WO2011162933A1

WO2011162933A1 - Anti-integrin immunoconjugate dosing regimens

Info

Publication number: WO2011162933A1
Application number: PCT/US2011/039129
Authority: WO
Inventors: Albert Qin; James J. O'leary; Joanne Elizabeth Sarah Schindler
Original assignee: Immunogen, Inc.
Priority date: 2010-06-04
Filing date: 2011-06-03
Publication date: 2011-12-29

Abstract

Methods of administering immunoconjugates that bind to integrin are provided. The methods comprise administering an anti-integrin immunoconjugate to a person in need thereof, for example, a cancer patient, at a therapeutically effective dosing regimen that results in minimal adverse effects.

Description

ΑΝΤΊ-INTEGRIN IMMUNOCONJUGATE DOSING REGIMENS

Field of the Invention

[0001] The field of the invention generally relates to methods of administering anti-alphaV integrin immunoconjugates for the treatment of diseases, such as cancer. The methods provide dosing regimens that minimize unwanted side-effects.

Background of the Invention

(0002] Leukocyte antigen alphaV integrin ("alphaV integrin") is also known as CD51, MSK8, Vitronectin receptor subunit alpha, or VNRA. Recent studies have identified a role for integrins in angiogenic processes, and there is considerable evidence to suggest that tumor growth is dependent on angiogenesis. Therefore, integrins are a promising therapeutic target for cancers.

[0003] During angiogenesis, a number of integrins that are expressed on the surface of activated endothelial cells regulate critical adhesive interactions with a variety of extracellular matrix (ECM) proteins to regulate distinct biological events such as cell migration, proliferation and differentiation. Specifically, the closely related but distinct integrins alphaVbeta3 and alphaVbeta5 have been shown to mediate independent pathways in the angiogenic process. An antibody generated against alphaVbeta3 blocked basic fibroblast growth factor (bFGF) induced angiogenesis, whereas an antibody specific to alphaVbeta5 inhibited vascular endothelial growth factor (VEGF) induced angiogenesis (Eliceiri, et al., J. Clin. Invest. 103: 1227-1230 (1999); Friedlander et al., Science 270: 1500-1502 (1995)). Therefore, integrins, and especially the alpha V subunit containing integrins, are reasonable therapeutic targets for diseases that involve angiogenesis such as disease of the eye and neoplastic disease, tissue remodeling such as restenosis, and proliferation of certain cell types particularly epithelial and squamous cell carcinomas.

[0004] Antibodies are emerging as a promising method to treat such cancers. In addition, immunoconjugates, which comprise an antibody conjugated to another compound, for example, a cytotoxin, are also being investigated as potential therapeutics. In particular, immunoconjugates comprising maytansinoids, which are plant derived anti-fungal and anti-tumor agents, have been shown to have some beneficial activities. The isolation of three ansa macrolides from ethanolic extracts of Maytenus ovatus and Maytenus buchananii was first reported by S. M. Kupchan et al. and is the subject of U.S. Pat. No. 3,896,11 1 along with demonstration of their anti-leukemic effects in murine models at the microgram/kg dose range. Maytansinoids, however, have unacceptable toxicity, causing both central and peripheral neuropathies, and side effects: particularly nausea, vomiting, diarrhea, elevations of hepatic function tests and, less commonly, weakness and lethargy. This overall toxicity is reduced to some extent by the conjugation of maytansinoids to antibodies because an antibody conjugate has a toxicity which is several orders of magnitude lower on antigen-negative cells compared to antigen-positive cells. However, immunoconjugates comprising maytansinoids have still been associated with unacceptable levels of adverse side effects. For example, animals injected with high dosages of anti-alphaV integrin immunoconjugates comprising a maytansinoids showed a drop in body weight which is indicative of intolerance to the immunoconjugate. The cause of this toxicity, for example, whether it could be related to Cmax or AUC, and its effect in humans was not known. Therefore, the toxicity profile was not known or predictable in humans.

[0005] As a result, there is still a need to identify particular dosage regimens of anti-alphaV integrin immunoconjugates that are therapeutically effective in humans but avoid adverse effects.

BRIEF SUMMARY OF THE INVENTION

[0006] Methods of administering an anti-integrin immunoconjugate at a therapeutically effective dosing regimen that minimizes unwanted side-effects are provided herein. Thus, described herein are methods for treating a patient having cancer comprising administering to the patient an effective dose of an immunoconjugate which binds to alphaV integrin, wherein the immunoconjugate is administered at a dose of about 5 to about 3₅₀ nig/m².

[0007] In some embodimenets, the immunoconjugate comprises an antibody that competitively inhibits the binding of an antibody with the sequences of SEQ ID NOs:4-6 and 7-9 to alpha V integrin. In some embodiments, the antibody is CNT09₅. In some embodiments, the immunoconjugate comprises a maytansinoid. In some embodiments, the maytansinoid is DM4. In some embodiments, the immunoconjugate comprises a linker that is SPDB. In some embodiments, the immunoconjugate is IMGN388.

[0008] The dose can be administered at about 30 mg/m². In other embodiments, the dose is about 45 mg/m². In some embodiments, the dose is about 60 mg/m². In some embddiments, the dose is about 80 mg m². In some embodiments, the dose is about 105 mg/m². In some embodiments, the dose is about 130 mg/m². In some embodiments, the dose is about 145 mg/m². In some embodiments, the dose is about 160 mg/m². In some embodiments, the dose is about 200 mg/m². In some embodiments, the dose is about 250 mg/m². In some embodiments, the dose is about 300 mg/m². In some embodiments, the dose is about 350 mg/m².

[0009] According to the methods described herein, the immunoconjugate can be administered about once every 3 weeks. In other embodiments, the immunoconjugate is administered about once every 2 weeks, (n some embodiments, the immunoconjugate is administered about once every 1 week. In some embodiments, the immunoconjugate is administered about twice a week. In some embodiments, the immunoconjugate is administered in a 3-week (i.e. 21-day) dosing cycle, for example on days 1 and 8 of a 21-day cycle or on days 1, 8, and 1₅ of a 21-day cycle. In some embodiments, the immunoconjugate is administered in a 4-week (i.e. 28 day) dosing cycle.

[0010J According to the methods described herein, the administration can produce an AUC of about 400-1000 hr^g/mL. In some embodiments, the administration can produce an AUC of about 400-10,000 hrpg/mL. In addition, the admmistration can produce an Cmax of about 10-100 g/r L. In some embodiments, the administration can produce an Cmax of about 10-200 μg/mL.

[0011] In some embodiments, immunoconjugate is administered intravenously.

[0012] The methods described herein can be used to treat a cancer that is selected from the group consisting of renal carcinoma, neuroendocrine carcinoma, retroperitoneal sarcoma, colon cancer, adrenal carcmoma, Kaposi's sarcoma, peritoneal mesothelioma, prostate cancer, rectal cancer, ovarian carcinoma, fibrous histiocytoma, small bowel adenocarcinoma, non-small cell lung cancer (NSCLC), prostate cancer, ovarian carcinoma, gastric cancer, breast cancer, uterine cancer, neuroendocrine carcinoma, or bladder cancer. In some embodiments, the cancer expresses alphaV integrin. In some embodiments, the cancer is metastatic.

[0013] In some embodiments, the method further comprises administering a second anti-cancer agent to the patient. The anti-cancer agent can be chemotherapeutic agent.

[0014] In some embodiments, the method further comprises administering a steroid to the patient. The steroid can be dexamethasone.

[0015] The methods described herein can result in a stable disease. The methods described herein can result in a decrease in tumor size. The methods described herein can result in a decrease in angiogenesis. The methods described herein can result in decreased adverse effects.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0016] Figure 1 depicts the chemical structure of IMGN388.

[0017] Figure 2 depicts a graph illustrating the effect of IMGN388 administered at 0.₅ mg/kg, 1 mg/kg, 3 mg/kg, and 10 mg kg on the volume of tumors derived from human lung carcinoma in nude rats. The effect of PBS is shown for comparison.

[0018] Figure 3 depicts a graph illustrating the effect of IMGN388 administered at 1 mg/kg (weekly x 5), 3 mg/kg (weekly x 5), 10 mg kg (weekly x 5), and 15 mg kg (every 2 weeks x 3) on the volume of tumors derived from human breast carcinoma in nude rats. The effects of docetaxel administered at 10 mg/kg every 10 days x 4 and PBS are shown for comparison.

[0019] Figure 4 depicts integrin staining by immunohistochemistry on a tumor sample from a human patient with NSCLC. The staining was classified as strong (intensity) and heterogeneous (25-75% stained cells). [0020] Figure ₅ depicts graphs illustrating the maximal observed plasma concentration (Cmax) and exposure (AUC) observed in human patients treated with IMGN388.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a new dosing regimen for integrin binding imm unocongugates.

I. Definitions

[0022] To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

[0023] The terms "alphaV integrin," "alphaV subunit integrin", and "alphaV subunit containing integrin" are used interchangeably herein to mean alphaV transmembrane glycoprotein subunits of a functional integrin heterodimer and include all of the variants, isoforms and species homologs of alphaV. The terms encompass "full-length," unprocessed alphaV integrin as well as any form of alphaV integrin that results from processing in the cell. The term also encompasses naturally occurring variants of alphaV integrin, e.g., splice variants, allelic variants, and isoforms. The alphaV integrin polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. Accordingly, antibodies of the invention may, in certain cases, cross-react with alphaV from species other than human, or other proteins which are structurally related to human alphaV (e.g., human alphaV homologs). In other cases, the antibodies may be completely specific for human alphaV and not exhibit species or other types of cross-reactivity.

[0024] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be of any the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g. IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0025] A "blocking" antibody or an "antagonist" antibody is one which inhibits or reduces biological activity of the antigen it binds, such as alphaV integrin. In some embodiments, blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. The biological activity can be reduced by 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%.

[0026] The term "anti-alphaV integrin antibody" or "an antibody that binds to alphaV integrin" refers to an antibody that is capable of binding alphaV integrin with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent in targeting alphaV integrin. The extent of binding of an anti-alphaV integrin antibody to an unrelated, non-alphaV integrin protein can be less than about 10% of the binding of the antibody to alphaV integrin as measured, e.g., by a radioimmunoassay (RIA). In certain embodiments, an antibody that binds to alphaV integrin has a dissociation constant (Kd) of <1 μΜ, <100 nM, <10 nM, <1 nM, or <0.1 nM.

[0027J The term "antibody fragment" refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments.

[0028] A "monoclonal antibody" refers to a homogeneous antibody population involved in the highly specific recognition and binding of a single antigenic determinant, or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies directed against different antigenic determinants. The term "monoclonal antibody" encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single chain (scFv) mutants, fusion proteins comprising an antibody portion, and any other modified immunoglobulin molecule comprising an antigen recognition site. Furthermore, "monoclonal antibody" refers to such antibodies made in any number of manners including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0029] The term "humanized antibody" refers to forms of non-human (e.g. murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, humanized antibodies are human immunoglobulins in which residues from the complementary determining region (CDR) are replaced by residues from the CDR of a non-human species (e.g. mouse, rat, rabbit, hamster) that have the desired specificity, affinity, and capability (Jones et al., 1986, Nature, 321 :522-525; Riechmann et al., 1988, Nature, 332:323-327; Verhoeyen et al., 1988, Science, 239:1534-1536). In some instances, the Fv framework region (FR) residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species that has the desired specificity, affinity, and capability. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or capability. In general, the humanized antibody will comprise substantially all of at least one, and typically two or three, variable domains containing all or substantially all of the CDR regions that correspond to the non-human immunoglobulin whereas all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. 5,225,539.

[0030] A "variable region" of an antibody refers to the variable region of the antibody light chain or the variable region of the antibody heavy chain, either alone or in combination. The variable regions of the heavy and light chain each consist of four framework regions (FR) connected by three complementarity determining regions (CDRs) also known as hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda Md.)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

[0031] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 -107 of the light chain and residues 1-1 13 of the heavy chain) (e.g, Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (19 1)).

[0032] The amino acid position numbering as in Kabat, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). Using this numbering system, the actual linear amino acid sequence can contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain can include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues can be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence. Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the insertions at H3₅A and H35B; if neither 35A nor 3₅B is present, the loop ends at 32; if only 3₅A is present, the loop ends at 33; if both 3₅A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.

Loop Kabat AbM Chothia

LI L24-L34 L24-L34 L24-L34

L2 L50-L56 L50-L56 L50-L56

L3 L89-L97 L89-L97 L89-L97

HI H31-H35B H26-H35B H26-H32..34

(Kabat Numbering)

HI H31-H35 H26-H35 H26-H32

(Chothia Numbering)

H2 H50-H65 H50-H58 H52-H56

H3 H95-H102 H95-H102 H95-H102

[0033] The term "human antibody" means an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and/or antibodies comprising at least one human heavy and/or light chain polypeptide such as, for example, an antibody comprising murine light chain and human heavy chain polypeptides.

[0034] The term "chimeric antibodies" refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g. mouse, rat, rabbit, etc) with the desired specificity, affinity, and capability while the constant regions are homologous to the sequences in antibodies derived from another (usually human) to avoid eliciting an immune response in that species.

[0035] The term "epitope" or "antigenic determinant" are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. [0036] "Binding affinity" generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity" refers to intrinsic binding affinity which reflects a 1: 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative embodiments are described in the following.

[0037] "Or better" when used herein to refer to binding affinity refers to a stronger binding between a molecule and its binding partner. "Or better" when used herein refers to a stronger binding, represented by a smaller numerical Kd value. For example, an antibody which has an affinity for an antigen of "0.6 nM or better", the antibody's affinity for the antigen is <0.6 nM, i.e. 0.₅9 nM, 0.58 nM, 0.57 nM etc. or any value less than 0.6 nM.

[0038] By "specifically binds," it is generally meant that an antibody binds to an epitope via its antigen binding domain, and that the binding entails some complementarity between the antigen binding domain and the epitope. According to this definition, an antibody is said to "specifically bind" to an epitope when it binds to that epitope, via its antigen binding domain more readily than it would bind to a random, unrelated epitope. The term "specificity" is used herein to qualify the relative affinity by which a certain antibody binds to a certain epitope. For example, antibody "A" may be deemed to have a higher specificity for a given epitope than antibody "B," or antibody "A" may be said to bind to epitope "C" with a higher specificity than it has for related epitope "D. "

[0039] By "preferentially binds," it is meant that the antibody specifically binds to an epitope more readily than it would bind to a related, similar, homologous, or analogous epitope. Thus, an antibody which "preferentially binds" to a given epitope would more likely bind to that epitope than to a related epitope, even though such an antibody may cross-react with the related epitope.

[0040] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope to the extent that it blocks, to some degree, binding of the reference antibody to the epitope. Competitive inhibition may be determined by any method known in the art, for example, competition ELISA assays. An antibody may be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0041] The phrase "substantially similar," or "substantially the same", as used herein, denotes a sufficiently high degree of similarity between two numeric values (generally one associated with an antibody of the invention and the other associated with a reference/comparator antibody) such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., Kd values). The difference between said two values can be less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10% as a function of the value for the reference/comparator antibody.

[0042] A polypeptide, antibody, polynucleotide, vector, cell, or composition which is "isolated" is a polypeptide, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cell or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, an antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

[0043] As used herein, "substantially pure" refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

1 0441 The term "immunoconjugate" or "conjugate" as used herein refers to a compound or a derivative thereof that is linked to a cell binding agent (i.e., an anti-alphaV integrin antibody or f agment thereof) and is defined by a generic formula: C-L-A, wherein C = cytotoxin, L = linker, and A = anti- alphaV integrin antibody or antibody fragment. Immunoconjugates can also be defined by the generic formula in reverse order: A-L-C.

[0045] A "linker" is any chemical moiety that is capable of linking a compound, usually a drug, such as a maytansinoid, to a cell-binding agent such as an anti alphaV integrin antibody or a fragment thereof in a stable, covalent manner. Linkers can be susceptible to or be substantially resistant to acid-induced cleavage, light-induced cleavage, peptidase-induced cleavage, esterase-induced cleavage, and disulfide bond cleavage, at conditions under which the compound or the antibody remains active. Suitable linkers are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. Linkers also include charged linkers, and hydrophilic forms thereof as described herein and know in the art.

[0046] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. "Tumor" and "neoplasm" refer to one or more cells that result from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions. Examples of "cancer" or "tumorigenic" diseases which can be treated and/or prevented include renal carcinoma, neuroendocrine carcinoma, retroperitoneal sarcoma, colon cancer, adrenal carcinoma, Kaposi's sarcoma, peritoneal mesothelioma, prostate cancer, rectal cancer, ovarian carcinoma, fibrous histiocytoma, small bowel adenocarcinoma, non-small cell lung cancer (NSCLC), prostate cancer, ovarian carcinoma, gastric cancer, breast cancer, uterine cancer, neuroendocrind carcinoma, or bladder cancer. The cancer can be a cancer that expresses alphaV integrin.

[0047] The terms "cancer cell," "tumor cell," and grammatical equivalents refer to the total population of cells derived from a tumor or a pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the term "tumor cell" will be modified by the term "non-tumorigenic" when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

[0048] The term "subject" refers to any animal (e.g., a mammal), including, but not limited to humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms "subject" and "patient" are used interchangeably herein in reference to a human subject.

[0049] Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

[0050] The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The formulation can be sterile.

[0051] An "effective amount" of an antibody as disclosed herein is an amount sufficient to carry out a specifically stated purpose. An "effective amount" can be determined empirically and in a routine manner, in relation to the stated purpose.

[0052] The term "therapeutically effective amount" refers to an amount of an antibody or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of cancer, the therapeutically effective amount of the drug can reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent or stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the cancer. See the definition herein of "treating". To the extent the drug can prevent growth and/or kill existing cancer cells, it can be cytostatic and/or cytotoxic. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0053] A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Chemotherapeutic agents include, for example, antagonists of CD20 such as Rituximab and cyclophosphamide, doxorubicin, vincristine, predinisone, fludarabine, etoposide, methotrexate, lenalidomide, chlorambucil, bentamustine and/or modified versions of such chemotherapeutics.

[0100] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and 2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder. Thus, those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In certain embodiments, a subject is successfully "treated" for cancer according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity, tumorgenic frequency, or tumorgenic capacity, of a tumor; reduction in the number or frequency of cancer stem cells in a tumor; differentiation of tumorigenic cells to a non-tumorigenic state; or some combination of effects.

[0101 ] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention are based upon antibodies, in certain embodiments, the polypeptides can occur as single chains or associated chains.

[0102] The terms "identical" or percent "identity" in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences. One such non- limiting example of a sequence alignment algorithm is the algorithm described in Karlin et al, 1990, Proc. Natl. Acad. Sci., 87:2264-2268, as modified in Karlin et al., 1993, Proc. Natl. Acad. Sci., 90:5873- 5877, and incorporated into the NBLAST and XBLAST programs (Altschul et al., 1991, Nucleic Acids Res., 25:3389-3402). In certain embodiments, Gapped BLAST can be used as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. BLAST-2, WU-BLAST-2 (Altschul et al., 1996, Methods in Enzymology, 266:460-480), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in GCG software (e.g., using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1 , 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) can be used to determine the percent identity between two amino acid sequences (e.g., using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 1₄, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Miller (CABIOS, 4: 1 1-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. Appropriate parameters for maximal alignment by particular alignment software can be determined by one skilled in the art. In certain embodiments, the default parameters of the alignment software are used. In certain embodiments, the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be longer than the percent identity of the second sequence to the first sequence.

[0103] As a non-limiting example, whether any particular polynucleotide has a certain percentage sequence identity (e.g., is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical) to a reference sequence can, in certain embodiments, be determined using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482 489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed. [0104] In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. Identity can exist over a region of the sequences that is at least about 10, about 20, about 40-60 residues in length or any integral value therebetween, and can be over a longer region than 60-80 residues, for example, at least about 90-100 residues, and in some embodiments, the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a nucleotide sequence for example.

[0105] A "conservative amino acid substitution" is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. In some embodiments, conservative substitutions in the sequences of the polypeptides and antibodies of the invention do not abrogate the binding of the polypeptide or antibody containing the amino acid sequence, to the antigen(s), i.e., the alphaV integrin to which the polypeptide or antibody binds. Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate antigen binding are well- known in the art (see, e.g., Brummell et al., Biochem. 32: 1 180-1 187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0106] As used in the present disclosure and claims, the singular forms "a," "an," and "the" include plural forms unless the context clearly dictates otherwise.

[0107] It is understood that wherever embodiments are described herein with the language "comprising," otherwise analogous embodiments described in terms of "consisting of and/or "consisting essentially of are also provided.

[0108] The term "and/or" as used in a phrase such as "A and/or B" herein is intended to include both "A and B," "A or B," "A," and "B." Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

II. AlphaV integrin binding agents

[0109] The methods described herein provide methods of administering sequences that specifically bind alpha V integrin ("alphaV integrin binding agents"). In certain embodiments, the alphaV integrin binding agents are antibodies, immunoconjugates or polypeptides. The amino acid sequence for human alphaV integrin is known in the art and is also provided herein as represented by SEQ ID NO: 1.

Human CD37:

MAFPPRRRLRLGPRGLPLLLSGLLLPLCRAFNLDVDSPAEYSGPEGSYFGFAVDFFVPSASSRMFL

LVGAP ANTTQPGIVEGGQVLKCDWSSTRRCQPIEFDATGNRDYAKDDPLEFKSHQ FGASVRS

KQDKILACAPLYHWRTE QEREPVGTCFLQDGT TVEYAPCRSQDIDADGQGFCQGGFSIDFT

KADRVLLGGPGSFYWQGQLISDQVAEIVSKYDPNVYSIKYNNQLATRTAQAIFDDSYLGYSVAV

GDFNGDGIDDFVSGVPRAARTLGMVYIYDG NMSSLYNFTGEQMAAYFGFSVAATDINGDDYA

DVFIGAPLFMDRGSDGKLQEVGQVSVSLQRASGDFQTTKLNGFEVFARFGSAIAPLGDLDQDGF

NDIAIAAPYGGED KGIVYIFNGRSTGLNAVPSQILEGQWAARSMPPSFGYSMKGATDIDKNGYP

DLIVGAFGVDRAILYRARPVITVNAGLEVYPSILNQDNKTCSLPGTALKVSCFNVRFCLKADGKG

VLPR LNFQVELLLDKLKQKGAIRRALFLYSRSPSHS NMTISRGGLMQCEELIAYLRDESEFRDK

LTPITIFMEYRLDYRTAADTTGLQPILNQFTPANISRQAHILLDCGEDNVCKPBCLEVSVDSDQKKIY

IGDDNPLTLIVKAQNQGEGAYEAELIVSIPLQADFIGVVRN EALARLSCAFKTENQTRQVVCDL

GNPMKAGTQLLAGLRFSVHQQSEMDTSVKFDLQIQSSNLFDKVSPVVSHKVDLAVLAAVEIRGV

SSPDHVFLPIPNWEHBiENPETEEDVGPVVQHIYELRNNGPSSFSKAiMLHLQWPYKYN NTLLYIL

HYDIDGPMNCTSDMEINPLRIKI S SLQTTEKNDTVAGQGERDFtLITKRDLALSEGDIHTLGCGVAQ

CLi VCQVGRLDRGKSAILYVKSLLWTETFMNKENQNHSYSLKSSASFNVIEFPYKNLPIEDITNS

TLVTT VTWGIQPAPMPVPVWVIILAVLAGLLLLAVLVFVMYRMGFFKRVRPPQEEQEREQLQP

HENGEGNSET (SEQ IDNO:l)

[0110] Thus, in some embodiments, the alphaV integrin binding agents can bind to an epitope of SEQ ID NO:l .

[0111] In some embodiments, the alphaV integrin binding agents are humanized antibodies. In other embodiments, the alphaV integrin binding agent is fully human.

[0112] In certain embodiments, the alphaV integrin-binding agents have one or more of the following effects: induce stable disease, inhibit proliferation of tumor cells, reduce the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, inhibit tumor growth, increase survival, trigger cell death of tumor cells, differentiate tumorigenic cells to a non-tumorigenic state, or prevent metastasis of tumor cells.

[0113] In certain embodiments, the alphaV integrin-binding agents are capable of inducing complement dependent cytotoxicity. In certain embodiments, the alphaV integrin-binding agents are capable of inducing antibody dependent cell mediated cytotoxicity (ADCC). In some embodiments, the alphaV integrin-binding agents are capable of inducing apoptosis.

|0114] In some embodiments, the alphaV integrin-binding agents are capable of reducing tumor volume. The ability of a alphaV integrin-binding agent to reduce tumor volume can be assessed, for example, by measuring a %T/C value, which is the median tumor volume of treated subjects divided by the median tumor volume of the control subjects. In certain embodiments, immunoconjugates or other agents that specifically bind human alphaV integrin trigger cell death via a cytotoxic agent. For example, in certain embodiments, an antibody to a human alphaV integrin antibody is conjugated to a maytansinoid that is activated in tumor cells expressing the alphaV integrin by protein internalization. In certain embodiments, the alphaV integrin-binding agents are capable of inhibiting tumor growth. In certain embodiments, the alphaV integrin-binding agents are capable of inhibiting tumor growth in vivo (e.g., in a xenograft mouse model and/or in a human having cancer).

[0115] The alphaV integrin-binding agents include CNTO95. The production and characterization of CNTO95 have been described in detail in International Published Application No. WO 02/12501, U.S. Published Application No. 2003/040044, and U.S. Published Application No. 2006/0127407, each of which is incorporated by reference herein in its entirety. Thus, the alphaV integrin-binding agents also include alphaV integrin-binding agents that comprise the heavy and light chain CDR sequences of CNT095. The variable heavy chain, variable light chain, and CDR sequences of CNTO95 are shown in Table 1 below.

Table 1 : Variable chain and CDR amino acid sequences of CNTO95.

[0116] The alphaV integrin binding molecules can be antibodies or antigen binding fragments that specifically bind to alphaV integrin that comprise the CDRs of CNT095 with up to four (i.e. 0, 1 , 2, 3, or 4) conservative amino acid substitutions per CDR. Polypeptides can comprise one of the individual variable light chains or variable heavy chains described herein. Antibodies and polypeptides can also comprise both a variable light chain and a variable heavy chain.

[0117] Also provided are polypeptides that comprise a polypeptide having at least about 90% sequence identity to SEQ ID NO: 1 or SEQ ID NO:2. In certain embodiments, the polypeptide comprises a polypeptide having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: l or SEQ ID NO:2. Thus, in certain embodiments, the polypeptide comprises (a) a polypeptide having at least about 95% sequence identity to SEQ ID NO: l and/or (b) a polypeptide having at least about 95% sequence identity to SEQ ID NO:2. In certain embodiments, the polypeptide comprises (a) a polypeptide having the amino acid sequence of SEQ ID NO: l; and/or (b) a polypeptide having the amino acid sequence of SEQ ID NO:2. In certain embodiments, the polypeptide is an antibody and/or the polypeptide specifically binds alphaV integrin. In certain embodiments, the polypeptide is a murine, chimeric, or humanized antibody that specifically binds alphaV integrin. In certain embodiments, the polypeptide having a certain percentage of sequence identity to SEQ ID NO: l or SEQ ID NO:2 differs from SEQ ID NO: l or SEQ ID NO:2 by conservative amino acid substitutions only.

[0 18] Polypeptides can comprise one of the individual light chains or heavy chains described herein. Antibodies and polypeptides can also comprise both a light chain and a heavy chain.

[0119] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein ( 1975) Nature 256:495. Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is immunized as described above to elicit the production by lymphocytes of antibodies that will specifically bind to an immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies directed specifically against a chosen antigen as determined by immunoprecipitation, immunoblotting, or by an in vitro binding assay (e.g. radioimmunoassay (RIA); enzyme-linked immunosorbent assay (ELISA)) can then be propagated either in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an animal. The monoclonal antibodies can then be purified from the culture medium or ascites fluid as described for polyclonal antibodies above.

[0120] Alternatively monoclonal antibodies can also be made using recombinant DNA methods as described in U.S. Patent 4,816,567. The polynucleotides encoding a monoclonal antibody are isolated from mature B-cells or hybridoma cell, such as by RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequence is determined using conventional procedures. The isolated polynucleotides encoding the heavy and light chains are then cloned into suitable expression vectors, which when transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, monoclonal antibodies are generated by the host cells. Also, recombinant monoclonal antibodies or fragments thereof of the desired species can be isolated from phage display libraries expressing CDRs of the desired species as described (McCafferty et al., 1990, Nature, 348:552- 554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J. Mol. Biol., 222:581-597).

[0121] The polynucleotide(s) encoding a monoclonal antibody can further be modified in a number of different manners using recombinant DNA technology to generate alternative antibodies. In some embodiments, the constant domains of the light and heavy chains of, for example, a mouse monoclonal antibody can be substituted 1) for those regions of, for example, a human antibody to generate a chimeric antibody or 2) for a non-immunoglobulin polypeptide to generate a fusion antibody. In some embodiments, the constant regions are truncated or removed to generate the desired antibody fragment of a monoclonal antibody. Site-directed or high-density mutagenesis of the variable region can be used to optimize specificity, affinity, etc. of a monoclonal antibody.

[0122] In some embodiments, the monoclonal antibody against the human alphaV integrin is a humanized antibody. In certain embodiments, such antibodies are used therapeutically to reduce antigenicity and HAMA (human anti-mouse antibody) responses when administered to a human subject. Humanized antibodies can be produced using various techniques known in the art. In certain alternative embodiments, the antibody to alphaV integrin is a human antibody.

[0123] Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immunized individual that produce an antibody directed against a target antigen can be generated (See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boemer et al., 1991, J. Immunol., 147 (1):86- 95; and U.S. Patent 5,750,373). Also, the human antibody can be selected from a phage library, where that phage library expresses human antibodies, as described, for example, in Vaughan et al., 1996, Nat. Biotech., 14:309-314, Sheets et al., 1998, Proc. Nat'l. Acad. Sci., 95:6157-6162, Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381, and Marks et al, 1991, J. Mol. Biol, 222:581). Techniques for the generation and use of antibody phage libraries are also described in U.S. Patent Nos. 5,969, 108, 6,172,197, 5,885,793, 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al, 2007, J. Mol. Bio, doi: 10.1016/j.jmb.2007.12.018 (each of which is incorporated by reference in its entirety). Affinity maturation strategies and chain shuffling strategies (Marks et al, 1992, Bio/Technology 10:779-783, incorporated by reference in its entirety) are known in the art and can be employed to generate high affinity human antibodies.

[0124] Humanized antibodies can also be made in transgenic mice containing human immunoglobulin loci that are capable upon immunization of producing the full repertoire of human antibodies in the absence of endogenous immunoglobulin production. This approach is described in U.S. Patents 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0125] This invention also encompasses bispecific antibodies that specifically recognize a alphaV integrin. Bispecific antibodies are antibodies that are capable of specifically recognizing and binding at least two different epitopes. The different epitopes can either be within the same molecule (e.g. the same alphaV integrin) or on different molecules such that both, for example, the antibodies can specifically recognize and bind a alphaV integrin as well as, for example, 1) an effector molecule on a leukocyte such as a T-cell receptor (e.g. CD3) or Fc receptor (e.g. CD64, CD32, or CD16) or 2) a cytotoxic agent as described in detail below.

[0126] Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in a polypeptide of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or Fc receptors for IgG so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies possess an antigen- binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Techniques for making bispecific antibodies are common in the art (Millstein et al., 1983, Nature 305:537-539; Brennan et al„ 1985, Science 229:81; Suresh et al, 1986, Methods in Enzymol. 121 : 120; Traunecker et al., 1991, EMBO J. 10:3655-3659; Shalaby et al., 1992, J. Exp. Med. 175:217-225; ostelny et al., 1992, J. Immunol. 148: 1547-1553; Gruber et al., 1994, J. Immunol. 152:5368; and U.S. Patent 5,731, 168 . Antibodies with more than two valencies are also contemplated. For example, trispecific antibodies can be prepared (Tutt et al., J. Immunol. 147:60 (1991)). Thus, in certain embodiments the antibodies to alphaV integrin are multispecific.

(0127] In certain embodiments are provided an antibody fragment to, for example, increase tumor penetration. Various techniques are known for the production of antibody fragments. Traditionally, these fragments are derived via proteolytic digestion of intact antibodies (for example Morimoto et al., 1993, Journal of Biochemical and Biophysical Methods 24: 107-1 17; Brennan et al., 1985, Science, 229:81). In certain embodiments, antibody fragments are produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli or other host cells, thus allowing the production of large amounts of these fragments. Such antibody fragments can also be isolated from the antibody phage libraries discussed above. The antibody fragment can also be linear antibodies as described in U.S. Patent 5,641,870, for example, and can be monospecific or bispecific. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner.

[0128] According to the present invention, techniques can be adapted for the production of single- chain antibodies specific to alphaV integrin (see U.S. Pat. No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (Huse, et al., Science 246:1275-1281 (1989)) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for alphaV integrin, or derivatives, fragments, analogs or homologs thereof. Antibody fragments can be produced by techniques in the art including, but not limited to: (a) a F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (b) a Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment, (c) a Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent, and (d) Fv fragments.

[0129] It can further be desirable, especially in the case of antibody fragments, to modify an antibody in order to increase its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the antibody fragment by mutation of the appropriate region in the antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the antibody fragment at either end or in the middle (e.g., by DNA or peptide synthesis).

[0130] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0131] For the purposes of the present invention, it should be appreciated that modified antibodies can comprise any type of variable region that provides for the association of the antibody with the polypeptides of a human alphaV integrin. In this regard, the variable region can comprise or be derived from any type of mammal that can be induced to mount a humoral response and generate immunoglobulins against the desired tumor associated antigen. As such, the variable region of the modified antibodies can be, for example, of human, murine, non-human primate (e.g. cynomolgus monkeys, macaques, etc.) or lupine origin. In some embodiments both the variable and constant regions of the modified immunoglobulins are human. In other embodiments the variable regions of compatible antibodies (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding properties or reduce the immunogenicity of the molecule. In this respect, variable regions useful in the present invention can be humanized or otherwise altered through the inclusion of imported amino acid sequences.

[0132] In certain embodiments, the variable domains in both the heavy and light chains are altered by at least partial replacement of one or more CDRs and, if necessary, by partial f amework region replacement and sequence changing. Although the CDRs can be derived from an antibody of the same class or even subclass as the antibody from which the framework regions are derived, it is envisaged that the CDRs will be derived from an antibody of different class and possibly from an antibody from a different species. It is not alway necessary to replace all of the CDRs with the complete CDRs from the donor variable region to transfer the antigen binding capacity of one variable domain to another. Rather, in some cases it is only necessary to transfer those residues that are necessary to maintain the activity of the antigen binding site. Given the explanations set forth in U.S. Pat. Nos. 5,585,089, 5,693,761 and 5,693,762, it will be well within the competence of those skilled in the art, either by carrying out routine experimentation or by trial and error testing to obtain a functional antibody with reduced immunogenicity.

[0133] Alterations to the variable region notwithstanding, those skilled in the art will appreciate that the modified antibodies of this invention will comprise antibodies (e.g., full-length antibodies or immunoreactive fragments thereof) in which at least a fraction of one or more of the constant region domains has been deleted or otherwise altered so as to provide desired biochemical characteristics such as increased tumor localization or reduced serum half-life when compared with an antibody of approximately the same immunogenicity comprising a native or unaltered constant region. In some embodiments, the constant region of the modified antibodies will comprise a human constant region. Modifications to the constant region compatible with this invention comprise additions, deletions or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein can comprise alterations or modifications to one or more of the three heavy chain constant domains (CHI, CH2 or CH3) and/or to the light chain constant domain (CL). In some embodiments, modified constant regions wherein one or more domains are partially or entirely deleted are contemplated. In some embodiments, the modified antibodies will comprise domain deleted constructs or variants wherein the entire CH2 domain has been removed (ACH2 constructs). In some embodiments, the omitted constant region domain will be replaced by a short amino acid spacer (e.g. 10 residues) that provides some of the molecular flexibility typically imparted by the absent constant region.

[0134] Besides their configuration, it is known in the art that the constant region mediates several effector functions. For example, binding of the C 1 component of complement to antibodies activates the complement system. Activation of complement is important in the opsonisation and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. Further, antibodies bind to cells via the Fc region, with a Fc receptor site on the antibody Fc region binding to a Fc receptor (FcR) on a cell. There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibody to Fc receptors on cell surfaces triggers a number of important and diverse biological responses including engulfrnent and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (called antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, placental transfer and control of immunoglobulin production. [0135] In certain embodiments, the alphaV integrin-binding antibodies provide for altered effector functions that, in turn, affect the biological profile of the administered antibody. For example, the deletion or inactivation (through point mutations or other means) of a constant region domain can reduce Fc receptor binding of the circulating modified antibody thereby increasing tumor localization. In other cases, it can be that constant region modifications, consistent with this invention, moderate complement binding and thus reduce the serum half life and nonspecific association of a conjugated cytotoxin. Yet other modifications of the constant region can be used to eliminate disulfide linkages or oligosaccharide moieties that allow for enhanced localization due to increased antigen specificity or antibody flexibility. Similarly, modifications to the constant region in accordance with this invention can easily be made using well known biochemical or molecular engineering techniques well within the purview of the skilled artisan.

[0136] In certain embodiments, a alphaV integrin-binding agent that is an antibody does not have one or more effector functions. For instance, in some embodiments, the antibody has no antibody-dependent cellular cytotoxicity (ADCC) activity and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the antibody does not bind to an Fc receptor and/or complement factors. In certain embodiments, the antibody has no effector function.

[0137] It will be noted that in certain embodiments, the modified antibodies can be engineered to fuse the CH3 domain directly to the hinge region of the respective modified antibodies. In other constructs it can be desirable to provide a peptide spacer between the hinge region and the modified CH2 and/or CH3 domains. For example, compatible constructs could be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region with a 5-20 amino acid spacer. Such a spacer can be added, for instance, to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers can, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic, or even omitted altogether, so as to maintain the desired biochemical qualities of the modified antibodies.

[0138] Besides the deletion of whole constant region domains, it will be appreciated that the antibodies of the present invention can be provided by the partial deletion or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain can be enough to substantially reduce Fc binding and thereby increase tumor localization. Similarly, it can be desirable to simply delete that part of one or more constant region domains that control the effector function (e.g. complement CLQ binding) to be modulated. Such partial deletions of the constant regions can improve selected characteristics of the antibody (serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed antibodies can be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it can be possible to disrupt the activity provided by a conserved binding site (e.g. Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified antibody. Certain embodiments can comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function or provide for more cytotoxin or carbohydrate attachment. In such embodiments it can be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0139] The present invention further embraces variants and equivalents which are substantially homologous to the chimeric, humanized and human antibodies, or antibody fragments thereof, set forth herein. These can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.

[0140] The polypeptides of the present invention can be recombinant polypeptides, natural polypeptides, or synthetic polypeptides comprising an antibody, or fragment thereof, against a human alphaV integrin. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Thus, the invention further includes variations of the polypeptides which show substantial activity or which include regions of an antibody, or fragment thereof, against alphaV integrin protein. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.

[0141] The polypeptides and analogs can be further modified to contain additional chemical moieties not normally part of the protein. Those derivatized moieties can improve the solubility, the biological half life or absorption of the protein. The moieties can also reduce or eliminate any desirable side effects of the proteins and the like. An overview for those moieties can be found in REMINGTON'S PHARMACEUTICAL SCIENCES, 20th ed. Mack Publishing Co., Easton, PA (2000).

[0142] The isolated polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthetic methods to constructing a DNA sequence encoding isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof. See, e.g. Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81 :5662-5066 (1984) and U.S. Pat. No. 4,588,585. [0143] In some embodiments a DNA sequence encoding a polypeptide of interest would be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back- translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0144] Once assembled (by synthesis, site-directed mutagenesis or another method), the polynucleotide sequences encoding a particular isolated polypeptide of interest will be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0145] In certain embodiments, recombinant expression vectors are used to amplify and express DNA encoding antibodies, or fragments thereof, against human alphaV integrin. Recombinant expression vectors are replicable DNA constructs which have synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an anti-alphaV integrin antibody, or fragment thereof, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences, as described in detail below. Such regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0146] The choice of expression control sequence and expression vector will depend upon the choice of host. A wide variety of expression host vector combinations can be employed. Useful expression vectors for eukaryotic hosts, include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from Esherichia coli, including pCR 1, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13 and filamentous single-stranded DNA phages.

[0147] Suitable host cells for expression of a alphaV integrin-binding polypeptide or antibody (or a alphaV integrin protein to use as an antigen) include prokaryotes, yeast, insect or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems could also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985), the relevant disclosure of which is hereby incorporated by reference. Additional information regarding methods of protein production, including antibody production, can be found, e.g., in U.S. Patent Publication No. 2008/0187954, U.S. Patent Nos. 6,413,746 and 6,660,501, and International Patent Publication No. WO 04009823, each of which is hereby incorporated by reference herein in its entirety.

[0148] Various mammalian or insect cell culture systems are also advantageously employed to express recombinant protein. Expression of recombinant proteins in mammalian cells can be performed because such proteins are generally correctly folded, appropriately modified and completely functional. Examples of suitable mammalian host cell lines include the COS-7 lines of monkey kidney cells, described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing an appropriate vector including, for example, L cells, CI 27, 3T3, Chinese hamster ovary (CHO), HeLa and BH cell lines. Mammalian expression vectors can comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5' or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). [0149] The proteins produced by a transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza coat sequence and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, nuclear magnetic resonance and x-ray crystallography.

[0150] For example, supernatants from systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Mil!ipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matr ices comprising sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a alphaV integrin-binding agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

[0151] Recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0152] Methods known in the art for purifying antibodies and other proteins also include, for example, those described in U.S. Patent Publication No. 2008/0312425, 2008/0177048, and 2009/0187005, each of which is hereby incorporated by reference herein in its entirety.

[0153] In certain embodiments, the alphaV integrin-binding agent is a polypeptide that is not an antibody. A variety of methods for identifying and producing non-antibody polypeptides that bind with high affinity to a protein target are known in the art. See, e.g., Skerra, Curr. Opin. Biotechnol., 18:295- 304 (2007), Hosse et al., Protein Science, 15: 14-27 (2006), Gill et al., Curr. Opin. Biotechnol., 17:653-658 (2006), Nygren, FEBS J., 275:2668-76 (2008), and Skerra, FEBS J., 275:2677-83 (2008), each of which is incorporated by reference herein in its entirety. In certain embodiments, phage display technology has been used to identify/produce the alphaV integrin-binding polypeptide. In certain embodiments, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin.

[0154] In some embodiments, the agent is a non-protein molecule. In certain embodiments, the agent is a small molecule. Combinatorial chemistry libraries and techniques useful in the identification of nonprotein alphaV integrin-binding agents are known to those skilled in the art. See, e.g., Kennedy et al., J. Comb. Chem, 10:345-354 (2008), Dolle et al, J. Comb. Chem., 9:855-902 (2007), and Bhattacharyya, Curr. Med. Chem., 8: 1383-404 (2001), each of which is incorporated by reference herein in its entirety. In certain further embodiments, the agent is a carbohydrate, a glycosaminoglycan, a glycoprotein, or a proteoglycan.

[0155] In certain embodiments, the agent is a nucleic acid aptamer. Aptamers are polynucleotide molecules that have been selected (e.g., from random or mutagenized pools) on the basis of their ability to bind to another molecule. In some embodiments, the aptamer comprises a DNA polynucleotide. In certain alternative embodiments, the aptamer comprises an RNA polynucleotide. In certain embodiments, the aptamer comprises one or more modified nucleic acid residues. Methods of generating and screening nucleic acid aptamers for binding to proteins are well known in the art. See, e.g., U.S. Patent No. 5,270,163, U.S. Patent No. 5,683,867, U.S. Patent No. 5,763,595, U.S. Patent No. 6,344,321, U.S. Patent No. 7,368,236, U.S. Patent No. 5,582,981, U.S. Patent No. 5,756,291, U.S. Patent No. 5,840,867, U.S. Patent No. 7,312,325, U.S. Patent No. 7,329,742, International Patent Publication No. WO 02/077262, International Patent Publication No. WO 03/070984, U.S. Patent Application Publication No. 2005/0239134, U.S. Patent Application Publication No. 2005/0124565, and U.S. Patent Application Publication No. 2008/0227735, each of which is incorporated by reference herein in its entirety.

III. Immunoconjugates

[0156] Methods for administering conjugates comprising the anti-alphaV integrin antibodies, antibody fragments, and their functional equivalents as disclosed herein, linked or conjugated to a drug or prodrug (also referred to herein as immunoconjugates) are also described herein. Suitable drugs or prodrugs are known in the art. The drugs or prodrugs can be cytotoxic agents. The cytotoxic agent used in the cytotoxic conjugate of the present invention can be any compound that results in the death of a cell, or induces cell death, or in some manner decreases cell viability, and includes, for example, maytansinoids and maytansinoid analogs. Other suitable cytotoxic agents are for example benzodiazepines, taxoids, CC-1065 and CC-1065 analogs, duocarmycins and duocarmycin analogs, enediynes, such as calicheamicins, dolastatin and dolastatin analogs including auristatins, tomaymycin derivaties, leptomycin derivaties, methotrexate, cisplatin, carboplatin, daunombicin, doxorubicin, vincristine, vinblastine, melphalan, mitomycin C, chlorambucil and morpholino doxorubicin.

[0157] Such conjugates can be prepared by using a linking group in order to link a drug or prodrug to the antibody or functional equivalent. Suitable linking groups are well known in the art and include, for example, disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups.

[0158] The drug or prodrug can, for example, be linked to the anti-alphaV integrin antibody or fragment thereof through a disulfide bond. The linker molecule or crosslinking agent comprises a reactive chemical group that can react with the anti-alphaV integrin antibody or fragment thereof. The reactive chemical groups for reaction with the cell-binding agent can be JV-succinimidyl esters and N- sulfosuccinimidyl esters. Additionally the linker molecule comprises a reactive chemical group, which can be a dithiopyridyl group that can react with the drug to form a disulfide bond. Linker molecules include, for example, JV-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) (see, e.g., Carlsson et al., Biochem. J., 173: 723-737 (1978)), JV-succinimidyl 4-(2-pyridyldifhio)butanoate (SPDB) (see, e.g., U.S. Patent No. 4,563,304), JV-succinimidyl 4-(2-pyridyldithio)2-sulfobutanoate (sulfo-SPDB) (see US Publication No. 20090274713) , JV-succinimidyl 4-(2-pyridyldithio) pentanoate (SPP) (see, e.g., CAS Registry number 341498-08-6), 2-iminothiolane, or acetylsuccinic anhydride. For example, the antibody or cell binding agent can be modified with crosslinking reagents and the antibody or cell binding agent containing free or protected thiol groups thus derived is then reacted with a disulfide- or thiol-containing maytansinoid to produce conjugates. The conjugates can be purified by chromatography, including but not limited to HPLC, size-exclusion, adsorption, ion exchange and affinity capture, dialysis or tangential flow filtration.

[0159] In another aspect of the present invention, the anti-alphaV integrin antibody is linked to cytotoxic drugs via disulfide bonds and a polyethylene glycol spacer in enhancing the potency, solubility or the efficacy of the immunoconjugate. Such cleavable hydrophilic linkers are described in WO2009/0134976. The additional benefit of this linker design is the desired high monomer ratio and the minimal aggregation of the antibody-drug conjugate. Specifically contemplated in this aspect are conjugates of cell-binding agents and drugs linked via disulfide group (-S-S-) bearing polyethylene glycol spacers ((CH CH₂0 with a narrow range of drug load of 2-8 are described that show relatively high potent biological activity toward cancer cells and have the desired biochemical properties of high conjugation yield and high monomer ratio with minimal protein aggregation.

[0160] Antibody-maytansinoid conjugates with non-cleavable links can also be prepared. Such crosslinkers are described in the art (see US Publication No. 20050169933) and include but are not limited to, JV-succinimidyl 4-(maleimidomethyl) cyclohexanecarboxylate (SMCC). In some embodiments, the antibody is modified with crosslinking reagents such as succinimidyl 4-(N-maleimidomethyl)- cyclohexane-l-carboxylate (SMCC), sulfo-SMCC, maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfo-MBS or succinimidyl-iodoacetate, as described in the literature, to introduce 1-10 reactive groups (Yoshitake et al, Eur. J. Biochem., 101 :395-399 (1979); Hashida et al, J. Applied Biochem., 56-63 ( 1984); and Liu et al, Biochem., 18:690-697 (1979)). The modified antibody is then reacted with the thiol- containing maytansinoid derivative to produce a conjugate. The conjugate can be purified by gel filtration through a Sephadex G25 column or by dialysis or tangential flow filtration. The modified antibodies are treated with the thiol-containing maytansinoid (1 to 2 molar equivalent/maleimido group) and antibody- maytansinoid conjugates are purified by gel filtration through a Sephadex G-25 column, chromatography on a ceramic hydroxyapatite column, dialysis or tangential flow filtration or a combination of methods thereof. Typically, an average of 1 -10 maytansinoids per antibody are linked. One method is to modify antibodies with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (SMCC) to introduce maleimido groups followed by reaction of the modified antibody with a thiol-containing maytansinoid to give a thioether-linked conjugate. Again conjugates with 1 to 10 drag molecules per antibody molecule result. Maytansinoid conjugates of antibodies, antibody fragments, and other proteins are made in the same way.

[0161] In another aspect of the invention, the alphaV integrin antibody is linked to the drug via a non-cleavable bond through the intermediacy of a PEG spacer. Suitable crosslinking reagents comprising hydrophilic PEG chains that form linkers between a drug and the anti-alpbaV integrin antibody or fragment are also well known in the art, or are commercially available (for example from Quanta Biodesign, Powell, Ohio). Suitable PEG-containing crosslinkers can also be synthesized from commercially available PEGs themselves using standard synthetic chemistry techniques known to one skilled in the art. The drugs can be reacted with bifunctional PEG-containing cross linkers to give compounds of the following formula, Z -Xr-(-CH₂-Ci¾-0-)„-Y_p-D, by methods described in detail in US Patent Publication 20090274713 and in WO2009/0134976, which can then react with the cell binding agent to provide a conjugate. Alternatively, the cell binding can be modified with the bifunctional PEG crosslinker to introduce a thiol-reactive group (such as a maleimide or haloacetamide) which can then be treated with a thiol-containing maytansinoid to provide a conjugate. In another method, the cell binding can be modified with the bifunctional PEG crosslinker to introduce a thiol moiety which can then be treated with a thiol-reactive maytansinoid (such as a maytansinoid bearing a maleimide or haloacetamide), to provide a conjugate.

[0162] Examples of suitable PEG-containing linkers include linkers having an W-succinimidyl ester or iV-sulfosuccinimidyl ester moiety for reaction with the anti-alphaV integrin antibody or fragment thereof, as well as a maleimido- or haloacetyl-based moiety for reaction with the compound. A PEG spacer can be incorporated into any crosslinker known in the art by the methods described herein.

[0163] Many of the linkers disclosed herein are described in detail in U.S. Patent Publication Nos. 20050169933 and 20090274713, and in WO2009/0134976; the contents of which are entirely incorporated herein by reference.

[0164] The present invention includes aspects wherein about 2 to about 8 drug molecules ("drug load"), for example, maytansinoid, are linked to an anti-alphaV integrin antibody or fragment thereof, the anti-tumor effect of the conjugate is much more efficacious as compared to a drug load of a lesser or higher number of drugs linked to the same cell binding agent. "Drug load", as used herein, refers to the number of drug molecules (e.g., a maytansinoid) that can be attached to a cell binding agent (e.g., an anti- alphaV integrin antibody or fragment thereof). In one aspect the number of drug molecules that can be attached to a cell binding agent can average from about 2 to about 8 (e.g., 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1 , 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1). In some embodiments, the number of drgu molecules attached to a cell binding agent can average from about 3 to about 4. N² -deacetyl-W² -(3-mercapto-l-oxopropyl)- maytansine (DM1) and N² -deacetyl-N² -(4-mercapto-4-methyl-l-oxopentyl) maytansine (DM4) can be used.

[0165] The anti-alphaV integrin antibody or fragment thereof can be modified by reacting a bifiinctional crosslinking reagent with the anti-alphaV integrin antibody or fragment thereof, thereby resulting in the covalent attachment of a linker molecule to the anti-alphaV integrin antibody or fragment thereof. As used herein, a "bifiinctional crosslinking reagent" is any chemical moiety that covalently links a cell-binding agent to a drug, such as the drugs described herein. In another method, a portion of the linking moiety is provided by the drug. In this respect, the drug comprises a linking moiety that is part of a larger linker molecule that is used to join the cell-binding agent to the drug. For example, to form the maytansinoid DM1, the side chain at the C-3 hydroxyl group of maytansine is modified to have a free sulfhydryl group (SH). This thiolated form of maytansine can react with a modified cell-binding agent to form a conjugate. Therefore, the final linker is assembled from two components, one of which is provided by the crosslinking reagent, while the other is provided by the side chain from D 1.

[0166] The drug molecules can also be linked to the antibody molecules through an intermediary carrier molecule such as serum albumin.

[0167] As used herein, the expression "linked to a cell-binding agent" or "linked to an anti-alphaV integrin antibody or fragment" refers to the conjugate molecule comprising at least one drug derivative bound to a cell-binding agent anti-alphaV integrin antibody or fragment via a suitable linking group, or a precursor thereof. One linking group is SMCC.

[0168] In certain embodiments, cytotoxic agents useful in the present invention are maytansinoids and maytansinoid analogs. Examples of suitable maytansinoids include esters of maytansinol and maytansinol analogs. Included are any drugs that inhibit microtubule formation and that are highly toxic to mammalian cells, as are maytansinol and maytansinol analogs.

[0169] Examples of suitable maytansinol esters include those having a modified aromatic ring and those having modifications at other positions. Such suitable maytansinoids are disclosed in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331 ,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 7,276,497 and 7,473,796.

[0170] In a certain embodiment, the immunoconjugates of the invention utilize the thiol-containing maytansinoid (DM1), formally termed N¹ -deacetyl-iV² -(3-mercapto-l- oxopropyl)-maytansine, as the cytotoxic agent. DM1 is represented by the following structural formula (III):

[0171] In another emb

utilize the thiol-containing maytansinoid N¹ -deacetyk/V² (4-methyl-4-mercapto-l- oxopentyl)-maytansine (e.g., DM4) as the cytotoxic agent. DM4 is represented by the following structural formula (IV):

[0172] Another maytansinoid comprising a side chain that contains a sterically hindered thiol bond is N² -deacetyl-jV-² (4-mercapto-l-oxopentyl)-maytansine (termed DM3), represented by the following structural formula (V):

[0173] Each of the maytansinoids taught in US Patent No. 5,208,020 and 7,276,497, can also be used in the conjugate of the present invention. In this regard, the entire disclosure of 5,208,020 and 7,276,697 is incorporated herein by reference.

[0174] Many positions on maytansinoids can serve as the position to chemically link the linking moiety. For example, the C-3 position having a hydroxyl group, the C-14 position modified with hydroxymethyl, the C-15 position modified with hydroxy and the C-20 position having a hydroxy group are all expected to be useful. In some embodiments, the C-3 position serves as the position to chemically link the linking moiety, and in some particular embodiments, the C-3 position of maytansinol serves as the position to chemically link the linking moiety.

[0175]

Ab-PEG4-Mal-DM1

Ab-SIA-DMl (X)

Ab-sulfo-SPDB-DM4 (χτττ) [0176] Several descriptions for producing such antibody-maytansinoid conjugates are provided in U.S. Patent Nos. 6,333,410, 6,441 ,163, 6,716,821, and 7,368,565, each of which is incorporated herein in its entirety.

[0177] In general, a solution of an antibody in aqueous buffer can be incubated with a molar excess of maytansinoids having a disulfide moiety that bears a reactive group. The reaction mixture can be quenched by addition of excess amine (such as ethanolamine, taurine, etc.). The maytansinoid-antibody conjugate can then be purified by gel filtration. [0178] The number of maytansinoid molecules bound per antibody molecule can be determined by measuring spectrophotometrically the ratio of the absorbance at 252 nm and 280 nm. The average number of maytansinoid molecules antibody can be, for example, 1-10 or 2-5. The average number of maytansinoid molecules/antibody can be, for example about 3 to about 4. The average number of maytansinoid molecules/antibody can be about 3.5.

[0179] Conjugates of antibodies with maytansinoid or other drugs can be evaluated for their ability to suppress proliferation of various unwanted cell lines in vitro. For example, cell lines such as the human lymphoma cell line Daudi and the human lymphoma cell line Ramos, can easily be used for the assessment of cytotoxicity of these compounds. Cells to be evaluated can be exposed to the compounds for 4 to 5 days and the surviving fractions of cells measured in direct assays by known methods. IC₅o values can then be calculated from the results of the assays.

[0180] The immunoconjugates can, according to some embodiments described herein, be internalized into cells. The immunocongugate, therefore, can exert a therapeutic effect when it is taken up by, or internalized, by a alphaV integrin-expressing cell. In some particular embodiments, the immunoconjugate comprises an antibody, antibody fragment, or polypeptide, linked to a cytotoxic agent by a cleavable linker, and the cytotoxic agent is cleaved from the antibody, antibody fragment, or polypeptide, wherein it is internalized by a alphaV integrin-expressing cell.

[0181] In some embodiments, the immunoconjugates are capable of reducing tumor volume. For example, in some embodiments, treatment with an immunoconjugate results in a %T/C value that is less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some particular embodiments, the immunoconjugates can reduce tumor size in a BJAB xenograft model and/or a SU-DHL-4 xenograft model. In some embodiments, the immunoconjugates are capable of inhibiting metastases. In some embodiments, the immunoconjugates are capable of inhibiting angiogenesis. In some embodiments, the immunoconjugates are capable of inhibiting osteoclasts.

ΠΙ. Methods of administering AlphaV Integrin-Binding Agents

[0182] The alphaV integrin-binding agents (including antibodies, immunoconjugates, and polypeptides) of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as the treatment of cancer. In certain embodiments, the agents are useful for inhibiting tumor growth, inducing differentiation, inhibiting metastases, reducing tumor volume, and/or reducing the tumorigenicity of a tumor. In certain embodiments, the agents are useful for inhibiting angiogenesis. In some embodiments, the agents are useful for anti-osteoclastic effects. The methods of use can be in vivo methods. In certain embodiments, the alphaV integrin-binding agent or antibody or immunoconjugate, or polypeptide is an antagonist of the human alphaV integrin to which it binds. [0183] According to the methods described herein, the alphaV integrin-binding agents can be administered at particular dosages. For example, the alphaV integrin-binding agents can be administered at a dose of about 5 to about 375 mg/m². The alphaV integrin-binding agents can be administered at a dose of about 5 to about 350 mg/m². Thus, in some embodiments, the alphaV integrin-binding agents are administered at about 5 mg m². Thus, in some embodiments, the alphaV integrin-binding agents are administered at about 10 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 20 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 30 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 45 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 60 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 80 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 105 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 130 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 145 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 160 mg/m². In in some embodiments, the alphaV integrin-binding agents are administered at about 200 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 250 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 300 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 310 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 350 mg/m². In some embodiments, the alphaV integrin-binding agents are administered at about 375 mg m².

[0184] Furthermoe, the alphaV integrin-binding agents can be administered at particular dose interval. For example, the the alphaV integrin-binding agents can be administered from about four times a week to about once every four weeks. Thus, in some embodiments, the alphaV integrin-binding agents are administered about once every three weeks. In some embodiments, the alphaV integrin-binding agents are administered about once every two and a half weeks. In some embodiments, the alphaV integrin-binding agents are administered about once every two weeks. In some embodiments, the alphaV integrin-binding agents are administered about once every ten days. In some embodiments, the alphaV integrin-binding agents are administered about once every week. In some embodiments, the alphaV integrin-binding agents are administered about once every five days. In some embodiments, the alphaV integrin-binding agents are administered about once every four days. In some embodiments, the alphaV integrin-binding agents are administered about once every three days. In some embodiments, the alphaV integrin-binding agents are administered about once every two days. In some embodiments, the alphaV integrin-binding agents are administered about twice a week. In some embodiments, the alphaV integrin- binding agents are administered about three times a week. [0185] The alphaV integrin-binding agents can also be administered in an about 3-week (i.e. about 21 -day) cycle. For example, the alphaV integrin-binding agents can be administered twice in about 3 weeks. Thus, in some embodiments, the alphaV integrin-binding agents can be administered at about days 1 and 8 of a 21 -day cycle. In other embodiments, the alphaV integrin-binding agents can be administered three times in about 3 weeks. Thus, in some embodiments, the alphaV integrin-binding agents can be administered at about days 1, 8, and 15 of a 21-day cycle.

[0186] The alphaV integrin-binding agents can also be administered in an about 4-week (i.e. about 28-day) cycle. For example, the alphaV integrin-binding agents can be administered three times in about 4 weeks. Thus, in some embodiments, the alphaV integrin-binding agents can be administered at about days 1 , 8, and 15 of a 28-day cycle.

[0187] In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in a particular Cmax. For example, the alphaV integrin-binding agents can be administered at a dose that results in a Cmax of about 0.5 to about 240 g mL. Thus, in some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 0.5 to about 240 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 200 g mL. Thus, in some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 10 to about 150 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 100 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 75 μg/mL.

[0188] In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 240 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 200 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 150 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 100 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 80 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 5 to about 70 g mL.

[0189] In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 10 to about 250 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 10 to about 240 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 10 to about 200 g mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 10 to about 150 μ¾Ίη In some embodiments, the alphaV integrm-binding agents are administered at a dose that results in a Cmax of about 10 to about 100 g/mL. In some embodiments, the alphaV integrm-binding agents are administered at a dose that results in a Cmax of about 10 to about 80 g/mL. In some embodiments, the alphaV integrm-binding agents are administered at a dose that results in a Cmax of about 10 to about 70 μg/mL.

(0190] In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 1₅ to about 240 μ /mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 200 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 150 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 100 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 80 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 15 to about 70 g/mL.

[0191] In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 to about 240 g mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 to about 200 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 to about 150 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 to about 100 μ¾Ληί. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 to about 80 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 to about 70 μ^ηιΐ_^.

[0192] In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 to about 240 μ /ηΛ. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 to about 200 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 to about 150 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 to about 100 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 to about 80 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 to about 70 μg mL.

[0193] Furthermore, in some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 240 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 200 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 150 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 100 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 90 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 80 μ§/πύ_^. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 70 g/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 60 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about ₅0 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 40 μg mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 30 μg/mL. In some embodiments, the alphaV integrin-binding agents are administered at a dose that results in a Cmax of about 20 μg/mL.

[0194] In certain embodiments, the alphaV integrin-binding agents can be administered at a dose that results in a particular AUC. For example, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 50 hr^g/mL to about 13,000 hr^g mL. Thus, in some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 250 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 500 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 750 hr^g/mL. In some embodiments, the integrin- binding agents can be administered at a dose that results in an AUC of about 1 ,000 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 1,500 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 2,000 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 2,500 hr^g mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 3,000 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 3,500 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 4,000 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 5,000 hr- g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 7,500 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 10,000 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 12,500 hr^g/mL. In some embodiments, the integrin-binding agents can be administered at a dose that results in an AUC of about 13,000 hr^g/mL. [0195] In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 0 hr^«pg/mL to about 13,000 hr^»pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 400 hr-pg/mL to about 10,000 hr^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,000 hr^«pg/mL to about ₅,000 hr'pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,000

In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 2,000 hr^g/mL to about 4,000 hr^»pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 400 hr»pg/mL to about 1,000 hr-pg/mL.

[0196] In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 100 hr»pg/mL to about 13,000 hr-pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 400 hr^«pg/niL to about 10,000 hr«pg mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,000 hr^g/mL to about 5,000 hr^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,000 hr'pg/mL to about 4,000 hr^«pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 2,000 hr>pg/mL to about 3,000 hr^g/mL.

[0197] In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 500 hr'pg/mL to about 13,000 hr^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 500 hr'pg/mL to about 10,000 hr^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 500 hr^g/mL to about 5,000 hr^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 500 lu^ /mL to about 4,000 hr-pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 500 hr^g/mL to about 3,000 hr^g/mL.

[0198] In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,500 hr»pg mL to about 13,000 hr-pg/mL. In some embodiments, the alphaV integrin-binding agents can be admmistered at a dose that results in an AUC of about 1,500 hr^g/mL to about 10,000 hr'pg/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,500 hr^«pg/inL to about 5,000 hi^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,500 hr^g/mL to about 4,000 hr^g/mL. In some embodiments, the alphaV integrin-binding agents can be administered at a dose that results in an AUC of about 1,500 hr^g/mL to about 3,000 hr^«pg/mL. [0199] In certain embodiments, the disease treated with the alphaV integrin-binding agent or antagonist (e.g., an anti-alphaV integrin antibody) is a cancer. In certain embodiments, the cancer is characterized by alphaV integrin expressing cells to which the alphaV integrin-binding agent (e.g., antibody) binds. In certain embodiments, a tumor overexpresses the human alphaV integrin.

[0200] The present invention provides for methods of treating cancer comprising administering a therapeutically effective amount of a alphaV integrin-binding agent to a subject (e.g., a subject in need of treatment). Cancers that can be treated by the methods encompassed by the invention include, but are not limited to, neoplasms, tumors, metastases, or any disease or disorder characterized by uncontrolled cell growth. The cancer can be a primary or metastatic cancer. Specific examples of cancers that can be treated by the methods encompassed by the invention include, but are not limited to, cancer of the head, neck, eye, mouth, throat, esophagus, chest, bone, lung, colon, rectum, stomach, prostate, breast, ovaries, kidney, liver, pancreas, and brain. Additional cancers include, but are not limited to, the following: leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplasia syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to Hodgkin's disease, non-Hodgkin's lymphoma; myelomas such as multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedullary plasmacytoma; Waldenstrom's macroglobulinemia; bone cancer and connective tissue sarcomas such as bone sarcoma, myeloma bone disease, osteosarcoma, chondrosarcoma, Ewing's sarcoma, Paget's disease of bone, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors such as but not limited to, glioma, astrocytoma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including adenocarcinoma and intraductal carcinoma, and papillary breast cancer; adrenal cancer including pheochromocytoma and adrenocortical carcinoma; thyroid cancer; pancreatic cancer; pituitary cancers; eye cancers not limited to ocular melanoma, choroidal melanoma, cilliary body melanoma, and retinoblastoma; vaginal cancers; vulvar cancer; cervical cancers; uterine cancers not limited to endometrial carcinoma and uterine sarcoma; ovarian cancers; esophageal and other head and neck cancers such as but not limited to, squamous cancer, adenocarcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers; colon cancers; rectal cancers; liver cancers such as hepatocellular carcinoma and hepatoblastoma, gallbladder cancers; cholangiocarcinomas; lung cancers such as non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers, choriocarcinoma (yolk-sac tumor), prostate cancers; penal cancers; oral cancers not limited to squamous cell carcinoma; basal cancers; salivary gland cancers; renal cell cancer and other kidney cancers; and bladder cancers not limited to transitional cell carcinoma (for a review of such disorders, see DeVita, V. T., Hellman, S., & Rosenberg, S. A. Cancer: Principles and practice of oncology. Philadelphia: J. B. Lippincott Company; 6th Edition, 2001 ). Pre-malignant conditions may also be treated by the methods and compositions of the invention. Such cancers may include, but not be limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis, and myelodysplastic syndromes.

[0201] In certain embodiments, the cancer is a cancer selected from the group consisting of renal carcinoma, neuroendocrine carcinoma, retroperitoneal sarcoma, colon cancer, adrenal carcinoma, Kaposi's sarcoma, peritoneal mesothelioma, prostate cancer, rectal cancer, ovarian carcinoma, fibrous histiocytoma, small bowel adenocarcinoma, non-small cell lung cancer (NSCLC), prostate cancer, ovarian carcinoma, gastric cancer, breast cancer, uterine cancer, neuroendocrind carcinoma, or bladder cancer.

[0202J In certain embodiments, the method of inhibiting tumor growth comprises administering to a subject a therapeutically effective amount of a alphaV integrin-binding agent. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or has had a tumor removed.

[0203] In addition, the invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering a therapeutically effective amount of a alphaV integrin-binding agent to the subject. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the agent.

[0204] The present invention further provides pharmaceutical compositions comprising one or more of the alphaV integrin-binding agents described herein. In certain embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable vehicle. These pharmaceutical compositions find use in inhibiting tumor growth and treating cancer in human patients.

[0205] In certain embodiments, formulations are prepared for storage and use by combining a purified antibody or agent of the present invention with a pharmaceutically acceptable vehicle (e.g. carrier, excipient) (Remington, The Science and Practice of Pharmacy 20th Edition Mack Publishing, 2000). Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (e.g. octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanoI; and m-cresol); low molecular weight polypeptides (e.g. less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosacchandes, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG).

(0206] The pharmaceutical compositions described herein can be administered in any number of ways for either local or systemic treatment. Administration can be topical (such as to mucous membranes including vaginal and rectal delivery) such as transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal); oral; or parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g., intrathecal or intraventricular) administration. In some paritcular embodiments, the administration is intravenous.

[0207] An antibody or immunoconjugate can be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound. In some embodiments, the second compound is a steroid. In some embodiments, the methods encompass administration of a steroid and an immunoconjugate that results in a reduction of headaches as compared to administration of the immunoconjugate alone.

[0208] The steroid can be administered at the same time as the immunoconjugate, prior to the administration of the immunoconjugate, and/or after the administration of the immunoconjugate. In some embodiments, the steroid is administered within about a week, about five days, about three days, about two days, or about one day prior to the administration of the immunoconjugate. In some embodiments, the steroid is administered within one day of the administration of the immunoconjugae. In some embodiments, the steroid is administered multiple times. In some embodiments, the steroid is administered about one day prior to the administration of the immunoconjugate and on the same day as the administration of the immunoconjugate. The steroid can be administered via any number of ways, including for example, topical, pulmonary, oral, parenteral, or intracranial administration. In some embodiments, the administration is oral. In some embodiments, the administration is intravenous. In some embodiments, the administration is both oral and intravenous.

[0209] An antibody or immunoconjugate can also be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with an analgesic, or other medications that prevent or treat headaches. For example, acetaminophin and or dephenhydramine can be administered in addition to the administration of the antibody or immunoconjugate. The analgesic can be administered prior to, at the same time, or after the administration of the immunoconjugate and can be via any appropriate administration route. In some embodiments, the analgesic is administered orally. [0210] In some embodiments, the methods comprise administration of a first compound that is an antibody or imm noconjugate, a second compound that is a steroid, and a third compound that is an analgesic. In some embodiments, the methods comprise administration of a first compound that is IMGN388, a second compound that is dexamethasone, and a third compound that is acetaminophin and/or diphenydramine.

[0211] An antibody or immunoconjugate can be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen can have complementary activities to the ADC of the combination such that they do not adversely affect each other. Pharmaceutical compositions comprising the alphaV integrin-binding agent and the second anticancer agent are also provided.

[0212] For the treatment of the disease, the appropriate dosage of an antibody or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the antibody or agent is administered for therapeutic or preventative purposes, previous therapy, patient's clinical history, and so on all at the discretion of the treating physician. The antibody or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g. reduction in tumor size).

[0213] The combination therapy can provide "synergy" and prove "synergistic", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect can be attained when the active ingredients are: (1) co-formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect can be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

* # *

[0214] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present disclosure. Examples

[02i5] It is understood that the examples and embodiments described herein are for illustrative puiposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application

Example 1

Production and Characterization of IMGN388

[0216] IMGN388 is an immunoconjugate containing CNT095 linked to DM4 via an SPDB ((2- pyridyl)-4-dithiobutanoic acid N-hydroxy succinimid ester) linkage. The structure of IMGN388 is shown in Figure 1. An average of 3.5 molecules of DM4 are conjugated to the monoclonal antibody in the preparation of IMGN388. CNT095 is a fully human alphaV integrin targeting monoclonal antibody which has been previously described in International Published Application No. WO 02/12501, U.S. Published Application No. 2003/0040044, and U.S. Published Application No. 2006/0127407, each of which is incorporated by reference herein in its entirety. Briefly, the cell line that is the source of CNTO95 was developed using DNA technology to transfect human genes into a myeloma host cell for expression of the antibody. Cells secreting large amounts of CNT095 were identified by screening supernatants from candidate cell lines followed by rounds of subcloning to generate stably-producing, homogenous cell lines.

[0217] The integrin target of IMGN388 has been found by immunohistochemical staining to be present on a wide range of human solid tumors, with high expression observed in lung carcinomas, renal cell carcinomas, thyroid carcinomas, bladder carcinomas, melanomas, and sarcomas. The binding affinity of IMGN388 was in the range of 1 to 8 nM (EC50 values) for several human tumor cell lines, as determined by flow cytometry. Thus, IMGN388 retains the binding affinity of the unconjugated antibody.

[0218] The activity of IMGN388 has been evaluated in xenograft models in nude rats using a variety of human tumor cell lines. The antibody portion of IMGN388 does not cross-react with the murine integrin ortholog, but does bind to the rat ortholog, albeit at an affinity approximately 40-fold less than to the human integrin molecule. In one study, rats bearing established A549 human non-small cell lung tumors were treated with IMGN388 at 0.5, 1, 3, or 10 mg/kg, given weekly for six weeks. The response to IMGN388 was dose-dependent, with the minimum efficacious dose found to be 1 mg/kg. Tumor regressions were observed in animals treated at 1, 3 or 10 mg/kg with 5 complete responses and one partial response in the 7 animals treated at 10 mg/kg. Figure 2. In addition, rats bearing MDA-MB- 23 1.0T.F2 breast carcinomas were treated with IMGN388 at 1, 3, or 10 mg/kg (weekly x 5) or with 15 mg/kg (every 2 weeks x 3), and regression of the breast carcinomas were observed. Figure 3. IMGN388 also demonstrated efficacy against established human tumors of colon (HT-29), large cell lung (H460), pancreatic (AsPC-1), and ovarian (A2780, S OV-3) carcinomas in nude rat models. Additionally, IMGN388 has been found to inhibit angiogenesis using an in vivo model of basic fibroblast growth factor- induced angiogenesis in nude rats.

[0219] Thus, the anti-tumor effects of IMGN388 can be attributed to two distinct mechanisms of action: direct tumor cell killing and anti-angiogenic activity. These studies indicate that IMGN388 could be useful in the clinical treatment of numerous tumors. However, toxicities have been observed in animals treated with maytansinoid conjugated anti-alphaV integrin antibodies. For example, anti-alphaV integrin immunoconjugates caused weight loss in rats. In addition, cynomolgus monkeys receiving anti- alphaV integrin immunoconjugates at 5 mg/kg administered every three weeks for two to four doses showed moderate skin toxicity and some sciatic nerve degeneration. At higher doses, monkeys showed severe skin toxicity, bone marrow suppression, corneal pigmentation, and sciatic nerve degeneration.

[0220] Thus, it was necessary to identify a dosage regimen that would provide therapeutic benefits while minimizing adverse effects in humans.

Example 2

Evaluation of Safety and Pharmacokinetics of IMGN388 in Humans

[0221] In order to evaluate the safety and pharmacokinetics of IMGN388 in humans, patients with histologically confirmed solid tumors that were metastatic or unresectable were identified. As shown in Table 2 below, patients with a variety of cancer types were identified. The "other" tumors listed in the table included 1 patient each with adrenal carcinoma, fibrous histiocytoma, gastric cancer, Kaposi's sarcoma, peritoneal mesothelioma, renal cell carcinoma, retroperitoneal sarcoma, small bowel adenocarcinoma; bladder, cervical, thyroid and uterine cancer.

Table 2. Cancer Types of Patients Studied

Type Number of Patients

Ovarian Carcinoma 6

Colorectal Cancer 6

Neuroendocrine Carcinoma 4

NSCLC 6

Prostate Cancer 3

Breast Cancer 4

Pancreatic Cancer 2

Melanoma 3

Endometrial 2

Others 12 [0222] The expression of the integrin target was evaluated by immunohistochemistry on archived tumor samples from the patients. Cellular membrane staining was evaluated. The intensity of the staining was scored as 0 = negative, 1= weak, 2 = moderate, 3 = strong, or 3+ = very strong. The uniformity (i.e. number of stained cells) was scored as 0 = negative, Focal = <2₅%, Heterogeneous ("hetero") = 25-75%, or Homogenous ("homo") = >75%. Figure 4 provides an example of one staining (patient 0212) that was characterized as 3/Hetero. Notably, integrin was observed to be present on the majority of tumor samples from patients enrolled in the study.

Table 3: IHC Evaluation of Integrin Target Expression.

[0223] IMGN388 was administered as an intravenous (IV) infusion once every 3 weeks at dosages of 5, 10, 20, 30, 45, 60, 80, 105, 130, and 160 mg m². Plasma concentrations were determined using ELISA methods, and analyses were performed using standard algorithms of the non-compartmental PK analysis (WinNonLin 5.2.1, Pharsight). The resulting measurements for 30 mg/m² (0.8 mg/kg), 45 mg/m² (1.2 mg/kg), 60 mg/m² (1.6 mg/kg), 80 mg/m² (2.2 mg/kg), 105 mg/m² (2.8 mg/kg), 130 mg/m² (3.5 mg/kg), and 160 mg/m² (4.3 mg/kg) are shown in Table 4 below. In addition, maximal observed plasma concentration and AUC are shown in Figure 5.

Table 4. Pharmacokinetic Parameters of IMGN388

*The IMGN388 plasma concentration value on day 8 of 0.7 μg/mL was set as the limit of the quantitation for one patient and used for calculation of pharmacokinetic parameters.

[0224] Maximal plasma concentration and exposure (AUC) increased linearly through the 105 mg/m² dose level, and elimination half-life was similar at all dose levels examined (approximately 28 hours). In addition, the samples were used to test for the presence of humoral responses against the antibody component of IMGN388 (HAHA) or against the DM4 component (HADA) and neither was observed.

[0225] Patients were evaluated for adverse events that were considered at least possibly related to study dosing. There were no Grade 4 adverse events, and only 2 patients reported Grade 3 events: 1 patient treated at 45 mg/m² had grade 3 headache and confusion; another patient treated at 80 mg/m² had grade 3 nausea and vomiting. The adverse events are summarized in Table 5 below. Grade 1 or 2 adverse events that were reported only once in patients who received the drug at 30 mg/m² or less were not shown in this Table.

Table 5: Grade 1 or 2 Adverse Events

Neuropathy

Flushing 1

Mucosal 1

Inflammation

Thrombo1

cytopenia

Neutropenia 1

Increased 1

AST*

Increased 1

GGT *

Increased 1

Glucose

Blood Urine 1

Hematochezia 1

Muscle 1

Spasms

*AST indicates aspartate aminotransferase and GGT indicates gamma-glutamyltransferase.

[0226] These data indicate that IMGN388 is well tolerated at the implicated dosage regimens.

Example 3

Steroid Prophylaxis for Headache Prevention

As described above, one patient experienced a grade 3 headache and confusion 24 hours after the first infusion of IMGN388 at a dose of 45 mg/m². In order to prevent headaches, prophylactic steroid administration was implemented. On the day prior to administration of IMGN388, patients received dexamethasone 8 mg (or similar) by mouth BID (twice a day). On the day of IMGN388 administration, and approximately one hour prior to the infusion, patients received dexamethasone 10 mg IV (or similar). In addition, pre-medication with 500-650 mg acetaminophen by mouth, and/or 25 mg or 50 mg diphenhydramine (or similar) 30-60 minutes before the start of the infusion is recommended. No further events have occurred. These results indicate that administration of steroids in addition to administration of IMGN388 can decrease headaches compared to the administration of IMGN388 alone.

Evaluation of Anti-Tumor Activity of IMGN388 in Humans

[0227] The anti-tumor activity of IMGN388 in human patients were also evaluated in this study.

Notably, stable disease has been reported in some patients, and these results are summarized in Table 6. Table 6: Patients Treated with IMGN388 Treatment with Stable Disease (SD) for 3 Cycles or Better

Patient Dose Tumor Type Target Lines of Prior Response Cycles on

(mg/m²) Expression Chemotherapy IMGN388

0203 45 NSCLC 2/Hetero 2 SD 6

0116 60 Uterine 3/Hetero 1 SD 3

0208 80 Breast Not 10 SD 4 available

0210 80 Prostate 3/Homo 2 SD 6

0211 105 Neuroendocrine Not 2 SD 7 available

0212 105 NSCLC 3/Hetero 5 SD 5

[0228] Thus, patients receiving 45, 60, 80, and 105 rag/m2 of IMGN388 who previously progressed on at least one other line of therapy showed stable disease. These results indicate that IMGN388 is, not only well tolerated, but also therapeutically effective at the implicated dosage regimens. The maximum- tolerated dose has been declared at 130 mg m² when IMGN388 is administered on Day 1 of a 21 -day cycle.

****

[0229] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections sets forth one or more, but not all, exemplary embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0230] The present invention has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0231] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0232] The breadth and scope of the present invention should not be limited by any of the above- described exemplary embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a patient having cancer comprising administering to the patient an effective dose of an immunoconjugate which binds to alphaV integrin, wherein the immunoconjugate is administered at a dose of about 5 to about 375 mg/m²,

2. The method of claim 1, wherein the immunoconjugate is administered at a dose of about 5 mg/m², about 10 mg/m², about 20 mg/m², about 30 mg/m², about 45 mg/m², about 60 mg/m², about 80 mg/m², about 105 mg/m², about 130 mg m², about 145 mg/m², about 160 mg/m², about 200 mg/m², about 250 mg/m², about 300 mg/m², about 310 mg/m², about 350 mg/m² , or about 375 mg/m²,

3. The method of claim 1 or 2, wherein the immunoconjugate comprises an antibody that competitively inhibits the binding of an antibody with the sequences of SEQ ID NOs:4-6 and 7-9 to alpha V integrin.

4. The method of claim 3, wherein the antibody is CNT095.

5. The method of any one of claims 1-4, wherein the immunoconjugate comprises a maytansinoid.

6. The method of claim 5, wherein the maytansinoid is DM4.

7. The method of any one of claims 1-6, wherein the immunoconjugate comprises a linker that is SPDB.

8. The method of any one of claims 1-7, wherein the immunoconjugate is IMGN388.

9. The method of any one of claims 1-8, wherein the immunoconjugate is administered about once every 3 weeks, about once every 2 weeks, about once every 1 week, or about twice a week.

10. The method of any one of claims 1-8, wherein the immunoconguate is administered in an about 3- week cycle and the immunoconjugate is administered at about days 1 and 8 of the cycle or at about days 1 , 8, and 15 of the cycle.

1 1. The method of any one of claims 1-8, wherein the immunoconguate is administered in an about 4- week cycle.

12. The method of any one of claims 1-1 1 , wherein the administration produces an AUC of about 500-13,000 hr^g/mL, about 500-10,000 hr^g/mL, about 500-5,000 hr^g/mL, or about 500- 4,000 hr^g/mL.

13. The method of any one of claims 1-12, wherein the administration produces a Cmax of about 10- 250 μg/mL, about 10-240 μg/mL, about 10-200 μg/mL, about 10-150 μ§/ιηΤ, or about 10-100 μg/mL.

14. The method of any one of claims 1-13, wherein the immunoconjugate is administered intravenously.

15. The method of any one of claims 1-14, wherein said cancer is selected from the group consisting of adenocarcinoma, thyroid cancer, sarcoma, cervical cancer, mesothelioma, endometrial cancer, melanoma, colorectal cancer, renal carcinoma, neuroendocrine carcinoma, retroperitoneal sarcoma, colon cancer, adrenal carcinoma, Kaposi's sarcoma, peritoneal mesothelioma, prostate cancer, rectal cancer, ovarian carcinoma, fibrous histiocytoma, small bowel adenocarcinoma, non- small cell lung cancer ( SCLC), prostate cancer, gastric cancer, breast cancer, uterine cancer, neuroendocrine carcinoma, or bladder cancer.

16. The method of any one of claims 1-15, wherein the cancer expresses alphaV integrin.

17. The method of any one of claims 1-16, wherein the cancer is metastatic.

18. The method of any one of claims 1-17, further comprising administering a second anti-cancer agent to the patient.

19. The method of claim 18, wherein said second anti-cancer agent is a chemotherapeutic agent.

20. The method of any one of claims 1-19, further comprising administering a steroid to the patient.

21. The method of claim 20, wherein the steroid is dexamethasone.

22. The method of any one of claims 1-21, wherein the administration results in a decrease in tumor size, a decrease in angiogenesis, and/or a decrease in adverse effects.