JP2006516636A - Anti-integrin αvβ3 antibody preparation and use thereof - Google Patents

Abstract

The present invention is a liquid preparation of an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ , exhibits stability, antibody fragmentation is undetectable to low level, and aggregation is not detected. It relates to a formulation that is at a low to possible level and that loses little or no biological activity of the antibody or antibody fragment, even during long-term storage. In particular, the present invention relates to a liquid formulation of an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ that is substantially free of surfactants, inorganic salts and / or other excipients. The present invention uses the liquid preparation of the present invention to treat inflammatory disorders, autoimmune disorders, disorders related to abnormal expression and / or activity of integrin α _v β ₃ , disorders related to abnormal bone metabolism, abnormal It also relates to methods for preventing, treating, managing or ameliorating disorders associated with angiogenesis as well as cancer.

Description

1. Introduction The present invention is a high-concentration liquid formulation of an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ , exhibits stability, antibody fragmentation is undetectable to low level, Relates to formulations in which the biological activity of the antibody or antibody fragment is not lost or hardly lost, even during long-term storage, even though it is undetectable to low levels. In particular, the present invention relates to liquid formulations of antibodies or fragments thereof that immunospecifically bind to integrin α _v β ₃ that are substantially free of surfactants, inorganic salts, or both. The present invention, integrin alpha _v with a high concentration liquid formulations of antibodies or fragments thereof that bind to beta ₃ immunospecifically, inflammatory diseases, autoimmune diseases, integrin alpha _v beta ₃ of aberrant expression and / or activity And disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, and methods for preventing, treating, managing or ameliorating cancer.

  This application has priority based on US Provisional Application No. 60 / 443,777 filed Jan. 30, 2003 and US Provisional Application No. 60 / 443,810 filed Jan. 30, 2003; And insist on this. This US provisional application is also incorporated herein by reference in its entirety.

2. BACKGROUND OF THE INVENTION Currently, many antibodies are provided as lyophilized formulations. Antibody lyophilized formulations have many limitations, including the long lyophilization process, which results in high manufacturing costs. Furthermore, the lyophilized formulation must be reconstituted aseptically and accurately by medical personnel prior to administration to the patient. The reconstruction step itself requires a certain operating procedure. (1) Sterile diluent (ie, water for intravenous administration and 5% aqueous glucose solution for intramuscular administration) is slowly added aseptically and slowly to a vial containing lyophilized antibody so that the vial does not foam Must be swirled very gently for 30 seconds; (2) the reconstituted antibody must be left at room temperature for a minimum of 20 minutes until the solution is clear; and (3) the reconstituted formulation Must be administered within 6 hours after reconstitution. Such reconstitution procedures are difficult to handle and may not be properly reconstituted due to time restrictions after reconstitution or if the reconstituted dose is not used within 6 hours This has to be discarded, resulting in considerable waste and can be very inconvenient when administering this formulation to a patient.

Therefore, there is a need for a liquid formulation of antibodies (particularly anti-integrin α _v β ₃ antibody) at a similar or higher concentration than the lyophilized formulation so that the formulation does not need to be reconstituted prior to administration. This makes it much faster and easier for medical personnel to administer antibodies to patients.

Conventional liquid antibody formulations have a short shelf life and can lose their biological activity during storage due to chemical and physical instabilities. Chemical instability can be caused by deamidation, optical inactivation, hydrolysis, oxidation, beta shedding or disulfide exchange, and physical instability is caused by antibody denaturation, aggregation, precipitation, or adsorption. There is a possibility. Among these, aggregation, deamidation, and oxidation are known to be the most common causes of antibody degradation (Wang et al., 1988, J. of Parenteral Science & Technology 42 (Suppl): S4 -S26; Cleland et al., 1993, Critical Reviews in Therapeutic Drug Carrier Systems 10 (4): 307-377). Accordingly, there is a need for stable liquid formulations of antibodies, particularly stable liquid anti-integrin α _v β ₃ antibodies.

3. SUMMARY OF THE INVENTION The present invention is a high concentration liquid formulation of an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ , which is manufactured, prepared, prepared in the absence of a surfactant and / or an inorganic salt. Of a formulation that is stable during transport and storage, with antibody fragmentation and / or aggregation at an undetectable to low level, with little or no loss of biological activity of the antibody or antibody fragment. Based in part on development. The liquid formulation of the present invention may have one or more symptoms associated with inflammatory and autoimmune diseases, including but not limited to rheumatoid arthritis, shoulder periarthritis, multiple sclerosis, psoriasis, myasthenia gravis Vasculitis, pemphigus vulgaris, ulcerative colitis, Crohn's disease, Hashimoto's thyroiditis, systemic lupus erythematosus, scleroderma, polymyositis, osteolysis, ankylosing spondylitis, psoriatic arthritis, and Sjogren's syndrome Facilitate administration of antibodies or fragments thereof that immunospecifically bind to integrin α _v β ₃ for prevention, treatment, management, and / or amelioration. The liquid formulations of the present invention are also for the prevention, treatment, management and amelioration of one or more symptoms associated with cancer, such as, but not limited to, glioblastoma, bone cancer, breast cancer and colon cancer. Facilitating administration of antibodies or fragments thereof that immunospecifically bind to integrin α _v β ₃ . The liquid formulations of the present invention may also include one or more symptoms associated with abnormal bone metabolism such as, but not limited to, degenerative joint disease (osteoarthritis), gouty arthritis, cartilage calcification, and bone. For prevention, treatment, management and improvement of tumors and tumor-like injuries (including but not limited to primary tumors of bone, secondary or metastatic tumors of bone, neuroarthritis and sarcoidosis arthropathy), Facilitates administration of antibodies or fragments thereof that immunospecifically bind to integrin α _v β ₃ . In particular, the liquid formulations of the present invention allow the integrin α in a sterile dosage form without the need for accurate and aseptic reconstitution of the antibody or antibody fragment prior to administration, as required in a lyophilized dosage form. _v an antibody or antibody fragment that binds to the beta ₃ immunospecifically, it is possible to medical personnel administering quickly.

The present invention relates to a liquid preparation substantially free from a surfactant and / or an inorganic salt, comprising histidine and an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ at a concentration of 50 mg / ml or more. A formulation comprising is provided. The present invention is a liquid formulation substantially free of surfactant and / or inorganic salt, having a pH in the range of about 5.0 to about 7.0, preferably about 6.0, and histidine, Also provided is a formulation comprising an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ at a concentration of 50 mg / ml or greater. The liquid formulations of the present invention may further comprise one or more excipients such as sugars, amino acids (eg, arginine, lysine and methionine), and polyols. In a preferred embodiment, the liquid formulation of the present invention is a formulation comprising histidine and an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ at a concentration of 95 mg / ml or more, comprising a surfactant and Includes formulations that are substantially free of salt.

The present invention is a stable liquid formulation of Vitaxin® (see, eg, Wu et al., 1998, PNAS USA 95 (11): 6037-6042), wherein antibodies are produced during manufacture, preparation, transportation, and long-term storage. Including formulations in which the aggregation and / or fragmentation of is not detectable to low levels and the biological activity of Vitaxin® is not lost or hardly lost. The present invention is a stable liquid preparation of an antibody that immunospecifically binds to integrin α _v β ₃ , which has a longer in vivo half-life than known antibodies (eg, Vitaxin®). Including formulations in which antibody aggregation and / or fragmentation is undetectable to low levels and the biological activity of the antibody or antibody fragment is not lost or hardly lost. The present invention also relates to an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ , variable heavy (VH) having the amino acid sequence of LM609 or VITAXIN® VH and / or VL domain and A stable liquid formulation of an antibody comprising a variable light (VL) domain, wherein antibody aggregation and / or fragmentation is at an undetectable to low level and the biological activity of the antibody or antibody fragment is totally Includes formulations that are not lost or rarely lost. The present invention further relates to an antibody or fragment thereof that immunospecifically binds to integrin α _v β ₃ and having one or more VH CDR and / or VL CDR amino acid sequences listed in Table 1. A stable liquid formulation of an antibody or antibody fragment comprising a sex determining region (CDR) and / or one or more VL CDRs, wherein antibody aggregation and / or fragmentation is at an undetectable to low level, Formulations in which the biological activity of the antibody fragment is not lost or hardly lost.

The present invention is a liquid formulation of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and is stable at 38 ° C. to 42 ° C. when evaluated by high performance size exclusion chromatography (HPSEC). Contains formulation. The liquid preparation of the present invention, when evaluated by HSPEC, is at least 15 days in the temperature range of 38 ° C. to 42 ° C., but within 25 days, at least 6 months in the range of 20 ° C. to 24 ° C., but 1.5 years Within a range of 2 ° C. to 8 ° C. (especially 4 ° C.) for at least 1.5 years, at least 2 years, at least 2.5 years, or at least 3 years. The invention also includes liquid formulations of antibodies or fragments thereof that immunospecifically bind to integrin α _v β ₃ , wherein antibody aggregation is undetectable to low levels as measured by HPSEC. In a preferred embodiment, the liquid formulation of the present invention is stable for at least 15 days at 38-42 ° C. and has an undetectable to low level of antibody aggregation as measured by HPSEC, and further includes, for example, ELISA, When measured in an antibody binding assay, the biological activity of the antibody or antibody fragment of the formulation is not lost or hardly lost compared to the reference antibody.

The present invention provides a method for preparing a liquid formulation of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ , wherein the purified fraction of the antibody is analyzed with an appropriate molecular weight (MW) cutoff value (eg, Using a semipermeable membrane with a 30 kD cutoff for complete antibody molecules and F (ab ′) ₂ fragments; and a 10 kD cutoff for antibody fragments such as Fab fragments), a final antibody concentration of about 15 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 150 mg / ml, about Concentrate to 175 mg / ml, or about 200 mg / ml, and diafilter the concentrated antibody fraction in formulation buffer using the same membrane The method comprising. The formulation buffer of the present invention comprises histidine at a concentration of about 1 mM to about 100 mM, preferably about 5 mM to about 50 mM, more preferably about 10 mM to about 25 mM. The pH of the formulation is in the range of about 5.0 to about 7.0, preferably 5.5 to about 6.5, more preferably about 5.8 to about 6.2, most preferably about 6.0. sell. In order to obtain a pH appropriate for a particular antibody, first, histidine (and glycine if glycine is added) in water so that a buffered aqueous solution with a pH higher than the desired pH is obtained. It is preferred to dissolve and then add HCl to lower the pH to the desired value. In this way, formation of inorganic salt (for example, formation of NaCl when histidine hydrochloride is used as a histidine supply source and pH is raised to a desired value by adding NaOH) can be avoided.

The liquid preparation of the present invention may be sterilized by aseptic filtration using a 0.2 μ filter. The sterile liquid formulation of the present invention is associated with inflammatory diseases, autoimmune diseases, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, abnormal expression and / or activity of integrin α _v β ₃ A subject can be administered to prevent, treat, manage or ameliorate a disorder, cancer, or one or more symptoms thereof. The liquid preparation of the present invention can be used for other treatments (for example, prophylactic or therapeutic agents other than antibodies that immunospecifically bind to integrin α _v β ₃ , such as anti-inflammatory agents, anticancer agents, bone metabolism regulators, immunomodulators, And an anti-angiogenic agent). The present invention also provides a kit comprising a liquid formulation of an antibody or antibody fragment that immunospecifically binds to, for example, integrin α _v β ₃ for use by medical personnel. The present invention further provides administration of the liquid formulation of the present invention to cause inflammatory diseases, autoimmune diseases, abnormal bone metabolism related disorders, abnormal angiogenesis related disorders, integrin α _v β ₃ abnormalities. Methods of preventing, treating, managing or ameliorating disorders associated with expression and / or activity, and cancer are provided. The liquid preparation of the present invention can be used for inflammatory diseases, autoimmune diseases, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, disorders associated with abnormal expression and / or activity of integrin α _v β _3. Or can be administered to a subject parenterally (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), or oral administration to prevent, treat, manage or ameliorate cancer.

The liquid formulations of the present invention also include disorders associated with abnormal expression of integrin α _v β ₃ , inflammatory diseases, autoimmune diseases, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, and cancer It can also be used for diagnosis, detection or monitoring.

3.1 Terminology As used herein, all liquid formulations of antibodies and / or antibody fragments that immunospecifically bind to integrin α _v β ₃ are collectively referred to as “liquid formulations of the present invention”, “ liquid antibody formulation ", referred to as" antibody formulations of the present invention "," integrin alpha _v antibody or liquid formulations of the fragments that bind to a beta ₃ immunospecifically "or" anti-integrin alpha _v beta ₃ antibody of the liquid formulation ".

As used herein, the term “abnormal” refers to deviations from a standard, eg, an average healthy subject and / or average population of healthy subjects. The term “abnormal expression” as used herein refers to an abnormal gene product (eg, RNA, protein, polypeptide or peptide) by a normal healthy cell or subject, or a population of normal healthy cells or subjects. Refers to expression. Such abnormal expression may be the result of gene amplification. In certain embodiments, the term “abnormal expression” refers to integrin α by a cell or subject as compared to the expression of a gene product by a normal healthy cell or subject, or a population of normal healthy cells or subjects. _v refers to beta ₃ of aberrant expression, the expression of integrin alpha _v beta ₃ gene product in unusual at the location of a cell or a subject, the expression of integrin alpha _v beta ₃ gene product in a modified level of a cell or a subject, It includes expression of a mutant integrin α _v β ₃ gene product, or a combination thereof. In another embodiment, “abnormal expression” refers to a cell or test compared to the expression of an integrin α _v β ₃ gene product by a normal healthy cell or subject, or a population of normal healthy cells or subjects. By overexpression of the integrin α _v β ₃ gene product and / or integrin α _v β ₃ gene product by the body. According to this embodiment, overexpression can be the result of gene amplification. The term “abnormal activity” as used herein refers to an altered level of a gene product in a cell or subject as compared to a normal healthy cell or subject, or a population of normal healthy cells or subjects. , Means increased activity by the gene product or loss of binding of the gene product. In certain embodiments, “abnormal activity” refers to integrin α _v β ₃ activity that deviates from that normally found in normal healthy cells or subjects, or a population of normal healthy cells or subjects. In another embodiment, the term “abnormal activity” refers to the integrin α _v β ₃ activity as compared to the activity normally found in normal healthy cells or subjects, or a population of normal healthy cells or subjects. Means increase. Examples of integrin α _v β ₃ activity include, but are not limited to, fibrinogen, fibronectin, von Willebrand factor, vitronectin, which mediate platelet aggregation and endothelial cell adhesion to ECM proteins and promote vascular authenticity. Receptors for Tsp (thrombospondin), osteopontin, and Bsp1 (bone sialoprotein 1) are included. In certain embodiments, the integrin α _v β ₃ exhibits an abnormal activity, such as, but not limited to, the formation of a stronger bond with its ligand. Examples of integrin α _v β ₃ ligands include, but are not limited to, vitronectin, osteopontin, bone sialoprotein, echistatin, RGD-containing peptides, RGD mimetics and inhibitory antibodies (eg, Dresner-Pollak et al., J. Cell Biochem. 56 (3): 323-30; Duong et al., Front. Biosci. 1 (3): d757-68).

  As used herein, the term “about” as used with respect to a given numerical value or range is a value or range within 20%, preferably within 10%, more preferably within 5% of the given value or range.

  The term “angiogenesis” as used herein means the growth (proliferation) of new blood vessels. Thus, “abnormal angiogenesis” or “abnormal angiogenesis” means an alteration (eg, increase or decrease) in the activity of angiogenesis, ie, angiogenesis that deviates from the normal angiogenesis process, eg, limited. Although not, there are an increase in angiogenic activity in the body, angiogenesis at an abnormal position in the body, and the like. The disease or disorder may be caused entirely by abnormal angiogenesis or may be exacerbated by abnormal angiogenesis. Diseases or disorders associated with abnormal angiogenesis resulting from excessive angiogenesis include, but are not limited to, cancer, tumors, rheumatoid arthritis, psoriasis, rosacea, and cancer cell metastasis. Diseases or disorders associated with abnormal angiogenesis resulting from insufficient angiogenesis include, but are not limited to, coronary artery disease, stroke and delayed wound healing. In some embodiments, “abnormal angiogenesis” means increased or excessive activity of angiogenesis.

  As used herein, the term “analog” for proteinaceous agents (eg, proteins, polypeptides, peptides and antibodies) retains similar or the same function as a second proteinaceous agent (but not necessarily It means a proteinaceous drug that does not contain the same or the same amino acid sequence as the second proteinaceous drug) or that retains a structure similar to or the same as the second proteinaceous drug. A proteinaceous drug having a similar amino acid sequence has the following three conditions: (a) the proteinaceous drug is at least 30%, at least 35%, at least 40%, at least 45% of the amino acid sequence of the second proteinaceous drug Amino acids having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% identity (B) the proteinaceous agent is at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 of the second proteinaceous agent Consecutive amino acid residues, at least 25 consecutive amino acid residues At least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous Coded by a nucleotide sequence that hybridizes under stringent conditions with a nucleotide sequence encoding amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, or at least 150 contiguous amino acid residues And (c) the proteinaceous agent is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60 with the nucleotide sequence encoding the second proteinaceous agent. %, At least 6 %, At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% encoded by a nucleotide sequence having identity It means two proteinaceous drugs. A proteinaceous drug similar or having the same structure as the second proteinaceous drug means a proteinaceous drug having a secondary, tertiary or quaternary structure similar to the second proteinaceous drug. The structure of a proteinaceous drug can be determined by methods known to those skilled in the art and include, but are not limited to, amino acid sequencing of peptides, X-ray crystallography, nuclear magnetic resonance, circular dichroism And crystallographic electron microscopy.

  To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (eg, the first for optimal alignment with a second amino acid or nucleic acid sequence). Gaps can be introduced in the amino acid or nucleic acid sequence of The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (ie,% identity = number of identical positions overlapping / total number of positions × 100%). In certain embodiments, the two sequences are the same length.

  The percent identity between two sequences can also be determined using a mathematical algorithm. A suitable non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is described in Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268, and Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated in the NBLAST and XBLAST programs described in Altschul et al., 1990, J. Mol. Biol. 215: 403. By performing a BLAST nucleotide search using the NBLAST nucleotide program parameter set (for example, score = 100, word length = 12), a nucleotide sequence homologous to the nucleic acid molecule of the present invention can be obtained. By performing the BLAST protein search using the XBLAST program parameter set (for example, score = 50, word length = 3), an amino acid sequence homologous to the protein molecule of the present invention can be obtained. To obtain an alignment with gaps inserted for comparison purposes, Gapped BLAST is used as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search that detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) may be used (see, eg, NCBI website). Another preferred non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm described in Myers and Miller, 1988, CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for amino acid sequence comparison, the PAM120 weight residue table, gap length penalty 12, and gap penalty 4 may be used.

  The percent identity between two sequences can be determined using techniques similar to those described above, with or without gaps. To calculate percent identity, typically only exact matches are counted.

  As used herein, the term “analog” with respect to a non-proteinaceous analog is similar or has the same function as the first organic or inorganic molecule and is structurally similar to the first organic or inorganic molecule. The second organic or inorganic molecule.

The term "antibody fragment" as used herein, refers to a fragment of an antibody that immunospecifically binds to integrin α _v β _3. Antibody fragments can be generated by any technique known to those of skill in the art. For example, Fab and F (ab ′) ₂ fragments are produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papa (for producing Fab fragments) or pepsin (for producing F (ab ′) ₂ fragments). Yes. The F (ab ′) ₂ fragment contains the complete light chain, the variable region, the CH1 region of the heavy chain and the hinge region. Antibody fragments can also be generated by recombinant DNA techniques. An antibody fragment can be one or more complementarity determining regions (CDRs) of an antibody, or one or more antigen-binding fragments of an antibody.

As used herein, the term “antibody” refers to a monoclonal antibody, bispecific antibody, multispecific antibody, human antibody, humanized antibody, chimeric antibody, camelized antibody, single chain Fv (scFv), single Mean chain antibody, disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the invention). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (ie, molecules that contain an antigen binding site). The immunoglobulin molecule can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ ) or subclass. There may be.

As used herein, “an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ ” and similar terms specifically refers to an integrin α _v β ₃ polypeptide or a fragment of an integrin α _v β ₃ polypeptide. It means an antibody or antibody fragment that binds and does not specifically bind to other polypeptides. Preferably, an antibody or antibody fragment that immunospecifically binds to an integrin α _v β ₃ polypeptide or fragment thereof does not cross-react with other antigens. Antibodies or antibody fragments that immunospecifically bind to an integrin α _v β ₃ polypeptide or fragment thereof can be identified, for example, by immunoassays or other techniques known to those of skill in the art. For example, Paul ed for a discussion specificity of the ^{antibody, 1989, Fundamental Immunology, 2 nd} ed., Raven Press, see New York at pages 332-336. Preferably, an antibody or antibody fragment that immunospecifically binds to an integrin α _v β ₃ polypeptide or fragment thereof only antagonizes integrin α _v β ₃ and contains other activities, such as α _v but not β ₃ It does not significantly antagonize the activity of non-integrins (eg, the fibronectin receptor, integrin α _v β ₁ ).

  As used herein, the term “derivative” with respect to proteinaceous agents (eg, proteins, polypeptides, peptides and antibodies) refers to an amino acid sequence that has been modified by the introduction of amino acid residue substitutions, deletions and / or additions. It means a proteinaceous drug containing. The term “derivative” as used herein also refers to a proteinaceous agent that has been modified by covalent attachment of any type of molecule to the proteinaceous agent. For example, but not limited to, antibodies can be glycosylated, acetylated, PEGylated, phosphorylated, amidated, derivatized (with known protecting / blocking groups), proteolytic cleavage, cellular It can be modified by binding to sex ligands or other proteins. Derivatives of proteinaceous drugs can be prepared by chemical modification using techniques known to those skilled in the art, such as, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic of tunicamycin Includes synthesis. Furthermore, the proteinaceous drug derivative may contain one or more non-classical amino acids. A derivative of a proteinaceous drug retains a similar or identical function as the original proteinaceous drug from which it was derived.

  As used herein, the term “derivative” with respect to a non-protein derivative means a second organic or inorganic molecule formed based on the structure of the first organic or inorganic molecule. Derivatives of organic molecules include, but are not limited to, molecules that have been modified, for example, by addition or deletion of hydroxyl, methyl, ethyl, carboxyl, or amino groups. The organic molecule may also be esterified, alkylated and / or phosphorylated.

  As used herein, the terms “disorder” or “disease” are used interchangeably and refer to the condition of a subject. In particular, the term “autoimmune disease” is used interchangeably with “autoimmune disorder” and refers to cells, tissues and / or caused by the subject's immunological response to the subject's own cells, tissues and / or organs. Refers to a symptom of a subject characterized by organ damage. The term “inflammatory disease” is used interchangeably with “inflammatory disorder” and refers to a symptom of a subject characterized by inflammation, preferably chronic inflammation. The autoimmune disorder may or may not be associated with inflammation. Furthermore, the inflammation may or may not be caused by an autoimmune disorder. Certain symptoms can be characteristic of more than one disorder, for example, certain diseases can be characterized by both autoimmune and inflammatory disorders.

The term “effective amount” as used herein for treatment refers to a disease or disorder (eg, inflammatory disease, autoimmune disease, disorder associated with abnormal bone metabolism, disorder associated with abnormal angiogenesis, integrin α _v disorders related to beta ₃ of aberrant expression and / or activity, or cancer) or or improved to reduce the severity and / or duration of the symptoms, to prevent the progression of the disease or disorder, or the disease Sufficient to guide the regression of the disorder, prevent the recurrence, onset or development of the syndrome associated with the disease or disorder, or to increase or improve the preventive or therapeutic effect of another treatment (eg, prophylactic or therapeutic agent) Means the amount of a therapeutic agent.

As used herein with respect to detecting expression of integrin α _v β ₃ , the term “effective amount” refers to an antibody, antibody fragment, or antibody or antibody fragment sufficient to detect integrin α _v β ₃ expression. The amount of the formulation containing

The term “epitope” as used herein refers to that portion of an integrin α _v β ₃ polypeptide that has antigenic or immunogenic activity in the body of an animal, preferably a mammal, and most preferably a human. An epitope having immunogenic activity is a portion of an integrin α _v β ₃ polypeptide that elicits an antibody response in the animal body. Antigenic active epitopes are those integrin α _v β to which the antibody immunospecifically binds as determined by methods known to those skilled in the art (eg, immunoassays described herein (see Section 5.4.3)). ₃ Polypeptide parts. Antigenic epitopes need not necessarily be immunogenic.

  As used herein, the term “excipient” refers to a diluent for a drug, an excipient that exerts the advantageous physical properties of the formulation, such as increased protein stability, increased protein solubility and reduced viscosity. Examples of dosage forms refer to inert substances commonly used as vehicles, preservatives, binders, or stabilizers. Protein (eg, serum albumin), amino acid (eg, aspartic acid, glutamic acid, lysine, arginine, glycine, histidine), surfactant (eg, SDS, polysorbate, nonionic surfactant), sugar (eg, glucose, sucrose) , Maltose, trehalose, etc.), polyols (eg, mannitol, sorbitol), fatty acids and phospholipids (eg, alkyl sulfonate, caprylic acid), but are not limited thereto. See also Remington's Pharmaceutical Sciences (Joseph P. Remington, 18th Edition, Mack Publishing Co., Easton, PA) for more information on excipients. This document is incorporated herein by reference in its entirety.

As used herein, the term “fragment” refers to at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues of another polypeptide or protein amino acid sequence. A group, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino groups , At least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues; at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues At least 150 consecutive amino acid residues, at least 1 5 contiguous amino acid residues, refers at least 200 contiguous amino acid residues, or at least 250 amino acids sequence of contiguous amino acid residues, peptides, polypeptides, or proteins (including antibodies). In certain embodiments, a protein or polypeptide fragment retains at least one function of the protein or polypeptide. In another embodiment, the protein or polypeptide fragment retains at least 2, 3, or 4 functions of the protein or polypeptide. It is preferable that the antibody fragment that binds to integrin α _v β ₃ immunospecifically retains the ability to bind to integrin α _v β ₃ . A “functional fragment” is a fragment that retains at least one function of a protein or polypeptide.

  As used herein, the term “fusion protein” refers to the amino acid sequence of a first protein, polypeptide or functional fragment, analog or derivative thereof and the amino acid sequence of a heterologous protein or polypeptide (ie, the first A second protein, polypeptide or functional fragment, analog or derivative thereof, or a first protein, polypeptide or functional fragment, similar to these, different from the protein or functional fragment, analog or derivative thereof Body or derivative and a second protein, polypeptide or functional fragment, analog or derivative thereof) that is not naturally conjugated). In one embodiment, the fusion protein comprises a prophylactic or therapeutic agent fused to a heterologous protein, polypeptide or peptide. According to this embodiment, these heterologous proteins, polypeptides or peptides may or may not be of a different type than the prophylactic or therapeutic agent.

As used herein, the terms “high concentration” and “concentrated antibody” refer to 50 mg / ml or more for an antibody or fragment thereof that specifically binds to integrin α _v β ₃ in an antibody formulation. Refers to high, preferably 95 mg / ml or higher concentrations.

  As used herein, the term “host cell” includes a cell of a subject that has been transfected or transformed with a nucleic acid molecule and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule due to mutations or environmental effects that may occur during generational inheritance, or due to integration of the nucleic acid molecule into the host cell genome. There is.

  As used herein, “hybridize under stringent conditions” means at least 30% (preferably at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60% of each other. Nucleotide sequences having identity typically remain hybridized to each other, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%) Describe hybridization and washing conditions. Such stringent conditions are known to those skilled in the art and can be found in “Current Protocols in Molecular Biology”, John Wiley & Sons, N. Y. (1989), 6.3.1-6.3.6. Generally, stringent conditions are selected to be about 5-10 ° C. lower than the thermal melting point (Tm) for the specific sequence at a specific ionic strength pH. T is the temperature at which 50% of the probes complementary to the target hybridize to the target sequence in equilibrium (under conditions of specific ionic strength, pH and nucleic acid concentration) (the target sequence is present in excess). Therefore, at that Tm, 50% of the probe is occupied in equilibrium). Stringent conditions are sodium ions with a salt concentration of less than about 1.0M, typically about 0.01-1.0M sodium ion concentration (or other salt) at pH 7.0-8.3. The temperature is at least about 30 ° C. for short probes (eg, 10-50 nucleotides) and at least about 60 ° C. for long probes (eg, 50 nucleotides or more). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least twice, preferably 10 times background hybridization.

  In one non-limiting example, stringent hybridization conditions include hybridization at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC) followed by 0.1 × SSC, 0.2 Consists of one or more washes at about 68 ° C. in% SDS. In a preferred non-limiting example, stringent hybridization conditions include hybridization at about 45 ° C. in 6 × SSC, followed by about 50-65 ° C. in 0.2 × SSC, 0.1% SDS. One or more washings (ie, one or more washings at 50 ° C., 55 ° C., 60 ° C. or 65 ° C.). It can be seen that the nucleic acids of the present invention do not contain nucleic acid molecules that hybridize exclusively with nucleotide sequences composed solely of A or T nucleotides under these conditions.

As used herein, the term “immunomodulatory agent” and variants thereof, including but not limited to “immunomodulating agent” or “immunomodulant”, refer to an agent that modulates the host immune system (action (Medicine). In certain embodiments, an immunomodulatory agent is an agent that shifts one aspect of a subject's immune response. In certain embodiments, an immunomodulatory agent is an agent that suppresses or reduces the subject's immune system (ie, an immunosuppressive agent). In other specific embodiments, an immunomodulatory agent is an agent that activates or elevates the subject's immune system (ie, an immunostimulatory agent). According to the present invention, the immunomodulatory agent used in the combination therapies of the present invention does not include an antibody that immunospecifically binds to integrin α _v β _3. Immunomodulators include, but are not limited to, small molecules, peptides, polypeptides, proteins, nucleic acids (eg, but not limited to antisense nucleotide sequences, triple helices, and biologically active proteins, And DNA or RNA nucleotides comprising nucleotide sequences encoding the polypeptide or peptide), antibodies, synthetic or natural inorganic molecules, mimetics, and synthetic or natural organic molecules.

As used herein, the term “combination” refers to the use of multiple treatments (eg, prophylactic and / or therapeutic agents). In use of the term “combination”, diseases or disorders (eg, inflammatory diseases, autoimmune diseases, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, integrin α _v β ₃ The order in which treatments (eg, prophylactic and / or therapeutic agents) are administered to a subject having a disorder associated with abnormal expression and / or activity, or cancer) is not limited. A first treatment (eg, a prophylactic or therapeutic agent) is administered (eg, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour) prior to administration of a second treatment (eg, a prophylactic or therapeutic agent). 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before) And at the same time (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, After 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks) disease or disorder (eg, inflammatory disease, autoimmune disease, disorder associated with abnormal bone metabolism, abnormal Disorders associated with angiogenesis, abnormal expression and / or activity of integrin α _v β ₃ Can be administered to a subject having a sex-related disorder or cancer).

As used herein, the term “inorganic salt” refers to any compound that does not contain carbon that results from substituting hydrogen or some or all of an acid with a metal or a group that acts like a metal. It may also be used as a tonicity adjusting compound in pharmaceutical compositions and preparations of biological materials. The most common inorganic salts are NaCl, KCl, NaH ₂ PO ₄ and the like.

As used herein, the term “α _v β ₃ ” or “integrin α _v β ₃ ” refers to a heterodimer of integrin subunit α _v and integrin subunit β ₃ , Subunit analogs, derivatives or fragments, and heterodimeric integrin α _v β ₃ , refers to fusion proteins comprising heterodimeric subunit analogs, derivatives or fragments. The integrin α _v β ₃ may be derived from any species. The nucleotide and / or amino acid sequence of integrin α _v β ₃ can be found in the literature or in public databases, or the nucleotide and / or amino acid sequence is determined using cloning and sequencing techniques known to those skilled in the art. be able to. For example, the nucleotide sequence of human integrin α _v β ₃ can be found in the GenBank database (see, eg, accession number No. NM — 002210 for α _v and accession number No. L28832 for β ₃ ). The amino acid sequence of human α _v β ₃ can be found in the GenBank database (see, eg, registration number No. AAA61631 for α _{v and} registration number No. S44360 for β ₃ ). In a preferred embodiment, the integrin α _v β ₃ is human integrin α _v β ₃ , an analog, derivative or fragment thereof. Antibodies or antibody fragments that immunospecifically bind to integrin alpha _v beta ₃ is a fusion protein, dimer integrin alpha _v beta _3, an analog thereof, binds to a part of a fusion protein comprising a derivative or fragment.

  The term “isolated” or “purified” as used herein with respect to a proteinaceous agent (eg, a peptide, polypeptide, fusion protein, or antibody) refers to the cell from which it is derived or from the tissue source. It means a proteinaceous drug that is substantially free of chemical substances or contaminating proteins, or substantially free of chemical precursors or other chemical substances. The term “substantially free of cellular material” refers to the preparation of a proteinaceous drug wherein the proteinaceous drug is isolated or recombinantly produced from the cell and separated from the cellular components of the cell. Means a thing. Accordingly, proteinaceous agents that are substantially free of cellular material are present in amounts less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (on a dry weight basis). Proteinaceous drug preparations containing a heterologous protein, polypeptide, peptide or antibody (also referred to herein as a “contaminating protein”). When the proteinaceous agent is produced recombinantly, it is preferably substantially free of medium, ie, the medium is about 20%, 10%, 5%, 1% of the volume of the protein preparation. , 0.5%, or less than 0.1%. When the proteinaceous drug is prepared by chemical synthesis, it is preferably substantially free of chemical precursors or other chemical substances, that is, chemical precursors or other chemical substances involved in the synthesis of proteinaceous drugs. Separated from. Accordingly, such proteinaceous drug preparations have less than about 30%, 20%, 10%, 5%, 1%, 0.5%, or 0.1% (dry weight basis) of chemical precursors or Contains compounds other than the proteinaceous drug of interest. In a preferred embodiment, the antibody of the present invention is isolated. In certain embodiments, a proteinaceous agent (eg, a peptide, polypeptide, fusion protein, or antibody) separates the proteinaceous agent from other substances by applying the steps shown in FIG. Will be done. "

  The term “isolated” or “purified” as used herein with respect to a nucleic acid molecule means a nucleic acid molecule that has been separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Furthermore, an “isolated” nucleic acid molecule, such as a cDNA molecule, is substantially free of other cellular material or media when produced recombinantly, and is chemically synthesized. Is substantially free of chemical precursors or other chemicals. In a preferred embodiment, a nucleic acid molecule encoding an antibody of the invention is isolated.

  The term “isolated” or “purified” as used herein with respect to organic or inorganic molecules (both small molecules or macromolecules) other than proteinaceous drugs or nucleic acids substantially refers to different organic or inorganic molecules. It means organic or inorganic molecules that are not included. Preferably, the organic or inorganic molecule is 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5% of the second different organic or inorganic molecule. It does not contain 99.9% or 100%. In preferred embodiments, the organic and / or inorganic molecules are isolated or purified.

  As used herein, the phrase “aggregation is undetectable to low level” refers to less than 5%, less than 4%, less than 3%, 2% in protein weight as measured by high performance size exclusion chromatography (HPSEC). Less than, less than 1% and most preferably less than 0.5% aggregates.

  As used herein, the term “fragmentation is undetectable to low level” refers to a protein equal to or greater than 80%, 85%, 90%, 95%, 98%, or 99% of the total protein. For example, it is included in a single peak when measured by HPSEC, or included in two peaks (heavy chain and light chain) by reducing capillary gel electrophoresis (rCGE) (these are non-degraded antibodies or non-degraded antibodies thereof) Other singles with proteins greater than 5%, greater than 4%, greater than 3%, greater than 2%, greater than 1%, or greater than 0.5% of each total protein A sample that does not contain a peak. As used herein, the term “reducing capillary gel electrophoresis (CGE)” refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in an antibody or fragment thereof.

  As used herein, the terms “manage” and “management” mean a beneficial effect elicited from a treatment (eg, a prophylactic or therapeutic agent) that does not result in cure of the disease in the subject. In certain embodiments, a subject is administered one or more treatments (eg, prophylactic or therapeutic agents) to “manage” the disorder to prevent the progression or worsening of the disorder.

As used herein, the terms “non-responsive” and “refractory” refer to diseases or disorders (eg, inflammatory diseases, autoimmune diseases, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ ). Presently available treatments (such as, but not limited to, prophylactic or therapeutic agents) for disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, or cancer), Clinically, means a patient treated with the prophylactic or therapeutic agent that is not sufficient to alleviate one or more symptoms associated with the disease or disorder. Typically, such patients suffer from severe persistent activity disease and require further treatment to ameliorate symptoms or symptoms associated with the disease or disorder.

  As used herein, the term “pharmaceutically acceptable” has been approved by the federal or state regulatory authorities for use in animals, and particularly in humans, or the American Pharmacopoeia Association, European Pharmacy. Means being listed on the association or other widely recognized pharmacopoeia associations.

  In this specification, the term “polyol” refers to a saccharide containing more —OH groups than a normal saccharide.

As used herein, the term “prophylactic agent” refers to a disease or disorder (eg, autoimmune disease, inflammatory disease, disorder associated with abnormal bone metabolism, disorder associated with abnormal angiogenesis, or cancer) or Any drug used for the prevention of one or more symptoms. In certain embodiments, the term “prophylactic agent” refers to an antibody or antibody fragment (eg, VITAXIN®) that immunospecifically binds to integrin α _v β ₃ . According to these embodiments, the antibody or antibody fragment can also be a component of the liquid formulation of the present invention. In certain other embodiments, the term “prophylactic agent” does not refer to an antibody or antibody fragment (eg, VITAXIN®) that immunospecifically binds to integrin α _v β ₃ . A prophylactic agent is a disease or disorder (eg, an autoimmune disease, inflammatory disease, cancer, a disorder characterized by abnormal bone metabolism, a disorder characterized by abnormal expression and / or activity of integrin α _v β ₃ , or Drugs that are known to be useful, preventive or prevent the onset, onset, progression, and / or severity of disorders characterized by abnormal angiogenesis It is preferable that Prophylactic agents can be characterized as different agents based on one or more effects they have in vitro and / or in vivo. For example, anti-angiogenic agents are also characterized as immunomodulators.

As used herein, the terms “prevent” and “prevention” result from administration of a treatment (eg, a prophylactic or therapeutic agent) or a combination therapy (eg, administration of a combination of prophylactic agents), Disease or disorder in a subject (eg, autoimmune disorder, inflammatory disorder, disorder characterized by abnormal bone metabolism, disorder characterized by abnormal expression and / or activity of integrin α _v β ₃ , abnormal angiogenesis Or the prevention of recurrence, development or onset of one or more symptoms thereof.

As used herein, the term “prophylactically effective amount” refers to a disease or disorder (eg, inflammatory diseases, autoimmune diseases, disorders characterized by abnormal bone metabolism, abnormal expression of integrin α _v β ₃ and Prevention of the onset, recurrence, onset or progression of a disorder characterized by activity, disorder characterized by abnormal angiogenesis, or one or more symptoms, recurrence, onset or progression, or other treatments (eg prophylactic drugs). Of a treatment sufficient to produce an increase or improvement in efficacy (eg, a prophylactic agent (eg, an antibody or fragment that immunospecifically binds to integrin α _v β ₃ , or a liquid formulation of the invention comprising the antibody or antibody fragment)) It means quantity.

  As used herein, the term “sugar” refers to a molecular class that is a derivative of a polyhydric alcohol. Sugars are commonly referred to as carbohydrates and can contain different amounts of sugar (saccharide) units such as, for example, monosaccharides, disaccharides, and polysaccharides.

  As used herein, “side effects” include unwanted adverse effects of prophylactic or therapeutic agents. An adverse effect is not always desired, but an undesirable effect is not always a disadvantage. The adverse effects from prophylactic or therapeutic agents can be harmful, uncomfortable or dangerous.

Side effects caused by the administration of Remicade ^™ include, but are not limited to, the risk of severe infections and hypersensitivity reactions. Other side effects include fever, chills, itching and urticaria, chest pain, hypotension, high blood pressure, cardiopulmonary reactions such as dyspnea, non-specific symptoms, thermal myalgia and / or joint pain, rash, face, head or It ranges from lip edema, dysphagia, sore throat, and headache. Still other side effects include but are not limited to abdominal hernia, splenic infarction, splenomegaly, dizziness, upper motor nerve injury, lupus erythematosus syndrome, rheumatoid nodule, earwax hypersecretion, abdominal pain, diarrhea, gastric ulcer, intestinal obstruction, Bowel perforation, bowel stenosis, nausea, pancreatitis, nausea, back pain, fracture, tendon injury or disorder, heart failure, myocardial ischemia, lymphoma, thrombocytopenia, cellulitis, anxiety, confusion, delirium, depression, somnolence, suicide There are aspirations, anemia, abscesses, bacterial infections, and sepsis.

Side effects caused by the administration of ENBREL ^™ include, but are not limited to, the risk of sepsis that can lead to severe infections and death. Adverse side effects include severe infections such as pyelonephritis, bronchitis, septic arthritis, abdominal abscess, cellulitis, osteomyelitis, wound infection, pneumonia, foot abscess, leg ulcer, diarrhea, sinusitis, sepsis , Headache, nausea, rhinitis, dizziness, sore throat, cough, asthenia, abdominal pain, rash, peripheral edema, respiratory distress, dyspepsia, sinusitis, nausea, oral ulcers, hair loss and pneumonia, and other less frequent side effects , E.g., heart failure, myocardial infarction, myocardial ischemia, cerebral ischemia, hypertension, hypotension, pancreatitis, gastrointestinal bleeding, bursitis, depression, dyspnea, deep vein thrombosis, pulmonary embolism, membranous glomerulonephritis, It ranges from polymyositis and thrombophlebitis. Side effects resulting from the administration of methotrexate include, but are not limited to, severe toxic reactions that can be fatal, such as unexpectedly severe myelosuppression, gastrointestinal toxicity, liver toxicity, fibrosis and cirrhosis after prolonged use. There are severe skin reactions that can lead to lung disease, diarrhea and ulcerative stomatitis, malignant lymphoma, and death.

  Side effects of chemotherapy include but are not limited to early and late forming diarrhea and bloating, nausea, vomiting, loss of appetite, leukopenia, anemia, neutropenia, asthenia Abdominal cramps, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, xerostomia, and renal failure, and constipation, nerve and muscle action, kidney and bladder Temporary or permanent damage, fluid-like symptoms, fluid stagnation, and temporary or permanent infertility. Side effects of radiation therapy include, but are not limited to, fatigue, dry mouth, and loss of appetite. Other side effects include but are not limited to early and late diarrhea and gastrointestinal toxicities such as bloating, nausea, vomiting, anorexia, leukopenia, anemia, neutropenia, asthenia, abdominal Convulsions, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, xerostomia, and renal failure. Side effects of biological treatment / immunotherapy include, but are not limited to, rash or swelling at the site of administration, flu-like symptoms such as fever, chills and fatigue, gastrointestinal problems and allergic reactions. Side effects of hormonal treatment include, but are not limited to, nausea, fertility problems, depression, loss of appetite, eye problems, headache, and weight fluctuation. Additional undesirable effects typically experienced by treated patients are known in the art. Many of them are described in the Physicians' Desk Reference (56th edition, 2002, 57th edition, 2003).

  The term “small molecule” and similar terms include peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organics having a molecular weight of less than about 10,000 grams per mole. Or inorganic compounds (ie, including heterogeneous organic compounds and / or organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1000 grams per mole, moles These include, but are not limited to, organic or inorganic compounds having a molecular weight of less than about 500 grams per cent, and salts, esters, and other pharmaceutically acceptable forms of these compounds.

As used herein, the terms “stability” and “stable” refer to a given manufacture, preparation, transport, and storage for a liquid formulation that includes an antibody or antibody fragment that immunospecifically binds to integrin α _v β _3. Refers to the resistance of the antibody or antibody fragment in a formulation to degradation or fragmentation under conditions. The stability of the antibody or antibody fragment can be assessed by the degree of degradation or fragmentation compared to the reference formulation as measured by HPSEC. The reference formulation is a reference standard frozen at −70 ° C. consisting of 10 mg / ml antibody or antibody fragment (eg VITAXIN®) in histidine-HCl buffer (pH 6.0) containing 150 mM NaCl, This reference formulation usually shows a single monomer peak (over 97% region) by HPSEC. Alternatively, the reference formulation is a reference standard frozen at −70 ° C. consisting of 10 mg / ml antibody or antibody fragment (eg, VITAXIN®) in histidine-HCl buffer (pH 6.0). Usually shows a single monomer peak (over 97% region) by HPSEC. The overall stability of a formulation comprising an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ can be determined, for example, by ELISA using an isolated integrin α _v β ₃ molecule or cells expressing it. It can be assessed by various immunological assays including radioimmunoassays.

As used herein, the terms “subject” and “patient” are used interchangeably. As used herein, the term “subject” refers to animals, preferably non-primates (eg, cows, pigs, horses, cats, dogs, rats, and mice), and primates (eg, chimpanzees, macaques, etc.). Mammals including monkeys and humans), and more preferably humans. In one embodiment, the subject is an immunocompromised or immunosuppressed mammal, preferably a human (eg, an HIV patient). In another embodiment, the subject is not a mammal, preferably a human, having a lymphocyte count of about 500 cells / mm ³ or less. In another embodiment, the subject is a mammal, preferably a human, that has been or has been treated with one or more TNF-α antagonists. In another embodiment, the subject is a mammal, preferably a human, who has been or has been treated with one or more TNF-α antagonists and methotrexate. In another embodiment, the subject is a mammal, preferably a human, that is not currently treated with a TNF-α antagonist or methotrexate. In another embodiment, the subject is a mammal, preferably a human, having an inflammatory disorder or autoimmune disorder that is refractory to treatment with a TNF-α antagonist, a non-steroidal anti-inflammatory agent or methotrexate alone. In yet another embodiment, the subject is a mammal, preferably a human, having a cancer that is refractory to treatment with one or more chemotherapeutic agents and / or radiation therapy. In preferred embodiments, the subject is a human.

As used herein, the term “substantially free of surfactant” is a formulation of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and is less than 0.0005%,. It refers to a preparation containing less than 0003% or less than 0.0001% surfactant.

As used herein, the term “substantially free of inorganic salts” refers to a preparation of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and is less than 0.0005%, 0.0003 % Or less than 0.0001% of an inorganic salt.

As used herein, the term “synergism” refers to a combination of treatments (eg, use of prophylactic or therapeutic agents) that is more effective than the additive effects of two or more single treatments. For example, the synergistic action of a combination of prophylactic or therapeutic agents can reduce the dose of one or more agents and / or cause a disease or disorder (eg, inflammatory disease, autoimmune disease, abnormal bone metabolism). Reducing the number of times the drug is administered to a subject having a characteristic disorder, a disorder characterized by abnormal expression and / or activity of integrin α _v β ₃ , a disorder characterized by abnormal angiogenesis, or cancer) It is possible. The ability to reduce the dose of a prophylactic or therapeutic agent and / or reduce the number of doses of the drug reduces the toxicity associated with the administration of the drug to a subject and also causes a disease or disorder (eg, inflammation) Diseases, autoimmune diseases, disorders characterized by abnormal bone metabolism, disorders characterized by abnormal expression and / or activity of integrin α _v β ₃ , disorders characterized by abnormal angiogenesis, or cancer) It does not reduce the efficacy of the drug in prevention, management or treatment. Moreover, due to synergism, diseases or disorders (inflammatory diseases, autoimmune diseases, disorders related to abnormal bone metabolism, disorders related to abnormal expression and / or activity of integrin α _v β ₃ , abnormal angiogenesis) The efficacy of treatment in the prevention or treatment of disorders related to cancer or cancer) can be improved. Finally, the synergistic effect of a combination of treatments (eg, prophylactic or therapeutic agents) can avoid or reduce adverse or undesirable side effects associated with the use of monotherapy.

As used herein, “therapeutic agent” refers to a disease or disorder (eg, inflammatory disease, autoimmune disease, disorder characterized by abnormal bone metabolism, abnormal expression and / or activity of integrin α _v β ₃ ). Or a disorder characterized by abnormal angiogenesis, or cancer) or one or more symptoms thereof, or a drug that can be used to treat (ameliorate). In certain embodiments, the term “therapeutic agent” refers to an antibody or fragment thereof (eg, VITAXIN®) that immunospecifically binds to integrin α _v β ₃ . According to these embodiments, the antibody or antibody fragment may be a component of the liquid formulation of the present invention. In certain other embodiments, the term “therapeutic agent” does not refer to an antibody or fragment thereof (eg, VITAXIN®) that immunospecifically binds to integrin α _v β ₃ . Preferably, the therapeutic agent is a disease or disorder (eg, inflammatory disease, autoimmune disease, disorder characterized by abnormal bone metabolism, disorder characterized by abnormal expression and / or activity of integrin α _v β ₃ , abnormal Is a drug that is known, used, or currently used to treat or ameliorate a disorder characterized by angiogenesis or cancer) or one or more symptoms thereof . A therapeutic agent may be characterized as another agent based on one or more effects that the agent has in vitro and / or in vivo. For example, anti-inflammatory agents can be characterized as immunomodulators.

As used herein, a “therapeutically effective amount” refers to a disease or disorder (eg, inflammatory disease, autoimmune disease, disorder characterized by abnormal bone metabolism, abnormal expression of integrin α _v β ₃ and / or One or more symptoms associated with the disease or disorder, reducing the severity of the disorder characterized by activity, disorder characterized by abnormal angiogenesis, or cancer), reducing the duration of the disease or disorder Improve disease, prevent progression of the disease or disorder, cause regression of the disease or disorder, or immunologically bind to other therapies (eg, other therapeutic agents such as integrin α _v β ₃₎ Or a fragment thereof, or the amount of treatment (eg, therapeutic agent) sufficient to increase or improve the therapeutic effect of the antibody or the liquid formulation of the invention comprising the antibody or antibody fragment).

As used herein, “therapies or therapy” refers to a disease or disorder (eg, inflammatory disease, autoimmunity) known to medical personnel (eg, doctors or nurses) or skilled researchers in the field. Disease, disorder characterized by abnormal bone metabolism, disorder characterized by abnormal expression and / or activity of integrin α _v β ₃ , disorder characterized by abnormal angiogenesis, or cancer) or one or more symptoms thereof Any protocol, method and / or agent that can be used in the prevention, treatment, management or amelioration of can be referred to. In certain embodiments, the term “treatment” refers to chemotherapy, radiation therapy, hormonal therapy, biological therapy, and / or a disease or disorder (eg, inflammatory disease, autoimmunity) known to healthcare professionals. Other useful for the treatment of diseases, disorders characterized by abnormal bone metabolism, disorders characterized by abnormal expression and / or activity of integrin α _v β ₃ , disorders characterized by abnormal angiogenesis, or cancer) Means treatment.

As used herein, “treat” and “treatment” refer to one or more treatments (eg, without limitation, administration of one or more therapeutic or prophylactic agents, radiation therapy, hormonal therapy, surgery, Diseases or disorders caused by physical therapy and any other methods or drugs that can be used (inflammatory diseases, autoimmune diseases, disorders characterized by abnormal bone metabolism, abnormal expression of integrin α _v β ₃ And / or a disorder characterized by activity, a disorder characterized by abnormal angiogenesis, or cancer) progression, severity, and / or duration, or one or more symptoms of the disease or disorder This is an improvement. In certain embodiments, the term refers to one or more associated with an autoimmune or inflammatory disorder resulting from administration of one or more prophylactic or therapeutic agents to a subject suffering from an autoimmune or inflammatory disorder. Improves swelling of joints, organs or tissues, or pain. In other embodiments, such terms refer to a reduction in the human PASI score. In another embodiment, such terms refer to an improvement in a human systemic assessment score. In other embodiments, such terms include inhibition or reduction of cancer cell growth, inhibition or reduction of tumor cell expansion (metastasis), development of one or more symptoms associated with cancer, inhibition or reduction of expression or progression, Or it means a reduction in tumor size. In other embodiments, such terms refer to inhibition or reduction of bone loss or bone resorption. In yet another embodiment, such terms refer to the inhibition or reduction of abnormal angiogenesis.

As used herein, the term “no or little loss of biological activity” is measured by a variety of immunological assays including, but not limited to, ELISA and radioimmunoassay. It is the refers to specifically binding the included antibodies activity of the antibody or fragment thereof to integrin α _v β _3. In one embodiment, an antibody or antibody fragment of a formulation of the invention has the ability to immunospecifically bind to integrin α _v β ₃ (ie, vitronectin receptor) known to those of skill in the art or as described herein. About 50%, preferably 55%, 60%, 65%, 70%, 75%, 80%, as compared to a reference antibody or antibody fragment (eg, VITAXIN®) Hold 85%, 90%, 95% or 98%. For example, an ELISA-based assay can be used to compare the ability of an antibody or antibody fragment to immunospecifically bind to integrin α _v β ₃ with a VITAXIN® reference standard. In this assay, referred to as a VnR binding ELISA, the plate was coated with integrin α _v β ₃ isolated from human placenta and the set concentration of VITAXIN® reference standard binding signal was Compare to the binding signal of the test antibody or antibody fragment. As used herein, “reference standard” refers to a −70 ° C. consisting of 10 mg / ml antibody or antibody fragment (eg, VITAXIN®) in histidine-HCl buffer (pH 6.0) containing 150 mM NaCl. Refers to an antibody or antibody fragment (eg, VITAXIN®) frozen at <RTIgt;, </ RTI> and usually shows a single monomer peak (over 97% region) by HPSEC.

4). BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing an overview of preparing a purified antibody that immunospecifically binds to integrin α _v β ₃ .

5. DETAILED DESCRIPTION OF THE INVENTION The liquid formulations of the present invention provide ready-to-use formulations of antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ . This liquid formulation does not require reconstitution of the formulation accurately and aseptically for administration to a subject and does not wait for a while for the solution to become clear before applying the formulation to the subject. This simplifies the operating procedure for medical personnel to administer the formulation to the subject. Moreover, due to its high stability during storage, the formulations of the present invention can be used for the biology of antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ by protein aggregation and / or fragmentation during long-term storage. An antibody or antibody fragment that immunospecifically binds to integrin α _v β _{3 can be} included at a concentration ranging from about 15 mg / ml to about 300 mg / ml without causing adverse effects on pharmacological activity. Such stability not only ensures the effectiveness of the antibody or antibody fragment, but also reduces the potential risk of causing adverse effects on the subject. Furthermore, the use of fewer ingredients in the formulation also reduces the risk of contaminants. Furthermore, the manufacturing process of the liquid preparation of the present invention is simplified, and all the steps of manufacturing the liquid preparation are performed in an aqueous solution and do not require a drying process such as freeze drying or freeze drying. More efficient than the manufacturing process. This formulation is therefore more cost effective.

5.1 Antibody Formulation The liquid formulation of the present invention provides an antibody formulation that is substantially free of surfactants, inorganic salts, and / or other excipients and yet exhibits high stability during long-term storage. In certain embodiments, such antibody formulations are homogeneous. The formulations of the present invention comprise histidine and an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ at a concentration of about 15 mg / ml to about 300 mg / ml. In one embodiment, the formulations of the present invention do not contain other ingredients other than water or a suitable solvent. In certain embodiments, an antibody or antibody fragment that binds to integrin alpha _v beta ₃ contained in the liquid formulations immunospecifically of the present invention is VITAXIN (R) or an antigen-binding fragment thereof. In another embodiment, the antibody or antibody fragment that binds to integrin alpha _v beta ₃ contained in the liquid formulations immunospecifically of the present invention is VITAXIN not ® or an antigen-binding fragment thereof. In a preferred embodiment, the antibody or antibody fragment that immunospecifically binds to the integrin α _v β ₃ contained in the liquid formulation of the present invention is one or more VH CDRs and / or one or more VLs shown in Table 1 above An antibody or antibody fragment comprising a CDR. In another embodiment, the antibody or antibody fragment that binds to integrin alpha _v beta ₃ contained in the liquid formulations immunospecifically of the present invention, another portion, for example, without limitation, a heterologous polypeptide, another Antibody or antibody fragment, a marker sequence, a diagnostic agent, a therapeutic agent, a radioactive metal ion, a polymer, albumin and a solid support. In yet another embodiment, the liquid formulation of the invention comprises two or more antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein at least one of the antibodies or antibody fragments is VITAXIN®. ) Or an antigen-binding fragment thereof.

The concentration of the antibody or antibody fragment that binds to integrin alpha _v beta ₃ which is included in the liquid formulations immunospecifically of the present invention is at least 15 mg / ml, at least 20 mg / ml, at least 25 mg / ml, at least 30 mg / ml, At least 35 mg / ml, at least 40 mg / ml, at least 45 mg / ml, at least 50 mg / ml, at least 55 mg / m, at least 60 mg / ml, at least 65 mg / ml, at least 70 mg / ml, at least 75 mg / ml, at least 80 mg / ml, At least 85 mg / ml, at least 90 mg / ml, at least 95 mg / ml, at least 100 mg / ml, at least 105 mg / ml, at least 110 mg / ml, at least 115 mg / ml, at least 120 mg / ml, at least 125 mg / ml, at least 130 mg / ml, at least 135 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, at least 250 mg / ml, at least 275 mg / ml, Or at least 300 mg / ml. In certain embodiments, the concentration of antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ contained in a liquid formulation of the invention is about 75 mg / ml, about 100 mg / ml, about 125 mg / ml. About 150 mg / ml, about 175 mg / ml, about 200 mg / ml, about 225 mg / ml, about 250 mg / ml, about 275 mg / ml, or about 300 mg / ml. In another embodiment, the concentration of antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ contained in the liquid formulation of the invention is 15-500 mg / ml, 50-300 mg / ml, 50- 250 mg / ml, 50-200 mg / ml, 50-175 mg / ml, 50-150 mg / ml, 50-125 mg / ml, or 50-100 mg / ml.

  The concentration of histidine contained in the liquid formulation of the present invention ranges from 1 mM to 100 mM, preferably from 5 mM to 50 mM, more preferably from 10 mM to about 25 mM. In certain embodiments, the concentration of histidine contained in the liquid formulation of the invention is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. The histidine can be in the form of L-histidine, D-histidine, or a mixture thereof, with L-histidine being most preferred. Histidine may be in the form of a hydrate. Histidine may be used in the form of a pharmaceutically acceptable salt such as hydrochloride (eg, monohydrochloride and dihydrochloride), hydrobromide, sulfate, acetate, and the like. The purity of histidine should be at least 98%, preferably at least 99%, and most preferably at least 99.5%. The term “purity” as used herein with respect to histidine refers to the chemical purity of histidine as understood in the art, eg, The Merck Index, 13th edition, edited by O'Neil et al. (Merck & Co. , 2001).

  The pH of the formulation should be different from the isoelectric point of the specific antibody or antibody fragment used in the formulation (eg, the isoelectric point of VITAXIN® is in the range of 8.65 to 8.89); It may range from about 5.0 to about 7, preferably from about 5.5 to about 6.5, more preferably from about 5.8 to about 6.2, and most preferably about 6.0.

In addition to antibodies or antibody fragments that immunospecifically bind to histidine and integrin α _v β ₃ , the formulations of the present invention comprise less than 150 mM, less than 100 mM, less than 75 mM, less than 50 mM, less than 25 mM, less than 10 mM, less than 5.0 mM. Or glycine at a concentration of less than 2.0 mM. In certain embodiments, the formulations of the invention have 1-150 mM, 1-100 mM, 1-75 mM, 1-50 mM, 1-25 mM, 1-10 mM, 1-5.0 mM, or 1-2.0 mM. It may further comprise glycine at a concentration. The amount of glycine in the formulation should avoid significant buffering to avoid precipitation of the antibody at its isoelectric point. Glycine may be used in the form of a pharmaceutically acceptable salt such as hydrochloride, hydrobromide, sulfate, acetate and the like. The purity of glycine should be at least 98%, preferably at least 99%, most preferably 99.5%. The term “purity” as used herein with respect to glycine refers to the chemical purity of glycine as understood in the art, eg, The Merck Index, 13th edition, edited by O'Neil et al. (Merck & Co. , 2001). In certain embodiments, glycine is not included in the liquid formulation of the present invention.

  Optionally, the formulations of the present invention may further comprise other excipients such as sugars (eg sucrose, mannose, trehalose etc.) and polyols (eg mannitol, sorbitol etc.). In one embodiment, the other excipient is sugar. In certain embodiments, the sugar is sucrose and its concentration range is between about 1% and about 20%, preferably between about 5% and about 15%, more preferably between about 8% and 10%. is there. In another embodiment, the sugar is sucrose and the concentration is 1%, 3%, 5%, 8%, 10%, 15%, or 20% of the formulation. In another embodiment, the other excipient is a polyol. However, it is preferable that the liquid preparation of the present invention does not contain mannitol. In certain embodiments, the polyol is a polysorbate (eg, Tween 20) and the concentration range is between about 0.001% and about 1% of the formulation, preferably between about 0.01% and about 0.1%. It is. In certain embodiments, the polyol is a polysorbate (eg, Tween 20) and the concentration is 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08% of the formulation. , 0.1%, 0.5%, or 1%.

The liquid formulations of the present invention are stable for at least 15 days in the temperature range of 38 ° C. to 42 ° C. as measured by high performance size exclusion chromatography (HPSEC), although in certain embodiments this is not more than 25 days. 20 ° C to 24 ° C, at least 6 months, 2 ° C to 8 ° C (especially at 4 ° C), at least 6 months, at least 1 year, at least 1.5 years, at least 2 years, at least 2.5 years, at least It is stable for at least 2 years, at least 3 years, at least 4 years, or at least 5 years at −20 ° C. for 3 years, or at least 4 years. That is, the liquid formulations of the present invention have undetectable or low levels of aggregation and / or fragmentation as defined herein after storage for a specific period of time as detailed above. Preferably, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, and most preferably 0, as measured by HPSEC after storage for a specific period as detailed above Less than 5% form aggregates. Furthermore, the liquid preparation of the present invention can be used for enzyme-linked immunosorbent assay (ELISA) for measuring the ability of an antibody or antibody fragment to specifically bind to integrin α _v β ₃ during long-term storage under the above conditions, for example. Little biological activity of the antibody or antibody fragment is lost when assessed by various immunological assays, including radioimmunoassay. The liquid preparation of the present invention is 80% or more, 85% or more, 90% or more, 95% of the initial biological activity before storage (for example, the ability to bind to integrin α _v β ₃ ) after storage for the specified period. As mentioned above, it keeps 98% or more, 99% or more, or 99.5% or more. In some embodiments, the liquid formulation of the present invention binds to its biological activity (eg, binding to integrin α _v β ₃₎ after storage for the specified period of time as compared to a reference antibody representing the antibody prior to storage. At least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more, or at least 99.5%.

The liquid formulations of the present invention can be prepared as unit dosage forms. For example, one unit dose per vial may have a concentration of about 15 mg / ml to about 300 mg / ml, about 50 mg / ml to about 300 mg / ml, about 75 mg / ml to about 300 mg / ml, about 95 mg / ml to about 300 mg / ml. ml, about 100 mg / ml to about 300 mg / ml, about 150 mg / ml to about 300 mg / ml, about 200 mg / ml to about 300 mg / ml, about 100 mg / ml to about 200 mg / ml, about 100 mg / ml to about 150 mg / ml 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml of antibodies or antibody fragments that immunospecifically bind to integrin α _v β _{3 at} various concentrations ranging from about 100 mg / ml to about 175 mg / ml, May contain 7ml, 8ml, 9ml, 10ml, 15ml, or 20ml Yes. If necessary, these preparations can be adjusted to the desired concentration by adding sterile diluent to each vial.

The present invention encompasses a stable liquid formulation comprising a single antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ . The invention also encompasses a stable liquid formulation comprising two or more antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ . In certain embodiments, a stable liquid formulation of the invention comprises VITAXIN® or a fragment thereof that immunospecifically binds to integrin α _v β ₃ . In another embodiment, a stable liquid formulation of the invention comprises two or more antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ , one of which is VITAXIN®. ) Or an antigen-binding fragment thereof. In another embodiment, a stable liquid formulation of the invention comprises two or more antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein the antibodies or antibody fragments are VITAXIN®. Or an antigen-binding fragment thereof.

5.1.1 It is to be appreciated that antibodies that function to integrin alpha _V beta ₃ as antibodies immunospecific integrin alpha _V beta ₃ and immunospecifically binds and antagonists are known in the art. Examples of known antibodies that immunospecifically bind to integrin α _v β ₃ include, but are not limited to, 11D2 (Searle), mouse monoclonal LM609 (Scripps; incorporated herein by reference in its entirety, WO 89/015155 and US Pat. No. 5,753,230), also known as humanized monoclonal antibody MEDI-522 (VITAXIN®, MedImmune, Inc., Gaithersburg, MD); Wu et al., 1998 , PNAAS USA 95 (11): 6037-6042; International Publication Nos. WO 90/33919 and WO 00/78815, each of which is incorporated herein by reference in its entirety), 17661-37E and 17661-37E 1- 5 (US Biological), MON2032 (CalTag), ab7166 (BV3) and ab7167 (BV4) (Abcam), Fine-WOW-1 (Kiosses et al, Nature Cell Biology 3:. 316-320), and the like.

Antibodies that immunospecifically bind to integrin α _V β ₃ are not limited, but include monoclonal antibodies, bispecific antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, camelized antibodies , Single chain Fv (scFv), single chain antibody, disulfide-linked Fv (sdFv), and anti-idiotype (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the invention). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (ie, molecules that contain an antigen binding site). In particular, the antibodies of the present invention include immunoglobulin molecules, immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that immunospecifically binds to integrin α _v β ₃ . The immunoglobulin molecules of the invention can be of any type (eg IgG, IgE, IgM, IgD, IgA and IgY), class (eg IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ ) or subclass. It may be. In a preferred embodiment, antibodies and antibody fragments that bind to integrin alpha _V beta ₃ and immunospecific is an antagonist of integrin α _V β _3. In one embodiment, antibodies and antibody fragments that integrin alpha _V beta ₃ and immunospecifically binds the integrin alpha _V beta _1, integrin alpha _V beta _5, not the antagonists of the integrin alpha _V beta ₆ or integrin alpha _V beta _8, . In another preferred embodiment, antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ inhibit or reduce angiogenesis. In one embodiment, antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ are prepared using assays known to those skilled in the art, such as the chicken chorioallantoic membrane (CAM) angiogenesis assay (eg, Nguyen et al., Microvascular Res. 1994). , 47: 31-40) and Matrigel plug assay (see, eg, Kragh et al., International J. of Oncology, 22: 305-311 (2003)) at least 90%, at least 80% compared to the negative control, Inhibit or reduce at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of angiogenesis.

Antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ include any of birds and mammals (eg, humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens) It may be of animal origin. Preferably, the antibody that immunospecifically binds to integrin α _v β ₃ is a human or humanized monoclonal antibody. As used herein, a “human” antibody includes an antibody having the amino acid sequence of a human immunoglobulin and is also isolated from a mouse that is isolated from a human immunoglobulin library or expresses an antibody from a human gene. Antibody.

An antibody that immunospecifically binds to integrin α _v β ₃ may be monospecific, bispecific, trispecific or even multispecific. Multispecific antibodies may be specific for different epitopes of integrin alpha _V beta _3, both the specific integrin alpha _V beta ₃ epitope heterologous epitope (such as a heterologous polypeptide or solid support material) There may be. For example, International Publication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793; Tutt et al., J. Immunol. 147: 60-69 (1991); US Pat. No. 4,474,893. 4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J. Immunol. 148: 1547-1553. Please refer.

The present invention encompasses antibodies and antibody fragments that have a high binding affinity for the integrin alpha _V beta _3. In certain embodiments, the binding rate constant or _{k on} rate of integrin alpha _V beta ₃ that immunospecifically binds to an antibody or antibody fragment (antibody (Ab) + antigen ^{(Ag) kon → Ab-Ag} ) is at least 10 ⁵ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁵ M ⁻¹ s ⁻¹ , at least 10 ⁶ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁶ M ⁻¹ s ⁻¹ , at least 10 ⁷ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁷ M ⁻¹ s ⁻¹ , or at least 10 ⁸ M ⁻¹ s ⁻¹ . In a preferred embodiment, _{k on} integrin alpha _V beta ₃ that immunospecifically binds to an antibody or antibody fragment is at least ^{2 × 10 5 M -1 s -1} , at least 5 × ¹⁰ 5 ^M -1 ^{s -1} , At least 10 ⁶ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁶ M ⁻¹ s ⁻¹ , at least 10 ⁷ M ⁻¹ s ⁻¹ , at least 5 × 10 ⁷ M ⁻¹ s ⁻¹ , or at least 10 ⁸ M ⁻¹ s ⁻¹ .

In other embodiments, the k _off rate (antibody (Ab) + antigen (Ag) ^koff ← Ab−Ag) of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is 10 ⁻¹ s ^−. Less than ^1, less than 5 × 10 ⁻¹ s ^−1, less than 10 ⁻² s ^−1, less than 5 × 10 ⁻² s ^−1, less than 10 ⁻³ s ^−1, less than 5 × 10 ⁻³ s ⁻¹ , 10 Less than ⁻⁴ s ^−1, less than 5 × 10 ⁻⁴ s ^−1, less than 10 ⁻⁵ s ^−1, less than 5 × 10 ⁻⁵ s ^−1, less than 10 ⁻⁶ s ⁻¹ , and 5 × 10 ⁻⁶ s ⁻ Less than ^1, less than 10 ⁻⁷ s ^−1, less than 5 × 10 ⁻⁷ s ^−1, less than 10 ⁻⁸ s ^−1, less than 5 × 10 ⁻⁸ s ^−1, less than 10 ⁻⁹ s ⁻¹ , 5 × 10 It is less than ⁻⁹ s ⁻¹ or less than 10 ⁻¹⁰ s ⁻¹ . In a preferred embodiment, _{k on} rate of integrin alpha _V beta ₃ that immunospecifically binds to an antibody or antibody fragment is less than 5 × ¹⁰ -4 ^{s -1,} less than ^{10 -5 s -1, 5 × 10} - Less than ⁵ s ^−1, less than 10 ⁻⁶ s ^−1, less than 5 × 10 ⁻⁶ s ^−1, less than 10 ⁻⁷ s ^−1, less than 5 × 10 ⁻⁷ s ^−1, less than 10 ⁻⁸ s ⁻¹ , Less than 5 × 10 ⁻⁸ s ^−1, less than 10 ⁻⁹ s ^−1, less than 5 × 10 ⁻⁹ s ⁻¹ , or less than 10 ⁻¹⁰ s ⁻¹ .

In other embodiments, the affinity constant or K _a (k _on / k _off ) of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is at least 10 ² M ⁻¹ , at least 5 × 10. ² M ⁻¹ , at least 10 ³ M ⁻¹ , at least 5 × 10 ³ M ⁻¹ , at least 10 ⁴ M ⁻¹ , at least 5 × 10 ⁴ M ⁻¹ , at least 10 ⁵ M ⁻¹ , at least 5 × 10 ⁵ M ⁻¹ , at least 10 ⁶ M ⁻¹ , at least 5 × 10 ⁶ M ⁻¹ , at least 10 ⁷ M ⁻¹ , at least 5 × 10 ⁷ M ⁻¹ , at least 10 ⁸ M ⁻¹ , at least 5 × 10 ⁸ M ⁻¹ , At least 10 ⁹ M ⁻¹ , at least 5 × 10 ⁹ M ⁻¹ , at least 10 ¹⁰ M ⁻¹ , at least 5 × 10 ¹⁰ M ⁻¹ , at least 10 ¹¹ M ⁻¹ , at least 5 × 10 ¹¹ M ⁻¹ , at least 10 ¹² M ⁻¹ , at least 5 × 10 ¹² M ⁻¹ , at least 10 ¹³ M ⁻¹ , at least 5 × 10 ¹³ M ⁻¹ , at least 10 ¹⁴ M ⁻¹ , at least 5 × 10 ¹⁴ M ⁻¹ , at least 10 ¹⁵ M ⁻¹ , or at least 5 × 10 ¹⁵ M ⁻¹ . In still other embodiments, the dissociation constant or K _d (k _off / k _on ) of an antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ is less than 10 ⁻² M, 5 × 10 ^−2. Less than M, less than 10 ⁻³ M, less than 5 × 10 ⁻³ M, less than 10 ⁻⁴ M, less than 5 × 10 ⁻⁴ M, less than 10 ⁻⁵ M, less than 5 × 10 ⁻⁵ M, less than 10 ⁻⁶ M Less than 5 × 10 ⁻⁶ M, less than 10 ⁻⁷ M, less than 5 × 10 ⁻⁷ M, less than 10 ⁻⁸ M, less than 5 × 10 ⁻⁸ M, less than 10 ⁻⁹ M, less than 5 × 10 ⁻⁹ M Less than 10 ⁻¹⁰ M, less than 5 × 10 ⁻¹⁰ M, less than 10 ⁻¹¹ M, less than 5 × 10 ⁻¹¹ M, less than 10 ⁻¹² M, less than 5 × 10 ⁻¹² M, less than 10 ⁻¹³ M, 5 × ¹⁰ than ^{-13 M,} less than ^{10 -14} M, 5 × ^{10 -14} less than M, 10 Is ¹⁵ M, or 5 × ^{10 -15} M.

In certain embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is LM609 or an antigen-binding fragment thereof (eg, one or more complementarity determining regions (CDRs) of LM609). LM609 is an amino acid sequence disclosed in, for example, International Publication No. WO 89/05155 (the entire text of which is incorporated herein by reference), or American Type Culture Collection (ATCC (registered trademark)) (10801 University Boulevard, Manassas, Virginia 20110-2209) having the amino acid sequence of the monoclonal antibody produced by the cell line deposited under accession number HB9537. In another embodiment, the antibody that immunospecifically binds to integrin α _v β ₃ is not LM609 or an antigen-binding fragment of LM609.

In a preferred embodiment, the antibody that immunospecifically binds to integrin α _v β ₃ is VITAXIN® or an antigen-binding fragment thereof (eg, one or more CDRs of VITAXIN®). VITAXIN® is disclosed, for example, in International Publication Nos. WO 98/33919, WO 00/78815 and WO 02/070007, and US Patent Application No. 09 / 339,222, each of which is referred to herein. The full text is incorporated. In another embodiment, the antibody that immunospecifically binds to integrin α _v β ₃ is not VITAXIN® or an antigen-binding fragment of VITAXIN®.

The present invention also provides antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , comprising the VH domain having the amino acid sequence of the variable heavy chain (VH) domain of LM609 or VITAXIN®. Includes antibodies and antibody fragments. The invention also encompasses the use of antibodies and antibody fragments that immunospecifically bind to integrin α _V β ₃ comprising a VH CDR having any one of the amino acid sequences of VH CDRs listed in Table 1 above.

In one embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR1 having the amino acid sequence of SEQ ID NO: 1. In other embodiments, the antibodies and antibody fragments that immunospecifically bind to integrin α _V β ₃ comprise VH CDR2 having the amino acid sequence of SEQ ID NO: 2. In other embodiments, the antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ comprise VH CDR3 having the amino acid sequence of SEQ ID NO: 3. In other embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises a combination of VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and VH CDR2 having the amino acid sequence of SEQ ID NO: 2. Including. In other embodiments, antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ comprise a combination of VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and VH CDR3 having the amino acid sequence of SEQ ID NO: 3. Including. In other embodiments, antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ comprise a combination of VH CDR2 having the amino acid sequence of SEQ ID NO: 2 and VH CDR3 having the amino acid sequence of SEQ ID NO: 3. Including. In a preferred embodiment, the antibody that immunospecifically binds to integrin α _v β ₃ comprises VH CDR1 having the amino acid sequence of SEQ ID NO: 1, VH CDR2 having the amino acid sequence of SEQ ID NO: 2, and the amino acid sequence of SEQ ID NO: 3. Including VH CDR3.

The present invention also provides antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , comprising the VL domain having the amino acid sequence of the variable light chain (VL) domain of LM609 or VITAXIN®. Includes the use of antibodies and antibody fragments. The invention also encompasses antibodies and antibody fragments that immunospecifically bind to integrin α _V β ₃ comprising a VL CDR having the amino acid sequence of any one VL CDR listed in Table 1.

In one embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VL CDR1 having the amino acid sequence of SEQ ID NO: 4. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VL CDR2 having the amino acid sequence of SEQ ID NO: 5. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VL CDR3 having the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ comprises VL CDR1 having the amino acid sequence of SEQ ID NO: 4 and VL CDR2 having the amino acid sequence of SEQ ID NO: 5. In a preferred embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ comprises VL CDR1 having the amino acid sequence of SEQ ID NO: 4 and VL CDR3 having the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ comprises VL CDR2 having the amino acid sequence of SEQ ID NO: 5 and VL CDR3 having the amino acid sequence of SEQ ID NO: 6. In a preferred embodiment, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises a VL CDR1 having the amino acid sequence of SEQ ID NO: 4, a VL CDR2 having the amino acid sequence of SEQ ID NO: 5, and SEQ ID NO: 6. VL CDR3 having the amino acid sequence of

The present invention also provides antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein a VH domain disclosed herein is combined with a VL domain or other VL domain disclosed herein. Including the above-mentioned antibodies and antibody fragments. The present invention further includes antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein the VL domain disclosed herein is combined with a VH domain or other VH domain disclosed herein Including the above-mentioned antibodies and antibody fragments.

The invention also encompasses antibodies and antibody fragments that immunospecifically bind to integrin α _V β ₃ comprising one or more VH CDRs and one or more VL CDRs listed in Table 1. In particular, the present invention relates to an antibody that immunospecifically binds to integrin α _V β ₃ , comprising VH CDR1 and VL CDR1; VH CDR1 and VL CDR2; VH CDR1 and VL CDR3; VH CDR2 and VL CDR1; VH CDR2 and VL CDR2; VH CDR2 and VL CDR3; VH CDR3 and VH CDR1; VH CDR3 and VL CDR2; VH CDR3 and VL CDR3; VH1 CDR1, VH CDR2 and VL CDR1; VH CDR1, VH CDR1, VH CDR1, VH CDR1, VH CDR1, VH CDR1, VH CDR1, VH CDR1, VH VH CDR2, VH CDR3 and VL CDR1; VH CDR2, VH CDR3 and VL CDR2; VH CDR2, VH CDR2 and VL CDR3; VH CDR1, VL CDR1 and VL CDR2, VH CDR1; VH CDR1; DR1 and VL CDR3; VH CDR2, VL CDR1 and VL CDR2; VH CDR2, VL CDR1 and VL CDR3; VH CDR3, VL CDR1 and VL CDR2; VH CDR3, VL CDR1 and VL CDR3; VH CDR3; VH CDR3; VH CDR3; VL CDR1; VH CDR1, VH CDR2, VH CDR3 and VL CDR2; VH CDR1, VH CDR2, VH CDR3 and VL CDR3; VH CDR1, VH CDR2, VL CDR1 and VL CDR2; VH CDR2; VH CDR2; VH CDR2; VH CDR1, VH CDR3, VL CDR1 and VL CDR2; VH CDR1, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR1 and VL CDR VH CDR2, VH CDR3, VL CDR1 and VL CDR3; VH CDR2, VH CDR3, VL CDR2 and VL CDR3; VH CDR1, VH CDR2, VH CDR3, VL CDR1 and VL CDR2, VH CDR2; VH CDR2; CDR1 and VL CDR3; VH CDR1, VH CDR2, VL CDR1, VL CDR2 and VL CDR3; VL CDR1, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; VH CDR2, VH CDR3, VH CDR3, VH CDR3, VH CDR3, VH CDR3, VH CDR3, VH CDR3 Alternatively, the antibody or antibody fragment comprising any combination of VH CDR and VL CDR listed in Table 1 above is provided.

In one embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ comprises VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and VL CDR1 having the amino acid sequence of SEQ ID NO: 4. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and VL CDR2 having the amino acid sequence of SEQ ID NO: 5. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR1 having the amino acid sequence of SEQ ID NO: 1 and VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR2 having the amino acid sequence of SEQ ID NO: 2 and VL CDR1 having the amino acid sequence of SEQ ID NO: 4. In other embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR2 having the amino acid sequence of SEQ ID NO: 2 and VL CDR2 having the amino acid sequence of SEQ ID NO: 5. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR2 having the amino acid sequence of SEQ ID NO: 2 and VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR3 having the amino acid sequence of SEQ ID NO: 3 and VL CDR1 having the amino acid sequence of SEQ ID NO: 4. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR3 having the amino acid sequence of SEQ ID NO: 3 and VL CDR2 having the amino acid sequence of SEQ ID NO: 5. In a preferred embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises VH CDR3 having the amino acid sequence of SEQ ID NO: 3 and VL CDR3 having the amino acid sequence of SEQ ID NO: 6.

The present invention also encompasses generally isolated nucleic acid molecules that encode antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ . In certain embodiments, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and encodes the antibody having the amino acid sequence of LM609 or VITAXIN®. To do. In one embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and has a VH domain amino acid sequence of LM609 or VITAXIN®. It encodes the antibody or antibody fragment containing the domain. In another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and is deposited in the ATCC® as accession number HB9537. Encodes the antibody or antibody fragment comprising a VH domain having the amino acid sequence of the VH domain of a monoclonal antibody produced by In another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and has the amino acid sequence of the LM domain of LM609 or VITAXIN®. It encodes the antibody or antibody fragment containing the VL domain. In another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and is deposited in the ATCC® as accession number HB9537. The antibody or antibody fragment containing the VL domain having the amino acid sequence of the VL domain of the monoclonal antibody produced by In another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and comprises VL CDR1 having the amino acid sequence of VL CDR1 listed in Table 1. It encodes the antibody or antibody fragment comprising. In another embodiment, the isolated nucleic acid molecule is an antibody that immunospecifically binds to integrin α _V β ₃ and comprises the VL CDR2 having an amino acid sequence of VL CDR2 listed in Table 1. Code.

The present invention is an antibody or antibody fragment that immunospecifically binds to integrin α _V β _3, and encodes the above antibody or antibody fragment comprising VH CDR having any amino acid sequence of VH CDR listed in Table 1. An isolated nucleic acid molecule. In particular, the present invention relates to an antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ , which has one of the amino acid sequences of VH CDRs listed in Table 1, 2, 3, 4, 5, Or an isolated nucleic acid molecule encoding the antibody or antibody fragment comprising the VH CDR or more. In one embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and comprises VH CDR1 having the amino acid sequence of VH CDR1 listed in Table 1. It encodes the antibody or antibody fragment. In another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and comprises VH CDR2 having the amino acid sequence of VH CDR2 listed in Table 1. It encodes the antibody or antibody fragment comprising. In yet another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and has a VH CDR3 amino acid sequence listed in Table 1. Which encodes the antibody or antibody fragment.

The present invention is an antibody or antibody fragment that immunospecifically binds to integrin α _V β _3, and encodes the above antibody or antibody fragment comprising the VL CDR having any amino acid sequence of the VL CDRs listed in Table 1. An isolated nucleic acid molecule. In particular, the present invention relates to an antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ , having 1, 2, 3, or more amino acid sequences of any of the VL CDRs listed in Table 1. It includes an isolated nucleic acid molecule that encodes the antibody or antibody fragment comprising a VL CDR. In one embodiment, an isolated nucleic acid molecule encodes an antibody or antibody fragment that integrin alpha _V beta ₃ and immunospecifically binds includes a VL CDR1 having the amino acid sequence of the VL CDRl listed in Table 1 It encodes the antibody or antibody fragment. In another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and comprises VL CDR2 having the amino acid sequence of VL CDR2 listed in Table 1. It encodes the antibody or antibody fragment comprising. In yet another embodiment, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and has the amino acid sequence of VL CDR3 listed in Table 1. Which encodes the antibody or antibody fragment.

In other embodiments, the isolated nucleic acid molecule is an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and has the amino acid sequence of the LM609 or VITAXIN® VH domain. It encodes the above antibody or antibody fragment comprising a VH domain and a VL domain having the amino acid sequence of the LM609 or VITAXIN® VL domain. In other embodiments, the isolated nucleic acid molecule comprises a VH CDR1, VL CDR1, VH CDR2, VL CDR2, VH CDR3, VL CDR3 having an amino acid sequence listed in Table 1, or any combination thereof encoding integrin alpha _V beta ₃ that immunospecifically binds to an antibody or antibody fragment which contains.

The present invention also provides antibodies and antibody fragments that integrin alpha _V beta ₃ and immunospecifically binds, VH domain described herein to integrin alpha _V beta ₃ and immunospecifically bind, VH CDR, VL Includes the use of the above antibodies and antibody fragments comprising a domain or derivative of a VL CDR. Using standard techniques known to those skilled in the art, mutations (eg, additions, deletions) in the nucleotide sequence encoding an antibody of the invention, including, for example, site-directed mutagenesis resulting in amino acid substitutions and PCR-mediated mutagenesis. Loss and / or substitution) can be introduced. Preferably, the derivative has less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions compared to the original molecule, 4 Includes fewer than 3 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions. In a preferred embodiment, the derivative was made at one or more putative non-essential amino acid residues (ie, amino acid residues that are not important for the antibody to immunospecifically bind to the integrin α _v β ₃ ). Has conservative amino acid substitutions. A “conservative amino acid substitution” is a substitution that replaces an amino acid residue with an amino acid residue having a similarly charged side chain. A family of amino acid residues having similar charged side chains has been defined in the art. These families include basic side chains (eg lysine, arginine, histidine), acidic side chains (eg aspartic acid, glutamic acid), uncharged polar side chains (eg glycine, asparagine, glutamine, serine, threonine, tyrosine) Cysteine), nonpolar side chains (eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (eg threonine, valine, isoleucine) and aromatic side chains (eg Amino acids having tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations are introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resulting mutants are screened for biological activity to identify mutants that retain activity. May be. Following mutagenesis, the encoded antibody or antibody fragment can be expressed to confirm the activity of the antibody or antibody fragment.

The present invention relates to antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein one or more amino acid residue substitutions are made in the variable light chain (VL) domain and / or variable heavy chain (VH) domain. The antibodies and antibody fragments comprising the amino acid sequence of LM609 or VITAXIN (registered trademark). The present invention also relates to antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein one or more VL CDRs and / or one or more VH CDRs have one or more amino acid residue substitutions, LM609 or The antibodies and antibody fragments comprising the amino acid sequence of VITAXIN® are included. An antibody or antibody fragment engineered to contain substitutions in the LM609 or VITAXIN® VH domain, VH CDR, VL domain and / or VL CDR, for example, immunospecifically binds to its integrin α _V β ₃ for their ability to (e.g., without limitation, by immunoassays including ELISA and BIAcore), or inflammatory disorder, autoimmune disease, abnormal bone metabolism related disorders, integrin alpha _v beta ₃ of aberrant expression and The ability to prevent, manage, treat, or ameliorate a disorder associated with activity, a disorder associated with abnormal angiogenesis, or cancer, or symptoms thereof, can be tested in vitro and in vivo.

In certain embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is a nucleotide sequence encoding a monoclonal antibody produced by a cell line deposited with ATCC® under accession number HB9537. In stringent conditions (eg, 6 × sodium chloride / sodium citrate (SSC) at about 45 ° C. followed by hybridization with 0.2 × SSC / 0.1% SDS. One or more washes at about 50-65 ° C.) under highly stringent conditions (eg, hybridization with filter-bound DNA at about 45 ° C. in 6 × SSC, followed by 0.1 × One or more washes in SSC / 0.2% SDS at about 68 ° C.), or other stringers known to those skilled in the art Hybridization conditions (e.g. Ausubel, FM et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York 6.3.1- (See pages 6.3.6 and 2.10.3).

In certain embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is produced under stringent conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)). Hybridization with filter-bound DNA followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50-65 ° C. under highly stringent conditions (eg, 6 × Hybridization with filter-bound DNA in SSC at about 45 ° C. followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68 ° C.), or other known to those skilled in the art (See, for example, Ausubel, FM, et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wil (See pages 6.3.1-6.3.6 and 2.10.3 of ey & Sons, Inc., New York) and is encoded by a nucleotide sequence that hybridizes to a nucleotide sequence encoding LM609 or VITAXIN®.

In certain embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is produced under stringent conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)). Hybridization with filter-bound DNA followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50-65 ° C. under highly stringent conditions (eg, 6 × Hybridization with filter-bound DNA in SSC at about 45 ° C. followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68 ° C.), or other known to those skilled in the art (See, for example, Ausubel, FM, et al., 1989, Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wil (see pages 6.3.1-6.3.6 and 2.10.3 of ey & Sons, Inc., New York) nucleotides that hybridize to nucleotide sequences encoding the VH and / or VL domains of LM609 or VITAXIN® The amino acid sequence of the VH domain or VL domain encoded by the sequence is included.

In another embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is under stringent conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)). Hybridization with filter-bound DNA followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50-65 ° C. under highly stringent conditions (eg, 6 × Hybridization with filter-bound DNA in SSC at about 45 ° C. followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68 ° C.), or other known to those skilled in the art Hybridizes to nucleotide sequences encoding one or more of the VH CDRs or VL CDRs listed in Table 1 under stringent hybridization conditions of It comprises the amino acid sequence or amino acid sequence of the VL CDR of VH CDR encoded by that nucleotide sequence.

In another embodiment, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is under stringent conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)). Hybridization with filter-bound DNA, followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50-65 ° C., under highly stringent conditions (eg, 6 × Hybridization with filter-bound DNA in SSC at about 45 ° C. followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68 ° C.), or other known to those skilled in the art Under the stringent hybridization conditions of the cell line produced by the cell line deposited with ATCC® under accession number HB9537. The VH CDR or VL nucleotide sequence which hybridizes to a nucleotide sequence encoding any one of the CDR clonal antibody comprises the amino acid sequence or amino acid sequence of the VL CDR of VH CDR encoded.

In another embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is under stringent conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)). Hybridization with filter-bound DNA followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50-65 ° C. under highly stringent conditions (eg, 6 × Hybridization with filter-bound DNA in SSC at about 45 ° C. followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68 ° C.), or other known to those skilled in the art Hybridize to a nucleotide sequence encoding any one of the VH CDRs and VL CDRs listed in Table 1 under stringent hybridization conditions. Comprising the amino acid sequence of amino acid sequences and VL CDR of VH CDR encoded by a nucleotide sequence that size.

In another embodiment, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is under stringent conditions (eg, at about 45 ° C. in 6 × sodium chloride / sodium citrate (SSC)). Hybridization with filter-bound DNA, followed by one or more washes in 0.2 × SSC / 0.1% SDS at about 50-65 ° C., under highly stringent conditions (eg, 6 × Hybridization with filter-bound DNA in SSC at about 45 ° C. followed by one or more washes in 0.1 × SSC / 0.2% SDS at about 68 ° C.), or other known to those skilled in the art Under the stringent hybridization conditions of the cell line produced by the cell line deposited with ATCC® under accession number HB9537. Comprising the amino acid sequence and amino acid sequence of the VL CDR of VH CDR encoded by nucleotide sequence which hybridizes to a nucleotide sequence encoding a clonal antibody.

In certain embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises an amino acid sequence of a monoclonal antibody produced by a cell line deposited with ATCC® under accession number HB9537; At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, Or at least 99% identical amino acid sequences. In other embodiments, an antibody that immunospecifically binds to integrin α _v β ₃ has a VITAXIN® amino acid sequence of at least 35%, at least 40%, at least 45%, at least 50%, at least 55 %, At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequences. The determination of percent identity between two amino acid sequences can be determined by any method known to one of skill in the art, including BLAST protein searches.

In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ has a VH domain of VITAXIN® and at least 35%, at least 40%, at least 45%, at least 50%. Amino acid sequences of VH domains that are at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical Including. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises a VH domain of a monoclonal antibody produced by a cell line deposited with ATCC® under accession number HB9537; At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, Or at least 99% identical amino acid sequence of the VH domain.

In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises at least 35%, at least 40%, at least 45%, at least, one or more of the VH CDRs listed in Table 1. One or more VHs that are 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical Contains the amino acid sequence of the CDR. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is any of the monoclonal antibody VH CDRs produced by the cell line deposited with ATCC® under accession number HB9537. And at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90 %, At least 95%, or at least 99% identical amino acid sequence of one or more VH CDRs.

In other embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ has a VL domain of VITAXIN® and at least 35%, at least 40%, at least 45%, at least 50%. VL domain amino acid sequences that are at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical Including. In other embodiments, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ comprises a monoclonal antibody VL domain produced by a cell line deposited with ATCC® under accession number HB9537; At least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95 %, Or at least 99% identical VL domain amino acid sequence.

In other embodiments, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ has at least 35%, at least 40%, at least 45%, at least 50% of any of the VL CDRs listed in Table 1. One or more VL CDRs that are%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical Of the amino acid sequence. In another embodiment, the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is a monoclonal antibody VL CDR of the monoclonal antibody produced by the cell line deposited with ATCC® under accession number HB 9537. Any one and at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least It comprises one or more VL CDR amino acid sequences that are 90%, at least 95%, or at least 99% identical.

The present invention encompasses antibodies that compete with the antibodies and antibody fragments described herein for binding to integrin α _v β ₃ . In certain embodiments, the invention encompasses antibodies and antibody fragments that compete with LM609 or antigen-binding fragments thereof for binding to integrin α _v β ₃ . Preferred embodiments include antibodies and antibody fragments that compete with VITAXIN® or antigen-binding fragments thereof for binding to integrin α _v β ₃ .

The present invention also provides a VH domain that competes with a protein, polypeptide or peptide comprising (or consisting of) the VH domain of LM609 or VITAXIN ^™ or the VH domain of LM609 or VITAXIN ^™ for binding to integrin α _v β _3. It includes a plurality of proteins, polypeptides or peptides comprising. The present invention also provides a VL domain that competes with a protein, polypeptide or peptide comprising (or consisting of) the ^LM domain of LM609 or VITAXIN ^™ or the VL domain of LM609 or VITAXIN ^™ for binding to integrin α _V β _3. It includes a plurality of proteins, polypeptides or peptides comprising.

The present invention also relates to a VH CDR listed in Table 1 or a protein, polypeptide or polypeptide comprising (or consisting of) at least one VH CDR comprising the VH CDR listed in Table 1 for binding to integrin α _V β ₃ Monoclonal antibody VH CDRs produced by a cell line deposited with ATCC ^™ under accession number HB9537 for binding to a plurality of proteins, polypeptides or peptides containing at least one VH CDR competing with the peptide, or integrin α _V β ₃ VH CDRs that compete with The present invention also competes with proteins, polypeptides or peptides comprising (or consisting of) the VL CDRs listed in Table 1 or VH CDRs comprising the VL CDRs listed in Table 1 for binding to integrin α _V β ₃ A plurality of proteins, polypeptides or peptides containing VH CDRs, or at least compete with the monoclonal antibody VL CDRs produced by the cell line deposited with ATCC® under accession number HB9537 for binding to integrin α _V β ₃ It includes a plurality of proteins, polypeptides or peptides containing one VL CDR.

Antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ include modified derivatives, ie, modified by covalent attachment of any type of molecule to the antibody. For example, without limitation, antibody derivatives include, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, cellular ligands or others. Antibodies or antibody fragments that have been modified by linking to other proteins, etc. Any of a number of chemical modifications can be performed by known techniques, including specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Furthermore, the derivative may contain one or more non-classical amino acids.

The present invention also provides an antibody fragment that immunospecifically binds to integrin α _v β ₃ and comprises the above-described antibody fragment comprising a framework region known to those skilled in the art. As a non-limiting example, the framework regions are generated from or derived from human germline immunoglobulin sequences using methods known in the art, eg, using polymerase chain reaction (PCR). can do. Human germline immunoglobulin sequences are described, for example, on the NCBI website (Kawasaki et al., 2001, Eur. J. Immunol. 31: 1017-1028; Schable and Zachau, 1993, Biol. Chem. Hoppe Seyler 374: 1001-1022; Matsuda et al. 1998, J. Exp. Med., 188: 1973-1975). Preferably, the fragment region of the antibody of the present invention is human. In certain embodiments, an antibody that immunospecifically binds to integrin α _v β ₃ comprises a framework region of VITAXIN®.

The present invention also relates to antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ and having one or more mutations (eg, one or more amino acid residue substitutions) in the framework region. The above antibody comprising the amino acid sequence of In certain embodiments, antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ have VITAXIN® with one or more amino acid residue substitutions in the VH and / or VL domain framework. Of the amino acid sequence.

The present invention also relates to antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ , wherein one or more mutations (eg, one or more amino acid residue substitutions) are made in the variable region and framework region. The above antibodies and antibody fragments containing the amino acid sequence of VITAXIN (registered trademark) are also included.

The invention also encompasses antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ comprising constant regions known to those of skill in the art. For example, Wu & An, 2003, Methods Mol. Biol., 207, 213-233; Wu, 2003, Methods Mol. Biol., 207, 197-212; International Publication WO 86/05807; International Publication WO 89/01036; And U.S. Pat. No. 5,122,464. Preferably, the constant region of the antibody of the present invention is human.

The invention also encompasses fusion proteins comprising an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ and a heterologous polypeptide. Preferably, the heterologous polypeptide fused to the antibody or antibody fragment is useful for targeting the antibody or antibody fragment to platelets, monocytes, macrophages, endothelial cells, osteoclasts, activated T cells, and / or B cells. It is.

5.1.1.1. Methods for identifying antibodies immunospecific for integrin α _v β _{3 The} present invention relates to antibodies and antibody fragments immunospecific for integrin α _v β ₃ , in particular VITAXIN® and / or LM609. Methods are provided for identifying antibodies and antibody fragments that specifically bind to the same epitope. Mutation of residues 171, 173 and / or 174 of human β ₃ chain has been found to disrupt the binding of VITAXIN® antibody and / or LM609 antibody to integrin α _v β ₃ heterodimer Yes. VITAXIN® and LM609 do not bind to mouse integrin α _v β ₃ , but VITAXIN® and LM609 have a mouse β chain amino acid region corresponding to amino acids 164 to 202 of human β chain. It has been found to bind to the modified mouse integrin α _v β ₃ substituted with 164-202. In certain embodiments, amino acid substitutions, for example, to destroy the order to change the ligand specificity of the integrin alpha _v beta ₃ and / or heterodimerization of these subunits chains, integrin alpha _v beta ₃ sub Made in the unit. Preferably, the integrin α _v β ₃ is human. In certain embodiments, such amino acid substitutions disrupt the specific interaction with specific antagonists of integrin alpha _v beta ₃ and a specific integrin alpha _v beta ₃ epitope. In a preferred embodiment, these amino acid substitutions are within the region of the integrin subunit conferring ligand binding specificity, preferably LM609 and / or VITAXIN® ligand binding specificity, in particular human β ₃ 164-202. Made in the second residue. Alternatively, mouse beta chain residues corresponding to 164-202 th residues of the human beta ₃ chain is replaced with a 164 to 202-th residue of the human beta _3. Such mice - human chimeric can bind to human beta ₃ regions 164-202 are used to screen for antagonists that does not bind to murine integrin α _v β _3.

In preferred embodiments, these amino acid substitutions are made in the β ₃ subunit. In certain embodiments, human β ₃ residues are substituted with rat residues as described in Table 2. In one embodiment, substitution of human residue Glu at position 171 with rat residue Gln (“mutant A”) disrupts integrin α _v β ₃ binding to LM609. This same change breaks the binding to VITAXIN®. In another embodiment, substitution of human residues Leu and Glu to rat residues Ile and Lys (“mutant B”) at positions 173 and 174, respectively, disrupts binding to VITAXIN®, increasing the binding to the anti-rat β ₃ antibody. In yet another embodiment, substitutions at each position 179 and position 182 to rat residue Thr and Ser human residues Asp and Thr ( "variant C") is to confer binding specificity for anti-rat beta ₃ antibody . Combinations of mutations A and mutant C (3 one substituted residues) is to confer binding specificity for mouse anti-rat beta ₃ antibody, disrupt binding to VITAXIN (TM). In certain preferred embodiments, amino acids 171, 173, and 174 can be substituted to break the bond to VITAXIN®. In an alternative preferred embodiment, amino acids 171, 173, 174, 179 and 182 disrupt the binding of integrin α _v β _{3 to} LM609 and humanized anti-integrin α _v β ₃ antibody (eg, VITAXIN®). Can be substituted to Such substitution is a preferred example and not a limitation. Such substituted subunits are exemplary only and not limiting. Any integrin α _v β ₃ region identified to be responsible for antibody binding can be substituted residues to characterize the binding specificity of various antibodies and to screen for antibodies with the same similar binding specificity; It can be altered by deleted residues or inserted residues.

Integrin alpha _v beta ₃ amino acid substituted subunits, wildtype integrin alpha _v monoclonal antibody binds to beta ₃ do not bind to modified forms, or human alpha _v beta ₃ regions that are substituted with the corresponding regions from Is used to screen for antibodies with specific affinity for a particular epitope by identifying monoclonal antibodies that bind to mouse α _v β ₃ integrin with no binding to wild type mouse integrin α _v β ₃ be able to. Furthermore, the present invention provides a method for identifying a monoclonal antibody that binds to heterodimerized α _v β ₃ but does not bind to either the α _v chain or the β ₃ chain when not included in the heterodimer. I will provide a. Such screening uses, for example, cell lines that do not express wild-type integrin α _v β ₃ to recombinantly express mutant α _v β ₃ or individual α _v or β ₃ chains, It can be achieved by any conventional method for assaying antibody specificity known in the art. Antibodies identified from such screening methods include integrin α _v β ₃ mediated diseases and disorders such as, but not limited to, inflammatory diseases, autoimmune diseases, bone metabolism related disorders, angiogenesis It may be useful for the prevention, management and treatment of disorders associated with, disorders associated with abnormal expression and / or activity of α _v β ₃ , and cancer. It is also contemplated that such antibodies can be used in the methods and compositions contemplated by the present invention. Preferably, these antibodies are either LM609 or VITAXIN®, and are LM609 or VITAXIN® having 1, 2, 2, 5, 8 or 10 amino acid substitutions, deletions or insertions. It is neither an antibody with a CDR (ie, one, two, three, four or five of a CDR or a heavy chain CDR3) nor their antigen binding site.

5.1.1.2 Antibodies that immunospecifically bind to integrin α _v β ₃ having an increased half-life The present invention immunospecifically binds to integrin α _v β ₃ having a long in vivo half-life Antibodies and antibody fragments are provided. In particular, the present invention relates to antibodies and antibody fragments that immunospecifically bind to integrin α _v β ₃ and are longer than 3 days, longer than 7 days in animals, preferably mammals, and most preferably humans. Longer than 10 days, preferably longer than 15 days, longer than 25 days, longer than 30 days, longer than 35 days, longer than 40 days, longer than 45 days, longer than 2 months, longer than 3 months, longer than 4 months Provided are the above antibodies and antibody fragments having a half-life that is long or longer than 5 months.

  In order to prolong the in vivo serum circulation of antibodies (eg, monoclonal antibodies, and single chain antibodies) or antibody fragments (eg, Fab fragments), inert polymer molecules such as, for example, high molecular weight polyethylene glycol (PEG) Can be conjugated to the antibody with or without a multifunctional linker via a position-directed conjugation of PEG to the N or C terminus of the antibody or via the ε-amino group present on the lysine residue . Linear or branched polymer derivatization that minimizes loss of biological activity may be utilized. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation between the PEG molecule and the antibody. Unreacted PEG can be separated from antibody-PEG conjugates by size exclusion or ion exchange chromatography. PEG-derivatized antibodies or antibody fragments can be tested for binding activity as well as in vivo efficacy using methods known to those skilled in the art, for example, by the immunoassays described herein.

  Antibodies with increased in vivo half-life also have one or more amino acid modifications (ie substitutions, insertions or deletions) in the IgG constant domain or FcRn binding fragment thereof (preferably an Fc or hinge-Fc domain fragment). It can also be produced by introduction. See, eg, International Publication Nos. WO 98/23289 and WO 97/34631; and US Pat. No. 6,277,375, each of which is hereby incorporated by reference in its entirety.

  Furthermore, the antibody or antibody fragment can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half-life in vivo. The technique is well known in the art. See, for example, International Publications WO 93/15199, WO 93/15200, and WO 01/77137, and European Patent EP 413,622, each of which is incorporated herein by reference in its entirety.

5.1.1.3 Antibody Conjugates The present invention includes one or more moieties, such as, but not limited to, peptides, proteins, fusion proteins, nucleic acid molecules, small molecules, pseudo drugs, synthetic drugs, inorganic molecules, And the use of antibodies or antibody fragments conjugated to organic molecules and the like.

The present invention provides antibodies or fragments thereof to heterologous polypeptides or proteins (or fragments thereof, preferably at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70. An antibody or recombinantly fused or chemically conjugated (including covalent and non-covalent conjugation), a polypeptide of at least 80, at least 90 or at least 100 amino acids) Includes the use of antibody fragments. The fusion need not be direct, but can occur through a linker sequence. For example, an antibody or antibody fragment can be synthesized in a specific cell type either in vitro or in vivo by fusing or conjugating the antibody or antibody fragment with an antibody specific for a specific cell surface receptor. Peptides can be targeted. Antibodies or antibody fragments fused or conjugated to heterologous polypeptides can also be used in purification methods using in vitro immunoassays, methods known in the art. For example, International Publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett. 39: 91-99; US Pat. No. 5,474,981; -1432; Fell et al., 1991, J. Immunol. 146: 2446-2452, which are hereby incorporated by reference in their entirety.

The invention further includes a composition comprising a heterologous protein, peptide or polypeptide fused or conjugated to an antibody fragment. For example, the heterologous polypeptide is fused to an antigen-binding fragment of an antibody (eg, Fab fragment, Fd fragment, Fv fragment, F (ab) ₂ fragment, VH domain, VL domain, VH CDR, VL CDR, or fragment thereof) or Can be conjugated. Methods for fusing or conjugating polypeptides to antibodies or antibody fragments are known in the art. For example, US Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,477,851, and 5,112,946; European Patent EP307 , 434 and EP 367,166; International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154: 5590-5600; Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89: 11337-11341, which are incorporated by reference in their entirety.

For example, additional fusion proteins of VITAXIN® or anti-integrin α _v β ₃ antibodies or antibody fragments may be used for gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively “DNA shuffling”). It can be generated using the technique of). DNA shuffling can be used to alter the activity of antibodies of the invention (eg, antibodies or antibody fragments having higher affinity and lower dissociation rate). For review, US Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458, and Patten et al., 1997, Curr. Opinion. Biotechnol. 8: 724-33; Harayama, 1998, Trends Biotechnol. 16 (2): 76-82; Hansson et al., 1999, J. Mol. Biol. 287: 265-76; Lorenzo and Blasco, 1998, Biotechniques 24 ( 2): 308-313, the contents of which are incorporated herein by reference in their entirety. The antibody or fragment thereof, or the encoded antibody or fragment thereof can be altered by subjecting it to random mutagenesis by error-prone PCR, random nucleotide insertion, or other methods prior to recombination. One or more portions of a polynucleotide encoding an antibody or fragment thereof that immunospecifically binds to integrin α _V β ₃ , one or more components, motifs, sections, portions, domains of one or more heterologous molecules; Can be recombined with fragments.

  Furthermore, the antibody or antibody fragment can be fused to a marker sequence such as a peptide to facilitate purification. In certain embodiments, the marker amino acid sequence is a hexahistidine peptide, such as a tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among many commercially available. It is. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86: 821-824, for example, hexahistidine peptides are advantageous for purification of fusion proteins. Other peptide tags useful for purification include, but are not limited to, hemagglutinin “HA” tags (Wilson et al., 1984, Cell 37: 767) and “flag” tags corresponding to epitopes derived from influenza hemagglutinin proteins. Is mentioned.

In another embodiment, an antibody or antibody fragment of the present invention (including variants thereof) is conjugated to a diagnostic or detectable agent. Such antibodies may be used as part of a clinical trial procedure, such as determining the efficacy of a particular treatment, disorders, abnormalities associated with inflammatory diseases, autoimmune diseases, abnormal expression and / or activity of integrin α _v β _3. It may be useful to monitor or predict the onset, development, progression and / or severity of disorders associated with normal bone metabolism, disorders associated with abnormal angiogenesis, or cancer. Such diagnosis and detection includes, but is not limited to, various enzymes such as horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; but not limited to streptavidin / biotin, avidin / biotin, etc. Prosthetic groups; but not limited to umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; but not limited to luminol, etc. Non-limiting bioluminescent materials such as, but not limited to, luciferase, luciferin, and aequorin; but not limited to iodine ( ¹³¹ I, ¹²⁵ I, ¹²³ I, ^{1 21} I), carbon ( ¹⁴ C), sulfur ( ³⁵ S), tritium ( ³ H), indium ( ¹¹⁵ In, ¹¹³ In, ¹¹² In, ¹¹¹ In), and technetium ( ⁹⁹ Tc), thallium ( ²⁰¹ Ti), gallium ^{(68 Ga,} 67 ^Ga), palladium ⁽¹⁰³ Pd), molybdenum ⁽⁹⁹ Mo), xenon ⁽¹³³ Xe), fluorine ^{(18 F), 153 Sm,} 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb , ¹⁶⁶ Ho, ⁹⁰ Y, ⁴⁷ Sc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁴² Pr, ¹⁰⁵ Rh, ⁹⁷ Ru, ⁶⁸ Ge, ⁵⁷ Co, ⁶⁵ Zn, ⁸⁵ Sr, ³² P, ¹⁵³ Gd, ¹⁶⁹ Yb, ⁵¹ Cr, ⁵⁴ Radioactive materials such as Mn, ⁷⁵ Se, ¹¹³ Sn, and ¹¹⁷ Tin; various Detectable including, but not limited to, positron emitting metals, non-radioactive paramagnetic metal ions, and molecules radiolabeled or conjugated to specific radioisotopes using positron emission tomography of This can be accomplished by coupling an antibody or antibody fragment to the substance.

  The invention further encompasses the use of antibodies or antibody fragments conjugated to a therapeutic moiety. The antibody or antibody fragment can be coupled to a therapeutic moiety such as, for example, a cytotoxin such as a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion such as an alpha emitter. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples of these include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dioxyanthracindione, mitoxantrone, mitromycin, actino Included are mycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogs or homologues thereof. Therapeutic moieties include antimetabolites (eg, methotrexate, 6-mercaptopurine, and 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine), alkylating agents (eg, mecloetamine, thiotepa, chlorambucil, melphalan, carmustine ( BCNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine, platinum (II) (DDP), cisplatin); anthracyclines (eg, daunorubicin (formerly daunomycin)) Antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mitramycin, and anthramycin (AMC)); auristatin Children (eg, auristatin PHE, bryostatin 1, solastatin 10 (Woyke et al., Antimicrob. Agents Chemother. 46: 3802-8 (2002), Woyke et al., Antimicrob. Agents Chemother. 45: 3580-4 (2001), Mohammad Anticancer Drugs 12: 735-40 (2001), Wall et al., Biochem. Biophys. Res. Commun. 266: 76-80 (1999), Mohammad et al., Int. J. Oncol. 15: 367-72 (1999) All of which are hereby incorporated by reference); mitotic inhibitors (eg, vincristine and vinblastine); hormones (eg, glucocorticoids, progestatins, androgens, and estrogens); DNA repair enzyme inhibition Agents (eg, etoposide or topotecan); kinase inhibitors (eg, compound ST1571, imatinib mesylate (Kantarjian et al., Clin Cancer Res. 8 (7): 2167 76 (2002)), and US Pat. No. 6,245, 759, No. 6,399 No. 6,633, No. 6,383,790, No. 6,335,156, No. 6,271,242, No. 6,242,196, No. 6,218,410, No. 6,218,372 No. 6,057,300, No. 6,034,053, No. 5,985,877, No. 5,958,769, No. 5,925,376, No. 5,922,844, 5,911,995, 5,872,223, 5,863,904, 5,840,745, 5,728,868, 5,648,239, and Compounds disclosed in US Pat. No. 5,587,459), farnesyltransferase inhibitors (eg, R115777, BMS214662, and US Pat. Nos. 6,458,935, 6,451,812, 6,440,974). , Sixth 36,960, 6,432,959, 6,420,387, 6,414,145, 6,410,541, 6,410,539, 6,403, 581, No. 6,399,615, No. 6,387,905, No. 6,372,747, No. 6,369,034, No. 6,362,188, No. 6,342,765 No. 6,342,487, No. 6,300,501, No. 6,268,363, No. 6,265,422, No. 6,248,756, No. 6,239,140, 6,232,338, 6,228,865, 6,228,856, 6,225,322, 6,218,406, 6,211,193, 6, 187,786, 6,169,096, 6,159,984, 6,143,766, 6,133,303, 6,127,366, 6,124,465, 6,124,295, 6,103,723, 6, 093,737, 6,090,948, 6,080,870, 6,077,853, 6,071,935, 6,066,738, 6,063, 930, 6,054,466, 6,051,582, 6,051,574, and 6,040,305); topoisomerase inhibitors (eg, camptothecin, Irinotecan, SN38, topotecan, 9 aminocamptothecin, GG211 (GI 147111), DX 895lf, IST622, rubitecan, pyrazoloacridine, XR 5000, signtopin, U CE6, UCE1022, TAN 1518A, TAN1518B, KT6006, KT6528, ED110, NB506, ED110, NB506, Rebeccamycin, and Bulgarein), Hoechst dye 33342 and Hoechst dye 33258, nitidine, fagaronine ber, epiberineine ber ), Coralyne, betalapachone, BC-4-1, bisphosphonate (eg, alendronate, simadronate, clodronate, tiludronate, etidronate, ibandronate, neridronate, olpandronate, risedronate, pyridronate, pamidronate HMG-CoA reductase inhibitors (eg, lovastatin, simvastatin, atorvastatin) , Pravastatin, fluvastatin, statin, cerivastatin, rescol, lupitol, rosuvastatin and atorvastatin); antisense oligonucleotides (eg, US Pat. Nos. 6,277,832, 5,998,596, 5,885) 834, 5,734,033 and 5,618,709); adenosine deaminase inhibitors (eg, fludarabine phosphate and 2-chlorodeoxyadenosine); ibritumomab tiuxetane ( ibritumomab tiuxetan) (Zevalin®; tositumomab (BEXAR®)) and their pharmaceutically acceptable salts, solvates, inclusion compounds, and prodrugs.

  In addition, the antibody may be conjugated to a therapeutic or drug moiety that modifies a given biological response. The therapeutic or drug moiety should not be limited in interpretation to classic chemotherapeutic drugs. For example, the drug moiety may be a protein, polypeptide or peptide that retains the desired biological activity. Such proteins include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, or diphtheria toxin, TNF, alpha interferon, beta interferon, nerve growth factor, platelet derived growth factor, tissue plasminoid Genactivators, apoptotic drugs such as TNF-α, TNF-β, AIM I (see International Publication WO 97/33899), AIM II (see International Publication WO 97/34911), Fas ligand (Takahashi et al., 1994, J. Immunol., 6: 1567-1574), VEGF (see International Publication No. WO 99/23105), anti-angiogenic agents such as angiostatin, endostatin or a component of the coagulation pathway (eg tissue factor), or eg lymphokines (Eg, interleukin-γ (“IFN-γ”) in) -Leukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-5 ("IL-5"), interleukin-6 ("IL-6"), interleukin- 7 ("IL-7"), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 ("IL-15"), interleukin-23 ("IL-23 )), Granulocyte-macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”), or growth factors (eg, growth hormone (“GH”)) Or blood coagulant (eg, calcium, vitamin K, tissue factor, such as, but not limited to, Hageman factor (factor XII), high molecular weight kininogen HMWK), prekallikrein (PK), blood coagulation protein factor II (prothrombin), factor V, factor XIIa, factor VIII, factor XIIIa, factor XI, factor XIa, factor IX, factor IXa , Factor X, phospholipid, fibrin peptides A and B derived from α and β chains of fibrinogen, fibrin monomer).

In addition, radioactive metal ions, for example alpha emitters such as ²¹³ Bi, or radioactive metal ions including but not limited to ¹³¹ 1n, ¹³¹ LU, ¹³¹ Y, ¹³¹ Ho, ¹³¹ Sm, are bound to the polypeptide. The antibody or antibody fragment can be conjugated to a therapeutic moiety, such as a macrocyclic chelator useful to do so. In certain embodiments, the macrocyclic chelator is 1,4,7,10-tetraazacyclododecane-N, N, 'N ", N''' tetraacetic acid (which can be attached to the antibody via a linker). DOTA). Such linker moieties are generally known in the art and are described in Denardo et al., 1998, Clin Cancer Res. 4 (10): 2483-90; Peterson et al., 1999, Bioconjug. Chem. 10 (4): 553-7; and Zimmerman et al., 1999, Nucl. Med. Biol. 26 (8): 943-50. Each of which is incorporated herein by reference in its entirety.

  Techniques for attaching therapeutic moieties to antibodies or antibody fragments are well known, for example, Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, Monoclonal Antibodies And Cancer Therapy, Reisfeld et al., 243-56 (Alan R Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, Controlled Drug Delivery 2nd edition edited by Robinson et al., 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer. Therapy: A Review ", Monoclonal Antibodies 84: Biological And Clinical Applications, edited by Pinchera et al., 475-506 (1985);" Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ", Monoclonal Antibodies For Cancer See Detection And Therapy, Baldwin et al., 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol. Rev. 62: 119-58.

  Alternatively, an antibody or antibody fragment can be conjugated to a secondary antibody as described by Segal in US Pat. No. 4,676,980, which is hereby incorporated by reference in its entirety. Can also be formed.

  The antibodies and antibody fragments may also be bound to a solid support, which is particularly useful for immunoassay or purification of the target antigen. Such solid phase carriers include, but are not limited to glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

The therapeutic moiety or agent conjugated to an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ should be selected to achieve the desired prophylactic or therapeutic effect for the particular disorder in the subject. is there. In deciding which therapeutic moiety or drug to bind to an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ , the clinician or other medical professional determines the nature of the disease, the severity of the disease And the condition of the subject should be considered.

5.2. Method for Preparing Antibody Formulation The present invention provides a method for preparing a liquid formulation of an antibody that immunospecifically binds to integrin α _v β ₃ , or a derivative, analog or fragment thereof. FIG. 1 is a schematic diagram showing an outline of preparing a purified anti-integrin α _v β ₃ antibody. A method of preparing a liquid formulation of the invention comprises purifying an antibody or antibody fragment from a conditioned medium (either a single lot of culture or a pooled lot) and the antibody or antibody fragment. Fractions were obtained at final antibody concentrations of about 15 mg / ml, about 20 mg / ml, about 30 mg / ml, about 40 mg / ml, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg. / Ml, about 100 mg / ml, about 150 mg / ml, about 200 mg / ml, about 250 mg / ml, or about 300 mg / ml, and the concentrated antibody fraction using the same membrane as formulation buffer Diafiltration in. The conditioned medium containing an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is subjected to CUNO filtration, and the filtered antibody is subjected to HS50 cation exchange chromatography. Fractions from HS50 cation exchange chromatography are subsequently subjected to rProtein A affinity chromatography followed by low pH treatment. After low pH treatment, the antibody or antibody fragment fraction is subjected to super Q650 anion exchange chromatography followed by nanofiltration. The fraction of the antibody or antibody fragment obtained after nanofiltration is subsequently subjected to diafiltration to concentrate the fraction of the antibody or antibody fragment and use the same act as a formulation buffer.

  The formulation buffer of the present invention comprises histidine at a concentration of about 1 mM to about 100 mM, about 5 mM to about 50 mM, about 10 mM to about 30 mM, or about 10 mM to about 25 mM. In certain embodiments, the formulation buffer of the invention comprises histidine at a concentration of about 10 mM, about 12 mM, about 15 mM, about 20 mM, or about 25 mM. The formulation can further comprise glycine at a concentration of less than 150 mM, less than 100 mM, less than 75 mM, less than 50 mM, less than 10 mM, less than 3.0 mM, or less than 2.0 mM. The amount of glycine in the formulation should avoid significant buffering to avoid precipitation of the antibody at its isoelectric point. The pH of the formulation may range from about 5.0 to about 7.0, preferably from about 5.5 to about 6.5, more preferably from about 5.8 to about 6.2, and most preferably about 6.0. In order to obtain a pH appropriate for a particular antibody, first, histidine (and glycine if glycine is added) in water so that a buffered aqueous solution with a pH higher than the desired pH is obtained. It is preferred to dissolve and then add HCl to lower the pH to the desired value. In this way, formation of inorganic salts (for example, formation of NaCl when histidine hydrochloride is used as a histidine supply source and pH is raised to a desired value by adding NaOH) can be avoided.

The liquid formulations of the present invention can be prepared as unit dosage forms by preparing vials containing one aliquot of the liquid formulation for a single use. For example, a unit dose per vial is 1 ml of an antibody or antibody fragment that immunospecifically binds to different concentrations of integrin α _v β ₃ , ranging in concentration from about 15 mg / ml to about 300 mg / ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml may be contained. If necessary, these preparations can be adjusted to the desired concentration by adding sterile diluent to each vial.

The liquid preparation of the present invention can be sterilized by various sterilization methods including aseptic filtration and radiation irradiation. In the most preferred embodiment, the diafiltered antibody formulation is filter sterilized using a pre-sterilized 0.2 μm microfilter. The sterile liquid formulation of the present invention can be used to treat diseases or disorders (eg, inflammatory disorders, autoimmune disorders, disorders related to abnormal bone metabolism, disorders related to abnormal expression and / or activity of integrin α _v β ₃ , abnormalities). Angiogenesis-related disorder, or cancer), or one or more symptoms thereof, can be administered to a subject to prevent, treat, or ameliorate.

  It should be noted that although the present invention is directed to liquid formulations that are not lyophilized, for purposes of equivalents, the formulations of the present invention may be lyophilized if desired. Thus, although such lyophilized formulations are not preferred, the present invention also encompasses lyophilized forms of the formulations of the present invention.

5.3 Antibody Production Methods Antibodies and antibody fragments that immunospecifically bind to antigens are produced by any method known in the art for synthesizing antibodies, particularly by chemical synthesis or preferably by recombinant expression techniques. can do.

  Polyclonal antibodies immunospecific for the antigen can be produced by various methods known in the art. For example, human antigens can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for human antigens. Can do. Depending on the host species, various adjuvants may be used to increase the immune response, such as, but not limited to, Freund's adjuvant (complete and incomplete), aluminum hydroxide, etc. Surface active substances such as mineral gels, lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and BCG (bacille Calmette-Guerin) and Corynebacterium parvum ( potentially useful human adjuvants such as corynebacterium parvum). Such adjuvants are also known in the art.

  Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma technology, recombinant technology, and phage display technology, or combinations thereof. For example, monoclonal antibodies are known in the art and are described in, for example, Harlow et al., “Antibodies: A Laboratory Manual” (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al., “Monoclonal Antibodies and T-Cell. Hybridomas "563-681 (Elsevier, NY, 1981), and Harlow et al.," Using Antibodies: A Labolatory Manual "(Cold Spring Harbor Laboratory Press, 1999), which is incorporated by reference in its entirety. It can be produced using hybridoma technology including the method described above. The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody obtained from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method of making it.

  Methods for producing and screening for specific antibodies using hybridoma technology are routinely used and known in the art. Briefly, mice can be immunized with a non-mouse antigen and once an immune response is detected (eg, an antibody specific for that antigen is detected in mouse serum). ) Collect mouse spleen and isolate splenocytes. The splenocytes are then fused by known techniques with any suitable myeloma cells, for example, cells from cultured cell line SP20 available from ATCC. Hybridomas are selected and cloned by limiting dilution. In addition, RIMMS (repeated immunization multiple site) techniques may be used to immunize animals (Kilptrack et al., 1997, Hybridoma 16: 381-9, which is incorporated by reference in its entirety). The hybridoma clones are then assayed for cells secreting antibodies capable of binding to the polypeptides of the invention by methods known in the art. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

  The present invention provides a method for producing a monoclonal antibody by culturing a hybridoma cell that secretes the antibody of the present invention, and an antibody produced by the method. Preferably, the hybridoma in the above method is non- Spleen cells isolated from mice immunized with mouse antigen are fused with myeloma cells, and then the hybridoma obtained from the fusion is screened for hybridoma clones that secrete antibodies capable of binding to the antigen. It is what is done.

  Antibody fragments that recognize specific specific epitopes may be generated by any technique known to those of skill in the art. For example, the Fab and F (ab ′) 2 fragments of the present invention can be produced by using proteins such as papain (which produces Fab fragments) or pepsin (which produces F (ab ′) 2 fragments) It can be produced by cracking. The F (ab ') 2 fragment contains the variable region, the light chain constant region and the CH1 domain of the heavy chain. Furthermore, the antibodies of the present invention can also be generated using various phage display methods known in the art.

  In the phage display method, a functional antibody domain is displayed on the surface of a phage particle carrying a polynucleotide sequence encoding the domain. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (eg, human or mouse cDNA libraries of affected tissues). DNA encoding the VH and VL domains is recombined with the scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli to infect E. coli with helper phage. The phage used in these methods is typically a filamentous phage containing fd and M13, and the VH and VL domains are usually fused to either phage gene III or gene VIII by recombinant methods. Phage expressing an antigen binding domain that binds to a particular antigen can be selected or identified by the antigen, eg, using a labeled antigen or an antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the invention include Brinkman et al., 1995, J. Immunol. Methods 182: 41-50; Ames et al., 1995, J. Immunol. Methods 184: 177- Kettleborough et al., 1994, Eur. J. Immunol. 24: 952-958; Persic et al., 1997, Gene 187: 9-18; Burton et al., 1994, Advances in Immunology 57: 191-280; International Application No. PCT / GB 91/01134; International Publications WO 90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO 097/13844; and the United States Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5th 571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, 5,969, No. 108, No. 6,333,187, No. 5,824,529, No. 5,702,892, each of which is incorporated herein by reference in its entirety.

  As described in the above references, after phage selection, the antibody coding region from the phage is isolated and further used to make whole antibodies, including human antibodies, or any other desired antigen-binding fragment. They can then be expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Techniques for producing Fab, Fab ′ and F (ab ′) 2 fragments by recombinant methods are also described in International Publication No. WO 92/22324; Mullinax et al., 1992, BioTechniques 12 (6): 864-869; Sawai et al., 1995, Using methods known in the art, such as those disclosed in AJRI 34: 26-34; and Better et al., 1988, Science 240: 1041-1043 (the above references are incorporated by reference in their entirety), Can be used.

  Amplifying VH or VL sequences in scFv clones using PCR primers containing VH or VL nucleotide sequences, restriction enzyme cleavage sites, and flanking sequences that protect the restriction enzyme cleavage sites to generate complete antibodies Can do. Using cloning techniques known to those skilled in the art, the PCR amplified VH domain is cloned into a VH constant region, eg, a vector expressing a human γ4 constant region, and the PCR amplified VL domain is For example, it can be cloned into a vector expressing the human κ or λ constant region. Preferably, the vector expressing the VH or VL domain comprises a cloning site for a selectable marker such as the EF-1α promoter, secretion signal, variable domain, constant domain and neomycin. The VH and VL domains can also be cloned into a single vector that expresses the required constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into the cultured cell line and a stable, transient cultured cell line expressing full-length antibody, eg, IgG, using techniques known to those skilled in the art. Make it.

  For multiple uses, including in vivo use of antibodies in humans, and in vitro detection assays, it may be preferable to use humanized or chimeric antibodies. Completely human and humanized antibodies are particularly desirable for the treatment of human subjects. Human antibodies can be made by a variety of methods known in the art, including the phage display methods described above using antibody libraries derived from human immunoglobulin sequences. U.S. Pat. Nos. 4,444,887 and 4,716,111; and International Publications WO 98/46645, WO 98/50433, WO 98/24893, W098 / 16654, WO 96/34096, WO 96/33735 No. and WO 91/10741; each of which is incorporated herein by reference in its entirety.

  Human antibodies can also be produced using transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes may be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy and light chain genes, human variable regions, constant regions, and diversity regions may be introduced into mouse embryonic stem cells. The human immunoglobulin loci may be introduced by homologous recombination to deactivate the mouse heavy and light chain immunoglobulin genes separately or simultaneously. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are propagated and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized with the selected antigen, eg, all or part of a polypeptide of the invention, in the usual manner. Monoclonal antibodies against the antigen can be obtained from immunized transgenic mice using conventional hybridoma technology. Human immunoglobulin transgenes carried by the transgenic mice rearrange upon B cell differentiation and subsequently undergo class switching and somatic mutation. Therefore, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies using such techniques. For a review of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13: 65-93). For a detailed discussion of this technique for producing human and human monoclonal antibodies and protocols for producing such antibodies, see, eg, International Publication No. WO 98/24893, which is incorporated herein by reference in its entirety. WO96 / 34096, and WO96 / 33735; and US Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825, and 5,661. 016, 5,545,806, 5,814,318, and 5,939,598. In addition, companies such as Abgenix, Inc. (Freemont, CA) and Genpharm (San Jose, CA) can be contracted to provide human antibodies against selected antigens using techniques similar to those described above.

  A chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulins. Methods for producing chimeric antibodies are known in the art. For example, Morrison, 1985, Science 229: 1202; Oi et al., 1986, BioTechniques 4: 214; Gillies et al., 1989, J. Immunol. Methods 125: 191-202; and US Pat. No. 5,807,715, No. 4, 816,567, 4,816,397, and 6,331,415, which are incorporated herein by reference in their entirety.

A humanized antibody is an antibody or a variant thereof comprising a framework capable of binding to a predetermined antigen and having a substantially human immunoglobulin amino acid sequence and a CDR having a substantially non-human immunoglobulin amino acid sequence. Or a fragment thereof. Humanized antibodies comprise substantially at least one and typically two variable domains (Fab, Fab ′, F (ab ′) ₂ , Fabc, Fv), wherein all or substantially all of the CDR regions. All correspond to the CDR regions of a non-human immunoglobulin (ie donor antibody) and all or substantially all of the framework regions are framework regions of a human immunoglobulin consensus sequence. Preferably, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. Ordinarily, the antibody will contain both the light chain as well as at least the variable domain of a heavy chain. The antibody may also comprise the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. The humanized antibody may be selected from any class of immunoglobulins including IgM, IgG, IgD, IgA and IgE, and any isotype including IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ . Usually the constant domain is a complement fixing constant domain humanized antibodies are desired to exhibit cytotoxic activity, and the class is typically IgG _1. When such cytotoxic activity is not desirable, the constant domain is an IgG ₂ class. Humanized antibodies may contain sequences from one or more classes or isoforms, and the selection of specific constant domains to optimize the desired effector function is encompassed by ordinary skill in the art. The framework and CDR regions of a humanized antibody need not correspond exactly to the parent sequence; for example, the donor CDR or consensus framework is mutated by substitution, insertion or deletion of at least one residue. CDRs or framework residues in may not correspond to consensus or imported antibodies. But such mutations may not be widespread. Usually, humanized antibody residues that may correspond to residues of the parent framework and CDR sequences will be at least 75%, more often 90%, and most preferably greater than 95%. Humanized antibodies can be produced using various techniques known in the art, including but not limited to CDR grafts (European Patent EP239,400; International Publication WO91). And US Pat. Nos. 5,225,539, 5,530,101, 5,585,089), veneering or resurfacing (European Patent EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28 (4/5): 489-498; Studnicka et al., 1994, Protein Engineering 7 (6): 805-814; and Roguska et al., 1994, PNAS 91: 969-973), And chain shuffling (US Pat. No. 5,565,332) and, for example, US Pat. Nos. 6,407,213, 5,766,886, WO 93/17105, Tan et al., J. Immunol. 169: 1119-25 (2002), Caldas et al., Protein Eng. 13 (5): 353-60 (2000), Morea et al., Methods 20 (3): 267-79 (2000), Baca et al., J. Biol. Chem. 272 (16): 10678-84 (1997) Roguska et al., Protein Eng. 9 (10): 895-904 (1996), Couto et al., Cancer Res. 55 (23 Supp): 5973s-5977s (1995), Couto et al., Cancer Res. 55 (8): 1717-22 (1995), Sandhu JS, Gene 150 (2): 409-10 (1994), and Pedersen et al., J. Mol. Biol. 235 (3): 959-73 (1994). Can be mentioned. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions can be made by methods known in the art, for example, by modeling the interaction of CDRs with framework residues to identify framework residues important for antigen binding, and further by comparing sequences at specific positions. (See, for example, Queen et al., US Pat. No. 5,585,089, which is incorporated herein by reference in its entirety; and Riechmann et al., 1988, Nature 332). : 323).

  Single domain antibodies, eg, antibodies lacking the light chain, can be made by methods known in the art. Riechmann et al., 1999, J. Immuno. 231: 25-38; Nuttall et al., 2000, Curr. Pharm. Biotechnol. 1 (3): 253-263; Muylderman, 2001, J. Biotechnol. 74 (4): 277302; See US Pat. No. 6,005,079; and International Publication Nos. WO94 / 04678, WO94 / 25591, and WO01 / 44301, each incorporated herein by reference.

In addition, this time, antibodies that immunospecifically bind to an antigen (eg, integrin α _v β ₃ ) are used to generate anti-idiotypic antibodies that “mimic” the antigen using techniques known to those of skill in the art. (See, for example, Greenspan & Bona, 1989, FASEB J. 7 (5): 437-444; and Nissinoff, 1991, J. Immunol. 147 (8): 2429-2438).

5.3.1 Polynucleotide Sequence Encoding Antibody The present invention provides a polynucleotide comprising a nucleotide sequence encoding an antibody or antibody fragment that immunospecifically binds to an antigen. The present invention also provides a polynucleotide that hybridizes with a polynucleotide encoding an antibody of the present invention under high stringency, moderate or low stringency hybridization conditions, eg, as defined above. Include.

  A polynucleotide can be obtained and the nucleotide sequence of the polynucleotide determined by any method known in the art. The nucleotide sequence of an antibody immunospecific for a desired antigen can be obtained from, for example, literature or a database such as GenBank. Since the amino acid sequences of VITAXIN® are known, the nucleotide sequences encoding these antibodies can be converted using methods known in the art (ie, nucleotide codons known to encode specific amino acids). Can be determined (assembled to produce nucleic acid encoding the antibody). Such polynucleotides encoding antibodies can be assembled from chemically synthesized oligonucleotides (eg, as described in Kutmeier et al., 1994, BioTechniques 17: 242), which briefly describes the sequence encoding the antibody. With the synthesis of overlapping oligonucleotides containing a portion of these, annealing and ligation of these oligonucleotides, followed by PCR amplification of the ligated oligonucleotides.

  Alternatively, a polynucleotide encoding an antibody or antibody fragment can be made from nucleic acid from a suitable source. Even if a clone containing a nucleic acid encoding a specific antibody is not available, if the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized, or Expresses an antibody of appropriate origin (eg, an antibody cDNA library or a cDNA library made therefrom, a nucleic acid isolated therefrom, preferably poly A + RNA, a hybridoma cell selected to express an antibody of the invention) By PCR amplification using synthetic primers that hybridize to the 3 ′ and 5 ′ ends of the sequence from any tissue or cell) or using oligonucleotide probes specific for a particular gene sequence, eg, a cDNA library Identification and cloning of cDNA clone encoding the antibody from The Rukoto, can be obtained. Amplified nucleic acids generated by PCR can then be cloned into replicable cloning vectors using methods known in the art.

  Once the nucleotide sequence of an antibody or antibody fragment is determined, the nucleotide sequence of the antibody or antibody fragment can be converted into methods known in the art for genetic manipulation of nucleotide sequences, such as recombinant DNA techniques, site-directed mutation, PCR, etc. (Eg, Sambrook et al., 1990, “Molecular Cloning, A Laboratory Manual”, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., Ed., 1998, “Molecules. See, for example, techniques described in Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are incorporated herein by reference in their entirety. Thus, antibodies having different amino acid sequences can be generated, eg, creating amino acid substitutions, deletions, and / or insertions.

  In certain embodiments, one or more CDRs are inserted into the framework regions using conventional recombinant DNA techniques. The framework region may be a natural or consensus framework region, preferably a human framework region (see, eg, Chothia et al., 1998, J. Mol. Biol. 278 for a listing of human framework regions: 457-479). Preferably, a polynucleotide produced by a combination of framework regions and CDRs encodes an antibody that immunospecifically binds to a specific antigen. Preferably, as described above, one or more amino acid substitutions may occur within the framework region, and preferably the amino acid substitutions improve the binding of the antibody to its antigen. In addition, such methods may be used to generate antibody molecules that lack one or more intrachain disulfide bonds by causing amino acid substitution or deletion of one or more variable region cysteine residues that participate in intrachain disulfide bonds. . Other modifications to the polynucleotide are encompassed by the present invention and within the skill of the art.

5.3.2 Recombinant expression of antibodies Recombinant expression of the antibodies of the invention, derivatives, analogs or fragments thereof (eg heavy chain or light chain of antibodies of the invention, or single chain antibodies of the invention) This requires the construction of an expression vector containing the polynucleotide encoding the antibody. Once a polynucleotide is obtained that encodes an antibody molecule of the invention, an antibody heavy or light chain, or a portion thereof (preferably comprising, but not necessarily comprising, a heavy or light chain variable domain) Vectors for producing antibody molecules can be prepared by recombinant DNA techniques using techniques known in the art. See, for example, US Pat. No. 6,331,415, which is incorporated herein by reference in its entirety. Thus, a method for producing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody is described herein. Methods which are known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The present invention therefore encodes an antibody molecule of the invention, an antibody heavy or light chain, an antibody heavy or light chain variable domain or portion thereof, or a heavy or light chain CDR and functions as a promoter. A replicable vector comprising nucleotide sequences operably linked is provided. Such vectors may contain nucleotide sequences encoding the constant region of the antibody molecule (see, eg, International Publication No. WO86 / 05807; WO89 / 01036; and US Pat. No. 5,122,464) Variable domains may be cloned into such vectors to express the entire heavy chain, the entire light chain, or both the entire heavy chain and the entire light chain.

  The expression vector is introduced into a host cell by conventional techniques, and then the transfected cells are cultured by conventional techniques to produce the antibody of the present invention. Accordingly, the present invention relates to an antibody of the present invention or a fragment thereof, or a heavy chain or light chain thereof or a portion thereof, or a single chain antibody of the present invention, which is operably linked to a heterologous promoter. Host cells containing nucleotides. In a preferred embodiment for expressing a double chain antibody, a vector encoding both heavy and light chains is co-expressed in a host cell to express the entire immunoglobulin molecule, as described in detail below. Can do.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention (see, eg, US Pat. No. 5,807,715). Such a host expression system not only means a vehicle used to create and then purify the coding sequence of the invention, but also when transformed or transfected with an appropriate nucleotide coding sequence, the antibody molecule of the invention It also means cells that can be expressed in situ. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences (eg, E. coli and Bacillus subtilis); antibodies Yeast transformed with a recombinant yeast expression vector containing the coding sequence (eg, Saccharomyces pichia); insect cell line infected with a recombinant viral expression vector containing the antibody coding sequence (eg, baculovirus); Plant cell lines infected with a recombinant virus expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plasmid expression vector (eg, Ti plasmid) containing an antibody coding sequence Or mammal A mammalian cell line carrying a recombinant expression construct containing a promoter from the vesicle genome (eg, a metallothionein promoter) or a promoter from a mammalian virus (eg, adenovirus late promoter; vaccinia virus 7.5K promoter) (eg, , COS, CHO, BHK, 293, NSO, and 3T3 cells). Preferably, the recombinant antibody molecule is expressed using bacterial cells such as E. coli, and more preferably using eukaryotic cells, particularly to express the entire recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells are effective expression systems for antibodies when used in conjunction with vectors such as the major mid-early gene promoter element from human cytomegalovirus (Foecking et al., 1986, Gene 45: 101; and Cockett et al., 1990, Bio / Technology 8: 2). In certain embodiments, the expression of a nucleotide sequence encoding an antibody that immunospecifically binds to integrin α _v β ₃ is regulated by a constitutive promoter, inducible promoter or tissue specific promoter.

  In bacterial systems, a variety of expression vectors can be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when producing large quantities of such antibodies to produce pharmaceutical compositions of antibody molecules, vectors that direct high level expression of easily purified fusion protein products are desirable. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., 1983) in which antibody coding sequences can be individually ligated into the vector in frame with the lacZ coding region to produce a fusion protein. EMBO 12: 1791); pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13: 3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24: 5503-5509). A foreign polypeptide can also be expressed as a fusion protein with glutathione-5-transferase (GST) using the pGEX vector. In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to the matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors can be designed to contain thrombin or factor Xa protease cleavage sites to release the cloned target gene product from the GST moiety.

  In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be individually cloned into non-essential regions of the virus (eg, polyhedrin gene) and placed under the control of an AcNPV promoter (eg, polyhedrin promoter).

  In mammalian host cells, various virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated with an adenovirus transcription / translation control complex, eg, the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region (eg, region E1 or E3) of the viral genome can result in a recombinant virus that is viable in the infected host and can express antibody molecules (eg, Logan & Shenk, 1984, Proc Natl. Acad. Sci. USA 8 1: 355-359). A specific initiation signal may also be required for efficient translation of the inserted antibody coding sequence. These signals include the ATG initiation codon and adjacent sequences. In addition, the start codon must be synchronized with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. Expression efficiency can be enhanced by incorporating appropriate transcription enhancer elements, transcription terminators, etc. (see, eg, Bittner et al., 1987, Methods in Enzymol. 153: 51-544).

  In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (eg, glycosylation) and processing (eg, cleavage) of protein products can be important for the function of the protein. Different host cells have unique and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. For this purpose, host eukaryotic cells with cellular mechanisms for proper primary transcription processing, gene product glycosylation and phosphorylation can be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (internally completely immune Mouse myeloma cell lines that do not produce globulin chains), CRL7O3O, and HsS78Bst cells.

  Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, a cultured cell line that stably expresses an antibody molecule can be produced by genetic manipulation. Rather than using an expression vector containing the viral origin of replication, the host cell is used with DNA that is controlled by appropriate expression control elements (eg, promoters, enhancers, transcription terminators, polyadenylation sites, etc.) and selectable markers. Can be transformed. After the introduction of the foreign DNA, the genetically engineered cells may be grown in a rich medium for 1-2 days and then switched to a selective medium. The selectable marker in the recombinant plasmid confers resistance to selection and allows the cell to stably integrate the plasmid into its chromosome and proliferate to form a cell nest, which is then cloned. Can be expanded to cultured cell lines. Using this method, cell lines expressing antibody molecules can be advantageously produced by genetic manipulation. Cultured cell lines produced by such genetic manipulation are particularly useful for screening and evaluating compositions that interact directly or indirectly with antibody molecules.

  A variety of selection systems are available, including but not limited to herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 1992 , Proc. Natl. Acad. Sci. USA 48: 202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 8-17) genes, which are tk-, hgprt- or aprt-, respectively. Can be used in cells. In addition, antimetabolite resistance can be used as a basis for selection for the following genes: dhfr conferring methotrexate resistance (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt conferring resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); Aminoglycoside G-418 resistance Neo (Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32: 573-596; Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIB TECH 11 (5): 155-215); and hygro conferring hygromycin resistance (Santerre et al., 1984, Gene 30: 147). ). Recombinant DNA techniques known in the art can be routinely applied to select the desired recombinant clones, such as those described in Ausubel et al. (Ed.), “Current Protocols in Molecular Biology”, John Wiley & Sons, NY (1993); Kriegler, “Gene Transfer and Expression, A Laboratory Manual”, Stockton Press, NY (1990); and Dracopoli et al. (Ed.), “Current Protocols in Human Genetics”, John Wiley & Sons, Chapters 12 and 13 of NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150: 1, which are hereby incorporated by reference in their entirety.

  Antibody molecule expression levels can be increased by vector amplification (for review, see Bebbington and Hentschel, “The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning”, Vol. 3 (See Academic Press, New York, 1987). If the marker in the vector system expressing the antibody can be amplified, an increase in the level of inhibitor present in the host cell culture can increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody may also increase (Crouse et al., 1983, Mol. Cell. Biol. 3: 257).

  Host cells can be co-transfected with two expression vectors of the invention, a first vector encoding a heavy chain derived polypeptide and a second vector encoding a light chain derived polypeptide. . The two vectors may contain identical selectable markers that allow equivalent expression of heavy and light chain polypeptides. Alternatively, a single vector that encodes and expresses both heavy and light chain polypeptides may be utilized. In such situations, the light chain must be placed in front of the heavy chain to avoid an excess of toxic free heavy chains (Proudfoot, 1986, Nature 322: 52; and Kohler, 1980, Proc. Natl). Acad. Sci. USA 77: 2 197). The heavy and light chain coding sequences may include cDNA or genomic DNA.

  Once the antibody molecule of the invention is produced by recombinant expression, the antibody molecule can be obtained by any method known in the art for purifying immunoglobulin molecules, eg, by chromatography (eg, ion exchange, affinity Purification, in particular by affinity for specific antigens after protein A, and size separation column chromatography), by centrifugation, differential solubility, or by any other standard technique for purifying proteins. Can do. Furthermore, the antibodies of the invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

5.4. Methods for monitoring antibody formulation stability and aggregation Various methods for assessing protein formulation stability, including antibody formulations, are available, including methods based on the physical and chemical structure of the protein, There are methods based on biological activity. For example, methods such as charge transfer absorption, thermal analysis, fluorescence spectroscopy, circular dichroism, NMR, and HPSEC can be used to study protein denaturation. See, for example, Wang et al., 1988, J. of Parenteral Science & Technology 42 (supp): S4-S26.

  rCGE and HPSEC are the most common and simplest methods for assessing the formation of protein aggregation, proteolysis, and protein fragmentation. Therefore, the stability of the liquid preparation of the present invention can be evaluated by these methods.

For example, the stability of a liquid formulation of the invention can be assessed by HPSEC or rCGE, where the peak area occupancy represents an undegraded antibody or an undegraded antibody fragment. Specifically, about 250 μg of integrin α _v β ₃ immunospecifically bound antibody or antibody fragment (about 25 μl of a liquid preparation containing about 10 mg / ml antibody or antibody fragment) was added to a TASK SW x1 guard column (6. Inject onto a TOSOH TSK G3000SW _XL column (7.8 mm × 30 cm) attached to 0 mm × 4.0 cm). The antibody or antibody fragment elutes with 0.1 M disodium hydrogen phosphate containing 0.1 M sodium sulfate and 0.05% sodium azide at a flow rate of 0.8-1.0 ml / min. Eluting protein is detected using UV absorbance at 280 nm. The VITAXIN® standard reference sample was included in the assay as a control, and the results are relative to all other peaks except the volume peak observed at about 12-14 minutes as a percentage of the peak area exhibited by the product monomer. Shown in comparison. The peak eluting earlier than the monomer peak is recorded as the aggregation ratio.

  In the liquid preparation of the present invention, when measured by HPSEC or rCGE, the aggregation is not detectable to a low level, that is, the aggregation is 5% or less, 4% or less, 3% or less, 2% or less per protein weight. 1% or less, and most preferably 0.5% or less, and fragmentation is at an undetectable to low level, that is, the peak area representing the complete antibody or fragment thereof is 80% or more of the total peak area 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more, or 99.5% or more. In the case of SDS-PAGE, the density or radioactivity of each band labeled or stained with a radioisotope can be measured, and the% density or% radioactivity of the band representing the undegraded antibody or antibody fragment. Can be obtained.

The stability of the liquid formulation of the present invention can be assessed by any assay that measures the biological activity of the antibody or antibody fragment in the formulation. The biological activity of the antibody or antibody fragment includes, but is not limited to, antigen binding activity, complement activation activity, Fc receptor binding activity and the like. The antigen binding activity of an antibody or antibody fragment can be measured by any method known to those skilled in the art including, but not limited to, ELISA, radioimmunoassay, Western blot, and the like. Complement activation activity, in the presence of complement components, the integrin alpha _v beta ₃ that immunospecifically binds to an antibody or antibody fragment, in a system of reacting with a cell expressing the integrin alpha _v beta ₃ , By performing a C3a / C4a assay. See also Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd edition, 1988). (This document is incorporated herein by reference in its entirety). An ELISA-based assay can be used to compare the activity of an antibody or antibody fragment to immunospecifically bind to integrin α _v β ₃ with a VITAXIN® standard reference sample. In this assay, referred to as a VnR binding ELISA, the plate was coated with integrin α _v β ₃ isolated from human placenta and the binding signal exhibited by the set concentration of VITAXIN® standard reference sample was tested at the same concentration of test. Compare to the binding signal exhibited by the antibody or test antibody fragment.

  The purity of the antibody liquid formulation of the present invention may be measured by any method known to those skilled in the art, such as HPSEC. Sterilization of the antibody liquid preparation can be evaluated as follows. The test antibody liquid formulation is inoculated into sterile soy casein digest broth and liquid thioglycolate broth by filtering through a normal sterile filter with a 0.45 μm pore system. When using the Sterisure ™ or Steritest ™ method, each filter device is filled with approximately 100 ml of sterile soy casein digest broth or liquid thioglycolate broth by sterilization. When using conventional methods, the exposed filter is transferred by sterilization to 100 ml of sterile soy casein digest broth or liquid thioglycolate broth. The culture is incubated at the appropriate temperature and observed for evidence of bacterial or fungal growth three times over 14 days.

5.5 Prophylactic and therapeutic uses of antibody formulations The present invention also relates to inflammatory disorders, autoimmune disorders, abnormal expression of integrin α _v β ₃ and / or abnormal activity in a subject (preferably human). Administering a liquid antibody formulation of the present invention for preventing, treating, managing or alleviating disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer or symptoms associated therewith Relates to antibody-based therapy. The liquid preparation of the present invention comprises an antibody or fragment thereof in a solution containing histidine at a concentration of about 15 mg / ml to about 300 mg / ml, wherein the antibody or antibody fragment thereof is immunospecific for integrin α _v β _3. Join. The liquid formulation of the present invention may comprise a single antibody or a fragment thereof (eg, VITAXIN®) that immunospecifically binds to integrin α _v β ₃ . The liquid formulation of the present invention may also comprise two or more antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ . In certain embodiments, one of the antibodies or antibody fragments contained in such a liquid formulation is VITAXIN® or a fragment thereof. In another embodiment, one of the antibodies or antibody fragments contained in such a liquid formulation is not VITAXIN® or a fragment thereof. In yet another embodiment, a liquid formulation of the invention comprises an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ , wherein the antibody or antibody fragment is also a separate moiety (heterologous protein, peptide Or a polypeptide, another antibody or antibody fragment, marker sequence, diagnostic agent, therapeutic agent, radioactive metal ion and solid support, including but not limited to.

The liquid formulations of the present invention can be used locally or systemically in the body as a therapeutic agent. In particular, liquid formulations of the present invention can be used prevention of a disease or disorder associated with integrin alpha _V beta ₃ of aberrant expression and / or aberrant activity, therapeutic, in the management and mitigation. The formulations of the present invention can be used to modulate the activity of cells that express integrin α _v β ₃ . In certain embodiments, the formulations of the present invention are used to modulate various activities of the body including but not limited to angiogenesis, bone metabolism and immune function. The formulations of the present invention may also comprise one or more other treatments (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, abnormalities of integrin α _v β ₃ Treatment useful in the treatment, prevention, management or alleviation of disorders associated with expression and / or abnormal activity, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, cancer or one or more symptoms thereof Can be advantageously used in combination. Where one or more other treatments (eg, prophylactic or therapeutic agents) are used, they can be administered separately by any appropriate route in any suitable form.

The liquid formulation of the present invention provides a mammal (preferably human) with one or more other treatments (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders. Prevention of disorders associated with abnormal expression and / or abnormal activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, cancer or one or more symptoms thereof, It can be administered at the same time as a treatment useful in treatment, management or alleviation. The term “simultaneously” is not limited to the administration of exactly the same prophylactic or therapeutic / therapeutic, but rather this means that the liquid formulation of the present invention and the other agents / therapies are mammals within the same time interval. As a result, antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ contained in this liquid formulation provide increased benefits over when they are administered in other ways Therefore, it means that it can work together with other drugs / treatments. For example, liquid formulations of the present invention, as well as inflammatory disorders, autoimmune disorders, disorders associated with abnormal expression and / or abnormal activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, abnormal angiogenesis One or more other prophylactic or therapeutic agents useful for the prevention, treatment, management or alleviation of disorders or cancers associated with can be administered simultaneously or sequentially in any order at different times without delay Can be administered. However, if not administered at the same time, they should be administered sufficiently closely without delay to provide the desired therapeutic or prophylactic effect.

In various embodiments, the liquid formulations of the invention and one or more other therapies (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, integrin α _V Treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal expression and / or abnormal activity of β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer, Less than 1 hour interval, about 1 hour interval, about 1 hour to about 2 hour interval, about 2 hour to about 3 hour interval, about 3 hour to about 4 hour interval, about 4 hour to about 5 hour interval, about 5 hour to About 6 hours, about 6 hours to about 7 hours, about 7 hours to about 8 hours, about 8 hours to about 9 hours, about 9 hours to about 10 hours, about 10 hours to about 11 hours, About 11 hours to about 12 hours, 24 hours or less, or 48 hours or less Administered at intervals of In preferred embodiments, the liquid formulations of the invention and one or more other treatments (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, integrin α _V β Treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal expression and / or abnormal activity of ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer It is administered within the patient's visit. In other embodiments, the liquid formulations of the invention and one or more other therapies (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, integrin α _V Treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal expression and / or abnormal activity of β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer, It is administered at intervals of about 2 days to about 4 days, about 4 days to about 6 days, about 1 week, about 1 week to about 2 weeks, or more than 2 weeks. In preferred embodiments, the liquid formulations of the invention and one or more other treatments (eg, prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, aberrant expression of integrin α _v β ₃ and / or Or a disorder useful for the prevention, treatment, management or alleviation of disorders associated with abnormal activity, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer, and both agents are still active Administered in a time frame. One skilled in the art can determine such a time frame by determining the half-life of the administered drug.

In certain embodiments, the liquid formulations of the invention and one or more other therapies (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, integrin α _V Treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal expression and / or abnormal activity of β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer, The subject is administered periodically. Cyclic treatment includes administration of a first agent over a period of time, subsequent administration of a second agent and / or third agent over a period of time, and repeating this sequential administration. . Periodic treatment may reduce the development of resistance to one or more treatments, may avoid or reduce the side effects of one of these treatments, and / or improve the efficacy of this treatment.

In certain embodiments, the liquid formulations of the invention and one or more other therapies (eg, one or more other prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, integrin α _V Treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal expression and / or abnormal activity of β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer, It is administered in a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days, or about once every week. A cycle may include administration of a therapeutic or prophylactic agent by infusion over about 90 minutes per cycle, about 1 hour per cycle, and about 45 minutes per cycle. Each cycle may include a rest of at least 1 week, a rest of at least 2 weeks, a rest of at least about 3 weeks. The number of cycles administered is from about 1 cycle to about 12 cycles, more typically from about 2 cycles to about 10 cycles, and more typically from about 2 cycles to about 8 cycles.

In other embodiments, the liquid formulations of the invention and one or more other treatments (eg, prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, aberrant expression of integrin α _v β ₃ and Treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal activity, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, or cancer are continuous without prolonged rest periods It is administered in a metronomic manner, either by infusion or frequent administration. Such metronome-like administration can include dosing at regular intervals without a rest period. Typically, this prophylactic or therapeutic agent (especially a cytotoxic agent) is used at lower doses. Such dosing methods include chronic daily administration of relatively low doses over an extended period of time. In preferred embodiments, the use of lower doses can minimize toxic side effects and eliminate rest periods. In certain embodiments, the prophylactic and therapeutic agents are from about 24 hours to about 2 days, up to about 1 week, up to about 2 weeks, up to about 3 weeks, up to about 1 month, up to about 2 months, Delivered by chronic low dose or continuous infusion ranging from up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months.

In one embodiment, the liquid formulations of the present invention, the plasma concentration of immunospecific antibodies or antibody fragments against alpha _V beta _3, the desired level to continuously block the integrin alpha _V beta ₃ activity (e.g., From about 0.1 μg / ml to about 100 μg / ml). In certain embodiments, the plasma concentration of the antibody or antibody fragment is 0.2 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml. ml, 7 μg / ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml or 50 μg / ml Maintained. The desired plasma concentration in a subject will vary depending on a number of factors including, but not limited to, the nature of the disease or disorder, the severity of the disease or disorder, and the condition of the subject. Such a method of administration is particularly beneficial in the prevention, treatment, management and alleviation of chronic diseases or disorders.

In one embodiment, the liquid formulation α _V β ₃ of the present invention has a plasma concentration of an antibody or antibody fragment that immunospecifically binds to integrin α _V β ₃ at least 40%, preferably at least 50% of bone resorption, Abnormal using a dosing method that maintains a level that blocks at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% Administered to a subject having a disease or disorder associated with normal bone metabolism. In certain embodiments, the plasma concentration of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is about 0.1 μg / ml in a subject having a disease or disorder associated with abnormal bone metabolism. Maintained at about 100 μg / ml.

  In some embodiments, a liquid formulation of the invention is intermittently administered to a subject, wherein the liquid formulation comprises an antibody or antibody fragment conjugated to a moiety (eg, a therapeutic agent or toxin).

Prevention of inflammatory disorders, autoimmune disorders, disorders associated with abnormal expression and / or abnormal activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer, When used in combination with other treatments (eg, prophylactic and / or therapeutic agents) useful for treatment, management or alleviation, the liquid formulations and other treatments of the present invention may be additive or more preferably Can act synergistically. The invention relates to other treatments (eg prophylactic or therapeutic agents), preferably inflammatory disorders, autoimmune disorders, integrin α _v β ₃ , by the same route of administration or different routes of administration (eg oral and parenteral). The present invention combined with treatments useful for the prevention, treatment, management or alleviation of disorders associated with abnormal expression and / or abnormal activity, disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis or cancer Administration of a liquid formulation. In certain embodiments, a liquid formulation of the invention is administered concurrently with one or more treatments (eg, prophylactic or therapeutic agents) that potentially cause adverse side effects, including but not limited to toxicity. If so, these treatments (eg, prophylactic or therapeutic agents) can be advantageously administered at doses that fall below a threshold at which adverse side effects are eliminated.

5.5.1. Treatment of Disorders Associated With Abnormal Angiogenesis The liquid formulation of the present invention requires it to prevent, treat, manage or alleviate a disease or disorder associated with abnormal angiogenesis or one or more symptoms thereof. Can be administered to a subject. The liquid formulations of the present invention may also include one or more treatments (eg, prophylactic or therapeutic agents) to prevent, treat, manage or alleviate a disease or disorder associated with abnormal angiogenesis or one or more symptoms thereof. Can be administered to a subject in need thereof. Non-limiting examples of such treatments include anti-inflammatory agents (eg, non-steroidal anti-inflammatory and steroidal agents), bisphosphonates, HMG-CoA reductase inhibitors, immunomodulators and anti-angiogenesis. Including but not limited to agents. In certain embodiments, a subject to whom a liquid formulation of the invention is administered alone or in combination with another treatment (eg, a prophylactic or therapeutic agent) is a conventional disorder for a particular disorder associated with abnormal angiogenesis. Refractory to treatment.

In certain embodiments, the present invention provides a method of preventing, managing, treating or alleviating a disorder associated with abnormal angiogenesis or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. Administering a dose of the liquid formulation of the invention to a subject in need thereof. In another embodiment, the invention provides a method of preventing, managing, treating or alleviating a disorder associated with abnormal angiogenesis or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. The dosage of the liquid formulation of the present invention and one or more treatments other than antibodies or antibody fragments that immunospecifically bind to a prophylactically or therapeutically effective amount of integrin α _v β ₃ (eg, prophylactic or therapeutic agents) Administering to a subject in need thereof.

The liquid formulations of the present invention can be used as the first, second, third or fourth line of treatment for disorders associated with abnormal angiogenesis. The present invention provides a method for managing, treating or alleviating a disorder associated with abnormal angiogenesis or one or more symptoms thereof in a subject refractory to conventional treatment for such diseases. The method includes the step of administering to the subject a dose of a liquid formulation of the invention in a prophylactically or therapeutically effective amount. The invention also provides for managing, treating or alleviating a disorder associated with abnormal angiogenesis or one or more symptoms thereof in a subject refractory to existing single drug treatments for such diseases. Wherein the antibody or antibody binds immunospecifically to a prophylactically or therapeutically effective amount of a liquid formulation of the present invention and a prophylactically or therapeutically effective integrin α _v β ₃ Administering to the subject a dose of one or more treatments (eg, prophylactic or therapeutic agents) other than the fragment. The present invention also relates to abnormal angiogenesis where it can be demonstrated or demonstrated that conventional treatment is too toxic for the subject being treated (ie, produces unacceptable or intolerable side effects). Provide alternative methods for the management or treatment of disorders. Furthermore, the present invention provides a method for preventing the recurrence of disorders associated with abnormal angiogenesis in patients who are being treated by administering a liquid formulation or have no disease activity. In certain embodiments, a subject to whom a liquid formulation of the invention is administered alone or in combination with another treatment (eg, a prophylactic or therapeutic agent) is associated with a particular disease or disorder associated with abnormal angiogenesis. Refractory to conventional treatment.

  Diseases or disorders associated with abnormal angiogenesis include, but are not limited to: neoplastic diseases (non-limiting examples are tumor metastasis and leukemia); ocular angiogenesis diseases (non- Limited examples include age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity, vascular restenosis); skin diseases (non-limiting examples include infantile hemangiomas, common warts, psoriasis) , Basal and squamous cell carcinomas, cutaneous melanoma, capoji sarcoma, neurofibromatosis, recessive dystrophic epidermolysis bullosa); arthritis (non-limiting examples include rheumatoid arthritis, ankylosing spondylitis Systemic lupus erythematosus, psoriatic arthropathy, Reiter's syndrome and Sjogren's syndrome); gynecological diseases (non-limiting examples include endometriosis, preeclampsia during pregnancy, Nest, a carcinoma of the endometrium and neck); and cardiovascular disease (non-limiting examples, forms of atherosclerosis plaques, atherosclerosis and coronary artery disease).

5.5.2. Treatment of Disorders Related to Abnormal Bone Metabolism The liquid formulation of the present invention regulates bone metabolism or prevents, treats, manages or alleviates a disease or disorder related to abnormal bone metabolism or one or more symptoms thereof. Can be used to The liquid formulations of the present invention may also include one or more treatments (eg, prophylactic or therapeutic agents) to prevent, treat, manage or alleviate a disease or disorder associated with abnormal bone metabolism or one or more symptoms thereof. Can be administered to a subject in need thereof. Non-limiting examples of such treatments include, but are not limited to, those listed in Section 5.5.2.1 below. In certain embodiments, the present invention provides a method for preventing, managing, treating or alleviating a disorder associated with abnormal bone metabolism or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. Administering a dose of the liquid formulation of the invention to a subject in need thereof. In another embodiment, the invention provides a method for preventing, managing, treating or alleviating a disorder associated with abnormal bone metabolism or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. The dosage of the liquid formulation of the present invention and one or more treatments other than antibodies or antibody fragments that immunospecifically bind to a prophylactically or therapeutically effective amount of integrin α _v β ₃ (eg, prophylactic or therapeutic agents) Administering to a subject in need thereof.

The liquid formulations of the present invention can be used as a first, second, third or fourth line of treatment for disorders associated with abnormal bone metabolism. The present invention provides a method for managing, treating or alleviating a disorder associated with abnormal bone resorption or one or more symptoms thereof in a subject refractory to conventional treatment for such diseases. The method includes the step of administering to the subject a dose of a liquid formulation of the invention in a prophylactically or therapeutically effective amount. The present invention also provides for managing, treating or alleviating a disorder associated with abnormal bone metabolism or one or more symptoms thereof in a subject refractory to existing single drug treatments for such diseases. provides methods, the method, a prophylactically or therapeutically effective amount of dosage of a liquid formulation of the present invention, and prophylactically or therapeutically effective amount of an integrin alpha _V beta ₃ antibody or immunospecifically bind its Administering a dose of one or more treatments (eg, prophylactic or therapeutic agents) other than the fragment to the subject. The present invention also relates to abnormal bone metabolism, where conventional treatment has proven or can prove to be too toxic for the subject being treated (ie, produces unacceptable or intolerable side effects). Provide alternative methods for the management or treatment of disorders. Furthermore, the present invention provides a method for preventing the recurrence of disorders associated with abnormal bone metabolism in patients who are being treated by administering a liquid formulation or have no disease activity. In certain embodiments, a subject to whom a liquid formulation of the invention is administered alone or in combination with another treatment (eg, a prophylactic or therapeutic agent) is associated with a particular disease or disorder associated with abnormal bone metabolism. Refractory to conventional treatment. As used herein, the term “abnormal bone metabolism” is used interchangeably with “unusual bone metabolism” and deviates from its normal process (eg, bone tissue resorption and Abnormal growth of bone cells, but not limited thereto).

  Diseases or disorders associated with abnormal bone metabolism include, but are not limited to: rickets and osteomalacia; primary hyperparathyroidism (eg, solitary adenoma, multiple endocrine adenoma), Lithium treatment, familial hypocalciuria hypercalcemia, solid tumors with metastases (eg breast cancer), solid tumors with humoral mediation of hypercalcemia (eg lung or kidney cancer), hematological malignancy Tumors (eg, multiple myeloma, lymphoma, leukemia), vitamin D poisoning, sarcoidosis and other granulomatous diseases, idiopathic hypercalcemia of newborns, hyperthyroidism, vitamin A poisoning, aluminum poisoning, Hypercalcemia that can be caused by, but not limited to, milk-alkaline syndrome and renal failure; hereditary hypoparathyroidism, acquired hypoparathyroidism Hypocalcemia, which can be caused by chronic renal failure, vitamin D deficiency, oncolysis, rhabdomyolysis and fibro-osteitis after parathyroidectomy; but not limited to; osteoporosis; generalization in adults Related to increased risk of osteoporosis (Turner syndrome, Klinefelter syndrome, anorexia nervosa, hypothalamic amenorrhea, hyperprolactinemia, primary or secondary hypogonadal status , Cushing syndrome, hyperparathyroidism, thyroid poisoning, insulin-dependent diabetes, acromegaly, adrenal insufficiency, malnutrition, parenteral nutrition, malabsorption syndrome, gastrectomy, severe liver disease, Pernicious anemia, rheumatoid arthritis, ankylosing spondylitis, chronic otitis media (bone resorption induced by cholesteatoma), hypertrophic pulmonary osteoarthritis (HPOA), go Lamb-Stout disease, multiple myeloma, lymphoma and leukemia, malignant tumor-related parathyroid hormone-related production, mastocytosis, hemophilia, thalassemia, osteogenesis imperfecta, Marfan syndrome, hemochromatosis, hypophosphatasia, Includes glycogenosis, homocystinuria, Ehrers-Danlos syndrome, porphyria, Menkes disease, epidermolysis bullosa, chronic obstructive pulmonary disease, scoliosis, multiple sclerosis, sarcoidosis and amyloidosis Drug-related that can be caused by, but not limited to, glucocorticoids, cyclosporine, cytotoxic drugs, anticonvulsants, excessive thyroxine, aluminum, gonadotropin-releasing hormone agonist, heparin and lithium Osteoporosis; Paget's disease of bone; Marble bone disease (Albers-Sc) Oenberg bone disease); Concentrated dystrophy; Osteomyelosclerosis; Hereditary hyperphosphatasia; Progressive bone dysplasia (Camurati-Engelmann disease); Melorheostosis; (Osteopoikilosis); hyperostosis frontalis interna; fibrous osteodysplasia (Mccune-Albright syndrome); vertebral epithelial dysplasia; cartilage dysplasia; endochondromatosis; and osteochondromatosis .

The liquid formulations of the present invention can also be administered to a subject in need thereof to prevent, treat, manage or alleviate periodontal disease or one or more symptoms thereof. Periodontal diseases include, but are not limited to, gingivitis and periodontitis. The liquid formulations of the present invention may also be combined with one or more treatments (eg, prophylactic or therapeutic agents) to prevent, manage, treat or alleviate periodontal disease or one or more symptoms thereof. It can be administered to a subject in need. Non-limiting examples of such treatments include, but are not limited to: Mistoral II, folic acid, green tea extract, aloe vera gel, hachipropolis, vitamin K1, vitamin E, chamomile, coenzyme Q10, oral hygiene Improvement, antibiotics (eg, PERIOSTAT ™ (doxycycline hyclate), CO ₂ laser treatment, calculus removal, root surface smoothing and surgery. In certain embodiments, the invention may be used to treat periodontal disease or its A method is provided for preventing, managing, treating or alleviating one or more symptoms, the method comprising administering to a subject in need thereof a dose of a prophylactically effective or therapeutically effective amount of a liquid formulation of the invention. In another embodiment, the present invention provides a method for preventing, managing, treating or alleviating periodontal disease or one or more symptoms thereof, the method comprising: One or more treatments other than antibodies or antibody fragments that immunospecifically bind to a prophylactically effective or therapeutically effective amount of a liquid formulation of the invention and a prophylactically effective or therapeutically effective integrin α _v β ₃ Administering a dose of (eg, a prophylactic or therapeutic agent) to a subject in need thereof.

The liquid formulation of the present invention can be used as a first, second, third or fourth line of treatment for periodontal disease. The present invention provides a method for managing, treating or alleviating periodontal disease or one or more symptoms thereof in a subject refractory to conventional treatment for such disease, Administering to the subject a dose of a liquid formulation of the invention in a prophylactically or therapeutically effective amount. The present invention also provides a method for managing, treating or alleviating periodontal disease or one or more symptoms thereof in a subject refractory to existing single drug treatments for such diseases. this method, a prophylactically or therapeutically effective amount of dosage of a liquid formulation of the present invention, and prophylactically or therapeutically effective amount of an integrin alpha _V beta ₃ antibodies that immunospecifically bind to or one other than antibody fragments Or administering a dose of a plurality of treatments (eg, prophylactic or therapeutic agents) to the subject. The present invention also provides for the management of periodontal disease, where it can prove or prove that conventional treatment is too toxic for the subject being treated (i.e., produces unacceptable or intolerable side effects) or Provide alternative methods for treatment. Furthermore, the present invention provides a method for preventing recurrence of periodontal disease in a patient who is treated by administering a liquid formulation and has no disease activity.

The liquid formulations of the invention are also administered to a subject in need thereof to prevent, treat, manage or alleviate aseptic relaxation of one or more joint replacements (eg, hip or knee replacement) or one or more symptoms thereof. can do. The liquid formulations of the present invention may also be combined with one or more treatments (eg, prophylactic or therapeutic agents) to prevent, manage, treat or alleviate aseptic relaxation of joint replacement or one or more symptoms thereof. Can be administered to a subject in need thereof. Non-limiting examples of such treatments include, but are not limited to, anti-inflammatory agents, bisphosphonates, vitamin D compounds, anticoagulants, surgery and physical treatment. In certain embodiments, the present invention provides a method for preventing, managing, treating or alleviating aseptic relaxation of joint replacement or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount of the present invention. Administering a dose of the liquid formulation to a subject in need thereof. In another embodiment, the present invention provides a method for preventing, managing, treating or alleviating aseptic relaxation of joint replacement or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount of the present invention. And a dosage of one or more treatments (eg, prophylactic or therapeutic agents) other than an antibody or antibody fragment that immunospecifically binds to a prophylactically effective amount or a therapeutically effective amount of integrin α _v β ₃ Administering to a subject in need thereof.

The liquid formulations of the present invention can be used as the first, second, third or fourth line of treatment for aseptic relaxation of joint replacement. The present invention provides a method for managing, treating or alleviating aseptic relaxation of joint replacement or one or more symptoms thereof in a subject refractory to conventional treatment for such diseases. The method includes administering to the subject a dose of a liquid formulation of the invention in a prophylactically or therapeutically effective amount. The invention also provides a method for managing, treating or alleviating aseptic relaxation of joint replacement or one or more symptoms thereof in a subject refractory to existing single drug treatments for such diseases. The method comprises a prophylactically effective or therapeutically effective amount of a liquid formulation of the invention, and an antibody or antibody fragment that immunospecifically binds to a prophylactically effective or therapeutically effective integrin α _v β ₃ Administering a dose of one or more treatments (eg, prophylactic or therapeutic agents) to the subject. The present invention also provides for aseptic relaxation of joint replacement where it has been proven or can prove that conventional treatment is too toxic for the subject being treated (ie, produces unacceptable or intolerable side effects). Provide alternative methods for the management or treatment of Furthermore, the present invention provides a method for preventing the recurrence of aseptic relaxation of joint replacement in patients who are treated by administering a liquid formulation and have no disease activity.

The liquid formulations of the invention can be administered to a subject in need thereof to prevent, treat, manage or alleviate a disorder associated with abnormal bone resorption or one or more symptoms thereof. The liquid formulations of the present invention also include one or more treatments (eg, prophylactic or therapeutic agents) to prevent, manage, treat or alleviate disorders associated with abnormal bone resorption or one or more symptoms thereof. In combination, it can be administered to a subject in need thereof. Non-limiting examples of such treatments include sex hormones (eg, estrogen), bisphosphonates (eg, alendronate, etidronate, clodronate, ibandronate, pamidronate, risedronate, tiludronate and zoledronate), calcitonin And exercise programs. In certain embodiments, the invention provides a method for preventing, managing, treating or alleviating a disorder associated with abnormal bone resorption or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. Administering a dose of the liquid formulation of the invention to a subject in need thereof. In another embodiment, the present invention provides a method for preventing, managing, treating or alleviating a disorder associated with abnormal bone resorption or one or more symptoms thereof, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. The dosage of the liquid formulation of the present invention and one or more treatments other than antibodies or antibody fragments that immunospecifically bind to a prophylactically or therapeutically effective amount of integrin α _v β ₃ (eg, prophylactic or therapeutic agents) Administering to a subject in need thereof.

The liquid formulations of the present invention can be used as the first, second, third or fourth line of treatment for disorders associated with abnormal bone resorption. The present invention provides a method for managing, treating or alleviating a disorder associated with abnormal bone resorption or one or more symptoms thereof in a subject refractory to conventional treatment for such diseases. The method includes the step of administering to the subject a dose of a liquid formulation of the invention in a prophylactically or therapeutically effective amount. The present invention also provides for managing, treating or alleviating a disorder associated with abnormal bone resorption or one or more symptoms thereof in a subject refractory to existing single drug treatments for such diseases. provides methods, this method is prophylactically or therapeutically effective amount of dosage of a liquid formulation of the present invention, and prophylactically or therapeutically effective amount of an integrin alpha _V beta ₃ antibody or immunospecifically bind its Administering a dose of one or more treatments (eg, prophylactic or therapeutic agents) other than the fragment to the subject. The present invention also relates to abnormal bone resorption where conventional treatment has proven or proved to be too toxic for the subject being treated (ie, produces unacceptable or intolerable side effects). Provide alternative methods for the management or treatment of disorders. Furthermore, the present invention provides a method for preventing the recurrence of disorders associated with abnormal bone resorption in patients who are being treated by administering a liquid formulation or have no disease activity.

  Disorders associated with abnormal bone resorption include but are not limited to: Parathyroid-related disorders (non-limiting examples include primary hyperparathyroidism, lithium treatment and familial hypocalciuria Hypercalcemia); malignant tumor-related disorders (non-limiting examples are solid tumors with metastases, solid tumors with humoral mediation of hypercalcemia and hematological malignancies); vitamin D Related disorders (non-limiting examples are vitamin D addiction, sarcoidosis and other granulomatous diseases, idiopathic hypercalcemia of the newborn); and other diseases or disorders associated with high bone turnover (Non-limiting examples are hyperthyroidism, immobilization, thiazide and vitamin A addiction).

5.5.2.1. Agents for use in regulating bone metabolism The present invention provides a method for preventing, treating, managing or alleviating a disease or disorder associated with abnormal bone metabolism or one or more symptoms thereof, the method comprising: One or more treatments (eg, prophylactic or therapeutic agents) other than an antibody or antibody fragment that immunospecifically binds to a liquid formulation of the invention and integrin α _v β ₃ are administered to a subject in need thereof Includes steps. Therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules. Any drug or treatment known or used for or useful for modulating bone metabolism is in accordance with the invention described herein. It can be used in combination with the liquid preparation of the present invention. Examples of such drugs or treatments include but are not limited to: phosphate, aluminum hydroxide, aluminum carbonate gel, magnesium, vitamin D, calcitriol, vitamin D ₂ (ergocalciferol), vitamin D ₃ (Cholecalciferol), calcium, lithium, glucocorticoid, bisphosphonate or pharmaceutically acceptable salt or ester thereof (non-limiting examples include alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, Tiludronate and zoledronate), calcitonin, pricamycin (mitromycin), gallium nitrate, estrogen, progestin, estrogen antagonist (eg tamoxifen), estrogen Receptor modulator, androgen receptor modulator, cytotoxic agent or antiproliferative agent, matrix metalloproteinase inhibitor, epidermal derived growth factor, fibroblast derived growth factor or platelet derived growth factor inhibitor, VEGF inhibitor, growth factor or growth factor Antibodies to receptors, inhibitors of Flk-1 / KDR, Flt-1, Tck / Tie-2 or Tie-1, cathepsin K inhibitors, inhibitors of osteoclast proton ATPase, inhibitors of urokinase plasminogen activator (u-PA), tumors Specific antibody-interleukin-2 fusion protein, inhibitor of HMG-CoA reductase (non-limiting examples include prenylation inhibitors (non-limiting examples include lovastatin, prava Tachin, fluvastatin, statin, simvastatin, cerivastatin, rescol, lipitol, lovastatin and atorvastatin), farnesyl transferase inhibitor, geranylgeranyl transferase inhibitor or dual farnesyl / geranylgeranyl transferase inhibitor), parathyroid hormone or parathyroid hormone fragment (A non-limiting example is exogenous PTH analog, 1-34 PTH), growth hormone, molecule disclosed in US Pat. Nos. 6,472,402 and 6,482,411, renal dialysis , Surgery, or a combination thereof.

  Treatments for disorders associated with abnormal bone metabolism and their dosage, route of administration and recommended usage are known in the art, such as the Physician's Desk Reference (56th edition, 2002 and 57th edition, 2003) In the literature.

5.5.3. Cancer Treatment The liquid formulations of the present invention can be administered to a subject in need thereof to prevent, treat, manage or alleviate cancer or one or more symptoms thereof. The liquid formulations of the present invention may also contain one or more treatments (preferably treatments useful for the prevention, management or treatment of cancer (to prevent, treat, manage or alleviate cancer or one or more symptoms thereof). For example, without limitation, it may be administered to a subject in need thereof in combination with a prophylactic or therapeutic agent as exemplified in Section 5.5.3.1 below))). In certain embodiments, the present invention provides a method of preventing, managing, treating or alleviating cancer or one or more symptoms thereof, the method comprising a prophylactically effective or therapeutically effective dose of a liquid formulation of the present invention. Administering to a subject in need thereof. In another embodiment, the present invention provides a method for preventing, managing, treating or alleviating cancer or one or more symptoms thereof, wherein the method comprises a prophylactically effective or therapeutically effective dose of a liquid formulation of the present invention. And a dose of one or more treatments (eg, a prophylactic or therapeutic agent) other than an antibody or antibody fragment that immunospecifically binds to a prophylactically effective or therapeutically effective amount of integrin α _v β ₃ Administering to a subject.

  The liquid formulation of the present invention can be used as the first, second, third or fourth line of cancer treatment. The present invention provides a method for treating or alleviating cancer or one or more symptoms thereof in a subject refractory to conventional treatment for cancer, wherein the method comprises a prophylactically effective amount or a therapeutically effective amount. Administering a dose of the liquid formulation of the present invention to the subject. A cancer can be determined to be refractory to a therapeutic measure if at least some significant portion of the cancer cells have not died or their cell division has stopped in response to treatment. Such a determination is any method known in the art for assaying the effectiveness of a treatment against cancer cells, using the art accepted meaning for “refractory” in such cases. Can be performed either in vivo or in vitro. In certain embodiments, the cancer is refractory when the number of cancer cells does not significantly decrease or increase.

The present invention provides a method for managing, treating or ameliorating cancer or one or more symptoms thereof in a subject refractory to an existing single drug treatment for cancer, the method comprising: One or more treatments other than antibodies or antibody fragments that immunospecifically bind to an effective or therapeutically effective amount of a liquid formulation of the invention and a prophylactically or therapeutically effective amount of integrin α _v β ₃ (eg A dose of a prophylactic or therapeutic agent) to the subject. The present invention also provides a liquid formulation of the present invention in combination with other treatments (eg, radiation therapy, chemotherapy or surgery) for patients who are refractory to other treatments and are no longer taking these treatments. A method for managing, treating, or ameliorating cancer by administration is provided. The present invention also provides a method of managing or treating cancer patients who are immunosuppressed because they have previously received other cancer therapies. The present invention has demonstrated that chemotherapy, radiation therapy, hormonal therapy and / or biological therapy / immunotherapy is toxic (ie, produces unacceptable or intolerable side effects) for the subject being treated. Another method for the management, treatment or amelioration of cancer or one or more symptoms thereof is provided. Furthermore, the present invention provides a method for preventing recurrence of cancer in a patient who has been treated by administering a liquid formulation of the present invention or has no disease activity.

Cancers that can be treated by the methods of the present invention include, but are not limited to: any disease or disorder characterized by neoplasia, tumor, metastasis, or uncontrolled cell growth. The cancer can be primary or metastatic cancer. Cancer may not to or expressed may express integrin α _v β _3. In a preferred embodiment, the cancer that is to be managed, treated or ameliorated according to the methods of the present invention is a cancer that has metastasized to bone and expresses integrin α _v β ₃ . Examples of cancer that can be treated by the method according to the present invention include, but are not limited to, the head, neck, eyes, mouth, tongue, esophagus, chest, bone, lung, large intestine, rectum, preposition gland, Examples include breast, ovary, kidney, liver, pancreas and brain. Additional cancers include, but are not limited to: leukemia, such as, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia such as myeloblastic, promyelocytic Spherical, myelomonocytic, monocytic, erytroleukemia leikemias and spinal dysplasia symptoms; chronic leukemia, including but not limited to chronic myelocytic (granulocyte) leukemia, chronic lymph Leukemia, hairy cell leukemia; polycythemia vera; lymphoma, eg, but not limited to Hodgkin's disease, non-Hodgkin's disease; multiple myeloma, eg, but not limited to smoldering type ( smoldering) multiple myeloma, non-secretory myeloma, osteosclerotic myeloma, plasma cell leukemia, single life plasmacytoma and extramedullary plasmacytoma; Waldenstrae Macroglobulinemia; unspecified monoclonal hypergammaglobulinemia; benign monoclonal immunoglobulinemia; heavy chain disease; bone and connective tissue sarcoma, including but not limited to bone sarcoma, Bone marrow bone disease, osteosarcoma, chondrosarcoma, Ewing sarcoma, Paget's disease of bone, malignant giant cell tumor, fibrosarcoma of bone, cartilage, periosteal sarcoma, soft tissue sarcoma, hemangiosarcoma, fibrosarcoma, capage Sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, schwannoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as, but not limited to, glioma, astrocytoma, brainstem glioma, upper garment Cytoma, oligodendroglioma, non-glioma, acoustic schwannoma, craniopharynoma, medulloblastoma, meningioma, pineal tumor, pineoblastoma, primary brain lymphoma; breast cancer, eg, limited Not what Adenocarcinoma, adenocarcinoma, lobular (small cell) cancer, intraductal cancer, medullary breast cancer, mucinous breast cancer, tubular glandular breast cancer, papillary breast cancer, Paget's disease (including juvenile Paget's disease), and inflammatory breast cancer; For example, but not limited to pheochromocytoma and adrenocortical carcinoma; thyroid cancer, eg, but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and undifferentiated thyroid cancer; pancreatic cancer, eg , But not limited to, insulinoma, gastrinoma, glucagonoma, bipoma, somatostatin secreting tumor, and carcinoid or islet cell tumor; pituitary cancer, eg, but not limited to Cushing's disease, prolactin secreting tumor, acromegaly , And diabetes insipidus; eye cancers such as, but not limited to, eye melanomas such as iris melanoma, choroidal melanoma, and ciliary Melanoma, and retinoblastoma; vaginal cancer, such as but not limited to squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, such as but not limited to squamous cell carcinoma , Melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancer, eg, but not limited to squamous cell carcinoma, and adenocarcinoma; uterine cancer, eg, but not limited to, Endometrial cancer and uterine sarcoma; ovarian cancer, such as, but not limited to, ovarian epithelial cancer, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancer, such as but not limited to flat Epithelial cancer, adenocarcinoma, adenoid cystic carcinoma, mucinous epidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, wart cancer, and oat cell (small cell) cancer; gastric cancer, for example, limited Not adenocarcinoma, granulomatous (poly Ulcerative, superficially enlarged, widespread, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancer; rectal cancer; liver cancer, for example, but not limited to hepatocellular Cancer and hepatoblastoma, gallbladder cancer, eg, but not limited to adenocarcinoma; bile duct cancer, eg, but not limited to papillary cancer, nodular and widespread; lung cancer, eg, limited Non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large cell carcinoma and small cell lung cancer; testicular cancer, for example, but not limited to, embryonic tumor, seminoma Differentiation, classic (typical), spermatocytes, nonseminoma, embryonal cancer, teratoma cancer, choriocarcinoma (yolk sac tumor), prostate cancer, for example, but not limited to adenocarcinoma, smooth muscle And rhabdomyosarcoma; penile cancer; oral cancer, eg, not limited Squamous cell carcinoma; basal cancer; salivary gland cancer, such as, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoid cystic cancer; pharyngeal cancer, such as but not limited to squamous cell Cancer and warts; skin cancers such as, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial enlarged melanoma, nodular melanoma, melanoma malignant melanoma, terminal melanoma Melanoma; kidney cancer, such as, but not limited to, renal cell carcinoma, adenocarcinoma, adrenal tumor, fibrosarcoma, transitional cell carcinoma (renal pelvis and / or uterus); Wilms tumor; bladder cancer, eg, limited Although not, transitional cell carcinoma, squamous cell carcinoma, adenocarcinoma, carcinosarcoma. Furthermore, cancers include myxosarcoma, osteogenic sarcoma, endothelial sarcoma, lymphatic endothelial sarcoma, mesothelioma, periosteum, hemangioblastoma, epithelial cancer, cystadenocarcinoma, primary bronchial carcinoma, sweat gland carcinoma, sebaceous gland carcinoma (For a review of such disorders, see Fishman et al., 1985, Medicine, 2nd edition, JB Lippincott Co., Philadelphia, and Murphy et al., 1997, "Informed decision: See Informed Decisions: The Complete Book of Cancer Diagnosis, Treatment, and Recovery, Viking Penguin, Penguin Books USA, Inc., United States of America. It is also contemplated that the methods and compositions of the present invention can treat cancer caused by abnormal apoptosis. Such cancers include, but are not limited to, follicular lymphoma, carcinoma with p53 mutation, hormone-dependent cancer of breast, prostate and ovary, precancerous injury such as familial colon adenomatosis, and bone marrow Examples include dysplastic syndrome.

  In preferred embodiments, the cancers that are prevented, managed, treated, or ameliorated by the methods of the invention are prostate cancer, breast cancer, bone cancer, melanoma, lung cancer, and ovarian cancer. In another embodiment, a cancer that is prevented, managed, treated, or ameliorated by the methods of the present invention is a metastatic tumor, including but not limited to a tumor that has metastasized or may metastasize to bone (non-limiting examples). Is prostate cancer, breast cancer and lung cancer that has metastasized to or may metastasize to bone), tumor that has metastasized to or may metastasize to lung, metastasized to brain, or may metastasize There are tumors and tumors that have or may have spread to other organs or tissues of the subject.

5.5.3.1. Anti-cancer therapy The present invention provides a method for preventing, managing, treating or ameliorating cancer or one or more symptoms thereof, the method comprising, for a subject in need thereof, a liquid formulation of the present invention and an integrin α _v comprises administering beta ₃ and one or more therapeutic other than an antibody or antibody fragment that immunospecifically binds to (e.g., prophylactic or therapeutic agents). Examples of therapeutic or prophylactic agents include, but are not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, pseudo drugs, synthetic drugs, inorganic molecules, and organic molecules. . Any drug or treatment known to be useful for cancer or one or more symptoms thereof, or used or currently used for its prevention, treatment, management or amelioration (eg, chemotherapy, radiation) Therapy, hormonal therapy and / or biological therapy / immunotherapy) can be used in combination with the liquid formulations of the present invention in accordance with the invention described herein. Examples of such agents (ie anticancer agents) include, but are not limited to: angiogenesis inhibitors, topoisomerase inhibitors, and immunomodulators (chemotherapeutic agents), and non-therapeutic immunity Modulators, including but not limited to anti-T cell receptor antibodies (eg, anti-CD4 antibodies (eg, cM-T412 (Boeringer), IDEC-CE9.1® (IDEC And SKB), mAB 4162W94, orthoclone, and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (eg, Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti CD5 antibody (eg, anti-CD5 lysine-conjugated immune complex), anti-CD7 antibody (eg, CHH-380 (Novartis)), anti-CD8 anti-antibody Body, anti-CD40 ligand monoclonal antibody (eg IDEC-131 (IDEC)), anti-CD52 antibody (eg CAMPATH 1H (Ilex)), anti-CD2 antibody (eg MEDI-507 (MedImmune Inc.)) , International Publication Nos. WO02 / 098370 and WO02 / 069904)), anti-CD11a antibodies (eg, Xanelim (Genentech)), and anti-B7 antibodies (eg, IDEC-114 (IDEC))), anti-cytokine receptors Antibody (eg, anti-IFN receptor antibody, anti-IL-2 receptor antibody (eg, Protein Design Labs), anti-IL-4 receptor antibody, anti-IL-6 receptor antibody, anti-IL-10 Receptor antibodies, anti-IL-12 receptor antibodies), and anti-cytokine antibodies (eg, anti-IFN antibodies, anti-TNF-α) Antibodies, anti-IL-1β antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (eg, ABX-IL-8 (Abgenix)), anti-IL-12 antibodies; CTLA4-immunoglobulin; LFA-3TIP (Biogen, International Published WO 93/08656 and US Pat. No. 6,162,432); soluble cytokine receptors (eg, TNF-α receptor extracellular domain or antigen-binding fragment thereof, IL-1β receptor extracellular domain or antigen thereof) Binding fragments, and IL-6 receptor extracellular domain or antigen-binding fragment thereof), cytokines or fragments thereof (eg, interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-23, TN -Α, TNF-β, interferon (IFN) -α, IFN-β, IFN-γ, and GM-CSF), and anti-cytokine antibodies (eg, anti-IL-2 antibody, anti-IL-4 antibody, anti-IL- 6 antibody, anti-IL-10 antibody, anti-IL-12 antibody, anti-IL-15 antibody, anti-TNFα antibody, anti-IFN-γ), as well as an antibody that immunospecifically binds to a tumor-associated antigen (eg, Herceptin®) .

Examples of angiogenesis inhibitors (ie, anti-angiogenic agents) include, but are not limited to, angiostatin (plasminogen fragment); anti-angiogenic anti-thrombin III; Angiozyme; ABT-627; 12-9566; Benefin; Bevacizumab; BMS-275291; Cartilage-derived inhibitor (CDI); CAI; CD59 complement fragment; CEP-7055; Col 3; Combretastatin A-4; End Statins (Endostatin) (collagen XVIII fragment); fibronectin fragment; Gro-beta; halofuginone; heparinase; heparin hexasaccharide fragment; HMV833; human chorionic gonadotropin (hCG); IM-862; / Γ; interferon-inducible protein (IP-10); interleukin-12; kringle 5 (plasminogen fragment); marimastat Metalloproteinase inhibitor (TIMP); 2-methoxyestradiol; MMI 270 (CGS 27023A); MoAb IMC-1C11; Neovastat; NM-3; Panzem; PI-88; placental ribonuclease inhibitor; Plasminogen activator inhibitor; platelet factor-4 (PF4); purinostat (Prinomastat); prolactin 16KD fragment; proliferin-related protein (PRP); PTK 787 / ZK 222594; retinoid; sorimastat (Solimastat); Squalamine; SS 3304; SU 5416; SU6668; SU11248; tetrahydrocortisol-S; tetrathiomolybdate; thalidomide; thrombospondin-1 (TSP-1); TNP-470; transforming growth factor-β ( TGF-b); vasculostatin; vasostatin (calreticulin fragment); ZD 6126; ZD 64 74; HMG-CoA reductase inhibitors (3-hydroxy-3-methyl-gratalyl coenzyme A reductase inhibitors) such as, but not limited to, lovastatin, pravastatin, fluvastatin, statin, simvastatin, and atorvastatin; Transferase inhibitors (FTI); and bisphosphonates such as, but not limited to, alendronate, clodronate, etidronate, ibandronate, pamidronate, risedronate, tiludronate, and zolendronate. In certain embodiments, the anti-angiogenic agent does not comprise an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ .

Specific examples of anti-cancer agents that can be utilized in the methods of the present invention include, but are not limited to, acivicin; acralvicin; acodazole hydrochloride; acronine; adzelesin Aldesleukin; altretamine; ambomycin; amethronerone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin Asparaginase; Asperlin; azacitidine; azateidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisaanthrene hydrochloride (Bisantrene hydrochloride); bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; busulfan; calctinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil (chlorambucil) Cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; deca Dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone medialate; Docetaxel; doxorubicin (doxorubicin); doxorubicin hydrochloride (droloxifene); droxyxene citrate (drooxifene citrate); dromostanolone propionate (dromostanolone propionate); Duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; Epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide Etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea (Hydroxyurea); idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II or rIL2), interferon α-2a; interferon α-2b; interferon α -n1; interferon α-n3; interferon β-1a; interferon γ-1b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate (leuprolide) liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoxprocol; maytansine; Mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate; Methotorexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; Mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; Oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobromman; (Piposulfan); piroxantrone hydrochloride; plicamycin; promestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride (procarbazine) puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol; Safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine spiroplatin Streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; texantrone hydrochloride; teloxantrone hydrochloride Temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofuline tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate (trimetrexate) triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; glucuronate; Vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; Tartrate (vinorelbine tartrate); Binroshijin sulfate (vinrosidine sulfate); vinzolidine sulfate (vinzolidine sulfate); vorozole (vorozole); Zenipurachin (zeniplatin); zinostatin (zinostatin); zorubicin hydrochloride (zorubicin hydrochloride) and the like. Other anticancer drugs include, but are not limited to, 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecipepenol Adozelesin; aldesleukin; all TK antagonists; altretamine; ambamustine; amidox; amifoxine; aminolevulinic acid; amrubicin; ansacrine Anagacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitor; antagonist D; antagonist G; antarelix; antidorpomorphic morphogenic protein -1 (anti-dorsalizing morphogenic protein-1); Anti-estrogen; antineoplaston; antisense oligonucleotide; aphidicolin glycinate; apoptosis gene modulator; apoptosis regulator; aprinic acid; ara-CDP-DL-PTBA Arginine deaminase; aslaclacine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron Azatoxin; azatyrosine; baccatin III derivative; balanol; batimastat; BCR / ABL antagonist; benzochlorins; benzoylstaurosporin orine); β-lactam derivative; β-alethine; β-clamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnaphthide (Bisnafide); bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostinol C (Calphostin C); camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxamidotriazole; CaRest M3; CARN 700; cartilage extraction inhibitor; carzelesin; casein kinase Inhibitor Castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analog; Trimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; crisnatol; cryptophycin 8; crypto Ficin A derivative; curacin A; cyclopentanthraquinone; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; Kurikishimabu (dacliximab); Decitabine (decitabine); dehydropeptidase Logistics Dem Nin (dehydrodidemnin) B; deslorelin (deslorelin); dexamethasone (Dexame
dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didoxin; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol Dioxamycin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxyfluridine; (droloxifene); dronabinol; duocarmycin SA; ebeslen; ecomstin ( edelfcolin; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estrum Estrogen agonist; estrogen antagonist; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filretinide (Fingrastim); finasteride; flavopiridol; flavopiridol; fluzelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; fluorofenunorunicin hydrochloride; forfenimexestane ); Host riecin (fostriecin); fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; galilelic (Ganirelix); gelatinase inhibitor; gemcitabine; glutathione inhibitor; hepsulfam; heegulin; hexamethylenebisacetamide; hypericin; ibandronic acid; idarubicin Idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; Interferon agonist; interferon; interleukin; iobenguane; iododoxorubicin; ipomeanol (4-); iroplact; irosogladine; isobenga Isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamelaline-N-triacetate; lanreotide Leinamycin; lenograstim; lentinan sulfate; leptolstatin; retrosol; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin Levamisole; liarozole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lombricine; Lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lysofylline; Mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitor; matrix metalloproteinase inhibitor; menogaril; merbarone; ); Methioninase; metoclopramide; MIF inhibitor; mifepristone; (miltefosine); mirimostim; mismatched double-stranded RNA; mitogua zone (m) mitolactol; mitomycin analogue; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; Ramostine (molgramostim); monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A + mycobacterial cell wall sk); mopidamol; multidrug resistance gene inhibitor; treatment based on multiple tumor suppressor 1; mustard anticancer Drugs; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinarine; N-substituted benzamide; nafarelin; nagrestip; naloxone + pentazocine (naloxone + pentazocine) Napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridonic acid; neutral endopeptidase; nilutamide; nisamycin; Nitric oxide modulators; nitric oxide antioxidants; nitrullyn; O6-benzyl guanine; octreotide; oxicenone; oligonucleotides; onapristone; ondansetron Ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel; Ritaxel analogs; paclitaxel derivatives; parauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; Pegaspargase; peldesine; sodium pentosan polysulfate; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenyl acetate; Phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; Platinogen activator inhibitor; platinum complex; platinum compound; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propylbisacridone ( Prostaglandin J2; Proteasome inhibitor; Protein A-based immunomodulator; Protein kinase C inhibitor; Protein kinase C inhibitor (of microalgae); Protein tyrosine phosphatase inhibitor; Purine nucleoside phosphorylase inhibitor Drugs; purpurin; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene complex; raf antagonist; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitor (ras farn) esyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozyme; RII retinamide Rogletimide; rohitukine; romurtide; roquinimex; rubiginone BL; ruboxyl; safingol; saintopin; saintopin; ) A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotide; signaling inhibitor; signaling modulator; single-chain antigen Binding protein; Schizophyllan sozozoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin ) D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipiamide; stromelysin inhibitor; sulfinosine Superactive vasoactive intestinal peptide antagonist; suramin; suramin; swainsonine; synthetic glycosaminoglycan; tallimustine; 5-fluorouracil; leucovorin; Tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tegafur; tellurapyrylium; telomerase inhibitor; temoporfin; temozolomide; temozolomide; ); Tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; Thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topsentin; Toremifene; totipotent stem cell factor; translation inhibitor; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; triptorelin; tropisetron ( turosteride; tyrosine kinase inhibitor; tyrphostins; UBC inhibitor; ubenimex; urogenitial sinus-derived growth inhibitory factor; urokinase receptor antagonist Vapreotide; variolin B; vector system, erythrocyte gene therapy; veraresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; Vitaxin (vi bolozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer.

  The present invention also includes administering a liquid formulation of the present invention in combination with radiation therapy including destroying cancer cells using x-rays, gamma rays and other radiation sources. In a preferred method, radiation therapy is applied as external beam radiation therapy or teletherapy where radiation is directed from a teleoperated source. In other preferred embodiments, radiation therapy is applied as internal therapy or brachytherapy, where the radioactive source is placed in the body in close proximity to the cancer cells or tumor mass.

  In a specific embodiment, a breast cancer patient may apply a prophylactically or therapeutically effective amount of a liquid formulation of the present invention to a prophylactically or therapeutically effective amount of one or more other substances useful for breast cancer treatment, including but not limited to doxorubicin, epirubicin. , Doxorubicin and cyclosporine combination (AC), cyclosporine, doxorubicin and 5-fluorouracil combination (CAF), cyclosporine, epirubicin and 5-fluorouracil combination (CDF), Herceptin®, tamoxifen, tamoxifen and cytotoxic chemistry (Including combination therapy). In certain embodiments, metastatic breast cancer patients are administered a prophylactically or therapeutically effective amount of one or more liquid formulations of the present invention in combination with administration of effective amounts of taxanes (eg, docetaxel and paclitaxel). In other embodiments, a prophylactically or therapeutically effective amount of a liquid formulation of the present invention comprises a prophylactically or therapeutically effective amount of taxene, plus standard doxorubicin and cyclophosphamide for adjunct therapy of node positive breast cancer. In combination with the administration of

In a specific embodiment, a prostate cancer patient may receive a prophylactically or therapeutically effective amount of a liquid formulation of the present invention in a prophylactically or therapeutically effective amount of one or more other substances useful for prostate cancer treatment, including but not limited to external Radiotherapy, radioisotope (ie, I ¹²⁵ , palladium, iridium) interstitial insertions, leuprolide or other LHRH agonists, nonsteroidal antiandrogens (flutamide, nilutamide, bicalutamide), steroidal antiandrogens (cyproterone acetate), leuprolide and Ruftamide combination, estrogen such as DES, chlorotrianisene, ethinyl estradiol, conjugated estrogen USP, DES-diphosphate, radioisotope (eg strontium-89), external radiation therapy and strontium-89, as a second measure Hormone therapy For example, aminoglutethimide, hydrocortisone, flutamide cessation, progesterone, and ketoconazole, low-dose prednisone, or other chemotherapy regimens that have been reported to cause subjective improvement of symptoms and reduction of PSA levels (docetaxel, paclitaxel, est Ramastine / docetaxel, estramustine / etoposide, estramustine / vinblastine, and estramustine / paclitaxel.) In a specific embodiment, patients with ovarian cancer are prophylactically or therapeutically effective doses Of a liquid formulation of the present invention in a prophylactically or therapeutically effective amount of one or more other substances useful for the treatment of ovarian cancer (including but not limited to intraperitoneal radiation therapy, eg, P32 therapy, whole abdominal and pelvic radiation therapy, , Paclitaxel (Taxol) or taxel (Taxo) tere) and cisplatin or carboplatin, cyclophosphamide and cisplatin, cyclophosphamide and carboplatin, 5-FU and leucovorin, etoposide, liposomal doxorubicin, gemcitabine or topotecan) For patients with platinum resistant disease, a prophylactically or therapeutically effective amount of the liquid formulation of the present invention is administered in combination with taxol administration, and ifosfamide, cisplatin-based for patients with platinum resistant disease Treatment of patients with resistant ovarian cancer, including administration of hexamethylmelamine (HMM) as salvage chemotherapy after failure of the combination regimen, and tamoxifen in patients with detectable levels of cytoplasmic estrogen receptor on the tumor Is included. In a specific embodiment, a patient with osteosarcoma is treated with a prophylactically or therapeutically effective amount of the liquid formulation of the present invention in one or more other substances useful for prophylactic or therapeutically effective amount of osteosarcoma, including but not limited to Including doxorubicin, ifosfamide, cisplatin, high dose methotrexate, cyclophosphamide, etoposide, vincristine, dactinomycin, and surgery). In a specific embodiment, a bone metastasized cancer patient may be treated with a prophylactically or therapeutically effective amount of the liquid formulation of the present invention in a prophylactically or therapeutically effective amount of one or more substances (not particularly limited). Drugs or treatments used to treat basic malignancies (non-limiting examples are prostate or breast cancer hormone inhibitors that metastasize to bone, and surgery), and radiation therapy (non-limiting examples are strontium 89 And samarium 153, which are administered in combination with osteogenic radionuclides that show anti-tumor effects and relieve symptoms)).

In a more specific embodiment, the invention also includes administration of a liquid formulation of the invention in combination with administration of one or more treatments, including, but not limited to, Table 3 The anticancer agents shown are mentioned and are preferred for the prevention, management, treatment or amelioration of the aforementioned breast cancer, bone cancer, ovarian cancer and prostate cancer.

  Cancer treatment and its dosage, administration route and recommended usage are known in the art and are described in literature such as Physicinan's Desk Reference (56th edition, 2002, 57th edition, 2003).

5.5.4. Treatment of Inflammatory Diseases The liquid formulations of the present invention can be administered to a subject for the prevention, management, treatment or amelioration of an inflammatory disorder or one or more symptoms thereof. The liquid formulations of the present invention may also be used in one or more other treatments, preferably treatments useful for the prevention, management, treatment or amelioration of inflammatory diseases (eg, but not limited to Section 5.5.4 below). In combination with a prophylactic or therapeutic agent exemplified in .1) and the like, it may be administered to a subject in need of prevention, management, treatment or amelioration of an inflammatory disease or one or more symptoms thereof. In certain embodiments, the present invention provides a method for the prevention, management, treatment or amelioration of an inflammatory disease or one or more symptoms thereof, wherein the method provides a subject in need thereof a prophylactic or therapeutically effective amount. Administering a dose of the liquid formulation of the present invention. In another embodiment, the present invention provides a method for the prevention, management, treatment or amelioration of an inflammatory disease or one or more symptoms thereof, said method comprising a prophylactic or therapeutic effective amount for a subject in need thereof. Of a liquid formulation of the present invention and one or more other treatments (eg, prophylactic or therapeutic agents) other than antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ Including administering a dose.

The present invention provides one or more symptoms of an inflammatory disorder in a subject refractory to conventional treatments for such inflammatory disorders (eg, methotrexate and TNF-α antagonists such as REMICADE ^™ or ENBREL ^™ ). A method is provided for administering, treating or alleviating a subject comprising administering to the subject a prophylactically effective amount or a therapeutically effective amount of a liquid formulation of the present invention. The present invention also provides a method for managing, treating or alleviating one or more symptoms of an inflammatory disorder in a subject refractory to existing single drug treatments for such inflammatory disorders. and, the method, a prophylactically or therapeutically effective amount of the present invention a dose of a liquid formulation, and other than a prophylactically or therapeutically effective amount of an integrin alpha _V beta ₃ antibody or antibody fragment that immunospecifically binds to 1 Administering to the subject a dose of a species or multiple treatments (eg, prophylactic or therapeutic agents). The present invention also provides liquid formulations of the present invention in combination with any other treatment to patients who have proven refractory to other treatments but are no longer refractory to these treatments. A method for managing or treating an inflammatory disorder is provided. The present invention also treats inflammatory disorders where it can prove or prove that another treatment is too toxic for the subject being treated (ie, produces unacceptable or intolerable side effects). Provide an alternative method for For example, a liquid formulation of the invention can be administered to a subject refractory to a TNF antagonist or methotrexate. Furthermore, the present invention provides a method for preventing the recurrence of inflammatory disorders in patients who are being treated by administering a liquid formulation of the present invention or have no disease activity.

  Inflammatory diseases that can be treated by the methods encompassed by the present invention include, but are not limited to, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), allergic disorders, septic shock, lungs Fibrosis, undifferentiated spondyloarthritis, undifferentiated arthritis, arthritis, osteoarthritis, spondyloarthritis (eg, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome (reactive arthritis), inflammatory osteolysis, Wilson disease, and chronic Examples include chronic inflammation caused by viral or bacterial infections, as described in section 3.1 of this specification, several autoimmune disorders are associated with symptoms of inflammation.

  Anti-inflammatory therapies and their dosages, routes of administration and recommended usage are known in the art and are described in literature such as the Physician's Desk Reference (56th edition, 2002 and 57th edition, 2003) .

5.5.4.1. Anti-inflammatory treatment The present invention provides a method for preventing, managing, treating or alleviating an inflammatory disorder or one or more symptoms thereof, wherein the method is immunospecific for the liquid formulation of the present invention and the integrin α _v β _3. Administering to the subject in need thereof one or more therapies (eg, prophylactic or therapeutic agents) other than the binding antibody or antibody fragment. Any drug or treatment known, used for, or currently used for the prevention, management, treatment or alleviation of an inflammatory disorder or one or more symptoms thereof Can be used in combination with the liquid formulations of the present invention according to the invention described herein. Examples of such agents include, but are not limited to, immunomodulators, anti-angiogenic agents, anti-inflammatory agents and TNF-α antagonists.

  Specific examples of immunomodulators that can be administered to a subject with an inflammatory disorder in combination with the liquid formulations of the present invention include, but are not limited to: methotrexate, leflunamide, cyclophosphamide, sites Cytoxan, Immuran, cyclosporin A, minocycline, azathioprine, antibiotics (eg FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxy Spagarin, brequinar, malononitrile triamide (eg leflunamide), anti-T cell receptor antibody (eg anti-C D4 antibodies (eg, cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (eg, Nuvion (Prodig) Labs), OKT3 (Johnson & Johnson) or Rituxan (IDEC)), anti-CD5 antibody (eg, anti-CD5 lysine-linked immunoconjugate), anti-CD7 antibody (eg, CHH-380 (Novartis)), anti-CD8 antibody, anti-CD8 antibody CD40 ligand monoclonal antibody (eg IDEC-131 (IDEC)), anti-CD52 antibody (eg CAMPATH 1H (Ilex)), anti-CD2 antibody (eg MEDI-507 (MedImmune, Inc., International Publication Nos. WO 02/098370 and WO 02/069904), anti-CD11a antibodies (eg, Xanelim (Genentech)) and anti-B7 antibodies (eg, IDEC-114) ( IDEC)); anti-cytokine receptor antibodies (eg, anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (eg, Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL- 10 receptor antibody and anti-IL-12 receptor antibody), anti-cytokine antibody (for example, anti-IFN antibody, anti-TNF-α antibody, anti-IL-1β antibody, anti-IL-6 antibody, anti-IL-8 antibody (for example, ABX-) IL-8 (Abgenix)) and IL-12 antibody)); CTLA4-immunoglobulin; LFA-3TIP (Biogen, WO 93/08656 and US Pat. No. 6,162,432); soluble cytokine receptor (eg, cell of TNF-α receptor) Ectodomain or fragment thereof, IL-1β receptor extracellular domain or fragment thereof and IL-6 receptor extracellular domain or fragment thereof; cytokines or fragments thereof (eg, interleukin (IL) -2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, TNF-α, TNF-β, interferon ( IFN) -α, IFN-β, IFN-γ and GM-CSF); Antibodies (eg, anti-IL-2 antibody, anti-IL-4 antibody, anti-IL-6 antibody, anti-IL-10 antibody, anti-IL-12 antibody, anti-IL-15 antibody, anti-TNF-α antibody and anti-IFN- gamma antibody).

  Non-limiting examples of anti-angiogenic agents that can be administered to a subject having an inflammatory disorder in combination with a liquid formulation of the invention include: endostatin, angiostatin, apomygren, anti Angiogenic antithrombin III, 29 kDa N-terminal and 40 kDa C-terminal proteolytic fragments of fibronectin, uPA receptor antagonist, 16 kDa proteolytic fragment of prolactin, 7.8 kDa proteolytic fragment of platelet factor 4, platelet 4 Anti-angiogenic 24 amino acid fragment of the factor, an anti-angiogenic factor referred to as 13.40, an anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, an anti-angiogenic 20 amino acid of SPARC Peptide Hula , RGD-containing peptide and NGR-containing peptide, laminin, fibronectin, small anti-angiogenic peptide of procollagen and EGF, acidic fibroblast growth factor (aFGF) antagonist, basic fibroblast growth factor (bFGF) antagonist, blood vessel Endothelial growth factor (VEGF) antagonists as well as VEGF receptor (VEGFR) antagonists (eg, anti-VEGFR antibodies).

Non-limiting examples of TNF-α antagonists that can be administered to a subject having an inflammatory disorder in combination with a liquid formulation of the invention include: protein, polypeptide, peptide, fusion protein, TNF- Antibodies such as antibodies that immunospecifically bind to α (eg, human, humanized, chimeric, monoclonal, polyclonal, Fv, ScFv, Fab fragment, F (ab) ₂ fragment and antigen binding fragments thereof), nucleic acid molecules Small molecules that block, reduce, inhibit or neutralize the function, activity and / or expression of (eg, antisense molecules or triple helices), organic molecules, inorganic molecules, and TNF-α. In various embodiments, the TNF-α antagonist has a TNF-α function, activity and / or expression that is at least 10%, at least 15 compared to a control, such as phosphate buffered saline (PBS). %, At least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, Decrease by at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. Examples of antibodies that immunospecifically bind to TNF-α include, but are not limited to: Infliximab (REMICADE ™; Centacor), D2E7 (Abbott Laboratories / Knoll Pharmaceuticals Co., Mt. Olive, NJ ), CDP571 and CDP-870, also known as HUMICADE ™ (both from Celltech / Pharmacia, Slough, UK), and TN3-19.12 (Williams et al., 1994, Proc. Natl. Acad. Sci. USA 91 : 2762-2766; Thorbecke et al., 1992, Proc. Natl. Acad. Sci. USA 89: 7375-7379). The invention also encompasses the use of antibodies that immunospecifically bind to TNF-α disclosed in the following US patents in the compositions and methods of the invention: US Pat. No. 5,136,021; 5,147,638; 5,223,395; 5,231,024; 5,334,380; 5,360,716; 5,426, No. 181, No. 5,436,154; No. 5,610,279; No. 5,644,034; No. 5,656,272; No. 5,658,746; No. 5,698,195; No. 5,736,138; No. 5,741,488; No. 5,808,029; No. 5,919,452; No. 5,958,412 No. 5,959,087; No. 5,968,741; No. 5, No. 94,510; No. 6,036,978; No. 6,114,517 and No. 6,171,787, each of which is incorporated herein by reference in its entirety. . Examples of soluble TNF-α receptors include, but are not limited to: sTNF-R1 (Amgen), etanercept (ENBREL ™; Immunex) and its rat homologs RENBREL ™, TNFrI, TNFrII-derived TNF Soluble inhibitors of -α (Kohno et al., 1990, Proc. Natl. Acad. Sci. USA 87: 8331-8335), as well as TNF-α Inh (Seckinger et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5188 -5192).

  Other TNF-α antagonists encompassed by the present invention include, but are not limited to: IL-10 known to block TNF-α production through interferon-γ activated macrophages (Oswald et al. 1992, Proc. Natl. Acad. Sci. USA 89: 8676-8680), TNFR-IgG (Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539), mouse product TBP-1 ( Serono / Yeda), vaccine CytoTAb (Protherics), antisense molecule 104838 (ISIS), peptide RDP-58 (SangStat), thalidomide (Celgene), CDC-801 (Celgene), DPC-333 (Dupont), VX-745 ( Vertex), AGIX-4207 (AtheroGenics), ITF-2357 (Italfarmaco), NPI-13021-31 (Nereus), SCIO-469 (Scios), TACE targeter (Immunix / AHP), CLX-120500 (Calyx), Thiazolopyrim (Dynavax), Auranofin (Ridaura) (SmithKline Beecham Pharmaceuticals), Quinacrine (mepacrine dichlorohydrate), Tenidap (inid), Elix Large Scale Biological) and Uriach anti-p38 MAPK agent.

  Non-limiting examples of anti-inflammatory agents administered to a subject having an inflammatory disorder in combination with the liquid formulation of the present invention include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, β-agonists , Anticholinergic agents and methylxanthine. Examples of NSAIDs include, but are not limited to: aspirin, ibuprofen, celecoxib (CELEBREX ™), diclofenac (VOLTAREN ™), etodolac (LODINE ™), fenoprofen (NALFON ™ )), Indomethacin (INDOCIN ™), ketorolac (TORADOL ™), oxaprozin (DAYPRO ™), nabumetone (RELAFEN ™), sulindac (CLINERIL ™), tolmetin (TOLETIN ™) , Rofecoxib (VIOXX ™), naproxen (ALEVE ™, NAPROSYN ™), ketoprofen (ACTRON ™) and nabumetone (RELAFEN ™). Such NSAIDs function by inhibiting cyclooxygenase enzymes (eg, COX-1 and / or COX-2). Examples of steroidal anti-inflammatory drugs include, but are not limited to: glucocorticoids, dexamethasone (DECADRON ™), cortisone, hydrocortisone, prednisone (DELTASONE ™), prednisolone, triamcinolone, azalfidine and eicosanoids ( For example, prostaglandins, thromboxanes and leukotrienes).

  In certain embodiments, patients with osteoarthritis are prophylactically effective in combination with other agents or treatments useful for the prevention, treatment, management or alleviation of osteoarthritis, including but not limited to: Or a therapeutically effective amount of a liquid formulation of the invention is administered: an analgesic (non-limiting examples include doses of up to 4000 mg / d acetaminophen; phenacetin; and daily doses in the range of 200 mg to 300 mg NSAID (non-limiting examples include aspirin, diflunisal, diclofenac, etodolac, fenamate, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, methyl salicylate, nabumetone, Naproxen, oxaprozin, phenylbutazone, pyroxy Low dose NSAIDs (eg 1200 mg / d ibuprofen, 500 mg / d naproxen) are preferred and gastric mucosal protective agents (eg misoprostol, famotidine or Omeprazole) is preferably used concomitantly with NSAIDs); non-acetylated salicylates (including but not limited to salsalate); cyclooxygenase (Cox) -2 specific inhibitors (CSI) (including but not limited to celecoxib and rofecoxib) Intraarticular or periarticular injection of depot glucocorticoid preparation; intraarticular injection of hyaluronic acid; capsaicin cream; multiple irrigation of osteoarthritic knee to wash out fibrin, cartilage debris and other debris ; As well as joint replacement surgery. The liquid formulations of the present invention can also be used in combination with other non-pharmacological measurements in the prevention, treatment, management and alleviation of osteoarthritis, including but not limited to: reduced joint burden ( Non-limiting examples are inferior posture correction, supporting excessive lumbar lordosis, avoiding overloading of the joints involved, and avoiding extended uprights, keeling and squatting ); Application of heat to affected joints; aerobic exercise and other physical treatments.

  In certain embodiments, patients with rheumatoid arthritis have a prophylactically effective amount in combination with other drugs or treatments useful in the prevention, treatment, management and alleviation of rheumatoid arthritis, including but not limited to: Or a therapeutically effective amount of a liquid formulation of the invention is administered: NSAIDs (non-limiting examples include aspirin, diflunisal, diclofenac, etodolac, phenamates, fenoprofen, flubiprofen, ibuprofen, indomethacin, ketoprofen, salicylic acid Analgesics (non-limiting examples are acetaminophen, phenacetin and tramadol); CSI (including but not limited to methyl, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac and tolmethine); Sele Including but not limited to xibu and rofecoxib; glucocorticoids (preferably low dose oral glucocorticoids (eg <7.5 mg / d prednisone) or high dose glucocorticoids, monthly pulse or intra-articular glucocorticoids) ); Disease-modifying anti-rheumatic drugs (DMARD) (methotrexate, preferably given intermittently at low doses (eg, 7.5 mg to 30 mg) once a week), gold compounds (eg, gold salts), D-penicillamine, anti-malarial agents (including but not limited to chloroquine) and sulfasalazine); TNF-α neutralizing agents (including but not limited to etanercept and infliximab); immunosuppressants and cytotoxicity Agents (examples include azathioprine, lefur Including, but not limited to, namide, cyclosporine and cyclophosphamide), and surgery (eg, arthroplasty, total joint replacement, hand reconstruction surgery, open synovectomy or arthroscopic synovectomy) , And early wrist tenosynovectomy). The liquid formulations of the present invention can also be used in combination with other measurements in the prevention, treatment, management and alleviation of rheumatoid arthritis, including but not limited to: rest, desire for inflamed joints Splints, exercise, the use of various corrective and auxiliary devices to reduce movement, and other physical treatments. The liquid formulations of the present invention can also be used in combination with several non-traditional approaches in the prevention, treatment, management and alleviation of rheumatoid arthritis, including but not limited to: diets (eg, Dietary ω-6 essential fatty acids found in meat replaced with ω-3 fatty acids found in certain fish oils (eg, eicosapentaenoic acid), vaccines, hormones and topical preparations.

In certain embodiments, patients with chronic obstructive pulmonary disease (COPD) in combination with other drugs or treatments useful in the prevention, treatment, management and alleviation of COPD, including but not limited to: A prophylactically or therapeutically effective liquid formulation of the invention is administered: bronchodilators (short-acting and long-acting β ₂ -adrenergic agonists (examples of short-acting β ₂ agonists include albuterol) , Pirbuterol, terbutaline and metaproterenol; examples of long acting beta ₂ agonists include oral sustained release albuterol and inhaled salmeterol Non-limiting), anticholinergic agents (including but not limited to ipratropium bromide) And theophylline and its derivatives (therapeutic range for theophylline is preferably but not limited to 10 μg / mL to 20 μg / mL)); glucocorticoids; exogenous α ₁ AT (eg, 60 mg Α ₁ AT derived from pooled human plasma, administered intravenously at weekly doses / kg); oxygen; lung transplantation; lung volume reduction surgery; endotracheal intubation, ventilation support; annual influenza vaccine and 23-valent polysaccharide Streptococcus pneumoniae vaccination; exercise; and smoking cessation.

In certain embodiments, a patient with pulmonary fibrosis is combined with an effective amount of one or more other agents useful for treating pulmonary fibrosis, including but not limited to: A therapeutically effective amount of a liquid formulation of the present invention is administered: oxygen; corticosteroids (non-limiting examples are 1 mg / kg / d to 1.5 mg / kg / d (up to 100 mg / d) over 6 weeks) Starting with daily prednisone, gradually decreasing over 3 to 6 months to a minimum maintenance dose of 0.25 mg / kg / d); cytotoxic drugs (non-limiting examples are 100 mg to 120 mg oral cyclophosphamide once daily, and oral azathioprine from 3 mg / kg to 200 mg once daily; bronchodilators (non-limiting examples are short-acting Fine long-acting beta ₂ - adrenergic agonists, anticholinergic agents and theophylline and its derivatives); and antihistamines (non-limiting examples are diphenhydramine and doxylamine).

  In certain embodiments, a patient having asthma has a prophylactically effective amount or a therapeutically effective amount in combination with one or more other agents useful for treating asthma, including but not limited to: Liquid formulations of the present invention are administered: adrenergic stimulants (eg, catecholamines (eg, epinephrine, isoproterenol and isoetharine); resorcinol (eg, metaproterenol, terbutaline and fenoterol); and Saligenin (including but not limited to salbutamol). Inhalation is the preferred route of administration for adrenergic stimulants; methylxanthine (including but not limited to theophylline and its various salts); anticholinergic agents (atropine sulfate, methylatropine nitrate and ipratropium bromide). Glucocorticoids (examples include but are not limited to systemic or oral steroids and inhaled glucocorticoids); mast cell stabilizers (examples include cromolyn sodium) And ledoctriene modifiers (examples include but are not limited to Zileuton, zafirlukast and montelukast); immunosuppressants (examples include methotrexate and gold salts). Contains It is but are not limited to); and mucolytic agents (examples, not including but acetylcysteine limited to).

In certain embodiments, an allergic patient includes a prophylactically effective amount or a therapeutically effective amount in combination with one or more other agents useful for treating an effective amount of allergy, including but not limited to: Liquid formulations of the invention are administered: cromolyn; anti-mediator drugs (for example, including but not limited to antihistamines, see Table 4); sympathomimetic drugs (for example, α-adrenergic). Including but not limited to agonists and β-adrenergic drugs; theophylline and its derivatives; glucocorticoids; Including but not limited to immunotherapy).

5.5.5. Treatment of Autoimmune Disorders The liquid formulations of the present invention can be administered to a subject in need thereof to prevent, manage, treat or alleviate an autoimmune disorder or one or more symptoms thereof. The liquid formulations of the present invention may also include one or more other treatments (preferably, prevention, management or treatment of autoimmune disorders) to prevent, manage, treat or alleviate autoimmune disorders or one or more symptoms thereof. In combination with a treatment useful for, including but not limited to, prophylactic or therapeutic agents listed in Section 5.5.5.1 below) Can be administered to the body. In certain embodiments, the present invention provides a method for preventing, managing, treating or alleviating an autoimmune disorder or one or more symptoms thereof, the method comprising a prophylactically effective amount or a therapeutically effective amount of a liquid formulation of the present invention. Administering to a subject in need thereof. In another embodiment, the present invention provides a method for preventing, managing, treating or alleviating an autoimmune disorder or one or more symptoms thereof, comprising a prophylactically effective amount or a therapeutically effective amount of a liquid formulation of the present invention. And a dose of one or more treatments (eg, prophylactic or therapeutic agents) other than an antibody or antibody fragment that immunospecifically binds to a prophylactically or therapeutically effective amount of integrin α _v β ₃ Administering to a subject in need thereof.

The present invention provides a method for managing, treating or alleviating an autoimmune disorder or one or more symptoms thereof in a subject refractory to conventional treatments for such autoimmune disorders. Comprises administering to the subject a dose of a liquid formulation of the invention in a prophylactically or therapeutically effective amount. The present invention also provides a method for managing, treating or alleviating an autoimmune disorder or one or more symptoms thereof in a subject refractory to an existing single drug treatment for such autoimmune disorder. Provided, wherein the method comprises a dose of a liquid formulation of the present invention in a prophylactically or therapeutically effective amount, and an antibody or antibody fragment that immunospecifically binds to a prophylactically or therapeutically effective amount of integrin α _v β ₃ . Administering a dose of one or more treatments (eg, prophylactic or therapeutic agents) to the subject. The present invention also provides liquid formulations of the present invention in combination with any other treatment to patients who have proven refractory to other treatments but are no longer refractory to these treatments. Provides a method for managing, treating or alleviating an autoimmune disorder or one or more symptoms thereof. The present invention also manages autoimmune disorders where it can prove or prove that another treatment is too toxic for the subject being treated (ie, produces unacceptable or intolerable side effects). Or provide alternative methods for treatment. In particular, the present invention provides an alternative method for the management or treatment of autoimmune diseases where the patient is refractory to other treatments. Furthermore, the present invention provides a method for preventing the recurrence of an autoimmune disorder in a patient who has been treated by administering a liquid formulation of the present invention or has no disease activity.

  In autoimmune disorders, the immune system triggers to fight the immune response in the absence of foreign substances, and the body's normal protective immune system damages its own tissues by accidentally attacking itself cause. There are a number of different autoimmune disorders that affect the body in different ways. For example, the brain is affected in individuals with multiple sclerosis, the intestine is affected in individuals with Crohn's disease, and the synovium, bone and cartilage of various joints are affected in individuals with rheumatoid arthritis. Autoimmune disorders can cause the destruction of one or more types of body tissue, resulting in abnormal organ growth or changes in organ function. An autoimmune disorder may affect only one type of organ or tissue, or it may affect multiple organs and tissues. Organs and tissues that are commonly affected by autoimmune disorders include red blood cells, blood vessels, connective tissue, endocrine glands (eg, thyroid or pancreas), muscles, joints and skin. Examples of autoimmune disorders that can be treated by the methods of the present invention include, but are not limited to: alopecia areata, ankylosing spondylitis, antiphospholipid antibody syndrome, autoimmune Addison disease, adrenal gland Autoimmune disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune ovitis and autoimmune orchiditis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac pleu skin Celiac sprue-dermatitis, chronic fatigue immunodeficiency syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, scarring pemphigus, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid Lupus erythematosus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Grave s disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, Meniere's disease, mixed connective tissue disease , Multiple sclerosis, type 1 diabetes or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndrome, polymyalgia rheumatica , Polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, Reiter syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff -Man syndrome, systemic lupus erythematosus, lupus erythematosus, Takayasu's arteritis, temporal arteritis / giant cell arteritis, ulcerative colitis, uveitis, vasculitis (eg, shingles) Dermatitis vasculitis), vitiligo and Wegener's granulomatosis.

  Autoimmune therapies and their dosages, routes of administration and recommended usage are known in the art and are described in literature such as the Physician's Desk Reference (56th edition, 2002 and 57th edition, 2003). .

5.5.5.1. Other Therapies The present invention provides a method for preventing, managing, treating or alleviating an autoimmune disorder or one or more symptoms thereof, wherein the method is immunospecific for the liquid formulation of the present invention and the integrin α _v β _3. Administering to the subject in need thereof one or more therapies (eg, prophylactic or therapeutic agents) other than the binding antibody or antibody fragment. Any drug or treatment known, used for, or currently used for the prevention, management, treatment or alleviation of an autoimmune disorder or one or more symptoms thereof Can be used in combination with the liquid formulations of the present invention according to the invention described herein. Examples of such agents include, but are not limited to, immunomodulators, anti-inflammatory agents and TNF-α antagonists. Specific examples of immunomodulators, anti-inflammatory agents and TNF-α antagonists that can be used in combination with the liquid formulations of the invention for the prevention, management, treatment or alleviation of autoimmune disorders are described herein above. Is disclosed.

In certain embodiments, patients with multiple sclerosis (MS) are prevented in combination with other drugs or treatments useful in the prevention, treatment, management and alleviation of MS, including but not limited to: An effective or therapeutically effective amount of a liquid formulation of the invention is administered: IFN-β1b (Betaseron) (eg, 8 million international units (MIU) are administered by subcutaneous injection every other day); IFN- β1a (Avonex) (eg, 6.0 MIU is administered by intramuscular injection once a week); glatiramer acetate (Copaxone) (eg, 20 mg is administered by daily subcutaneous injection); mitoxantrone (eg, 12 mg / m ² is administered by intravenous infusion every 3 months); azathioprine (eg, ² mg to 3 mg per kg body weight is orally administered each day); Methotrexate (eg, 7.5 mg orally administered once a week); cyclophosphamide; intravenous immunoglobulin (eg, 0.15 to 0.2 g / kg body weight administered monthly for up to 2 years) Glucocorticoid; methylprednisolone (eg, administered at a high dose every other bimonthly); 2-chlorodeoxyadenosine (Cladribine); baclofen (eg, 15-15 mg / d to 80 mg / d in divided doses, or Orally at higher doses up to 240 mg / d or intrathecally via an indwelling catheter; cycloenzaprine hydrochloride (eg 5 mg to 10 mg twice a day or 1 day) 3 times); clonazepam (for example, 0.5 mg to 1.0 mg 3 times a day, including bedtime dose); (Eg, 0.1 mg to 0.2 mg 3 times a day, including a bedtime dose); carbamazepine (eg, 100 mg / d to 1200 mg / d in escalated doses); gabapentin (eg, 300 mg / d-3600 mg / d); dilantin (eg 300 mg / d to 400 mg / d); amitriptyline (eg 25 mg / d to 150 mg / d); baclofen (eg 10 mg / d to 80 mg / d); primidone (Eg, 125 mg to 250 mg twice a day or three times a day); ondansetron (eg, 4 mg to 8 mg twice a day or three times a day); isoniazid (eg, up to 1200 mg in divided doses) ); Oxybutynin (eg, 5 mg twice a day or three times a day); tolterodine (eg, 1 g-2 mg twice a day; propantheline (eg, 7.5 mg-15 mg four times a day); bethanechol (eg, 10 mg-50 mg three times a day or four times a day); terazosin hydrochloride (eg, , Sildenafil citrate (eg, 50 mg to 100 mg orally as needed); amantadine (eg, 100 mg twice daily); pemoline (eg, 37.5 mg 2 times a day) Times); high dose vitamins; calcium orotate; ganciclovir; antibiotics; and plasma exchange.

  In certain embodiments, patients with psoriasis have a prophylactically effective amount or a therapeutically effective amount in combination with other agents or treatments useful in the prevention, treatment, management and alleviation of psoriasis, including but not limited to: Of topical steroid creams or ointments; tar (e.g. Estar, Psorigel, Fototar cream, and 10% mixed in Nutraderm lotion or directly with 0.1% triamcinolone cream Occlusion; topical vitamin D analogs (non-limiting examples are calcipotriene ointments); ultraviolet light; PUVA (soralen + ultraviolet A); methotrexate (Eg in doses divided every 12 hours for up to 25 mg once a week or once a week for 3 doses); Adult retinoid (a non-limiting example is, for example, etretinate at a dosage of 0.5 mg / kg / d to 1 mg / kg / d); an immunomodulatory treatment (a non-limiting example is cyclosporine); sulfasalazine (Eg at a dosage of 1 g 3 times a day).

  In certain embodiments, patients with Crohn's disease include a prophylactically effective amount or treatment in combination with other drugs or treatments useful in the prevention, treatment, management and alleviation of Crohn's disease including, but not limited to: An effective amount of a liquid formulation of the invention is administered: an antidiarrheal (a non-limiting example is diphenoxylate with 2 mg to 4 mg of loperamide up to 4 times a day with 1 tablet of atropine up to 4 times a day ) 8 to 15 drops of opium tincture up to 4 times a day, 2g to 4g of cholestyramine once or twice a day or 5g of colestipol); an anticonvulsant (a non-limiting example is propane Terin 15 mg, dicyclomine 10 mg to 20 mg or hyoscyamine 0.125 mg given before meals); 5-amino Salicylic acid agents (non-limiting examples are sulfasalazine 1.5 g to 2 g twice a day, particularly high dosages (eg, 4 g of Pentasa 1 g and 4 days of Asacol 0.8 g to 1.2 g) ), Mesalamine (Asacol) and its sustained release form (Pentasa); corticosteroids; immunomodulatory drugs (non-limiting examples include azathioprine (1MG / KG to 2MG / KG), mercapto Purines (50MG-100MG), cyclosporine and methotrexate); antibiotics; TNF inhibitors (including but not limited to INFLIXMAB); immunosuppressants (including but not limited to tacrolimus, mycophenolate mofetil and thalidomide) Not); anti-inflammatory cytokines (includes IL-10 and IL-11) But not limited to); Nutrition; enteral therapy with elemental diet (e.g., VIVONEX over 4 weeks); and total parenteral nutrition (total parenteral nutrition).

  In certain embodiments, patients with lupus erythematosus are prophylactically effective or therapeutically effective in combination with other drugs or treatments useful in the prevention, treatment, management and alleviation of lupus erythematosus, including but not limited to: A liquid formulation of the invention is administered: antimalarial agents (including but not limited to hydroxychloroquine); glucocorticoids (eg, low dose, high dose or high dose intravenous + treatment that can be used Immunosuppressive agents (including but not limited to cyclophosphamide, chlorambucil and azathioprine); cytotoxic agents (including but not limited to methotrexate and mycophenolate mofetil); androgenic steroids (danazol) Including, but not limited to); and Coagulant (including but warfarin not limited to).

5.6. Method of administering antibody preparation The present invention provides an inflammatory disease, autoimmune disease, disorder related to abnormal bone metabolism, abnormality of integrin α _v β ₃ by administering an effective amount of the liquid preparation of the present invention to a subject. Methods of treating, preventing, and ameliorating disorders associated with normal expression and / or activity, disorders associated with abnormal angiogenesis, or cancer, or one or more symptoms thereof. Various delivery systems are known and can be used to administer the liquid formulations of the invention, or therapeutic or prophylactic agents. The method of administering the antibody liquid preparation or treatment (prophylactic or therapeutic agent) of the present invention includes parenteral administration (for example, intradermal administration, intramuscular administration, intraperitoneal administration, intravenous administration, and subcutaneous administration), hard administration. Intramembrane administration, topical administration, intrapulmonary administration, and mucosal administration (eg, intranasal and oral routes) are included, but are not limited to these. In certain embodiments, the liquid formulations of the invention are administered intramuscularly, intravenously, or subcutaneously. In a preferred embodiment, the liquid formulation of the present invention is administered subcutaneously. The formulation may be administered by any convenient route, for example, by injection or bolus injection, by absorption through epithelial cell layers or mucosal skin cell layers (eg, oral mucosa, rectal mucosa, and intestinal mucosa) Can also be administered with other agents having biological activity. It can be administered systemically or locally. In certain embodiments, the liquid formulations of the invention are administered intratumoral or at the site of inflammation.

In general, the antibody or antibody fragment that integrin alpha _v beta ₃ immunospecifically bind included in the liquid formulations of the present invention is of the same species origin or species reactivity as recipient of a liquid formulation of the present invention. Thus, in a preferred embodiment, the liquid formulation of the invention comprising an integrin alpha _v beta ₃ that immunospecifically binds to a human or humanized antibody contained in the liquid formulations of the present invention, to a human patient for therapy or prophylaxis Be administered.

The present invention provides that the liquid formulation of the present invention is packaged in sealed containers such as ampoules and sachettes indicating the amount of antibody or antibody fragment. The liquid formulation of the present invention is preferably present in a sealed container indicating the amount and concentration of the antibody or antibody fragment. The liquid formulation of the present invention has at least 15 mg / ml, 20 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, 100 mg / ml, 150 mg / ml 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, or 20 ml in a concentration of ml, 175 mg / ml, 200 mg / ml, 250 mg / ml, or 300 mg / ml, and Most preferably, an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is supplied in a sealed container in an amount of 1.2 ml. In certain embodiments of the invention, the liquid formulation of the invention is at least 15 mg / ml, 20 mg / ml, 25 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 175 mg / ml for intravenous injection. an antibody or antibody fragment (eg, VITAXIN® or an antigen-binding fragment thereof) that immunospecifically binds to integrin α _v β ₃ at a concentration of ml, 200 mg / ml, 250 mg / ml, or 300 mg / ml; For repeated subcutaneous administration, a concentration of at least 15 mg / ml, 20 mg / ml, 50 mg / ml, 80 mg / ml, 100 mg / ml, 150 mg / ml, 175 mg / ml, 200 mg / ml, 250 mg / ml, or 300 mg / ml in integrin alpha _v beta ₃ and immune antibodies specifically bind or Body fragments (e.g., VITAXIN (R) or an antigen-binding fragment thereof) is preferably supplied in a sealed container.

Inflammatory disease, autoimmune disease, disorder associated with abnormal bone metabolism, disorder associated with abnormal expression and / or activity of integrin α _v β ₃ , disorder associated with abnormal angiogenesis, or cancer, or 1 thereof The amount of the liquid formulation of the present invention effective to prevent, treat, manage or ameliorate the above symptoms can be determined by standard clinical techniques well known in the art or described herein. The exact dose to be used in this formulation will depend on the route of administration and the severity of the inflammatory disorder, autoimmune disorder or cancer and should be determined according to the judgment of the physician and the condition of each patient. Effective doses can also be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  For antibodies, proteins, polypeptides, peptides, and fusion proteins encompassed by the present invention, the dose administered to a patient is typically about 0.0001 mg / kg to 100 mg / kg per patient body weight. The dose administered to the patient is preferably 15 mg / kg to 50 mg / kg per patient body weight, more preferably 5 mg / kg to 25 mg / kg or 5 mg / kg to 15 mg / kg per patient body weight. In certain embodiments, the dosage to the patient is 5 mg / kg, 8 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg or 50 mg / kg per patient body weight. . Due to the immune response against foreign polypeptides, human antibodies generally have a longer half-life in the human body than antibodies from other species. Thus, administration of human antibodies can be performed at smaller doses and less frequently. Furthermore, the dosage, volume, and frequency of administration of the liquid formulation of the invention can increase the concentration of the antibody or antibody fragment in the formulation, enhance the affinity and / or binding activity of the antibody or antibody fragment, And / or can be decreased by increasing the half-life of the antibody or antibody fragment.

  Examples of small molecule doses include small molecular weights per kilogram of subject or sample, milligram quantities or microgram quantities (eg, from about 1 microgram per kilogram to about 500 milligrams per kilogram, 1 kilogram From about 100 micrograms per kilogram to about 5 milligrams per kilogram, or from about 1 microgram per kilogram to about 50 micrograms per kilogram).

In certain embodiments, an antibody or antibody fragment (eg, VITAXIN® or a fragment thereof) that immunospecifically binds to integrin α _v β ₃ in a liquid formulation of the invention is 0.1-20 mg / kg / week, preferably 1-15 mg / kg / week, more preferably 2-8 mg / week, even more preferably 3-7 mg / kg / week, most preferably 4-6 mg / kg / week, for inflammatory disorders, autoimmune disorders or Administer to a subject with cancer. In another embodiment, one or more doses of a prophylactically or therapeutically effective amount of a liquid formulation of the invention is administered to a subject, wherein the prophylactically or therapeutically effective amount is not the same at each dose.

In a specific embodiment, liquid formulations of the present invention, the plasma concentration of antibodies immunospecific for the integrin alpha _v beta _3, continuously blocking integrin alpha _v beta ₃ activity, preferred level (e.g., about 0 .1 to about 100 mg / kg). In specific embodiments, the plasma concentration of the antibody is 0.2 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml, 6 μg / ml, 7 μg / ml. maintained at ml, 8 μg / ml, 9 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 30 μg / ml, 35 μg / ml, 40 μg / ml, 45 μg / ml, or 50 μg / ml . The preferred plasma concentration in a subject depends on several factors, including but not limited to the nature of the disease or disorder, the severity of the disease or disorder, and the condition of the subject. Such dosage regimes are particularly beneficial for the prevention, treatment, management, and amelioration of chronic diseases or disorders.

In another embodiment, the liquid formulation of the present invention comprises at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% of bone resorption. %, At least 90%, or at least 95% associated with abnormal bone metabolism using a dosage regimen that maintains plasma concentrations of antibodies or antibody fragments immunospecific for integrin α _v β ₃ It is administered to a subject with a disease or disorder. In certain embodiments, the antibody or antibody fragment immunospecific for integrin α _v β ₃ is maintained at about 0.1 μg / ml to about 100 μg / ml in a patient with a disease or disorder associated with abnormal bone metabolism. The

In a specific embodiment, a liquid formulation comprising a conjugated antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is administered intermittently. As used herein, “conjugated antibody or antibody fragment” refers to other residues (including but not limited to heterologous peptides, polypeptides, other antibodies or antibody fragments, marker sequences, diagnostic agents, therapeutic agents, radioactive metal ions, Means an antibody or antibody fragment attached to a polymer, albumin, and solid support).

In another embodiment, the subject, preferably a human, the prophylactically or therapeutically effective amount of an antibody or antibody fragment that binds to integrin alpha _v beta ₃ in liquid formulation immunospecifically of the present invention (e.g., VITAXIN (registered Trademark) or antigen-binding fragments thereof), wherein a dose of a prophylactically or therapeutically effective amount of an antibody or antibody fragment in a liquid formulation of the invention administered to said subject is treated For example, 0.01 μg / kg, 0.02 μg / kg, 0.04 μg / kg, 0.05 μg / kg, 0.06 μg / kg, 0.08 μg / kg, 0.1 μg / kg, 0.2 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 0.75 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 4 μg / kg, 5 μg / kg, 10 μ / Kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg 75 μg / kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, or 125 μg / kg.

In another embodiment, the subject, preferably a human, has a prophylactically or therapeutically effective amount of an antibody or antibody fragment (eg, VITAXIN®) that immunospecifically binds to integrin α _v β ₃ in a liquid formulation of the invention. Or a fragment thereof), wherein a dose of a prophylactically or therapeutically effective amount of an antibody or antibody fragment in a liquid formulation of the invention administered to the subject is treated Accordingly, for example, 0.01 μg / kg, 0.02 μg / kg, 0.04 μg / kg, 0.05 μg / kg, 0.06 μg / kg, 0.08 μg / kg, 0.1 μg / kg,. 2 μg / kg, 0.25 μg / kg, 0.5 μg / kg, 0.75 μg / kg, 1 μg / kg, 1.5 μg / kg, 2 μg / kg, 4 μg / kg, 5 μg / kg, 10 μg / kg 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 35 μg / kg, 40 μg / kg, 45 μg / kg, 50 μg / kg, 55 μg / kg, 60 μg / kg, 65 μg / kg, 70 μg / kg, 75 μg / kg Increase by kg, 80 μg / kg, 85 μg / kg, 90 μg / kg, 95 μg / kg, 100 μg / kg, or 125 μg / kg.

  Prophylactic or therapeutic doses are described in the Physician's Desk Reference (56th edition, 2002, 57th edition, 2003).

5.7. Biological Assays Some embodiments of the liquid formulations of the present invention are preferably tested in vitro, cell culture systems, and animal model organisms (eg, rodent animal model systems) for the desired therapeutic activity. Used in humans. For example, an assay that can be used to determine whether administration of a liquid formulation of the present invention is indicated is a cell culture assay, in which a patient tissue sample is grown in culture and the liquid formulation of the present invention is Exposed to or contacted with, and the effect of such compositions on the tissue sample is observed. A tissue sample is obtained from the patient by biopsy. This test allows the identification of the therapeutically most effective prophylactic or therapeutic agent for each patient. In various specific embodiments, associated with autoimmune diseases, inflammatory diseases, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, abnormal blood vessel formation Or a representative cell of a type of cell involved in cancer (eg, endothelial cells, cancer cells, activated T cells, osteoclasts, and B cells). An assay can be performed to determine if the type of cell has the desired effect. A low level of contacted cell growth or survival indicates that the liquid formulation of the present invention is effective in treating, managing, preventing or ameliorating a patient's symptoms. Alternatively, instead of culturing patient cells, cells of tumor or malignant tumor cell lines or endothelial cell lines may be used to screen the liquid formulations of the present invention. Many assays standard in the art can be used to assess such survival and / or proliferation: known by direct cytometry, eg, by measuring 3H-thymidine incorporation. Cell proliferation can be measured by detecting transcriptional activity of genes (eg, fos and myc) or changes in cell cycle markers. Cell viability can be assessed by trypan blue staining, and differentiation can be assessed visually based on changes such as morphology.

  The binding specificity, affinity and functional activity of the antibody or antibody fragment of the liquid formulation of the present invention can be analyzed by various in vitro binding and cell adhesion assays known in the art, such as, but not limited to, international Patent Publications WO00 / 78815 and WO02 / 070007, US Patent No. 6,248,326, US Patent No. 6,472,403, Pecheur et al., The FASEB J. 16 (10): 1266-8 (2002), Almed et al., The Journal of Histochemistry & Cytochemistry 50: 1371-1379 (2002), all of which are incorporated herein by reference.

The binding specificity of the antibody or antibody fragment of the liquid formulation of the present invention should be assessed by measuring binding to α _v β ₃ and cross-reactivity with other α _v -or β ₃ -containing integrins. Can do. In particular, binding specificity measures binding to α _11b β ₃ (the main integrin expressed on platelets) and α _v β ₅ (an integrin commonly found in endothelial and connective tissue cells). Can be evaluated. Briefly, to examine cross-reactivity, integrins are coated on ELISA plates and measured for antibody binding activity against integrin α _v β ₃ and other integrins. Integrins α _v β ₃ and α _v β ₅ can be obtained by methods known in the art such as Cheresh, 1987, Proc. Natl. Acad. Sci. USA 84: 6471-6475, and Cheresh and Spiro, 1987, J. Biol. Can be isolated by affinity chromatography as described in Chem. 262: 17703-17711. In a specific embodiment, α _v β ₃ is isolated from octyl glucoside human placental lysate using an anti-α _v β ₃ antibody affinity column, and the integrin α _v using an anti-α _v affinity column. α _v β ₅ is isolated from the β ₃ deletion column flow-through. Antibody binding activity is assessed by ELISA using goat anti-human IgG-alkaline phosphatase conjugate. Purified human IgG ₁ antibody, can be used as a control.

In another embodiment, a competitive binding assay with the parent anti-integrin alpha _v beta ₃ antibody or antibody fragment against integrin alpha _v beta _3, the binding affinity and specificity is evaluated. Competitive binding is measured by an ELISA assay. Antibody binding is measured in the presence of increasing concentrations of antibody competitor. Alternatively, the control competitor antibody is a steroidal anti-inflammatory agent-human IgG _1.

In another embodiment, binding affinity and specificity are assessed by measuring the inhibitory activity of an antibody or antibody fragment on integrin α _v β ₃ binding to fibrinogen. Briefly, integrin α _v β ₃ is plated on an ELISA plate. The inhibitory activity of an antibody or antibody fragment can be quantified by measuring the amount of bound biotinylated fibrinogen in the presence of increasing concentrations of antibody or control antibody. Streptavidin alkaline phosphatase is used to detect bound fibrinogen.

In another embodiment, the specificity of antibody or antibody fragment binding is assessed by inhibition of integrin α _v β ₃ binding in a cell adhesion assay. Endothelial cell adhesion events are an important component of the angiogenic process, and inhibition of α _v β ₃ is known to reduce tumor angiogenesis and hence tumor base rate. Inhibition of α _v β ₃ mediated cell attachment by anti-integrin α _v β ₃ antibody in these assays exhibits inhibitory activity when the antibody is used in situ or in vivo. Briefly, integrin α _v β ₃ positive M21 melanoma cells grown in RPMI with 10% FBS are used in these cell binding assays. Cells are released from the culture plate by trypsinization and resuspended in adhesion buffer at a concentration of 4 × 10 ⁵ cells / ml. The antibody and control antibody are diluted to the desired concentration with 250 μl of adhesion buffer (10 mM Hepes, 2 mM MgCl2, 2 mM CaCl2, 0.2 mM MnCl2, and 1% BSA in Hepes-buffered saline pH 7.4) to fibrinogen. Add to pre-coated 48-well plate. Each well is coated with 200 μl fibrinogen at a concentration of 10 μg / ml for 1 hour at 37 ° C. For the assay, cells (250 μl) containing equal amounts of antibody or isotype matched control antibody (250 μl) are added to each of the wells, mixed gently and incubated at 37 ° C. for 20 minutes. Adhesion buffer was washed to remove unbound cells until no cells remained in control wells coated with BSA alone. Bound cells are stained with crystal violet, which is then extracted with 100 μl acetic acid (10%) and quantified by measuring the absorbance of the solubilized dye at 560 nm.

In another embodiment, the inhibitory activity of an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is tested in an endothelial cell migration assay. In this regard, the Transwell cell migration assay is used to assess Vitaxin's ability to inhibit endothelial cell migration (Choi et al., 1994, J. Vascular Surg. 19: 125-134 and Leavesly et al., 1993, J Cell. Biol. 121: 163-170). Briefly, low-passage human umbilical vein endothelial cells in log phase were collected by gentle trypsinization, washed, and 37 ° C. HBS containing 1% BSA (20 mM Hepes, 150 mM NaCl, 1.8 mM MgCl2, Resuspend in 1.8 mM CaCl 2, 5 mM KCl, and 5 mM glucose, pH 7.4) at a concentration of 2 × 10 ⁶ cells / ml. Dilute antibody to 10 μg / ml for stock solution. Antibody is diluted 1: 1 into cells (final antibody concentration = 5 μg / ml; final cell concentration = 1 × 10 ⁶ cells / ml) and incubated on ice for 10-30 minutes. The cell / antibody suspension (200 μl in each compartment) is then added to the upper compartment of the Transwell cell culture chamber, and the lower compartment is coated with 0.5 ml of 10 μg / ml vitronectin (in HBS). Vitronectin acts as a chemoattractant for endothelial cells. The chamber is placed at 37 ° C. for 4 hours to allow cell migration. Visualization of cell migration is performed by first removing the remaining cells in the upper compartment with a cotton swab. Cells that have migrated to the underside of the insert are stained with crystal violet for 30 minutes, then solubilized in acetic acid, and the absorbance of the dye is measured at a wavelength of 550 nm. The amount of absorbance is directly proportional to the number of cells that have migrated from the upper chamber to the lower chamber.

In a preferred embodiment, BIAcore kinetic analysis is used to measure the binding on and off rates of antibodies or antibody fragments to integrin α _v β ₃ . BIAcore kinetic analysis involves analyzing integrin α _v β ₃ binding and dissociation from a chip with an antibody or fragment thereof immobilized on its surface.

  Additional examples of in vitro assays such as Western blot analysis, flow cytometry analysis, cell adhesion assay to cortical bone and extracellular matrix proteins, cell migration assay, cell invasion assay, and cell proliferation assay are described in Pecheur et al. The FASEB J. 16 (10): 1266-8 (2002), the entire text of which is hereby incorporated by reference.

The liquid formulations of the present invention are used in humans after being tested in a suitable animal model system. Such animal model systems include, but are not limited to, rats, mice, chickens, cows, monkeys, pigs, dogs, rabbits and the like. Any animal system known in the art can be used. In a specific embodiment of the invention, the liquid formulation of the invention is tested in a mouse model system. Such model systems are widely used and known to those skilled in the art, for example, SCID mouse models in which mouse integrin α _v β ₃ is replaced with human integrin α _v β ₃ or transgenic mice, nude with human xenografts Mouse, animal models in which antibodies that specifically bind to integrin α _v β ₃ or fragments thereof recognize the same target as VITAXIN (registered trademark), such as hamsters and rabbits, are known. “Relevance of Tumor Models for Anticancer Drug Development” (1999, edited by Fiebig and Burger); “Contributions to Oncology” (1999, Karger); “Nude mice in oncology research (The Nude Mouse in Oncology Research ”(1991, edited by Boven and Winograd); and“ Anticancer Drug Development Guide ”(1997, edited by Teicher) (see Which is hereby incorporated by reference in its entirety. The liquid formulation of the present invention can be administered repeatedly. Some aspects of operation can vary.

  Use various animal models known in the art associated with the target disease or disorder (eg, inflammatory diseases, autoimmune diseases, abnormal bone metabolism and / or disorders associated with abnormal angiogenesis, or cancer) Although not particularly limited, International Patent Publication WO00 / 78815, US Patent No. 6,248,326, US Patent No. 6,472,403, Pecheur et al., The FASEB J. 16 (10): 1266-8 (2002), Almed et al., The Journal of Histochemistry & Cytochemistry 50: 1371-1379 (2002), all of which are incorporated herein by reference.

In certain embodiments, inhibition of tumor growth by a liquid formulation of the invention is tested in two animal models. The first model measures angiogenesis in the chick chorioallantoic membrane (CAM). This assay is a well-recognized model of in vivo angiogenesis because all tissues are undergoing angiogenesis. In particular, this assay measures growth factor-induced angiogenesis of chick CAM blood vessels toward a filter disk immersed in growth factors or to tissue grown on CAM. Inhibition of angiogenesis is based on the amount and extent of new blood vessel growth or based on the inhibition of tissue growth on the CAM. This assay has been described in detail by other investigators and has been used to measure angiogenesis as well as angiogenesis of tumor tissue (Ausprunk et al., 1980, Am. J. Physiol. 79: 597-618 (19759 Ossonski et al., Cancer Res. 40: 2300-2309; Brooks et al., 1994, Science 264: 569-571, and Brooks et al., 1994, Cell 79: 1157-1164. Briefly, growth factor-induced angiogenesis To do this, punch out the filter discs using a skin biopsy punch from # 1 Whatman Qualitative Circles, first sterilize the discs by exposure to UV light, with various concentrations of TNFα or HBSS as a negative control under aseptic conditions. Saturate (at least 1 hour) Saturated filter discs are placed on CAM to induce angiogenesis, embryos are treated with various amounts of vitaxin and control (antibody or purified human IgG ₁ ), Inhibits angiogenesis, treatment by intravenous injection Perform approximately 24 hours after placing the disc 48 hours later, dissect the CAM and score angiogenesis on a scale of 1 to 4. The HBSS saturated filter disc is used as a negative control to prepare the CAM. Shows angiogenesis that occurs in response to tissue damage and subtracts these CAM values as a background.Because VITAXIN® is a human IgG ₁ subclass, purified human IgG ₁ is used as a negative control for injection Is done.

In addition to the above CAM assay using growth factor induced angiogenesis, further assays can be performed using tumor induced angiogenesis. For these assays, an α _v β ₃ negative tumor fragment is transplanted into CAM to induce angiogenesis. Use of alpha _v beta ₃ negative tumors fragment inhibition of tumor growth, be for the inhibition of alpha _v beta _3-mediated angiogenesis by endothelial cells from CAM, interposed alpha _v beta ₃ present on a tumor To ensure that this is not an adhesive event. Inhibition of tumor growth is assessed by placing a single cell suspension of FG (8 × 10 ⁶ cells, pancreatic cancer) and Hep-3 cells (5 × 10 ⁵ cells, pharyngeal cancer) on 30 μl CAM. One week later, the tumor is removed and cut into approximately 50 mg fragments, at which time they are placed on a new CAM. 24 hours after the second round, embryos are injected intravenously with Vitaxin or human IgG ₁ as a negative control. Tumors are allowed to grow for about 7 days and then removed and weighed.

  In the second animal model, inhibition of Vx2 cancer cells in rabbits is used as a measure of the inhibitory effect of the liquid formulation of the present invention on tumors. Vx2 cancer is a transplantable cancer derived from Shope virus-induced papilloma. It was first described in 1940 and has since been widely used in research on tumor invasion, tumor-host interactions and angiogenesis. Vx2 cancer is inherently fibrous, aggressive, and exhibits the characteristics of undifferentiated cancer. Vx2 tumor growth is achieved by continuous transplantation in donor rabbits. Following subcutaneous implantation, the host repair mechanism has been reported to begin 2-4 days after the initial inflammatory response. This repair mechanism is characterized by the production of new connective tissue and the production of new capillaries. Newly formed capillaries are confined to the repair zone by day 4 but have spread to the external area of the tumor by day 8. These characteristics and pharmacokinetics of the liquid formulations of the present invention can be used to determine initial doses and treatment schedules for these experiments.

  The growth of Vx2 tumors in the animal model is used to examine the effect of the liquid formulation of the present invention after early administration on the growth of primary tumors in rabbits transplanted subcutaneously with Vx2 cancer. Briefly, a Vx2 tumor (50 mg) is implanted into the inside of the rabbit's thigh through skin and muscle incisions. Primary tumor measurements are made during the experiment up to 25 days.

In another embodiment, Balb / c nu / nu mice are used as animal models to investigate different diseases, particularly those associated with abnormal bone metabolism and / or abnormal angiogenesis. Different cell lines expressing various types of α _v β ₃ (eg, CHO, or cancer cell types such as breast cancer cells) can be injected intravenously into nude mice. See Pecheru et al. (Supra). For example, CHO cells are transfected with various cDNA constructs (eg, wild type or mutant) of α _v β ₃ and injected intravenously into nude mice. The effects of α _v β ₃ (which has varying levels of activity due to mutations) and α _v β ₃ antibodies on bone metastases can be assessed, for example, by radiography, histological observation or statistical analysis of bone tissue it can.

  In another embodiment, an animal (a healthy and already built animal model) in a space environment (eg, a space shuttle) can be used to evaluate an antibody of the invention. Astronauts who have been flying in space for a long time are similar to osteoporotic patients, but have been proven to reduce bone density 10 times faster than humans who have the benefit of the Earth's gravity. The middle animal is an ideal osteoporosis model for measuring the effect of an antibody or antibody fragment of the invention on osteoporosis or diseases associated with abnormal bone metabolism and / or abnormal angiogenesis.

  In another embodiment, SCID mice with subcutaneously implanted human bone fragments (SCID-human-bone model) are used as animal models to treat diseases associated with abnormal bone metabolism and / or abnormal angiogenesis. The action of the antibody or antibody fragment of the present invention is evaluated. For example, cancer cells (eg, human prostate cancer cells) are injected directly into human bone fragments of an animal model. At the same time, antibody treatment is started. The effect of an antibody or antibody fragment of the invention on bone metastasis or angiogenesis can be assessed by comparison with a control group. See Nemeth et al., 2002, Clinical & Experimental Metastasis, 19 (separate volume 1): 47.

  The anti-inflammatory activity of the liquid formulations of the present invention is known in the art and is described in “Arthritis and Allied Conditions: A Textbook of Rheumatology” in Crofford LJ and Wilder RL, “Arthritis and Allied Conditions: A Textbook of Rheumatology”. Measurements can be made using a variety of experimental animal models described in “Autoimmunity in Animals”, McCarty et al. (Eds.), Chapter 30 (Lee and Febiger, 1993). Experimental and spontaneous animal models of inflammatory arthritis and autoimmune rheumatic diseases can also be used to evaluate the anti-inflammatory activity of the liquid formulations of the present invention.

  Major animal models of arthritis or inflammatory diseases known and widely used in the art include: adjuvant-induced arthritis rat model, collagen-induced arthritis rat and mouse model, and antigen-induced arthritis rat, rabbit And hamster models, all Crofford LJ and Wilder RL, “Arthritis and Allied Conditions: A Textbook of Rheumatology”, “Arthritis and Autoimmunity in Animals”, McCarty et al. Chapter 30 (Lee and Febiger, 1993), which is hereby incorporated by reference in its entirety.

  The anti-inflammatory activity of the liquid formulation of the present invention can be evaluated using a carrageenin-induced arthritis rat model. Carrageenan-induced arthritis has been used in rabbits, dogs and pigs to study chronic arthritis or inflammation. Quantitative histomorphological evaluation is used to determine the therapeutic effect. A method for using such a carrageenin-induced arthritis rat model is described by Hansra P. et al., “Carrageenin-induced arthritis in rats”, Inflammation, 24 (2): 141-155 (2000). Zymosan-induced inflammation animal models are also commonly used as known and described in the art.

  The anti-inflammatory activity of the liquid formulation of the present invention also demonstrated inhibition of rat carrageenin-induced footpad edema by Winter CA et al. Can be evaluated using a modification of the method described in Exp. Biol. Med. 111, 544-547. This assay has been used as a primary in vivo screen for the anti-inflammatory activity of most NSAIDs and is expected to predict human effects. The anti-inflammatory activity of the liquid formulation of the present invention is expressed as a percent inhibition of the increase in hind paw weight of the test group compared to the vehicle-treated control group.

  In an embodiment of the invention where the experimental animal model used is an adjuvant-induced arthritis rat model, the body weight may be measured relative to the control group to measure the anti-inflammatory activity of the liquid formulation of the invention. it can. Alternatively, the effect of the liquid formulation of the present invention can be assessed using an assay that measures bone loss. Animal models such as ovariectomy-induced bone resorption mice, rat and rabbit models are known in the art for obtaining dynamic parameters of bone formation. Bone volume is measured in vivo by micro-computed tomography analysis and bone histology analysis using methods such as those described by Yositake et al. Or Yamamoto et al. Yoshitake et al., 1999, “Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption”, Proc. Natl. Acad. Sci. USA 96: 8156-8160; Yamamoto et al., 1998, “integrin ligand. Echistatin prevents bone loss in ovariectomized mice and rats ", Endocrinology 139 (3): 1411-1419, both of which are hereby incorporated by reference in their entirety.

  In addition, animal models of inflammatory bowel disease can also be used to evaluate the effects of the liquid formulations of the present invention (Kim et al., 1992, Scand. J. Gastroentrol. 27: 529-537; Strober, 1985, Dig. Dis. Sci. 30 (12 Suppl); 3S-10S). Ulcerative colitis and Crohn's disease are human inflammatory bowel diseases that can be induced in animals. Sulfated polysaccharides (including but not limited to amylopectin, carrageenan, amylopectin sulfate, and dextran sulfate) or chemical stimulants (but not limited to trinitrobenzene sulfonic acid (TNBS)) and acetic acid are orally administered to animals. Can induce inflammatory bowel disease.

  Asthma animal models can be used to assess the efficacy of the liquid formulations of the present invention. Examples of such models are associated with a strong neutrophil (TH1) and eosinophil (TH2) pulmonary mucosal inflammatory response, with TF effector cells migrating to the airway upon induction of air allergen in TH1 or TH2 recipient mice Mouse adoptive transfer model (Cohn et al., 1997, J. Exp. Med. 1861-1747).

  Animal models of autoimmune disease can also be used to evaluate the effects of the compositions of the invention and combination therapies. Animal models of autoimmune diseases (eg, type I diabetes, thyroid autoimmunity, systemic lupus erythematosus, and glomerulonephritis) have been developed (Flanders et al., 1999, Autoimmunity 29: 235-246; Krogh et al., 1999, Biochimie 81: 511-515; Foster, 1999, Semin. Nephrol. 19: 12-24).

  In addition, the invention disclosed herein for inflammatory diseases, autoimmune diseases, abnormal bone metabolism or disorders associated with abnormal angiogenesis, and / or cancer using any assay known to those of skill in the art The prophylactic or therapeutic utility of the liquid formulation can be evaluated. Assays known in the art (eg, those described above) can be used to assess the prophylactic and / or therapeutic oil requirements of the liquid formulations of the present invention when combined with one or more other treatments. .

  The effect of the liquid formulation of the present invention on peripheral blood lymphocytes can be followed / evaluated using standard methods known to those skilled in the art. A subject's peripheral blood lymphocyte count can be determined, for example, by obtaining a peripheral blood sample from the subject and using Ficoll-Hypack (Pharmacia) gradient centrifugation for other components of peripheral blood lymphocytes (eg, plasma). ) Can be quantified by separating the lymphocytes from) and counting the lymphocytes using trypan blue. A subject's peripheral blood T cell count can be determined, for example, by obtaining a peripheral blood sample from the subject and using Ficoll-Hypaque (Pharmacia) gradient centrifugation for other components of peripheral blood lymphocytes (eg, plasma). ) From lymphocytes, labeled with TTC antigens (eg, CD3, CD4 and CD8) bound to FITC or phycoerythri and quantified by measuring the number of T cells by FACS be able to.

Toxicity and / or efficacy of the prophylactic and / or therapeutic protocols of the present invention can be determined by standard pharmacological methods in cell cultures or experimental animals, such as LD ₅₀ (dose lethal to 50% of the population) and ED ₅₀ ( The therapeutically effective dose in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the LD ₅₀ / ED ₅₀ . The liquid preparation of the present invention having a large therapeutic index is preferred. The liquid formulations of the present invention that exhibit toxic side effects may be used, but in order to reduce the possibility of injury to uninfected cells and thus reduce side effects, such substances are targeted to the affected tissue site Care should be taken to design the delivery system to be optimized.

Data obtained from cell culture assays and animal studies can be used to prepare dose ranges for prophylactic and / or therapeutic agents for use in humans. The dosage of such agents lies preferably little in a range of circulating concentrations that include the ED ₅₀ at all no toxicity. The dosage varies within this range depending on the dosage form used and the route of administration used. For any substance used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Doses are prepared in animal models to achieve a circulating plasma concentration range that includes an IC50 (ie, the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information is used to accurately determine useful doses in humans. Plasma levels are measured, for example, by high performance liquid chromatography.

5.8. Use of liquid formulations in integrin α _v β ₃ expression analysis The liquid formulations of the present invention can be used to visualize the expression of integrin α _v β ₃ in cells or cell lines, and tissue sections and biopsies. In certain embodiments, analysis of tissue samples and biopsies requires the use of frozen tissue. In a preferred embodiment, tissue samples and biopsies are prepared for tissue processing and paraffin embedding using standard methods, but immunohistochemistry of integrin α _v β ₃ in the resulting paraffin-embedded tissue. Dyeing becomes possible. According to the present invention, such methods can facilitate the analysis of integrin α _v β ₃ expression in tissue samples from clinical trials, animal models and biopsies.

By measuring the expression and / or activity level of integrin α _v β ₃ in cells or cell lines and tissue sections and biopsies using the liquid formulation of the present invention, cancer (eg lung cancer, breast cancer, prostate cancer or ovarian cancer) ) Can also be evaluated.

The liquid formulations of the present invention comprising labeled antibodies, fragments, derivatives and analogs thereof are associated with various diseases and disorders (eg, but not limited to inflammatory diseases, autoimmune diseases, abnormal bone metabolism). Can be used for diagnostic purposes to detect, diagnose or monitor disorders, disorders associated with abnormal angiogenesis, or cancer). Such diagnostic techniques are known in the art (eg, Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; and Jalkanen et al., 1987, J. Cell. Biol. 105 : 3087-3096). Other antibody-based methods useful for detecting protein gene expression include immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA). Labels for official antibody assays are known in the art, eg, enzyme labels such as glucose oxidase; radioisotopes such as iodine ( ¹²⁵ I, ¹²¹ I), carbon ( ¹⁴ C), sulfur ( ³⁵ S). , Tritium ( ³ H), indium ( ¹²¹ In) and technetium ( ⁹⁹ Tc); luminescent labels such as luminol; and oral labels such as fluorescein and rhodamine and biotin. Other diagnostic techniques known in the art include, but are not limited to, those described in the following references: International Publication No. WO 01/58483, US Pat. No. 6,248,326, Pecheur et al., The FASEB J. 16 (10): 1266-8 (2002), Ahmed et al., The Journal of Histochemistry & Cytochemistry 50: 1371-1379 (2002) (the entire text of which is incorporated herein by reference) Incorporated). In a preferred embodiment, antibodies or antibody fragments that immunospecifically bind to integrin α _v β ₃ are used for diagnostic purposes to detect, diagnose or monitor a disease or disorder. The detection or diagnosis of a disease or disorder can be accomplished using techniques well known to those skilled in the art in an effective amount in an in vitro or in vivo assay (ie, an amount effective to detect the expression of integrin α _v β ₃ ). The liquid preparation of the present invention can be used. In preferred embodiments, the disease or disorder utilizes a liquid formulation of the invention in an effective amount in standard imaging techniques known to those skilled in the art in a subject, preferably a mammalian subject, most preferably a human subject. Is detected.

The present invention includes a method for detecting or diagnosing a disease or disorder, which method comprises the following steps: a) a labeled antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ . Administering to the subject an effective amount of a liquid formulation; b) to allow the labeled antibody or antibody fragment to be selectively concentrated at any desired site in the subject (eg, a cancer site) ( And waiting for a certain time interval after the administering step to allow unbound labeled antibody or antibody fragment to be cleared to background level); c) determining background level And d) detecting the labeled molecule in the subject, so that the background Detecting a labeled antibody or antibody fragment above the threshold level indicates the presence of the disease.

The invention includes a method of detecting or diagnosing or monitoring a disease or disorder, the method comprising the following steps: a) a liquid of the invention comprising an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ Administering an effective amount of the formulation to a subject; b) administering a second labeled antibody or antibody fragment that recognizes the antibody or antibody fragment of the liquid formulation of the invention; c) a labeled antibody or antibody To allow the fragment to selectively enrich at any desired site in the subject (eg, cancer site) (and to allow unbound labeled antibody or antibody fragment to be cleared to background levels). Waiting) for a certain time interval after the administering step; d) background E) detecting a labeled antibody or antibody fragment in the subject so that the detection of the labeled antibody or antibody fragment above the background level is the presence of the disease Showing steps.

In some embodiments, the tissue to be analyzed according to the method of the present invention is tissue from a cancer patient obtained during surgery. See Ahmed et al., The Journal of Histochemistry & Cytochemistry 50: 1371-1379 (2002). For example, surgically obtained tissue from an ovarian cancer patient is divided and frozen in a cylinder of frozen section encapsulated medium (OCT) by immersing it in isopentane cooled in dry ice. Frozen tissue sections are cut to 5 μm thickness and stored immediately at −20 ° C. when not in use. For staining, sections are fixed in cold acetone for 15 minutes and kept in Tris buffer (100 mM, pH 7.6). Endogenous peroxidase activity is removed with 3% H ₂ O ₂ in methanol and endogenous biotin activity is all taken for 10 minutes using a series of diluted egg whites (5% in distilled water) and skim milk powder (5% in distilled water). And blocking. Sections are incubated with α _v β ₃ Mab for 1 hour in Tris buffer (100 mM, pH 7.6). Antibody binding is amplified over 15 minutes each using biotin and streptavidin HRP, and the complex is visualized using diaminobenzidine (DAB). Nuclei are stained brightly with Mayer's hematoxylin, placed in sections, and covered with a cover glass. Isotype IgG1 can be diluted appropriately and replaced with antibody as a negative control. Sections are evaluated microscopically for positive DAB staining by a trained physiologist and the degree of staining for α _v β ₃ expression is scored in a blinded test.

The present invention includes a method for diagnosing or detecting a disease or disorder, wherein the method comprises a labeled antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ of a liquid formulation of the present invention. Imaging the subject at regular time intervals after administering an effective amount to the subject, wherein the time interval includes the labeled antibody or antibody fragment at a particular site in the subject (eg, cancer Detection of a labeled antibody or antibody fragment localized at this site in the subject is sufficient to allow selective enrichment at the site) Indicates existence.

In some embodiments, the disease or disorder monitor provides methods for diagnosing the disease or disorder, eg, one month after the first diagnosis, six months after the first diagnosis and one year after the first diagnosis. This is done by iterating later. In certain embodiments of the invention, tumor density facilitates detection of tumors using anti-integrin α _v β ₃ antibodies according to the methods of the invention.

  The presence of labeled antibody or antibody fragment can be detected in the patient using methods known in the art for in vivo scanning. These methods depend on the type of label used. One skilled in the art can determine an appropriate method for detecting a particular label. Methods and devices that can be used in the diagnostic methods of the present invention include, but are not limited to: whole body scans such as computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging. (MRI) and ultrasound tomography. In certain embodiments, the antibody or antibody fragment is labeled with a radioisotope and detected in a patient using a radiation responsive surgical device (Thurston et al., US Pat. No. 5,441,050). In another embodiment, the antibody or antibody fragment is labeled with a fluorescent compound and detected in the patient using a fluorescence responsive scanning device. In another embodiment, the antibody or antibody fragment is labeled with a positron emitting metal and detected in the patient using positron emission tomography. In yet another embodiment, the antibody or antibody fragment is labeled with a paramagnetic label and detected in the patient using magnetic resonance imaging (MRI).

5.9. Kit The present invention provides a drug pack or kit comprising one or more containers containing the liquid formulation of the present invention. In certain embodiments, liquid formulations of the invention include, but are not limited to, heterologous proteins, heterologous polypeptides, heterologous peptides, large molecules, small molecules, marker sequences, detectable diagnostic agents, therapeutics It includes an antibody or antibody fragment that is recombinantly fused or chemically bound to another component including a component, a drug component, a radioactive metal ion, a secondary antibody, and a solid phase carrier. The present invention also includes one or more first containers containing a liquid formulation of the present invention and an inflammatory disease, a disorder associated with abnormal bone metabolism, a disorder associated with abnormal angiogenesis, an integrin α _v β ₃ One or more second containers containing one or more other prophylactic or therapeutic agents useful for the prevention, management or treatment of disorders associated with abnormal expression and / or activity, autoimmune diseases, or cancer; A drug pack or kit is provided. In some cases, such containers are approved by this agency for manufacture, use or sale for human administration in a form prescribed by the agency that regulates the manufacture, use or sale of the drug or biologic. May be accompanied by a notification indicating that

The present invention provides kits that can be used in the above methods. In one embodiment, the kit comprises the liquid formulation of the present invention in one or more containers. In another embodiment, the kit comprises a liquid formulation of the invention in one or more containers, and further in one or more containers, an inflammatory disease, a disorder associated with abnormal bone metabolism. One or more other useful for the prevention, management or treatment of disorders associated with abnormal angiogenesis, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ , autoimmune diseases, or cancer Includes drugs or therapeutics. In certain embodiments, the antibody or antibody fragment included in the liquid formulation is VITAXIN® or an antigen-binding fragment. In yet another embodiment, the antibody or antibody fragment contained in the liquid formulation is not VITAXIN® or an antigen-binding fragment. The kit further provides instructions and methods for administration, prevention, treatment, management or amelioration of the disease (eg, using the liquid formulation of the present invention alone or in combination with another prophylactic or therapeutic agent). It is preferable to include side effect information and dose information related to

Equivalents Those skilled in the art will recognize a number of equivalents to the specific embodiments described herein or may be ascertained without further routine experimentation. Such equivalents are considered to be encompassed by the following claims.

  All published documents, patents and patent applications mentioned in this specification are as if each individual published document, patent or patent application was identified and individually indicated to be incorporated herein by reference. Similarly, the entire text is incorporated herein by reference.

  Citation and explanation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

It is a schematic diagram showing the outline for preparing integrin alpha _v beta ₃ in purified antibodies that immunospecifically bind.

[Sequence Listing]

Claims (93)

An antibody preparation comprising an aqueous carrier, histidine, and an antibody or antibody fragment that immunospecifically binds to 50 mg / ml or more of integrin α _v β ₃ and substantially free of a surfactant or an inorganic salt.   The preparation according to claim 1, which is sterile.   The formulation of claim 1, wherein the aqueous carrier is distilled water.   The formulation according to claim 1 or 2, wherein the formulation has a pH in the range of 5.0 to 7.0.   The formulation of claim 1 or 2, wherein the antibody or antibody fragment is at a concentration of at least 95 mg / ml.   6. The formulation of claim 5, wherein the antibody or antibody fragment is at a concentration of at least 100 mg / ml.   7. The formulation of claim 6, wherein the antibody or antibody fragment is at a concentration of at least 150 mg / ml.   The formulation of claim 1 or 2, further comprising glycine at a concentration in the range of about 1 to about 10 mM.   The formulation of claim 1 or 2, wherein the histidine is at a concentration in the range of about 5 to about 25 mM. An antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is stable in a storage period of at least 15 days at 40 ° C. as measured by high-speed size exclusion chromatography (HPSEC). The formulation described. _3. The formulation of claim 1 or 2, wherein the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is stable during storage at ambient temperature for at least 6 months as measured by HPSEC. _3. The formulation of claim 1 or 2, wherein the antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is stable for at least 1.5 years of storage at 4 ° C. as measured by HPSEC.   11. The formulation of claim 10, wherein less than 5% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC.   12. A formulation according to claim 11, wherein less than 5% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC.   13. The formulation of claim 12, wherein less than 5% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC.   14. The formulation of claim 13, wherein less than 2% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC.   15. A formulation according to claim 14, wherein less than 2% of the antibody or antibody fragment forms an aggregate during storage as measured by HPSEC.   16. A formulation according to claim 15, wherein less than 2% of the antibody or antibody fragment forms an aggregate during storage as measured by HPSEC.   17. The formulation of claim 16, wherein less than 1% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC.   18. A formulation according to claim 17, wherein less than 1% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC.   19. A formulation according to claim 18, wherein less than 1% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC. 11. The formulation of claim 10, wherein the antibody or fragment thereof retains at least 80% of the ability to bind to integrin α _v β ₃ compared to a reference antibody representing the antibody prior to storage. Antibody, or at least 80% to retain, formulation of claim 11, wherein the binding capacity of the integrin alpha _v beta ₃ in comparison with a reference antibody fragment thereof represent antibodies before storage. 13. The formulation of claim 12, wherein the antibody or fragment thereof retains at least 80% of the ability to bind to integrin α _v β ₃ compared to a reference antibody representing the antibody prior to storage. 23. The formulation of claim 22, wherein the antibody or fragment thereof retains at least 85% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 24. The formulation of claim 23, wherein the antibody or fragment thereof retains at least 85% of the ability to bind to integrin [alpha] _{v [} beta] ₃ compared to a reference antibody representing the antibody prior to storage. 25. The formulation of claim 24, wherein the antibody or fragment thereof retains at least 85% of the ability to bind integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 26. The formulation of claim 25, wherein the antibody or fragment thereof retains at least 90% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 27. The formulation of claim 26, wherein the antibody or fragment thereof retains at least 90% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 28. The formulation of claim 27, wherein the antibody or fragment thereof retains at least 90% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 29. The formulation of claim 28, wherein the antibody or fragment thereof retains at least 95% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 30. The formulation of claim 29, wherein the antibody or fragment thereof retains at least 95% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody prior to storage. 32. The formulation of claim 30, wherein the antibody or fragment thereof retains at least 95% of the ability to bind to integrin α _v β ₃ compared to a reference antibody representing the antibody prior to storage.   The formulation according to claim 1 or 2, further comprising an excipient.   35. The formulation of claim 34, wherein the excipient is sugar.   35. The formulation of claim 34, wherein the excipient is a polyol.   3. The formulation of claim 2, wherein the antibody or antibody fragment is MEDI-522 (Vitaxin®) or an antigen binding fragment of MEDI-522 (Vitaxin®).   6. The formulation of claim 5, wherein the antibody or antibody fragment is MEDI-522 (Vitaxin®) or an antigen-binding fragment of MEDI-522 (Vitaxin®).   A pharmaceutical dosage unit form suitable for parenteral administration to a human comprising the antibody formulation of claim 2 or 37 in a suitable container.   40. The pharmaceutical dosage unit form of claim 39, wherein the antibody formulation is administered intravenously, subcutaneously or intramuscularly.   A pharmaceutical dosage unit form suitable for aerosol administration to a human comprising the antibody formulation of claim 2 or 37 in a suitable container.   42. The pharmaceutical dosage unit form of claim 41, wherein the antibody formulation is administered intranasally.   38. An antibody preparation produced by lyophilizing the aqueous antibody preparation according to claim 1, 2 or 37.   A sealed container containing the preparation according to claim 1, 2, 3 or 37.   44. A sealed container comprising the formulation of claim 43.   46. The sealed container of claim 45, having a volume sufficient for reconstitution with a pharmaceutically acceptable carrier.   The sealed container according to claim 46, wherein the carrier is water for injection, USP or physiological saline.   48. The sealed container according to claim 47, wherein the container maintains an aseptic environment and can reconstitute the preparation without impairing sterility. In one or more containers, containing an aqueous carrier, histidine, and an antibody formulation comprising an antibody or fragment thereof that immunospecifically binds to 50 mg / ml or more of integrin α _v β ₃ and instructions for use of the formulation The kit described above, wherein the preparation is substantially free of a surfactant or an inorganic salt.   50. The kit of claim 49, wherein the formulation is sterile.   50. The kit of claim 49, wherein the aqueous carrier is distilled water.   51. The kit according to claim 49 or 50, wherein the antibody or antibody fragment is MEDI-522 (Vitaxin®) or an antigen-binding fragment of MEDI-522 (Vitaxin®).   51. A kit according to claim 49 or 50, wherein the formulation is produced by freezing an aqueous antibody formulation.   53. The kit of claim 52, wherein the formulation is produced by freezing an aqueous antibody formulation.   50. The kit of claim 49, wherein the antibody or antibody fragment is at a concentration of 95 mg / ml. Inflammatory diseases, autoimmune diseases, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, or cancer, or 1 thereof A method for preventing, managing, treating or ameliorating the above symptoms, which comprises administering to a subject in need thereof an effective or preventive or therapeutic amount of the antibody preparation according to claim 2. Inflammatory diseases, autoimmune diseases, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, or cancer, or 1 thereof 38. A method for preventing, managing, treating or ameliorating the above symptoms, comprising administering to a subject in need thereof an effective amount for preventing or treating the antibody preparation according to claim 37.   58. The method of claim 56 or 57, wherein the formulation has a pH in the range of 5.0 to 7.0.   57. The method of claim 56, wherein the antibody or antibody fragment is at a concentration of at least 95 mg / ml, 100 mg / ml, 125 mg / ml, or 150 mg / ml. 58. The method of claim 57, wherein MEDI-522 or a fragment thereof that immunospecifically binds to integrin [alpha] _{v [} beta] ₃ is at a concentration of at least 95 mg / ml, 100 mg / ml, 125 mg / ml, or 150 mg / ml.   58. The method of claim 56 or 57, further comprising glycine at a concentration in the range of about 1 to about 10 mM.   58. The method of claim 56 or 57, wherein the histidine is at a concentration in the range of about 5 to about 25 mM. 57. The antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ is stable in a storage period of at least 15 days at 40 ° C. as measured by high-speed size exclusion chromatography (HPSEC). Method. 58. The method of claim 57, wherein MEDI-522 or an antibody fragment that immunospecifically binds to integrin [alpha] _{v [} beta] ₃ is stable for a storage period of at least 15 days at 40 [deg.] C as measured by HPSEC.   64. The method of claim 63, wherein less than 5% of the antibody or antibody fragment forms an aggregate during the storage period as measured by HPSEC. 65. The method of claim 64, wherein less than 5% of MEDI-522 or antibody fragments that immunospecifically bind to integrin [alpha] _{v [} beta] ₃ form aggregates during storage as measured by HPSEC. 64. The method of claim 63, wherein the antibody or fragment thereof retains at least 80% of the ability to bind to integrin [alpha] _{v [} beta] _{3 as} compared to a reference antibody representing the antibody or antibody fragment prior to storage. Retains at least 80% of the binding capacity of the integrin alpha _v beta ₃ in comparison with a reference antibody MEDI-522 or antibody fragments that immunospecifically bind to integrin alpha _v beta ₃ each represents a MEDI-522 or fragments before storage 65. The method of claim 64.   58. The method of claim 56 or 57, further comprising an excipient.   70. The method of claim 69, wherein the excipient is a sugar or a polyol.   58. The method of claim 56 or 57, wherein the formulation is administered parenterally.   72. The method of claim 71, wherein the formulation is administered intramuscularly, subcutaneously or intravenously.   58. The method of claim 56 or 57, wherein the reconstituted formulation is administered orally or intranasally. The subject, further comprising, according to claim 56 or 57 A method according to administering a prophylactically or therapeutically effective amount of an integrin alpha _v beta prophylactic other than an antibody or antibody fragment that immunospecifically binds to ₃ or therapeutic agent.   75. The method according to claim 74, wherein the prophylactic or therapeutic agent is an anti-inflammatory agent, an immunomodulator, an agent having a bone metabolism regulating action, an anti-angiogenic agent, or an anticancer agent.   76. The method of claim 75, wherein the anti-inflammatory agent is an anti-TNFα agent.   58. The method of claim 56 or 57, wherein the cancer is prostate cancer, ovarian cancer, lung cancer, breast cancer, colon cancer or melanoma.   58. The method of claim 56 or 57, wherein the cancer has metastasized to bone.   58. The method of claim 56 or 57, wherein the inflammatory disease is arthritis, pulmonary fibrosis, osteoarthritis, or inflammatory osteolysis.   58. The method of claim 56 or 57, wherein the autoimmune disease is rheumatoid arthritis or Crohn's disease.   58. The method of claim 56 or 57, wherein the disease associated with abnormal bone metabolism is osteoporosis. Inflammatory diseases, autoimmune diseases, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, or cancer, or 1 thereof A method for preventing, managing, treating or improving the above symptoms,
(A) reconstituting a frozen antibody formulation with a pharmaceutically acceptable carrier such that the antibody or antibody fragment concentration is 50 mg / ml or more, the frozen antibody formulation comprising an aqueous carrier, histidine, and It is produced by freezing an aqueous antibody preparation containing an antibody or fragment thereof that immunospecifically binds to 50 mg / ml or more of integrin α _v β ₃ , and the aqueous antibody preparation contains a surfactant or an inorganic salt. Said step, which is substantially free, and (b) administering a prophylactic or therapeutically effective amount of the reconstituted antibody formulation to a subject in need, said reconstituted antibody formulation comprising an aqueous carrier, Histidine and 50 mg / ml or more of the antibody or antibody fragment, and the reconstituted antibody formulation is substantially free of surfactant or inorganic salt and is sterile. Step,
Including the above method. Inflammatory diseases, autoimmune diseases, disorders associated with abnormal expression and / or activity of integrin α _v β ₃ , disorders associated with abnormal bone metabolism, disorders associated with abnormal angiogenesis, or cancer, or 1 thereof A method for preventing, managing, treating or improving the above symptoms,
(A) reconstituting a frozen antibody formulation with a pharmaceutically acceptable carrier such that the antibody or antibody fragment concentration is 50 mg / ml or more, wherein the frozen antibody formulation comprises an aqueous carrier, histidine, and The aqueous antibody prepared by freezing an aqueous antibody preparation containing MEDI-522 (VITAXIN (registered trademark)) or a fragment thereof that immunospecifically binds to 50 mg / ml or more of integrin α _v β ₃ The formulation is substantially free of surfactants or inorganic salts, and (b) administering a prophylactically or therapeutically effective amount of the reconstituted antibody formulation to a subject in need thereof, comprising: the reconstruction antibody formulation also MEDI-522 binds an aqueous carrier, histidine, and immunospecifically 50 mg / ml or more integrin alpha _v beta ₃ Including its fragments, and reconstituted antibody formulation is free of surfactant or inorganic salts substantially a sterile, step,
Including the above method.   84. The method of claim 82 or 83, wherein the pharmaceutically acceptable carrier is water for injection, USP or saline.   84. The method of claim 82 or 83, wherein the reconstituted antibody formulation is administered parenterally.   88. The method of claim 85, wherein the reconstituted antibody formulation is administered intramuscularly, subcutaneously or intravenously.   84. The method of claim 82 or 83, wherein the reconstituted formulation is administered orally or intranasally. 84. The method of claim 82 or 83, further comprising administering a prophylactic or therapeutic agent other than an antibody or antibody fragment that immunospecifically binds to integrin α _v β ₃ .   84. The method of claim 82 or 83, wherein the cancer is prostate cancer, ovarian cancer, lung cancer, breast cancer, colon cancer or melanoma.   84. The method of claim 82 or 83, wherein the cancer has metastasized to bone.   84. The method of claim 82 or 83, wherein the inflammatory disease is arthritis, pulmonary fibrosis, osteoarthritis, or inflammatory osteolysis.   84. The method of claim 82 or 83, wherein the autoimmune disease is rheumatoid arthritis or Crohn's disease.   84. The method of claim 82 or 83, wherein the disease associated with abnormal bone metabolism is osteoporosis.

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