RU2361614C2 - APPLICATION OF ANTIBODIES AGAINST α5β1 FOR INHIBITION OF CANCER CELLS PROLIFERATION - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions concerns medicine, namely, to oncology and can be used for treatment of the cancer expressing intergrin α5β1. The ways under the invention include introduction of the liquid pharmaceutical composition containing 0.1-15 mg/ml of an antibody against α5β1 to the patient.

EFFECT: possibility to suppress proliferation and to prevent diffusion of a tumour at the expense of direct lysis of cancer cells at fixation by an intergrin α5β1 antibody on their surface.

22 cl, 2 dwg, 6 tbl, 9 ex

Description

FIELD OF THE INVENTION

The present invention relates to antibodies that specifically recognize α5β1 integrin expressed on the surface of cancer cells, and to methods of using antibodies to inhibit the proliferation of such cancer cells.

State of the art

The association of α5β1 integrin with tumor angiogenesis is well known (see, for example, published US patent application 2002/0172675 A1, filed May 7, 1999, which is incorporated herein by reference). α5β1 is a heterodimeric integrin that specifically binds to the ligand fibronectin. α5β1 is expressed on the surface of endothelial cells and mediates adhesion and migration to fibronectin. It was shown that the binding of α5β1 and fibronectin is an important event of tumor angiogenesis. Tumor angiogenesis begins with the release of one or more pro-angiogenic factors (e.g., FGF, VEGF, PDGF, etc.) that locally activate endothelial cells. These activated endothelial cells then form new blood vessels by binding, through their α5β1 integrin, to extracellular matrix fibronectin. Antibodies against α5β1 have been shown to inhibit angiogenesis in tumor models in vivo (see, for example, US 2002/017675 A1).

Cancer therapy directed against angiogenesis is based on inhibiting vascularization of tumors and, thus, preventing their growth and metastasis (for a review see, for example, Marx, "A Boost for Tumor Starvation," Science 301, 452 (2003); Sato , "Molecular Diagnosis of Tumor Angiogenesis and Anti-Angiogenic Cancer Therapy," Int. J. Clin. Oncol. 8, 200 (2003); Bissachi et al., "Anti-Angiogenesis and Angioprevention: Mechanisms, Problems and Perspectives," Cancer Detec. Prev. 27, 229 (2003)). More than 60 therapeutic substances against angiogenesis are currently in clinical development for cancer treatment. Although in some cases it is possible to make a tumor “starve”, preventing its vascularization, existing studies show that some types of cancer appear to be insensitive to antiangiogenic therapy (see Sato, supra). For example, clinical trials of a therapeutic antibody against VEGF, AVASTIN ™ (bevacicumumab) have been successful in the case of colon cancer, but not for breast cancer (see Marx, above). In addition, therapeutic methods directed against angiogenesis are not suitable for treatment at an early stage, when the process of vascularization of the tumor has not yet begun.

Because of its function in angiogenesis, α5β1-integrin has been proposed as a therapeutic target for numerous diseases mediated by angiogenic processes, including the growth of cancerous tumors. Chimeric and humanized antibodies to α5β1 have been developed that block specific binding to fibronectin. The chimeric α5β1 antibody, M200 (also known by its generic name volociximab), has been shown to induce apoptosis of activated endothelial cells in vitro regardless of growth factor stimuli.

Thus, there is a need for methods of treating cancer and methods for treating an early stage of cancer, capable of directly killing cancer cells before the start of the tumor vascularization process, or when targeted antiangiogenic therapy is ineffective.

SUMMARY OF THE INVENTION

The present invention relates to methods for the lysis of cancer cells using antibodies against α5β1. In a general embodiment, the method comprises contacting a cancer cell that expresses α5β1 on its surface with an anti-α5β1 antibody.

In one preferred embodiment, the present invention relates to a method for inhibiting the proliferation of a cancer (or tumor) cell that expresses on its surface an α5β1 integrin comprising contacting the tumor cell with an antibody that binds to the α5β1 integrin expressed on the surface of the tumor cell. In a preferred embodiment, the patient’s tumor cell is a non-treatable solid tumor tumor cell. In another preferred embodiment, the tumor cell is a cancer cell, where the cancer is selected from the group consisting of: bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, cancer ovary, kidney cancer, and spleen cancer.

In another embodiment, the present invention relates to a method for inducing the death of a tumor cell that expresses on its surface an α5β1 integrin comprising contacting the tumor cell with an antibody that binds to the α5β1 integrin expressed on the surface of the tumor cell. In a preferred embodiment, the patient's tumor cell is a non-treatable solid tumor cell. In another preferred embodiment, the tumor cell is a cancer cell, where the cancer is selected from the group consisting of: bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, cancer ovary, kidney cancer and spleen cancer.

In another embodiment, the invention relates to a method for inhibiting cancer cell proliferation in a patient, wherein the cancer cell expresses α5β1 integrin on its surface. In this embodiment, the method comprises administering to the patient a therapeutically effective amount of an antibody, wherein the antibody competitively inhibits the binding of M200 to α5β1 integrin on the surface of the cancer cell. In another embodiment, the antibody comprises a variable region with an amino acid sequence substantially identical to SEQ ID NOs: 2, 4, 6, and 8. In a preferred embodiment of the method, the antibody administered to a patient neutralizes at least one biological activity of α5β1 integrin. In another embodiment, an antibody administered to a patient comprises a therapeutic effector moiety (e.g., an antibody-drug conjugate). In yet another embodiment of the method, the antibody is administered to a patient sequentially or together with another chemotherapeutic agent. In another preferred embodiment of the method, the antibody is administered to a patient with an untreatable solid tumor sequentially or together with another chemotherapeutic agent.

In another embodiment, the present invention relates to a method for treating an individual who is suspected of having cancer that expresses α5β1 on the surface of the cells, where the cancer has not yet spread in the individual, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an antibody that binds to α5β1 -integrin. In a preferred embodiment, the cancer is selected from the group consisting of bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer and spleen cancer.

In another embodiment, the present invention relates to a method of treating an individual with a genetic predisposition to cancer that expresses α5β1, where the cancer has not yet spread in the individual, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising an antibody that binds to α5β1 integrin. In a preferred embodiment, the cancer is selected from the group consisting of bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer and spleen cancer.

Preferred anti-α5β1 antibodies used in the methods of the present invention include IIA1, M200, F200 and antibodies that specifically bind to the same α5β1 epitope as IIA1, M200 and F200 antibodies.

In another embodiment, anti-α5β1 antibodies that can be used in the methods of the invention include antibodies that competitively inhibit the binding of IIA1 and / or M200 to α5β1 integrin expressed on the surface of the tumor cell.

Other antibodies that can be used in the method according to the invention include antibodies containing the amino acid sequence of the variable region, essentially identical to SEQ ID NO: 2, 4, 6 and 8. The invention also includes antibodies containing amino acid sequences of the variable region, which are at least about 90%, 95%, 98% or preferably 99% or more identical to SEQ ID NO: 2, 4, 6, and 8.

In another embodiment, the invention relates to antibodies against α5β1, which are part of the pharmaceutical composition. The pharmaceutical compositions can be used in various methods described in the present invention. In various embodiments, pharmaceutical compositions containing anti-α5β1 antibodies can be administered in a therapeutically effective amount to an individual in various ways, including, but not limited to, oral, subcutaneous, topical, intravenous, intranasal, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal , rectally, intraocular, intraventricular or intrathecal. In a preferred embodiment, the pharmaceutical composition is a liquid composition comprising from about 1.0 mg / ml to 15 mg / ml anti-α5β1 antibody, about 22-27 mM citrate, about 145-165 mM sodium chloride, about 0.04% - 0.06% polysorbate (TWEEN®) 80 at a pH of approximately 5.5-7.5. In another preferred embodiment, the pharmaceutical composition is a liquid composition containing about 10 mg / ml anti-α5β1 antibody, about 25 mM citrate, about 150 mM sodium chloride, about 0.05% polysorbate (TWEEN®) 80, at a pH of about 6.5 . In a particularly preferred embodiment, the pharmaceutical composition is a liquid composition comprising about 10 mg / ml M-200, about 25 mM citrate, about 150 mM sodium chloride, about 0.05% polysorbate (TWEEN®) 80 at a pH of about 6.5. In another preferred embodiment, each of the pharmaceutical compositions described herein may further comprise a chemotherapeutic agent. In another embodiment, a pharmaceutical composition comprising an anti-α5β1 antibody may be administered to a patient together with a pharmaceutically effective amount of another chemotherapeutic agent.

The pharmaceutical compositions described above can be used in a method of treating a patient diagnosed with cancer or suspected of having cancer selected from the group consisting of: bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer, and spleen cancer, where the method comprises administering to a patient a therapeutically effective dose of a liquid composition containing about approximately 1.0 mg / ml to 15 mg / ml anti-α5β1 antibody, approximately 22-27 mM citrate, approximately 145-165 mm sodium chloride, approximately 0.04% -0.06% polysorbate (TWEEN®) 80 at pH approximately 5.5-7.5. In one embodiment of the methods of treatment, a therapeutically effective dose is administered at about 10 mg / kg. In a preferred embodiment, the patient receiving the pharmaceutical composition is diagnosed with kidney cancer or metastatic melanoma or is suspected of having kidney cancer or metastatic melanoma, and a therapeutically effective dose is approximately 10 mg / kg.

Brief Description of the Drawings

Figure 1 shows: (A) The nucleic acid sequence (SEQ ID NO: 1) and the amino acid sequence (SEQ ID NO: 2) V _H IIF1; (B) The nucleic acid sequence (SEQ ID NO: 3) and the amino acid sequence (SEQ ID NO: 4) V _L IIA1.

Figure 2 shows: (A) The nucleic acid sequence (SEQ ID NO: 5) and the amino acid sequence (SEQ ID NO: 6) V _H M200; (B) The nucleic acid sequence (SEQ ID NO: 7) and the amino acid sequence (SEQ ID NO: 4) V _L M200.

DETAILED DESCRIPTION OF THE INVENTION

For clarity and better understanding, the following detailed description illustrates embodiments and examples of the present invention. Embodiments and examples of the present description are not intended to limit the scope of the invention. One skilled in the art will appreciate that equivalent materials and / or methods can be used and / or obvious changes, variations or modifications can be made to any of the described embodiments and examples without departing from the scope of the appended claims.

All publications and patent applications cited in the description are hereby incorporated by reference, as if each individual publication or patent application was here expressly and individually specified as incorporated by reference.

Unless otherwise indicated, all terms used herein have the usual meanings set by those skilled in the art to which the invention pertains.

general review

The present invention is based on the unexpected discovery that α5β1 integrin is expressed on the surface of tumor epithelial cells of many types of cancer. In addition, it was found that targeted binding of antibodies to surface α5β1 leads to direct lysis of cancer cells. Since this method of attack on cancer cells and lysis of cancer cells is direct, it can be used to treat a tumor at a very early stage (for example, before the tumor spreads significantly). In addition, the method for direct lysis of cancer cells according to the invention can be particularly effective for treating cancers that express α5β1 on their surface but are not sensitive to antiangiogenic approaches. Types of cancer in this category include, but are not limited to, bladder cancer, breast cancer, kidney cancer, pancreatic cancer, lung cancer, prostate cancer, ovarian cancer, and metastatic melanoma.

The present invention relates to methods for lysing or otherwise preventing the proliferation of cancer cells using antibodies against α5β1. In a most general embodiment, the method comprises contacting a cancer cell that expresses α5β1 on its surface with an anti-α5β1 antibody and inducing through this death (for example, through apoptosis) the cancer cell.

The methods of the present invention can be used to lyse cancer cells in vivo (for example, in a patient) and thereby prevent or ameliorate tumor formation and growth. In addition, the method can be used to treat existing tumors and can be used in conjunction with other cancer therapies (e.g., chemotherapeutic agents or other molecular-based therapeutic agents). For example, a patient with cancer growth may be given an M200 antibody composition along with a chemotherapeutic compound, such as doxorubicin. Since M200 is a chimeric antibody with relatively low toxicity in humans, this combination treatment can provide comparable cancer cell lysis efficacy without the toxic side effects that occur with higher doses of the same chemotherapeutic agent.

The present invention relates to a method in which antibodies against α5β1 kill cancer cells or inhibit the proliferation of cancer cells directly, even in the absence of any tumor vasculature that may be sensitive to the antiangiogenic effect of such antibodies. Thus, the method is particularly effective for the prevention or therapeutic treatment of cancer that expresses α5β1 but does not form highly vascularized tumors and / or is not otherwise sensitive to therapeutic substances acting on angiogenesis, such as pancreatic cancer, kidney cancer, metastatic melanoma lung cancer and breast cancer.

In addition, due to the ability of the M200 antibodies to directly lyse cancer cells (and the other anti-α5β1 antibodies described herein), they can be used in the treatment of cancer at an early stage before the formation of vascularized tumors. A method for treating cancer at an early stage is especially important in connection with the emergence of new, more sensitive diagnostic tests for cancer, which were developed using information from genetic markers available from the sequence of the human genome. Apparently, a large number of types of cancer can be recognized and diagnosed at a very early stage, i.e. at the pretumor stage, when cancer cells may be present in the tissue and / or in the bloodstream, but the tumor structure has not yet been established, visualized by less sensitive non-genetic diagnostic methods. In this early diagnostic method, antiangiogenic therapy approaches may have little or no effect when the tumor does not have a vasculature, and conventional chemotherapeutic agents may produce too many toxic side effects to be warranted. Since the method of the invention leads to targeted antibody lysis of cancer cells that express α5β1 integrin on their surface, it is particularly effective for preventive treatment in the early stages of cancer.

Individuals for whom an early treatment method could probably be effective include, but are not limited to: 1) individuals whose pre-tumor tests indicated a high probability of developing and / or the presence of tumors (or micro-tumors); 2) an individual who has been exposed to a powerful carcinogenic stimulus and who is highly likely to develop a tumor; 3) an individual who has a high genetic predisposition to develop cancer, where cancer cells express α5β1 on their surface.

Antibodies against α5β1

The methods of the present invention use antibodies against α5β1 as agents for direct lysis of cancer cells. In the context of the present description, the term "antibody" refers to an immunoglobulin molecule that specifically binds to a particular antigen or is immunologically reactive, and includes polyclonal, monoclonal, genetically engineered and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies, antibody heteroconjugates (e.g., bispecific antibodies, diabodies, triates and tetra) and antigen-binding fragments of antibodies, including a fragment you Fab ', F (ab') _2, Fab, Fv, rIgG, and scFv. The term "scFv" refers to a single chain Fv antibody in which the variable domains of the heavy and light chains of a conventional antibody are connected to form a single chain. In addition, the term "antibody" in the context of the invention described herein includes mixtures of more than one antibody that reacts with a specific antigen (for example, a mixture of various types of monoclonal antibodies that react with α5β1 integrin).

Preferably, the anti-α5β1 antibodies used in the present invention are monoclonal antibodies. Monoclonal antibodies that can be used in the methods of the present invention can be obtained using a wide variety of methods known in this field, including using hybridoma methods, recombinant methods and phage display methods, or a combination thereof. For example, monoclonal antibodies can be prepared using hybridoma methods, including methods known in the art and described, for example, in Harlow and Lane, "Antibodies: A Laboratory Manual," Cold Spring Harbor Laboratory Press, New York (1988) ; Hammerling et al., In "Monoclonal Antibodies and T-Cell Hybridomas," Elsevier, New York (1981), pp. 563-681 (which are incorporated herein by reference in full). Obtaining antibodies by selection from libraries of recombinant antibodies in phage or similar vectors see, for example, Huse et al., Science 246: 1275-1281 (1989); Ward et al., Nature 341: 544-546 (1989) and Vaughan et al., Nature Biotechnol. 14: 309-314 (1996) or by immunizing an animal with an antigen or DNA encoding that antigen.

In preferred embodiments, the methods for direct lysis of cancer cells of the present invention can be carried out using previously characterized anti-α5β1, IIA1, M200 or F200 antibodies. IIA1 is the original IgGI mouse antibody that has been shown to inhibit the binding of α5β1 integrin to fibronectin (see, for example, U.S. Patent Application Publication No. 2002/0172675 A1, filed May 7, 1999, which is incorporated herein by reference). M200 is a chimeric IgG4 antibody derived from IIA1. F200 is a Fab fragment derived from M200. A description of the preparation of these antibodies, their functional characteristics and specific amino acid sequences is given in US patent applications No. 10/724274, filed November 26, 2003, and 10/830956, filed April 23, 2004, each of which is incorporated herein by reference. Both M200 and F200 have been shown to exhibit in vivo antiangiogenic efficacy in monkey or rabbit eye disease models (see U.S. Patent Applications 10/724274, filed November 26, 2003, and 10/830956, filed April 23, 2004).

Antibodies that can be used in the method of the present invention also include antibodies that specifically bind to the same α5β1 epitope as antibodies IIA1, M200 and F200. “Epitope” (or “antigenic determinant”) refers to the site on the antigen to which the antibody binds. Epitopes can be formed from contiguous amino acids or non-contiguous amino acids located side by side as a result of the formation of the tertiary structure of the protein. For example, an epitope on α5β1 integrin may contain amino acids on each of the α and β polypeptide chains that form a heterodimeric structure. Epitopes formed from contiguous amino acids are usually retained by denaturing solvents, while epitopes formed by a tertiary structure are usually lost by treatment with denaturing solvents. The epitope usually includes at least 3, usually 5 or 6-10 amino acids in a unique spatial conformation. Methods for determining the spatial conformation of epitopes include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).

Two antibodies are said to bind to the same protein epitope if mutations in a protein that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody. It can also be concluded that two antibodies bind to the same epitope if these two antibodies compete for binding to this protein, i.e. the binding of one antibody to this protein competitively inhibits, reduces or eliminates the binding of another antibody. Thus, the methods of the invention can be carried out with an antibody that has been determined to competitively inhibit the binding of IIA1, V200 (volociximab) or F200 to α5β1 integrin expressed on the surface of cancer cells.

Antibodies against α5β1 that can be used in the methods of the present invention are not limited to IIA1, M200, and F200, but may include antibodies containing a variable region, frame region, or amino acid sequence of complementarity determining regions (CDRs) identical to the amino acid sequence of CDR IIA1, M200 and F200. Finding and the length of the variable regions, framework regions and CDR are well known to those skilled in the art (see., E.g., Kabat et al., "Sequences of Proteins of Immunological Interest", 5 ^{th ed. National Institutes of Health,} Bethesda, MD ( 1991 )). In the context of the description, the variable region of the heavy chain ("V _H " or "VH") or light chain ("V _L " or "VL") of an antibody includes the heavy and light chains of an antigen binding fragment, for example Fv, scFv, dcFv or Fab . The variable regions of the light or heavy chains of the antibodies also contain framework regions interrupted by three hypervariable regions, also referred to as “complementarity determining regions” or “CDRs”. These CDRs are primarily responsible for binding to an antigen epitope.

“Substantially identical" variable, constant, scaffold regions or CDRs indicate an antibody region whose amino acid sequence is at least about 85-90% and preferably at least 95% identical to the variable or constant region of a natural or unchanged antibody. The terms “identical” or percent “identity,” in the context of two or more amino acid or nucleotide sequences, mean two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides (i.e., approximately 60% identity, approximately 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity in the specified area, with comparison and matching for maximum match in the comparison window or area) when measured using BLAST or BLAST 2.0 sequence comparison algorithms with the default parameters described below, or by manually matching and visual control (see, for example, the BLAST description in the National Center for Biotechnology Information (NCBI) website, located at www.ncbi.nlm.nih.gov.).

Identical or essentially identical sequences include sequences with deletions and / or additions, as well as sequences with substitutions, as well as natural, for example polymorphic or allelic, variants and human-created variants, such as conservatively modified variants. Well-known algorithms for measuring sequence identity may be responsible for omissions, etc. Preferably, sequence identity exists in an area of at least about 25 amino acids or nucleotides in length, or more preferably in an area of between 50-100 amino acids or nucleotides in length.

The amino acid sequences of an anti-α5β1 antibody that can be used in the methods of the present invention are not limited to those found in natural antibodies; antibodies can be redesigned to obtain the desired characteristics using recombinant DNA methods. Such "genetically modified antibodies" include antibodies in which the amino acid sequence has been changed relative to the sequence of the original (i.e., unchanged) antibody. Possible variations range from a change in just one or a small number of amino acids to a complete redesign, for example, a variable or constant region. To improve or alter the functional characteristics of a therapeutic antibody, such as immunogenicity, pharmacokinetic properties (e.g. serum half-life), complement fixation, interaction with membranes, and other effector functions in the constant region, changes can be made through site-directed mutagenesis. To improve the characteristics of antigen binding, changes in the variable region of antibodies can usually be made.

In one of the preferred embodiments, the chimeric antibody M200 can be used in the methods of direct lysis of cancer cells of the invention. The term "chimeric antibody" refers to an immunoglobulin molecule in which (a) its constant region or part thereof is changed, replaced or exchanged so that the antigen-binding site (variable region) is associated with a constant region of another or altered class, another effector function and / or another species, or a completely different molecule that gives new properties to this chimeric antibody, for example, toxin, hormone, growth factor, drug, etc .; or (b) this variable region or part thereof is altered, replaced, or exchanged for a variable region having a different or altered antigen specificity. Methods for producing chimeric antibodies are well known to those skilled in the art. See, for example, Morrison et al .. Science 229: 1202-1207 (1985); Oi et al., BioTechniques 4: 214-221 (1986); Gillies et al., J. Immunol. Methods 125: 191-202 (1989) and US Patent Nos. 5807715; 4816567 and 4816397, each of which is incorporated herein by reference in full.

In another preferred embodiment, humanized anti-α5β1 antibodies can be used in the methods for direct lysis of cancer cells of the invention. Additional human sequences in humanized antibodies further reduce the possible immunogenicity of the antibody when used as a therapeutic agent for humans. Humanized versions of IIA1 are described in US patent applications 10 / 724,274, filed November 26, 2003, and 10 / 830,956, filed April 23, 2004, each of which is incorporated herein by reference. The term “humanized antibody” refers to an immunoglobulin comprising a framework region of a human antibody, at least one and preferably all of the CDRs of a non-human antibody antibody, in which any constant region present is substantially identical to the constant region of a human immunoglobulin, i.e. at least about 85-90% identical and preferably at least 95% identical. Thus, all parts of a humanized immunoglobulin, with the exception of CDRs, are essentially identical to the corresponding parts of one or more natural sequences of a human immunoglobulin. Thus, such humanized antibodies are chimeric antibodies in which substantially less than one intact variable region of a human immunoglobulin has been replaced by a corresponding sequence of another species. Frame residues in human framework regions can be replaced with the corresponding residue from the CDR donor antibody to change, preferably to improve antigen binding. These framework substitutions can be identified by methods well known in the art, for example, modeling the interactions of CDRs and framework residues to identify framework residues important for antibody binding, and sequence comparisons to identify unusual framework residues at specific positions. See for example. Queen et al., U.S. Patent Nos. 5530101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (each of which is incorporated herein by reference in its entirety). Antibodies can be humanized by various methods known in the art, including, for example, by CDR transplantation (EP 239400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5530101 and 5585089), lining or surface modification of antibodies ( EP 592 106; EP 519596; Padlan, Mol. Immunol., 28: 489-498 (1991); Studnicka et al., Prot. Eng. 7: 805-814 (1994); Roguska et al., Proc. Natl. Acad Sci. USA 91: 969-973 (1994) and chain shuffling (US Pat. No. 5,565,332), each of which is incorporated herein by reference in its entirety.

In another embodiment, human antibodies (i.e., antibodies containing both variable and constant regions of a human) against α5β1 can be used for therapeutic treatment of human patients in accordance with the methods of the invention. Human antibodies can be made or prepared by various methods known in the art, including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See U.S. Patent Nos. 4,444,887 and 4,716,111 and PCT Publication WO 98/46645; WO 98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735 and WO 91/10741, each of which is hereby incorporated by reference in its entirety. Human antibodies can also be obtained using transgenic mice that are not able to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For a review of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13: 65-93 (1995). For a detailed discussion of this technology for the production of human antibodies and human monoclonal antibodies and the protocols for producing such antibodies, see, for example, PCT publication WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European patent No. 0598877; U.S. Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5916771 and 5939597, which are hereby incorporated by reference in full. Fully human antibodies that recognize the selected epitope can also be obtained using a method called "controlled selection". In this approach, a selected non-human monoclonal antibody, such as a murine antibody, is used to control the selection of a fully human antibody recognizing the same epitope (Jespers et al., Biotechnology 12: 899-903 (1988).

In an alternative embodiment, primatized antibodies (i.e., antibodies containing variable regions of a monkey and constant regions of a human) can be used for therapeutic treatment in accordance with the methods of the present invention. Methods for preparing primatized antibodies are known in the art. See, for example, US Pat. Nos. 5,658,570; 5681722 and 5693780, which are hereby incorporated by reference in full.

Antibody function

In the methods of the invention, functional antibodies are used that exhibit specific binding to α5β1 integrin. Testing the avidity of antibodies for specific binding to an antigen enables a person skilled in the art to identify antibodies that specifically recognize one or more epitopes of α5β1 integrin. Antibodies are defined as specifically binding if 1) they exhibit a threshold level of binding activity; and / or 2) they lack significant cross-linking with related polypeptide molecules.

First, anti-α5β1 antibodies that can be used for the methods described here specifically bind (or "specifically react" or "specifically immunoreact") if they bind to an α5β1-integrin polypeptide, peptide or epitope with binding affinity (K _a ) 10 ⁶ mol ^-1 or more, preferably 10 ⁷ mol ^-1 or more, more preferably 10 ⁸ mol ^-1 or more, and most preferably 10 ⁹ mol ^-1 or more. Antibody binding affinity can be readily determined by one of skill in the art, for example, Sketchard analysis (Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) or surface plasmon resonance using BIAcore. A variety of antibody binding assays that can be used to characterize anti-α5β1 antibodies are described in US patent applications 10/724274, filed November 26, 2003, and 10/830956, filed April 23, 2004, each of which is incorporated herein by reference.

Secondly, antibodies specifically bind if they lack significant cross-linking with related polypeptides. Antibodies lack significant cross-linking with related polypeptide molecules, for example, if they detect the α5β1 integrin polypeptide and not the known related polypeptides using standard Western blot analysis (see, for example, Current Protocols in Molecular Biology, eds Ausubel et al., 1995). Examples of known related polypeptides include orthologs, proteins of the same species that are members of the integrin protein family, or mutant α5β1-integrin polypeptides, where the mutation changes the epitope of the anti-α5β1 antibody. In addition, antibodies can be screened against known related polypeptides to isolate a population that specifically binds to α5β1 integrin. For example, antibodies induced to human α5β1 integrin polypeptides are adsorbed by related polypeptides attached to an insoluble matrix; antibodies specific for human α5β1 integrin polypeptides will flow through this matrix under the correct buffer conditions. Such screening allows the isolation of polyclonal and monoclonal antibodies that lack cross-linking with closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and Lane (eds.). Cold Spring Harbor Laboratory Press, 1988; Current Protocols in Immunology, Cooligan et al. ( eds.) National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art (see Fundamental Immunology, Paul (eds.). Raven Press, 1993; Getzoff et al .. Adv. In Immunol. 43: 1-98, 1988; Monoclonal Antibodies: Principles and Practice, Coding, JW (eds.), Academic Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984). Representative examples of such assays include: competitive immunoelectrophoresis, radioimmunoassay, radioimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot analysis, inhibition assay or competitive assay, and sandwich assay. For a review of immunological processes and procedures immunoassays see. Basic and Clinical Immunology (Stites & Terr eds., 7 ^th ed. 1991).

The specific binding of antibodies against α5β1, which can be used in the methods of the present invention, can be obtained by a selection method for α5β1 binding. Typically, polyclonal antibodies induced to specifically bind to a particular protein, or their polymorphic variants, alleles, orthologs, conservatively modified variants, splicing variants, can be selected to obtain only those antibodies that are specifically immunoreactive with the selected protein (for example, α5β1 integrin ), but not with other proteins. Selection for specific binding is achieved by subtraction (subtraction) of antibodies in which there is no cross-linking with other molecules. A variety of immunoassay formats can be used to select antibodies that specifically bind to the α5β1 integrin polypeptide. For example, ELISA solid phase immunoassays can be used to select antibodies that specifically bind to a protein (see, for example, Harlow and Lane, "Antibodies: A Laboratory Manual" (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

Antibody Drug Conjugates

In some embodiments, an anti-α5β1 antibody conjugated to an effector molecule can be used in a method for lysing cancer cells. An effector molecule can be any number of molecules, including labeling molecules, such as radioactive labels or fluorescent labels, or preferably it can be a therapeutic component. This effector moiety (or “effector component”) can be coupled (or linked or conjugated) to an anti-α5β1 antibody either covalently, via a linker or chemical bond, or non-covalently, via ionic, van der Waals, electrostatic or hydrogen bonds.

In one aspect, such a therapeutic component of the molecule is a small molecule that modulates the activity of α5β1 integrin. In another aspect, the therapeutic component of the molecule affects the activity of molecules or cells associated with or in close proximity to α5β1 integrin. For example, this therapeutic component of the molecule may be a cytotoxic agent. The term "cytotoxic agent" means in this context a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., I ¹²⁵ , Y ^90, and Re ¹⁸⁶ ), chemotherapeutic agents, and toxins, such as active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof. Suitable toxins and their corresponding fragments include diphtheria toxin A chain, exotoxin A chain, ricin A chain, Abrin A chain, curcin, crotin, phenomycin, enomycin, auristatins (e.g., auristatin E or auristatin F) and t .P. Targeting the therapeutic part of the molecule to α5β1-integrin expressed on the surface of cancer cells not only serves to increase the local concentration of the therapeutic component of the molecule in the cancerous area, but also reduces the side effects that may be associated with this therapeutic component of the molecule.

Examples of chemotherapeutic agents that can be used as therapeutic components of anti-α5β1 antibody molecules of the invention include, but are not limited to, adriamycin, doxorubicin, doxil, epirubicin, 5-fluorouracil, cytosine arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan, cytoxin, taxoids, for example, paclitaxel (TAXOL ™, Bristol-Myers Squibb Oncology, Princeton, NY) and doxetaxel (TAXOTERE ™, Rhone-Poulenc Rorer), toxotere, methotrexate, cisplatin belfin, melfin etoposide, ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycin, esperamycins (see US patent No. 4675187), 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin chloramphenol, VP-16, and other related nitrogen analogues of mustard gas. Hormonal agents that regulate or inhibit the action of hormones on tumors, such as tamoxifen and onapriston, are also included in this definition.

Alternatively, a method of the invention can be carried out in which the chemotherapeutic agents described above are administered in a preparation together with an anti-α5β1 antibody, although not in the form of a conjugate.

Medical indications

The present invention relates to methods for lysing cancer cells, inhibiting the proliferation and / or metastasis of cancer cells, targeting α5β1 integrin to the surface of the cancer cells using an antibody. Cancer is a physiological condition, usually characterized by cells undergoing uncontrolled growth. Cancer includes all malignant neoplasms, including, but not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia. More specific examples of cancers that express α5β1 on their cell surface that can be targeted using the methods described here include, but are not limited to, bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer and spleen cancer.

A tumor is a proliferating cell mass devoid of normal growth regulators. The tumor may be benign or malignant and may include precancerous or cancerous cells and tissues. Cancer cells usually form tumors as cancer progresses. Tumor growth is accompanied by an increase in vascular density. This tumor vasculature provides the required nutrition that allows the tumor to grow. For anti-angiogenic therapeutic antibodies against α5β1 and anti-VEGF (VEGF = vascular endothelial growth factor), efficacy in delaying tumor growth has been shown in a variety of cancer models (see, for example, Kirn et al., J Clin Invest., 110 (7): 933-41 (2002); Ferrara, Nat Rev Cancer, 2 (10): 795-803 (2002)). The anti-α5β1, M200 antibody has been shown to inhibit angiogenesis in in vitro and in vivo models of rabbit and monkey angiogenic eye diseases (e.g., progression of macular degeneration; see U.S. Patent Application No. 10/724274, filed November 26, 2003, and 10/830956, filed April 23, 2004, each of which is incorporated herein by reference).

The invention is based on the unexpected discovery that for many types of cancer, α5β1-integrin is expressed on the surface of tumor epithelial cells (along with endothelial cells of the tumor vasculature) and that targeted binding of α5β1 on the surface by antibodies leads to direct lysis of these cancer cells. Thus, cancer treatment is characterized by proliferation of epithelial cells expressing α5β1, can be carried out with antibodies against α5β1 and / or cells can be targeted by antibodies against α5β1 even in the absence of any formation of a tumor vasculature. Since this method of attacking cancer cells and lysis of cancer cells is direct, it can be used to treat cancer at a very early stage (for example, before the tumor spreads significantly).

Patients in whom such treatment at an early stage is likely to be effective include, but are not limited to: 1) patients in whom pre-tumor diagnostic tests indicate a high probability of developing and / or the presence of tumors (or micro-tumors); 2) patients exposed to a very strong carcinogenic environment, the likelihood of tumor progression is high; 3) a subject whose genetic predisposition (for example, detected using a genetic marker of cancer) makes it highly likely that cancer will develop in which cancer cells express α5β1 on their surface. For example, in a patient with a genetic predisposition to breast cancer (for example, a BRCA-gene-positive) or who has been found to have any pre-tumor cancer marker (for example, micrometastases detected by PCR), this method of preventative direct lysis of early cancer cells a step comprising administering a pharmaceutical composition of an anti-α5β1 antibody will be particularly effective.

Genes indicating an increased likelihood of developing cancer, and tumor markers indicating the likelihood of developing a tumor, can be found in the biomedical literature on cancer and in databases. In addition, lists of carcinogens and their exposure levels, which significantly increase the risk of developing cancer, are available in the biomedical literature well known to those skilled in the art. One of skill in the art can use these sources along with the well-known methods for visualizing the expression of α5β1 integrin on cancer cells, described below, to determine whether the methods for treating early stage cancer of the invention can be used in an individual or not. As described in Example 2 below, flow cytometric analysis reveals α5β1 integrin that is expressed on the surface of cell lines obtained from at least the following types of cancer: bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma , pancreatic cancer, prostate cancer, ovarian cancer, and kidney cancer.

In addition, the direct cancer cell lysis method of the invention can be particularly effective for treating cancer that expresses α5β1 but does not show sensitivity to antiangiogenic approaches. Cancer in this case includes, but is not limited to, bladder cancer, breast cancer, fibrosarcoma, kidney cancer, pancreatic cancer, prostate cancer, ovarian cancer, lung cancer, and metastatic melanoma. In a particularly preferred embodiment, the methods of the invention can be used to treat any of the above cancers where the solid tumors of the patient are not treatable. The anti-α5β1 antibody M200 (volociximab) has been shown to show some efficacy in treating human patients with various types of cancer, with solid, untreatable tumors, including colorectal cancer (CRC), melanoma (MEL), and cancer kidney (RCC), liver cancer (NSS), lung cancer (NSCLC), pancreatic cancer (PC), parotid cancer (RANO), and breast cancer (BC).

One of skill in the art can determine the type of cancer that expresses α5β1 on the surface of epithelial cells using an anti-α5β1 antibody (e.g., IIA1 or M200) to probe tumor biopsy samples in accordance with standard immunohistochemistry methods. As described in Example 2 below, immunohistochemical analysis (IHC) of tumor biopsy samples taken from patients with melanoma, lung cancer, kidney cancer, pancreatic and breast cancer showed in all cases the expression of α5β1 integrin on tumor epithelial cells. It is expected that other types of cancer expressing α5β1 on the surface of tumor epithelial cells using IHC (or other methods) will be detected, and the authors believe that such types of cancer will be sensitive to the cancer cell lysis method of the invention.

In addition to IHC tumor samples, cancer cell lines can be screened for expression of α5β1 on the cell surface using an anti-α5β1 antibody and standard flow cytometry methods well known to those skilled in the art. As described in detail in Example 2 below, flow cytometric screening revealed the expression of α5β1 on surface 21 of a well-known cancer cell line. Based on this result, these cell lines are sensitive to direct cell lysis with anti-α5β1 antibodies in accordance with the methods of the invention. In addition, if it is determined that a cancer cell line expresses α5β1 on their surfaces, and this cell line corresponds to cancer cells present in a cancer patient, originates or is derived from cancer cells present in a cancer patient, the methods of the invention can be used for treating such a patient. For example, the NW231 cell line, which was found in Example 2 to express α5β1 integrin on its surface, originated from breast tumors. Thus, one skilled in the art will be able to directly determine that an anti-α5β1 antibody can be used to treat a patient with breast cancer, in accordance with the methods described herein.

Compositions, formulations and administration of antibodies

Antibodies against α5β1, which can be used in the methods of the invention, can be used in isolated and purified form and directly contact with cancer cells or tumors. Methods for purifying antibodies against α5β1 (e.g., IIA1 and F200) are described in US Patent Applications No. 10/724274, filed November 26, 2003, and 10/830956, filed April 23, 2004, each of which is incorporated herein by reference. F200 can also be obtained as a Fab'-NAC fragment in accordance with the methods described in US patent application 60/583127, filed June 25, 2004, which is incorporated herein by reference. F200-Fab'-NAC has increased stability in liquid and lyophilized forms of this antibody. Purity and homogeneity can be determined by standard analytical chemistry methods, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. It is believed that the antibody that predominates in the preparation is essentially purified. For example, an antibody solution that detects substantially one band in an electrophoretic gel is substantially purified.

Preferably, the antibody used in the pharmaceutical compositions of the invention is at least 85% pure, more preferably at least 95% pure and most preferably at least 99% pure.

In preferred embodiments, a method for direct lysis of cancer cells is carried out in which purified anti-α5β1 antibodies are formulated as a pharmaceutical composition which is administered to a subject in a therapeutically effective amount. As used herein, a “therapeutically effective amount” means an amount of a pharmaceutical formulation or composition that is sufficient to cure, alleviate, ameliorate, or at least partially delay the development of cancer and / or its symptoms and / or complications. Clinical methods for determining a therapeutically effective amount of an anti-α5β1 antibody for treating cancer are well known to those skilled in the art and can be determined empirically using routine experimentation. For example, in the context of cancer treatment, a “therapeutically effective amount” is an amount capable of causing one or more of the following effects: (1) inhibiting, to some extent, tumor growth, including attenuation and complete growth retardation; (2) reduction in the number of cancer cells; (3) reduction in tumor size; (4) inhibition (i.e., reduction, retardation or complete cessation) of peripheral organ cancer cell infiltration; (5) inhibition (i.e., reduction, retardation, or complete cessation) of cancer cell metastasis; (6) enhancing the anti-cancer immune response, which may, but not necessarily, lead to regression or rejection of the tumor; and / or (7) relief, to some extent, of one or more symptoms associated with the violation.

Pharmaceutical compositions for administration will typically contain an anti-α5β1 antibody dissolved in a pharmaceutically acceptable carrier or excipient, preferably in an aqueous carrier. Acceptable carriers, excipients or stabilizers for the pharmaceutical composition are carriers, excipients or stabilizers that are non-toxic to the cell or mammal exposed to them in the doses and concentrations used. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids, antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextran; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt forming counterions, such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG) and PLURONICS ™. A variety of aqueous vehicles may be used, for example, buffered saline and the like. These solutions should be sterile and they usually should not contain unwanted material. The pharmaceutical compositions may be sterilized by conventional, well known sterilization methods.

These pharmaceutical compositions may also contain pharmaceutically acceptable excipients (additives) that are necessary to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity correcting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and other pharmaceutically acceptable salts, which are believed to include both acid addition salts and base addition salts. These pharmaceutical compositions may also include one or more of the following components: carrier proteins, such as serum albumin; buffer; filters, such as microcrystalline cellulose, lactose, corn starch or other starches; binding agents; sweeteners and other taste and odor improving agents; dyes and polyethylene glycol. The concentration of antibodies in these formulations can vary widely and will be selected, primarily, depending on volumes, viscosities of fluids, body weight, etc., in accordance with the particular chosen route of administration and the needs of the patient (for example, "Remingtons Pharmaceutical Sciences "(15 ^th ed., 1980) and Goodman &Gillman," The Pharmacological Basis of Therapeutics "(Hardman et al., Eds., 1996)).

In a preferred embodiment of the methods of the invention, the anti-α5β1 antibody is prepared in the form of a pharmaceutical composition comprising a solution of approximately 1.0 mg / ml - 15 mg / ml of the antibody, approximately 22 mm - 28 mm citrate, approximately 135-165 mm sodium chloride, 0, 04% -0.06% polysorbate (TWEEN®) 80, at pH 5.5-7.5. Preferably, the pH range of the liquid formulation is approximately pH 6.0 to pH 7.0, and most preferably approximately pH 6.3 to pH 6.7. In a particularly preferred embodiment, the pharmaceutical composition comprises a solution of about 10.0 mg / ml antibody, about 25 mM citrate, about 150 mM sodium chloride, about 0.05% polysorbate (TWEEN®) 80, at a pH of about 6.5. In other embodiments, the above pharmaceutical composition of an anti-α5β1 antibody may further comprise a chemotherapeutic agent or, alternatively, may be administered to a patient together with a pharmaceutically effective amount of a chemotherapeutic agent.

Preferably, the liquid formulation of this pharmaceutical composition is a stable, colorless or transparent, slightly opalescent solution, detecting no more than 10% and, preferably, 5% or less degraded antibody monomer as measured by SEC-HPLC. Preferably, not more than 10%, and preferably 5% or less, of hydrolytic cleavage is observed, and not more than 10%, and preferably 5% or less, of an antibody aggregate is formed. Preferably, the concentration, pH and osmolarity of this formulation has no more than ± 10% change. Efficiency is in the range of 70-130%, preferably 80-120% of the control.

The administration of pharmaceutical compositions containing antibodies against α5β1 to an individual can be carried out in various ways, including, but not limited to, oral, subcutaneous, intravenous, intranasal, topical, percutaneous, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, intraocular, intraventricular or intrathecal . It is understood that when administered orally, antibodies must be protected from digestion. This is usually accomplished either by complexing these molecules with a composition to confer resistance to acid and enzymatic hydrolysis, or by packing these molecules in a suitable stable carrier, such as a liposome or protective barrier. Digestion protection agents are well known in the art.

The pharmaceutical compositions may be administered in various unit dosage forms, depending on the route of administration. For example, unit dosage forms for oral administration include, but are not limited to, powder, tablets, pills, capsules, and troches.

The exact administration dose in a particular embodiment of the method of the invention will depend on the purpose of the treatment and can be determined by one skilled in the art using well-known methods (e.g., Ansel et al., "Pharmaceutical Dosage Forms and Drug Delivery;" Lieberman, "Pharmaceutical Dosage Forms "(vol. 1-3, 1992), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd," The Art, Science and Technology of Pharmaceutical Compounding "(1999); and Pickar," Dosage Calculations "(1999) ) As is known in the art, corrections for the degradation of a drug by a tumor, systemic delivery relative to localized delivery, and the rate of synthesis of a new protease, as well as age, body weight, general condition, gender, diet, time of administration, drug interactions, and severity of the condition, can be necessary and will be determined by routine experimentation by specialists in this field.

In one embodiment of the invention, the anti-α5β1 antibody pharmaceutical compositions are administered to a patient based on the weight of the antibody (in mg) per patient's body weight (in kg). Thus, preferred dose levels include at least about 0.5 mg / kg, 1.0 mg / kg, 2.5 mg / kg, 5.0 mg / kg, 10.0 mg / kg and 15 mg. / kg Preferably, the dose is administered to the patient as an intravenous infusion over a period of 1 hour. Additional doses can be administered over an extended period of time, so that the patient establishes a steady state concentration in the serum. For example, an infusion of 10 mg / kg may be performed once a week for a year.

In one preferred embodiment, the level and dosage regimen is chosen so as to ensure that this dose provides a maximum serum concentration lower than the safe maximum serum concentrations observed in monkey pharmacokinetic studies (e.g., dog-headed monkey) . For example, in dog-headed monkeys, the maximum level of M200 at a dose of 50 mg / kg after 4 weekly doses was 1862 μg / ml (range: 1000-2606 μg / ml). The monkeys did not show toxicity at these serum concentrations. In addition, or alternatively, the dose could be chosen so that throughout the experiment, the serum level was> 1 μg / ml, i.e. a concentration that gives 80% inhibition of the binding of α5β1 to fibronectin in an in vitro activity assay.

According to one embodiment of the cancer cell therapeutic lysis methods of the invention, a pharmaceutical composition comprising an anti-α5β1 antibody is administered to a patient intravenously at a fixed dose, typically about 0.1-10 mg per patient per day. In embodiments in which this pharmaceutical composition is administered into an isolated area, such as a body cavity or organ lumen, rather than into the bloodstream, fixed doses of 0.1 mg to about 100 mg per patient per day can be used. Significantly higher doses are possible for embodiments in which topical administration is desired. Actual methods for preparing parenterally administered compositions will be known or apparent to those skilled in the art, for example, "Remington's Pharmaceutical Sciences" and Goodman & Gillman, "The Pharmacologial Basis of Therapeutics," above.

The pharmaceutical compositions used in the cancer cell lysis method of the invention may be administered as part of a therapeutic or prophylactic treatment. In a therapeutic method, a pharmaceutical composition is administered to a patient already suffering from cancer in an amount sufficient to cure or at least partially delay the progression of the disease and its complications. Usually, in the context of therapeutic treatment, the success of this therapy can be measured as a decrease in tumor size or a decrease in tumor growth rate. An amount sufficient to accomplish this is defined as a “therapeutically effective dose”. Amounts effective for this use will depend on the severity of the cancer and the general health of the patient. Single or multiple administrations of pharmaceutical compositions may be used depending on the dose and frequency of administrations carried by a particular patient.

A method for treating an early stage of cancer is aimed at preventing or slowing down the development of cancer in a subject who is suspected of developing the disease, or at a very early stage of the disease. The specific dose needed to treat an early stage will depend on the medical condition and history of a particular patient, on the particular cancer to be prevented, as well as other factors such as age, weight, gender, route of administration, effectiveness, etc. An early treatment method can also be used prophylactically, for example, in a patient who previously had cancer, to prevent recurrence of this cancer, or in a patient who is suspected of having a significant likelihood of developing cancer. For example, a patient with a genetic predisposition for breast cancer in which any precancerous cancer marker has been detected (e.g., micrometastases detected by PCR) would be particularly suitable for an early treatment method.

In an alternative embodiment of the present invention, a method for direct lysis of cancer cells can be carried out in which a chemotherapeutic agent is administered along with an anti-α5β1 antibody. Representative chemotherapeutic agents useful in this embodiment are described above. This combination therapy method may be particularly preferred at an early stage or in the context of prophylactic treatment, when the patient is deprived of the symptoms of a fully developed disease. At this early stage, or in a preventive context, many patients do not agree to undergo the toxic effects that accompany the use of only a chemotherapeutic agent. By administering a lower dose of a standard chemotherapeutic agent together with a relatively non-toxic anti-α5β1 antibody, this cancer can be treated prophylactically, or at a very early stage, using an administration regimen that is effective, but much more well tolerated for the patient.

The following examples are intended to illustrate and not to limit the invention described herein.

Examples

Example 1

The expression of α5β1 on tumor samples detected by immunohistochemical analysis (IHC)

Tumor biopsy samples taken from patients with melanoma, lung cancer, kidney cancer, pancreatic cancer, and breast cancer were examined for expression of α5β1 integrin through immunohistochemistry (IHC).

Materials and methods

Frozen tissue samples (obtained from Mayo Clinic or Cleveland Clinic) were frozen at the optimum temperature for the preparation of sections (OCT) and stored at -70 ° C. Sections of cryostat tissue (7 μm) were fixed in a mixture of 75% acetone / 25% ethanol for 1 minute. Samples were incubated with either a mouse anti-α5β1 IIA1 antibody (5 μg / ml) or with a control mouse IgGI (anti-trinitrophenyl, ATCC 1B7.11 hybridoma clone) for 30 minutes. Antibody binding was determined using a biotinylated secondary antibody, a goat anti-mouse IgG antibody (3 μg / ml, 30 minutes; Jackson ImmunoResearch) and developed using a Vectastain Elite ABC kit (Vector Laboratories) and stable DAB (diaminobenzidine and H ₂ O ₂ ; Research Genetics). Staining was performed using DAKO Autostainer at room temperature.

results

As shown in table 1, almost all investigated sections of the tumors stained positively for α5β1 on the vasculature of the tumors. Unexpectedly, a significant part of the tumor samples also found positive staining for expression of α5β1 integrin on the tumor epithelium itself. These results indicate that the anti-α5β1 antibody will directly infect cancer cells, along with its introduction into the newly formed vasculature.

Table 1 IHC results for tumor samples stained with anti-α5β1 IIA1 antibody Tumor type (number of samples) Tumor vasculature Tumor epithelium Neg. 1+ 2+ 3+ 4+ Neg. 1+ 2+ 3+ 4+ Kidney (n = 39) 2 2 13 eighteen four 27 8 four 0 0 Pancreas (n = 32) 0 one 17 12 2 fifteen 2 7 7 one Easy (n = 39) 0 one 0 28 one 19 fourteen 3 2 one Colon (n = 21) 2 2 3 8 6 19 0 one one 0 Melanoma (n = 19) 3 0 2 13 one 13 5 one 0 0 Ovary, metastases in omentum (n = 4) 0 0 one 3 0 2 2 0 0 0 Bladder (n = 8) 0 0 four 3 one 5 2 one 0 0

Example 2

Expression of α5β1 on cancer cell lines visualized by flow cytometry

A set of 24 cancer cell lines was examined by flow cytometry for expression of α5β1 integrin on the cell surface. As shown in table 2, these 24 cell lines were obtained from many different types of cancer, including: bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, melanoma, pancreatic cancer, prostate cancer, cancer ovary, kidney cancer, and spleen cancer.

Materials and methods

Cells were removed from 5 mM EDTA in Tris-HCl (pH 8.0) and blocked by centrifugation in Hanks balanced salt solution containing 3% heat inactivated FBS, 1% normal goat serum (Sigma) and 1% BSA, at 4 ° C. within 5 minutes. Cells were incubated for 30-60 minutes at 4 ° C with a murine anti-α5β1, IIA1 antibody (10 μg / ml) in FACS buffer (PBS containing 0.1% BSA). Excess monoclonal antibody was removed by centrifugation, and the cells were washed twice with FACS buffer before suspension in PE anti-mouse IgG antibody (H + L) (Southern Biotech, dilution 1: 400) for 30-60 minutes at 4 ° C. After washing, the cells were resuspended in FACS buffer containing propidium iodide (1 μg / ml). The average fluorescence intensity (MFI) was measured on a FACSCalibur instrument (Becton Dickinson). MFI background was ~ 5.

results

As shown in Table 2, significant detectable surface expression of α5β1 integrin was observed for 21 of the 24 evaluated cell lines. In three cancer cell lines CHL-1, COLO 357 and C32, MFI values were found that were very close to the background, showing minimal or no expression of α5β1 integrin. 21 lines of cells expressing α5β1-integrin on their surface should be sensitive to direct cell lysis with anti-α5β1 antibody. In addition, the cancer from which these cell lines were derived may be sensitive to treatment with a therapeutic anti-α5β1 antibody.

table 2 Results of FACS analysis of various cancer cell lines using IIA1 and in vitro proliferation analysis using M200 Cell line Cancer nature Surface expression of α5β1 (MFI) Growth Inhibition,% - serum + serum A549 Lung 71.41 40 0 H460 Lung 87.5 40 0 SW839 Bud 65.8 thirty 0 MIA RASA-2 Pancreas 61.9 twenty 0 NST-116 Colon 42.5 fifteen 0 786-0 Bud 144.4 10 0 Ekvx Lung 36.7 10 0 SW1990 Spleen nd ¹ 10 0 SN12C Bud 129.1 5 0 A498 Bud 67.1 5 0 A375 Melanoma 156.1 5 0 A2058 Melanoma 36,2 0 0 CHL-1 Melanoma 6.8 0 0 TK-10 Bud 36.6 0 0 COLO 357 Pancreas 7.8 0 0 NT144 Melanoma 102.1 0 0 S8161 Melanoma 122 0 0 NT1376 Bladder 111.9 0 0 DU145 Prostate 87.5 0 0 UACC-62 Melanoma 88.5 0 0 NT1080 Fibrosarcoma 421.7 0 0 ES-2 Ovary 132.8 0 0 Capan-1 Pancreas nd ¹ 0 0 Capan-2 Pancreas nd ¹ 0 0 AS PC-1 Pancreas nd ¹ 0 0 C32 Melanoma 5.7 0 0 LOX melanoma 261.6 40 ² 35 Nw231 Breast 132.2 10 ² 35 ¹ nd = not defined.
² Values of growth inhibition in% were determined on the 2nd day, and not on the 4th day of analysis.

Example 3

In Vitro Antibody M200 Inhibition of Cancer Cell Proliferation

A set of 28 cancer cell lines was evaluated for sensitivity to a chimeric anti-α5β1 antibody in a cell proliferation assay in the presence or absence of serum.

Materials and methods

Cancer cell lines were seeded at a density of 2500 cells per well in 96-well plates in IMDM supplemented with or without 10% FBS. At the time of plating, cells were stimulated with various concentrations of M200 or a non-functional blocking antibody against α5, VC5. After four days, cell viability was assessed using a non-radioactive cell proliferation assay (CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega)) in accordance with the manufacturer's instructions. All growth studies were performed at least 3 times in triplicate.

results

As shown in Table 2, the M200 antibody inhibited the growth of thirteen cancer cell lines in the absence of serum and two cell lines in the presence of serum. It was found that two of these cell lines, LOX and NW231, are sensitive to M200 under both conditions. Based on these results, the cancers from which these cell lines (melanoma and breast cancer) originate seem to respond to treatment with antibody M200.

Example 4

In vivo inhibition of tumor cell proliferation in NW231 and LOX xenograft models

NW231 and LOX cells were grown as orthotopic xenografts in SCID mice and stimulated with anti-α5β1 M200 and IIA1 antibodies by intraperitoneal injection.

Materials and methods

Mice with immunocompromised CB-17 SCID (strain C. B-Ighl / IcrTac-Prkdc) were obtained from Taconic Farms (Germantown, NY). Studies began using female mice at 6-10 weeks of age (weighing ~ 20 g). The animals were pre-injected with M200, IIA1 or control IgG intraperitoneally at a dose of 10 mg / kg 1 hour before inoculation of NW231 (1 × 10 ⁷ cells in IMDM) into the adipose tissue of the mammary gland. Dosing was continued for 3 weeks at a frequency of 3 times per week at 10 mg / kg. Tumor volume was measured twice a week using a caliper and calculated using the formula π / 6 × length × width × height. Clinical and mortality observations were performed daily in accordance with IACUC rules.

results

It was found that IIA1 reproducibly inhibits the growth of both the NW231 xenograft and the LOX xenograft in this model. It was found that M200 reproducibly inhibits tumor growth in these models. Since IIA1 does not recognize murine α5β1 in the vasculature of a xenograft tumor, the entire inhibitory effect of IIA1 on tumor growth can be attributed to the direct antiproliferative effect on cancer cells of this tumor.

Example 5

IIA1 in combination with DOXIL® prevents tumor insertion in the in vivo NW231 graft model

The following experiment was performed to determine the efficacy of an anti-α5β1-integrin, IIA1 antibody in combination with a chemotherapeutic agent, DOXIL®, to prevent the laying of human NW231 tumors in vivo. DOXIL® is a liposome-encapsulated form of doxorubicin-HCl, a cytotoxic anthracycline antibiotic isolated from Streptomyces peucetius. The NW231 cell line is derived from breast cancer and is a good model for exploring treatments for breast cancer.

Materials and methods

Four to six-week-old female SCID mice obtained from Taconic Farms and located in micro-isolator cells were inoculated into 1 × 10 ⁷ mammary fat accumulations in NW231 cells. Animals received either a TIB control (n = 20) or M200 (n = 20) at 5 mg / kg for the first injection at the time of tumor cell inoculation. Subsequent treatments were as 0.7 mg / kg per injection. DOXIL® treatment was performed 5 days after inoculation of tumor cells. The chemotherapeutic doses were 4 mg / kg for the first injection and 2 mg / kg for subsequent injections. Reagents were delivered by intraperitoneal injection for four doses. Tumor volume was measured twice a week and clinical and mortality observations were performed daily in accordance with IACUC rules.

results

Treatment with IIA1 showed a significant effect in slowing the laying of NW231 tumors in mice. At 24 days after implantation of NW231 tumors, the average tumor volume of the control treated TIB xenografts increased exponentially to ~ 425 mm ³ , while the treated IIA1 xenografts increased to an average volume of ~ 175 mm ³ .

DOXIL® treatment itself also showed a significant effect in preventing tumor lining. For DOXIL®-treated xenografts alone, the average tumor volume initially increased only to ~ 25 mm ³ during the first 45 days after implantation and then gradually increased to ~ 275 mm ³ on day 74.

Treatment of mice with combination therapy of IIA1 and doxil had an even higher inhibitory effect on the incidence rate of the tumor compared to any treatment alone. For treated IIA1 plus DOXIL® xenografts, the average tumor volume remained close to zero until day 54, then increased gradually, but only to ~ 125 mm ³ per day 74. These results show increased efficacy (ie combined tumor inhibitory effect) in vivo for combined treatment with anti-α5β1, IIA1 antibody and chemotherapeutic agent, DOXIL®.

Example 6

Efficiency of antibody M200 measured in a rabbit VX2 tumor model

Although M200 does not cross-react with mouse or rat α5β1 integrin, it does recognize the α5β1 integrin found in the rabbit. Thus, rabbit VX2 carcinoma may provide a good model for determining the efficacy of M200 in direct in vivo cancer cell lysis. VX2 is a widely recognized rabbit model for primary tumor treatment studies at various locations (see, for example, Chen JG, et al .. Lab Anim. 2004 Jan; 38 (1): 79-84; Purdie, TG et al., Phys Med Biol. 2001 Dec; 46 (12): 3161-75); Geschwind, JH, et al., Cancer Res. 2002 Jul 62 (1): 3909-3913).

A. Search (pilot) study of the effectiveness of the M200 in the VX2 model

An initial exploratory study was performed to determine the general parameters for the study of the effectiveness of M200 in a rabbit VX2 tumor model.

Tumor inoculation

Rabbits were inoculated on day 0 with a suspension of cells (100 μl) subcutaneously (into the left hind limb) and intramuscularly at a depth of approximately 3 mm (into the right hind limb). Intramuscular inoculation was performed as follows. During rabbit anesthesia with a ketamine / isoflurane mixture, an incision of ~ 2 cm was made parallel to the right femur with a scalpel on the front, lateral side of the femoral axis, ~ 1/3 of the total length of the femur distally from the hip joint. Muscle groups were divided to create a cavity with a depth of ~ 0.5 cm. One fragment of a VX2 tumor from an animal donor was placed in this cavity. The skin was securely covered with sterile surgical staples or sutures and a topical antibiotic was applied to the wound site.

Tumor Measurements

Starting from day 5, we measured the size of the tumor (length, width and height) in millimeters using an electronic Vernier circuit connected to a laptop (road) computer, at a minimum frequency of twice a week. For consistency, the same trained specialist performed tumor measurements throughout this study. Tumor volume was calculated using the formula: length × width × height × 0.52. After killing the animals, the tumors were carefully excised, cut and weighed. In addition, animals were weighed at least once a week. Representative samples of each tumor were canned in a sample cassette in formalin or OCT and quickly frozen in liquid nitrogen.

Passivation of VX2 tumors in vivo

Tumors VX2, used both in the pilot (search) study, and in the study of the effectiveness of M200, was supported by passivation in vivo once a month. Either a cell suspension (100 μl) or tumor pieces (~ 5-10 mm ³ ) were used for intramuscular inoculation in each hind limb. Animals and tumors were subjected to visual monitoring and tumors were removed before reaching a diameter of 2 cm for passaging in vivo. In vivo passaging was performed by killing the animal, removing the tumor and treating the tumor by dividing it into pieces of 5-10 mm ³ . These pieces were re-implanted into the next group of 2 rabbits.

IHC analysis of VX2 tumors from treated M200 rabbits

M200 was administered intravenously at a dose of 10 mg / kg to the tumor-bearing rabbit and this tumor was excised after 1 hour. Tumor sections were stained with: (i) a secondary anti-human Ig antibody specific for tumor bound M200; (ii) IIA1, followed by a secondary antibody against mouse Ig to detect total α5β1; or (iii) a control IgG and a secondary anti-mouse Ig antibody.

results

It was found that both subcutaneous tumors and intramuscular tumors were available for M200 injected intravenously in these animals, as evaluated by IHC. IHC analysis (immunohistochemical analysis) of stained sections revealed high levels of α5β1 expression both in VX2 tumor cells and in the vasculature of rabbit tumors.

B. Study of the effectiveness of M200 in the VX2 model

Based on the IHC results of this search (pilot) study, the rabbit VX2 model was used to evaluate in vivo efficacy of the M200.

Methods

Rabbits (30 in total) were inoculated, subcutaneously or intramuscularly, either with a suspension of VX2 cells (100 μl), or with tumor pieces (~ 5-10 mm ³ ). A test group of 20 rabbits was treated with M200 at 10 mg / kg intravenously, twice a week for 3 weeks. A control group of 10 animals was treated with control IgG delivered in the same manner as M200 was delivered. Measurement of tumors, termination, weighing, preservation of tumors and the duration of the study was carried out in accordance with the methods described above for search research. In addition, one milliliter of blood was taken from the ear vessel once a week for serum analysis.

results

A significant correlation was observed between inhibition of tumor growth and levels of M200 in the bloodstream in these animals. Typically, while maintaining M200 levels at or above 50 μg / ml, tumor growth is inhibited. Since M200 is immunogenic in rabbits, some of the test group rabbits generated an immune response to M200 as early as two weeks after administration of M200, and it was found that all these animals altered their serological specificity over time. In animals, test groups that generated an anti-idiotypic response leading to M200 clearance (i.e., M200 <50 μg / ml on day 14) showed greater tumor growth. Thus, the results observed using the VX2 carcinoma model show that M200 is able to inhibit tumor growth in a clear in vivo oncology model.

Example 7

Efficiency of M200 and IIA1 in the SCID mouse VX2 xenograft model

As described in Example 6, M200 was effective in the rabbit model of VX2 carcinoma. The next measurement of the ability of direct lysis of cancer cells with antibodies M200 and IIA1 was performed in a SCID mouse VX2 xenograft model. Since M200 and IIA1 do not recognize the murine α5β1 integrin present on the vasculature of the VX2 xenograft, any inhibitory effect on the growth of the VX2 xenograft can be attributed to the direct antiproliferative effect that M200 and IIA1 have on VX2 cancer cells.

Materials and methods

Mice with immunocompromised CB-17 SCID (strain C. B-Ighl / IcrTac-Prkdc) were obtained from Taconic Farms (Germantown, NY). Studies began using female mice at 6-10 weeks of age (weighing ~ 20 g). The animals were pre-injected with M200, IIA1, or control IgG intraperitoneally at a dose of 10 mg / kg one hour before inoculation of VX2 (1 × 10 ⁷ cells in modified Dulbecco's Isc media) into the accumulation of adipose tissue of the mammary gland. Dosing was continued for 3 weeks at a frequency of 3 times per week at 10 mg / kg. Tumor volume was measured twice a week using a caliper and calculated using the formula π / 6 × length × width × height. Clinical and mortality observations were performed daily in accordance with IACUC rules.

results

The administration of doses of M200 or IIA1 did not correlate with a reduced tumor growth rate of VX2 or an overall reduced tumor size compared to mice that were dosed with control IgG. These results suggest that neither M200 nor IIA1 were able to directly kill VX2 cells in this mouse xenograft model.

Example 8

Phase I study with an escalation of the M200 dose in human patients with untreatable solid tumors

A. Overview

A two-part open-label phase I study was performed to determine treatment actions using up to 6 dose levels, from 0.5 mg / kg to 15.0 mg / kg M200 (volociximab), in 21 human patients with various types of untreatable solid tumors. The total duration of treatment for the first part was 6 weeks with physical assessments for 45 days after the last dose. The second part was an extended study in which 6 out of 11 patients who showed a stable disease response in the first part continued to administer the dose for up to 52 weeks.

B. Study parameters and protocols

1. Patient selection

For this study, patients who met the following inclusion / exclusion criteria were selected:

but. At least one measurable damage in a routine computed tomography (CT) image or nuclear magnetic resonance image (MRI) image.

b. Estimated survival of ≥3 months; health status of the Western Cooperative Cancer Research Group (ECOG) ≤2.

from. The absence of tumors or metastasis of the central nervous system (CNS) (documented by screening the image of the head).

d. Lack of extensive surgery (“large” surgery) within 4 weeks before admission.

e. The absence of minor surgical intervention ("minor" surgery) within one week before admission; the absence of chemotherapy, immunotherapy or radiation therapy within 4 weeks before admission.

f. The absence of an active disorder with bleeding or thromboembolic events.

g. The absence of another clinically significant or unstable medical condition.

n Patients who have previously received murine or chimeric mAb therapy should be negative for screening for anti-M200 antibodies (ie, cross-reactive anti-mouse human antibody [NAMA] or human anti-chimeric antibody [NASA]).

A total of 22 patients were enrolled in this study. The types of tumors of these 22 patients included: colorectal cancer (4 patients; CRC), melanoma (4 patients; MEL), renal cell carcinoma (3 patients; RCC), esophageal cancer (2 patients; EC), hepatic cell carcinoma (2 patients; HSS), lung cancer (1 patient; NSCLC), prostate cancer (1 patient; PRO), thyroid cancer (1 patient; THY), pancreatic cancer (2 patients; PC), parotid cancer ( 1 patient; PARO) and breast cancer (1 patient; BC). 21 out of 22 patients received treatment, and 20 out of 21 treated patients completed all 5 dose administrations plus follow-up for 45 days. These treated patients (21 patients, 12 men, 9 women) had a median age of 59 years (range 29-81) and an average score of the Western Cooperative Cancer Research Group (ECOG) 1 (range 0-2).

2. The choice of levels and dosage regimens M200

The levels and dosage regimen were chosen in such a way as to ensure that the highest dose (15.0 mg / kg) would produce maximum serum concentrations that were sufficiently lower than the safe average maximum concentrations observed in dog-headed monkeys, and that all levels in serum concentrations for minimum doses of ≥1.0 mg / kg will produce serum concentrations of ≥1 μg / ml, a concentration that provides 80% inhibition of the binding of α5β1 to fibronectin in an in vitro activity assay.

The levels and dosage regimen used in this study were as follows. M200 was administered to 21 patients on days 1, 15, 22, 29 and 36. The dose and number of patients per level were: 0.5 mg / kg (1 patient), 1.0 mg / kg (2 patients), 2.5 mg / kg (3 patients), 5.0 mg / kg (3 patients), 10.0 mg / kg (6 patients) and 15 mg / kg (6 patients). This dose was administered as an intravenous infusion over 1 hour. The first and second doses were divided by a period of two weeks to allow sampling for pharmacokinetic (FC) data for individual doses. These PK measurements performed after the first dose were used to predict the maximum serum concentration of each patient after the fifth dose. If the predicted maximum serum concentration after the first dose was <750 μg / ml, then the patient received all 5 doses of M200. The remaining 3 doses were given once a week, followed by a 45-day evaluation period.

Human serum concentrations of M200 were predicted based on the above dose schedule using the same range of variability observed in dog-headed monkeys (i.e., 54% -140% average). It was predicted that at the highest dose in humans (15.0 mg / kg), the average maximum after 5 doses is 592 μg / ml with a predicted range of variability in humans of 320-829 μg / ml. Table 3 was compiled using FC data from monkey studies to predict the maximum and minimum serum concentrations (Cmax and Cmin, respectively) for each dose at each dose level in humans.

Table 3 Estimated maximum and minimum serum levels of M200 in humans Level Dose (mg / kg) FC value ^a Number of doses one 2 3 four 5 one 0.5 Cmax eleven eleven 12 13 13 Cmin ^b 1,5 1,6 2 2.1 2.2 2 1,0 Cmax 22 22 25 26 26 Cmin 3 3 four 5 5 3 2.5 Cmax 54 57 64 67 68 Cmin 9 10 13 fourteen fifteen four 5,0 Cmax 108 114 132 141 147 Cmin twenty 24 33 39 43 5 10.0 Cmax 216 236 282 317 346 Cmin 51 66 101 131 156 6 15.0 Cmax 324 366 450 524 592 Cmin 91 126 200 268 332 Range Cmax (54-140%) 175-453 197-512 243-629 283-733 320-829 ^a Values expressed in μg / ml
^b Calculated for values expected 1 week after the first dose.

Based on the above table, it was predicted that the final half-life in humans at 15.0 mg / kg is approximately 13 days and is shorter for lower doses. It was predicted that serum M200 accumulation was significant at dose levels of ≥ 5.0 mg / kg, with a steady state concentration achieved within 4 weeks for all doses <10.0 mg / kg and within 5 weeks for doses ≥10. 0 mg / kg.

3. Preparation and introduction of M200

Biological mass M200 was prepared in accordance with the existing Good Manufacturing Practices (cGMP) guidelines. The composition of the drug M200 used in this study was 10 mg / ml M200, 25 mm citrate, 150 mm sodium chloride, 0.05% polysorbate (TWEEN®) 80, with a pH of 6.5. This drug is a sterile, colorless, clear, slightly opalescent, non-preservative liquid for I.V. use. Each 20 ml vial for single use was filled to obtain 15 ml of M200 at a dose of 10.0 mg / ml. Each 10 ml vial for single use was filled to obtain 10 ml of M200 at a dose of 10.0 mg / ml. The intact vials were refrigerated at 2-8 ° C (36-46 ° F) and kept without freezing or shaking.

After preparation, M200 was administered within 6 hours if stored at room temperature (25 ° C), or 48 hours if the preparations were cooled in a refrigerator (between 2 and 8 ° C). After this time, the prepared solution was discarded.

A suitable dose of M200 for administration to a patient was calculated by multiplying the patient's weight (kg) by the appropriate dose level (mg / kg) for the patient. The patient’s weight before dosing on day 1 of the study was used to calculate the dose over the course of the study. For patients included in the dose groups of 0.5 mg / kg - 10.0 mg / kg, and for patients weighing ≤80 kg included in the dose group of 15.0 mg / kg, this dose was administered in a fixed total volume of 120 ml for one hour.

Although 120 ml of the diluted test drug was administered to the patient, an infusion bag was prepared with a total capacity of 150 ml. An additional 30 ml was used to start the infusion line and was not administered to the patient. Thus, the total dose of the investigational drug placed in the infusion bag was the dose of the patient (ie patient weight (kg) × dose level [mg / kg] multiplied by 1.25). The total volume in the infusion bag was adjusted to 150 ml by the addition of sodium chloride for injection, USP (0.9%).

Patients weighing> 80 kg, enrolled in a group with a dose of 15.0 mg / kg, received their dose, administered in a fixed total volume of 180 ml for one hour. Although 180 ml of the diluted test drug was administered to the patient, an infusion bag was prepared with a total capacity of 210 ml. An additional 30 ml was used to start the infusion line and was not administered to the patient. Thus, the total dose of the test drug in the infusion bag was the dose of the patient (ie patient weight (kg) × dose level [mg / kg] multiplied by 1.167). The total volume in the infusion bag was adjusted to 210 ml by the addition of sodium chloride for injection, USP (0.9%).

A pre-filled infusion line was connected to the patient’s IV access (e.g., heparin stopper). The infusate was administered at a rate of 2 ml / min (or 3 ml / min for patients weighing> 80 kg of the group with a dose of 15.0 mg / kg) for one hour using an infusion pump.

4. Primary endpoints and adverse events

The endpoints of the study included the maximum tolerated dose, dose-limiting toxicity, safety profile, immunogenicity, pharmacokinetics (PK), monocyte saturation (monocytes express the α5β1 receptor), and tumor response based on Response Evaluation Criteria in Solid Tumors (RECIST) criteria (see . Therasse P, et al., "New guidelines to evaluate the response to treatment in solid tumors," Journal of the National Cancer Institute, 92 (3): 205-216 (2000), which is incorporated herein by reference in its entirety. form).

Harmful events were categorized using the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE), version 3.0. Dose-limiting toxicity was defined as any adverse event of category-3 or category-4, with the exception of planned hospitalizations or not included in the surgical program. Harmful events of Category-3 or Category-4, which are believed to be unrelated to the M200, were excluded, if necessary, after a joint analysis with medical regulatory and regulatory institutions.

Patient ratings during this study include the following:

but. Screening (within 14 days of entry into the study): medical history and physical examination, health status of ECOG, routine chemical panel, blood test (CBC) with determination of leukocyte formula and platelets, sensitive C-reactive protein (CRP), urinalysis using microscopic analysis (UA / micro), electrocardiograms (ECG), chest X-ray (CXR), CT or MRI of the body, aimed at the disease, urinalysis for pregnancy within 48 hours before dosing and studies convolution antibodies (prothrombin time [PT] and partial thromboplastin time [PTT]), antibodies against M200 (ie, for detecting cross-reactive NAMAs or NASAs) for patients who have previously received murine or chimeric antibodies, CT heads for patients with tumors which usually metastasize to the brain or central nervous system, and, in some cases, biopsies to assess the presence of blood vessels in the tumor.

b. Laboratory assessments during the study: routine chemical panel, urate, serum amylase, SHS with a leukocyte formula and platelets, UA / micro.

from. Routine disease-targeted x-ray imaging (e.g., CT scans) before and after treatment with M200 was used to evaluate tumor response using univariate RECIST criteria. Target injuries were selected based on their size (damage with the longest diameter (LD)) and their suitability for accurate, repeatable measurements (imaging or clinically). The total LD was calculated for all target lesions and reported as the background total LD. This background sum LD was used as a reference to characterize the objective response of the tumor. The following reaction criteria for target lesions were used to evaluate the best overall response.

- Complete response (CR): disappearance of all target lesions confirmed by re-imaging 25-28 days after the first documented CR.

- Partial Reaction (PR): at least a 30% reduction in the amount of LD damage to the targets taken relative to the background amount of LD, confirmed by re-visualization 25-28 days after the first documented PR.

- Stable Disease (3D): neither a sufficient reduction for qualification as a PR, nor a sufficient increase for qualification as a PD when considering the smallest amount of LD.

- Progressive disease (PD): at least a 20% increase in the amount of LD target damage taken relative to the smallest amount of LD recorded since the start of treatment, or the appearance of one or more new lesions.

d. Blood sampling for serum FC measurements, immunogenicity assays (anti-M200 antibodies), vascular growth factors, and measurement of bound and unbound α5β1 on peripheral blood monocytes.

e. A 3 mm puncture biopsy of surface tumor metastasis was obtained and frozen after the last injection of M200 from patients who consented to this procedure (optional for inclusion in the study). This biopsy test could be used to assess the presence of blood vessels in the tumor after treatment with M200.

5. Pharmacokinetic measurements

The first human FC data at low doses were used to predict human serum levels at higher doses (as described above). Serum for measuring M200 concentrations was obtained immediately before the first dose and after the first dose and at 4, 24, 48 and 168 hours after completion of the first dose. Serum samples for measuring M200 concentrations were also taken immediately before the completion of each subsequent infusion, at the end and 4 hours after the completion of each subsequent infusion. These samples were separated and analyzed aliquots before the next dose and at the end of the study: at the end of the 1st dose, 168 hours after the 1st dose, immediately before the dose (minimum) and immediately after the dose (maximum) for 2 3rd, 4th, 5th and 5th doses. M200 serum levels were measured by ELISA.

Data on the concentration of M200 in plasma from time to time from each patient was subjected to FC analysis. The calculations included the following parameters: maximum (peak) (Cmax) and minimum (dip) (Cmin), half-life of the terminal phase (T _{1 / 2β} ), area under the concentration-time curve (AUC), eliminating clearance (CL) and volume of distribution (V). The total parameters were calculated using a computer in all patients of each treatment group. Changes in PK parameters were also evaluated as a function of dose and time.

6. Immunogenicity

Immunogenicity M200 was determined using ELISA with double bridge conjugation of antigens. Serum samples for antibodies against M200 were obtained from patients before treatment, before the second dose and upon exiting the study, 45 days after the last dose of M200. In addition, serum was taken for antibodies against M200, if the interval between doses was ≥2 weeks. Samples were stored and evaluated at the end of this study. Samples that were positive for antibodies to M200, re-analyzed for neutralizing antibodies to M200. Patients who were known to be exposed to murine or chimeric monoclonal antibodies were screened for the presence of anti-M200 antibodies before they received their first dose of M200.

7. Flow cytometric analysis

α5β1-integrin is also expressed on peripheral blood monocytes. To determine the dose at which the α5β1 sites on these circulating cells are saturated with M200, peripheral blood samples were taken before the first dose on study day 1, study days 2 and 8, immediately before each subsequent dose, and on study day 43. Saturation of α5β1 sites on monocytes determined by flow cytometry. Monocytes were identified using anti-CD14 antibodies. Bound M200 on the surface of monocytes was detected by the addition of labeled anti-human IgG4 antibody. Unoccupied (free) α5β1 on these cells was detected by the addition of labeled IIA1. The percentage of monocytes and lymphocytes expressing α5β1 was also evaluated.

C. Results - Dose escalation study

M200 was well tolerated by patients at doses up to 15 mg / kg, and dose limiting toxicities of M200 were not observed. The overall response for this study was as follows: stable disease (3D) in 11 patients and progressive disease (PD) in 10 patients. Table 4 shows the distribution of the result of the reaction according to the dose and type of tumor of the patients.

Table 4 Reaction results by dose and type of tumor 0.5 mg / kg 1.0 mg / kg 2.5 mg / kg 5.0 mg / kg 10.0 mg / kg 15.0 mg / kg CRC (PD) HCC (SD) CRC (PD) HCC (SD) CRS (SD) EU (PD) PC (SD) NSCLC (SD) EU (PD) CRC (SD) RCC (SD) MEL ophthalmic (PD) THY (PD) PARO (SD) PRO (PD) Sun (SD) MEL (PD) RCC (SD) MEL (SD) MEL (PD) RCC (PD)

Abbreviations: CRC = colorectal; MEL = melanoma; RCC = renal; EC = esophogeal; HSS = hepatic cell; NSCLC = pulmonary; PRO = prostate gland; THY = thyroid gland; PC = pancreas; PARO = parotid gland; BC = breast; SD = stable disease; PD = progressive disease

Harmful events were usually mild to moderate in intensity and included fatigue, nausea, constipation, headache, and anorexia. There were no serious or serious adverse events that were dose-limiting or, according to the researchers, were associated with M200. Three patients at dose levels of 0.5, mg / kg and 1.0 mg / kg were tested as positive for antibodies against M200, but no visible associated adverse events were observed. There were no patients in groups with higher doses giving a positive reaction when tested for antibodies against M200. One patient who received 0.5 mg / kg had a fever after the first dose, which was reported as a response to infusion. However, this patient did complete all 5 doses of M200 without subsequent bouts of fever or other symptoms of an infusion reaction.

The results of this study showed that M200 detects non-linear pharmacokinetics. Slower clearance was observed at higher concentrations with T _1/2 = 15.7 days at a dose level of 10 mg / kg. In addition, a dose of 10 mg / kg led to saturation of monocytes and had an average minimum level of 82 μg / ml two weeks after the 1st dose, which is higher than the minimum effective in vitro concentration of 2-3 μg / ml.

D. Expanded research

Six out of 11 patients who showed a stable disease response or better response entered an extended study and continued to administer doses. As shown in table 5 below, 5 out of 6 patients showed a stable disease (SD) or better response. One of these patients with renal cancer (RCC) in the 15.0 mg / kg dose group achieved a partial response.

Table 5 Expanded Research Results Group dose (number of patients; tumor type) Best reaction result Time to progression * (days) 2.5 mg / kg (1; NSCLC) SD 129 5 mg / kg (1; HCC) SD 122 10 mg / kg (1; CRC) PD 70 10 mg / kg (1; PARO) SD 143 15 mg / kg (1; RCC) PR 214 15 mg / kg (1; MEL) SD 172 + (in study) * Includes 43 days in a dose escalating study plus days of an extended study.
SD is defined as stabilization for ≥16 weeks (112 days)

Example 9

M200 Phase II open-label study in human patients with metastatic renal cell carcinoma

A. Overview

Based on the effectiveness of the M200 demonstrated by the phase I study of Example 8, an open, multicenter, one-sided, 2-stage study of the M200 phase II was undertaken to treat human patients. The primary goal of this study is to evaluate the efficacy (tumor response) of M200 in patients with metastatic renal cell carcinoma (RCC) as determined using Response Criteria for Solid Tumors (see RECIST, Example 8 above). The second objective of the present invention is also the evaluation of other criteria of effectiveness (namely, the time of disease progression and the duration of the reaction) and an additional assessment of the safety, immunogenicity and FC parameters of M200, which were originally evaluated in the phase I study of Example 8. An additional objective of the study is to measure markers in serum and plasma. This study should list up to 40 patients in eight research centers. Twenty patients should be included in stage 1 of this study. If one confirmed complete reaction (CR) or partial reaction (PR) is observed at 4 months (16 weeks) or if one stable disease (3D) is observed at 4 months, then an additional 20 patients must be recruited (stage 2). All patients will receive M200 (10 mg / kg) as an intravenous infusion for 30 minutes once every other week for up to 52 weeks or until the disease progresses, regardless of what happens first. A visit to exit the study will take place at 45 days after the last dose or at the time of early termination if the patient is unable to return. A follow-up visit will take place at 3 months after the last dose. If a visit is not possible, follow up should be done by telephone. Long-term follow-up will be done over the phone at 6 months after the last dose.

B. Study parameters and protocols

The phase II study is carried out in accordance with the parameters and protocols described in table 6 below.

Table 6 Phase II study parameters in patients with metastatic renal cell carcinoma Study Population: Men and women at least 18 years old with metastatic RCC clear cell histology The main criteria for inclusion / exclusion of a patient: Inclusion: men and women of at least 18 years of age with metastatic RCC of predominantly clear cell histology who have received 0-2 previous treatment regimens for metastatic disease; measurable disease in accordance with Response Criteria for Solid Tumors (RECIST); health status of the Western Cooperative Oncology Group (ECOG) ≤1; negative pregnancy test (only women with childbearing potential); pre-treatment laboratory levels that meet specific criteria; and signed informed consent, including permission to use protected health information (PHI). Exception: any of the following RCC histology: papillary, chromophobic, rectal duct or unclassified; known sensitivity to murine proteins or chimeric antibodies or other components of this product; the use of any study drug within 4 weeks before a screening survey or 5 half-lives of the study drug (depending on which of these periods is longer); systemic chemotherapy, immunotherapy, radiation therapy or therapy using monoclonal antibodies within 4 weeks of the introduction of M200; documented CNS tumor or CNS metastasis; a history of thromboembolic events and circulatory disorders within the past year and medical conditions that may be exacerbated by bleeding. Sample Size: The list can include up to 40 patients. Twenty patients will be included in stage one. If one confirmed complete reaction (CR) or partial reaction (PR) is observed at 4 months (16 weeks) or if one stable disease (SD) is observed at 4 months, 20 additional patients will be added to the list (stage 2). Dosage form and concentration / preparation: M200 is a sterile, colorless, transparent - slightly opalescent, preservative-free liquid for intravenous (IV) use. Each 10 ml vial for single use is filled to deliver 10 ml of M200 at 10 mg / ml. The composition of each bottle: 10 mg / ml M200, 25 mm citrate, 150 mm sodium chloride and 0.05% Polysorbate (Tween®) 80, with a pH of 6.5. Storage and filtering: Intact vials are stored in a refrigerator at 2-8 ° C (36-46 ° F). Do not freeze or shake. M200 should be introduced within 6 hours if it is stored at room temperature (25 ° C), or 48 hours if it is in the refrigerator (2-8 ° C). After this time, the prepared solution should be discarded. Dosage schedule / route of administration: All selected patients will receive 10 mg / kg M200 by intravenous (IV) infusion for 30 minutes every second week for a period of up to 52 weeks or until disease progression, whichever occurs first. The dose of M200 administered to the patient will be calculated by multiplying the patient's mass (kg) by the dose level (10 mg / kg). The patient’s weight before the dose is administered on day 0 of the study will be used to calculate the dose over the course of this study, provided that the weight does not change by more than 10%. A suitable volume of the M200 composition of 10 mg / ml will be recovered and diluted with 0.9% sodium chloride to a final volume of 120 ml for IV infusion into the patient over a period of 30 minutes. Duration of treatment and follow-up: All patients will receive M200 (10 mg / kg) intravenously once every other week for up to 52 weeks or until disease progression, whichever occurs first. A visit to exit the study will take place 45 days after the last dose or at the time of early termination if this patient is not able to return. A follow-up visit will take place at 3 months after the last dose. If this visit is not possible, follow-up will be conducted by telephone. Long-term follow-up will be done over the phone at 6 months after the last dose. Endpoints: The primary endpoint of this study is the proportion of patients with a confirmed tumor response at any time during this study. Secondary endpoints are: (1) time to disease progression; (2) the duration of the tumor reaction; (3) FC M200; and (4) immunogenicity. The diagnostic endpoint is the measurement of detectable serum biomarkers in plasma. Performance Measurements: Disease-targeted radiographic imaging every 8 weeks (2 months) to evaluate tumor response using a one-dimensional RECIST. On the screen, CT or MRI of the head, chest, abdomen and pelvis will be received. Every 8 weeks a full physical examination will be performed and scans of images of all injuries present on the screen will be received, plus all anatomical injuries aimed at the disease. For each PR or CR reaction, confirmatory radiographic tomography will be repeated one month later (28-35 days) after PR or CR. All measurable lesions of up to 5 lesions per organ maximum and a total of 10 lesions representative of all included organs should be identified as target lesions and recorded and measured at baseline (background). Measurable lesions are determined as follows: 2.0 cm in one direction using conventional CT / MRI and 1.0 cm in one direction using helical CT. The sum of the longest diameter (LD) for all target lesions is calculated and reported as the background sum of LD. This background sum LD is then used as the basis of the reflection (background) by which the target tumor response will be characterized. Security Measurements: The following safety measurements will be monitored: harmful events (AE); serious adverse events (SAE); physical examination results; signs of life and abnormal laboratory indicators. Pharmacokinetic measurements: Pharmacokinetic (FC) measurements will be performed in all patients at the beginning of the dose (within 15 minutes before the dose) and after the dose (within one hour after the end of the infusion): day 0, weeks 2, 4 and 6, once a every second month (weeks 8, 16, 24, 32, 40, 48), week 52, a visit to exit the study, and a 3-month follow-up (if possible). Samples obtained for RK can be used for analysis against Ab, if necessary. Immunogenicity: Anti-Ab tests will be performed in all patients within 15 minutes before dosing on day 0, week 8, at the visit to exit the study, and in a 3-month follow-up (if possible). Samples obtained for RK can be used for analysis against Ab, if necessary. Other measurements: A diagnostic analysis will evaluate the presence of biomarkers in serum and plasma. The following cancer markers are included in this analysis: CEA (carcinoembryonic antigen), CA 19-9, Sindecan, IGFBP-2 and LFL2. The following cytokines / growth factors are included: MIA, IL-6, TNF-α, PGF, VEGF, EGF and bFGF. Statistical methods: This study design will provide a power of at least 79.5% for detecting a total degree of response of ≥20% when up to 20% of responders exhibit stable disease (SD). Summary statistics and 95% confidence intervals will be provided for dichotomous endpoints. Kaplan-Mayer methods will be used to summarize the temporary variables.

It should be understood that the examples described above in no way serve to limit the true scope of the present invention, but are presented for illustrative purposes. All publications, sequences of access numbers, and patent applications cited in this specification are incorporated herein by reference, as if each individual publication or patent application was individually indicated to be incorporated by reference.

Claims (22)

1. A method of inhibiting the proliferation of cancer cells in a patient, where the cancer cells express α5β1-integrin on their surface, where the method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition containing a liquid composition comprising:
from about 1.0 to 15 mg / ml anti-α5β1 antibody;
approximately 22 to 27 mM citrate;
approximately 145 to 165 mM sodium chloride;
from about 0.04 to 0.06% polysorbate (TWEEN®) 80;
and whose pH is from about 5.5 to about 7.5. 2. The method according to claim 1, where the antibody neutralizes at least one biological activity of α5β1-integrin. 3. The method according to claim 1, where this anti-α5β1 antibody binds to the same α5β1 integrin epitope as the M200 antibody. 4. The method according to claim 1, where the anti-α5β1 antibody competitively inhibits the binding of M200 to α5β1 integrin expressed on the cell surface. 5. The method according to claim 1, where the anti-α5β1 antibody is selected from the group consisting of M200, F200 and IIA1. 6. The method according to claim 1, where the antibody competitively inhibits the binding of the second antibody to α5β1 integrin on the surface of the cancer cell, where the second antibody contains an amino acid sequence selected from SEQ ID NO: 2, 4, 6 and 8. 7. The method according to claim 1, where the therapeutically effective dose is approximately 10 mg / kg 8. The method of claim 1, wherein the cancer cell is selected from the group consisting of breast cancer cells, lung cancer cells, metastatic melanoma cells, pancreatic cancer cells, and kidney cancer cells. 9. The method according to claim 1, where the cancer is kidney cancer or metastatic melanoma. 10. The method according to claim 1, further comprising administering to the patient a chemotherapeutic agent, either sequentially or in combination with an antibody. 11. A method of treating cancer that expresses α5β1 in an individual in whom the tumor has not yet spread, comprising administering to the individual a therapeutically effective amount of a pharmaceutical composition comprising a liquid composition comprising:
from about 1.0 to 15 mg / ml anti-α5β1 antibody;
approximately 22 to 27 mM citrate;
approximately 145 to 165 mM sodium chloride;
from about 0.04 to 0.06% polysorbate (TWEEN®) 80;
and whose pH is from about 5.5 to about 7.5. 12. The method of claim 11, wherein the antibody binds to the same α5β1 integrin epitope as the M200 antibody. 13. The method according to claim 11, where the antibody competitively inhibits the binding of M200 with α5β1 integrin expressed on the surface of the cancer cell. 14. The method of claim 11, wherein the cancer is selected from the group consisting of bladder cancer, breast cancer, colon cancer, fibrosarcoma, lung cancer, metastatic melanoma, pancreatic cancer, prostate cancer, ovarian cancer, kidney cancer, and spleen cancer. 15. The method according to claim 11, where the cancer is kidney cancer or metastatic melanoma. 16. The method according to claim 11, where the therapeutically effective dose is approximately 10 mg / kg 17. The method according to claim 11, further comprising administering to the patient a chemotherapeutic agent, either sequentially or in combination with an antibody. 18. A pharmaceutical composition comprising a liquid composition comprising:
from about 1.0 to 15 mg / ml anti-α5β1 antibody;
approximately 22 to 27 mM citrate;
approximately 145 to 165 mM sodium chloride;
from about 0.04 to 0.06% polysorbate (TWEEN®) 80;
and whose pH is from about 5.5 to about 7.5. 19. The pharmaceutical composition of claim 18, wherein the concentration of the anti-α5β1 antibody is about 10 mg / ml. 20. The pharmaceutical composition of claim 18, wherein the anti-α5β1 antibody is selected from the group consisting of M200, F200, and IIA1. 21. The pharmaceutical composition of claim 18, wherein the anti-α5β1 antibody is M200. 22. The pharmaceutical composition of claim 18, wherein the composition further comprises a chemotherapeutic agent.

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