cytotoxic or, more generally, one or more biologically active components to a specific binder. See, for example, Ram et al. Chem Eur J 12: 2091-2095 (2000), incorporated herein by reference. Cytotoxic agents of this type improve antibody-mediated cytotoxicity and include, directly or indirectly, cytokines, radioisotopes, chemotherapy drugs (including drug precursors), bacterial toxins For example, Pseudomonas exotoxin, diphtheria toxin, etc.), plant toxin (e.g., lysine, gelonin, etc.), chemical binders (e.g., maytansinoid toxins, Carrechaemycin, etc.), Radioconjugates and enzyme conjugates (RNase conjugates, antibody-directed enzyme / prodrug therapy (ADEPT)). In one aspect, the cytotoxic agent may be "bound" to a component of a bispecific or multispecific antibody by binding the agent to one of the selective antigen recognition sites of the antibody. Alternatively, the cytotoxin protein can be expressed by conjugating a polynucleotide encoding a toxin to a polynucleotide encoding a binder, followed by fusing the fusion protein with a specific binder. As another alternative, the specific binding agent may be covalently modified to include the desired cytotoxin.
[107] Examples of such fusion proteins include immunogenic polypeptides, proteins with long circulating half-lives such as immunoglobulin constant regions, marker proteins, proteins or polypeptides that facilitate the purification of preferred specific binder polypeptides, and multimeric proteins (eg, dimers). There are polypeptides that promote the formation of leucine zipper motifs useful for body formation / stability.
[108] Insertional variants of this type generally have a natural molecule or substantial portion thereof that binds the N- or C-terminus to the secondary polypeptide or portion thereof. For example, fusion proteins typically use a leader sequence of a different species origin to recombinantly express the protein in a heterologous host. Other useful fusion proteins include adding immunologically active domains such as antibody epitopes to facilitate purification of the fusion protein. Inclusion of the cleavage site at or near the fusion junction promotes removal of the exogenous polypeptide after purification. Other useful fusions include binding of the active site, glycosylation domain, cell target signal or transmembrane region with the functional domain of the enzyme.
[109] Various commercially available fusion protein expression systems can be used in the present invention. Particularly useful systems are the glutathione-S-transferase (GST) system (available from Pharmacia), the maltose binding protein system (available from ENBI, Burberry, Mass.), And the plaque system (FLAG system, Connecticut New, USA). And 6xHis systems (obtained from Qiagen, Chatsworth, California). Such a system allows the production of recombinant peptibodies on a few additional amino acids that do not affect the antigenic capacity of the recombinant polypeptide. For example, the plaque system and the 6xHis system add only short sequences and are known to be incomplete antigenic and have no adverse effect on the folding of the native conformational polypeptide. Another N-terminal fusion that is considered useful is the fusion of Met-Lys dipeptide in the N-terminal region of a protein or peptide. Such fusions can effectively increase protein expression or activity.
[110] Particularly useful fusion constructs are, for example, specific binder peptides fused with hapten to enhance the immunogenicity of specific binder fusion constructs useful for the production of anti-genic antibodies of the invention. Fusion constructs to enhance immunogenicity are known to those of skill in the art and include, for example, an auxiliary antigen such as hsp70, a peptide sequence such as diphtheria toxin chain, or a fusion specific binder having a cytokine such as IL-2. It is useful for causing an immune response. In other embodiments, fusion constructs can be prepared to enhance targeting of antigen binding compositions that bind to specific sites or cells.
[111] Other fusion constructs including heterologous polypeptides with desirable properties for targeting (eg, Ig constant regions or antibodies or fragments thereof to enhance serum half-life) are also included. Polypeptide hybrids can be prepared with other fusion systems where it is desired to remove the fusion partner from the desired polypeptide. In one embodiment, the fusion partner is linked to a recombinant specific binder polypeptide with a peptide sequence comprising a specific recognition sequence for the protease. Examples of suitable sequences include those recognized for tobacco etch virus protease (obtained from Life Technologies, Gettysburg, Maryland) or factor Xa (obtained from New England Biolabs, Burberry, Mass.).
[112] The present invention relates to a heavy chain variable region having an amino acid sequence as described herein, in combination with a truncated tissue factor (tTF), which is a vascular targeting agent consisting of a truncated form of human coagulation-inducing protein that acts as a tumor vascular coagulant or Provided is a fusion polypeptide comprising all or a portion of a variable domain of an Ang-2 antibody, such as a light chain variable region having an amino acid sequence as described herein. Fusion of an anti-Ang-2 antibody or fragment thereof with tTF can facilitate the transport of anti-Ang-2 to target cells.
[113] Variants of Specific Binders
[114] Variants of specific binding agents of the present invention include insertional, deletional and / or substitutional variants. In one aspect of the invention, insertion variants are provided wherein one or more amino acid residues complement a specific binder amino acid sequence. The insert may be located at one or both of the protein termini or in an internal region of the specific binder amino acid sequence. Insertion variants having additional residues at one or both ends include, for example, fusion proteins and proteins comprising amino acid labels or labels. Insertional variants include specific binder polypeptides in which one or more amino acid residues are added to a specific binder amino acid sequence or fragment thereof.
[115] Variation products of the present invention also include mature specific binder products. Such specific binder products are those in which the leader or signal sequences have been removed, but the final protein has additional amino terminal residues compared to the wild type Ang-2 polypeptide. Additional amino terminal residues may comprise one or more residues derived from other proteins or not identical to those derived from a particular protein. Methionine residues and lysine residues comprising a specific binder product having an additional methionine residue at position-1 (Met- ¹ -specific binder) and additional to positions -2 and -1 (Met- ² -Lys ^-1 -specific binder) Specific binder products having are also included. Variants of specific binders with additional Met, Met-Lys, Lys residues (or generic one or more basic residues) are particularly effective for enhancing recombinant protein production in bacterial host cells.
[116] The present invention also encompasses specific binder variants having additional amino acid residues that occur with specific expression systems. For example, a commercially available vector that expresses the desired polypeptide as part of a glutathione-S-transferase (GST) fusion product is used to generate the desired polypeptide having an additional glycine residue at amino acid position-1 and then the desired polypeptide. The GST component was removed at. Also included are variants expressed with other vector systems, which variants generally comprise a poly-histidine marker mixed into an amino acid sequence at the carboxy and / or amino terminus of the sequence.
[117] Insertion variants also include fusion proteins as described above, wherein the amino and / or carboxy terminus of a specific binder polypeptide is fused to an amino acid sequence that is not recognized as part of another polypeptide, fragment thereof, or generally a specific protein sequence.
[118] In another aspect, the invention provides deletion variants in which one or more of the amino acid residues of a specific binder polypeptide are removed. The deletion may be formed by removing at one or both ends of the specific binder polypeptide or by removing one or more of the residues in the specific binder amino acid sequence. Deletion variants necessarily include all fragments of a specific binder polypeptide.
[119] Antibody fragments include those portions of an antibody that bind to an epitope on an antigen polypeptide. Examples of such fragments include, for example, Fab and F (ab ') ₂ produced by enzymatic or chemical cleavage of full length antibodies. Other binding fragments include those generated by recombinant DNA techniques such as recombinant plasmid expression comprising nucleic acid sequences encoding antibody variable regions. The invention also encompasses polypeptide fragments of Ang-2 binding agents having the ability to specifically bind Ang-2 polypeptides. Also included herein are fragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 or more consecutive amino acids of a peptide or polypeptide of the invention. Preferred polypeptide fragments exhibit unique or specific immunological properties for the antigen-binding agents of the invention. Fragments of the invention having desirable immunological properties are well known in the art and can be prepared by methods generally performed.
[120] In another aspect, the invention provides substitutional variants of specific binding agents of the invention. Substitution variants are generally considered to be "similar" to the original polypeptide or have a specific "identity rate" for the original polypeptide, and include polypeptides in which one or more of the amino acid residues of a polypeptide has been removed and replaced with a replacement residue. In one aspect, substitutions are conservative in nature, but the invention also encompasses non-conservative substitutions.
[121] Identity and similarity of related polypeptides can be readily determined by known methods. Such methods include Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press (1987); Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York (1991); And methods described in SIAM J. Applied Math., 48: 1073 (1988) to Carillo et al.
[122] Preferred methods for determining the relevance or identity of two polypeptides were designed such that the sequences tested matched many more. Methods of determining identity are described as publicly available computer programs. Preferred computer program methods for determining identity between two sequences are described in the GCG program package, including GAP, in Nucl. Acid. Res., 12: 387 (1984); Genetics Computer Group, obtained from University of Wisconsin, Madison, Wisconsin, USA], BLASTP, BLASTN and FASTA [J. Mol. Biol., 215: 403-410 (1990)]. The BLASTX program includes National Center for Biotechnology Information (NCBI) and other sources [NCB / NLM / NIH Bethesda, MD 20894 by BLAST Manual, Altschul et al .; See above (1990) by Altschul et al., Publicly available. The well known Smith Waterman algorithm can also be used to determine identity.
[123] Specific alignment schemes for aligning two amino acid sequences can only match short regions of the two sequences, and even though there is no significant relationship between the two sequences in the full length, these small regions have very high sequence identity. Thus, in certain embodiments, at least 10% of the full length target polypeptides compared to the selected alignment method (GAP program), ie, at least 40 consecutive amino acids of at least 400 amino acid sequences compared, at least 300 to about 400 amino acids compared Alignments spanning 30 consecutive amino acids in the sequence, at least 20 consecutive amino acids in the compared 200 to about 300 amino acid sequences and at least 10 amino acids in the compared about 100 to 200 amino acid sequences.
[124] For example, using the computer algorithm GAP (Genetics Computer Group, obtained from the University of Wisconsin, Madison, Wisconsin, USA), two sequences for determining sequence identity are aligned to optimally match relative amino acids (as determined by the algorithm). Same as "matched length"). In certain embodiments, the gap opening penalty (typically calculated as 3X average diagonals; as well as control matrices such as PAM 250 or BLOSUM 62; “average diagonals” is the average of the diagonals of the control matrix used; “diagonal” Face "is a score or number defined for each perfect amino acid match by a particular control matrix) and gap elongation penalty (generally 1/10 times the gap opening penalty) can be used in the algorithm. In certain embodiments, a standard control matrix [Athoff of Protein Sequence and Structure, 5 (3) (1978) by Dayhoff et al. On PAM control matrix; Henikoff et al. Proc. Natl. Acad. Sci USA, 89: 10915-10919 (1992), may also be used in the algorithm.
[125] In certain embodiments, the parameters for polypeptide sequence comparison include:
[126] Algorithm: J. Mol. Biol., 48: 443-453 (1970);
[127] Control matrix: BLOSUM 62, supra from Henikoff et al. (1992);
[128] Gap Penalty: 12
[129] Gap Length Penalty: 4
[130] Threshold of similarity: 0.
[131] It is effective to perform the GAP program with the above parameters. In certain embodiments, the above mentioned parameters are default parameters for polypeptide comparison (no penalty for terminal gap) using the GAP algorithm.
[132] In certain embodiments, the parameters for polynucleotide molecular sequence comparisons include:
[133] Algorithm: See Needleman et al., Supra (1970);
[134] Contrast matrix: correspondence = +10, noncorrespondence = 0
[135] Gap Penalty: 50
[136] Gap Length Penalty: 3.
[137] The GAP program is also effective to carry out with the above parameters. The aforementioned parameters are abbreviated parameters for polynucleotide molecule comparisons.
[138] Other typical algorithms, gap opening penalty, gap stretching penalty, control matrix, threshold of similarity, etc. can be used, including those described in Program Maunal, Wisconsin Package, Version 9, September, 1997. Certain choices for implementing the program will be apparent to those skilled in the art, depending on the specific comparisons to be performed such as DNA-to-DNA, protein-to-protein, protein-to-DNA; And additionally, such a comparison can be a comparison between a given pair of sequences (when GAP or BestFit is generally preferred) or a large database of sequences (when FASTA or BLASTA is preferred).
[139] Typical twenty amino acids and abbreviations thereof as used herein follow general use. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991), incorporated herein by reference for specific purposes.
[140] Amino acids have L or D stereochemistry (exceptionally, Gly has neither L nor D) The polypeptides and compositions of the present invention include combinations of stereochemistry. However, L stereochemistry is preferred. The present invention also provides reverse molecules in which the amino termini are opposite to the carboxy terminal sequence of the amino acid. For example, the inverse of a molecule having the normal sequence X ₁ -X ₂ -X ₃ is X ₃ -X ₂ -X ₁ . The present invention also provides retro-reverse molecules in which the amino terminal is opposite to the carboxy terminal sequence of amino acids and residues, which are generally “L” enantiomers, are replaced with “D” stereoisomeric forms.
[141] Stereoisomers of conventional 20 amino acids and non-natural amino acids such as α-, α-unsubstituted amino acids, N-alkyl amino acids, lactic acid and other unusual amino acids (e.g. D-amino acids) Suitable components for the polypeptide. Uncommon amino acids include but are not limited to: aminoadipic acid, beta-alanine, beta-aminopropionic acid, aminobutyric acid, piperidine acid, aminocaprioic acid, aminoheptanoic acid, aminoisobutyric acid, aminopimelic acid , Diaminobutyric acid, desmocin, diaminopimelic acid, diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, hydroxyproline, isodesmocin, allo-isoleucine , N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, norleucine, orithine, 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and others With similar amino acids Amino acids (eg, 4-hydroxyproline).
[142] Similarly, except for the other cases specified, the left end of the sequence of single-stranded polynucleotides is the 5 'end; The left direction of the double-stranded polynucleotide sequence is called the 5 'direction. The direction in which the initial RNA transcript is added from 5 'to 3' is called transcription direction; The sequence region on the DNA strand from the 5 'to 5' end of the RNA transcript and having the same sequence as the RNA is said to be "upstream"; The sequence region on the DNA strand from the 3 'to 3' end of the RNA transcript and having the same sequence as the RNA is called "downstream sequence".
[143] Conservative amino acid substitutions include non-naturally occurring amino acid residues synthesized by chemical peptide synthesis, rather than synthesis within a biological system. Such amino acid residues include inverted forms of peptidomimetics and amino acid components.
[144] Naturally occurring residues can be classified according to the general side chain properties as follows:
[145] 1) hydrophobicity: Met, Ala, Val, Leu, Ile;
[146] 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[147] 3) acidic: Asp, Glu;
[148] 4) basic: His, Lys, Arg;
[149] 5) residues that influence chain orientation: Gly, Pro; And
[150] 6) Aromatic: Trp, Tyr, Phe.
[151] For example, non-conservative substitutions include the replacement of a member of one of these classes with a member of another class. Such substituted residues may be introduced into regions of human antibodies that are homologous to non-human antibodies or into non-homologous regions of a molecule.
[152] In substitutions according to certain embodiments, hydrotherapy values of amino acids are contemplated. Each amino acid is determined hydrotherapy values based on its hydrophobicity and charge characteristics. This figure is as follows: 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); Aspartic acid salts (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).
[153] The importance of amino acid hydrotherapy values that confer interactive biological function on proteins is known in the art. J. Mol. Biol., 157: 105-131 (1982). It is known that certain amino acids can be substituted with other amino acids having similar hydrotherapy values and have similar biological activities. In certain embodiments substituting based on hydrotherapy values includes substitution of amino acids having hydrotherapy values within ± 2. In certain embodiments, those having numerical values within ± 1 are included and in certain embodiments, those within ± 0.5 are also included.
[154] It is understood in the art that analogous amino acids are effectively substituted on the basis of hydrophilicity, in particular biologically functional proteins or peptides resulting from such substitutions are used in immunological embodiments as in the case of the present invention. In certain embodiments, the very local mean hydrophilicity of a protein regulated by the hydrophilicity of adjacent amino acids relates to its immunogenicity and antigenicity, ie the biological properties of the protein.
[155] Hydrophilicity values of amino acid residues are shown below: arginine (+3.0); Lysine (+3.0); Asparagine salts (+3.0 ± 1); Glutamate (+3.0 ± 1); Serine (+0.3); 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); And tryptophan (-3.4). Substitution based on similar hydrophilicity values includes, in certain embodiments, substitutions of amino acids with hydrophilicity values within ± 2 and substitutions of amino acids with hydrophilicity values within ± 1 and amino acids within ± 0.5. Epitopes can also be identified from primary amino acid sequences on the basis of hydrophilicity. Such regions are referred to as "epitope core regions".
[156] Typical amino acid substitutions are shown in Table 1 below.
[157]
[158] Those skilled in the art can determine suitable variants of the polypeptides as described herein using well known techniques. In certain embodiments, one of ordinary skill in the art can identify appropriate portions of the molecule that can shift to target regions that are not believed to have a significant effect on activity without losing activity. In certain embodiments, one skilled in the art can identify residues and portions of molecules that are conserved among analogous polypeptides. In certain embodiments, even regions that have a significant effect on biological activity or structure can be made conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.
[159] In addition, those skilled in the art can review structure-acting studies that identify residues of similar polypeptides that are important for activity or structure. This comparison can predict the importance of amino acid residues in proteins that correspond to amino acid residues that are important for the activity or structure of the analogous protein. Those skilled in the art can substitute amino acid residues predicted to be important residues with chemically similar amino acids.
[160] Those skilled in the art can also analyze amino acid sequences and three-dimensional structures regarding the structure of analogous polypeptides. Those skilled in the art can use this information to predict the alignment of amino acid residues of an antibody with respect to the three-dimensional structure of the antibody. In certain embodiments, when the residues are involved in important interactions with other molecules, one of ordinary skill in the art can choose not to cause radical changes to the predicted amino acid residues on the surface of the protein. In addition, those skilled in the art can make experimental variants in which a single amino acid is substituted at each preferred amino acid residue. This variant can then be screened using activity assays known to those skilled in the art. Such variants can be used to collect information about suitable variants. For example, if changes in specific amino acid residues result in loss of activity, undesirably reduced or inadequate, then variants with such a variation are preferred. That is, one of ordinary skill in the art can readily determine the amino acids so that additional variations do not occur alone or in combination with other mutations, based on information gathered from general experiments.
[161] There have been many scientific presentations predicting secondary structure. Moult J., Curr. Op. in Biotech., 7 (4): 422-427 (1996), Biochemistry by Chou et al., 13 (2): 222-245 (1974); Chou et al. Biochemistry, 113 (2): 211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148 (1978); Ann, Chou et al. Rev. Biochem., 47: 251-276 and Chou et al. Biophys. J., 26: 367-384 (1979). In addition, computer programs that are helpful in predicting secondary structure are generally available. One way of estimating secondary structure is based on homology modeling. For example, two polypeptides or proteins with at least 30% sequence identity or at least 40% similarity often have similar structural topologies. Recent developments in protein structure databases (PDBs) have increased the predictability of secondary structures, including the number of possible folds in polypeptide or protein structures. Holm et al. Nucl. Acid. See Res., 27 (1): 244-247 (1999). There is a limited number of folds in a particular polypeptide or protein and it is suggested that once the critical number of structures is established, the prediction of the structure will be accurate [Brenner et al. Curr. Op. Struct. Biol., 7 (3): 369-376 (1997).
[162] An additional method of predicting secondary structure is "threading" [Jones, D., Curr. Opin. Struct. Biol., 7 (3): 377-87 (1997); Structure, 4 (1): 15-19 (1996) by Sippl et al., "Profile Analysis" [Bowie et al. Science, 253: 164-170 (1991); Gribskov et al. Meth. Enzym., 183: 146-159 (1990); Gribskov et al. Proc. Nat. Acad. Sci., 84 (13): 4355-4358 (1987) and "affinity associations" (see Holm, supra (1999) and Brenner, supra (1997)).
[163] In certain embodiments, antibody variants include glycosylation variants in which the number and / or type of glycosylation sites is substituted for the amino acid sequence of the morphopeptide. In certain embodiments, protein variants comprise more or fewer N-linked glycosylation sites than native proteins. The N-linked glycosylation site is characterized by the sequence Asn-X-Ser or Asn-X-Thr, wherein the amino acid residues represented by X are all amino acid residues except proline. Substitution of amino acid residues to generate such sequences provides a novel site to which N-linked carbohydrate chains can be added. Optionally, the N-linked carbohydrate chains present were removed with substitutions to remove these sequences. In addition, one or more N-linked glycosylation sites (typically naturally occurring sites) are removed to provide rearrangement of the N-linked carbohydrate chains resulting in one or more new N-binding sites. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues have been deleted or substituted with another amino acid (eg, serine) compared to the parent amino acid sequence. Cysteine variants are useful when the insoluble inclusion bodies have to be separated and then refolded into biologically active conformations. Cysteine variants generally have fewer cysteine residues than natural proteins and typically have numbers that minimize interactions due to unpaired cysteines.
[164] According to certain embodiments, amino acid substitutions may (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity to form protein complexes, and (4) bind affinity. And / or (5) convert or modify other functional properties related to the polypeptide. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be performed with naturally occurring sequences (in certain embodiments, portions of polypeptide external domains that form intermolecular contacts). In certain embodiments, conservative amino acid substitutions typically do not substantially change the structural characteristics of the sequence (eg, substituted amino acids do not destroy the helix occurring within the sequence or secondary structures that specify the sequence). Do not disintegrate). Examples of polypeptide secondary and tertiary structures recognized in the art include Proteins, Structures and Molecular Principles [Creighton, Ed., W. H. Freeman and Company, New York (1984)]; Introduction to Protein Structure [C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991); And Nature 354: 105 (1991) to Thornton et al.
[165] Specific binder molecules of the invention that are polypeptide or peptide substitution variants have about 10-12% of the original amino acid sequence substituted. For antibody variants, the light chains are 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40,39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29 , 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 Light chain has 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substituted amino acids.
[166] Derivatives of specific binders
[167] The present invention also provides derivatives of specific binder polypeptides. Derivatives include specific binder polypeptides by insertion, deletion or substitution of amino acid residues and other variations. Preferably, the variants are naturally covalent, and include, for example, chemical bonds with polymers, lipids, other organic and inorganic components. Derivatives of the invention can be prepared to increase the circulating half-life of specific binding polypeptides or to enhance the target's ability to target a desired cell, tissue or organ.
[168] The invention also includes derivative binders covalently modified to attach one or more of a water soluble polymer such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol (US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 and 4,179,337). Still other useful polymers known in the art are monomethoxy-polyethylene glycol, dextran, cellulose or other carbohydrate base polymers, poly- (N-vinyl pyrrolidone) -polyethylene glycols, propylene glycol homopolymers, polypropylene oxides / Ethylene oxide co-polymers, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohols as well as mixtures of such polymers. Particular preference is given to specific binder products which have been covalently modified with polyethylene glycol (PEG) subunits. The water soluble polymer may bind to a specific position that is, for example, the amino terminus of the specific binder product, or may randomly bind to one or more of the side chains of the polypeptide. PEG, which is used to enhance the therapeutic capacity for specific binding agents and in particular humanized antibodies, is described in US Pat. No. 6,133,426 to Gonzales et al., Published October 17, 2000.
[169] Target site for antibody mutagenesis
[170] Certain strategies can be used to tailor the inherent properties of Ang-2-specific antibodies, such as the affinity of the antibody for antibody targets. This strategy is a screening step designed to site-specific or random mutagenesis of polynucleotide molecules encoding antibodies to produce antibody variants, and then recover antibody variants that exhibit desirable changes (eg, increased or decreased activity). Used for
[171] In general, amino acid residues that are targeted for mutation strategies are present in CDRs. As described in this document, such regions include residues that substantially interact between Ang-2 and other amino acids that affect the spatial arrangement of the residues. However, amino acids in the conformational regions of the variable domains outside the CDR regions are shown to contribute to the antigen-binding properties of the antibody, which can be targeted to modulate these properties. See Hudson, Curr Opin Biotech, 9: 395-402 (1999) and references herein.
[172] Libraries of less effectively and more effectively screened antibody variants can be generated with limited random or site-specific mutagenesis for sites in CDRs corresponding to sites that are prone to "over-mutation" during the process of physical activity development. See Chowdhury and Pastan, Nature Biotech, 17: 568-572 [1999] and references herein. Known types of DNA elements that characterize hyper-mutation sites in this way include direct and converting repeats, specific cannula sequences, secondary structures, and palindroms. Census DNA sequences include tetra sequence Purine-G-pyrimidine-A / T (ie A or G-G-C or T-A or T) and serine codon AGY where Y is C or T.
[173] Accordingly, embodiments of the present invention include mutagenic strategies aimed at enhancing the affinity of the antibody for antibody targets. Such strategies include overall variable heavy and light chains, mutagenesis only in the CDR regions, mutagenesis of the census overmutation site in CDRs, mutagenesis in the framework regions or a combination of these methods ("mutagenicity" herein). "Is random or site-specific). Although the structures of the antibodies and antibody-ligand complexes of the invention have been addressed, techniques known to those skilled in the art, such as X-ray crystallography, to delineate CDR regions and to identify residues comprising binding sites of antibodies You can check it. Various methods based on the analysis and characterization of antibody crystal structures are known to those skilled in the art and can be used to infer CDR regions, although not critical. Examples of commonly used methods include Kabat, Chothia, AbM and contact definitions.
[174] The Kabat definition is based on sequence diversity and is a commonly used definition of CDR regions (see Johnson and Wu, Nucleic Acids Res, 28: 214-8 (2000)). Chothia definitions are based on the location of structural loop regions [Jhothia et al. J Mol Biol, 196: 901-17 (1986); See Nature, 342: 877-83 (1989), Chothia et al.]. The AbM definition is a compromise of the Kabat and Chothia definitions. AbM is well suited to the program for modeling antibody structures produced by Oxford Molecular Group [Proc Natl Acad Sci (USA) 86: 9268-9272 (1989); ABM ™, Rees et al., A computer program for modeling variable regions of antibodies, available from Oxford Moricula Limited, Oxford, UK. The AbM suite produced the tertiary structure of the antibody from primary sequencing by a combination of knowledge databases and original methods. Known additional definitions, such as contact definitions, have recently been introduced (see Mac Mall Biol, J: Mol Biol, 5: 732-45 (1996)). This definition is based on the analysis of available complex crystal structures.
[175] Typically, the CDR regions in the heavy chain are typically named H1, H2 and H3 and numbered sequentially to enumerate from amino terminus to carboxy terminus. CDR regions within the light chain are typically named L1, L2 and L3 and numbered sequentially to enumerate from amino terminus to carboxy terminus.
[176] CDR-H1 consists of approximately 10-12 residues in length and typically starts at the fourth residue after Cys according to the Chothia and AbM definitions or typically after the fifth residue according to the Kabat definition. H1 is typically present in front of Trp, typically Trp-Val and also Trp-Ile or Trp-Ala. The length of H1 consists of approximately 10-12 residues according to the AbM definition, while the last four residues are excluded according to the Chothia definition.
[177] CDR-H2 typically starts at the 15th residue following the H1 terminus according to the Kabat and AbM definitions. The residue before H2 is typically Leu-Glu-Trp-Ile-Gly but there are various variations. H2 is typically present before the amino acid sequence Lys / Arg-Leu / Ile / Val / Phe / Thr / Ala-Thr / Ser / Ile / Ala. According to the Kabat definition, the length of H2 consists of approximately 16 to 19 residues, and according to the AbM definition the length typically consists of 9 to 12 residues.
[178] CDR-H3 typically starts at the 33rd residue following the end of H2 and typically begins before the amino acid sequence (typically Cys-Ala-Arg). H3 is typically present before the amino acid sequence-Gly. The length of H3 consists of approximately 3 to 25 residues.
[179] CDR-L1 typically starts at approximately residue 24 and will typically start after Cys. The residue after CDR-L1 is always Trp and generally starts with the sequences Trp-Tyr-Gln, Trp-Leu-Gln, Trp-Phe-Gln or Trp-Tyr-Leu. The length of CDR-L1 consists of approximately 10 to 17 residues. The harsh CDR-L1 for the antibodies of the present invention follows the manner with exactly 15 amino acids following the Cys residue followed by Trp-Tyr-Gln.
[180] CDR-L2 starts at approximately 16th residue after the end of L1. Generally follow the residues Ile-Tyr, Val-Tyr, Ile-Lys or Ile-Phe. The length of CDR-L2 consists of approximately seven residues.
[181] CDR-L3 typically starts at the 33rd residue after the end of L2 and typically follows Cys. L3 is typically present before the amino acid sequence Phe-Gly-XXX-Gly. L3 consists of approximately 7 to 11 residues in length.
[182] Various methods of modifying antibodies are known in the art. For example, US Pat. No. 5,530,101 (Queen et al., June 25, 1996) discloses that the sequence of the humanized immunoglobulin heavy chain variable region outline is 65% to 95% of the sequence of the donor immunoglobulin heavy chain variable region outline. A method of producing the same humanized antibody is described. Each humanized immunoglobulin chain interacts with a CDR that affects binding affinity, such as amino acids within about 3 kHz or one or more amino acids very close to the CDRs in the donor immunoglobulin, as predicted by molecular modeling in addition to the CDRs. It includes amino acids of the donor immunoglobulin outline that can act. Heavy and light chains can each be designed using one or both of a variety of location criteria. When mixed with raw antibodies, the humanized immunoglobulins of the present invention are substantially non-immunogenic in the human body and have substantially the same binding capacity as donor immunoglobulins that bind antigens, such as proteins or other compounds comprising epitopes. . See also US Pat. No. 5,693,761 ("Polynucleotides encoding improved humanized immunoglobulins"), issued December 2, 1997; U.S. Patent No. 5,693,762 to Queen et al., Issued December 2, 1997 (“Humanized Immunoglobulins”); Related methods are described in US Pat. No. 5,585,089 ("Humanized Immunoglobulins"), published December 17, 1996, in Queen et al.
[183] In one embodiment, US Patent No. 5,565,332 to Hoogenboom et al., Published on October 15, 1996 ("Production of chimeric antibodies-a combinatorial approach"), has binding properties similar to antibodies, and parent antibodies, but with improved human properties. A method for producing an antibody fragment with is described. Humanized antibodies are obtained by chain shuffling using, for example, phage display technology, and polypeptides comprising the heavy or light chain variable domains of a non-human antibody specific for a desired antigen are human complementary (light or heavy chains). Mix with the variable domain repertoire of the chain. The hybrid pairs specific for the antigen of interest are identical and the human chains obtained from the selected pairs are mixed with a repertoire of human complementary variable domains (heavy or light). In other embodiments, the CDR components of the non-human antibody are mixed with a repertoire of components of the CDRs portion of the human antibody. From the resulting library of antibody polypeptide dimers, hybrids are selected and used in the second humanized shuffling step. Optionally, the second step excludes if the hybrid already exhibits effective human characteristics that exhibit therapeutic value. Modifications have also been made to improve human characteristics. Winter, FEBS Leets 430: 92-92 (1998).
[184] In another embodiment, US Pat. No. 6,054,297 to Carter et al., Published April 25, 2000, substitutes and / or replaces the corresponding human CDR amino acid sequence with a CDR amino acid sequence and / or replaces the corresponding human FR amino acid sequence with an FR amino acid sequence. A method for preparing a humanized antibody is described.
[185] In another embodiment, US Pat. No. 5,766,886 to Studnicka et al., Published on June 16, 1998 (“Modified antibody variable domains”), describes immunogenicity with respect to heterologous species without reducing the original affinity of the antigen binding domain. Methods of identifying amino acid residues of antibody variable domains modified to be reduced and methods of making modified antibody variable domains useful for administration to heterologous species are described. See also US Pat. No. 5,869,619 to studnicka, published February 9, 1999.
[186] As discussed, methods known in the art can be modified to enhance the binding affinity for an antigen and / or to reduce the immunogenicity of the antibody of the receptor. In one approach, humanized antibodies can be modified to remove glycosylation sites to enhance affinity for cognate antigens (see Co et al. Mol Immunol 30: 1361-1367 (1993)). Techniques such as "remodeling", "chimerization" and "veneering / resurfacing" can produce humanized antibodies with excellent therapeutic potential [Annas of Allergy, Asthama, et al., Vaswami et al. & Immunol 81: 105 (1998); See Prot Engineer 9: 895-904 (1996) by Roguska et al.]. See also US Pat. No. 6,072,035 to Hardman et al., Issued June 6, 2000, which describes methods for reforming antibodies. While this technique reduces the number of foreign residues to reduce antibody immunogenicity, it does not interfere with anti-genic and anti-isotyped responses following repeated administrations of the antibody. Alternative methods of reducing immunogenicity are described in J Immunol 62 (6): 3663-71 (1999) by Gilliland et al.
[187] In many instances, humanized antibodies lose antigen binding capacity. Thus, it is desirable to “reverse mutation” humanized antibodies to include one or more of the amino acids obtained from the original (most rodent) antibodies intended to restore the binding affinity of the antibody. See, eg, Mol Immunol 36: 709-17 (1999) by Saldanha et al.
[188] Non-peptide specific binder analogs / protein analogs
[189] Also contemplated are non-peptide specific binder analogs of peptides that provide stabilized structure or reduced biodegradation. Specific binder peptide similar analogs can be prepared based on inhibitory peptides selected by substituting one or more of the residues with a non-peptide component. Preferably, the non-peptide component provides a preferred, eg, stabilized, bioreactive conformation having the ability to maintain a natural conformation or to recognize and bind Ang-2. In one aspect, the resulting analogs exhibit enhanced binding affinity with Ang-2. One example of a method for preparing non-peptide analogues from specific binder peptides is described in Nagulman et al., Regul. Pept. 57: 359-370 (1995). If desired, the specific binder peptides of the present invention are, for example, glycosylated, amidated, carboxylated or phosphorylated or acid addition salts, amides, esters (especially C-terminal esters) and N- of the peptides of the present invention. Acyl derivatives can be generated and mutated. Specific binder peptides can also be mutated to form covalent or non-covalent complexes with other components to produce peptide derivatives. Covalently-linked complexes can be prepared by binding chemical components to the side chains of amino acids comprising specific binder peptides or to functional groups on the N- or C-terminus.
[190] In particular, specific binder peptides include, but are not limited to, radiolabels, fluorescent labels, enzymes (e.g., to promote coloring or fluorescence), substrates, solid matrices or carriers (e.g., biotin or avidin). It can be conjugated with a reporter group. The present invention provides a molecule comprising an antibody molecule, the molecule preferably further comprises a reporter selected from radiolabels, fluorescent labels, enzymes, substrates, solid matrices and carriers. Such labels are well known to those skilled in the art, for example biotin labels are particularly contemplated. The use of such labels is well known to those skilled in the art and is described in US Pat. No. 3,817,837; US Patent No. 3,850,752; US Patent No. 3,996,345; And US Pat. No. 4,227,437. Other useful labels include, but are not limited to, radioactive labels, fluorescent labels, and chemiluminescent labels. For example, US Pat. No. 3,817,837 for the use of such labels; US Patent No. 850,752; US Patent No. 3,939,350; And US Patent No. 3,996,345. All peptides of the present invention may comprise one, two or more of the above labels.
[191] How to prepare specific binder
[192] Specific binders of the invention that are proteins can be prepared by chemical synthesis in solution or on solid support according to conventional techniques. The current limitation on solid phase synthesis is that it consists of about 85-100 amino acids in length. However, chemical synthesis techniques can be used to chemically bind a series of smaller peptides to produce full length polypeptides. Various automated synthesizers are commonly available and can be used according to known protocols. For example, Stewart and Young (supra), each of which is incorporated herein by reference; J. Am. Chem. Soc., 105: 6442, (1983); Merrifield, Science 232: 341-347 (1986); Barany and Merrifield, The Peptides, Gross and Meienhofer, eds, Academic Press, New York, 1-284; Barany et al. Int. J. Pep. Protein Res., 30: 705-739 (1987); And US Pat. No. 5,424,398.
[193] The solid phase peptide synthesis method uses copoly (styrene-divinylbenzene) comprising 0.1-1.0 mM amine / g polymer. The method of synthesizing peptides uses 9-fluorenylmethyloxy-carbonyl (FMOC) or butyloxycarbonyl (t-BOC) protection of alpha-amino groups. Both methods synthesize stepwise and thus add a single amino acid to each step starting at the C-terminus of the peptide (see Curr. Prot. Immunol., Wiley Interscience, 1991, Unit 9, Coligan et al.). By chemical synthesis, synthetic peptides can remove t-BOC or FMOC amino acid blockers and react with acids (e.g., liquid HF-10% anisol treated at 0 ° C for about 0.25 to about 1 hour) at reduced temperature. Treatment to cleave in the polymer. After evaporation of the drug, the specific binder peptide was extracted from the polymer with lyophilized 1% acetic acid solution to yield the raw material. Purification was generally carried out by techniques such as gel purification on spandex G-15 using 5% acetic acid as solvent. The appropriate fraction of the column was lyophilized to yield homologous specific binder peptides or peptide derivatives, which were characterized by standard techniques such as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar fluorescence, solubility and solid phase. Quantification by Edman degradation.
[194] Chemical synthesis of anti-Ang-2 antibodies, derivatives, variants and fragments thereof as well as other protein-based Ang-2 binders allows for the incorporation of non-naturally occurring amino acids into the formulation.
[195] Recombinant DNA technology is a convenient method for preparing the full-length antibodies and large protein specific binders or fragments thereof of the present invention. The cDNA molecule encoding the antibody or fragment can be inserted into an expression vector and inserted into a host cell to produce the antibody or fragment. It has been understood that the cDNA encoding such antibodies can vary the “original” cDNA (translated from mRNA) such that codon degeneracy occurs or that the codon is preferably used in various host cells.
[196] In general, DNA molecules encoding peptides or peptibodies can be obtained by the methods described in the Examples. If it is desired to obtain Fab molecules or CDRs relating to the original antibody molecules, suitable libraries (phage display libraries; lymphocyte libraries, etc.) can be screened using standard techniques to identify and clone relevant Fabs / CDRs. . Probes used for screening are full length or truncated Fab probes encoding the Fab portion of the original antibody, probes for one or more CDRs of the Fab portion of the original antibody, or other suitable probes. Where DNA fragments are used as probes, typical hybridization conditions are as described in Ausubel et al. Current Protocols in Molecular Biology, Current Protocols Press [1994]. After hybridization, probed blots are washed under suitable and stringent conditions depending on factors such as probe size, homology of the expected probes for cloning and the number of screened clones. Examples of very stringent screening are 0.1 × SSC and 0.1% SDS at temperatures 50-65 ° C.
[197] Various expression vectors / host systems can be used to include and express polynucleotide molecules encoding specific binder polypeptides of the invention. Such systems include, but are not limited to: microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; Yeast transformed with yeast expression vector; Insect cell systems infected with virus expression vectors (eg, baculovirus); Plant cell systems transfected with virus expression vectors (eg Cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (eg Ti or pBR322 plasmid); Or animal cell systems.
[198] Mammalian cells useful for the production of recombinant specific binder proteins include, but are not limited to: VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (eg, COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells as well as hybridoma cell lines as described herein. Mammalian cells are preferred for preparing specific binding agents, such as antibodies and antibody fragments, which are typically glycosylated and require proper refolding for activity. Preferred mammalian cells include CHO cells, hybridoma cells and myeloid cells.
[199] Some typical protocols for recombinant expression of specific binders are described herein below.
[200] The term "expression vector" refers to a plasmid, phage, virus or vector that expresses a polypeptide in a DNA (RNA) sequence. The expression vector may comprise a transcription unit comprising a combination of the following: (1) a gene that regulates gene expression, such as a promoter or enhancer, (2) encoding a binder that is transcribed into mRNA and translated into a protein Structures or sequences and (3) suitable transcription initiation and termination sequences. Structural units used in yeast or eukaryotic expression systems preferably comprise a leader sequence which allows the translated protein to be secreted extracellularly in the host cell. Optionally, if a recombinant specific binder protein without a leader sequence or transport sequence is expressed, it comprises an amino terminal methionine residue. Such residues may or may not be substantially cleaved from the expressed recombinant protein to obtain the final specific binder peptide product.
[201] For example, specific binding agents can be recombinantly expressed in yeast using a commercially available expression system (eg, a Peacha expression system; available from Invitrogen, San Diego, Calif.) According to the manufacturer's instructions. . This system follows a pre-pro-alpha sequence that is directly secreted, but the transcript of the insert is driven by a methanol induced alcohol oxidase (AOXI) promoter.
[202] Specific binder peptides secreted in yeast growth media were purified by methods used to purify peptides from bacterial and mammalian cell supernatants.
[203] Optionally, the cDNA encoding the specific binder peptide can be cloned into the baculovirus expression vector pVL1393 (available from Faminzen, San Diego, Calif.). Such vectors can be used according to the manufacturer's instructions (available from Paminegen) to infect Spodoptera frugiperda cells with sF9 protein-free medium and produce recombinant proteins. Specific binder proteins were purified and concentrated in medium using a heparin-sepharose column (available from Pharmacia).
[204] Optionally, the peptide will be expressed in the insect system. Insect systems suitable for protein expression are known to those skilled in the art. As one of the insect systems, autographa california nuclear polyhedrasis virus (AcNPV) can be used as a vector for expressing foreign genes in Spodoptera pulperifera cells or cabbage larvae (trichoplusia larvae). have. Specific binder peptide coding sequences can be cloned into noncritical regions of the virus, such as polyhedrin genes, and placed under the control of a polyhedral promoter. Successful insertion of specific binder peptides will inactivate the polyhedral genes and produce recombinant viruses lacking coat protein coating. Recombinant viruses can be used to infect S. luciferda cells or cabbage larvae expressing peptides. Smith et al. J. Virol. 46: 584 (1983); Engelhard et al. Proc. Nat. Acad. Sci. (USA) 91: 3224-7 (1994).
[205] In another embodiment, the DNA sequence encoding the specific binder peptide can be amplified by PCR and cloned into a suitable vector, eg, pGEX-3X (available from Pharmacia). The pGEX vector was designed to produce a fusion protein comprising glutathione-S-transferase (GST) encoded by this vector and a specific binder protein encoded by a DNA fragment inserted into the cloning site of the vector. Primers used for PCR can be prepared, for example, to include appropriate cleavage sites. Recombinant specific binder fusion proteins can be removed from the GST portion of the fusion protein if the specific binder fusion component is used only for ease of expression or if it is undesirable to attach to the peptide of interest. pGEX-3X / specific binder peptide constructs were transformed into E. coli XL-1 blue cells (available from Stratagen, La Jolla, Calif.), and each of these transformants was isolated and grown. Plasmid DNA from each transformant was purified and partially sequenced using an automated sequencer to confirm the presence of the desired specific binder that encodes the nucleic acid insert in the proper orientation.
[206] The expression of polynucleotides encoding anti-Ang-2 antibodies and fragments thereof using the recombinant systems described above must be "re-folded" (which properly causes various disulfide bonds) to be biologically activated. Or fragments thereof. Representative refolding procedures for these antibodies are described in the Examples herein and in the following sections.
[207] Specific binders made from bacterial cells can be produced in bacteria as insoluble inclusion bodies, which can be purified as follows: Host cells can be killed by centrifugation as follows: 0.15 M NaCl, 10 mM Tris, pH 8, Washed with 1 mM EDTA and treated with 0.1 mg / ml of lysozyme (obtained from Sigma, St. Louis, MO) for 15 minutes at room temperature. The filtrate can be removed by sonication and cell debris can be pelleted by centrifugation at 12,000 x g for 10 minutes. Specific binders containing pellets are resuspended in 50 mM Tris and 10 mM EDTA at pH 8 and centrifuged at 6000 × g for 30 minutes to form a layer over 50% glycerol. The pellet can be resuspended in standard phosphate buffered saline (PBS) without Mg ⁺⁺ and Ca ⁺⁺ . It can be further purified by fractionation of the resuspended pellet in an SDS-polyacrylamide gel (see Sambrook et al., Which is incorporated herein by reference) in which a specific binder is modified. Gels were submerged in 0.4 M KCl to specify the protein, which protein can be cut out of gel-developing buffer lacking SDS and eluted. If GST / fusion proteins are produced as soluble proteins in bacteria, they can be purified using the GST purification module (available from Pharmacia).
[208] Mammalian host systems for expressing recombinant proteins are well known to those skilled in the art. Host cell strains may be selected according to the particular ability to produce specific post-translational modifications useful for processing the expressed protein or for supplying protein activity. Such modifications of polypeptides include, but are not limited to: acetylation, carboxylation, glycosylation, phosphosylation, lipidation and acylation. Hybridoma cell lines and other host cells, as well as CHO, HeLa, MDCK, 293, WI38, have specific intracellular organs and specific mechanisms for post-translational activity, which allows for the correct modification and processing of introduced foreign proteins. Can be chosen to ensure.
[209] Numerous selection systems can be used to recover transformed cells for recombinant protein production. Such screening systems include, but are not limited to: HSV thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, and adenine phosphoribosyltransferra in tk-, hgprt- or aprt- cells, respectively. 1st gene. In addition, anti-metabolite resistance is determined by DHFR which confers resistance to methotrexate; Gpt conferring resistance to mycophenolic acid; Neo conferring resistance to aminoglycoside G418 and conferring resistance to chlorsulfuron; And screening for hygro conferring resistance to hygromycin. Additionally, it is useful to include trpB in cells that use indole instead of tryptophan as selectable genes, or hisD in cells that use histinol instead of histidine. Markers that confer visual indicators for transformant identification include anthocyanins, β-glucuronidase and its substrate, GUS and luciferase, its substrate and luciferin.
[210] Purification and Refolding of Specific Binders
[211] In some cases, the specific binders produced by the processes described above are required to be oxidized and "folded back" in the appropriate tertiary structure and form disulfide bonds to be biologically active. Refolding can be accomplished using a number of procedures well known in the art. Such methods include, for example, exposing the solubilized polypeptide agent to a pH of at least 7, in the presence of chaotropic agents. The selection of chaotrope is similar to the selection used for inclusion body solubilization, but chaotropes are generally used at low concentrations. Typical chaotropic agents are guanidines. In most cases, the refolded / oxidized solution also contains certain proportions of its oxidation forms in addition to the reducing agent to produce specific redox potentials that take into account disulfide shuffling that occurs for cysteine crosslinking. Some commonly used redox pairs are cysteine / cytamine, glutathione / dithiobis GSH, copper chloride, dithiothritol DTT / dithiane DTT and 2-mercaptoethanol (bME: 2-mercapto ethanol) / dithio Include bME. In many cases, co-solvents have been used to increase the refolding efficiency. Commonly used cosolvents include polyethylene glycol, glycerol and arginine of various molecular weights.
[212] It is preferred to purify the specific binder protein or variants thereof of the present invention. Protein purification techniques are well known to those skilled in the art. Such techniques include initial fractionation of polypeptide and non-polypeptide fractions at certain stages. The desired polypeptide having a specific binder polypeptide isolated from other proteins can be further purified using chromatography and electrophoresis techniques to partially or completely purify (or purify for homogeneity). In particular, analytical methods suitable for the preparation of pure specific binder peptides include ion-exchange chromatography, exclusion chromatography; Polyacrylamide gel electrophoresis; There is an isoelectric focusing method. Particularly efficient methods for purifying peptides are rapid protein liquid chromatography or HPLC.
[213] Certain aspects of the present invention relate to the purification of encoded specific binder proteins or peptides and the substantial purification in certain embodiments. As used herein, the term “purified specific binder protein or peptide” refers to a composition that is separable from other components, wherein the specific binder protein or peptide has been purified to a degree suitable for its naturally-acquisable state. Therefore, purified specific binder proteins or peptides also represent specific binder proteins or peptides that are not present in a naturally occurring environment.
[214] In general, "purified" refers to a specific binder composition fractionated to remove various other components, which compositions retain substantially the biological activity expressed. When the term "substantially purified" is used, the term refers to about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the specific binder protein or peptide in the composition. Specific binder compositions that form the major components of the composition as constituting the protein are shown.
[215] Various methods for measuring the degree of purification of specific binders are well known to those skilled in the art in light of the present specification. Such methods include, for example: determining the specific binding activity of an active fraction, determining the amount of specific binder polypeptide in the fraction by SDS / PAGE analysis. A preferred method of measuring the purity of a specific binder fraction is a method of estimating the degree of purification by estimating the binding activity of the fraction compared to the binding activity of the initial extract (which is measured herein as "number of folded tablets"). The actual unit used to represent the amount of activity depends on the particular assay technique selected depending on the purification and whether or not the expressed specific binder protein or peptide exhibits detectable binding activity or not.
[216] Various techniques suitable for the purification of specific binder proteins are well known to those skilled in the art. The purification method includes, for example: Precipitation or heat denaturation by ammonium sulfate, PEG, antibodies (immunoprecipitation) and similar methods followed by centrifugation; Chromatography steps such as affinity chromatography (eg, Protein-A-Sepharose), ion exchange chromatography, gel filtration chromatography, reverse phase chromatography, hydroxylapatite chromatography, and affinity chromatography; Isoelectric focusing method; Gel electrophoresis; And a combination of the above and other techniques. As is generally known in the art, the various purification steps may be reversed in the order in which they are processed or certain steps may be omitted, which is still a suitable method for preparing substantially specific specific binders.
[217] Specific binders of the present invention are not always a general requirement provided in most steps of purification. In addition, substantially fewer specific binder products are expected to be practical in certain embodiments. Upon binding, partial purification was carried out using a few purification steps or partial purification using other purification steps generally having the same purification scheme. For example, cation-exchange column chromatography performed using HPLC apparatus is generally expected to result in larger "folding" purification than the same technique using low pressure chromatography systems. Methods that exhibit relatively lower grades of purification have the advantage of maintaining the total recovery of the specific binder protein product or the binding activity of the expressed specific binder protein.
[218] It is known that the transport of polypeptides can vary considerably from time to time by different conditions of SDS / PAGE (see Capaldi et al. Biochem Biophys Res Comm, 76: 425 (1977)). Therefore, under different electrophoretic conditions, the apparent molecular weight of the purified or partially purified specific binder expression product is expected to vary.
[219] Combine black
[220] In general, immunological binding assays were used to specifically bind the capture agent and often to immobilize the sample target antigen. The capture agent is a component that specifically binds to the sample. In an embodiment of the invention, the capture agent is an antibody or fragment thereof that specifically binds Ang-2. Such immunological binding assays are well known in the art. [1993] Asai et al., Obtained from Academic Press, Inc., New York, USA. 37, Antibodies in Cell Biology].
[221] Often, immunological binding assays utilize a label that signals the presence of a binding complex formed with a capture agent and an antigen. The labeling agent may be one of the molecules comprising the bound complex; That is, it may be a labeled specific binder or a labeled anti-specific binder antibody. Optionally, the labeling agent is a tertiary molecule that binds to the bound complex and may generally be another antibody. For example, the labeling agent can be an anti-specific binder antibody containing a label. Secondary antibodies specific for the bound complex may lack binding but can be bound by quaternary molecules specific for antibodies that are secondary antibody members. For example, secondary antibodies can be modified with detectable components such as biotin and then bound to quaternary molecules such as enzyme-labeled streptavidin. Other proteins capable of specifically binding immunoglobulin constant regions, such as A protein or G protein, were used as markers. The binding protein is a normal component of the Streptococcus bacterial cell wall, which exhibits a strong non-immunogenic reactivity with immunoglobulin constant regions of various species [Akerstrom, J Immunol, 135: 2589-2542 (1985, incorporated herein by reference). ); Chaubert, Mod Pathol, 10: 585-591 (1997).
[222] Throughout the entire assay, incubation and / or wash steps are required after combining each reagent. The constant temperature step may vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time depends on the assay format, sample, volume and concentration of solution. Although the assay can be run over the full range of temperatures, it is generally run at room temperature.
[223] A. Non-Competitive Coupling Test
[224] Immunological binding assays can be in a non-economical manner. The assay includes the amount of captured sample measured directly. For example, in a preferred "sandwich" assay, the capture agent (antibody) can be bound directly to the immobilized solid substrate. The immobilized antibody is then captured (bound) to the antigen present in the test sample. Therefore, the immobilized protein was then bound to a labeling agent, such as a secondary antigen with a label. In another preferred "sandwich" assay, the secondary antibody has no label, but the secondary antibody can be bound by a labeled antibody specific for the antibody species from which it was derived. Secondary antibodies can also be modified with detectable components such as biotin, which can specifically bind to tertiary labeled molecules such as streptavidin [New York Cold Spring, USA, hereby incorporated by reference]. See Harlow and Lane, Antibodies, ALaboratory Manual, Ch 14, obtained from Burr Laboratories (1988).
[225] B. Competitive Combination Test:
[226] Immunological binding assays can be in a competitive manner. The amount of sample present in the sample was indirectly measured by measuring the amount of the sample present in the sample that was competitively separated from or replaced by the capture agent. In a preferred competitive binding assay, generally known amounts of labeled specimens were added to a sample, and then the sample was contacted with an antibody (trapping agent). The amount of labeled sample bound to the antibody is inversely proportional to the concentration of the sample present in the sample (see Harlow and Lane, Antibodies, A Laboratory Manual, Ch 14, pp. 579-583, incorporated herein by reference).
[227] In another preferred competitive binding assay, antibodies were immobilized on a solid substrate. The amount of protein bound to the antibody can be selectively determined by measuring the amount of protein present in the protein / antibody complex or by measuring the amount of remaining uncomplexed protein. The amount of protein was measured by feeding the labeled protein. See Antibodies, A Laboratory Manual, Ch14, supra, of Harlow and Lane, cited herein by reference.
[228] In another preferred competitive binding assay, hapten inhibition was utilized. Specimens known herein were immobilized on a solid substrate. A known amount of antibody was added to the sample and the sample was brought into contact with the fixed sample. The amount of antibody bound to the immobilized sample is inversely proportional to the amount of sample present in the sample. The amount of immobilized antibody was determined by measuring the fraction remaining in the solution or the immobilized antibody fraction. Antibodies were detected by labeling the antibody directly or by continuously adding labeled components that specifically bind to the antibody as described above.
[229] C. Use of competitive binding tests
[230] Competitive binding assays allow those skilled in the art to determine whether the enzyme complex or protein complex recognized by the specific binding agents of the invention is a protein that is required and not a cross-reacting molecule or the antibody is antigen specific and unrelated to the antigen. It was used to determine cross-reactivity to determine if it did not bind to. For this type of assay, antigen was immobilized on a solid support and an unknown protein mixture was added to the assay to complete binding of the specific binder to the immobilized protein. The completed molecule also bound one or more antigens not related to the antigen. To measure the ability of a protein to competitively bind to an immobilized antigen and the cross-reactivity of the protein mixture, the binding capacity by the same protein immobilized on a solid support was compared.
[231] D. Different binding assays
[232] The present invention also provides Western blotting for detecting or quantifying Ang-2 present in a sample. Generally, the technique involves separating sample proteins by gel electrophoresis based on molecular weight and transporting them to a suitable solid support, such as a nitrocellulose filter, nylon filter or nylon derived filter. The sample was incubated with an antibody or fragment thereof that specifically binds to Ang-2 to detect the resulting complex. The antibody was selectively detected using a labeled antibody that was either directly labeled or specifically bound to the antibody.
[233] Binding assays for detecting Ang-2 specific binders that separate Ang-2 that binds to Ang-2 receptors are described in the Examples herein.
[234] Diagnostic assay
[235] An antibody or fragment thereof of the invention diagnoses a disease or condition characterized by expressing Ang-2 or a subunit or observes a patient being treated with an Ang-2 inducer, fragment thereof, an inhibitor of Ang-2 activity or an agonist Useful for assays to do so. Diagnostic assays for Ang-2 include methods that utilize labels and specific binding agents to detect Ang-2 in human body fluids or extracts of cells or tissues. The peptibody of the present invention can be used with or without modification. In a preferred diagnostic assay, specific binders are labeled, for example, by attaching a label or reporter molecule. Various labeling and reporter molecules are known, some of which have already been described herein. The present invention is particularly useful for diagnosing human diseases.
[236] Various protocols are known in the art for measuring Ang-2 proteins using polyclonal antibodies or monoclonal antibodies specific for each protein. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescence activated cell sorting (FACS). Two-site monoclonal-based immune assays utilizing monoclonal antibodies that respond to two non-interfering epitopes on Ang-2 are preferred, but competitive binding assays were used. See, eg, J. Exp Med, 158: 1211 (1983) by Maddox et al. For such assays.
[237] To provide a basis for diagnosis, normal and reference values for human Ang-2 expression were generally established. Such determination can be accomplished by binding a cell extract or body fluid of a normal subject (preferably human) with a specific binding agent, eg, an antibody, to Ang-2, under conditions suitable for complex formation well known in the art. . The reference amount of the complex formation can be quantified by comparing specific binders that bind known amounts of Ang-2 protein with both disease and control samples. The reference value obtained from a normal sample can then be compared with the value obtained from a sample of a subject potentially affected by the disease. Deviations between baseline and subject values suggest a role for Ang-2 in disease states.
[238] For diagnostic applications, general specific binders of certain embodiments of the invention have been labeled with detectable components. The detectable component can generate a detectable signal directly or indirectly. For example, detectable components include radioisotopes such as ³ H, ¹⁴ C, ³² P, ³⁵ S or ¹²⁵ I, fluorescent or chemiluminescent compounds such as fluorescein isothiocyanate, rhodamine or luciferin; Or enzymes such as alkaline phosphatase, β-galactosidase or horseradish peroxidase (see Meth Enz, 184: 138-163 (1990) by Bayer et al., Incorporated herein by reference).
[239] disease
[240] The present invention provides specific binders that bind Ang-2 useful for treating human disease and pathological symptoms. Agents that modulate Ang-2 binding activity or other intracellular activity have been used with other therapeutic agents to increase their therapeutic effect or reduce potential side effects.
[241] In one aspect, the invention provides methods and reagents useful in treating conditions and conditions in which the activity level of Ang-2 in the cell is out of range or exhibits undesirable levels. Such diseases include cancer and hyperproliferative symptoms such as hyperplasia, psoriasis, contact dermatitis, immune disorders and infertility.
[242] The present invention also provides a method of treating cancer in an animal, including the human body, comprising administering to the animal an effective amount of a specific binding agent that inhibits or reduces Ang-2 activity. Moreover, the present invention relates to methods for inhibiting cancer cell proliferation in biological systems, including intracellular proliferation, invasiveness and metastasis processes. The method involves the use of a compound of the invention as an inhibitor of cancer cell proliferation. Preferably, the method was used to inhibit or reduce cancer cell proliferation, invasiveness, metastasis or tumor development in living animals such as mammals. In addition, the methods of the present invention can be readily modified for use in assay systems that not only identify compounds that affect cancer cell proliferation but also assay, for example, the proliferation of cancer cells and their properties.
[243] Cancers treatable with the methods of the present invention preferably occur in mammals. Mammals include, for example: pets or animals such as dogs and cats, as well as laboratory animals such as humans and other primates, rats, mice, and rabbits, and domestic animals such as horses, pigs, sheep, and cattle do.
[244] Tumors and neoplasms include the growth of tissue cells in which the proliferation of the cells is not inhibited or advanced. Some of these growths are benign, while others are termed malignant tumors, causing the death of the organism. Malignant neoplasms or cancers are distinguished from benign proliferation in that they invade and metastasize to surrounding tissues in addition to exhibiting aggressive intracellular proliferation. Moreover, malignant neoplasms are characterized by a drastic reduction (radical dedifferentiation) of their organisation and differentiation, which is suitable for each other and their surrounding tissues. The property is named or "degeneration."
[245] Neoplasms treatable by the present invention also include solid tumors such as carcinomas and sarcomas. Carcinomas include their malignant neoplasms derived from epithelial cells that infiltrate (penetrate) and metastasize surrounding cells. Adenocarcinomas are carcinomas derived from adenocarcinomas or form recognizable adenomas. Other broad categories or cancers include sarcomas, which are tumors of these cells inserted into fibrillar material, such as connective tissue of the embryo, or of homogeneous material. The invention is also easy to treat myeloid or lymphoid cancer, including leukemias, lymphomas and other cancers, which do not generally exist as tumor mass but are distributed in the pulmonary or lymphatic reticuloendothelial system.
[246] Types of cancer or tumor cells that can be treated with the present invention include, for example: ACTH-producing tumors, acute lymphocytic leukemia, acute nonlymphocytic leukemia, adrenal cortical cancer, bladder cancer, brain cancer, breast cancer, cervical cancer , Chronic lymphocytic leukemia, Chronic myeloid leukemia, Colorectal cancer, T-cell leukemia of the skin, Endometrial cancer, Esophageal cancer, Ewing sarcoma, Gallbladder cancer, Cytoblastic leukemia, Head cancer, Cervical cancer, Staphylococcal lymphoma, Kaposi's sarcoma, Kidney cancer, Liver cancer, lung cancer (bovine and non-small cell), malignant peritoneal effusion, malignant pleural effusion, melanoma, mesothelioma, multiple myeloma, neuroblastoma, glioma, non-Hodgkin's lymphoma, osteosarcoma, ovarian cancer, ovarian (embryonic) cancer , Pancreatic cancer, penile cancer, prostate cancer, retinoblastoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, trophoblastic neoplasm, uterine cancer, vaginal cancer, vulvar cancer and bilms Amount.
[247] The present invention particularly relates to the treatment of certain types of cancer defined experimentally. In these exemplary treatments, in vivo and ex vivo models of baseline conditions were used. These methods were used to identify agents that would be expected to be efficacious as therapeutic in vivo treatments. However, it is to be understood that the methods of the present invention are not limited to treating such tumor types and may extend to solid tumors derived from any other organ system. Invasiveness or metastasis of cancers involved in Ang-2 expression or activity may be inhibited or specifically induced by retrograde by the present invention.
[248] In addition, the present invention may be practiced by including another anti-cancer chemotherapeutic agent, such as a conventional chemotherapeutic agent, in combination with a specific binding agent of the invention, such as an antibody. Combinations of specific binders and other agents can elevate the chemotherapy protocol. Numerous chemotherapeutic protocols can be imparted per se from the point of view of a skilled practitioner as integratable in the methods of the invention. Chemotherapeutic agents including alkylating agents, anti-metabolic agents, hormones and antagonists, radioisotopes as well as natural products were used. For example, the compounds of the present invention may be treated with antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustard such as cyclophosphamide, pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxol ( taxol) and its natural derivatives and synthetic derivatives and analogs. As another example, in the case of mixed tumors such as adenocarcinoma of the breast, including gonadotropin-dependent cells and gonadotropin-independent cells, the compounds may be selected from leuprolide or goserelin (synthetic peptide of LH-RH). Analogues). Other anti-neoplastic protocols include the use of tetracycline compounds in conjunction with other therapeutic modalities such as, for example, surgical procedures, radiation, and the like, which are also referred to herein as "anti-neoplastic modality aids." Therefore, the method of the present invention can be used in conjunction with these common curing methods which have the advantage of reducing side effects and increasing efficiency.
[249] Therefore, the present invention provides compositions and methods useful for treating a variety of cancers, including solid tumors and leukemia. Types of cancer to be treated include, but are not limited to: adenocarcinoma of the breast, prostate, and colon; Bronchial cancer of all forms of lungs; Bone marrow volume; Melanoma; Liver cancer; Neuroblastoma; Papilloma; Afudoma; Isolated species; New species; Malignant carcinoid syndrome; Carcinoid heart disease; Carcinoma (eg, Walker, basal cells, basal flatness, Brown-Pearce, ducts, Ehrlich tumors, Krebs 2, Megkel cells, viscous, non-lung lungs Small cells, oat cells, papillary, hard carcinoma, bronchioles, tracheal support, squamous epithelium and transitional cells]; Organizational disorder; leukemia ; Malignant histiocytosis; Hodgkin's disease; Immunoproliferative pulmonary cell carcinoma; Non-Hodgkin's lymphoma; Plasmacytoma; Reticuloendothesis; Melanoma; Chondrocytes; Chondroma; Chondrosarcoma; Fibrous species; Fibrosarcoma; Giant cell tumor; Histiocytoma; Lipoma; Liposarcosis; Mesothelioma; Myxoma; Myxedema; Osteomas; Osteosarcoma; Chordoma; Two species; Undifferentiated germ cell tumor; Hamartoma; Mesenchymal species; Mesothelioma; Myoma; Enamel blastoma; Chalky species; Tooth species; Teratoma; Thymoma; And gout cell tumors. Moreover, the following types of cancer were also treated: adenoma; Cholangiocarcinoma; Cholesterin species; Cyclindroma; Cyst cancer; Cyst adenoma; Granuloma cell tumor; Glioblastoma; Liver cancer; Han Sun-Jong; Islet cell tumor; Leidihicinoma; Papilloma; Sertoliocytosis; Follicular cell tumor; Leiomyoma; Leiomyosarcoma; Myoblastoma; Myoma; Myoma; Rhabdomyomas; Rhabdomyosarcoma; Superspecies; Ganglion neuroma; Glioma; Mesothelioma; Meningioma; Neurofibrillary edema; Neuroblastoma; Neuroepithelioma; Neurofibroma; Neuroma; Adrenal ganglion; Non-chromogenic apoptosis; Angiokeratoma; Vascular lymphangioproliferation; Eosinophilia; Angiosclerosis; Hemangioma; Glomerular angioma; Hemangioendothelioma; Hemangioma; Hemangioblastoma; Angiosarcoma; Lymphangioma; Lymphangiomyoma; Lymphangiosarcoma; Pineal carcinoma; Carcinosarcoma; Chondrosarcoma; Frond cystic sarcoma; Fibrosarcoma; Angiosarcoma; Leiomyosarcoma; Leukocytoma; Liposarcoma; Lymphangiosarcoma; Myoma; Myxedema; Ovarian Cancer; Rhabdomyosarcoma; Sarcoma; Neoplasm; Neurofibromatosis; And cervical dysplasia.
[250] Another aspect of the invention relates to the use of the materials and methods of the invention to prevent and / or treat hyperproliferative symptoms of skin, including psoriasis and contact dermatitis or other hyperproliferative diseases. Patients suffering from psoriasis and contact dermatitis have demonstrated increased Ang-2 activity in these lesions [Ogoshi et al. J. Inv. Dermatol, 110: 818-23 (1998). Preferably, specific binding agents specific for Ang-2 are used in combination with other pharmaceutical agents to treat the human body expressing such clinical symptoms. In particular, the binder can be delivered using a variety of carriers via the route of administration described herein and other routes of administration well known to those skilled in the art.
[251] Another aspect of the invention provides angiogenesis as well as disorders / diseases in female reproductive tracts such as endometriosis, uterine fibroids and other symptoms associated with dysfunctional vasodilation (including microvascular proliferation of the endometrium) during the female reproductive cycle. Including various retinopathy (diabetic retinopathy and age-related macular degeneration) treatments.
[252] Another aspect of the present invention also includes cerebral arteriovenous malformations (AVMs), damage and healing of gastrointestinal mucosa, ulceration of gastroduodenal mucosa in patients who have had peptic ulcers, and ischemia due to seizures, wide in liver disease It relates to abnormal vasculature treatment, including portal hypertension in patients with wide-spectrum pulmonary vascular disorders and non-hepatic portal hypertension.
[253] Another aspect of the invention relates to the prevention of cancer utilizing the compositions and methods provided by the invention. Such reagents include specific binders for Ang-2.
[254] Pharmaceutical composition
[255] Pharmaceutical compositions of Ang-2 specific binders are within the scope of the present invention. Pharmaceutical compositions comprising antibodies are described in detail, for example, in US Pat. No. 6,171,586 to Lam et al., Issued January 9, 2001. Such compositions include therapeutically and prophylactically effective amounts of specific binding agents, such as antibodies or fragments, variants, derivatives thereof or mixtures thereof, as described herein in admixtures having pharmaceutically acceptable formulations. In a preferred embodiment, the pharmaceutical composition comprises a specific binder antagonist that partially or completely modulates at least one biological activity of Ang-2 with a blend having a pharmaceutically acceptable formulation. In general, specific binders were sufficiently purified for administration to animals.
[256] Pharmaceutical compositions may be formulated to modify, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, aroma, sterilization, stability, rate of dissolution or release, adsorption or permeation of the composition. It includes. Suitable formulation materials include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); Antimicrobial agents; Antioxidants (eg, ascorbic acid, sodium sulfate or sodium hydrogen sulfide); Buffer solutions (eg, borate, bicarbonate, tris-HCl, citrate, phosphate and other organic acids); Volume control agents (eg mannitol or glycine); Chelating agents [eg, ethylenediamine tetraacetic acid (EDTA)]; Complexing agents (caffeine polyvinylpyrrolidone, β-cyclodextine or hydroxypropyl-β-cyclodextrin); Filler; Monosaccharides; Disaccharides and other carbohydrates (eg, glucose, mannose, or dextrins); Proteins (eg, serum albumin, gelatin or immunoglobulins); Pigment; Flavoring and diluents; Emulsifiers; Hydrophilic polymers (eg, polyvinylpyrrolidone); Low molecular weight polypeptides; Salt-forming counterions (eg sodium); Preservatives (eg, benzalkonium chloride, benzoic acid, salicylic acid, chimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); Solvents (eg glycerin, propylene glycol or polyethylene glycol); Sugar alcohols (eg, mannitol or sorbitol); Suspending agent; Surfactants or wetting agents (eg, polysorbates such as pluronic, PEG, sorbitan esters, polysorbate 20, polysorbate 80, tritone, tromethamine, lecithin, cholesterol and tiloxapal); Stability enhancers (sucrose or sorbitol); Tonicity enhancers [alkali metal halide compounds (preferably sodium, potassium chloride, mannitol sorbitol)]; Transport excipients; Diluent; Prosthetics; And / or pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences, 18 th Edition 1990, edited by A. R. Gennaro, obtained from the Mac Publishing Company, which is incorporated herein by reference.
[257] The optimal pharmaceutical composition can be determined by one skilled in the art, for example, depending on the intended route of administration, mode of transport and dosage required. See, eg, Remington's Pharmaceutical Sciences, which is incorporated herein by reference. The composition affects the rate of separation, in vivo release rate, stability and physical state of the specific binder in vivo.
[258] In pharmaceutical compositions, the primary excipient or carrier is substantially aqueous or non-aqueous. For example, a suitable excipient or carrier can be distilled water, physiological saline or artificial cerebrospinal fluid, which is usually another substance that can be supplemented with a composition for parenteral administration if possible. Neutral buffered saline or physiological saline mixed with serum albumin is an additional example of an excipient. Other typical pharmaceutical compositions include tris buffer solutions of about pH 7.0-8.5 or acetate buffers of about pH 4.0-5.5, which further comprise sorbitol or a suitable substituent thereof. In an embodiment of the invention, the binder composition is stored in the form of a lyophilized cake or aqueous solution by mixing the optimal formulation with the optional composition having the desired purity (see Remington's Pharmaceutical Sciences, cited above). To prepare. Moreover, the binder product was formulated as a lyophilized product using a suitable prosthetic such as sucrose.
[259] Pharmaceutical compositions can be selected for parenteral administration. Optionally, the composition is selected for intestinal administration or inhalation such as oral, ear, eye, rectal or vaginal. The preparation of such pharmaceutically acceptable compositions is within the skill of the art.
[260] The formulation component is present at an acceptable concentration at the site of administration. For example, buffers were used to maintain the composition at physiological pH or slightly lower pH, generally from about pH 5 to about pH 8.
[261] When intended for parenteral administration, the therapeutic compositions for use in the present invention are in pyrogen-free form, which is a parenterally acceptable aqueous solution comprising the specific binder required in a pharmaceutically acceptable excipient. Particularly suitable excipients for parenteral injection are sterile distilled water which formulates the binder as sterile, isotonic, which is suitably preserved. Still other formulations are injectable microparticles, bio-corrosive particles, high molecular compounds (polylactic acid and polyglycolic acid) that are transported via depot injection and then provide controlled release and sustained release products. Formulations of the molecule as needed, together with agents such as beads or liposomes. Hyaluronic acid can also be used, which has the effect of increasing the duration in circulation. Other suitable means for injecting the desired molecular weight include implantable drug transport devices.
[262] In another aspect, pharmaceutical formulations suitable for parenteral administration are formulated in aqueous solutions, preferably in physiologically compatible buffer solutions such as Hanks' solution, Ringer's solution or physiological buffered saline. Aqueous injection suspensions include substrates that increase the viscosity of the suspension, such as carboxymethylsodium cellulose, sorbitol or textan. In addition, suspensions of the active compounds were prepared as suitable oily injection suspensions. Suitable lipophilic solvents or excipients include fatty oils such as sesame oil or synthetic fatty acids such as ethyl oxylate, triglycerides or liposomes. Non-lipid polycationic amino polymers were also used for transport. Optionally, the suspension also contains suitable stabilizers or agents which increase the solubility of the compound and allow for the preparation of highly concentrated solutions.
[263] In yet another embodiment, a pharmaceutical composition for inhalation is prepared. For example, the binder is formulated as a dry powder for inhalation. In addition, polypeptide or nucleic acid molecule inhalation solutions were formulated with propellants for erosol transport. In yet another embodiment, the solution is sprayed. Moreover, lung administration is described in PCT Application No. PCT / US94 / 001875, which describes waste transportation of chemically modified proteins.
[264] It is also expected that certain formulations may be administered orally. In embodiments of the present invention, the binder molecule administered in this manner may be formulated with or without a carrier customarily used in compounding with solid pharmaceutical formulations such as tablets and capsules. For example, capsules have been prepared to release the active portion of the formulation to the gastrointestinal tract when bioavailability is maximized and systemic degradation is minimized. Additional agents may be included to promote absorption of the binder molecule. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, disintegrant tablets and binders were also used.
[265] In addition, pharmaceutical compositions for oral administration may be formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated into tablets, pills, sugars, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
[266] Oral pharmaceutical formulations can be obtained by treating the reaction mixture of granules (optionally after grinding) and combining the active compound with a solid prosthetic to obtain tablets or sugar cores. If desired, suitable suppositories can be added. Suitable prosthetics include carbohydrate or protein fillers such as sugars, including lactose, sucrose, mannitol and sorbitol; Corn starch, wheat, rice, potatoes or other plants; Celluloses such as methyl cellulose, hydroxypropylmethyl-cellulose or carboxymethylcellulose sodium; Gums including arabic and tragacanth; And proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, agar and salts thereof such as alginic acid or sodium alginate are added.
[267] The sugar core is used in conjugation with a suitable coating such as concentrated sugar solution, which also contains arabic gum, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacca solution, and suitable Organic solvents or solvent mixtures. Dyestuffs and pigments were added to the tablets or sugar coatings to identify the product or to indicate the amount of active compound such as dosage.
[268] Pharmaceutical preparations that can be used orally also include soft sealed capsules made of gelatin and coatings such as glycerol or sorbitol, as well as push-fit capsules made of gelatin. Push-fit capsules may comprise an active ingredient mixed with a binder or filler such as lactose or starch, a lubricant such as talc or magnesium stearate and optionally a stabilizer. In soft capsules, the active compounds are dissolved or suspended in suitable liquids, such as fatty oils, liquids or liquid polyethylene glycols, with or without stabilizers.
[269] Another pharmaceutical composition comprises an effective amount of the binder in a mixture with a non-toxic prosthetic agent suitable for preparing into a tablet. By dissolving the tablets in sterile water, or other suitable excipients, solutions may be prepared in pharmaceutical formulation units. Suitable prosthetic agents include, but are not limited to: inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; Binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.
[270] Moreover, it will be apparent to those skilled in the art that pharmaceutical compositions include formulations containing binder molecules in sustained-release or controlled release formulations. Techniques for formulating various other sustained or controlled release means such as liposome carriers, bio-corrosive microparticles or porous beads and depot injections are also well known to those skilled in the art. See, for example, patent number PCT / US93 / 00829, which describes controlled release of porous polymeric microparticles for transporting pharmaceutical compositions. Additional examples of sustained release formulations include semipermeable polymer matrices in the form of specified pharmaceuticals, such as, for example, films or microcapsules. Sustained release matrices include polyesters, hydrogels, polylactides (see US Pat. No. 3,773,919 and EP 58,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate [Sidman et al. Biopolymers 22: 547-556 (1983)], poly (2-hydroxyethyl-methylacrylate) [see J Biomed Master Res, Langer et al., 15 167-277 (1981)] and Chem Tech of Langer et al. , 12: 98-105 (1982)] ethylene vinyl acetate (see Langer et al., Incorporated herein by reference) or poly-D (-)-3-hydroxybutyric acid (European Patent No. EP 133,988). Reference). Sustained release compositions also include liposomes that can be prepared by several methods known in the art. See, eg, Proc Natl Acad Sci (US), Eppstein et al., 82: 3688-3692 (1985); European Patent No. EP 36,676; European Patent No. EP 88,046; See European Patent No. EP 143,949.
[271] In general, pharmaceutical compositions used for in vivo administration must be sterile. This can be accomplished by filtration through sterile filtration membranes. When the composition is lyophilized, it is treated before lyophilization and reconstitution or after sterilization using this method. Compositions for parenteral administration are stored in lyophilized form or in solution. In addition, parenteral compositions are generally stored in containers with sterile access ports, such as vials or intravenous solution bags with stoppers that can be inserted into a hypodermic needle.
[272] Once the pharmaceutical composition is formulated, it can be stored in sterile vials such as solutions, suspensions, gels, emulsions, solids or dehydrogenated or lyophilized powders. Such formulations are stored in an easy-to-use form or in a form that requires reconstitution (eg, lyophilized) prior to administration.
[273] In certain embodiments, the invention relates to a kit for providing a single-dose dosage unit. The kits include both primary containers containing dried protein and secondary containers containing aqueous formulations. Kits included within the scope of the present invention also contain single and multi-threaded prefield syringes (eg, liquid syringes and lgosyringes).
[274] The effective amount of the pharmaceutical composition used therapeutically depends, for example, on the treatment situation and the subject. Therefore, those skilled in the art will appreciate that the dosage range suitable for treatment is partially transported, the indication that the binder molecule is used, the route and size of administration (weight, body surface or organ size) and the symptoms of the patient (age and overall health status). Will vary. Thus, the clinician can titrate the dosage and change the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 mg / kg up to about 100 mg / kg or more, depending on the factors mentioned above. In another embodiment, the dosage range is 0.1 mg / kg up to about 100 mg / kg; Or 1 mg / kg up to about 100 mg / kg; Or 5 mg / kg up to about 100 mg / kg.
[275] For a compound, a therapeutically effective amount can be assessed for the first time in an animal model such as mouse, rat, rabbit, dog or pig or in a cell culture assay. Animal models were also used to assess suitable concentration ranges and routes of administration. This information can then be used to determine useful dosages and routes for administration to the human body.
[276] The correct dosage was determined in light of the factors relevant to the subject in need of treatment. Dosage and administration were adjusted to provide sufficient concentration of the active compound or to maintain the desired effect. Factors that may be considered include gastric severity of the disease state, the overall health state of the subject, age, weight and the sex, time and frequency of administration of the subject, drug combination, response sensitivity, and response to treatment. Long-acting pharmaceutical compositions are administered every 3 to 4 days, every week, or every 2 weeks depending on the half-life and rate of elimination of the particular formulation.
[277] The number of doses depends on the pharmacokinetic parameters of the binder molecule of the formulation used. Typically, the composition is administered until the dosage achieves the desired effect. Therefore, the compositions were administered by single or multidose (same or different concentrations / dosages) or by continuous infusion over time. Moreover, improvement of suitable dosages is usually made. Appropriate doses were identified using appropriate dose-response data.
[278] The route of administration of the pharmaceutical composition is consistent with known methods, including, for example: oral; Intravenous, intraperitoneal, intracranial (brain parenchyma), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional route, intramedullary, intradural, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal tract Injection into the topical, sublingual, urethral, vaginal or rectal organs; Sustained release system; Or injection using an implant device. If desired, the composition may be administered by bolus injection or continuously by infusion or implantation device.
[279] Alternatively or additionally, the composition was administered topically through the insertion of a membrane, sponge or other suitable material in which the required molecules were absorbed or encapsulated. When using an insertion device, the device was inserted into an appropriate tissue or organ and the required molecules were transported by diffusion, sustained mass or continuous administration.
[280] In some cases, it is desirable to use the pharmaceutical composition in a disassembly manner. In this case, the cells, tissues, or organs removed from the patient were subsequently transplanted back to the patient after exposure to the pharmaceutical composition.
[281] In other cases, a binder of the present invention, such as a polypeptide, may be transported to specific cells genetically produced using the methods described herein to express and secrete a polypeptide. The cell may be an animal or human cell, and may be autologous, heterologous or heterologous. Optionally, cells multiply indefinitely. Cells were encapsulated to reduce the chance of immune response and to avoid infiltration of surrounding tissues. Encapsulating materials are generally biocompatible, semipermeable polymeric inclusions or membranes, which release protein products but prevent the destruction of cells by other harmful factors from the patient's immune system or surrounding tissue.
[282] Combination Therapy
[283] Specific binding agents of the present invention can be utilized in combination with other therapies in treating Ang-2 beds. Such other therapies include, for example, radiation therapy, chemotherapeutic agents as well as other growth factors.
[284] Chemotherapy treatments can utilize anti-neoplastic agents, including, for example, sulfated agents, including: nitrogen mustard such as mechlorethamine, cyclophosphamide, phosphamide, melphalan and chloroambucil ; Nitrosoureas such as carmustine (BCNU), lomustine (CCNU) and semustine (methyl-CCNU); Ethyleneimine / methylmelamine such as triethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine); Alkyl sulfonates such as busulfan; Triazines such as dacarbazine (DTIC); Folic acid analogues such as methotrexate and trimerectate, pyrimidine analogues such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine) 5-azacity Dine, 2, 2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathiopurine, 2'-deoxycoformycin (pentostatin), erythrohydrate Anti-metabolites including oxynonyl adenine (EHNA), fludarabine phosphate and 2-chlorodeoxyadenosine (cladravin, 2-CdA); Natural products, including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine and vinorelbine, taxotere, esturamustine and esturamustine phosphate; Ppipodophyllotoxins such as etoposide and teniposide; Antibiotics such as acticomycin D, daunomycin (rubidomycin), doxorubicin, mitoxanthrone, idarubicin, bleomycin, pilamycin (mithramycin), mitomycin C, and actinomycin; Enzymes such as L-asparaginase; Biological response modifiers such as interferon-alpha, IL-2, G-CSF and GM-CSF; Platinum coordination complexes such as cisplatin and carboplatin, anthraceneoneone such as mitosantron, substituted ureas such as hydroxyurea, methylhydridine derivatives including N-methylhydrazine (MIH) and procarbazine, mitotan Heterogeneous mixtures including adrenal cortical inhibitors such as (O, P'-DDD); Corticosteroid antagonists such as prednisone and equivalents, hormones and antagonists including dexamethasone and aminoglutetimides; Progestins such as caproic acid hydroxyprogesterone, medoxyprogesterone acetate and megestrol acetate; Estrone, such as diethylstilbestrol and ethynylestradiol equivalents; Antiestrogens such as tamoxifen; Androgens including propionic acid testosterone and fluoxymesterone / equivalents; Anti-androgens such as flutamide, gonadotropin-releasing hormone analogs, and leuprolides; And nonsteroidal anti-androgens such as flutamide.
[285] Combination therapy with growth factors includes: cytokines, lymphokines, growth factors or M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL -5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17 , Hematopoietic factors such as IL-18, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor and erythropoietin. Other compositions include known angiopoietins, such as Ang-1, -2, -4, -Y, and / or human Ang-like polypeptides and / or vascular endothelial growth factor (VEGF). ) May be included. Growth factors include: angiogenin, osteomorphogenic protein-1, osteomorphogenic protein-2, osteomorphogenic protein-3, osteomorphogenic protein-4, osteomorphogenic protein-5, bonemorph Developmental protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11, bone morphogenic protein-12, bone morphogenic Protein-13, Osteomorphic Protein-14, Osteomorphic Protein-15, Osteomorphic Protein Receptor IA, Osteomorphic Protein Receptor IB, Brain-derived Neurotrophic Factor, Cilia Neutrophil, Cilia Neutrophil Receptor, Cytokine Induced neutrophil chemotactic factor-1, cytokine-induced neutrophil, chemotactic factor-2, cytokine-induced neutrophil chemotactic factor-2, endothelial growth factor, endothelin-1, epidermal growth factor, epidermal neutrophil Attractant, Fibroblast Growth Factor-4, Fibroblast Growth Factor-5, Fibroblast Growth Factor-6, Fibroblast Growth Factor-7, Fibroblast Growth Factor-8, Fibroblast Growth Factor-8b, Fibroblast Growth Factor- 8c, fibroblast growth factor-9, fibroblast growth factor-10, acidic fibroblast growth factor, basic fibroblast growth factor, glial cell-derived neutrophil factor receptor-1, glial cell-derived neutrophil factor receptor-2, Growth-related protein, growth-related protein-2, growth-related protein-2, growth-related protein-3, heparin-binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor , Insulin-like growth factor Ⅱ, insulin-like growth factor binding protein, keratinocyte factor, leukemia inhibitor, leukemia inhibitor Body-1, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotropin-4, placental growth factor, placental growth factor-2, platelet-derived endothelial growth factor, platelet derived growth factor, platelet derived Growth factor A chain, platelet-derived growth factor AA, platelet-derived growth factor AB, platelet-derived growth factor B chain, platelet-derived growth factor BB, platelet-derived growth factor receptor-1, platelet-derived growth factor receptor-2, Pre B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor-1, transforming growth factor-2, transforming growth factor-3, transforming growth factor-1.2, transforming growth factor-4 , Transforming growth factor-5, latent transforming growth factor-1, transforming growth factor binding protein I, transforming growth factor binding protein II, transforming growth factor binding protein III, agent Type I tumor necrosis factor receptor, type II tumor necrosis factor receptor, urokinase-type plasminogen activating receptor, endothelial growth factor and chimeric protein and biological or immunological active fragments thereof.
[286] Immunotherapy
[287] In general, immunotherapies rely on the use of molecules and immune effector cells to target and destroy cancer cells. Immune agonists are antibodies of the invention, for example, that recognize some markers on the surface of a target cell. Antibodies may be provided alone as therapeutic agents or supplement other cells to accurately affect cell death. Antibodies are also provided simply as targeting agents by conjugation to drugs or toxins (chemotherapy, radionuclide lysine A chain, cholera toxin, pertussis toxin, etc.).
[288] In accordance with the present invention, mutant forms of Ang-2 were targeted with antibodies or antibody conjugates of the present invention by immunotherapy. In particular, the antibody compositions of the present invention are expected to be used in combination therapy approaches in combination with Ang-2 targeted therapy.
[289] Passive immunotherapy has proven to be particularly effective against numerous cancers. See, for example, patent number WO 98/39027.
[290] The following examples are for illustrative purposes only and do not limit the scope of the invention.
[291] Example 1
[292] Ang-2 expression in pathological and normal tissues
[293] In situ hybridization was used to express Ang-2 in normal and pathological tissues. Human Ang-2 sequence fragments (Genbank Accession No. AF 004327, nucleotides 1274-1726) and mouse Ang-2 sequence fragments (Genbank Accession No. AF004326, nucleotides 1135-1588) were obtained from human or mouse fetal lung cDNA. It was amplified using reverse transcriptase-PCR and cloned into pGEM-T plasmid (available from Promega Corporation) and confirmed by sequencing. ³³ P labeled antisense RNA probes were transcribed from linear plasmid templates using ³³ P-UTP and RNA polymerase. Paraffin embedded tissues to block formaldehyde-immobilization were cut to 5 μm and placed on charged slides. Prior to in situ hybridization, tissue was permeated with 0.2 M HCL, then digested with protease K and acetylated with triethanolamine and anhydrous acetate. Sections were hybridized overnight at 55 ° C. with radiolabeled probes, then digested with RNase and washed very strictly at about 0.1 × SSC at 55 ° C. The slides were immersed in Kodak NTB2 emulsion and then counterstained by exposure and color development at 4 ° C. for 2-3 weeks. Sections were examined with darkfield illumination and standard illumination to simultaneously evaluate tissue morphology and hybridization signals.
[294] This indicates that in normal postnatal human life, Ang-2 expression is limited to very few tissues including angiogenic vasculature such as ovaries, placenta and uterus. Ang-2 was not expressed in the heart, brain, kidney, liver, lungs, pancreas, spleen, muscle, tonsils, thymus, appendix, lymph nodes, gallbladder, prostate or testes of normal adult humans. In five main mice (not including adult monkey or human body), the kidney showed significant Ang-2 expression in the rectum. To determine whether this expression is a remnant of embryogenic development, this experiment was repeated on kidneys derived from mice up to 1 year old using the conditions described above and mouse Ang-2 probes. Ang-2 expression decreased during postnatal development but was still evident in the kidneys of mice 1 year old.
[295] Ang-2 expression was also detected in virtually all tumor types tested, including: primary human tumors such as colorectal cancer (5), breast cancer (10), lung cancer (8), and glioblastoma (1) ; Metastatic human tumors such as ovarian cancer (2), lung cancer (2) and breast cancer (2) that metastasize to the brain; And C6 (rat glioma), HT29 (human colorectal cancer), Colo-205 (human colorectal cancer), HCT116 (human colorectal cancer), A431 (human papilloma carcinoma), A673 (human rhabdomyosarcoma), HT1080 (human fiber Sarcoma), rodent tumor models such as PC-3 (human prostate cancer), B16F10 (mouse melanoma), MethA (mouse sarcoma) and Lewis lung cancer mets. In addition, Ang-2 expression was detected in the immature retina of the neovascular and hypoxic mouse models growing in the Matrigel plug in response to VEGF.
[296] Example 2
[297] Production of Recombinant mAng-2 Protein and Rabbit Polyclonal Anti-Ang-2 Antiserum
[298] Full-length His-labeled mouse Ang-2 cDNA by mouse using PCR primers for full-length human Ang-2 (Clontech Advantage PCR Kit, Cat ## 1905-01) Mouse 15-day embryo cDNA Obtained from the library (Marathon-Ready-cDNA, Cat. No. # 7459-1, available from Clontech Incorporated). The PCR product was bound to the CMV promoter expression vector and the resulting plasmid was HT1080 human fibrosarcoma cells (ATCC Accession No. CCL-121) using FuGENE6 transfection reagent (available from Roche, Cat. No. # 1814443). ) Transfected into). Stable clones were isolated by G418 selection. MAng-2-his expressing clones were screened by anti-His labeled ELISA and western blotting.
[299] Recombinant mAng-2 polypeptide was purified from the control medium of these cells (C.M.). C.M. comprising mAng-2-His. Was purified by a two step chromatography protocol. In brief, the conditioned medium was titrated to pH 8.9 by adding Tris buffer at pH 9.5 to a final concentration of about 20 mM. Additionally, detergent CHAPS was added so that the final concentration was 5 mM. Then C.M. Was added directly to the anion exchange column Q-Sepharose ff (available from Pharmacia). The column was then washed with a pH 8.0 tris of about 10 mM containing about 50 mM NaCl. Recombinant mAng-2-His was eluted in a single step using 10 mM pH 8.0 Tris containing about 350 mM NaCl and about 5 mM CHAPS.
[300] The eluate of the Q-Sepharose column was adjusted to about 4 mM imidazole and then added to a fixed metal affinity column (Ni-NTA superflow (available from Qiagen)). Bound proteins were eluted with PBS containing about 5 mM CHAPS and about 100 mM imidazole. The eluate was concentrated to approximately 1.0 mg / ml and then dialyzed against PBS. The purity of mAng-2-His is higher than 90% as determined by SDS-PAGE Coomassie staining.
[301] Rabbits were immunized with about 0.2 mg of mAng-2 / injection to produce antibodies. To rats 1: 1 ratio were injected with about 1 mL Hunter's TiterMax of ^® (Sigma) and mAng-2. After 4 weeks, each rabbit was injected or repeated immunization; After 2 weeks, the cells were further immunized, and after 7 weeks, serum was collected and titrated for mAng-2. At high serum titers, 50 mL of fresh production bleeds were collected weekly for six consecutive weeks. However, at low serum titers, rabbits were given additional immunization and 50 mL of freshly generated blood was collected weekly for six consecutive weeks beginning at 9 weeks. Six weeks after fresh blood production, rabbits were allowed to rest for six weeks. If more serum was needed, they were further immunized again one month after the last blood production.
[302] Using neutralizing ELISA (described below), two rabbits 5254 and 5255 anti-Ang-2 rabbit polyclonal antiserum were observed to neutralize mAng-2: Tie2 interaction.
[303] Example 3
[304] Molecular assays for measuring Ang-2 antibodies
[305] Molecular assays (affinity ELISA, neutralizing ELISA, and BIAcore) were developed to assess the effects of antibodies under the Ang-2: Tie-2 interaction by direct access to antibodies that bind Ang-2 and related family members. Such ex vivo and cell-based assays are described below.
[306] A. Affinity ELISA
[307] For initial screening of candidate anti-Ang-2 antibodies, purified human Ang-2 polypeptides (obtained from R & D Systems, Inc .; Catalog No. 623-AN; Ang-2 are two truncated variants) Supplied as a mixture of water) or mouse Ang-2 polypeptide (prepared as described above) was used. To confirm the binding assay, human 293T cells transfected with full-length human Ang-2 DNA from human Ang-2 (obtained from Ginhunter Corporation, Nashville Grasmere Park Drive 624, 37211, Tennessee, USA, catalog number Q401) And cultured in serum-free DMEM containing about 50 μg / ml bovine serum albumin (BSA).
[308] Using a microplate, approximately 100 μl / well of Ang-2 was added to each well and the plate was incubated for about 2 hours, followed by phosphate buffered saline (PBS: phosphate) containing about 0.1% of Tween-20. buffered saline) four times. The wells were then blocked using about 250 μl / well PBS containing about 5% BSA and the plates were incubated at room temperature for about 2 hours. After incubation, excess blocking solution is removed and approximately 100 μl of the candidate anti-Ang-2 antigen is serially diluted starting at a concentration of about 40 nM and subsequently diluted four-fold with PBS containing about 1% BSA. To each well. The plate was then incubated overnight at room temperature. After incubation, the plates were washed with PBS containing about 0.1% of Tween-20. After 4 additional washes, goat anti-human IgG (Fc) -HRP (obtained from Pierce Chemical Company) already diluted 1: 5000 with PBS containing 1% BSA (serum albumin), catalog No. # 31416) was added. This plate was incubated for approximately 1 hour at room temperature. The plate was then washed five times with PBS containing about 0.1% Tween-20, followed by about 100 μl / well of TMB (3,3 ＇, 5,5′-tetramethylbenzidine liquid substrate system; St. Missouri, USA Substrate No. T8665 obtained from Sigma Chemical Company, Lewis) substrate was added and the plate was incubated for about 5-15 minutes until blue color appeared. Then, the absorbance at about 370 mm was measured with a spectrophotometer.
[309] B. Neutralization ELISA
[310] Microplates bound to human Ang-2 polypeptides were prepared by the affinity ELISA described above. Candidate anti-Ang-2-antibodies are provided as Tie-2-Fc molecules containing only about 1% BSA and about 1 nM Tie2 (the extracellular portion where the Tie-2 moiety is soluble of the molecule; obtained from R & D Systems) Prepared by serial dilution with PBS solution containing one catalog number 313-TI) as described for the affinity ELISA. About 100 ml of antibody / Tie-2 solution was added to each well, and then the plate was incubated overnight at room temperature and washed five times with PBS containing about 0.1% Tween-20. After washing, about 100 μl / well of anti-Tie-2 antibody (obtained from Paminegen, Cat. No. # 557039) is added to a final concentration of about 1 μg / ml and the plate Incubated at room temperature for about 1 hour. Then about 100 μl / well of goat anti-mouse-IgG-HRP (obtained from Pierce Chemical Company, Cat. No. # 31432) was added diluted 1: 10,000 with PBS containing about 1% BSA. It was. After incubating the plate at room temperature for about 1 hour, the plate was washed five times with PBS containing about 0.1% of Tween-20. About 100 μl / well of TMB substrate (described above) was added and allowed to color. Then, the absorbance was measured at 370 nm with a spectrophotometer.
[311] C. Friendly BIAcore
[312] Each candidate of Biacore that material to Ang-2 peptides of 0.005% expansion buffer affinity black body P20 surfactant (Biacore, Inc. ray obtained from Inc.) and BIAcore ^® 2000 with PBS (New Jersey, USA, Piscataway Run from Corporation). Recombinant G protein (obtained from Repleygen, Needham, Mass., USA) was studied via an initial amine group using an amine binding kit (obtained from Biacore Inc.) according to the manufacturer's proposed protocol. (CM5 Sensor chip, available from BIAcore Inc.).
[313] A binding assay was first performed by attaching about 100 Ru of each candidate anti-Ang-2 antibody to the immobilized G protein, followed by varying concentrations of huAng-2 or mAng-2 (0-100 nM) for 3 minutes. The bound antibody surface was injected at a flow rate of 50 μl / min. BIA Evaluation 3.1 The antibody binding kinetics including k _a (set rate constant), k _d (dissociation rate constant), and K _D (dissociation equilibrium constant) were measured using a computer program (obtained from Biacore Inc.). . Low dissociation equilibrium constants indicate a high affinity of the peptibody for Ang-2.
[314] Example 4
[315] Production of Fully Human Ang-2 Antibodies by Phage Display
[316] Full human Ang-2 antibodies were subjected to target quest phage display Fab libraries (target quest incorporation) against human Ang-2 polypeptides (available from R & D Systems Inc., catalog 623-AN) according to the following protocol. Available from Tied).
[317] Human Ang-2 was immobilized on the surface of polystyrene magnetic beads according to the following two methods: (1) direct coating Ang-2 at 50 ug / ml at 4 ° C. overnight; And (2) indirect capture of Ang-2 with goat anti-Ang-2 antibody at 50 ug / ml overnight at 4 ° C. Bead surfaces were blocked with 2% milk (MPBS) dissolved in PBS. Human Fab phage libraries (obtained from Target Quest Inc.) were pre-screened to remove phage clones for uncoated magnetic beads or goat anti-Ang-2 antibodies. Ang-2-coated magnetic beads were then incubated with library phage for 1.5 hours at room temperature. After the phage binding step, six washes with MPBS containing about 0.1% Tween 20, followed by six washes with PBS containing about 0.1% Tween 20 and two washes with PBS. Binding phages were first eluted with about 100 ug / ml of human Tie2-Fc (obtained from R & D Systems, Minneapolis, Minn.) And then eluted with about 100 mM triethanolamine. Eluted phages were infected and amplified into E. coli TG1 cells (available from Amesham Pharmacia Biotech) and obtained for screening. A more rigorous wash was applied and the number of input phages was reduced to continuously increase the selection pressure. After tertiary screening, 18 specific Ang-2-binding Fab clones were identified that were all recognizing human Ang-2, mouse Ang-2 and rat Ang-2 measured using an ELISA affinity assay as described above. It was. About 10% of the phages also bind to human Ang-1. These clones were converted to IgG1 antibodies as described below.
[318] In order to obtain additional specific phage, secondary screening was carried out in a slightly different protocol using the same library. In this protocol, human Ang-2 was plated in NaHCO3 buffer at pH 9.6 in Nunc maxithorpe immunotubes at about 4 ° C. overnight. Ang-2 was plated at about 1.5, 0.74 and 0.3 ug / ml for the 1st, 2nd and 3rd runs, respectively. Dissolve in PBS before incubating with about 2 × 10 ¹² phage particles (about 50 replicates of each particular phage in the library) from the same phage display library (obtained from Target Quest) described above in about 4 ml of 2% MPBS. Approximately 2% milk (MPBS) was used to block the surface of the immunotubes. After the phage incubation step, the surface was washed 20 times with PBS and about 0.1% Tween 20 and then 20 times with PBS. Bound phages were eluted using 1 uM hAng-2 or 1 uM human Tie2 (obtained from R & D Systems, described above). Eluted phages were infected and amplified into E. coli TG1 cells (provided with phage libraries) and obtained for the next step, screening. Sixteen specific Ang-2-binding Fab clones were identified by PCR amplification of all phages that bind hAng-2 or Tie2 and analyzed by restriction cleavage. DNA of each clone was sequenced.
[319] The sequence encoding each heavy chain variable region from each phage was amplified with complementary primers. Primers were generated to bind the HindIII site, XbaI site, Kozak sequence and signal sequence (translated peptide is MDMRVPAQLLGLLLLWLRGARC; SEQ ID NO: 69) to the 5 'end of the variable region and the BsmBI site was 3 Added at the end. As an example of how to clone the heavy chain, the template phage DNA for clone 544 (Seq ID NO. 19) was added to the primer 2248-21 (GTG GTT GAG AGG TGC CAG ATG TCA) with the signal sequence of 7 amino acids at the end. Amplification using GGT CCA GCT GGT GCA G; SEQ ID NO: 70) and primer 2502-31 (ATT ACG TCT CAC AGT TCG TTT GAT CTC CAC; SEQ ID NO: 71) with BsmBI site added at the end of the variable region I was. The resulting product was subjected to primer 2148-98 (CCG CTC AGC TCC TGG GGC TCC TGC TAT TGT GGT TGA GAG GTG CCA GAT; SEQ ID NO: 9) with 9 amino acids added to the signal peptide (AQLLGLLLL; SEQ ID NO: 73). 72) and using primer 2502-31 followed by primer 2489-36 (CAG CAG AAG CTT CTA GAC CAC CAT GGA CAT GAG GGT CCC CGC TCA GCT CCT GGG; SEQ ID NO: 74) and primer 2502-31 By amplification. To the primers 2489-36 were added the HindIII site, XbaI site, Kozak sequence and signal sequence of the first 6 amino acids from 5 'to 3'. PCR products were digested with XbaI and BsmBI and cloned into mammalian expression vectors containing human IgG1 constant regions. This vector includes the SV40 promoter and DHFR selection.
[320] The light chain from each phage is of kappa or lambda class. In each light chain, complementary primers were generated by adding the HindIII site, XbaI site, Kozak sequence and signal sequence (see above) from 5 'to 3'. This chain with error free coding regions was cloned as a full length product. For example, primer 2627-69 (GTG GTT GAG AGG TGC CAG ATG TGA CAT TGT GAT GAC TCA GTC TCC) with the signal sequence of 7 amino acids at the end of the light chain from phage clone 536 (SEQ ID NO. 11) (SEQ ID NO: 75) and primers 2458-54 (CTT GTC GAC TTA TTA ACA CTC TCC CCT GTT G; SEQ ID NO: 76) with SalI site added after the stop codon were amplified as full length coding regions. This PCR product was then amplified with additional 5 'primers, 2148-98 and 2489-36, respectively, as described previously and paired with primers 2458-54 to complete the addition of the signal sequence and cloning site. The full length light chain was cloned into the mammalian expression vectors described above as XbaI-SalI fragments.
[321] Certain lambda clones have errors in their constant regions when compared to natural human constant region sequences. To correct this inconsistency, duplicate PCR was performed using DNA and variable region derived phages encoding complete lambda constant regions. Such clones were also cloned as XbaI-SalI fragments as described above.
[322] The kappa variable region was cloned separately from its constant region and the BsmBI site was added to the 3 'end of the PCR product. After cleavage of the PCR product with XbaI and BsmBI, the kappa chain variable regions were cloned into expression vectors containing human kappa constant regions.
[323] Paired light and heavy chain constructs from each converted phage were used to calculate CHO cells (ATCC Accession No. CRL 9096) using calcium phosphate transfection kit (available from Invitrogen Corp.) generally in accordance with the manufacturer's suggested protocol. Co-transfected into. The medium was replaced 14-16 hours after transfection and cells were transferred to tissue culture dishes for selection after about 48 hours according to the manufacturer's recommendations. Transfected cells were separated by HT selection for about 3 weeks, which is the time to trypsinize the transfected CHO cell colonies and bind to a pool of transfected cells.
[324] Small scale media were collected after 48 hours and assayed for antibody production by Western blotting analysis using anti-human Fc antibody, anti-human kappa antibody or anti-human lambda antibody. The selected cell populations were then inoculated with 2 × 10 ⁷ viable cells in four 850 cm 2 rotary bottles, respectively, using standard tissue culture sterilization techniques to obtain enough cells to prepare cryopreserved cell lines using DMSO. Passed under select pressure until After inoculation, cells were kept in a carousel with about 10% serum containing DMEM medium (Gibco / Bial, available from Incorporated) supplemented with glutamine and non-essential amino acids. Cells were maintained for 2 to 3 days until a cell population of about 80% was reached. At this point, the medium in the carousel was exchanged with a serum-free medium mixture (50% DMEM and 50% F12, available from Gibco) supplemented with glutamine and non-essential amino acids. After 7 days a conditioned medium was obtained and fresh serum-free medium was added for one or two additional harvests.
[325] G protein affinity chromatography was used to purify the antibody directly from the control medium following the manufacturer's procedure. Elution from the G protein column was done using low pH (about pH 3) buffer and the eluted antibody protein was neutralized using 1 M Tris at pH 8.5 and then concentrated using a 10 kD fractional molecular weight centrifuge concentrator. . The buffer was replaced with PBS in the concentrated antibody source.
[326] 31 antibodies were generated, each consisting of two heavy chains and two light chains (kappa or lambda) as described in Table 2.
[327]
[328]
[329] The four tables below show the sequences and SEQ ID NOs of the heavy and light chains (kappa and lambda) of 31 anti-Ang-2 antibodies converted to full length IgGl antibodies in phage. Complementary determinants (CDRs) of monoclonal antibodies can be prepared using techniques described in Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242; US Dept. Health and Human Services, 5 ^th ed.) By Kabat et al. Expected using a VBASE database. Fab regions are sequenced in a database (see http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ALIGNMENTS.html) with an impractical close germline sequence. After alignment, these sequences were visually compared. CDRs for each variable region (heavy or light) are shown in Table 7.
[330]
[331]
[332]
[333]
[334]
[335]
[336]
[337]
[338]
[339]
[340] Seventeen antibodies and negative control IgG1 (named RDB1) were tested using affinity and neutralizing ELISA (as described in Example 3 above) as well as Biacore neutralization assays to determine their affinity, neutralization and specificity capabilities. The results are shown in Table 8 below and were calculated using standard methods.
[341]
[342] Two antibodies, clones 536 and clone 545, were measured with the Biacore assay described above. Antibody binding was detected as described above in the Biacore assay and had a low K _D indicating greater affinity. The results are shown in Table 9 below.
[343]
[344] Example 5
[345] Therapeutic efficacy studies with anti-Ang-2 antibodies
[346] G Protein Purification Pharmacokinetics of rabbit anti-Ang-2 polyclonal antibodies were tested in mice. 24 mice were treated with polyclonal anti-Ang-2 rabbit antibody (1 mg / mouse). Four treated animals were sacrificed after antibody injection at 1 hour, 6 hours, 1 day, 3 days, 7 days and 14 days.
[347] The total rabbit IgG has a circulating half-life in serum of about 19 days and the anti-Ang-2 IgG component of total IgG has a half-life of about 8 days.
[348] To assess therapeutic efficacy, G protein purification after 1, 5, 6, 7, 8, 12, 13, 14, 15 and 18 days of xenograft in mice (10 animals / group) showing A431 tumor xenografts. Anti-mAng-2 polyclonal antibodies were administered intraperitoneally at 10 doses (about 10 mg IgG / mouse / dose). Tumor size was measured at 7, 12, 15, 19 and 21 days. Body weights were measured on days 0, 7, 15 and 21 and body weight was not affected by treatment. Anti-Ang-2 polyclonal antibody is p = 0.008 for control of non-immune purified polyclonal antiserum (10 mg IgG / mouse / dose) and excipient (PBS) (using repeated measures ANOVA) A431 tumor The results show that they inhibit about 50% growth of xenografts.
[349] To test the in vivo efficacy of fully human monoclonal anti-Ang-2 antibodies, mice (10 animals / group) representing A431 tumor xenografts were treated with anti-Ang-2 antibody clones 533, 537 or 544 or PBS. A negative control of human IgG1-kappa was administered intraperitoneally. The dose was about 420 ug protein / mouse at the first dose, about 140 ug protein / mouse at the next three doses, and about 55 ug protein / mouse at the next four doses, for a total of eight doses per mouse. Tumor size and body weight were recorded every two weeks. At the end of the study animals were sacrificed and serum was obtained to determine antibody concentration by ELISA. Tumors and normal tissues were collected in all groups.
[350] A marked difference in tumor growth between the anti-Ang-2-treated and control groups is shown in FIG. 1. A total of three anti-Ang-2 treatments inhibited tumor growth compared to the control (p <0.005 for the hIgG1 control, repeated measures ANOVA for a total of three antibodies in all treatments). In contrast, tumors in the control grew at a much higher rate.
[351] Example 6
[352] Epitope mapping
[353] Full length (amino acids 1-495), N-terminus (amino acids 1-254) and C-terminus (amino acids 255-495) human Ang-2 (hAng-2) proteins with CMV-derived mammals with a 6xHis label at the C-terminus It was cloned into a CMV-driven mammalian expression vector. The resulting three constructs and vector control were transiently expressed into 293T cells. Modulated media were then collected from the transfected cells and Ang-2 expression levels in the medium were detected by anti-6 × his ELISA and Western blotting.
[354] Binding epitopes of anti-Ang-2 antibodies and peptibodies were determined by their ability to bind three human hAng-2s by ELISA according to the following protocol: High-binding 96-well assay plates with 100 μl of control medium per well After coating it was incubated at 37 ℃ for 1 hour. The conditioned medium was removed and the plate blocked with 5% BSA dissolved in 200 μl PBS per well for 1 hour at room temperature. The blocking solution was then removed. 100 μl of antibody, peptibody or Tie2-Fc per well was added at 1 μg / ml in 1% BSA dissolved in PBS and incubated for 1 hour at room temperature. Wells were washed four times with 200 μl 0.1% Tween dissolved in PBS. 100 μl of HRP-conjugated goat anti-human IgG or goat anti-mouse IgG per well was added and incubated for 45 minutes at room temperature. The wells were then washed four times with 200 μl 0.1% Tween dissolved in PBS. Then 100 μl of TMB substrate was added per well. O.D. Was read at 370 nm.
[355] The results are shown in Figures 2A, 2B and 2C.

权利要求:
Claims (37)
[1" claim-type="Currently amended] A specific binder comprising one or more peptides and fragments thereof selected from the following.

[2" claim-type="Currently amended] The specific binding agent of claim 1, wherein the specific binding agent is an antibody.
[3" claim-type="Currently amended] The antibody of claim 2 is a polyclonal antibody, monoclonal antibody, chimeric antibody, humanized antibody or fully human antibody.
[4" claim-type="Currently amended] The antibody of claim 3, wherein the antibody is a single chain antibody.
[5" claim-type="Currently amended] A hybridoma producing the monoclonal antibody of claim 3.
[6" claim-type="Currently amended] A conjugate comprising the specific binder of claim 1.
[7" claim-type="Currently amended] A conjugate comprising the antibody of claim 2, 3 or 4.
[8" claim-type="Currently amended] A nucleic acid molecule encoding the specific binding agent of claim 1.
[9" claim-type="Currently amended] A nucleic acid molecule encoding the antibody of claim 2, 3 or 4.
[10" claim-type="Currently amended] A vector comprising the nucleic acid molecule of claim 8.
[11" claim-type="Currently amended] A host cell containing the vector of claim 10.
[12" claim-type="Currently amended] How to prepare a specific binder comprising the following steps
(a) one or more nucleic acids encoding the specific binding agent of claim 1
Transforming the host cell with the molecule;
(b) expressing a nucleic acid molecule in the host cell; And
(c) separating the specific binder.
[13" claim-type="Currently amended] Method for preparing an antibody comprising the following steps
(a) one encoding the antibody of claim 2, 3 or 4 or
Transforming the host cell with more nucleic acid molecules;
(b) expressing a nucleic acid molecule in the host cell; And
(c) separating the specific binder.
[14" claim-type="Currently amended] A method of inhibiting undesirable angiogenesis in a mammal comprising administering a therapeutically effective amount of the specific binding agent of claim 1.
[15" claim-type="Currently amended] A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the specific binding agent of claim 1.
[16" claim-type="Currently amended] A method of inhibiting undesired angiogenesis in a mammal comprising administering a therapeutically effective amount of the antibody of claim 2, 3 or 4.
[17" claim-type="Currently amended] A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the antibody of claim 2, 3 or 4.
[18" claim-type="Currently amended] A pharmaceutical composition comprising the specific binding agent of claim 1 and a pharmaceutically acceptable formulation.
[19" claim-type="Currently amended] A pharmaceutical composition comprising the antibody of claim 2, 3 or 4 and a pharmaceutically acceptable agent.
[20" claim-type="Currently amended] A method of modulating or inhibiting angiopoietin-2 activity, comprising administering the specific binding agent of claim 1.
[21" claim-type="Currently amended] A method of modulating or inhibiting angiopoietin-2 activity, comprising administering the antibody of claim 2, 3 or 4.
[22" claim-type="Currently amended] A method of controlling one or more vascular permeability or plasma leakage in a mammal comprising administering a therapeutically effective amount of the specific binding agent of claim 1.
[23" claim-type="Currently amended] A neovascular disease, obesity, hemangioblastoma, hemangioma, arteriosclerosis, inflammatory disease, inflammatory disorders, atherosclerosis, endometriosis, tumorigenicity in a mammal, comprising administering a therapeutically effective amount of the specific binding agent of claim 1 A method of treating one or more of the diseases, bone-related diseases or psoriasis.
[24" claim-type="Currently amended] A method of modulating one or more vascular permeability or plasma leakage in a mammal comprising administering a therapeutically effective amount of the antibody of claim 2, 3 or 4.
[25" claim-type="Currently amended] A neovascular disease, obesity, hemangioblastoma, hemangioma, atherosclerosis, inflammatory disease, inflammatory disorders, atherosclerosis in a mammal, comprising administering a therapeutically effective amount of the antibody of claim 2, 3 or 4 A method of treating one or more of sclerosis, endometriosis, neoplastic disease, bone-related disease or psoriasis.
[26" claim-type="Currently amended] A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the specific binding agent of claim 1 and a chemotherapeutic agent.
[27" claim-type="Currently amended] 27. The method of claim 26, wherein the specific binder and the chemotherapeutic agent are not administered simultaneously.
[28" claim-type="Currently amended] A method of treating cancer in a mammal comprising administering a therapeutically effective amount of the antibody of claim 2, 3 or 4 and a chemotherapeutic agent.
[29" claim-type="Currently amended] 27. The method of claim 26, wherein the specific binder and the chemotherapeutic agent are not administered simultaneously.
[30" claim-type="Currently amended] A specific binder comprising one of the following CDR 1.

[31" claim-type="Currently amended] A specific binding agent comprising one of the following CDR 2 's.

[32" claim-type="Currently amended] A specific binding agent comprising one of the following CDR 3 's.

[33" claim-type="Currently amended] 33. A nucleic acid molecule encoding the specific binder of any one of claims 30, 31 and 32.
[34" claim-type="Currently amended] (a) contacting the biological sample with the specific binder of claim 1, and
(b) measuring the degree of binding of the specific binder to the biological sample
Steps to
Comprising, angiopoietin-2 concentration in a biological sample.
[35" claim-type="Currently amended] (a) contacting the antibody of claim 20 with a biological sample, and
(b) measuring the degree of binding of the antibody to the biological sample, the method for detecting the concentration of angiopoietin-2 in the biological sample.
[36" claim-type="Currently amended] Antibodies containing heavy and light chains:
The heavy chain comprises a variable region of a heavy chain selected from

The light chain includes the variable region of the light chain selected from

[37" claim-type="Currently amended] A nucleic acid molecule encoding the antibody of claim 36.

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JP2005506067A|2005-03-03|

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EP1495053B1|2012-06-13|

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HK1152949A1|2014-04-25|

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KR20100049682A|2010-05-12|

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EP2272869A3|2011-05-11|

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EP1495053A4|2006-05-31|

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AU2002343498B2|2006-04-13|

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JP5044608B2|2012-10-10|

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CN101671393A|2010-03-17|

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EA010726B1|2008-10-30|

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CY1112993T1|2016-04-13|

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CA2461435A1|2003-04-17|

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MXPA04003345A|2004-07-08|

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US20030124129A1|2003-07-03|

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KR101089844B1|2011-12-05|

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RS51437B|2011-04-30|

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CN100595211C|2010-03-24|

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NO20041907L|2004-05-10|

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NZ575673A|2011-03-31|

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PL373911A1|2005-09-19|

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US7521053B2|2009-04-21|

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DK1495053T3|2012-09-24|

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SI1495053T1|2012-09-28|

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EP2272869B1|2013-11-20|

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BR0213224A|2007-03-27|

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HU0500994A2|2007-11-28|

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ES2389060T3|2012-10-22|

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EP2272869A2|2011-01-12|

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IL161021A|2013-08-29|

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JP4637480B2|2011-02-23|

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EP1495053A2|2005-01-12|

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AU2011200824A1|2011-03-17|

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:
2001-10-11|Priority to US32860401P

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2001-10-11|Priority to US?60/328,604?

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2002-10-10|Priority to US?10/269,805?

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2002-10-10|Priority to US10/269,805

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2002-10-11|Application filed by 암젠 인코포레이티드

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2002-10-11|Priority to PCT/US2002/032613

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2004-06-18|Publication of KR20040051606A

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2011-12-05|Application granted

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2011-12-05|Publication of KR101089844B1

优先权:

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申请号 | 申请日 | 专利标题

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US32860401P| true| 2001-10-11|2001-10-11|

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US?60/328,604?|2001-10-11|

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US?10/269,805?|2002-10-10|

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US10/269,805|US7521053B2|2001-10-11|2002-10-10|Angiopoietin-2 specific binding agents|

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PCT/US2002/032613|WO2003030833A2|2001-10-11|2002-10-11|Angiopoietin-2 specific binding agents|