Antibodies to Integrin Heterodimers and Uses Thereof - Patent application

Abstract

The present disclosure provides antibodies that bind to human ITGAv / B1, and methods of use thereof. In some aspects, the present disclosure is directed to a method of treating cancer in a subject, comprising administering to the subject an anti-ITGAv / B1 antibody.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 158,769, filed March 9, 2021, which is incorporated by reference in its entirety.

[0002] References to sequence listings submitted electronically via EFS-WEB The contents of the electronically submitted sequence listing (Name: 4756_001PC01_Seqlisting_ST25.txt, Size: 68,219 bytes, and Creation Date: March 7, 2022) submitted in this application are incorporated herein by reference in their entirety. [Background technology]

[0003] The αv family of integrins that bind RGD have been identified as regulators of transforming growth factor β1 (TGFβ1), a pleiotropic cytokine that can function as a tumor-promoting factor. αv family integrins can activate TGFβ1 in vivo and indirectly regulate tumor progression through the local production of active TGFβ1. TGFβ1 is a potent activator of fibroblast transdifferentiation into myofibroblasts. Thus, αv family integrins activate TGFβ1 and induce myofibroblastogenesis, resulting in myofibroblast-dependent activities, including matrix remodeling, matrix stiffening, and cancer promotion. Here, TGFβ1 induces fibroblasts to adopt a contractile wound-repair phenotype and transdifferentiate in the tumor microenvironment, resulting in cancer-associated fibroblasts (CAFs) or tumor-associated fibroblasts (TAFs). In addition, TGFβ1 can induce some non-fibroblastic cells, including adipocytes and circulating myeloid-derived suppressor cells, to transdifferentiate into cancer-associated myofibroblasts.

[0004] By increasing the contraction of the extracellular matrix, myofibroblasts also increase the likelihood of activating latent TGFβ1. For example, myofibroblasts secrete a vast number of proteins that further enhance cancer progression, including ECM proteases, growth factors, cytokines, and chemokines. Similarly, TGFβ1-activated fibroblasts can secrete osteopontin, an RGD-containing integrin ligand that is involved in promoting tumor growth, EMT, and metastasis. Thus, increased osteopontin correlates with increased metastasis, and often poor survival, in multiple types of cancer, including laryngeal squamous cell carcinoma, melanoma, nasopharyngeal carcinoma, and breast cancer.

[0005] TGFβ1 also promotes angiogenesis, where local activation of TGFβ by αv integrins promotes the development of blood vessels within tumors. In addition, different αv integrins are also upregulated in endothelial cells of new blood vessels, promoting their migration, and integrins αvβ3, αvβ5, and αvβ8 all regulate angiogenesis.

[0006] Activation of TGFβ by any αv integrin can affect local inflammation and immune cells, promote immunosuppressive effects on various effector T cells, and induce tumor-promoting phenotypes in both neutrophils and macrophages. Here, TGFβ1 promotes the formation of tumor-promoting M2 tumor-associated macrophages and N2 tumor-associated neutrophils. Macrophages that present as having "M1" exhibit anti-tumor properties, however, "M2" exhibits tumor-promoting features, increasing the transcription of TGFβ1, TGFBRI, and TGFBRII in carcinoma cells.

[0007] TGFβ1 also alters the inflammatory gene expression profile of macrophages by stimulating monocyte recruitment, increasing metastasis-associated interleukin-6 (IL-6), and suppressing cytokines such as IL-10, and the chemoattractants CCL3 and CCL4. TGFβ1 stimulation of macrophages also promotes angiogenesis under hypoxic conditions by increasing the production of VEGF, MMP-9, and expression of the VEGF receptor Flk-1. Furthermore, high levels of M2 macrophages also correlate with poor survival from various cancers. These include pancreatic and cervical cancer, spread of gastric cancer, and recurrence after chemotherapy. Furthermore, multiple studies suggest that M2 cells promote metastasis. Summary of the Invention [Problem to be solved by the invention]

[0008] Blocking TGFβ receptors with inhibitory antibodies would facilitate systemic targeting with adverse side effects.However, suitable targeted therapy has not yet been developed that minimizes off-target effects and provides local control of TGFβ activation.As a means of controlling TGFβ1 activity in cancer and fibrosis, therapeutic antibodies that specifically target αv integrin are described herein. [Means for solving the problem]

[0009] Some embodiments of the present disclosure are directed to bispecific antibodies, or antigen-binding portions thereof, that specifically bind to integrin-αv heterodimers and inhibit integrin-αv-mediated activation of TGFβ.

[0010] In some embodiments, the bispecific antibody or antigen-binding portion comprises at least a first paratope and a second paratope, and the first paratope binds to a first epitope on the integrin αvβ1 heterodimer. In some embodiments, the second paratope binds to a second epitope on the integrin αvβ1 heterodimer. In some embodiments, the first epitope and the second epitope are not the same.

[0011] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain. In some embodiments, the first heavy chain and the second heavy chain are different. In some embodiments, the first heavy chain and the second heavy chain are different and the first light chain and the second light chain are the same.

[0012] In some embodiments, the first heavy chain comprises a first variable heavy chain region ("VH1") comprising variable heavy chain complementarity determining region (VH1-CDR)1, VH1-CDR2, and VH1-CDR3, wherein VH1-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 5, 15, and 25. In some embodiments, VH1-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 4, 14, and 24. In some embodiments, VH1-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 3, 13, and 23. In some embodiments, the first light chain comprises a first variable light chain region ("VL1") comprising VL1-CDR1, VL1-CDR2, and VL1-CDR3, wherein VL1-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 10, 20, and 30. In some embodiments, VL1-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 9, 19, and 29. In some embodiments, VL1-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 8, 18, and 28.

[0013] In some embodiments, the bispecific antibody or antigen-binding portion thereof according to any one of claims 1 to 13 comprises (i) a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 3, (ii) a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 4, (iii) a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 5, (iv) a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, (v) a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and (vi) a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0014] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises (i) a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, (ii) a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, (iii) a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, (iv) a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, (v) a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and (vi) a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0015] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises (i) a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, (ii) a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, (iii) a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5, (iv) a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28, (v) a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and (vi) a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0016] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises (i) a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, (ii) a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, (iii) a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25, (iv) a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, (v) a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and (vi) a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30. In some embodiments, the second heavy chain comprises a second variable heavy chain region ("VH2") comprising VH2-CDR1, VH2-CDR2, and VH2-CDR3, wherein VH2-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 5, 15, and 25. In some embodiments, VH2-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 4, 14, and 24. In some embodiments, VH2-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 3, 13, and 23. In some embodiments, the second light chain comprises a second variable light chain region ("VL1") comprising VL2-CDR1, VL2-CDR2, and VL2-CDR3, wherein VL2-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 20 and 30. In some embodiments, VL2-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 19 and 29. In some embodiments, VH2-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 18 and 28.

[0017] In some embodiments, the bispecific antibody, or antigen-binding portion thereof, comprises: (a) a first variable heavy chain region (VH1) comprising (i) a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, (ii) a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5; (b) a first variable heavy chain region (VH1) comprising (i) a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:18, (ii) a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:19, and (iii) a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:20; (c) a second variable heavy chain region (VH2) comprising (i) a VH2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, (ii) a VH2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and (iii) a VH2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15; and (d) a second variable light chain region (VL2) comprising (i) a VL2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, (ii) a VL2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and (iii) a VL2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0018] In some embodiments, the bispecific antibody, or antigen-binding portion thereof, comprises: (a) a first variable heavy chain region (VH1) comprising (i) a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, (ii) a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, and (iii) a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5; (b) a first variable heavy chain region (VH1) comprising (i) a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28, (ii) a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and (iii) a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30; (c) a second variable heavy chain region (VH2) comprising (i) a VH2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 23, (ii) a VH2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 24, and (iii) a VH2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 25; and (d) a second variable light chain region (VL2) comprising (i) a VL2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 28, (ii) a VL2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 29, and (iii) a VL2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 30.

[0019] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a first variable heavy chain region (VH1) and a first variable light chain region (VL1), wherein VH1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 2, 12, or 22.

[0020] In some embodiments, VL1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:7, 17, or 27.

[0021] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a second variable heavy chain region (VH2) and a second variable light chain region (VL2), wherein VH2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 2, 12, or 22.

[0022] In some embodiments, VL2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:7, 17, or 27.

[0023] In some embodiments, (a) VH1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and (b) VL1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17. (c) VH2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:12; and (d) VL2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17.

[0024] In some embodiments, (a) VH1 comprises the amino acid sequence set forth in SEQ ID NO:2, (b) VL1 comprises the amino acid sequence set forth in SEQ ID NO:17, (c) VH2 comprises the amino acid sequence set forth in SEQ ID NO:12, and (d) VL2 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0025] In some embodiments, (a) VH1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and (b) VL1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:27. (c) VH2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22; and (d) VL2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:27.

[0026] In some embodiments, (a) VH1 comprises the amino acid sequence set forth in SEQ ID NO:2, (b) VL1 comprises the amino acid sequence set forth in SEQ ID NO:27, (c) VH2 comprises the amino acid sequence set forth in SEQ ID NO:22, and (d) VL2 comprises the amino acid sequence set forth in SEQ ID NO:27.

[0027] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a first heavy chain (H1) and a first light chain (L1), wherein H1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 11, 21, 31, 34, or 37.

[0028] In some embodiments, L1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:6, 16, or 26.

[0029] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a second heavy chain (H2) and a second light chain (L2), wherein H2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 11, 21, 31, 34, or 37.

[0030] In some embodiments, L2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:6, 16, or 26.

[0031] In some embodiments, the first heavy chain is associated with the second heavy chain. In some embodiments, the first heavy chain is associated with the second heavy chain by a covalent bond. In some embodiments, each of the first heavy chain and the second heavy chain comprises an IgG constant region or an IgG constant region that comprises one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions promote heterodimerization of the first heavy chain and the second heavy chain.

[0032] In some embodiments, a first heavy chain comprises one or more amino acid substitutions in the constant region of the first heavy chain that create a knob, and a second heavy chain comprises one or more amino acid substitutions, or an antigen-binding portion thereof, in the constant region of the second heavy chain that create a hole, and the knob of the first heavy chain associates with the hole of the second heavy chain.

[0033] In some embodiments, the first heavy chain comprises one or more amino acid substitutions in the constant region of the first heavy chain that create a hole, and the second heavy chain comprises one or more amino acid substitutions in the constant region of the second heavy chain that create a knob, and the hole of the first heavy chain associates with the knob of the second heavy chain.

[0034] In some embodiments, (a) H1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:31; and (b) L1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16. and (c) H2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 34, and (d) L2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, (a) H1 comprises the amino acid sequence set forth in SEQ ID NO: 31, (b) L1 comprises the amino acid sequence set forth in SEQ ID NO: 16, (c) H2 comprises the amino acid sequence set forth in SEQ ID NO: 34, and (d) L2 comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0035] In some embodiments, (a) H1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:31; and (b) L1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. and (c) H2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 37, and (d) L2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26. In some embodiments, (a) H1 comprises the amino acid sequence set forth in SEQ ID NO: 31, (b) L1 comprises the amino acid sequence set forth in SEQ ID NO: 26, (c) H2 comprises the amino acid sequence set forth in SEQ ID NO: 37, and (d) L2 comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0036] In some embodiments, the bispecific antibody has one or more properties selected from the group consisting of: (a) the bispecific antibody inhibits the binding of integrin-αvβ1 to LAP-TGFβ1; (b) the bispecific antibody can bind to an integrin αv selected from αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and any combination thereof; (c) the bispecific antibody inhibits cell adhesion; (d) the bispecific antibody inhibits tumor growth and / or metastasis; (e) the bispecific antibody increases progression-free survival; (f) the bispecific antibody increases overall survival; and (g) any combination thereof. In some embodiments, the bispecific antibody or antigen-binding portion thereof is capable of (i) inhibiting the binding of integrin-αvβ1 to LAP-TGFβ1, and (ii) inhibiting cell adhesion. In some embodiments, the bispecific antibody or antigen-binding portion thereof is capable of binding to an integrin αvβ1 selected from αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and any combination thereof. -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 K below M D and binds to integrin-αv.

[0037] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") comprising a variable heavy chain complementarity determining region (VH-CDR)1, a VH-CDR2, and a VH-CDR3, and a variable light chain region ("VL") comprising a VL-CDR1, a VL-CDR2, and a VL-CDR3, wherein VH-CDR3 comprises the amino acid sequence of SEQ ID NO:5, 15, or 25. In some embodiments, VH-CDR2 comprises the amino acid sequence of SEQ ID NO:4, 14, or 24. In some embodiments, VH-CDR1 comprises the amino acid sequence of SEQ ID NO:3, 13, or 23. In some embodiments, VL-CDR3 comprises the amino acid sequence of SEQ ID NO:10, 20, or 30. In some embodiments, VL-CDR2 comprises the amino acid sequence of SEQ ID NO:9, 19, or 29. In some embodiments, the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8, 18, or 28.

[0038] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein (i) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, (ii) VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, (iii) VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5, (iv) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, (v) VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and (vi) VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10.

[0039] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein (i) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, (ii) VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, (iii) VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, (iv) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, (v) VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and (vi) VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0040] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein (i) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23, (ii) VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, (iii) VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25, (iv) VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28, (v) VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, and (vi) VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:30.

[0041] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region ("VH") and a light chain variable region ("VL"), wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 12, and 22.

[0042] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain variable region ("VH") and a light chain variable region ("VL"), wherein the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7, 17, and 27.

[0043] In some embodiments, (a) the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:2; and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NO:7; (b) the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:12. or (c) the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 17; or (b) the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 22, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 27.

[0044] In some embodiments, (a) the VH comprises the amino acid sequence set forth in SEQ ID NO:2 and the VL comprises the amino acid sequence set forth in SEQ ID NO:7, (b) the VH comprises the amino acid sequence set forth in SEQ ID NO:12 and the VL comprises the amino acid sequence set forth in SEQ ID NO:17, or (c) the VH comprises the amino acid sequence set forth in SEQ ID NO:22 and the VL comprises the amino acid sequence set forth in SEQ ID NO:27.

[0045] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain ("HC") and a light chain ("LC"), wherein the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1, 11, and 21.

[0046] In some embodiments, the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 6, 16, and 16. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain ("HC") and a light chain ("LC"), wherein the HC comprises an amino acid sequence selected from SEQ ID NOs: 1, 11, and 21. In some embodiments, the LC comprises an amino acid sequence selected from SEQ ID NOs: 6, 16, and 26.

[0047] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain ("HC") and a light chain ("LC"), wherein (a) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6, (b) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about or (c) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16; or (b) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21; and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 26.

[0048] In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain ("HC") and a light chain ("LC"), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO:1 and the LC comprises the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain ("HC") and a light chain ("LC"), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO:11 and the LC comprises the amino acid sequence set forth in SEQ ID NO:16. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain ("HC") and a light chain ("LC"), wherein the HC comprises the amino acid sequence set forth in SEQ ID NO:21 and the LC comprises the amino acid sequence set forth in SEQ ID NO:26.

[0049] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 2, 12, or 22. In some embodiments, the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7, 17, or 27.

[0050] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7.

[0051] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:12, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17.

[0052] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:22, and the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:27.

[0053] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, the antibody or antigen-binding portion thereof comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:2 and the VL comprises the amino acid sequence set forth in SEQ ID NO:7.

[0054] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, the antibody or antigen-binding portion thereof comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 12 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0055] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, the antibody or antigen-binding portion thereof comprising a variable heavy chain region ("VH") and a variable light chain region ("VL"), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO:22 and the VL comprises the amino acid sequence set forth in SEQ ID NO:27.

[0056] In some embodiments, the antibody or antigen-binding portion thereof is an antigen-binding portion, which is a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide-linked Fv, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0057] Some embodiments of the present disclosure are directed to bispecific antibodies comprising an antibody or antigen-binding portion thereof disclosed herein, wherein the bispecific antibody is capable of inhibiting integrin-αv-mediated activation of TGFβ.

[0058] Some embodiments of the present disclosure are directed to bispecific antibodies as disclosed herein, or multispecific antibodies comprising an antibody or antigen-binding portion thereof as disclosed herein, which can inhibit integrin-αv-mediated activation of TGFβ.

[0059] Some aspects of the present disclosure are directed to an antibody drug conjugate comprising a bispecific antibody or antigen-binding portion thereof disclosed herein, an antibody or antigen-binding portion thereof disclosed herein, or a multispecific antibody disclosed herein. In some aspects, the bispecific antibody or antigen-binding portion thereof, the antibody or antigen-binding portion thereof, or the multispecific antibody is fused to a detectable marker.

[0060] Some aspects of the present disclosure are directed to a polynucleotide or set of polynucleotides that encode a bispecific antibody disclosed herein, an antibody or antigen-binding portion thereof disclosed herein, or a multispecific antibody disclosed herein.

[0061] Some embodiments of the present disclosure are directed to a set of polynucleotides comprising: (i) a first polynucleotide that encodes a first heavy chain disclosed herein, (ii) a second polynucleotide that encodes a second heavy chain disclosed herein, and (iii) a third polynucleotide that encodes a first light chain disclosed herein.

[0062] Some aspects of the disclosure are directed to a vector or set of vectors that comprise a polynucleotide of the set of polynucleotides disclosed herein.

[0063] Some embodiments of the present disclosure are directed to a cell comprising a bispecific antibody disclosed herein, any one of the antibodies or antigen-binding portions thereof disclosed herein, a multispecific antibody disclosed herein, or a polynucleotide of the set of polynucleotides disclosed herein.

[0064] Some aspects of the present disclosure are directed to pharmaceutical compositions comprising a polynucleotide of a bispecific antibody disclosed herein, an antibody or antigen-binding portion thereof disclosed herein, a multispecific antibody disclosed herein, or a set of polynucleotides disclosed herein, and a pharmaceutically acceptable excipient.

[0065] Some aspects of the present disclosure are directed to methods of making a bispecific antibody or antigen-binding portion thereof, an antibody or antigen-binding portion thereof, or a multispecific antibody or antigen-binding portion thereof, comprising culturing a host cell as disclosed herein under suitable conditions and isolating the bispecific antibody.

[0066] Some aspects of the present disclosure are directed to a method of treating cancer in a subject in need thereof, comprising administering to the subject a bispecific antibody or antigen-binding portion thereof disclosed herein, an antibody or antigen-binding portion thereof disclosed herein, a multispecific antibody or antigen-binding portion thereof disclosed herein, an antibody drug conjugate disclosed herein, a polynucleotide of the set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein. In some aspects, administration of the bispecific antibody reduces or inhibits metastasis of the cancer in the subject.

[0067] Some aspects of the present disclosure are directed to methods of reducing or inhibiting metastasis of cancer in a subject in need thereof, comprising administering to the subject a bispecific antibody or antigen-binding portion thereof disclosed herein, an antibody or antigen-binding portion thereof disclosed herein, a multispecific antibody or antigen-binding portion thereof disclosed herein, an antibody drug conjugate disclosed herein, a polynucleotide of the set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0068] Some embodiments of the present disclosure are directed to a method of inhibiting integrin-αvβ1-mediated TGFβ activation in a subject in need thereof comprising administering to the subject a bispecific antibody or antigen-binding portion thereof disclosed herein, an antibody or antigen-binding portion thereof disclosed herein, a multispecific antibody or antigen-binding portion thereof disclosed herein, an antibody drug conjugate disclosed herein, a polynucleotide of the set of polynucleotides disclosed herein, a set of polynucleotides disclosed herein, a vector or set of vectors disclosed herein, a cell disclosed herein, or a pharmaceutical composition disclosed herein.

[0069] In some embodiments, the subject is suffering from cancer. In some embodiments, the cancer comprises a tumor. In some embodiments, the cancer comprises small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), squamous NSCLC, non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer, clear cell carcinoma, ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer, renal cell carcinoma (RCC), prostate cancer, hormone refractory prostate adenocarcinoma, thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma polymorphism), cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma (hepatocellular carcinoma), breast cancer, colon cancer, head and neck cancer (or cancer), head and neck squamous cell carcinoma (HNSCC), gastric cancer , germ cell tumors, pediatric sarcomas, sinonasal natural killer, melanoma, metastatic malignant melanoma, cutaneous or intraocular malignant melanoma, mesothelioma, bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, cancer of the fallopian tubes, cancer of the endometrium, cancer of the cervix, cancer of the vagina, cancer of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, soft tissue sarcomas, cancer of the urethra, cancer of the penis, childhood solid tumors, cancer of the ureter, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain cancer, brain stem glioma, pituitary adenoma , Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers or cancers of viral origin, tumors associated with or of human papillomavirus (HPV) origin, acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML, myeloblastic leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroleukemia, megakaryoblastic leukemia, isolated granulocytic leukemia sarcoma, chloroma, Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic, peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder,The cancer is selected from the group consisting of true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphocytic lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC), lymphoplasmacytic lymphoma with Waldenstrom's macroglobulinemia (LPL), myeloma, IgG myeloma, light chain myeloma, non-secretory myeloma, smoldering myeloma (indolent myeloma), solitary plasmacytoma, multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma, and any combination of the above cancers.

[0070] In some embodiments, the method further comprises administering to the subject an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy comprises chemotherapy, immunotherapy, surgery, radiation therapy, or any combination thereof. In some embodiments, the additional anti-cancer therapy comprises a standard of care treatment.

[0071] In some embodiments, the additional anti-cancer therapy comprises a checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy comprises an inhibitor of inducible T cell costimulator (ICOS), CD137 (4-1BB), CD134 (OX40), NKG2A, CD27, CD96, glucocorticoid-induced TNFR-related protein (GITR), and herpes virus entry mediator (HVEM), programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), CTLA-4, B- and T-lymphocyte attenuating factor (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), adenosine The antibodies or antigen-binding portions thereof specifically bind to a protein selected from A2a receptor (A2aR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immunoreceptor with Ig and ITIM domains (TIGIT), and V-domain Ig suppressor of T cell activation (VISTA), KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CEACAM-1, CD52, HER2, and any combination thereof. In some embodiments, the anti-PD-1 antibody comprises nivolumab or pembrolizumab. [Brief description of the drawings]

[0072] [Figure 1A] 1A-1B are diagrams of titrations of anti-integrin-αvβ1 clone 10392 (FIG. 1A) and clone 10404 (FIG. 1B). [Figure 1B] 1A-1B are diagrams of titrations of anti-integrin-αvβ1 clone 10392 (FIG. 1A) and clone 10404 (FIG. 1B). [Figure 2A] Figures 2A-2B are bar graphs summarizing the epitope binning of anti-integrin-αvβ1 Fab 10404 (Figure 2A) and Fab 10392 (Figure 2B). All numbers above the bar graphs represent percentage values, MFI indicates mean fluorescence from the mean, and error bars indicate standard deviation. [Figure 2B] Figures 2A-2B are bar graphs summarizing the epitope binning of anti-integrin-αvβ1 Fab 10404 (Figure 2A) and Fab 10392 (Figure 2B). All numbers above the bar graphs represent percentage values, MFI indicates mean fluorescence from the mean, and error bars indicate standard deviation. [Figure 3A] 3A-3B are scatter plots showing IgG affinity titration validation of anti-integrin-αvβ1 Fab 10392 (FIG. 3A) and Fab 10404 (FIG. 3B) against different integrin CHO cell lines. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 3B] 3A-3B are scatter plots showing IgG affinity titration validation of anti-integrin-αvβ1 Fab 10392 (FIG. 3A) and Fab 10404 (FIG. 3B) against different integrin CHO cell lines. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 4] FIG. 4 is a scatter plot showing IgG affinity titration validation of different modalities of antibody clones against recombinant human integrin-αvβ1. [Diagram 5] FIG. 5 is a scatter plot showing IgG affinity titration validation of different modalities of antibody clones against recombinant mouse integrin-αvβ1. [Figure 6A] Figures 6A-6C show size-exclusion chromatography graphs of IgG clones 10392 (Figure 6A), 10404 (Figure 6B), and 11867 (Figure 6C). [Figure 6B] Figures 6A-6C show size-exclusion chromatography graphs of IgG clones 10392 (Figure 6A), 10404 (Figure 6B), and 11867 (Figure 6C). [Figure 6C] Figures 6A-6C show size-exclusion chromatography graphs of IgG clones 10392 (Figure 6A), 10404 (Figure 6B), and 11867 (Figure 6C). [Figure 7]7A-7B are PAGE-SDS gel images showing an assessment of IgG purity after expression and purification for IgG clones 10404 (FIG. 7A) and 10392 (FIG. 7B). [Figure 8A] 8A-8B are bar graphs showing freeze-thaw analysis of clone 11867 and different IgG modalities. FIG. 8A provides a graph showing concentration values ​​determined by spectrophotometry at absorbance 280 wavelength. FIG. 8B provides a graph showing flow cytometry mean fluorescence intensity (MFI) values. IgG binding activity was measured using AvB1 CHO cells and all samples were normalized to "no treatment" for the respective group. [Figure 8B] 8A-8B are bar graphs showing freeze-thaw analysis of clone 11867 and different IgG modalities. FIG. 8A provides a graph showing concentration values ​​determined by spectrophotometry at absorbance 280 wavelength. FIG. 8B provides a graph showing flow cytometry mean fluorescence intensity (MFI) values. IgG binding activity was measured using AvB1 CHO cells and all samples were normalized to "no treatment" for the respective group. [Figure 9] FIG. 9 is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of clone IgG 10404, 10392, and 10404 / 10392. [Figure 10A]FIG. 10A is a diagram of different bispecific IgGs, null indicates non-selective (random CDR) double IgG arms. FIG. 10B is a bar graph summary of Fab binding activity of integrin-αvβ1 CHO cells in the presence of bispecific IgG 10404 / 10392. FIG. 10C is a bar graph summary of bispecific IgG binding activity of integrin-αvβ1 CHO cells in the presence of different Fab clones. FIG. 10D is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of different clone IgGs. FIG. 10E is a flow cytometric titration graph of integrin-αvβ1 CHO cells. FIG. 10F is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 10B] FIG. 10A is a diagram of different bispecific IgGs, null indicates non-selective (random CDR) double IgG arms. FIG. 10B is a bar graph summary of Fab binding activity of integrin-αvβ1 CHO cells in the presence of bispecific IgG 10404 / 10392. FIG. 10C is a bar graph summary of bispecific IgG binding activity of integrin-αvβ1 CHO cells in the presence of different Fab clones. FIG. 10D is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of different clone IgGs. FIG. 10E is a flow cytometric titration graph of integrin-αvβ1 CHO cells. FIG. 10F is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 10C]FIG. 10A is a diagram of different bispecific IgGs, null indicates non-selective (random CDR) double IgG arms. FIG. 10B is a bar graph summary of Fab binding activity of integrin-αvβ1 CHO cells in the presence of bispecific IgG 10404 / 10392. FIG. 10C is a bar graph summary of bispecific IgG binding activity of integrin-αvβ1 CHO cells in the presence of different Fab clones. FIG. 10D is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of different clone IgGs. FIG. 10E is a flow cytometric titration graph of integrin-αvβ1 CHO cells. FIG. 10F is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 10D] FIG. 10A is a diagram of different bispecific IgGs, null indicates non-selective (random CDR) double IgG arms. FIG. 10B is a bar graph summary of Fab binding activity of integrin-αvβ1 CHO cells in the presence of bispecific IgG 10404 / 10392. FIG. 10C is a bar graph summary of bispecific IgG binding activity of integrin-αvβ1 CHO cells in the presence of different Fab clones. FIG. 10D is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of different clone IgGs. FIG. 10E is a flow cytometric titration graph of integrin-αvβ1 CHO cells. FIG. 10F is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 10E]FIG. 10A is a diagram of different bispecific IgGs, null indicates non-selective (random CDR) double IgG arms. FIG. 10B is a bar graph summary of Fab binding activity of integrin-αvβ1 CHO cells in the presence of bispecific IgG 10404 / 10392. FIG. 10C is a bar graph summary of bispecific IgG binding activity of integrin-αvβ1 CHO cells in the presence of different Fab clones. FIG. 10D is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of different clone IgGs. FIG. 10E is a flow cytometric titration graph of integrin-αvβ1 CHO cells. FIG. 10F is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 10F] FIG. 10A is a diagram of different bispecific IgGs, null indicates non-selective (random CDR) double IgG arms. FIG. 10B is a bar graph summary of Fab binding activity of integrin-αvβ1 CHO cells in the presence of bispecific IgG 10404 / 10392. FIG. 10C is a bar graph summary of bispecific IgG binding activity of integrin-αvβ1 CHO cells in the presence of different Fab clones. FIG. 10D is a bar graph summary of integrin-αvβ1 CHO cell adhesion to LAP-TGFβ in the presence of different clone IgGs. FIG. 10E is a flow cytometric titration graph of integrin-αvβ1 CHO cells. FIG. 10F is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 11A]11A-11F show data of the comparison of inhibitory properties from different 10404 and 10392 clone IgG modalities. FIG. 11A is a table summarizing the inhibitory values ​​of cell adhesion to LAP-TGFβ for different clone IgGs using different integrin CHO cell lines. FIG. 11B-11F are graphs summarizing the inhibitory titration curves of cell adhesion to LAP-TGFβ for clone IgGs αvβ1 (FIG. 11B), αvβ6 (FIG. 11C), αvβ3 (FIG. 11D), αvβ8 (FIG. 11E), and αvβ5 (FIG. 11F) using different integrin CHO cell lines. Error bars indicate standard deviation. [Figure 11B] 11A-11F show data of the comparison of inhibitory properties from different 10404 and 10392 clone IgG modalities. FIG. 11A is a table summarizing the inhibitory values ​​of cell adhesion to LAP-TGFβ for different clone IgGs using different integrin CHO cell lines. FIG. 11B-11F are graphs summarizing the inhibitory titration curves of cell adhesion to LAP-TGFβ for clone IgGs αvβ1 (FIG. 11B), αvβ6 (FIG. 11C), αvβ3 (FIG. 11D), αvβ8 (FIG. 11E), and αvβ5 (FIG. 11F) using different integrin CHO cell lines. Error bars indicate standard deviation. [Figure 12A]Figure 12A is a summary bar graph showing epitope binding competition of different IgG antibodies in the presence of 10404 Fab protein using integrin-αvβ1 CHO cells. Figure 12B is a summary bar graph of IgG inhibition of cell adhesion to different LAP-TGFβ using different integrin CHO cell lines. For each cell line, the different IgG antibodies (11883, 11855, 11857, 11874, 11914, and 11867) used in the experiment are shown from left to right. Figures 12C-12I are size exclusion chromatography graphs of trastuzumab (Figure 12C), 10404 (Figure 12D), 11855 (Figure 12E), 11857 ​​(Figure 12F), 11867 (Figure 12G), 11883 (Figure 12H), and 11914 (Figure 12I) IgG clones. Figure 12J is a table summarizing the affinity and inhibitory values ​​of different clone IgG. Figure 12K is a flow cytometric titration graph of integrin-αvβ1 CHO cells. Figure 12L is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ using different clone IgG. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 12B]Figure 12A is a summary bar graph showing epitope binding competition of different IgG antibodies in the presence of 10404 Fab protein using integrin-αvβ1 CHO cells. Figure 12B is a summary bar graph of IgG inhibition of cell adhesion to different LAP-TGFβ using different integrin CHO cell lines. For each cell line, the different IgG antibodies (11883, 11855, 11857, 11874, 11914, and 11867) used in the experiment are shown from left to right. Figures 12C-12I are size exclusion chromatography graphs of trastuzumab (Figure 12C), 10404 (Figure 12D), 11855 (Figure 12E), 11857 ​​(Figure 12F), 11867 (Figure 12G), 11883 (Figure 12H), and 11914 (Figure 12I) IgG clones. Figure 12J is a table summarizing the affinity and inhibitory values ​​of different clone IgG. Figure 12K is a flow cytometric titration graph of integrin-αvβ1 CHO cells. Figure 12L is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ using different clone IgG. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 12C]Figure 12A is a summary bar graph showing epitope binding competition of different IgG antibodies in the presence of 10404 Fab protein using integrin-αvβ1 CHO cells. Figure 12B is a summary bar graph of IgG inhibition of cell adhesion to different LAP-TGFβ using different integrin CHO cell lines. For each cell line, the different IgG antibodies (11883, 11855, 11857, 11874, 11914, and 11867) used in the experiment are shown from left to right. Figures 12C-12I are size exclusion chromatography graphs of trastuzumab (Figure 12C), 10404 (Figure 12D), 11855 (Figure 12E), 11857 ​​(Figure 12F), 11867 (Figure 12G), 11883 (Figure 12H), and 11914 (Figure 12I) IgG clones. Figure 12J is a table summarizing the affinity and inhibitory values ​​of different clone IgG. Figure 12K is a flow cytometric titration graph of integrin-αvβ1 CHO cells. Figure 12L is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ using different clone IgG. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 12D]Figure 12A is a summary bar graph showing epitope binding competition of different IgG antibodies in the presence of 10404 Fab protein using integrin-αvβ1 CHO cells. Figure 12B is a summary bar graph of IgG inhibition of cell adhesion to different LAP-TGFβ using different integrin CHO cell lines. For each cell line, the different IgG antibodies (11883, 11855, 11857, 11874, 11914, and 11867) used in the experiment are shown from left to right. Figures 12C-12I are size exclusion chromatography graphs of trastuzumab (Figure 12C), 10404 (Figure 12D), 11855 (Figure 12E), 11857 ​​(Figure 12F), 11867 (Figure 12G), 11883 (Figure 12H), and 11914 (Figure 12I) IgG clones. Figure 12J is a table summarizing the affinity and inhibitory values ​​of different clone IgG. Figure 12K is a flow cytometric titration graph of integrin-αvβ1 CHO cells. Figure 12L is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ using different clone IgG. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 12E]Figure 12A is a summary bar graph showing epitope binding competition of different IgG antibodies in the presence of 10404 Fab protein using integrin-αvβ1 CHO cells. Figure 12B is a summary bar graph of IgG inhibition of cell adhesion to different LAP-TGFβ using different integrin CHO cell lines. For each cell line, the different IgG antibodies (11883, 11855, 11857, 11874, 11914, and 11867) used in the experiment are shown from left to right. Figures 12C-12I are size exclusion chromatography graphs of trastuzumab (Figure 12C), 10404 (Figure 12D), 11855 (Figure 12E), 11857 ​​(Figure 12F), 11867 (Figure 12G), 11883 (Figure 12H), and 11914 (Figure 12I) IgG clones. Figure 12J is a table summarizing the affinity and inhibitory values ​​of different clone IgG. Figure 12K is a flow cytometric titration graph of integrin-αvβ1 CHO cells. Figure 12L is a graph of the inhibition titration of integrin-αvβ1 CHO cells for adhesion to LAP-TGFβ using different clone IgG. MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. [Figure 13] Figures 13A and 13C-13F are summary graphs showing titration curves of 11867 and 10392 IgG modalities against integrin CHO cell lines αvβ1 (Figure 13A), αvβ3 (Figure 13C), αvβ5 (Figure 13D), αvβ6 (Figure 13E), and αvβ8 (Figure 13F). MFI indicates mean fluorescence from the mean and error bars indicate standard deviation. Figure 13B is a summary table of affinity values ​​of different integrin CHO cell lines. [Figure 14]14A and 14C-14F are graphs summarizing the inhibitory titration curves of cell adhesion to LAP-TGFβ of different clone IgGs using the integrin CHO cell lines αvβ1 (FIG. 14A), αvβ3 (FIG. 14C), αvβ5 (FIG. 14D), αvβ6 (FIG. 14E), and αvβ8 (FIG. 14F). Error bars indicate standard deviation. FIG. 14B is a table summarizing the inhibitory values ​​of cell adhesion to LAP-TGFβ of different clone IgGs using different integrin CHO cell lines. [Figure 15A] Figure 15A is a graph of a flow cytometry summary of αv- and β1-subunit expression levels using 11876 / 10392 dual IgG, showing the log2 values ​​of fold change in MFI signal of selective subunits relative to isotype control antibody. Figure 15B is a bar graph of a summary of lung cell proliferation at the endpoint time point on day 4 in the presence of 11876 / 10392 dual IgG. Figures 15C-15G are time series graphs showing cell proliferation of lung cancer cell lines A549 (Figure 15C), H292 (Figure 15D), H661 (Figure 15E), H460 (Figure 15F), and H1563 (Figure 15G) in the presence of 11876 / 10392 dual IgG. Error bars indicate standard deviation. [Figure 15B] Figure 15A is a graph of a flow cytometry summary of αv- and β1-subunit expression levels using 11876 / 10392 dual IgG, showing the log2 values ​​of fold change in MFI signal of selective subunits relative to isotype control antibody. Figure 15B is a bar graph of a summary of lung cell proliferation at the endpoint time point on day 4 in the presence of 11876 / 10392 dual IgG. Figures 15C-15G are time series graphs showing cell proliferation of lung cancer cell lines A549 (Figure 15C), H292 (Figure 15D), H661 (Figure 15E), H460 (Figure 15F), and H1563 (Figure 15G) in the presence of 11876 / 10392 dual IgG. Error bars indicate standard deviation. [Figure 15C]Figure 15A is a graph of a flow cytometry summary of αv- and β1-subunit expression levels using 11876 / 10392 dual IgG, showing the log2 values ​​of fold change in MFI signal of selective subunits relative to isotype control antibody. Figure 15B is a bar graph of a summary of lung cell proliferation at the endpoint time point on day 4 in the presence of 11876 / 10392 dual IgG. Figures 15C-15G are time series graphs showing cell proliferation of lung cancer cell lines A549 (Figure 15C), H292 (Figure 15D), H661 (Figure 15E), H460 (Figure 15F), and H1563 (Figure 15G) in the presence of 11876 / 10392 dual IgG. Error bars indicate standard deviation. [Figure 16] Figure 16A is a titration curve graph showing inhibitory titration evaluation in different lung cancer cell lines using 11867 / 10392 dual IgG. Error bars indicate standard deviation. Figure 16B is a summary table of IC50 values ​​obtained from dual IgG 11867 / 10392 in different lung cancer cell lines. [Figure 17A] Figures 17A-17P are graphical representations of the evaluation of cell migration inhibition using dual IgG 11867 / 10392. Figures 17A (A549), 17E (H460), 17I (H661), and 17M (H1563) are time-lapse graphs of wound healing assays using different lung cancer cell lines, with error bars indicating standard deviation. For each experiment, Figures 17B, 17F, 17J, and 17N show representative microscopic images of untreated cells, Figures 17C, 17G, 17K, and 17O show representative microscopic images of control cells, and Figures 17D, 17H, 17L, and 17P show representative microscopic images of dual IgG 11867 / 10392 treated cells. [Figure 17B]Figures 17A-17P are graphical representations of the evaluation of cell migration inhibition using dual IgG 11867 / 10392. Figures 17A (A549), 17E (H460), 17I (H661), and 17M (H1563) are time-lapse graphs of wound healing assays using different lung cancer cell lines, with error bars indicating standard deviation. For each experiment, Figures 17B, 17F, 17J, and 17N show representative microscopic images of untreated cells, Figures 17C, 17G, 17K, and 17O show representative microscopic images of control cells, and Figures 17D, 17H, 17L, and 17P show representative microscopic images of dual IgG 11867 / 10392 treated cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] The present disclosure relates to antibodies, bispecific antibodies, multispecific antibodies, and antigen-binding fragments thereof that specifically bind to integrin-αv heterodimers and inhibit integrin-αv-mediated activation of TGFβ. Some embodiments of the present disclosure are directed to antibodies, or antigen-binding portions thereof, that specifically bind to integrin-αv heterodimers and inhibit integrin-αv-mediated activation of TGFβ. Some embodiments of the present disclosure are directed to bispecific antibodies, or antigen-binding portions thereof, that specifically bind to integrin-αv heterodimers and inhibit integrin-αv-mediated activation of TGFβ. In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises at least a first paratope and a second paratope, the first paratope binds to a first epitope on an integrin αv heterodimer (e.g., integrin-αvβ1) heterodimer, and the second paratope binds to a second epitope on an integrin αv heterodimer (e.g., integrin-αvβ1) heterodimer. Other embodiments of the present disclosure relate to a method of treating a subject in need thereof, comprising administering to the subject an antibody, bispecific antibody, multispecific antibody, or antigen-binding fragment thereof that specifically binds to an integrin-αv heterodimer and inhibits integrin-αv-mediated activation of TGFβ. In some embodiments, the subject has cancer, and the antibody, bispecific antibody, multispecific antibody, or antigen-binding fragment thereof treats the cancer in the subject.

[0074] I. Terminology In order that the description herein may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0075] It should be noted that the term "a" or "an" entity refers to one or more of that entity; for example, a "nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0076] Furthermore, "and / or", when used herein, shall be interpreted as a specific disclosure that each of the two specified features or components may or may not be accompanied by each other. Thus, when the term "and / or" is used herein in a phrase such as "A and / or B", it is intended to include "A and B", "A or B", "A (single)", and "B (single)". Similarly, when the term "and / or" is used in a phrase such as "A, B, and / or C", it is intended to include 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 (single); B (single); and C (single).

[0077] Where an embodiment is described herein using the word "comprising," it is understood that analogous embodiments separately described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure pertains. See, for example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill in the art with a general dictionary for many of the terms used in this disclosure.

[0079] Units, prefixes, and symbols are shown in the format accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. Nucleotide sequences are written from left to right in a 5' to 3' orientation unless otherwise indicated. Amino acid sequences are written from left to right in an amino to carboxy orientation. The headings provided herein are not intended to limit the various aspects of the disclosure, which may be obtained by reference to the specification as a whole. Thus, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0080] The term "about" is used herein to mean approximately, roughly, around, or within a range thereof. When the term "about" is used in conjunction with a numerical range, the term modifies that range by extending the boundaries above and below the numerical values ​​set forth. In general, the term "about" can modify the numerical value above and below the stated value, for example, 10 percent above or below (higher or lower).

[0081] The term "antibody" refers, in some embodiments, to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). As used herein, the term "bispecific antibody" refers to an antibody comprising at least two antigen-binding domains, i.e., at least two paratopes. Thus, in some embodiments, a bispecific antibody comprises at least two heavy chain variable regions (VH1 and VH2) and at least two light chain variable regions (VL1 and VL2. In some embodiments, the at least two heavy chain variable regions are the same or different. In some embodiments, the at least two light chain variable regions are the same or different. As used herein, a "multispecific antibody" refers to an antibody comprising at least three antigen-binding domains, i.e., at least three paratopes.

[0082] In some antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In some antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, such as various cells of the immune system (e.g., effector cells) and the first component (Clq) of the traditional complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, the amino acids of the variable region are numbered using the Kabat numbering system, and the amino acids of the constant region are numbered using the EU system.

[0083] "IgG antibodies", such as human IgG1, IgG2, IgG3 and IgG4 antibodies, as used herein, in some embodiments have the structure of a naturally occurring IgG antibody, i.e., it has the same number of heavy and light chains and disulfide bonds as a naturally occurring IgG antibody of the same subclass. For example, an anti-integrin-αv heterodimeric IgG1, IgG2, IgG3 or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), in which these two HCs and LCs are linked by disulfide bridges in the same number and positions as occur in naturally occurring IgG1, IgG2, IgG3 and IgG4 antibodies, respectively (unless the antibody has been mutated to modify the disulfide bridges).

[0084] Antibodies usually bind to their cognate antigens. -5 ~10 -11 The dissociation constant (K D ) specifically binds with high affinity as reflected by approximately 10 -4 Any K greater than M D As used herein, an antibody that "specifically binds" to an antigen is one that binds to the antigen and substantially the same antigen in a 10 -7 M or less, 10 -8 M or less, 5×10 -9 M or less, or 10 -8 M to 10 -10 K between M and below D An antibody that specifically binds to a given antigen is an antibody that binds to a given antigen with high affinity, meaning that it has high affinity but does not bind to unrelated antigens. An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity to the given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97% or at least 99% sequence identity to the sequence of the given antigen. As an example, an antibody that specifically binds to a human integrin αv heterodimer (e.g., integrin-αvβ1) may in some embodiments cross-react with integrin αv heterodimer (e.g., integrin-αvβ1) antigens from a particular primate species (e.g., cynomolgus monkey integrin αv heterodimer (e.g., integrin-αvβ1)), but may not cross-react with integrin αv heterodimer (e.g., integrin-αvβ1) antigens from other species, or with antigens other than integrin αv heterodimer (e.g., integrin-αvβ1).

[0085] Immunoglobulins may be from any of the well-known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM. The IgG isotype is divided into subclasses in certain species: IgG1, IgG2, IgG3 and IgG4 in humans and IgG1, IgG2a, IgG2b and IgG3 in mice. In some embodiments, the anti-integrin-αv heterodimeric antibodies described herein are of the IgG1 subtype. Immunoglobulins, such as IgG1, exist in several allotypes that differ from each other by at most a few amino acids. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and fully synthetic antibodies.

[0086] As used herein, the term "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a human integrin αv heterodimer (e.g., integrin-αvβ1)). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody, e.g., an anti-integrin-αv heterodimer antibody described herein, include (i) a V L , V H (ii) a F(ab')2 fragment (a fragment derived from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a V H and an Fd fragment consisting of the CH1 domain, (iv) a V of one arm of an antibody. L and V H (v) an Fv fragment consisting of a V H(vi) an isolated complementarity determining region (CDR); and (vii) a combination of two or more isolated CDRs, which may be connected by a synthetic linker, if desired. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be connected using recombinant methods with a synthetic linker that allows the VL and VH domain pair to be made as a single protein chain forming a monovalent molecule (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0087] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148:1547-1553 (1992). In some embodiments, the bispecific antibodies disclosed herein are produced by modifying a first heavy chain in a manner that increases the affinity of the first heavy chain for a second heavy chain, for example, through alteration of the amino acid sequence or one or more amino acid side chains in the constant region of the first heavy chain. In some embodiments, the modifications are made to both the first heavy chain and the second heavy chain. In some embodiments, the modifications promote heterodimerization of the first and second heavy chains. Any modification that increases the affinity of the first and second heavy chains can be used. In certain embodiments, the first heavy chain is modified to contain a knob motif and the second heavy chain is modified to contain a hole motif.

[0088] The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogenous antibodies, i.e., the individual antibodies contained within the population are substantially similar and bind to the same epitope (e.g., the antibodies exhibit a single binding specificity and affinity), except for possible variants that may arise during the production of the monoclonal antibody, although such variants are generally present in minor amounts. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogenous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. The term "human monoclonal antibody" refers to an antibody derived from a substantially homogenous population of antibodies that exhibits a single binding specificity and has variable and optionally constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse having a genome that includes a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0089] As used herein, the term "recombinant human antibody" includes all human antibodies prepared, expressed, generated, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal of human immunoglobulin genes or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express the antibody, e.g., from transfectomas, (c) antibodies isolated from recombination, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, generated, or isolated by any other means, including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies utilize certain human germline immunoglobulin sequences and contain variable and constant regions encoded by germline genes, but include subsequent rearrangements and mutations that occur, e.g., during antibody maturation. As known in the art (see, e.g., Lonberg (2005) Nature Biotech. 23(9):1117-1125), the variable regions contain antigen-binding domains encoded by different genes that rearrange to form antibodies specific to foreign antigens. In addition to rearrangement, the variable regions can be further modified by multiple single amino acid changes (also called somatic mutation or hypermutation) to increase the affinity of the antibody to the foreign antigen. The constant regions change further in response to the antigen (i.e., isotype switching). Thus, the rearranged and somatically mutated nucleic acid molecules encoding light and heavy immunoglobulin polypeptides in response to the antigen may not have sequence identity to the original nucleic acid molecule, but instead will be substantially identical or similar (i.e., have at least 80% identity).

[0090] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The anti-integrin-αv heterodimeric antibody described herein may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-specific mutagenesis or by in vivo somatic mutation). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used interchangeably.

[0091] A "humanized" antibody refers to an antibody in which some, most or all of the amino acids outside the CDR domain of a non-human antibody are replaced with the corresponding amino acids from a human immunoglobulin. In some embodiments of a humanized form of an antibody, some, most or all of the amino acids outside the CDR domain are replaced with amino acids from a human immunoglobulin, while some, most or all of the amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions or substitutions or modifications of amino acids are permitted as long as they do not abrogate the ability of the antibody to bind to a particular antigen. A "humanized" antibody retains antigen specificity similar to that of the original antibody.

[0092] "Chimeric antibody" refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species, such as an antibody whose variable region is derived from a murine antibody and whose constant region is derived from a human antibody.

[0093] As used herein, "isotype" refers to the antibody class (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE antibodies) that is encoded by heavy chain constant region genes.

[0094] "Allotype" refers to naturally occurring variants within a particular isotype group, which variants differ by a few amino acids (see, for example, Jefferis et al. (2009) mAbs 1:1). The anti-integrin-αv heterodimeric antibodies described herein can be of any allotype. Antibodies referred to herein as "IgG1f", "IgG1.1f" or "IgG1.3f" isotypes are IgG1, effectorless IgG1.1 and effectorless IgG1.3 antibodies with allotype "f", i.e., 214R, 356E and 358M according to the EU index in Kabat, respectively.

[0095] The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used herein synonymously with the term "an antibody that specifically binds to an antigen."

[0096] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other proteins and cellular material.

[0097] As used herein, an antibody that "binds to an integrin αv heterodimer (e.g., integrin-αvβ1)" refers to an antibody that has an EC of about 25 μg / mL or less, about 23 μg / mL or less, about 20 μg / mL or less, about 15 μg / mL or less, about 10 μg / mL or less, about 5 μg / mL or less, about 3 μg / mL or less, about 2 μg / mL or less, about 1 μg / mL or less, about 0.5 μg / mL or less, about 0.45 μg / mL or less, about 0.4 μg / mL or less, about 0.35 μg / mL or less, or about 0.3 μg / mL or less in a binding assay using, for example, CHO cells transfected with human integrin αv heterodimer (e.g., integrin-αvβ1) or tumor cells expressing an integrin αv heterodimer (e.g., integrin-αvβ1) in an art-recognized manner. 50In some embodiments, the term "anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody is intended to refer to an antibody that interacts with an integrin αv heterodimer (e.g., integrin-αvβ1) at about 200 nM or less, about 175 nM or less, about 160 nM or less, about 150 nM or less, about 125 nM or less, about 110 nM or less, about 100 nM or less, about 80 nM or less, about 75 nM or less, about 60 nM or less, about 50 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, about 9 nM or less, about 8 nM or less, about 7 nM or less, about 8 nM or less, about 9 ... M or less, about 6nM or less, about 5nM or less, about 4nM or less, about 3nM or less, about 2nM or less, about 1.9nM or less, about 1.8nM or less, about 1.7nM or less, about 1.6nM or less, about 1.5nM or less, about 1.4nM or less, about 1.3nM or less, about 1.2n EC of M or less, about 1.1 nM or less, about 1.0 nM or less, about 0.9 nM or less, about 0.8 nM or less, about 0.7 nM or less, about 0.6 nM or less, about 0.5 nM or less, about 0.4 nM or less, about 0.3 nM or less, about 0.2 nM or less, or about 0.1 nM or less 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1), e.g., expressed on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds to human integrin αv heterodimer (e.g., integrin-αvβ1) expressed, for example, on CHO cells. In certain embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC 50 and binds, for example, to human integrin αv heterodimers (eg, integrin-αvβ1) expressed on CHO cells.

[0098] "Effector function" refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical event that results from it. Exemplary "effector functions" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and downregulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain).

[0099] The term "epitope" or "antigenic determinant" refers to a site on an antigen (e.g., an integrin αv heterodimer (e.g., integrin-αvβ1)) to which an immunoglobulin or antibody specifically binds, e.g., as defined by the particular method used to identify it. Epitopes can be formed both from contiguous amino acids (usually linear epitopes) or non-contiguous amino acids juxtaposed by tertiary folding of a protein (usually conformational epitopes). Epitopes formed from contiguous amino acids are usually, but not necessarily, retained upon exposure to denaturing solvents, whereas epitopes formed from tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes usually include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining the epitope bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, in which overlapping or consecutive peptides (e.g., derived from an integrin αv heterodimer (e.g., integrin-αvβ1)) are tested for reactivity with a given antibody (e.g., an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody). Methods for determining the spatial conformation of an epitope include techniques in the art and described herein, such as X-ray crystallography, X-ray cocrystallography, antigen mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS (see, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris, Ed. (1996)). The term "epitope mapping" refers to the process of identifying molecular determinants of antibody-antigen recognition.

[0100] With respect to two or more antibodies, the term "binds to the same epitope" means that the antibodies bind to the same segment of amino acid residues as determined by a given method. Techniques for determining whether the antibodies bind to the "same epitope on integrin αv heterodimer (e.g., integrin-αvβ1)" using the antibodies described herein include epitope mapping methods such as, for example, X-ray analysis of crystals of antigen:antibody complexes that provide atomic resolution of the epitope and hydrogen / deuterium exchange mass spectrometry (HDX-MS). Other methods monitor the binding of antibodies to antigen fragments or to mutated variations of the antigen, where loss of binding due to modification of amino acid residues in the antigen sequence is often considered an indication of epitope components. Additionally, computational combinatorial methods for epitope mapping may be used. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. Antibodies with identical VH and VL or identical CDR1, 2 and 3 sequences are predicted to bind to the same epitope.

[0101] An antibody that "competes with another antibody for binding to a target" refers to an antibody that inhibits (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent an antibody inhibits the binding of the other antibody to the target, may be examined using known competition experiments, such as BIACORE® surface plasmon resonance (SPR) analysis. In some embodiments, an antibody competes with or inhibits the binding of another antibody to the target by at least 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may vary depending on which antibody is the "blocking antibody" (i.e., the cold antibody that is first incubated with the target). Competitive assays can be performed, for example, as described in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006; doi:10.1101 / pdb.prot4277 or in Chapter 11 of "Using Antibodies" by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Two antibodies "cross-compete" if they block each other by at least 50% in both directions, regardless of whether one antibody or the other antibody contacts the antigen first in the competition experiment.

[0102] Competitive binding assays to determine whether two antibodies compete or cross-compete for binding include, for example, competition for binding to cells expressing integrin αv heterodimers (e.g., integrin-αvβ1) by flow cytometry, such as that described in the Examples. Other methods include SPR (e.g., BIACORE®), solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see Stahli et al., Methods in Enzymology 9:242 (1983)); solid-phase direct biotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid-phase direct labeling RIA using 1-125 label (see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid-phase direct biotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and direct labeling RIA (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0103] As used herein, the term "paratope" refers to one or more amino acids present in an antibody or antigen-binding fragment thereof that interacts with an epitope on an antigen.

[0104] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to antibody binding to an epitope on a given antigen. Typically, an antibody (i) binds to an epitope of approximately 10% or more of the antigen, as determined, for example, by surface plasmon resonance (SPR) techniques in a BIACORE® 2000 using a given antigen, e.g., recombinant human integrin αv heterodimer (e.g., integrin-αvβ1), as the analyte and an antibody as the ligand, or Scatchard analysis of the binding of the antibody to antigen-positive cells. -7 Less than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 The equilibrium dissociation constant (K D ) and (ii) binds to a given antigen with an affinity that is at least 2-fold greater than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). Thus, an antibody that "specifically binds to human integrin αv heterodimer (e.g., integrin-αvβ1)" is one that specifically binds to a given antigen with an affinity that is at least 2-fold greater than its affinity for binding to a nonspecific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). -7 M or less, for example, about 10 -8 M, 10 -9 M or 10 -10 K less than or even smaller than M D and refers to an antibody that binds to the human integrin αv heterodimer (e.g., integrin-αvβ1).

[0105] As used herein, the term "k assoc " or "k a " is intended to refer to the association rate of a particular antibody-antigen interaction, whereas, as used herein, the term "k dis " or "k d " refers to the off-rate of a particular antibody-antigen interaction. D " as used herein refers to the dissociation constant, which is k a k for d (i.e., k d / k a ) and expressed as molar concentration (M). DValues ​​can be determined using methods well established in the art. D Available methods for determining include surface plasmon resonance, the BIACORE® system, or flow cytometry and Scatchard analysis.

[0106] As used herein, the term "high affinity" with respect to an IgG antibody means that the antibody has a high affinity for the target antigen, -8 M or less, 10 -9 M or less or 10 -10 K below M D However, "high affinity" binding may vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody having a binding affinity of 10 -10 M or less or 10 -8 K below M D It refers to having.

[0107] The term "EC 50 " refers to the concentration of an antibody or antigen-binding portion thereof that induces 50% of the maximal response, i.e., a response that is halfway between the maximal response and the baseline.

[0108] The term "naturally occurring" as applied to an object herein refers to the fact that the object can be found in nature.For example, naturally occurring polypeptide or polynucleotide sequences exist in organisms (including viruses) that can be isolated from sources in nature and are not intentionally modified by humans in laboratories.

[0109] A "polypeptide" refers to a chain containing at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues in a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0110] The term "nucleic acid molecule" as used herein includes DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded, and may be cDNA.

[0111] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with 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, tryptophan), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody is replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummel et al., Biochem. 32:1180-1187 (1993); Kobayashi et al. Protein Eng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0112] With respect to nucleic acids, the term "substantial homology" indicates that two nucleic acids or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80%, at least about 90%-95%, or at least about 98%-99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand.

[0113] With respect to polypeptides, the term "substantial homology" indicates that two polypeptides or their designated sequences, when optimally aligned and compared, are identical, with appropriate amino acid insertions or deletions, in at least about 80%, at least about 90%-95%, or at least about 98%-99.5% of the amino acids.

[0114] The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions x 100) and takes into account the number of gaps, the length of each gap, that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0115] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at worldwideweb.gcg.com) using a NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) as incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. Furthermore, percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), 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 or 6.

[0116] The nucleic acid and protein sequences described herein can further be used as a "query sequence" to perform a search against public databases, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. To obtain nucleotide sequences homologous to the nucleic acid molecules described herein, BLAST nucleotide searches can be performed using the NBLAST program, score=100, wordlength=12. To obtain amino acid sequences homologous to the protein molecules described herein, BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See worldwideweb.ncbi.nlm.nih.gov.

[0117] Nucleic acids may be present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" when they are purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others known in the art. See F. Ausubel, et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0118] Nucleic acids, for example cDNA, may be mutated according to standard techniques for providing gene sequences. For coding sequences, these mutations may affect the desired amino acid sequence. In particular, DNA sequences that are substantially homologous to or derived from natural V, D, J, constant, switch and other such sequences described herein are considered (wherein "derived" indicates that the sequence is identical to or modified from another sequence).

[0119] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. Since the plasmid is the most commonly used form of vector, "plasmid" and "vector" may be used interchangeably herein. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, are also included.

[0120] The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell that contains a nucleic acid that does not naturally occur in the cell and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in later generations, either by mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0121] "Immune response" is understood in the art and generally refers to a biological response in a vertebrate to a foreign substance or an abnormal, e.g., cancerous, cell, which protects the organism from these substances and the diseases caused by them. Immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, and complement) produced by any of these cells or the liver, which results in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune response includes, for example, the activation or inhibition of T cells, e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells, or the activation or inhibition of any other cells of the immune system, e.g., NK cells.

[0122] An "immunomodulator" or "immunoregulator" refers to a substance that may be involved in the regulation, control or modification of an immune response, e.g., a substance that targets components of a signal transduction pathway. "Regulation," "control," or "modulation" of an immune response refers to any change in the cells of the immune system or in the activity of such cells (e.g., effector T cells, such as Th1 cells). Such regulation includes stimulation or suppression of the immune system and may be indicated by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other change that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced function in the tumor microenvironment. In some embodiments, an immunomodulator targets a molecule on the surface of a T cell. An "immunomodulatory target" or "immunoregulatory target" is a molecule, e.g., a cell surface molecule, that is targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered by the binding of the substance, agent, moiety, compound, or molecule. Immunomodulatory targets include, for example, cell surface receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands").

[0123] "Immunotherapy" refers to the treatment of a subject suffering from or at risk of developing or suffering from a recurrence of a disease by methods involving inducing, enhancing, suppressing or otherwise modifying the immune system or immune response.

[0124] As used herein, the term "linked" refers to the association of two or more molecules. Linkages can be covalent or non-covalent. Linkages can also be genetic (i.e., recombinantly fused). Such linkages can be achieved using a variety of art-recognized techniques, such as chemical conjugation and recombinant protein production.

[0125] As used herein, "administering" refers to the physical introduction of a composition containing a therapeutic agent into a subject using any of a variety of methods and delivery systems. Alternative routes of administration of the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. Alternatively, the antibodies described herein may be administered by a non-parenteral route, such as a topical, epithelial or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually or topically. Administering may be performed, for example, once, multiple times and / or over one or more extended periods of time.

[0126] As used herein, the phrase "inhibiting tumor growth" includes any measurable decrease in tumor growth, such as at least about 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99% or 100% inhibition of tumor growth. In some embodiments, inhibition of tumor growth is measured as tumor growth inhibition percentage (TGI%). TGI% is calculated according to the formula: [1-(T t / T0) / (C t / C0))] / [(C t -C0) / C t ]*100[Formula 1](In the formula, T t = individual tumor size of treated animals at time "t", T0 = individual tumor size of treated animals at the first measurement, C tThe TGI can be determined by calculating the TGI at time "t" calculated from all treated animals according to: TGI = median tumor size of control animals at time "t", C0 = median tumor size of control animals at the first measurement.

[0127] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can lead to the formation of malignant tumors or cells that can invade adjacent tissues and metastasize to distant sites in the body via the lymphatic system or bloodstream.

[0128] The terms "treat", "treating" and "treatment" as used herein refer to any type of intervention or process performed on a subject or administration of an active agent to a subject with the goal of reversing, alleviating, ameliorating, inhibiting, or slowing or preventing the progression, occurrence, severity or recurrence of symptoms, complications, conditions or biochemical manifestations associated with a disease, or enhancing overall survival. Treatment may be of subjects with disease or subjects without disease (e.g., for prophylaxis).

[0129] The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease regression as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time that a patient diagnosed with a disease is still alive from the date of diagnosis or treatment initiation of disease, e.g., cancer), or prevention of functional impairment or disability due to disease affliction. A therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount" or "prophylactically effective dosage," which is the amount of drug that inhibits the occurrence or recurrence of disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing disease or suffering from recurrence of disease. The ability of a therapeutic agent to promote disease regression or inhibit the onset or recurrence of disease can be evaluated using a variety of methods, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, and by assaying the activity of the agent in in vitro assays.

[0130] As an example, an anti-cancer drug is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administering an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of disease symptom-free periods, an increase in overall survival, prevention of functional or disabling disorders due to disease afflictions, or otherwise remission of disease symptoms in a patient. Furthermore, the terms "effective" and "effectiveness" in relation to treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cell, organ and / or organism level resulting from administration of the drug.

[0131] As an example of tumor treatment, a therapeutically effective amount or dosage of a drug inhibits cell growth or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to untreated subjects. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%. The ability of a compound to inhibit tumor growth can be evaluated using the assays described below. Alternatively, the properties of the composition can be evaluated by examining the ability of the compound to inhibit cell growth, and such inhibition can be measured in vitro by any assay. In some embodiments described herein, tumor regression can be observed and can last for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

[0132] The term "patient" includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment.

[0133] As used herein, the term "subject" includes any human or non-human animal.For example, the methods and compositions described herein can be used to treat subjects with cancer.The term "non-human animal" includes all vertebrates, such as non-human primates, sheep, dogs, cows, chickens, and other mammals and non-mammals, amphibians, reptiles, and the like.

[0134] As used herein, the terms "ug" and "uM" are used synonymously with "μg" and "μM", respectively.

[0135] The various aspects described herein are described in further detail in the following subsections.

[0136] II. Compositions of the Disclosure Certain embodiments of the present disclosure are directed to isolated antibodies or antigen-binding portions thereof that specifically bind to integrin-αv heterodimers (e.g., integrin-αvβ1) and inhibit, prevent or reduce integrin-αv-mediated activation of TGFβ. Some embodiments of the present disclosure are directed to bispecific antibodies that specifically bind to integrin-αv heterodimers (e.g., integrin-αvβ1) and inhibit, prevent or reduce integrin-αv-mediated activation of TGFβ. Some embodiments of the present disclosure are directed to methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject an antibody or antigen-binding portion thereof or a bispecific antibody that specifically binds to integrin-αv heterodimers (e.g., integrin-αvβ1) and inhibits, prevents or reduces integrin-αv-mediated activation of TGFβ.

[0137] In some embodiments, integrin-αv-mediated TGFβ activation is reduced by at least about 1-fold, at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, or at least about 10-fold compared to the level of integrin-αv-mediated TGFβ activation prior to contact with an antibody, or antigen-binding portion thereof, or bispecific antibody of the present disclosure. In some embodiments, integrin-αv-mediated TGFβ activation is completely eliminated after contact with an antibody, or antigen-binding portion thereof, or bispecific antibody of the present disclosure.

[0138] II.A. Anti-integrin-αv heterodimer antibodies Certain embodiments of the present disclosure are directed to isolated antibodies or antigen-binding portions thereof that specifically bind to integrin-αv heterodimers (e.g., integrin-αvβ1) and inhibit, prevent or reduce integrin-αv-mediated activation of TGFβ. In some embodiments, the antibodies or antigen-binding portions thereof comprise a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR3 comprises the amino acid sequence of SEQ ID NO:5, 15, or 15. In some embodiments, VH-CDR2 comprises the amino acid sequence of SEQ ID NO:4, 14, or 24. In some embodiments, VH-CDR1 comprises the amino acid sequence of SEQ ID NO:3, 13, or 23. In some embodiments, the VL-CDR3 comprises the amino acid sequence of SEQ ID NO: 10, 20, or 30. In some embodiments, the VL-CDR2 comprises the amino acid sequence of SEQ ID NO: 9, 19, or 29. In some embodiments, the VL-CDR1 comprises the amino acid sequence of SEQ ID NO: 8, 18, or 28.

[0139] [Table 1] [Table 2] [Table 3]

[0140] Some embodiments of the present disclosure are directed to an antibody or antigen-binding portion thereof that cross-competes with a reference antibody or antigen-binding portion thereof for binding to an integrin-αv heterodimer, wherein the reference antibody comprises a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, VL-CDR3, and wherein VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5, 15, or 15.

[0141] Some embodiments of the present disclosure are directed to an antibody or antigen-binding portion thereof that binds to the same epitope on the integrin-αv heterodimer as a reference antibody or antigen-binding portion thereof, wherein the reference antibody comprises a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, and wherein VH-CDR3 comprises the amino acid sequence of SEQ ID NO:5, 15, or 15.

[0142] Some embodiments of the present disclosure are directed to an antibody or antigen-binding portion thereof that binds to an epitope on an integrin-αv heterodimer having at least one amino acid residue that overlaps with the epitope recognized by a reference antibody or antigen-binding portion thereof, wherein the reference antibody comprises a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, and wherein VH-CDR3 comprises the amino acid sequence of SEQ ID NO: 5, 15, or 15.

[0143] In some embodiments, the VH-CDR2 of the reference antibody comprises the amino acid sequence of SEQ ID NO: 4, 14, or 24. In some embodiments, the VH-CDR1 of the reference antibody comprises the amino acid sequence of SEQ ID NO: 3, 13, or 23. In some embodiments, the VL-CDR3 of the reference antibody comprises the amino acid sequence of SEQ ID NO: 10, 20, or 30. In some embodiments, the VL-CDR2 of the reference antibody comprises the amino acid sequence of SEQ ID NO: 9, 19, or 29. In some embodiments, the VL-CDR1 of the reference antibody comprises the amino acid sequence of SEQ ID NO: 8, 18, or 28.

[0144] In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10.

[0145] In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0146] In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:30.

[0147] In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a heavy chain variable region ("VH") and a light chain variable region ("VL"), wherein the VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 2, 12, and 22. In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a heavy chain variable region ("VH") and a light chain variable region ("VL"), wherein the VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 7, 17, and 27. In some embodiments, the reference antibody or antigen-binding portion thereof comprises an amino acid sequence selected from SEQ ID NOs: 7, 17, and 27.

[0148] In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 2 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the reference antibody, or antigen-binding portion thereof, comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 22 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0149] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a VH comprising VH-CDR1, VH-CDR2, and VH-CDR3, and a VL comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10.

[0150] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a VH comprising VH-CDR1, VH-CDR2, and VH-CDR3, and a VL comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 20.

[0151] Some embodiments of the present disclosure are directed to an isolated antibody or antigen-binding portion thereof that specifically binds to an integrin-αv heterodimer, comprising a VH comprising VH-CDR1, VH-CDR2, and VH-CDR3, and a VL comprising VL-CDR1, VL-CDR2, and VL-CDR3, wherein VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23, VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24, VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25, VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28, VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:30.

[0152] In some embodiments, the antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 2 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 12 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the antibody or antigen-binding portion thereof comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 22 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 27.

[0153] In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain ("HC") and a light chain ("LC"), wherein the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 11, and 21. In some embodiments, the HC comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 point mutations compared to the amino acid sequence set forth in SEQ ID NOs: 1, 11, or 21. In some embodiments, the HC comprises one point mutation compared to the amino acid sequence set forth in SEQ ID NO: 1, 11, or 21. In some embodiments, the HC comprises two point mutations compared to the amino acid sequence set forth in SEQ ID NO: 1, 11, or 21. In some embodiments, the HC comprises three point mutations compared to the amino acid sequence set forth in SEQ ID NO: 1, 11, or 21. In some embodiments, the HC comprises four point mutations compared to the amino acid sequence set forth in SEQ ID NO: 1, 11, or 21. In some embodiments, the HC comprises five point mutations compared to the amino acid sequence set forth in SEQ ID NO: 1, 11, or 21. In some embodiments, the HC comprises ten point mutations compared to the amino acid sequence set forth in SEQ ID NO: 1, 11, or 21.

[0154] In some embodiments, the HC comprises an amino acid sequence selected from SEQ ID NOs: 1, 11, and 21. In particular embodiments, the HC comprises the amino acid sequence set forth in SEQ ID NO: 1. In particular embodiments, the HC comprises the amino acid sequence set forth in SEQ ID NO: 11. In particular embodiments, the HC comprises the amino acid sequence set forth in SEQ ID NO: 21.

[0155] In some embodiments, the antibody or antigen-binding portion thereof comprises a HC and a LC, wherein the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 6, 16, and 26. In some embodiments, the LC comprises at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 point mutations compared to the amino acid sequence set forth in SEQ ID NOs: 6, 16, or 26. In some embodiments, the LC comprises one point mutation compared to the amino acid sequence set forth in SEQ ID NOs: 6, 16, or 26. In some embodiments, the LC comprises two point mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, 16, or 26. In some embodiments, the LC comprises three point mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, 16, or 26. In some embodiments, the LC comprises four point mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, 16, or 26. In some embodiments, the LC comprises five point mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, 16, or 26. In some embodiments, the LC comprises ten point mutations compared to the amino acid sequence set forth in SEQ ID NO: 6, 16, or 26.

[0156] In some embodiments, the LC comprises an amino acid sequence selected from SEQ ID NOs: 1, 11, and 21. In certain embodiments, the LC comprises the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the LC comprises the amino acid sequence set forth in SEQ ID NO: 16. In certain embodiments, the LC comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0157] In some embodiments, the antibody or antigen-binding portion thereof comprises a HC and a LC, wherein the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:1, 11, and 21, and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs:6, 16, and 26. In some embodiments, the antibody or antigen-binding portion thereof comprises a HC and a LC, wherein the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1, and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6. In some embodiments, the antibody or antigen-binding portion thereof comprises a HC and a LC, wherein the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:11, and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.In some embodiments, the antibody or antigen-binding portion thereof comprises a HC and a LC, wherein the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21, and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26.

[0158] In some embodiments, the antibody, or antigen-binding portion thereof, comprises an HC and an LC, wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 1 and the LC comprises the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the antibody, or antigen-binding portion thereof, comprises an HC and an LC, wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 11 and the LC comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody, or antigen-binding portion thereof, comprises an HC and an LC, wherein the HC comprises the amino acid sequence set forth in SEQ ID NO: 21 and the LC comprises the amino acid sequence set forth in SEQ ID NO: 26.

[0159] In some embodiments, the antibody is an antigen-binding portion of an antibody. In some embodiments, the antigen-binding portion is a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide-linked Fv, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

[0160] In some aspects, the antibody or antigen-binding portion thereof reduces or inhibits tumor cell proliferation. In some aspects, the antibody or antigen-binding portion thereof reduces or inhibits tumor cell migration. In some aspects, the antibody or antigen-binding portion thereof reduces or inhibits tumor cell metastasis. In some aspects, the antibody or antigen-binding portion thereof increases tumor cell death. In some aspects, the antibody or antigen-binding portion thereof is capable of inhibiting tumor growth in a subject in need thereof. In some aspects, the antibody or antigen-binding portion thereof is capable of reducing tumor volume in a subject in need thereof. In some aspects, the antibody or antigen-binding portion thereof is capable of increasing progression-free survival in a subject in need thereof. In some aspects, the antibody or antigen-binding portion thereof is capable of increasing overall survival in a subject in need thereof.

[0161] In some embodiments, the anti-integrin αv heterodimeric antibodies described herein are capable of binding with high affinity, e.g., at least 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, 10 -12 M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, or 10 -9 M~10 -7 K of M D In some embodiments, the anti-integrin αv heterodimer antibody binds to a human integrin αv heterodimer (e.g., integrin-αvβ1) at a binding affinity of 10 or more, as determined by, e.g., surface plasmon resonance, e.g., using BIACORE™ (e.g., as described in the Examples). -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M (1nM) or less, 10 -10M or less, 10 -12 M~10 -7 M, 10 -11 M~10 -7 M, 10 -10 M~10 -7 M, 10 -9 M~10 -7 M, or 10 -8 M~10 -7 K of M D and binds to human integrin αv heterodimers (e.g., integrin-αvβ1). In some embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody has an EC50 of 100 nM or less, 10 nM or less, 1 nM or less, 100 nM to 0.01 nM, 100 nM to 0.1 nM, 100 nM to 1 nM, or 10 nM to 1 nM, or 10 ug / mL or less, 5 ug / mL or less, 1 ug / mL or less, 0.9 ug / mL or less, 0.8 ug / mL or less, 0.7 ug / mL or less, 0.6 ug / mL or less, 0.5 ug / mL or less, 0.4 ug / mL or less, 0.3 ug / mL or less, 0.2 ug / mL or less, 0.1 ug / mL or less, 0.05 ug / mL or less, or 0.01 ug / mL or less, as determined, e.g., by ELISA, e.g., an integrin αv heterodimer (e.g., integrin-αvβ1) ELISA kit. 50 E-commerce 50 and binds to human integrin αv heterodimers (e.g., integrin-αvβ1).

[0162] In some embodiments, the anti-integrin α heterodimer antibody is at a concentration of about 5×10 -4 M or less, approximately 1×10 -4 M or less, 5×10 -5 M or less, approximately 1×10 -5 M or less, approximately 1×10 -6 M or less, approximately 1×10 -7 M or less, or about 1 x 10 -8 K below M D , specifically binds to human integrin αv heterodimers (e.g., integrin-αvβ1), and has a K Dis measured by surface plasmon resonance (Biacore) analysis. In some embodiments, the anti-integrin αv heterodimer antibody has a binding affinity of at least about 1×10 3 ms -1 , at least about 5 × 10 3 ms -1 , at least about 1 x 10 4 ms -1 , at least about 5 × 10 4 ms -1 , at least about 1 x 10 5 ms -1 , at least about 5 × 10 5 ms -1 , or at least about 1 × 10 6 ms -1 The binding rate constant (k a ) specifically binds to human integrin αv heterodimers (e.g., integrin-αvβ1), and k a is measured by surface plasmon resonance (Biacore) analysis.

[0163] In some embodiments, the anti-integrin α heterodimer antibody has a cytotoxicity of about 0.1 s -1 Below, 0.05s -1 Below, 0.01s -1 Below, 5 x 10 -3 s -1 Below, 1×10 -3 s -1 Below, 5 x 10 -4 s -1 Below, 1×10 -4 s -1 Below, 5 x 10 -5 s -1 or less, or 1×10 -5 s -1 The dissociation rate constant (k d ) specifically binds to human integrin αv heterodimers (e.g., integrin-αvβ1) and has a K D is measured by surface plasmon resonance (Biacore) analysis.

[0164] The VH domain or one or more CDRs described herein can be linked with a constant domain to form a heavy chain, e.g., a full-length heavy chain.Similarly, the VL domain or one or more CDRs described herein can be linked with a constant domain to form a light chain, e.g., a full-length light chain.A full-length heavy chain (optionally excluding C-terminal lysine (K) or C-terminal glycine and lysine (GK) because they may be absent) and a full-length light chain can be combined to form a full-length antibody.

[0165] The VH domains described herein may be fused to the constant domain of a human IgG, e.g., IgG1, IgG2, IgG3 or IgG4, either naturally occurring or, e.g., modified as further described herein. For example, the VH domain may be fused to a human IgG, e.g., IgG1, constant region, e.g., the amino acid sequence of the wild-type human IgG1 constant domain: [ka] (SEQ ID NO:40) or an allotypic variant of SEQ ID NO: 40, comprising the amino acid sequence of any VH domain described herein fused to the amino acid sequence: [ka] (SEQ ID NO: 41, allotype-specific amino acid residues are shown in bold and underlined).

[0166] The VH domain of the anti-integrin αv heterodimeric antibody may comprise an effectorless constant region, such as the effectorless human IgG1 constant domain amino acid sequence: [ka] (SEQ ID NO: 42, "IgG1.1f", containing the underlined substitutions L234A, L235E, G237A, A330S and P331S) or [ka] (SEQ ID NO: 43, "IgG1.3f", with the substitutions L234A, L235E and G237A shown underlined).

[0167] For example, allotypic variants of IgG1 include K97R, D239E, and / or L241M (underlined and bolded above) numbered in SEQ ID NO: 40. In some embodiments, the constant region of an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody may further include one or more mutations or substitutions at amino acids L117, A118, G120, A213, and P214 (underlined above) numbered in SEQ ID NOs: 41, 42, and 43, or L234, A235, G237, A330, and P331 according to EU numbering. In some embodiments, the constant region of an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody comprises one or more mutations or substitutions at amino acids L117A, A118E, G120A, A213S, and P214S, or L234A, L235E, G237A, A330S, and P331S according to EU numbering, of SEQ ID NO: 40. The constant region of an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody may also comprise one or more mutations or substitutions at L117A, A118E, and G120A, or L234A, L235E, and G237A according to EU numbering, of SEQ ID NO: 40.

[0168] Alternatively, the VH domain of the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody can be selected from the group consisting of a human IgG4 constant region, e.g., the following human IgG4 amino acid sequence or a variant thereof: ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 44, including S228P).

[0169] The VL domains described herein can be fused to the constant domain of a human kappa or lambda light chain. For example, the VL domain of an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody can be fused to the following human IgG1 kappa light chain amino acid sequence: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 45).

[0170] In some embodiments, the heavy chain constant region includes a lysine or another amino acid at the C-terminus, e.g., it includes the following last amino acid in the heavy chain: LSPGK (SEQ ID NO: 46). In some embodiments, the heavy chain constant region lacks one or more amino acids at the C-terminus, e.g., has the C-terminal sequence LSPG (SEQ ID NO: 47) or LSP (SEQ ID NO: 48).

[0171] An anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody may comprise a heavy chain variable region comprising CDR1, CDR2 and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2 and CDR3 sequences, wherein one or more of these CDR sequences comprise a designated amino acid sequence based on the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein or a conservatively modified form thereof, and wherein the antibody retains the desired functional properties of the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein.

[0172] Conservative amino acid substitutions may be made in parts of the antibody other than or in addition to the CDRs. For example, conservative amino acid modifications may be made in the framework regions or Fc region. The variable region or heavy or light chain may contain 1, 2, 3, 4, 5, 1-2, 1-3, 1-4, 1-5, 1-10, 1-15, 1-20, 1-25, or 1-50 conservative amino acid substitutions compared to the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody sequences provided herein. In some embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody contains a combination of conservative and non-conservative amino acid modifications.

[0173] Engineered and modified antibodies are also provided that can be prepared using an antibody having one or more of the VH and / or VL sequences disclosed herein as starting material to engineer a modified antibody, which can have altered properties from the starting antibody. The antibody can be engineered by modifying one or more residues in one or both variable regions (i.e., VH and / or VL), e.g., in one or more CDR regions and / or in one or more framework regions. Additionally or alternatively, the antibody can be engineered by modifying residues in the constant region(s), e.g., to alter the effector function(s) of the antibody.

[0174] One type of variable region engineering that can be carried out is CDR grafting.Antibody interacts with target antigen mainly through the amino acid residues located in six heavy and light chain complementarity determining regions (CDRs).Therefore, the amino acid sequences in CDRs are more diverse between individual antibodies than the sequences outside CDRs. Because CDR sequences are involved in most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors containing CDR sequences from a particular naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0175] Thus, some embodiments described herein relate to isolated monoclonal antibodies or antigen-binding portions thereof that comprise a heavy chain variable region that comprises the CDR1, CDR2, and CDR3 sequences described herein, and a light chain variable region that comprises the CDR1, CDR2, and CDR3 sequences described herein.Such antibodies thus comprise the VH and VL CDR sequences of the monoclonal antibodies described herein, and may further comprise different framework sequences from these antibodies.

[0176] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops" I. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V H "Eur. J. Immunol. 24:827-836, the contents of each of which are expressly incorporated herein by reference.

[0177] In some embodiments, the framework sequence for use in the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody described herein is structurally similar to the framework sequence used by the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody described herein. The VH CDR1, CDR2 and CDR3 sequences and the VL CDR1, CDR2 and CDR3 sequences can be grafted onto framework regions that have the same sequence as found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations when compared to the germline sequence. For example, in certain cases, it has been found to be beneficial to mutate residues in framework regions to maintain or enhance the antigen-binding ability of antibodies (see, for example, U.S. Patent Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.).

[0178] The engineered anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein include those in which modifications have been made to framework residues in VH and / or VL, for example to improve the properties of the antibody. Typically, such framework modifications are made to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutations may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. Somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-mediated mutagenesis, to return the framework region sequence to its germline configuration. Such "backmutated" antibodies are also intended to be encompassed. Another type of framework modification involves mutating one or more residues in the framework region or even in one or more CDR regions to remove T cell epitopes, thereby reducing the immunogenic potential of the antibody. This approach is also called "deimmunization" and is described in more detail in U.S. Patent Publication No. 20030153043 by Carr et al.

[0179] Another type of variable region modification is to mutate amino acid residues in the VH and / or VL CDR1, CDR2 and / or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s) and their effect on antibody binding or other functional properties of interest can be evaluated in in vitro or in vivo assays described herein and provided in the examples. In some embodiments, conservative modifications (as discussed above) are introduced. Mutations can be amino acid substitutions, additions, or deletions. Moreover, usually no more than 1, 2, 3, 4 or 5 residues in the CDR regions are changed.

[0180] Methionine residues in the CDRs of an antibody can be oxidized, resulting in the possibility of chemical degradation and a resulting reduction in the efficacy of the antibody.Therefore, an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody is also provided in which one or more methionine residues in the heavy and / or light chain CDRs are replaced with amino acid residues that are not susceptible to oxidative degradation.

[0181] Similarly, deamidation sites may also be removed from anti-integrin αv heterodimer (eg, integrin-αvβ1) antibodies, particularly in the CDRs.

[0182] The anti-integrin αv heterodimer (e.g., integrin-αvβ1) variable regions described herein can be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc, which can be, for example, any allotype or isoallotype of IgG1: G1m, G1m1(a), G1m2(x), G1m3(f), G1m17(z); any allotype or isoallotype of IgG2: G2m, G2m23(n); any allotype or isoallotype of IgG3: Groups: G3m, G3m21(g1), G3m28(g5), G3m11(b0), G3m5(b1), G3m13(b3), G3m14(b4), G3m10(b5), G3m15(s), G3m16(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v) and K: Km, Km1, Km2, Km3 (see, e.g., Jefferis et al. (2009) mAbs 1:1).

[0183] In general, the variable regions described herein may be linked to an Fc that typically includes one or more modifications to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, antigen-dependent cellular cytotoxicity, and / or antigen-dependent cellular phagocytosis. Additionally, the antibodies described herein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation, to alter one or more functional properties of the antibody. Each of these aspects is described in detail below. The numbering of the residues in the Fc region is that of the EU index of Kabat.

[0184] The Fc region includes domains derived from the constant region of immunoglobulins, including fragments, analogs, variants, mutants, or derivatives of the constant region. Suitable immunoglobulins include IgG1, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally occurring or synthetically produced polypeptide that is homologous to the immunoglobulin C-terminal region, and may include CH1 domain, hinge, CH2 domain, CH3 domain, or CH4 domain, either separately or in combination.

[0185] Ig molecules interact with multiple classes of cell receptors. For example, IgG molecules interact with three classes of Fcγ receptors (FcγR), namely FcγRI, FcγRII and FcγRIII, which are specific for IgG class antibodies. It has been reported that the sequences important for IgG binding to FcγR receptors are located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by its ability to bind to Fc receptors (FcR).

[0186] In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion, and / or insertion) to provide desirable structural properties and / or biological activity compared to a parent Fc sequence (e.g., an unmodified Fc polypeptide, which is subsequently modified to generate the variant).

[0187] In general, a variant of a constant region or portion thereof, such as a CH1, CL, hinge, CH2 or CH3 domain, may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations and / or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutation or 1-10 or 1-5 mutations, or may comprise an amino acid sequence that is at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to that of the corresponding wild-type region or domain (CH1, CL, hinge, CH2 or CH3 domain, respectively), provided that the heavy chain constant region containing the particular variant retains the required biological activity.

[0188] For example, modifications can be made in the Fc region to generate Fc variants that, compared to the parent Fc, (a) mediate increased or decreased antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP), (b) mediate increased or decreased complement-mediated cytotoxicity (CDC), (c) have increased or decreased affinity for C1q, and / or (d) have increased or decreased affinity for Fc receptors. Such Fc region variants generally contain at least one amino acid modification in the Fc region. Combining amino acid modifications may be particularly desirable. For example, the variant Fc region may contain substitutions in 2, 3, 4, 5, etc. of the specific Fc region positions identified herein.

[0189] The variant Fc region may also include sequence changes in which amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal can avoid reaction with other cysteine-containing proteins present in the host cell used to produce the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein. Even when cysteine ​​residues are removed, the single-chain Fc domain can still form a dimeric Fc domain that is held together non-covalently. In some embodiments, the Fc region can be modified to be more compatible with a selected host cell. For example, the PA sequence near the N-terminus of a typical native Fc region may be removed, which can be recognized by digestive enzymes in E. coli, such as proline iminopeptidase. In some embodiments, one or more glycosylation sites within the Fc domain can be removed. Residues that are typically glycosylated (e.g., asparagine) can result in a cytolytic response. Such residues may be deleted or substituted with non-glycosylated residues (e.g., alanine). In some embodiments, sites involved in interaction with complement, such as the C1q binding site, may be removed from the Fc region. For example, the EKK sequence of human IgG1 may be deleted or substituted. In some embodiments, sites that affect binding to Fc receptors, preferably sites other than the salvage receptor binding site, may be removed. In some embodiments, the Fc region may be modified to remove ADCC sites. ADCC sites are known in the art, for example, see Molec. Immunol. 29 (5): 633-9 (1992) for ADCC sites in IgG1. Specific examples of mutant Fc domains are disclosed, for example, in WO97 / 34631, WO96 / 32478 and WO07 / 041635.

[0190] In some embodiments, the hinge region of Fc is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of Fc is altered, e.g., to facilitate assembly of light and heavy chains or to increase or decrease the stability of the antibody. In some embodiments, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the interface region of the CH2-CH3 domain of the Fc-hinge fragment so that the antibody has impaired Staphylococcus protein A (SpA) binding compared to the native Fc-hinge domain. This approach is further described in U.S. Patent No. 6,165,745 by Ward et al.

[0191] In some embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to modify the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, 322, 330, and / or 331 can be replaced with different amino acid residues so that the antibody has a reduced affinity for the effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand for which the affinity is modified can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0192] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be replaced with a different amino acid residue such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0193] In another example, one or more amino acid residues within amino acid positions 231-239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication WO 94 / 29351 by Bodmer et al.

[0194] In another example, the Fc region can be modified to enhance affinity to Fcγ and increase macrophage-mediated phagocytosis. See, for example, Richard et al., Mo. Cancer. Ther. 7(8):2517-27 (2008), which is incorporated herein by reference in its entirety. In certain embodiments, the Fc region can be modified to increase affinity to FcγRIIa compared to inhibitory FcγRIIb. One particular point mutation, G236A (numbering according to the EU index), has been identified as having increased affinity to FcγRIIa compared to inhibitory FcγRIIb. This increased affinity to FcRIIa correlated with increased macrophage-mediated phagocytosis compared to native IgG1. In some embodiments, the Fc region of the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody comprises one or more mutations or combinations of mutations selected from G236A, I332E, S239 / I332E, I332E / G236A, and S239D / I332E / G236A. Other modifications to the Fc region may increase antibody-dependent cellular cytotoxicity (ADCC), for example, by increasing affinity to activating receptors, for example, FcγRI and / or FcγRIIIa. For example, the G236A substitution, as well as the combination of a G236A substitution and a modification that improves affinity to an activating receptor (e.g., FcγRI and / or FcγRIIIa), including but not limited to, substitutions at 332 and 239, results in substantially improved ADCC compared to the parent wild-type antibody. See US Pat. No. 9,040,041, which is incorporated herein by reference in its entirety.

[0195] In another embodiment, the Fc region is selected from the group consisting of the amino acids 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 352, 354, 355, 356, 358, 352, 354, 355, 356, 358, 362, 263, 264, 265, 267, 268, 269, 270, 27 , 322, 324, 325, 326, 327, 329, 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438, or 439 to reduce antibody-dependent cellular cytotoxicity (ADCC) and / or to reduce affinity for Fcγ receptors. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T and 267E / 268F / 324T. Other modifications to enhance FcγR and complement interactions include, but are not limited to, the substitutions 298A, 333A, 334A, 326A, 247I, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 305I and 396L. These and other modifications are reviewed in Strohl (2009) Current Opinion in Biotechnology 20:685-691.

[0196] Fc modifications that increase binding to Fcγ receptors include those at amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 38 and / or an amino acid modification at any one or more of positions 4, 327, 329, 330, 335, 337, 338, 340, 360, 373, 376, 379, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439, where the numbering of the residues in the Fc region is that of the EU index in Kabat (WO00 / 42072).

[0197] Optionally, the Fc region may include non-naturally occurring amino acid residues in additional and / or alternative positions (see, e.g., U.S. Pat. Nos. 5,624,821, 6,277,375, 6,737,056, 6,194,551, 7,317,091, 8,101,720, 9,040,041, PCX Patent Publication No. WO 00 / 42362). 072, WO01 / 58957, WO02 / 06919, WO04 / 016750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925 and WO06 / 020114).

[0198] The affinity and binding properties of an Fc region for its ligand can be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)) or kinetics (e.g., BIACORE analysis) and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (e.g., gel filtration). These and other methods may utilize a label on the component or components being examined and / or may use a variety of detection methods, including, but not limited to, chromatographic, fluorescent, luminescent or isotopic labels. A detailed description of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0199] In some embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this can be done by increasing the binding affinity of the Fc region to FcRn. For example, as described in U.S. Pat. No. 6,277,375, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, 436. Specific exemplary substitutions include one or more of the following: T252L, T254S and / or T256F. Alternatively, to increase biological half-life, the antibody can be modified in the CH1 or CL region to include a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including, for example, 259I, 308F, 428L, 428M, 434S, 4341 1, 434F, 434Y, and 434X1.Other mutations that enhance Fc binding to FcRn include 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, and 434A (Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 311S, 433 R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S(Dall Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281:23514-23524). Other modifications to modulate FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.

[0200] In some embodiments, hybrid IgG isotypes with specific biological characteristics can be used. For example, IgG1 / IgG3 hybrid mutants can be constructed by replacing IgG1 positions in the CH2 and / or CH3 regions with amino acids derived from IgG3 at positions where the two isotypes differ. In this way, hybrid mutant IgG antibodies can be constructed that contain one or more substitutions, such as 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 422I, 435R and 436F. In some embodiments described herein, IgG1 / IgG2 hybrid mutants can be constructed by replacing IgG2 positions in the CH2 and / or CH3 regions with amino acids derived from IgG1 at positions where the two isotypes differ. In this way, hybrid mutant IgG antibodies can be constructed that contain one or more substitutions, for example one or more of the following amino acid substitutions: 233E, 234L, 235L, -236G (referring to the insertion of a glycine at position 236) and 327A.

[0201] Furthermore, the binding sites for FcγRI, FcγRII, FcγRIII, and FcRn in human IgG1 have been mapped and mutants with improved binding have been described (see Shields, RL et al., (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. In addition, combination mutants T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A have been shown to improve FcγRIII binding and have been shown to exhibit enhanced FcγRIIIa binding and ADCC activity (Shields et al., 2001). Other IgG1 mutants with strongly enhanced binding to FcγRIIIa have been identified, including mutants with S239D / I332E and S239D / I332E / A330L mutations, which showed the greatest increase in affinity for FcγRIIIa, reduced FcγRIIb binding, and potent cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2 / neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E mutant showed enhanced ability to deplete B cells in monkeys (Lazar et al., 2006). Furthermore, IgG1 mutants containing the L235V, F243L, R292P, Y300L and P396L mutations were identified that showed enhanced binding to FcγRIIIa and concomitant enhanced ADCC activity in transgenic mice expressing human FcγRIIIa in models of B cell malignancies and breast cancer ( Stavenhagen et al., 2007 , Nordstrom et al., 2011 ).Other Fc mutants that can be used include: S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L and M428L / N434S.

[0202] Specific mutations at positions 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330, and 332 have been shown to improve binding to FcγRIIa and / or reduce binding to FcγRIIb, resulting in enhanced ADCC and / or ADCP activity (Richards et al., Mol. Cancer Ther. 7(8):2517-2527; US Patent No. 9,040,041). In particular, Fc variants that selectively improve binding to one or more human activating receptors relative to FcγRIIb or that selectively improve binding to FcγRIIb relative to one or more activating receptors may contain substitutions selected from the group consisting of 234G, 234I, 235D, 235E, 235I, 235Y, 236A, 236S, 239D, 267D, 267E, 267Q, 268D, 268E, 293R, 295E, 324G, 324I, 327H, 328A, 328F, 328I, 330I, 330L, 330Y, 332D, and 332E. Additional substitutions that can be further combined include other substitutions that modulate FcγR affinity and complement activity, including, but not limited to, 298A, 298T, 326A, 326D, 326E, 326W, 326Y, 333A, 333S, 334L, and 334A (US Pat. No. 6,737,056; Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604; US Pat. No. 6,528,624; Idusogie et al., 2001, J. Immunology 166:2571-2572). Preferred variants that may be particularly useful for combining with other Fc variants include those that include the substitutions 298A, 326A, 333A, and 334A. Additional substitutions that can be combined with FcγR-selective variants include 247L, 255L, 270E, 392T, 396L, and 421K (U.S. Application No. 10 / 754,922, U.S. Application No. 10 / 902,588), and 280H, 280Q, and 280Y (U.S. Application No. 10 / 370,749).

[0203] If an IgG4 constant domain is used, it may contain the substitution S228P, which mimics the hinge sequence in IgG1, thereby stabilizing the IgG4 molecule.

[0204] II.B. Bispecific and Multispecific Antibodies Some embodiments of the present disclosure are directed to bispecific or multispecific antibodies that specifically bind to an integrin-αv heterodimer (e.g., integrin-αvβ1). In some embodiments, the bispecific or multispecific antibody binds to an integrin-αv heterodimer (e.g., integrin-αvβ1) and a second antigen. In some embodiments, the second antigen is not an integrin-αv heterodimer (e.g., integrin-αvβ1). In some embodiments, the bispecific or multispecific antibody binds to a first epitope on an integrin-αv heterodimer (e.g., integrin-αvβ1) and a second epitope of an integrin-αv heterodimer (e.g., integrin-αvβ1). In some embodiments, the first epitope and the second epitope do not overlap.

[0205] Any of the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein can be used to form bispecific or multispecific antibodies. An anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody or an antigen-binding portion thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or a receptor ligand), to generate a bispecific or multispecific molecule that binds to at least two different binding sites or target molecules. In some embodiments, the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody is linked to any protein that can be used as a potential target for combination treatment, such as an antibody or scFv that specifically binds to a protein described herein (e.g., an antibody against PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-2, IL-8, B7-H4, Fas Ligand, CXCR4, mesothelin, CD27, CD96, VISTA, or GITR, or pegylated IL-2 or pegylated IL-10). The antibodies described herein may in fact be derivatized or linked to two or more other functional molecules to generate multispecific molecules that bind to three or more different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" herein. To generate the bispecific molecules described herein, the antibodies described herein can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent attachment or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic, resulting in a bispecific molecule.

[0206] Thus, provided herein are bispecific molecules that comprise a first binding specificity for at least one integrin αv heterodimer (e.g., integrin-αvβ1) and a second binding specificity for a second target epitope. In some embodiments described herein where the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity.

[0207] In some embodiments, the bispecific molecules described herein comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab')2, Fv or single chain Fv (scFV). As described by Ladner et al. in U.S. Pat. No. 4,946,778, an antibody can also be a light or heavy chain dimer or any minimal fragment thereof, such as an Fv or single chain construct.

[0208] Although human monoclonal antibodies are preferred, other antibodies that can be utilized in the bispecific molecules described herein include murine, chimeric and humanized monoclonal antibodies.

[0209] The bispecific molecules described herein can be prepared by conjugating the component binding specificities using methods known in the art.For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other.When the binding specificity is a protein or peptide, various coupling or cross-linking agents can be used for covalent conjugation. Exemplary cross-linkers include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132, Brennan et al. (1985) Science 229:81-83) and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Some conjugation agents include SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0210] When the binding specificities are antibodies, they can be conjugated by sulfhydryl bonding of the C-terminal hinge regions of the two heavy chains. In some embodiments, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.

[0211] Alternatively, both binding specificities may be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb fusion protein, a mAb x Fab fusion protein, a mAb x (scFv)2 fusion protein, a Fab x F(ab')2 fusion protein, or a ligand x Fab fusion protein. The bispecific antibody may comprise an antibody comprising an scFv at the C-terminus of each heavy chain. The bispecific molecules described herein may be single-chain molecules comprising one single-chain antibody and a binding determinant, or may be single-chain bispecific molecules comprising two binding determinants. The bispecific molecule may comprise at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Pat. No. 5,260,203, U.S. Pat. No. 5,455,030, U.S. Pat. No. 4,881,175, U.S. Pat. No. 5,132,405, U.S. Pat. No. 5,091,513, U.S. Pat. No. 5,476,786, U.S. Pat. No. 5,013,653, U.S. Pat. No. 5,258,498 and U.S. Pat. No. 5,482,858.

[0212] Binding of a bispecific molecule to its specific target may be confirmed using art-recognized methods such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassays (e.g., growth inhibition), or Western blot assays. Each of these assays generally detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest.

[0213] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises at least a first paratope and a second paratope, and the first paratope binds to a first epitope on an integrin αv heterodimer (e.g., integrin-αvβ1) heterodimer. In some embodiments, the second paratope binds to a second epitope on an integrin αv heterodimer (e.g., integrin-αvβ1) heterodimer. In some embodiments, the first epitope and the second epitope are not the same. In some embodiments, the first epitope and the second epitope do not overlap.

[0214] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a first heavy chain, a first light chain, a second heavy chain, and a second light chain. In some embodiments, the first heavy chain and the second heavy chain are different. In some embodiments, the first light chain and the second light chain are different. In some embodiments, the first heavy chain and the second heavy chain are different and the first light chain and the second light chain are the same. In some embodiments, the first heavy chain and the second heavy chain are the same and the first light chain and the second light chain are different.

[0215] The first heavy chain, the second heavy chain, the first light chain, and the second light chain may comprise any heavy or light chain disclosed herein. In some embodiments, the first heavy chain comprises a first variable heavy chain region ("VH1") comprising variable heavy chain complementarity determining region (VH1-CDR)1, VH1-CDR2, and VH1-CDR3, and VH1-CDR3 comprises an amino acid sequence selected from SEQ ID NO:5, 15, and 25. In some embodiments, VH1-CDR2 comprises an amino acid sequence selected from SEQ ID NO:4, 14, and 24. In some embodiments, VH1-CDR1 comprises an amino acid sequence selected from SEQ ID NO:3, 13, and 23. In some embodiments, the first light chain comprises a first variable light chain region ("VL1") comprising VL1-CDR1, VL1-CDR2, and VL1-CDR3, wherein VL1-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 10, 20, and 30. In some embodiments, VL1-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 9, 19, and 29. In some embodiments, VL1-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 8, 18, and 28.

[0216] In certain embodiments, the bispecific antibody or antigen-binding portion thereof comprises a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5, a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:18, a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:19, and a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:20.

[0217] In certain embodiments, the bispecific antibody or antigen-binding portion thereof comprises a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 13, a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 15, a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 18, a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20.

[0218] In certain embodiments, the bispecific antibody or antigen-binding portion thereof comprises a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5, a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28, a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0219] In certain embodiments, the bispecific antibody or antigen-binding portion thereof comprises a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25, a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28, a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0220] In some embodiments, the second heavy chain comprises a second variable heavy chain region ("VH2") comprising VH2-CDR1, VH2-CDR2, and VH2-CDR3, wherein VH2-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 5, 15, and 25. In some embodiments, VH2-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 4, 14, and 24. In some embodiments, VH2-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 3, 13, and 23. In some embodiments, the second light chain comprises a second variable light chain region ("VL1") comprising VL2-CDR1, VL2-CDR2, and VL2-CDR3, wherein VL2-CDR3 comprises an amino acid sequence selected from SEQ ID NOs: 20 and 30. In some embodiments, VL2-CDR2 comprises an amino acid sequence selected from SEQ ID NOs: 19 and 29. In some embodiments, VH2-CDR1 comprises an amino acid sequence selected from SEQ ID NOs: 18 and 28.

[0221] In certain embodiments, the bispecific antibody or antigen-binding portion thereof comprises a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5, a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:18, a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:19, a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:20, a VH2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:13, a VH2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:14, a VH2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:15, a VL2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:18, a VL2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:19, and a VL2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:20.

[0222] In certain embodiments, the bispecific antibody or antigen-binding portion thereof comprises a VH1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:3, a VH1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:4, a VH1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:5, a VL1-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28, a VL1-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, a VL1-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30, a VH2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:23, a VH2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:24, a VH2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25, a VL2-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:28, a VL2-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:29, and a VL2-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:30.

[0223] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a first variable heavy chain region (VH1) and a first variable light chain region (VL1), wherein VH1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 2, 12, or 22. In some embodiments, VL1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 7, 17, or 27.

[0224] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a second variable heavy chain region (VH2) and a second variable light chain region (VL2), wherein VH2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 2, 12, or 22. In some embodiments, VL2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 7, 17, or 27.

[0225] In some embodiments, (a) VH1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and (b) VL1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17. (c) VH2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12, and (d) VL2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, VH1 comprises the amino acid sequence set forth in SEQ ID NO: 2, VL1 comprises the amino acid sequence set forth in SEQ ID NO: 17, VH2 comprises the amino acid sequence set forth in SEQ ID NO: 12, and VL2 comprises the amino acid sequence set forth in SEQ ID NO: 17.

[0226] In some embodiments, (a) VH1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2; and (b) VL1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:27. (c) VH2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 22, and (d) VL2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 27. In some embodiments, VH1 comprises the amino acid sequence set forth in SEQ ID NO: 2, VL1 comprises the amino acid sequence set forth in SEQ ID NO: 27, VH2 comprises the amino acid sequence set forth in SEQ ID NO: 22, and VL2 comprises the amino acid sequence set forth in SEQ ID NO: 27.

[0227] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a first heavy chain (H1) and a first light chain (L1), wherein H1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 11, 21, 31, 34, or 37. In some embodiments, L1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 6, 16, or 26.

[0228] In some embodiments, the bispecific antibody or antigen-binding portion thereof comprises a second heavy chain (H2) and a second light chain (L2), wherein H2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 1, 11, 21, 31, 34, or 37. In some embodiments, L2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NOs: 6, 16, or 26.

[0229] In some embodiments, the first heavy chain is associated with the second heavy chain. In some embodiments, the first heavy chain is associated with the second heavy chain by one or more covalent bonds. In some embodiments, each of the first heavy chain and the second heavy chain comprises an IgG constant region or an IgG constant region comprising one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions promote heterodimerization of the first heavy chain and the second heavy chain. Any amino acid substitution can be used to promote heterodimerization. In some embodiments, the first heavy chain comprises one or more amino acid substitutions in the constant region of the first heavy chain that create a knob, and the second heavy chain comprises one or more amino acid substitutions or antigen-binding portions thereof in the constant region of the second heavy chain that create a hole, and the knob of the first heavy chain is associated with the hole of the second heavy chain. In some embodiments, the first heavy chain comprises one or more amino acid substitutions in the constant region of the first heavy chain that create a hole, and the second heavy chain comprises one or more amino acid substitutions in the constant region of the second heavy chain that create a knob, and the hole of the first heavy chain associates with the knob of the second heavy chain.

[0230] In some embodiments, (a) H1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:31; and (b) L1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16. (c) H2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:34; and (d) L2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.

[0231] In some embodiments, H1 comprises the amino acid sequence set forth in SEQ ID NO:31, L1 comprises the amino acid sequence set forth in SEQ ID NO:16, H2 comprises the amino acid sequence set forth in SEQ ID NO:34, and L2 comprises the amino acid sequence set forth in SEQ ID NO:16.

[0232] In some embodiments, (a) H1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:31; and (b) L1 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26. (c) H2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:37; and (d) L2 comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26.

[0233] In some embodiments, H1 comprises the amino acid sequence set forth in SEQ ID NO:31, L1 comprises the amino acid sequence set forth in SEQ ID NO:26, H2 comprises the amino acid sequence set forth in SEQ ID NO:37, and L2 comprises the amino acid sequence set forth in SEQ ID NO:26.

[0234] In some embodiments, the bispecific antibody inhibits the binding of an integrin αv heterodimer (e.g., integrin-αvβ1) to LAP-TGFβ1. In some embodiments, the binding of an integrin αv heterodimer (e.g., integrin-αvβ1) to LAP-TGFβ1 is reduced to less than about 90%, less than about 80%, less than about 70%, less than about 60%, 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%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% compared to the level of binding of an integrin αv heterodimer (e.g., integrin-αvβ1) to LAP-TGFβ1 in the absence of the bispecific antibody.

[0235] In some embodiments, the bispecific antibody is capable of binding to an integrin αv heterodimer selected from αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, and any combination thereof.

[0236] In some embodiments, the bispecific antibody inhibits cell adhesion. In some embodiments, the bispecific antibody inhibits tumor growth and / or metastasis. In some embodiments, the bispecific antibody increases progression-free survival. In some embodiments, the bispecific antibody increases overall survival.

[0237] II.C. Immunoconjugates, Antibody Derivatives and Diagnostics The anti-integrin αv antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose, the antibodies (or binding fragments thereof) can be conjugated with a suitable detectable agent to form an immunoconjugate. For diagnostic purposes, suitable agents are detectable labels, including radioisotopes for whole body imaging and radioisotopes, enzymes, fluorescent labels and other suitable antibody tags for sample testing.

[0238] Detectable labels that can be linked to any of the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies described herein include particle labels that include metal sols, such as colloidal gold, e.g., I presented with N2S2, N3S, or N4 type peptidic chelators. 125 or Tc 99 The enzyme labels may be any of a variety of types currently used in the field of in vitro diagnostics, including isotopes such as , chromophores including fluorescent markers, luminescent markers, phosphorescent markers, etc., as well as enzyme labels that convert a given substrate into a detectable marker and polynucleotide tags that are revealed after amplification, such as by polymerase chain reaction. Suitable enzyme labels include horseradish peroxidase and alkaline phosphatase, etc. For example, the label can be the enzyme alkaline phosphatase, which is detected by measuring the presence or formation of chemiluminescence following conversion of 1,2 dioxetane substrates such as adamantyl methoxyphosphoryloxyphenyl dioxetane (AMPPD), disodium 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decan}-4-yl)phenyl phosphate (CSPD) and CDP and CDP-STAR® or other luminescent substrates known to those skilled in the art, e.g., chelates of suitable lanthanides such as terbium(III) and europium(III). The means of detection will be determined by the label chosen. Appearance of the label or its reaction products can be achieved with the naked eye, if the label is particulate and accumulates at appropriate levels, or using instruments such as spectrophotometers, luminometers, fluorometers, and the like, all following standard practices.

[0239] In some embodiments, the conjugation method produces substantially (or nearly) non-immunogenic bonds, such as peptide (i.e., amide) bonds, sulfide bonds (steric hindrance), disulfide bonds, hydrazone bonds and ether bonds. These bonds are nearly non-immunogenic and show reasonable stability in serum (see, for example, Senter, PD, Curr. Opin. Chem. Biol. 13 (2009) 235-244, WO2009 / 059278, WO95 / 17886).

[0240] Depending on the biochemical nature of the moiety and the antibody, different conjugation strategies can be used. If the moiety is naturally occurring or recombinant of 50-500 amino acids, standard procedures are found in textbooks describing chemical reactions for the synthesis of protein conjugates and can be easily followed by the skilled artisan (see, for example, Hackenberger, CPR, and Schwarzer, D., Angew. Chem. Int. Ed. Engl. 47 (2008) 10030-10074). In some embodiments, reaction of maleimide moieties with cysteine ​​residues in the antibody or moiety is used. This is a particularly suitable coupling chemistry, for example, when Fab or Fab' fragments of antibodies are used. Alternatively, in some embodiments, coupling to the C-terminus of the antibody or moiety is performed. C-terminal modification of proteins, for example Fab fragments, can be performed as described (Sunbul, M. and Yin, J., Org. Biomol. Chem. 7 (2009) 3361-3371).

[0241] Generally, site-specific reactions and covalent couplings are based on converting natural amino acids into amino acids with reactivity that is orthogonal to the reactivity of other functional groups present. For example, certain cysteines in rare sequence contexts can be enzymatically converted to aldehydes (see Frese, MA, and Dierks, T., ChemBioChem. 10 (2009) 425-427). It is also possible to obtain the desired amino acid modifications by utilizing the specific enzymatic reactivity of certain enzymes with natural amino acids in a given sequence context (see, for example, Taki, M. et al., Prot. Eng. Des. Sel. 17 (2004) 119-126; Gautier, A. et al. Chem. Biol. 15 (2008) 128-136. Protease-catalyzed C--N bond formation is used in Bordusa, F., Highlights in Bioorganic Chemistry (2004) 389-403). Site-specific reactions and covalent couplings can also be achieved by selective reaction of the terminal amino acid with an appropriate modifying reagent.

[0242] The reactivity of the N-terminal cysteine ​​with benzonitrile (see Ren. H. et al., Angew. Chem. Int. Ed. Engl. 48 (2009) 9658-9662) can be used to achieve site-specific covalent coupling.

[0243] Native chemical ligation can also rely on a C-terminal cysteine ​​residue (Taylor, E. Vogel; Imperiali, B, Nucleic Acids and Molecular Biology (2009), 22 (Protein Engineering), 65-96).

[0244] US6437095B1 describes a conjugation method based on the rapid reaction of cysteines in a stretch of negatively charged amino acids with cysteines located in a stretch of positively charged amino acids.

[0245] The moiety may also be a synthetic peptide or peptidomimetic.When a polypeptide is chemically synthesized, amino acids with orthogonal chemical reactivity can be incorporated during such synthesis (see, for example, de Graaf. AJ et al., Bioconjug. Chem. 20 (2009) 1281-1295).Since a wide variety of orthogonal functional groups are at stake and can be introduced into synthetic peptides, the conjugation of such peptides with linkers is a standard chemical reaction.

[0246] To obtain single-labeled polypeptide, the conjugate with 1:1 stoichiometry may be separated from other conjugation by-products by chromatography.This procedure can be facilitated by using dye-labeled binding pair members and charged linkers.By using this type of label and highly negatively charged binding pair members, mono-conjugated polypeptides are easily separated from unlabeled polypeptides and polypeptides with more than one linker, because the difference in charge and molecular weight can be used for separation.Fluorescent dyes can be useful for purifying complexes from unbound components, such as labeled monovalent binding agents.

[0247] In some embodiments, the moiety that binds to an anti-integrin αv heterodimer (eg, integrin-αvβ1) antibody is selected from the group consisting of a binding moiety, a labeling moiety, and a biologically active moiety.

[0248] The anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody described herein can also be conjugated with a therapeutic agent to form an immunoconjugate, e.g., an antibody-drug conjugate (ADC).Suitable therapeutic agents include antimetabolites, alkylating agents, DNA minor groove binders, DNA intercalators, DNA crosslinkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents.In ADC, the antibody and therapeutic agent are preferably conjugated via a cleavable linker, such as a peptidyl, disulfide or hydrazone linker. In some embodiments, the linker is a peptidyl linker such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 108), Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser, or Glu. ADCs can be prepared as described in U.S. Pat. Nos. 7,087,600, 6,989,452, and 7,129,261, PCT Publication Nos. WO02 / 096910, WO07 / 038658, WO07 / 051081, WO07 / 059404, WO08 / 083312, and WO08 / 103693, U.S. Patent Publication Nos. 20060024317, 20060004081, and 20060247295.

[0249] In some embodiments, the therapeutic agent is selected from the group consisting of a cytotoxin, a non-cytotoxic drug, a radioactive agent, a second antibody, an enzyme, an anti-neoplastic agent, and any combination thereof.

[0250] In some embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and a cytotoxin. The cytotoxin may be selected from any cytotoxin known in the art. In some embodiments, the cytotoxin is selected from the group consisting of dolastatin, monomethylauristatin E (MMAE), maytansine, duocarmycin, calicheamicin, pyrrolobenzodiazepine, duocarmycin, centanamycin, SN38, doxorubicin, derivatives thereof, synthetic analogs thereof, and any combination thereof. In certain embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and a cytotoxin A. In other embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and a non-cytotoxic drug.

[0251] In some embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and a radiopharmaceutical. In some embodiments, the radiopharmaceutical is a radionucleotide. In certain embodiments, the radiopharmaceutical comprises radioactive iodine. In certain embodiments, the radiopharmaceutical comprises 131-iodine. In other embodiments, the radiopharmaceutical comprises the radioactive isotope yttrium-90.

[0252] In some embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and a second antibody. The second antibody may be any antibody described in this disclosure, including, but not limited to, an antibody that specifically binds to a protein selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-2, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, VISTA, CD96, CD27, GITR, and any combination thereof. In one embodiment, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and an anti-PD-1 antibody. In another embodiment, the immunoconjugate comprises an anti-integrin αv heterodimer (eg, integrin-αvβ1) antibody and nivolumab.

[0253] In one embodiment, the immunoconjugate comprises an anti-integrin αv heterodimer (eg, integrin-αvβ1) antibody and pegylated IL-2 or pegylated IL-10.

[0254] In certain embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and an enzyme. In some embodiments, the enzyme comprises glucose oxidase. In some embodiments, the enzyme comprises peroxidase. In some embodiments, the enzyme comprises myeloperoxidase. In some embodiments, the enzyme comprises glucose oxidase. In some embodiments, the enzyme comprises horseradish peroxidase.

[0255] In certain embodiments, the immunoconjugate comprises an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody and an anti-neoplastic agent. The anti-neoplastic agent can be any such agent known in the art. In some embodiments, the anti-neoplastic agent is epirubicin. In some embodiments, the anti-neoplastic agent is a superantigen. In certain embodiments, the superantigen is staphylococcal enterotoxin A (SEA / E-120, estafenatox).

[0256] Anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies, such as those described herein, can also be used to detect integrin αv heterodimers (e.g., integrin-αvβ1). The antibodies can be used, for example, in ELISA assays or flow cytometry.

[0257] Other uses of anti-integrin αv heterodimer (eg, integrin-αvβ1) antibodies, eg, as monotherapy or in combination therapy, are provided elsewhere herein, eg, in the section relating to combination treatments.

[0258] II.D. Nucleic acid molecules Another embodiment described herein pertains to a nucleic acid molecule encoding an anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibody described herein. The nucleic acid may be present in a whole cell, in a cell lysate, or in a partially purified or substantially pure form when purified from other cellular components or other contaminants, e.g., other cellular nucleic acids (e.g., other chromosomal DNA, e.g., chromosomal DNA that is naturally linked to the isolated DNA) or proteins, by standard techniques including alkaline / SDS treatment, CsCl banding, column chromatography, restriction enzymes, agarose gel electrophoresis, and others known in the art. A nucleic acid is "isolated" or "substantially purified." See F. Ausubel, et al., ed. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids described herein may be, for example, DNA or RNA, and may or may not contain intron sequences. In some embodiments, the nucleic acid is a cDNA molecule.

[0259] The nucleic acids described herein can be obtained using standard molecular biology techniques.For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes as described further below), the cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques.For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acids encoding the antibodies can be retrieved from the library.

[0260] Some nucleic acid molecules described herein encode the VH and VL sequences of an anti-integrin αv heterodimer antibody.

[0261] The nucleic acid molecules described herein may be modified to delete particular sequences, for example, restriction enzyme recognition sequences, or to be codon optimized.

[0262] The method for producing the anti-integrin αv heterodimer (e.g., integrin-αvβ1) antibodies disclosed herein may include expressing the heavy and light chains in a cell line that contains nucleotide sequences encoding the heavy and light chains with a signal peptide. Host cells that contain these nucleotide sequences are encompassed herein.

[0263] Once the DNA fragments encoding the VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes into full-length antibody chain genes, into Fab fragment genes, or into scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" shall mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in frame.

[0264] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding a heavy chain constant region (hinge, CH1, CH2, and / or CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, e.g., an IgG1 region. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0265] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the DNA encoding the VL with another DNA molecule encoding a light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0266] To generate an scFv gene, a DNA fragment encoding the VH and VL is operably linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3, such that the VH and VL sequences can be expressed as a contiguous single-chain protein with the VL and VH regions connected by the flexible linker (see, e.g., Bird et al., (1988) Science 242:423-426; Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0267] Additionally, nucleic acid molecules with conservative substitutions (ie, substitutions that do not change the resulting amino acid sequence upon translation of the nucleic acid molecule), eg, for codon optimization, are provided herein.

[0268] II.E. Pharmaceutical Compositions Further provided are compositions, e.g., pharmaceutical compositions, containing one or a combination of the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies described herein, or combinations with antibodies against other targets, or antigen-binding portion(s) thereof, formulated together with a pharma- ceutically acceptable carrier. Such compositions may contain one or a combination of (e.g., two or more different) antibodies or immunoconjugates or bispecific molecules described herein. For example, the pharmaceutical compositions described herein may contain a combination of antibodies (including immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or have complementary activities.

[0269] In some embodiments, the composition comprises an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1-300 mg / ml, or 100-300 mg / ml.

[0270] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents.For example, the combination therapy can include the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody described herein in combination with at least one other anti-cancer agent and / or an immunomodulator, such as, for example, a T cell stimulating (e.g., activating) agent.Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody described herein.

[0271] In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is combined with at least one other agent selected from chemotherapeutic agents, small molecule drugs, and antibodies that stimulate an immune response to a given cancer. In some cases ..., for example, an anti-CTLA-4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody (e.g., BMS986178, or MDX-1803), an anti-CD137 antibody, an anti-LAG-3 antibody, an anti-GITR antibody, an anti-KIR antibody, an anti-TGFβ antibody, an anti-IL-10 antibody, a long-acting IL-10 molecule (e.g., an IL-10-Fc fusion, or a pegylated IL-10, e.g., ARMO and AM0010 from BioSciences), long-acting IL-2 (e.g., a pegylated IL-2 molecule, e.g., NKTR-214 from Nektar, see US8,252,275, WO12 / 065086, and WO15 / 125159), an anti-VISTA antibody, an anti-CD96 antibody, an anti-IL-8 antibody, an anti-B7-H4, an anti-Fas ligand antibody, an anti-CXCR4 antibody, an anti-mesothelin antibody, an anti-CD27 antibody, an anti-MICA / B antibody, or any combination thereof.

[0272] In other embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is formulated with a second antibody. In some embodiments, the second antibody specifically binds to a protein selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG3, TIGIT, TIM3, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-2, VISTA, CD96, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, MICA / B, and any combination thereof.

[0273] In some embodiments, the second antibody is an anti-PD-1 antibody. The anti-PD-1 antibody can be any antibody that binds to PD-1 and inhibits the interaction of PD-1 and PD-L1. In some embodiments, the anti-PD-1 antibody is any anti-PD-1 antibody disclosed herein. In some embodiments, the second antibody is nivolumab. In some embodiments, the second antibody is pembrolizumab.

[0274] In some embodiments, the second antibody is an anti-PD-L1 antibody. The anti-PD-L1 antibody can be any antibody that binds to PD-L1 and inhibits the interaction of PD-1 and PD-L1. In some embodiments, the anti-PD-L1 antibody is any anti-PD-L1 antibody disclosed herein. In some embodiments, the second antibody is atezolizumab. In some embodiments, the second antibody is durvalumab. In some embodiments, the second antibody is avelumab.

[0275] In some embodiments, the second antibody is an anti-CTLA-4 antibody. The anti-CTLA-4 antibody can be any antibody that binds to and inhibits the activity of CTLA-4. In some embodiments, the anti-CTLA-4 antibody is any anti-CTLA-4 antibody disclosed herein. In some embodiments, the second antibody is tremelimumab. In some embodiments, the second antibody is ipilimumab.

[0276] In some embodiments, the second antibody is an anti-LAG3 antibody. The anti-LAG3 antibody can be any antibody that binds to LAG-3 and inhibits its activity. In some embodiments, the anti-LAG3 antibody is any anti-LAG3 antibody disclosed herein. In some embodiments, the second antibody is 25F7.

[0277] In some embodiments, the second antibody is an anti-CD137 antibody. The anti-CD137 antibody can be any antibody that binds to and inhibits the activity of CD137. In some embodiments, the anti-CD137 antibody is any anti-CD137 antibody disclosed herein. In some embodiments, the second antibody is urelumab.

[0278] In some embodiments, the second antibody is an anti-KIR antibody. The anti-KIR antibody can be any antibody that binds to KIR and inhibits its activity. In some embodiments, the anti-KIR antibody is any anti-KIR antibody disclosed herein. In some embodiments, the second antibody is lirilumab.

[0279] In some embodiments, the second antibody is an anti-GITR antibody. The anti-GITR antibody can be any antibody that binds to GITR and inhibits its activity. In some embodiments, the anti-GITR antibody is any anti-GITR antibody disclosed herein. In some embodiments, the second antibody is MK4166. In some embodiments, the second antibody is TRX518.

[0280] In some embodiments, the second antibody is an anti-CD96 antibody.

[0281] In some embodiments, the second antibody is an anti-TIM3 antibody.

[0282] In some embodiments, the second antibody is an anti-VISTA antibody.

[0283] In some embodiments, the second antibody is an anti-NKG2a antibody.

[0284] In some embodiments, the second antibody is an anti-ICOS antibody.

[0285] In some embodiments, the second antibody is an anti-OX40 antibody.

[0286] In some embodiments, the second antibody is an anti-IL8 antibody, e.g., HuMax®-IL8 (BMS-986253).

[0287] In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is formulated with a long-acting IL-10 molecule. In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is formulated with an IL-10-Fc fusion molecule. In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is formulated with a pegylated IL-10, e.g., AM0010 from ARMO BioSciences.

[0288] In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is formulated with a long-acting IL-2. In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is formulated with a pegylated IL-2 molecule, e.g., Nektar's NKTR-214. See US8,252,275, WO12 / 065086, and WO15 / 125159.

[0289] In some embodiments, the composition of the present disclosure further comprises a bulking agent. The bulking agent may be selected from the group consisting of NaCl, mannitol, glycine, alanine, and any combination thereof. In other embodiments, the composition of the present disclosure comprises a stabilizing agent. The stabilizing agent may be selected from the group consisting of sucrose, trehalose, raffinose, arginine, or any combination thereof. In other embodiments, the composition of the present disclosure comprises a surfactant. The surfactant may be selected from the group consisting of polysorbate 80 (PS80), polysorbate 20 (PS20), and any combination thereof. In certain embodiments, the composition further comprises a chelating agent. The chelating agent may be selected from the group consisting of diethylenetriaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid, nitrilotriacetic acid, and any combination thereof.

[0290] In other embodiments, the composition comprises a third antibody. In some embodiments, the third antibody is any antibody disclosed herein.

[0291] In one embodiment, the composition further comprises NaCl, mannitol, pentetic acid (DTPA), sucrose, PS80, and any combination thereof.

[0292] As used herein, "pharmaceutical acceptable carriers" include any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Subcutaneous injection options are based on Halozyme Therapeutics' ENHANZE® drug delivery technology, which involves co-formulating antibodies with recombinant human hyaluronidase enzyme (rHuPH20), thereby eliminating traditional limitations on the volume of biologics and drugs that can be delivered subcutaneously due to the extracellular matrix (U.S. Patent No. 7,767,429). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0293] The pharmaceutical compounds described herein may include one or more pharma- ceutically acceptable salts. A "pharma- ceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like, and non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, calcium, and the like, and those derived from non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine, and the like.

[0294] The pharmaceutical compositions described herein may also contain pharma- ceutical acceptable antioxidants. Examples of pharma-ceutical acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0295] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0296] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures, as above, and by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents in the composition, such as sugars, sodium chloride, etc. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0297] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharma-ceutical active substances is known in the art.Except where any conventional media or agent is incompatible with active compound, its use in the pharmaceutical compositions described herein is considered.The pharmaceutical compositions may or may not contain preservatives.Supplementary active compounds can be incorporated into the composition.

[0298] Therapeutic compositions must usually be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, the compositions can contain isotonic agents, for example, polyalcohols such as sugars, mannitol, sorbitol, or sodium chloride. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0299] Sterile injection solution can be prepared by incorporating the active compound in the required amount in suitable solvent with one or combination of the above-listed components as necessary, followed by sterilization precision filtration.Generally, dispersion is prepared by incorporating active compound in a sterile vehicle that contains basic dispersion medium and other components required from those listed herein.In the case of sterile powder for preparing sterile injection solution, the preparation method includes vacuum drying and freeze-drying (lyophilization), which obtains the powder of active ingredient and any additional desired ingredients from its previously sterile-filtered solution.

[0300] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form varies depending on the subject being treated and the specific mode of administration.The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect.In general, in combination with a pharma-ceutically acceptable carrier, this amount will be in the range of about 0.01 percent to about 99 percent of active ingredient, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent of active ingredient, out of 100 percent.

[0301] The dosage regimen is adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions into dosage unit forms for ease of administration and uniformity of dosage form. As used herein, dosage unit forms refer to physically discrete units suitable as unitary dosages for the subject being treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications of dosage unit forms described herein are determined and directly influenced by (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the technical field of compounding such active compounds for the treatment of susceptibility in individuals.

[0302] The antibody can be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies and non-human antibodies. The dosage and frequency of administration can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals for a long period of time. Some patients continue to receive treatment for life. In therapeutic applications, relatively high dosages at relatively short intervals are sometimes required until the progression of the disease is reduced or terminated, or until the patient shows partial or complete remission of the symptoms of the disease. The patient may then be administered a preventive administration regimen.

[0303] The actual dosage level of the active ingredient in the pharmaceutical compositions described herein may be varied to obtain an amount of the active ingredient that is not toxic to the patient and is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration. The dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular composition described herein or its ester, salt or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated and similar factors well known in the pharmaceutical arts.

[0304] A "therapeutically effective dose" of an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody described herein may result in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. In the context of cancer, a therapeutically effective dose may result in an increase in survival, e.g., overall survival, and / or prevention of further progression of physical symptoms associated with cancer. Symptoms of cancer are well known in the art and include, for example, unusual lentigo features, changes in the appearance of lentigines including asymmetry, border, color and / or diameter, newly pigmented skin areas, abnormal lentigines, dark areas under the fingernails, breast lumps, nipple changes, breast cysts, breast pain, death, weight loss, weakness, excessive fatigue, eating disorders, loss of appetite, chronic cough, worsening shortness of breath, hemoptysis, hematuria, blood in the stool, nausea, vomiting, liver metastases, lung metastases, bone metastases, abdominal bloating, flatulence, fluid in the peritoneal cavity, vaginal bleeding, constipation, abdominal distension, perforation of the colon, acute peritonitis (infection, fever, pain), pain, vomiting of blood, profuse sweating, fever, hypertension, anemia, diarrhea, jaundice, dizziness, chills, muscle cramps, colon metastases, lung metastases, bladder metastases, liver metastases, bone metastases, kidney metastases and pancreatic metastases, difficulty swallowing, and the like.

[0305] Therapeutically effective doses, as may be desired when early or preceding signs of disease exist, can prevent or delay the occurrence of cancer. Laboratory tests used in the diagnosis of cancer include chemistry (including measuring integrin-αv heterodimer (e.g., integrin-αvβ1) levels), hematology, serology and radiology. Therefore, any clinical or biochemical assay that monitors any of the above may be used to determine whether a particular treatment is a therapeutically effective dose for treating cancer.

[0306] The compositions described herein can be administered by one or more routes of administration using one or more of a variety of methods known in the art. The route and / or mode of administration varies depending on the desired outcome. The routes of administration of the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies described herein include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. As used herein, the phrase "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraarticular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0307] Alternatively, the antibodies described herein can potentially be administered by non-parenteral routes, such as topical, epithelial or mucosal routes of administration, for example, intranasal, oral, vaginal, rectal, sublingual or topical.

[0308] Active compound can be prepared with carrier that protects compound from rapid release, such as sustained release preparation, including implant, transdermal patch and microencapsulated delivery system.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoester and polylactic acid can be used.Many methods for preparing such preparations are generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0309] The therapeutic composition can be administered using medical devices known in the art.For example, in some embodiments, the therapeutic composition described herein can be administered using needleless hypodermic injection devices, such as the devices disclosed in U.S. Patent No. 5,399,163; No. 5,383,851; No. 5,312,335; No. 5,064,413; No. 4,941,880; No. 4,790,824; or No. 4,596,556. Examples of known indwelling agents and modules for use with the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies described herein include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medicine at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicine through the skin; U.S. Patent No. 4,447,233, which discloses a medicine infusion pump for delivering medicine at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system.These patents are incorporated herein by reference.

[0310] In some embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies described herein can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds described herein cross the BBB (if desired, e.g., for brain cancer), they can be formulated, for example, in liposomes. For methods of making liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thus enhancing targeted drug delivery (see, for example, VV Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.); mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134); p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090), and are described in K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; see also JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0311] III. Methods of the Disclosure III.A. Uses and Methods Certain aspects of the present disclosure are directed to a method of treating a subject comprising administering to the subject an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody disclosed herein, a polynucleotide encoding an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody, a vector comprising the polynucleotide, a host cell comprising the polynucleotide, an immunoconjugate comprising an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody, or any combination thereof.

[0312] Certain aspects of the present disclosure are directed to a method of treating cancer in a subject in need thereof, comprising administering to the subject an effective dose of a composition (e.g., an antibody, a polynucleotide, a vector, a host cell, an immunoconjugate, or a pharmaceutical composition) disclosed herein. In other aspects, the present disclosure is directed to a method of inhibiting integrin-αv heterodimer (e.g., integrin-αvβ1) shedding by tumor cells in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the present disclosure is directed to a method of reducing integrin-αv heterodimer (e.g., integrin-αvβ1) shedding in serum and / or retaining integrin-αv heterodimer (e.g., integrin-αvβ1) on the cell surface in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the disclosure is directed to a method of killing tumor cells in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the disclosure is directed to a method of reducing tumor size in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In other aspects, the disclosure is directed to inhibiting tumor metastasis in a subject in need thereof, comprising administering to the subject an effective dose of a composition disclosed herein. In some aspects, the subject is a human.

[0313] The composition of the present disclosure can be administered using any pharma- ceutically acceptable route. In some aspects, the composition (e.g., antibody, polynucleotide, vector, host cell, immunoconjugate, or pharmaceutical composition) is administered intravenously, intraperitoneally, intramuscularly, intraarterially, intrathecally, intralymphatically, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intrasternal, topically, epidermally, mucosally, or any combination thereof. In some aspects, the composition is administered intravenously. In some aspects, the composition is administered subcutaneously.

[0314] In certain embodiments, the method reduces the size of a cancer in a subject, such as the size of a tumor. In some embodiments, the size of a cancer is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%.

[0315] In some embodiments, the method increases the overall survival of the subject. In some embodiments, the overall survival is increased compared to the average overall survival of subjects with the same cancer but treated with a different therapy. In certain embodiments, the overall survival is increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 3-fold, at least about 5-fold. In some embodiments, the overall survival is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 1 month, at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or at least about 10 years.

[0316] In some embodiments, the method increases the progression-free survival of the subject. In some embodiments, the overall survival is increased compared to the average progression-free survival of subjects with the same cancer but treated with a different therapy. In certain embodiments, the progression-free survival is increased by at least about 10%, at least about 20%, at least about 25%, at least about 50%, at least about 2-fold, at least about 3-fold, at least about 5-fold. In some embodiments, the overall survival is increased by at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 1 month, at least about 12 months, at least about 15 months, at least about 18 months, at least about 21 months, at least about 2 years, at least about 3 years, at least about 4 years, at least about 5 years, or at least about 10 years.

[0317] In some aspects, the method increases the response rate in the subject. In certain aspects, the method induces a complete response in the subject. In some aspects, the method induces a partial response in the subject.

[0318] In some embodiments, the method comprises administering an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody (or polynucleotide, vector, host cell, or immunoconjugate) disclosed herein and a second therapy. In some embodiments, the second therapy is administered before the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody. In some embodiments, the second therapy is administered after the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody. In some embodiments, the second therapy is administered simultaneously with the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody. In certain embodiments, the anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody and the second therapy are administered separately. In other embodiments, the anti-integrin-αv heterodimer (eg, integrin-αvβ1) antibody and the second therapy are administered in a single formulation.

[0319] The second therapy can be any other therapy known in the art. In some embodiments, the second therapy comprises immunotherapy. In some embodiments, the second therapy comprises chemotherapy. In some embodiments, the second therapy comprises radiation therapy. In some embodiments, the second therapy comprises surgery. In some embodiments, the second therapy comprises administering a second therapeutic agent.

[0320] In certain embodiments, the second therapeutic agent comprises a second antibody. In some embodiments, the second therapeutic agent is selected from the group consisting of inducible T cell costimulator (ICOS), CD137 (4-1BB), CD134 (OX40), NKG2A, CD27, glucocorticoid-induced TNFR-related protein (GITR), and herpes virus entry mediator (HVEM), programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), CTLA-4, B- and T-lymphocyte attenuating factor (BTLA), T cell immunoglobulin and mucin domain-3 (TIM-3), lymphocyte activation gene-3 (LAG-3), adenosine A2a receptor. (A2aR), killer cell lectin-like receptor G1 (KLRG-1), natural killer cell receptor 2B4 (CD244), CD160, T cell immunoreceptor with Ig and ITIM domains (TIGIT), and receptor for V-domain Ig suppressor of T cell activation (VISTA), NKG2a, KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CEACAM-1, CD96, CD52, HER2, and any combination thereof.

[0321] XI.A. Anti-PD-1 antibody In some embodiments, the second antibody is an anti-PD-1 antibody. Anti-PD-1 antibodies known in the art can be used in the compositions and methods described herein. Various human monoclonal antibodies that specifically bind to PD-1 with high affinity are disclosed in U.S. Patent No. 8,008,449. The anti-PD-1 human antibodies disclosed in U.S. Patent No. 8,008,449 have been shown to exhibit one or more of the following characteristics: (a) a 1×10 mAb binding affinity as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D(b) binds to human PD-1 in a mixed lymphocyte reaction (MLR) assay, (b) does not substantially bind to human CD28, CTLA-4, or ICOS, (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay, (d) increases interferon-γ production in an MLR assay, (e) increases IL-2 secretion in an MLR assay, (f) binds to human PD-1 and cynomolgus PD-1, (g) inhibits binding of PD-L1 and / or PD-L2 to PD-1, (h) stimulates an antigen-specific memory response, (i) stimulates an antibody response, and (j) inhibits tumor cell growth in vivo. Anti-PD-1 antibodies that can be used in the present disclosure include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments at least five, of the foregoing characteristics.

[0322] Other anti-PD-1 monoclonal antibodies are described in, e.g., U.S. Patent Nos. 6,808,710, 7,488,802, 8,168,757, and 8,354,509, U.S. Application No. 2016 / 0272708, and PCT Application Nos. WO2012 / 145493, WO2008 / 1567, and the like. No. 12, WO2015 / 112900, WO2012 / 145493, WO2015 / 112800, WO2014 / 206107, WO2015 / 35606, WO2015 / 085847, WO2014 / 179664, WO2017 / 020291, WO2017 / 020858, WO2016 / 197367, WO2017 / 024515, WO2017 / 025051, WO2017 / 123557, WO2016 / 106159, WO2 014 / 194302, WO2017 / 040790, WO2017 / 133540, WO2017 / 132827, WO2017 / 024465, W Nos. 5,133,354, 5,163, 5,172, 5,182, 5,192, 5,102, 5,118, 5,122, 5,146, 5,163, 5,182, 5,192, 5,102 ...

[0323] In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (OPDIVO®, also known as 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck, also known as KEYTRUDA®, lambrolizumab, and MK-3475, see WO2008 / 156712), PDR001 (Novartis, see WO2015 / 112900), MEDI-0680 (AstraZeneca, also known as AMP-514, see WO2012 / 145493), cemiplimab (Regeneron, also known as REGN-2810, see WO2015 / 112800), JS001 (TAIZHOU JUNSHI PHARMA, Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene, see WO2015 / 35606 and US2015 / 0079109), INCSHR1210 (Jiangsu Hengrui Medicine, also known as SHR-1210, see WO2015 / 085847, Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical, also known as ANB011, see WO2014 / 179664), GLS-010 (Wuxi / Harbin Gloria Pharmaceuticals, also known as WBP3055, Si-Yang Liu et al., J. Hematol. Oncol.10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics, see WO2014 / 194302), AGEN2034 (Agenus, see WO2017 / 040790), MGA012 (Macrogenics, see WO2017 / 19846), and IBI308 (Innovent, see WO2017 / 024465, WO2017 / 025016, WO2017 / 132825, and WO2017 / 133540).

[0324] In one embodiment, the anti-PD-1 antibody is nivolumab, a fully human IgG4(S228P)PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of anti-tumor T cell function (U.S. Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

[0325] In another embodiment, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.

[0326] Anti-PD-1 antibodies that can be used in the disclosed compositions and methods also include isolated antibodies that specifically bind to human PD-1 and cross-compete with any of the anti-PD-1 antibodies disclosed herein, such as nivolumab, for binding to human PD-1 (see, e.g., U.S. Patent Nos. 8,008,449 and 8,779,105, WO2013 / 173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as any of the anti-PD-1 antibodies described herein, such as nivolumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibody, such as nivolumab, given their binding to the same epitope region of PD-1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0327] In certain embodiments, the antibody that cross-competes with the human PD-1 antibody, nivolumab, or binds to the same epitope region as it for binding to human PD-1 is a monoclonal antibody.For administration to human subjects, these cross-competing antibodies are chimeric antibodies, genetically engineered antibodies, or humanized or human antibodies.Such chimeric, genetically engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0328] The anti-PD-1 antibodies that can be used in the disclosed compositions and methods also include antigen-binding portions of the above-mentioned antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0329] Anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are those that bind to PD-1 with high specificity and affinity, block binding of PD-L1 and / or PD-L2, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-1 "antibody" includes an antigen-binding portion or fragment that binds to the PD-1 receptor and exhibits functional properties similar to those of a full antibody with respect to inhibiting ligand binding and upregulating the immune system. In certain embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

[0330] XI.B. Anti-PD-L1 antibody In some embodiments, the second antibody is an anti-PD-L1 antibody. Anti-PD-L1 antibodies known in the art can be used in the compositions and methods of the disclosure. Examples of anti-PD-L1 antibodies useful in the compositions and methods of the disclosure include the antibodies disclosed in U.S. Patent No. 9,580,507. The anti-PD-L1 human monoclonal antibody disclosed in U.S. Patent No. 9,580,507 has been demonstrated to exhibit one or more of the following characteristics: (a) a PD-L1 affinity of 1×10 as determined by surface plasmon resonance using a Biacore biosensor system; -7 K below M D (b) bind to human PD-L1 in a mixed lymphocyte reaction (MLR) assay, (c) increase interferon-γ production in an MLR assay, (d) increase IL-2 secretion in an MLR assay, (e) stimulate antibody responses, and (f) reverse the effects of regulatory T cells on T cells, effector cells, and / or dendritic cells. Anti-PD-L1 antibodies that can be used in the present disclosure include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments at least five, of the foregoing characteristics.

[0331] In certain embodiments, the anti-PD-L1 antibody is selected from the group consisting of BMS-936559 (also known as 12A4, MDX-1105, see, e.g., U.S. Patent No. 7,943,743 and WO 2013 / 173223), atezolizumab (Roche, also known as TECENTRIQ®, MPDL3280A, RG7446, see U.S. Patent No. 8,217,149, and see Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca, IMFINZI™, also known as MEDI-4736, see WO2011 / 066389), avelumab (Pfizer, BAVENCIO®, also known as MSB-0010718C, see WO2013 / 079174), STI-1014 (Sorrento, see WO2013 / 181634), CX-072 (Cytomx, see WO2016 / 149201), KN035 (3D Med / Alphamab, see Zhang et al., Cell Discov. 7:3 (March 2017), LY3300054 (Eli Lilly Co, see, e.g., WO2017 / 034916), and CK-301 (Checkpoint Therapeutics, see Gorelik et al., AACR:Abstract 4606 (Apr 2016)).

[0332] In a particular embodiment, the PD-L1 antibody is atezolizumab (TECENTRIQ®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

[0333] In a particular embodiment, the PD-L1 antibody is durvalumab (IMFINZI™). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

[0334] In a particular embodiment, the PD-L1 antibody is avelumab (BAVENCIO®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

[0335] In other embodiments, the anti-PD-L1 monoclonal antibody is selected from the group consisting of 28-8, 28-1, 28-12, 29-8, 5H1, or any combination thereof.

[0336] Anti-PD-L1 antibodies that can be used in the disclosed compositions and methods also include isolated antibodies that specifically bind human PD-L1 and cross-compete with any of the anti-PD-L1 antibodies disclosed herein, such as atezolizumab, durvalumab, and / or avelumab, for binding to human PD-L1. In some embodiments, the anti-PD-L1 antibodies bind to the same epitope as any of the anti-PD-L1 antibodies described herein, such as atezolizumab, durvalumab, and / or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that they bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to those of the reference antibodies, such as atezolizumab and / or avelumab, given their binding to the same epitope region of PD-L1. Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and / or avelumab in standard PD-L1 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0337] In certain embodiments, the antibodies that cross-compete with or bind to the same epitope region as human PD-L1 antibodies, such as atezolizumab, durvalumab, and / or avelumab, for binding to human PD-L1 are monoclonal antibodies. For administration to human subjects, these cross-competing antibodies are chimeric, engineered, or humanized or human antibodies. Such chimeric, engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0338] The anti-PD-L1 antibodies that can be used in the disclosed compositions and methods also include antigen-binding portions of the above-mentioned antibodies. It has been well shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0339] Anti-PD-L1 antibodies suitable for use in the disclosed compositions or methods are those that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effects of the PD-1 signaling pathway. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits functional properties similar to those of the full antibody with respect to inhibiting receptor binding and upregulating the immune system. In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab, and / or avelumab for binding to human PD-L1.

[0340] XI.C. Anti-CTLA-4 antibody In some embodiments, the second antibody is an anti-CTLA-4 antibody. Any anti-CTLA-4 antibody known in the art can be used in the compositions and methods of the present disclosure. The anti-CTLA-4 antibody of the present disclosure binds to human CTLA-4 so as to disrupt the interaction of CTLA-4 with human B7 receptor. Because the interaction of CTLA-4 with B7 transmits a signal that leads to the inactivation of T cells that have CTLA-4 receptor, disrupting this interaction effectively induces, enhances, or prolongs the activation of such T cells, thereby inducing, enhances, or prolongs immune response.

[0341] A human monoclonal antibody that specifically binds to CTLA-4 with high affinity is disclosed in U.S. Patent No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are described in, for example, U.S. Patent Nos. 5,977,318, 6,051,227, 6,682,736, and 7,034,121, as well as International Publication Nos. WO2012 / 122444, WO2007 / 113648, WO2016 / 196237, and WO2000 / 037504, each of which is incorporated herein by reference in its entirety. The anti-CTLA-4 human monoclonal antibody disclosed in U.S. Patent No. 6,984,720 has been demonstrated to exhibit one or more of the following characteristics: (a) a CTLA-4 affinity of at least about 10, as determined by Biacore analysis; 7 M -1 , or about 10 9 M -1 , or about 10 10 M -1 ~10 11 M -1 , or a higher equilibrium binding constant (K a (b) specifically binds to human CTLA-4 with a binding affinity reflected by at least about 10 3 , about 10 4 , or about 10 5 m -1 s -1 The kinetic binding constant (k a ), (c) at least about 10 3 , about 104 , or about 10 5 m -1 s -1 The kinetic dissociation constant (k d ), and (d) inhibiting the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86). Anti-CTLA-4 antibodies useful in the present disclosure include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the aforementioned characteristics.

[0342] In certain embodiments, the CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as YERVOY®, MDX-010, 10D1, see U.S. Pat. No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc., see WO2016 / 196237), and tremelimumab (AstraZeneca, also known as ticilimumab, CP-675,206, see WO2000 / 037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). In a specific embodiment, the anti-CTLA-4 antibody is ipilimumab.

[0343] In a specific embodiment, the CTLA-4 antibody for use in the compositions and methods disclosed herein is ipilimumab. Ipilimumab is a fully human IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.

[0344] In a specific embodiment, the CTLA-4 antibody is tremelimumab.

[0345] In a specific embodiment, the CTLA-4 antibody is MK-1308.

[0346] In a specific embodiment, the CTLA-4 antibody is AGEN-1884.

[0347] In some embodiments, the CTLA-4 antibody is non-fucosylated or hypofucosylated. In some embodiments, the CTLA-4 antibody exhibits enhanced ADCC and / or ADCP activity. In some embodiments, the CTLA-4 antibody is BMS-986218, described in PCT / US18 / 19868.

[0348] Anti-CTLA-4 antibodies that can be used in the disclosed compositions and methods also include isolated antibodies that specifically bind to human CTLA-4 and cross-compete with any of the anti-CTLA-4 antibodies disclosed herein, such as ipilimumab and / or tremelimumab, for binding to human CTLA-4. In some embodiments, the anti-CTLA-4 antibody binds to the same epitope as any of the anti-CTLA-4 antibodies described herein, such as ipilimumab and / or tremelimumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region. Given that these cross-competing antibodies bind to the same epitope region of CTLA-4, they are expected to have functional properties very similar to those of the reference antibodies, such as ipilimumab and / or tremelimumab. Cross-competing antibodies can be readily identified based on their ability to cross-compete with ipilimumab and / or tremelimumab in standard CTLA-4 binding assays, such as Biacore analysis, ELISA assays, or flow cytometry (see, e.g., WO2013 / 173223).

[0349] In certain embodiments, the antibody that cross-competes with human CTLA-4 antibodies such as ipilimumab and / or tremelimumab for binding to human CTLA-4 or binds to the same epitope region as human CTLA-4 antibodies is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric antibodies, genetically engineered antibodies, or humanized or human antibodies. Such chimeric, genetically engineered, humanized, or human monoclonal antibodies can be prepared and isolated by methods well known in the art.

[0350] Anti-CTLA-4 antibodies that can be used in the disclosed compositions and methods also include antigen-binding portions of the above-mentioned antibodies. It has been well demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.

[0351] Anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are those that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and disrupt the interaction of CTLA-4 with the human B7 receptor. In any of the compositions or methods disclosed herein, the anti-CTLA-4 "antibody" includes an antigen-binding portion or fragment that binds to CTLA-4 and exhibits functional properties similar to those of a complete antibody in terms of inhibiting the interaction of CTLA-4 with the human B7 receptor and upregulating the immune system. In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and / or tremelimumab for binding to human CTLA-4.

[0352] XI.D. Anti-LAG-3 antibody In some embodiments, the second antibody is an anti-LAG-3 antibody. The anti-LAG-3 antibody of the present disclosure binds to human LAG-3. Antibodies that bind to LAG-3 are disclosed in International Publication No. WO / 2015 / 042246 and US Publication Nos. 2014 / 0093511 and 2011 / 0150892.

[0353] An exemplary LAG-3 antibody useful in the present disclosure is 25F7 (described in U.S. Publication No. 2011 / 0150892). An additional exemplary LAG-3 antibody useful in the present disclosure is BMS-986016. In one embodiment, an anti-LAG-3 antibody useful in the composition cross-competes with 25F7 or BMS-986016. In another embodiment, an anti-LAG-3 antibody useful in the composition binds to the same epitope as 25F7 or BMS-986016. In other embodiments, an anti-LAG-3 antibody comprises the six CDRs of 25F7 or BMS-986016.

[0354] XI.E. Anti-CD137 antibody In some embodiments, the second antibody is an anti-CD137 antibody.Anti-CD137 antibody specifically binds to and activates immune cells expressing CD137, stimulating immune response against tumor cells, particularly cytotoxic T cell response.Antibodies that bind to CD137 are disclosed in US Publication No. 2005 / 0095244, and in U.S. Patent Nos. 7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685, 6,355,476, 6,362,325, 6,974,863 and 6,210,669.

[0355] In some embodiments, the anti-CD137 antibody is urelumab (BMS-663513) (20H4.9-IgG4 [10C7 or BMS-663513]) described in U.S. Patent No. 7,288,638. In some embodiments, the anti-CD137 antibody is BMS-663031 (20H4.9-IgG1) described in U.S. Patent No. 7,288,638. In some embodiments, the anti-CD137 antibody is 4E9 or BMS-554271 described in U.S. Patent No. 6,887,673. In some embodiments, the anti-CD137 antibody is an antibody disclosed in U.S. Patent No. 7,214,493, U.S. Patent No. 6,303,121, U.S. Patent No. 6,569,997, U.S. Patent No. 6,905,685, or U.S. Patent No. 6,355,476. In some embodiments, the anti-CD137 antibody is 1D8 or BMS-469492, 3H3 or BMS-469497, or 3E1, as described in U.S. Patent No. 6,362,325. In some embodiments, the anti-CD137 antibody is an antibody disclosed in issued U.S. Patent No. 6,974,863 (e.g., 53A2). In some embodiments, the anti-CD137 antibody is an antibody disclosed in issued U.S. Patent No. 6,210,669 (e.g., 1D8, 3B8, or 3E1). In some embodiments, the antibody is Pfizer's PF-05082566 (PF-2566). In other embodiments, the anti-CD137 antibody useful in the present disclosure cross-competes with the anti-CD137 antibody disclosed herein. In some embodiments, the anti-CD137 antibody binds to the same epitope as the anti-CD137 antibody disclosed herein. In other embodiments, an anti-CD137 antibody useful in this disclosure comprises the six CDRs of an anti-CD137 antibody disclosed herein.

[0356] XI.F. Anti-KIR antibody In some embodiments, the second antibody is an anti-KIR3 antibody. Antibodies that specifically bind to KIR block the interaction between killer cell immunoglobulin-like receptors (KIR) on NK cells and their ligands. Blocking these receptors promotes the activation of NK cells and potentially the latter's destruction of tumor cells. Examples of anti-KIR antibodies are disclosed in International Publication Nos. WO / 2014 / 055648, WO2005 / 003168, WO2005 / 009465, WO2006 / 072625, WO2006 / 072626, WO2007 / 042573, WO2008 / 084106, WO2010 / 065939, WO2012 / 071411, and WO / 2012 / 160448.

[0357] One anti-KIR antibody useful in the present disclosure is lirilumab (also referred to as BMS-986015, IPH2102, or the S241P variant of 1-7F9), first described in International Publication WO2008 / 084106. An additional anti-KIR antibody useful in the present disclosure is 1-7F9 (also referred to as IPH2101), described in International Publication WO2006 / 003179. In one embodiment, the anti-KIR antibody of the present composition cross-competes with lirilumab or I-7F9 for binding to KIR. In another embodiment, the anti-KIR antibody binds to the same epitope as lirilumab or I-7F9. In other embodiments, the anti-KIR antibody comprises the six CDRs of lirilumab or I-7F9.

[0358] XI.G. Anti-GITR antibody In some embodiments, the second antibody is an anti-GITR antibody. The anti-GITR antibody can be any anti-GITR antibody that specifically binds to the human GITR target and activates glucocorticoid-induced tumor necrosis factor receptor (GITR). GITR is a member of the TNF receptor superfamily that is expressed on the surface of multiple types of immune cells, including regulatory T cells, effector T cells, B cells, natural killer (NK) cells, and activated dendritic cells ("anti-GITR agonist antibodies"). Specifically, GITR activation increases the proliferation and function of effector T cells, as well as abrogates the suppression induced by activated regulatory T cells. In addition, GITR stimulation promotes anti-tumor immunity by increasing the activity of other immune cells, such as NK cells, antigen-presenting cells, and B cells. Examples of anti-GITR antibodies are disclosed in International Publication Nos. WO / 2015 / 031667, WO2015 / 184,099, WO2015 / 026,684, WO11 / 028683, and WO / 2006 / 105021, U.S. Patent Nos. 7,812,135 and 8,388,967, and U.S. Publication Nos. 2009 / 0136494, 2014 / 0220002, 2013 / 0183321, and 2014 / 0348841.

[0359] In one embodiment, the anti-GITR antibody useful in the present disclosure is TRX518 (e.g., as described in Schaer et al. Curr Opin Immunol. (2012) Apr; 24(2): 217-224 and WO / 2006 / 105021). In another embodiment, the anti-GITR antibody is selected from MK4166, MK1248, and the antibodies described in WO11 / 028683 and US 8,709,424, e.g., comprising a VH chain comprising SEQ ID NO: 104, and a VL chain comprising SEQ ID NO: 105 (the SEQ ID NOs are from WO11 / 028683 or US 8,709,424). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in WO2015 / 031667, e.g., an antibody comprising VH CDR1-3 comprising SEQ ID NOs: 31, 71, and 63 of WO2015 / 031667, and VL CDR1-3 comprising SEQ ID NOs: 5, 14, and 30 of WO2015 / 031667, respectively. In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in WO2015 / 184099, e.g., antibody Hum231#1 or Hum231#2, or CDRs thereof, or derivatives thereof (e.g., pab1967, pab1975, or pab1979). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in JP2008278814, WO09 / 009116, WO2013 / 039954, US20140072566, US20140072565, US20140065152, or WO2015 / 026684, or is INBRX-110 (INHIBRx), LKZ-145 (Novartis), or MEDI-1873 (MedImmune). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody described in PCT / US2015 / 033991 (e.g., an antibody comprising the variable regions of 28F3, 18E10, or 19D3). For example, the anti-GITR antibody is an antibody comprising the following VH and VL chains, or their CDRs: VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWGQGTTVTVS (SEQ ID NO: 78), and VL: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIK (SEQ ID NO: 79); or VH: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGFHWVRQAPGKGLEWVAVIWYAGSNKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGQLDYYYYYVMDVWGQGTTVTVSS (SEQ ID NO: 80), and VL: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 81); or VH: VQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYAGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGRIAVAFYYSMDVWGQGTTVTVSS (SEQ ID NO: 82), and VL: DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPYTFGQGTKLEIK (SEQ ID NO: 83)

[0360] In certain embodiments, antibodies comprising the above VH and VL light chain pairs, or their CDRs, comprise a heavy chain constant region of the IgG1 isotype, either wild type or mutated, e.g., to be effectorless. In one embodiment, the anti-GITR antibody comprises the following heavy and light chain amino acid sequences:

[0361] Heavy Chain: QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVIWYEGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGGSMVRGDYYYGMDVWG QGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 84), and

[0362] Light chain: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 85), or

[0363] Heavy Chain: qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvaviwyegsnkyyadsvkgrftisrdnskntlylqmnslraedtavyycarggsmvrgdyyygmdvwgq gttvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktht cppcpapeaegapsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpssiektiskakgqprepqvytlppsreemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspg (SEQ ID NO: 86), and

[0364] Light chain: AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 87)

[0365] In certain embodiments, the anti-GITR antibody cross-competes with the anti-GITR antibody described herein, for example, TRX518, MK4166, or an antibody comprising the VH and VL domain amino acid sequences described herein. In some embodiments, the anti-GITR antibody binds to the same epitope as the anti-GITR antibody described herein, for example, TRX518, MK4166, or an antibody comprising the VH and VL domain amino acid sequences described herein. In certain embodiments, the anti-GITR antibody comprises the six CDRs of TRX518, MK4166, or an antibody comprising the VH and VL domain amino acid sequences described herein.

[0366] XI.H. Anti-TIM3 antibody In some embodiments, the second antibody is an anti-TIM3 antibody. In some embodiments, the anti-TIM3 antibody is one described in International Publication Nos. WO2018013818, WO / 2015 / 117002 (e.g., MGB453, Novartis), WO / 2016 / 161270 (e.g., TSR-022, Tesaro / AnaptysBio), WO2011155607, WO2016 / 144803 (e.g., STI-600, Sorrento), and the like. Therapeutics), WO2016 / 071448, WO17055399, WO17055404, WO17178493, WO18036561, WO18039020 (e.g., Ly-3221367, Eli Lilly), WO2017205721, WO17079112, WO17079115, WO17079116, WO11159877, WO13006490, WO2016068802, WO2016068803, WO2016 / 111947, or WO / 2017 / 031242.

[0367] XI.I. Anti-OX40 antibody In some embodiments, the second antibody is an anti-OX40 (also known as CD134, TNFRSF4, ACT35, and / or TXGP1L) antibody. In some embodiments, the anti-OX40 antibody is BMS-986178 (Bristol-Myers Squibb Company), described in International Publication No. WO20160196228. In some embodiments, the anti-OX40 antibody is selected from the anti-OX40 antibodies described in International Publication Nos. WO95012673, WO199942585, WO14148895, WO15153513, WO15153514, WO13038191, WO16057667, WO03106498, WO12027328, WO13028231, WO16200836, WO17063162, WO17134292, WO17096179, WO17096281, and WO17096182.

[0368] XI.J. Anti-NKG2A antibody In some embodiments, the second antibody is an anti-NKG2A antibody. NKG2A is a member of the C-type lectin receptor family that is expressed on natural killer (NK) cells and a subset of T lymphocytes. Specifically, NKG2A is mainly expressed on tumor-infiltrating innate immune effector NK cells, as well as on some CD8+ T cells. Its natural ligand, human leukocyte antigen E (HLA-E), is expressed on solid tumors and hematological tumors. NKG2A is an inhibitory receptor that binds to HLA-E.

[0369] In some embodiments, the anti-NKG2A antibody is BMS-986315, a human monoclonal antibody that blocks the interaction of NKG2A with its ligand HLA-E, thus allowing activation of an anti-tumor immune response. In some embodiments, the anti-NKG2A antibody is a checkpoint inhibitor that activates T cells, NK cells, and / or tumor-infiltrating immune cells. In some embodiments, the anti-NKG2A antibody is disclosed in, for example, WO2006 / 070286 (Innate Pharma SA, University of Genova), U.S. Pat. No. 8,993,319 (Innate Pharma SA, University of Genova), WO2007 / 042573 (Innate Pharma S / A, Novo Nordisk A / S, University of Genova), U.S. Pat. No. 9,447,185 (Innate Pharma S / A, Novo Nordisk A / S, University of Genova), WO2008 / 009545 (Novo Nordisk A / S), U.S. Pat. Nos. 8,206,709, 8,901,283, and 9,683,041 (Novo Nordisk A / S), WO2009 / 092805 (Novo Nordisk A / S), U.S. Pat. Nos. 8,796,427 and 9,422,368 (Novo Nordisk A / S), WO2016 / 134371 (Ohio State Innovation Foundation), WO2016 / 032334 (Janssen), WO2016 / 041947 (Innate), WO2016 / 041945 (Academisch Ziekenhuis Leiden HODN LUMC), WO2016 / 041947 (Innate Pharma), and the anti-NKG2A antibodies described in WO2016 / 041945 (Innate Pharma).

[0370] XI.K. Anti-ICOS antibody In some embodiments, the second antibody is an anti-ICOS antibody. ICOS is an immune checkpoint protein that is a member of the CD28 superfamily. ICOS is a 55-60 kDa type I transmembrane protein expressed on T cells after T cell activation and costimulates T cell activation after binding to its ligand, ICOS-L (B7H2). ICOS is also known as inducible T cell costimulator, CVID1, AILIM, inducible costimulator, CD278, activation-induced lymphocyte immune mediating molecule, and CD278 antigen.

[0371] In some embodiments, the anti-ICOS antibody is BMS-986226, a humanized IgG monoclonal antibody that binds to and stimulates human ICOS. In some embodiments, the anti-ICOS antibody is disclosed in, for example, WO2016 / 154177 (Jounce Therapeutics, Inc.), WO2008 / 137915 (MedImmune), WO2012 / 131004 (INSERM, French National Institute of Health and Medical Research), EP3147297 (INSERM, French National Institute of Health and Medical Research), WO2011 / 041613 (Memorial Sloan Kettering Cancer Center), EP2482849 (Memorial Sloan Kettering Cancer Center), WO1999 / 15553 (Robert Koch Institute), U.S. Patent Nos. 7,259,247 and 7,722,872 (Robert Kotch Institute), WO1998 / 038216 (Japan Tobacco Research Center ... WO2012 / 131004 (INSERM, French National Institute of Health and Medical Research), WO2012 / 131004 (INSERM, French National Institute of Health and Medical Research), WO2012 / 131004 (IN Nos. 7,045,615, 7,112,655, and 8,389,690 (Japan Tobacco Inc.), U.S. Pat. Nos. 9,738,718 and 9,771,424 (GlaxoSmithKline), and anti-ICOS antibodies described in WO2017 / 220988 (Kymab Limited).

[0372] XI.L. Anti-TIGIT antibody In some embodiments, the second antibody is an anti-TIGIT antibody. In some embodiments, the anti-TIGIT antibody is BMS-986207. In some embodiments, the anti-TIGIT antibody is clone 22G2 described in WO2016 / 106302. In some embodiments, the anti-TIGIT antibody is MTIG7192A / RG6058 / RO7092284 or clone 4.1D3 described in WO2017 / 053748. In some embodiments, the anti-TIGIT antibody is selected from, for example, the anti-TIGIT antibodies described in WO2016 / 106302 (Bristol-Myers Squibb Company) and WO2017 / 053748 (Genentech).

[0373] XI.M. Additional Anticancer Agents In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-IL-10 antibody. In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with a long-acting IL-10 molecule. In some embodiments, the long-acting IL-10 molecule can be an IL-10-Fc fusion molecule. In some embodiments, the long-acting IL-10 molecule can be pegylated IL-10, e.g., AM0010 (ARMO BioSciences).

[0374] In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-IL-2 antibody. In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with a long-acting IL-2 molecule. In some embodiments, the long-acting IL-2 can be pegylated IL-2, e.g., NKTR-214 (Nektar, see US8,252,275, WO12 / 065086, and WO15 / 125159).

[0375] In some embodiments, an anti-integrin-αv heterodimer (eg, integrin-αvβ1) antibody is used in combination with an anti-VISTA antibody.

[0376] In some embodiments, an anti-integrin-αv heterodimer (eg, integrin-αvβ1) antibody is used in combination with an anti-CD96 antibody.

[0377] In some embodiments, an anti-integrin-αv heterodimer (eg, integrin-αvβ1) antibody is used in combination with an anti-IL-8 antibody, such as HuMax®-IL8.

[0378] In some embodiments, an anti-integrin-αv heterodimer (eg, integrin-αvβ1) antibody is used in combination with an anti-TGFβ antibody.

[0379] In some other embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-B7-H4 antibody. In certain embodiments, the anti-B7-H4 antibody is the anti-B7-H4 disclosed in International Publication No. WO / 2009 / 073533.

[0380] In certain embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-Fas Ligand antibody. In certain embodiments, the anti-Fas Ligand antibody is an anti-Fas Ligand disclosed in International Publication No. WO / 2009 / 073533.

[0381] In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-CXCR4 antibody. In certain embodiments, the anti-CXCR4 antibody is an anti-CXCR4 disclosed in U.S. Publication No. 2014 / 0322208 (e.g., ulcuplumab (BMS-936564)).

[0382] In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-mesothelin antibody. In certain embodiments, the anti-mesothelin antibody is an anti-mesothelin antibody disclosed in U.S. Patent No. 8,399,623.

[0383] In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-HER2 antibody. In certain embodiments, the anti-HER2 antibody is Herceptin (U.S. Pat. No. 5,821,337), trastuzumab, or ado-trastuzumab emtansine (Kadcyla, e.g., WO / 2001 / 000244).

[0384] In embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-CD27 antibody. In embodiments, the anti-CD-27 antibody is varlilumab (also known as "CDX-1127" and "1F5"), a human IgG1 antibody that is an agonist of human CD27, e.g., as disclosed in U.S. Patent No. 9,169,325.

[0385] In some embodiments, an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody is used in combination with an anti-CD73 antibody. In a particular embodiment, the anti-CD73 antibody is CD73.4.IgG2C219S.IgG1.1f.

[0386] In certain embodiments, the second treatment comprises administering a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent induces integrin-αv heterodimer (e.g., integrin-αvβ1) expression on tumor cells. In some embodiments, the chemotherapeutic agent is selected from a proteasome inhibitor, an immunomodulatory drug (IMiD), a Bet inhibitor, and any combination thereof. In some embodiments, the proteasome inhibitor is selected from bortezomib, ixazomib, carfilzomib, oprozomib, and marizomib. In certain embodiments, the proteasome inhibitor comprises bortezomib.

[0387] In some embodiments, the second therapy comprises radiation therapy. Any radiation therapy known in the art can be used as the second therapy.

[0388] In some embodiments, the second therapy comprises administering an agent that activates innate immune cells. In some embodiments, the agent that activates innate immune cells comprises an NLRP3 agonist. In some embodiments, the NLRP3 agonist comprises monosodium urate monohydrate (MSU) and / or alum, which is a vaccine adjuvant. In some embodiments, the agent that activates innate immune cells is a toll-like receptor 7 (TLR7) agonist. In some embodiments, the TLR7 agonist comprises imiquimod (R837), GS-9620 (see Tsai et al., J. Virology doi:10.1128 / JVI.02166-16 (Feb. 8, 2017)), ORN R-2336 (Miltenyl Biotec), or any combination thereof.

[0389] In some embodiments, the second therapy comprises natural killer (NK) cells, CD8 + The method includes administering an agent that enhances survival of T cells, T cells, or both. In some embodiments, the agent comprises IL-2. In certain embodiments, the agent comprises pegylated IL-2.

[0390] In certain embodiments, the second therapy is selected from the group consisting of doxorubicin (ADRIAMYCIN®), cisplatin, carboplatin, bleomycin sulfate, carmustine, chlorambucil (LEUKERAN®), cyclophosphamide (CYTOXAN®, NEOSAR®), lenalidomide (REVLIMID®), bortezomib (VELCADE®), dexamethasone, mitoxantrone, etoposide, cytarabine, bendamustine (TREANDA®), rituximab, rifampicin ... The present invention includes administering an agent selected from the group consisting of cimetuzumab (RITUXAN®), ifosfamide, vincristine (ONCOVIN®), fludarabine (FLUDARA®), thalidomide (THALOMID®), alemtuzumab (CAMPATH®), ofatumumab (ARZERRA®), everolimus (AFINITOR®, ZORTRESS®), carfilzomib (KYPROLISTM), and any combination thereof.

[0391] XI.N.Cancer Anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies can enhance immune responses against cancerous cells in patients with cancer. Provided herein is a method for treating a subject with cancer, comprising administering to the subject an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody as described herein, such that the subject is treated, e.g., the growth of cancerous tumors is inhibited or reduced, and / or the tumor regresses, and / or survival is prolonged. Anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies can be used with another agent, e.g., another immunogenic agent, standard cancer treatment, or another antibody, as described below.

[0392] Thus, provided herein is a method of treating cancer in a subject, for example by inhibiting the growth of tumor cells, comprising administering to the subject a therapeutically effective amount of an anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody described herein, such as MICA.36, MICA.52, MICA.54, MICA.2, and 71C2, or an antigen-binding portion thereof, having a wild-type IgG constant region or a constant region mutant with altered effector function. The antibody can be a human anti-integrin-αv heterodimer (e.g., integrin-αvβ1) antibody (e.g., any of the human anti-human integrin-αv heterodimer (e.g., integrin-αvβ1) antibodies described herein). Cancers whose growth can be inhibited using the antibodies of the present disclosure include cancers that are typically responsive to immunotherapy and those that are typically not responsive to immunotherapy. Cancers that can be treated also include integrin-αv heterodimer (e.g., integrin-αvβ1) positive cancers. The cancer may be a cancer involving a solid tumor or a hematological malignancy (liquid tumor).Non-limiting examples of cancers for treatment include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC) and non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, gastric cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma and head and neck cancer (or carcinoma), gastric cancer (gastric cancer), germ cell tumors, childhood sarcoma, sinonasal natural killer, melanoma (e.g. metastatic melanoma such as cutaneous or intraocular melanoma), bone cancer, skin cancer, uterine cancer, cancer of the anal region, testicular cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the rectum, solid tumors of childhood , cancer of the ureter, carcinoma of the renal pelvis, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, tumors of the spinal axis, brain cancer, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers including those induced by asbestos, virus-associated cancers or cancers of viral origin (e.g., human papillomavirus (HPV-associated or originating tumors)), and any combination of the foregoing cancers.

[0393] Non-limiting examples of cancers for treatment include hematological malignancies derived from either of the two major blood cell lineages, i.e., myeloid cell lines (producing granulocytes, erythrocytes, platelets, macrophages, and mast cells) or lymphoid cell lines (producing B, T, NK, and plasma cells), such as all types of leukemias, lymphomas, and myelomas, including acute, chronic, lymphocytic, and / or myeloid leukemias, such as acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CML), anaplastic AML (MO), myeloblastic leukemia (MYE ... Leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant with eosinophilia [M4E]), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), solitary granulocytic sarcoma, and chloroma; lymphomas, e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytic lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) Large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, angiocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic and lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, B-cell lymphoma lymphoma, lymphoblastic lymphoma (LBL), hematopoietic neoplasms of lymphocytic lineage, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome), and lymphoplasmacytic lymphoma with Waldenström's macroglobulinemia (LPL);Myelomas, e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, and multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma; hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; tumors of the central and peripheral nerves, including seminoma, teratocarcinoma, astrocytoma, and schwannoma; fibrosarcoma tumors of mesenchymal origin, including rhabdomyosarcoma, osteosarcoma, and osteosarcoma; and other tumors, including melanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, follicular thyroid carcinoma, and teratocarcinoma; hematopoietic tumors of lymphoid lineage, such as T-cell disorders, such as T-cell and B-cell tumors, including but not limited to T-prolymphocytic leukemia (T-PLL), including small cell and cerebriform cell types; large granular lymphocytic leukemia (LGL), T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / retrothymic T-cell lymphoma (pleomorphic and immunoblastic subtypes); angiocentric (nasal) T-cell lymphoma; and any combination of the above cancers;

[0394] The methods described herein may also be used for the treatment of metastatic cancer, unresectable refractory cancer (e.g., cancer refractory to previous immunotherapy, e.g., using blocking CTLA-4 or PD-1 antibodies), and / or recurrent cancer.

[0395] In some embodiments, the subject has a cancer selected from non-small cell lung cancer (NSCLC), head a...

Claims

1. 1. An isolated antibody, or antigen-binding portion thereof, that specifically binds to an integrin-αv heterodimer, comprising a variable heavy chain region ("VH") comprising variable heavy chain complementarity determining region (VH-CDR)1, VH-CDR2, and VH-CDR3, and a variable light chain region ("VL") comprising VL-CDR1, VL-CDR2, and VL-CDR3; (a) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3; VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4; VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8; VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:10; (b) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18; VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:19, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:20; or (c) VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23; VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24; VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28; VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29, and VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:30 , an isolated antibody or antigen-binding portion thereof.

2. a. VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3; b. VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:4; c. VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:5; d. VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:8; e. VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9; and f. VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 10; 2. An isolated antibody or antigen-binding portion thereof according to claim 1.

3. a. VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 13; b. VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14; c. VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 15; d. VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 18; e. VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 19; and f. VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:20; 2. An isolated antibody or antigen-binding portion thereof according to claim 1.

4. a. VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:23; b. VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:24; c. VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:25; d. VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:28; e. VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:29; and f. VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:30; 2. An isolated antibody or antigen-binding portion thereof according to claim 1.

5. a. VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:2, and VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NO:7; b. VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO: 12, and VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NO: 17, or c. VH comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence selected from SEQ ID NO:22, and VL comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to an amino acid sequence set forth in SEQ ID NO:27; The antibody or antigen-binding portion thereof described in claim 1.

6. a. VH comprises the amino acid sequence set forth in SEQ ID NO:2 and VL comprises the amino acid sequence set forth in SEQ ID NO:7; b. the VH comprises the amino acid sequence set forth in SEQ ID NO: 12 and the VL comprises the amino acid sequence set forth in SEQ ID NO: 17, or c. The VH comprises the amino acid sequence set forth in SEQ ID NO:22 and the VL comprises the amino acid sequence set forth in SEQ ID NO:27; An antibody or antigen-binding portion thereof according to claim 1 or 5.

7. an antibody, or antigen-binding portion thereof, comprising a heavy chain ("HC") and a light chain ("LC"); (a) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:1; and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:6; (b) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:11, and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16; or (c) the HC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:21; and the LC comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26; An antibody or antigen-binding portion thereof according to any one of claims 1, 5 and 6.

8. An antibody or antigen-binding portion thereof according to any one of claims 1 to 7, which is an antigen-binding portion.

9. 9. The antibody or antigen-binding portion of claim 8, which is a Fab, Fab', F(ab')2, single chain Fv (scFv), disulfide linked Fv, IgNar, intrabody, IgGΔCH2, minibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc.

10. A bispecific antibody comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 8, wherein the bispecific antibody is capable of inhibiting integrin-αv-mediated activation of TGFβ.

11. A polynucleotide or set of polynucleotides encoding a bispecific antibody according to claim 10, or an antibody or antigen-binding portion thereof according to any one of claims 1 to 9.

12. A vector or a set of vectors comprising the polynucleotide or set of polynucleotides according to claim 11.

13. A cell comprising a bispecific antibody according to claim 10, an antibody or antigen-binding portion thereof according to any one of claims 1 to 9, or a polynucleotide or set of polynucleotides according to claim 11.

14. A pharmaceutical composition comprising a bispecific antibody described in claim 10, an antibody or antigen-binding portion thereof described in any one of claims 1 to 9, or a polynucleotide or set of polynucleotides described in claim 11, and a pharma- ceutically acceptable excipient.

15. A composition for treating cancer in a subject in need thereof, comprising a bispecific antibody or antigen-binding portion thereof described in claim 10, an antibody or antigen-binding portion thereof described in any one of claims 1 to 9, or a polynucleotide or set of polynucleotides described in claim 11.

Images