Anti-alphavbeta8 integrin antibodies and methods of use

Abstract

Provided herein are anti-alpha v beta 8 antibodies, or antigen binding portions thereof, and methods of making and using the same. Further provided herein are methods of treating cancer with anti-[ alpha] v [beta] 8 and PD-1 axis antagonists.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 456,246, filed March 31, 2023, and U.S. Provisional Patent Application No. 63 / 536,342, filed September 1, 2023, each entitled “ANTI-ALPHA V BETA 8 INTEGRIN ANTIBODIES AND METHODS OF USE”, the contents of each of which are incorporated herein by reference in their entirety for all purposes.

[0003] By referencing and incorporating into the sequence list

[0004] This application is submitted together with an electronic sequence list. This sequence list is provided as a text file titled 146392064840SeqList.xml, created on March 28, 2024, and is 157,697 bytes in size. Information from the electronic sequence list is incorporated herein by reference in its entirety. Technical Field

[0005] This invention relates to anti-αvβ8 antibodies and methods of using them. Background Technology

[0006] Transforming growth factor β (TGFβ) exists as an inactive dormant complex, with most cells producing its cytokines and / or expressing its receptors. TGFβ acts locally and is highly dependent on the cellular and tissue microenvironment. TGFβ can promote cancer progression through highly modulated and differentiated effects on multiple cell types within the microenvironment. It is also associated with reduced survival rates in cancer patients and a lack of response to checkpoint inhibitors and other anticancer drugs.

[0007] The TGFβ complex must be activated to release active cytokines. Certain integrins (such as αvβ8) and proteases convert latent TGFβ into active cytokines. Antibodies need to bind to integrin αvβ8 and reduce ligand binding function. Summary of the Invention

[0008] This invention provides an anti-αvβ8 antibody and its method of use.

[0009] This article provides an antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety thereof exhibits at least one of the following properties: (a) binding to human αvβ8 with 1 nM or less KD; (b) binding to mouse αvβ8 with 1 nM or less KD; (c) binding to cynomolgus monkey αvβ8 with 1 nM or less KD; (d) inhibiting αvβ8-mediated activation by presentation of human leucine-rich repeat protein 32 (LRRC32), LRRC32 and / or latent TGFβ-binding protein (LTBP) and / or its associated LTGFβ1 and LTGFβ3; and and / or (e) blocking the binding of TGFβ peptide to αvβ8. In some embodiments, the antibody or its antigen-binding portion comprises: a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3 and a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, wherein: (a) the CDR-L1, CDR-L2, and CDR-L2 sequences are derived from the VL domain of SEQ ID NO:152, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:153; (b) the CDR-L1, CDR-L2, and CDR-L2 sequences are derived from the VL domain of SEQ ID NO:154, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:155; (c) the CDR-L1, CDR-L2, and CDR-L3 sequences are derived from the VH domain of SEQ ID NO:155. (d) The CDR-L1, CDR-L2, and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:156, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:157; (e) The CDR-L1, CDR-L2, and CDR-L2 sequences are derived from the VL domain of SEQ ID NO:158, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:159;(f) The CDR-L1, CDR-L2, and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:164, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:165; (g) The CDR-L1, CDR-L2, and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:166, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:167; (h) CDR-L1 according to SEQ ID NO:7, CDR-L2 according to SEQ ID NO:8, CDR-L3 according to SEQ ID NO:9, CDR-H1 according to SEQ ID NO:10, CDR-H2 according to SEQ ID NO:11, and CDR-H3 according to SEQ ID NO:12; (i) CDR-L1 according to SEQ ID NO:164. NO:13, CDR-L2 according to SEQ ID NO:14, CDR-L3 according to SEQ ID NO:15, CDR-H1 according to SEQ ID NO:16, CDR-H2 according to SEQ ID NO:17, and CDR-H3 according to SEQ ID NO:18; (j) CDR-L1 according to SEQ ID NO:19, CDR-L2 according to SEQ ID NO:20, CDR-L3 according to SEQ ID NO:21, CDR-H1 according to SEQ ID NO:22, CDR-H2 according to SEQ ID NO:23, and CDR-H3 according to SEQ ID NO:24; (k) CDR-L1 according to SEQ ID NO:25, CDR-L2 according to SEQ ID NO:26, CDR-L3 according to SEQ ID NO:27, CDR-H1 according to SEQ ID NO:28, CDR-H2 according to SEQ ID NO:29, and CDR-H3 according to SEQ ID NO:30; (l) CDR-L1 according to SEQ ID NO:15, CDR-L2 according to SEQ ID NO:16, CDR-H2 according to SEQ ID NO:17, and CDR-H3 according to SEQ ID NO:30. IDNO:31, CDR-L2 according to SEQ ID NO:32, CDR-L3 according to SEQ ID NO:33, CDR-H1 according to SEQ ID NO:34, CDR-H2 according to SEQ ID NO:35, and CDR-H3 according to SEQ ID NO:36;(m) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:42; (n) The sequences CDR-L1, CDR-L2, and CDR-H3 originate from the VL domain of SEQ ID NO:150, and the sequences CDR-H1, CDR-H2, and CDR-H3 originate from the VH domain of SEQ ID NO:151; (o) CDR-L1 according to SEQ ID NO:1, CDR-L2 according to SEQ ID NO:2, CDR-L3 according to SEQ ID NO:3, CDR-H1 according to SEQ ID NO:4, CDR-H2 according to SEQ ID NO:5, and CDR-H3 according to SEQ ID NO:6; (p) The CDR-L1, CDR-L2, and CDR-H3 sequences are derived from the VL domain of SEQ ID NO:162, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:163; or (q) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:42.

[0010] This article further provides an antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety comprises: a heavy chain variable domain (VH) containing CDR-H1, CDR-H2, and CDR-H3 and a light chain variable domain (VL) containing CDR-L1, CDR-L2, and CDR-L3, wherein: (a) the sequences of CDR-L1, CDR-L2, and CDR-L2 are derived from the VL domain of SEQ ID NO:152, and the sequences of CDR-H1, CDR-H2, and CDR-H3 are derived from the VH domain of SEQ ID NO:153; (b) the sequences of CDR-L1, CDR-L2, and CDR-L3 are derived from the VL domain of SEQ ID NO:154, and the sequences of CDR-H1, CDR-H2, and CDR-H3 are derived from the VH domain of SEQ ID NO:155; (c) (d) The CDR-L1, CDR-L2, and CDR-L3 sequences originate from the VL domain of SEQ ID NO:156, and the CDR-H1, CDR-H2, and CDR-H3 sequences originate from the VH domain of SEQ ID NO:157; (e) The CDR-L1, CDR-L2, and CDR-L2 sequences originate from the VL domain of SEQ ID NO:158, and the CDR-H1, CDR-H2, and CDR-H3 sequences originate from the VH domain of SEQ ID NO:159; (f) The CDR-L1, CDR-L2, and CDR-L3 sequences originate from the VL domain of SEQ ID NO:160, and the CDR-H1, CDR-H2, and CDR-H3 sequences originate from the VH domain of SEQ ID NO:161; The sequences are derived from the VL domain of SEQ ID NO:164, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:165; (g) The CDR-L1, CDR-L2, and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:166, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:167;(h) CDR-L1 according to SEQ ID NO:7, CDR-L2 according to SEQ ID NO:8, CDR-L3 according to SEQ ID NO:9, CDR-H1 according to SEQ ID NO:10, CDR-H2 according to SEQ ID NO:11, and CDR-H3 according to SEQ ID NO:12; (i) CDR-L1 according to SEQ ID NO:13, CDR-L2 according to SEQ ID NO:14, CDR-L3 according to SEQ ID NO:15, CDR-H1 according to SEQ ID NO:16, CDR-H2 according to SEQ ID NO:17, and CDR-H3 according to SEQ ID NO:18; (j) CDR-L1 according to SEQ ID NO:19, CDR-L2 according to SEQ ID NO:20, CDR-L3 according to SEQ ID NO:21, CDR-H1 according to SEQ ID NO:22, CDR-H2 according to SEQ ID NO:23, and CDR-H3 according to SEQ ID NO:24; (k) CDR-L1 according to SEQ ID NO:25, CDR-L2 according to SEQ ID NO:26, CDR-L3 according to SEQ ID NO:27, CDR-H1 according to SEQ ID NO:28, CDR-H2 according to SEQ ID NO:29, and CDR-H3 according to SEQ ID NO:30; (l) CDR-L1 according to SEQ ID NO:31, CDR-L2 according to SEQ ID NO:32, CDR-L3 according to SEQ ID NO:33, CDR-H1 according to SEQ ID NO:34, CDR-H2 according to SEQ ID NO:35, and CDR-H3 according to SEQ ID NO:36; (m) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:42; (n) The CDR-L1, CDR-L2, and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:150, and the CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:151.(o) CDR-L1 according to SEQ ID NO:1, CDR-L2 according to SEQ ID NO:2, CDR-L3 according to SEQ ID NO:3, CDR-H1 according to SEQ ID NO:4, CDR-H2 according to SEQ ID NO:5, and CDR-H3 according to SEQ ID NO:6; (p) CDR-L1, CDR-L2, and CDR-H3 sequences are derived from the VL domain of SEQ ID NO:162, and CDR-H1, CDR-H2, and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:163; or (q) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:42.

[0011] In some embodiments, the antibody or its antigen-binding portion is a monoclonal antibody. In some embodiments, the antibody or its antigen-binding portion is a humanized antibody or a chimeric antibody. In some embodiments, the antibody or its antigen-binding portion is an antibody fragment that specifically binds to human αvβ8. In some embodiments, the antibody or its antigen-binding portion comprises a sequence selected from the group consisting of: (a) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:152 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:153; (b) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:154 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:155; (c) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:156 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:157; (d) a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:158 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:159; (e) a sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:152 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:159; (f) A VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:160 and a VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:161; (g) A VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:164 and a VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:165; (g) A VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:166 and a VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:167; and (h) A VL sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:162 and a VH sequence with at least 95% sequence identity to the amino acid sequence of SEQ ID NO:163.

[0012] In some embodiments, the antibody or its antigen-binding portion comprises a sequence selected from the group consisting of: (a) a VL sequence comprising the amino acid sequence shown in SEQ ID NO:152 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:153; (b) a VL sequence comprising the amino acid sequence shown in SEQ ID NO:154 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:155; (c) a VL sequence comprising the amino acid sequence shown in SEQ ID NO:156 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:157; (d) a VL sequence comprising the amino acid sequence shown in SEQ ID NO:158 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:159; (e) a VL sequence comprising the amino acid sequence shown in SEQ ID NO:160 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:161; (f) a VL sequence comprising the amino acid sequence shown in SEQ ID NO:164. (g) A VH sequence containing the amino acid sequence shown in SEQ ID NO:165; (h) A VL sequence containing the amino acid sequence shown in SEQ ID NO:166 and a VH sequence containing the amino acid sequence shown in SEQ ID NO:167; (h) A VL sequence containing the amino acid sequence shown in SEQ ID NO:150 and a VH sequence containing the amino acid sequence shown in SEQ ID NO:151; and (i) A VL sequence containing the amino acid sequence shown in SEQ ID NO:162 and a VH sequence containing the amino acid sequence shown in SEQ ID NO:163.

[0013] In some embodiments, the antibody or its antigen-binding portion is a full-length antibody of the IgG1 isotype. In some embodiments, the antibody or its antigen-binding portion comprises a variant IgG1 Fc region having reduced effector function. In some embodiments, the Fc region comprises amino acid substitutions L234A / L235A according to the EU index number of Kabat. In some embodiments, the Fc region comprises amino acid substitutions P329G according to the EU index number of Kabat. In some embodiments, the antibody binds to human αvβ8 with a KD of 1 nM or less as measured by surface plasmon resonance. In some embodiments, the antibody or its antigen-binding portion comprises: (a) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:201 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:200; or (b) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:203 and a light chain exhibiting 95% sequence identity with the amino acid sequence shown in SEQ ID NO:202. In some embodiments, the antibody or its antigen-binding portion comprises: (a) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:201 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:200; or (b) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:221 and a light chain exhibiting 95% sequence identity with the amino acid sequence shown in SEQ ID NO:202. In some embodiments, the antibody or its antigen-binding portion comprises: (a) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:220 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:200; or (b) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:203 and a light chain exhibiting 95% sequence identity with the amino acid sequence shown in SEQ ID NO:202. In some embodiments, the antibody or its antigen-binding portion comprises: (a) the heavy chain of SEQ ID NO:201 and the light chain of SEQ ID NO:200; or (b) the heavy chain of SEQ ID NO:203 and the light chain of SEQ ID NO:202.In some embodiments, the antibody or its antigen-binding portion comprises: (a) the heavy chain of SEQ ID NO:220 and the light chain of SEQ ID NO:200; or (b) the heavy chain of SEQ ID NO:221 and the light chain of SEQ ID NO:202.

[0014] This document further provides an isolated nucleic acid encoding any of the aforementioned antibodies. This document further provides a vector comprising the nucleic acid. This document further provides a host cell comprising the nucleic acid or the vector. This document further provides a method for generating an antibody that binds to human αvβ8, the method comprising culturing the host cell under conditions suitable for the expression of the antibody. In some embodiments, the method further comprises recovering the antibody from the host cell. This document further provides an antibody generated by this method.

[0015] This article further provides a pharmaceutical composition comprising any of the aforementioned antibodies or their antigen-binding portion and a pharmaceutical carrier.

[0016] This article further provides a method for treating cancer in an individual who requires such treatment, the method comprising administering to the individual an effective amount of one of the aforementioned antibodies or its antigen-binding portion, or the aforementioned pharmaceutical composition.

[0017] This article further provides a method for treating cancer in an individual who requires such treatment, the method comprising administering: (a) an effective amount of one of the aforementioned antibodies or an antigen-binding fragment thereof or the aforementioned pharmaceutical composition, and (b) an effective amount of a PD-1 axis antagonist. In some embodiments, the PD-1 axis antagonist is an anti-PD-L1 antibody. In some embodiments, the PD-1 axis antagonist is atezolizumab. In some embodiments, the method further comprises assessing the level of αvβ6 expressed by the cancer.

[0018] In some embodiments of the aforementioned method, the cancer is selected from the list of ovarian cancer, triple-negative breast cancer, non-small cell lung cancer, colorectal cancer, bile duct cancer, endometrial cancer, renal papillary carcinoma, and bladder cancer. Attached Figure Description

[0019] Representative embodiments of the present invention are disclosed with reference to the following drawings. It should be understood that the depicted embodiments are not limited to the precise details shown.

[0020] Figure 1 shows a schematic diagram of the screening method for αVβ8 antibodies.

[0021] Figures 2A and 2B show the IC50 of rabbit αvβ8 antibody compared to C6D4 mIgG2a LALALG control.

[0022] Figures 3A to 3C show the binding patterns of rabbit αvβ8 antibodies (rb.avb8-65, Figure 3A, and rb.avb8-92, Figure 3B) against cynomolgus monkey, human, or mouse αvβ8, compared to the control antibody (hu.C6D4, Figure 3C). Figure 3D shows the binding patterns of rabbit αvβ8 antibodies (rb.avb8-65, middle; and rb.avb8-92, right) in the presence of two metal ions (e.g., divalent cations), compared to the control antibody (C6D4).

[0023] Figures 4A and 4B show the sequence alignment of rabbit and humanized αvβ8 antibodies. CDR sequences are underlined and identified according to the Kabat numbering system. Figure 4A shows the sequence alignment of the light chain variable region. Figure 4B shows the sequence alignment of the heavy chain variable region.

[0024] Figures 5A through 5D show the results of cryo-EM, highlighting the interaction between rb.αvβ8-65 (anti-αvβ8 integrin antibody) and αvβ8 integrin (Figures 5A and 5B (rotated 90° relative to Figure 5A)) and the interaction between latent TGFβ1 (L-TGFβ1) and αvβ8 integrin (Figures 5C and 5D (rotated 90° relative to Figure 5A)). Figures 5A through 5D show that Fab65 binding blocks the interaction between latent TGFβ1 and αvβ8. Figures 5C and 5D show the location of the L-TGFb1 RGDLXXI / L motif inserted into the interface between the αV and β8 subunits. Figures 5A and 5B show that FAB65 occupies a similar position to L-TGFb1 relative to αvβ8, thus effectively blocking the L-TGFb1 binding site.

[0025] Figures 5E to 5H are enlarged views of the interface between rb.αvβ8-65 (anti-αvβ8 integrin antibody) and αvβ8 integrin (Figures 5E and 5G), and the interface between L-TGFβ1 and αvβ8 integrin (Figures 5F and 5H). Several residues in αvβ8 integrin that interact with rb.αvβ8-65 or L-TGFβ1 are highlighted. Figures 5E and 5G show that residues F177 and D218 of αV form a specific contact with CDRH2; K119, Q120, E121, and D148 of αV form a specific contact with CDRL1; N219 of β8 forms a specific contact with CDRH2; and R164 of β8 forms a specific contact with CDRL1.

[0026] Figures 5I to 5K are enlarged views of the interface between rb.αvβ8-65 (anti-αvβ8 integrin antibody) and αvβ8 integrin, showing the salt bridge formed between hu.αvβ8-65 (anti-αvβ8 integrin antibody) and αvβ8 integrin. Specific residues are highlighted.

[0027] Figure 5L shows the sequences of the αV and β8 subunits of αvβ8 and the EM structure of the interface between rb.αvβ8-65 (anti-αvβ8 integrin antibody) and αvβ8 integrin. Residues located within 5 Å of rb.αvβ8-65 are highlighted in the sequence of the αV subunits of αvβ8 (i.e., R115, 118M, 119K, 120Q, 121E, 123E, 147I, 148D, 149A, 150D, 154F, 177F, 178Y, 180Q, 212T, 213A, 214Q, 215A, and 218D) and the β8 subunits (i.e., 118H, 119N, 122E, 158I, 159S, 160I, 164R, 166H, 169C, 170S, 171D, 172Y, 206G, 207N, and 208I) and depicted as putative αvβ8 epitopes bound by Fab65.

[0028] Figure 5M illustrates the isotype specificity assessment of Fab65 binding to αV compared to other αV integrins. This alignment is based on low-sequence retention of the αV epitope. Fab65 is specific to αV and should not bind to other α integrins.

[0029] Figure 5N illustrates the isotype specificity assessment of the binding between Fab65 and β8 compared to other β8 integrins. This alignment is based on low-sequence retention of the β8 epitope. Fab65 is specific for β8 and should not bind to other β integrins. The backbone of Fab65 CDRL3 Gly95a forms hydrogen bonds with the backbone of αvβ8 Ser159 (Lys in αvβ6). Anti-selection against αvβ1, αvβ3, αvβ5, and αvβ6 is performed during antibody discovery.

[0030] Figures 6A and 6B show the binding diagrams of two separate experiments on the binding of humanized αvβ8 antibody (rb.aVb8-65) to cynomolgus monkey (cynomolgus monkey), human (hu) or mouse (mu) antigens.

[0031] Figures 7A and 7B show the binding diagrams of two separate experiments on the binding of humanized αvβ8 antibody (rb.aVb8-92) to cynomolgus monkey, human (hu), or mouse (mu) antigens.

[0032] Figure 8 shows surface plasmon sensing maps of the binding of anti-αvβ8-65 to recombinant αvβ8 protein from humans, cynomolgus monkeys, rats, and mice. The recombinant αvβ8 concentrations (from bottom to top) for each sensing map are 3.7 nM, 11.1 nM, 33.3 nM, and 100 nM, respectively. The solid black lines represent curves fitted using a 1:1 binding model, and the colored lines represent measured data.

[0033] Figures 9A and 9B show surface plasmon resonance (SPR) maps of the binding of αvβ8 to hC6D4 and hADWA11. Figure 9A shows the binding of hC6D4 (left) and αvβ8-65 (right) to human αvβ8 protein. The solid black line represents the curve fitted using a 1:1 binding model, and the colored lines represent the measured data. Recombinant αvβ8 concentrations (from bottom to top) were 0.8, 4, 20, and 100 nM. Format: Protein A capture, 37 °C, 100 µl / min, HBS-P pH 7.2, 0.5 mM CaCl2. Figure 9B shows the SPR maps of the binding of αvβ8-65 (RO7566802; left) and hADWA11-2.4 (right) to human αvβ8 protein. The solid black line represents the curve fitted using a 1:1 binding model, and the colored lines represent the measured data. The recombinant αvβ8 concentrations (from bottom to top) were 3.7, 11.1, 33.3, and 100 nM. Form: Protein A capture, 37 °C, 100 µl / min, HBS-P pH 7.2, 0.5 mM CaCl2.

[0034] Figure 10 shows surface plasmon resonance sensing maps of the binding of αvβ8-65 (RO7566802; left), hADWA11-2.4 (middle), and hC6D4 (right) to human αvβ8 protein. The solid black line represents the curve fitted using a 1:1 binding model, and the colored lines represent the measured data. The recombinant αvβ8 concentrations (from bottom to top) were 0.4, 2, 10, and 50 nM. Formulation: anti-mouse Fc capture, 37 °C, 100 µl / min, HBS-P pH 7.2, 0.5 mM CaCl2.

[0035] Figures 11A through 11D illustrate four experiments describing the ability of αvβ8 antibodies to block the binding of αvβ8 to TGFβ1 or TGFβ3. Figure 11A shows Experiment 1, which is a co-culture assay of LN-229 with 3T3-Nano-human GARP / LTGFb1 (WT). Figure 11B shows Experiment 2, which is a co-culture assay of LN-229 with 3T3-Nano-human GARP / LTGFb1 (WT). Figure 11C shows Experiment 3, which is a co-culture assay of LN-229 with 3T3-Nano-human GARP / LTGFb1 (non-releasable (NR)). Figure 11D shows Experiment 4, which is a co-culture assay of LN-229 with 3T3-Nano-human GARP / LTGFb3 (WT).

[0036] Figures 12A to 12H show the competition assays between αvβ8 and ADWA11 or C6D4 in EMT6 or HCC1159 cells. Figure 12A shows the competition assay between αvβ8 and ADWA11 (1 μg / mL) in the EMT6 cell line. Figure 12B shows the competition assay between αvβ8 and ADWA11 (40 μg / mL) in the EMT6 cell line. Figure 12C shows the competition assay between αvβ8 and C6D4 (1 μg / mL) in the EMT6 cell line. Figure 12D shows the competition assay between αvβ8 and C6D4 (40 μg / mL) in the EMT6 cell line. Figure 12E shows the competition assay between αvβ8 and ADWA11 (1 μg / mL) in the HCC1159 cell line. Figure 12F shows the competition assay between αvβ8 and ADWA11 (10 μg / mL) in the HCC1159 cell line. Figure 12G shows the competition assay between αvβ8 and C6D4 (1 μg / mL) in the HCC1159 cell line. Figure 12H shows the competition assay between αvβ8 and C6D4 (10 μg / mL) in the HCC1159 cell line.

[0037] Figure 13 shows the pharmacokinetics of the humanized αvβ8 antibody in SCID mice.

[0038] Figure 14 shows the pharmacokinetics of the humanized αvβ8 antibody in cynomolgus monkeys.

[0039] Figure 15 shows the pharmacokinetics of the humanized αvβ8 antibody in CD-1 mice at two doses.

[0040] Figures 16A and 16B show the percentage of pSMAD / SMAD in tumors and lymph nodes (LNs).

[0041] Figure 17 shows tumor volumes in mice that received one or both antibodies. GP120 antibody was used as a negative control. aPDL1 antibody (mu.C6D4) was administered alone or in combination with rb.aVb8-65 (c65) or rb.aVb8-92 (c92). Complete response (CR) is shown as a percentage.

[0042] Figures 18A and 18B show tumor volumes in mice of the EMT6 mouse model that received one or two antibodies. GP120 antibody was used as a negative control. aPDL1 antibody (mu.C6D4) was administered alone or in combination with hu.ADWA11, rb.aVb8-65 (c65), hu.aVb8-65, or hu.aVb8-92 (c92). Complete response (CR) is shown as a percentage.

[0043] Figures 19A and 19B show the calculation of complete response (CR) for the combination of anti-PD-L1 and ADWA11 compared to the combination of anti-PD-L1 and αvβ8-65. Figure 19A shows the percentage of CR for anti-PD-L1 + ADWA11 compared to anti-PD-L1 + αvβ8-65. Figure 19B shows the percentage of CR for anti-PD-L1 + ADWA11, a direct comparison, compared to anti-PD-L1 + αvβ8-65. Each point represents one study with 10 mice in each treatment group.

[0044] Figures 20A and 20B show tumor volumes in mice of the MC38 mouse model that received one or two antibodies. GP120 antibody was used as a negative control. aPDL1 antibody (mu.C6D4) was administered alone or in combination with hu.ADWA11, rb.aVb8-65 (c65), hu.aVB8-65, and hu.aVb8-92. Complete response (CR) is shown as a percentage.

[0045] Figure 21 shows the antitumor activity of Ch anti-αvβ8-65 and anti-αvβ8-ADWA11 in combination with anti-PD-L1 compared with isotype control and anti-PD-L1 alone.

[0046] Figures 22A, 22B, and 22C illustrate the comparison of the antitumor activity of Ch anti-αvβ8-65 and anti-αvβ8-ADWA11 by the following: (Figure 22A) the percentage of CR in the combination group compared to the anti-PD-L1 group alone across several studies; (Figure 22B) a direct comparison (i.e., using both molecules in the same study) of the percentage of CR in the combination group compared to the anti-PD-L1 group alone; and (Figure 22C) the percentage of mice with tumor regression at day 14, measured in studies testing both molecules. Detailed Implementation

[0047] I. Definition

[0048] For the purposes of this document, a “recipient human frame” is a frame as defined below, comprising the amino acid sequence of a light chain variable domain (VL) frame or a heavy chain variable domain (VH) frame derived from the human immunoglobulin frame or the human consensus frame. A recipient human frame “derived from” the human immunoglobulin frame or the human consensus frame may contain the same amino acid sequence as said human immunoglobulin frame or human consensus frame, or it may contain amino acid sequence variations. In some aspects, the number of amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some aspects, the VL recipient human frame is sequence-identical to the VL human immunoglobulin frame sequence or the human consensus frame sequence.

[0049] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise stated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of molecule X for its partner Y can generally be determined by the dissociation constant (K). D Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary methods for measuring binding affinity are described below.

[0050] The term "affinity maturation" refers to an antibody that has one or more variations in one or more complementarity-determining regions (CDRs) that result in an improved affinity of the antibody for the antigen compared to a parent antibody that does not have such variations.

[0051] The terms "anti-αvβ8 antibody" and "antibody that binds to αvβ8" refer to an antibody that binds to αvβ8 with sufficient affinity, making it usable as a diagnostic and / or therapeutic agent targeting αvβ8. In one respect, as measured, for example by surface plasmon resonance (SPR), anti-αvβ8 antibodies bind to unrelated, non-αvβ8 proteins to less than approximately 10% of the amount bound to αvβ8. In other respects, the dissociation constant (Ki) of antibodies binding to αvβ8... D The values ​​are ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10-9 Up to 10 -13 M). When the antibody K D When the concentration is 1 μM or lower, the antibody is said to "specifically bind" to αvβ8. In some respects, the anti-αvβ8 antibody binds to epitopes of αvβ8 that are conserved across different species of αvβ8. In the disclosed embodiments, the anti-αvβ8 antibody blocks the binding of αvβ8 to TGFβ1, including latent TGFβ1 and mature TGFβ1. As used herein, "αvβ8" and αvβ6 may or may not be written with Greek letters (e.g., avB8, αvB8, αvB6, or avβ6). Similarly, TGFβ1 may be written as "TGFB1" and TGFβ3 may be written as "TGFB3".

[0052] The term “antibody” in this article is used in the broadest sense and covers a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0053] "Antibody fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen bound by the complete antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bisomatic antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv and scFab); single-domain antibodies (dAb); and multispecific antibodies formed from antibody fragments. For a review of certain antibody fragments, see Holliger and Hudson, Nature Biotechnology 23:1126-1136 (2005). The fragments of anti-αvβ8 antibodies disclosed herein can be selected based on their ability to bind to αvβ8 and block the binding of αvβ8 to TGFβ1 and / or TGFβ3.

[0054] As used herein, the term "epitope" refers to a site on an antigen that binds to an antibody. Epitopes can be formed from a continuous stretch of amino acids (linear epitopes) or contain discontinuous amino acids (conformal epitopes), for example, due to the folding of the antigen, i.e., spatial proximity through tertiary folding of the protein antigen. Linear epitopes typically remain bound to anti-αvβ8 antibodies after the protein antigen is exposed to a denaturing agent, while conformational epitopes are typically destroyed after treatment with a denaturing agent. Epitopes contain at least 3, at least 4, at least 5, at least 6, at least 7, or 8 to 10 amino acids in a unique stereoconformation.

[0055] For the purposes of this article, “atezolizumab” is an Fc-engineered, humanized, non-glycosylated IgG1κ immunoglobulin that binds to PD-L1 and contains the heavy chain sequence of SEQ ID NO:218 and the light chain sequence of SEQ ID NO:219. Using the EU numbering of the amino acid residues in the Fc region, atezolizumab contains a single amino acid substitution (asparagine replaced by alanine) at position 297 of the heavy chain (N297A), which results in minimal binding of the non-glycosylated antibody to the Fc receptor. Atezolizumab is also described in WHO Drug Information (International Nonproprietary Names (INN)), recommended INN: List 112, Volume 28, Issue 4, 2014, pp. 488-489 and WHO Drug Information (International Nonproprietary Names (INN)), recommended INN: List 74, Volume 29, Issue 3, 2015, p. 387.

[0056] Antibodies that bind to a specific epitope, such as the αvβ8 epitope (i.e., those antibodies that bind to the same epitope), can be screened using methods conventional in the art. These methods include, for example, but are not limited to, alanine scanning, Western blotting (see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

[0057] Antigen structure-based antibody profiling (ASAP) (also known as modification-assisted profiling (MAP)) allows binning of a large number of monoclonal antibodies based on the binding profiles of each antibody from a large number of monoclonal antibodies that specifically bind to αvβ8 to a chemically or enzymatically modified antigen surface (see, for example, US 2004 / 0101920). Antibodies in each group bind to the same epitope, which can be a unique epitope that is significantly different from or partially overlaps with the epitope represented by another group.

[0058] Competitive binding can also be used to readily determine whether an antibody binds to the same epitope as the antigen or competes with a reference antibody for binding. For example, "antibody binding to the same epitope" in a competitive assay refers to an antibody that blocks 50% or more of the binding of the reference anti-αvβ8 antibody to its antigen, while conversely, the reference antibody blocks 50% or more of the binding of the antibody to its antigen in a competitive assay. Similarly, for example, to determine whether an antibody binds to the same epitope as a reference anti-αvβ8 antibody, the reference antibody is saturated with αvβ8. After removing excess reference anti-αvβ8 antibody, the binding ability of the anti-αvβ8 antibody in question to αvβ8 is evaluated. If the anti-αvβ8 antibody is able to bind to αvβ8 after saturation binding with the reference anti-αvβ8 antibody, it can be concluded that the anti-αvβ8 antibody in question binds to a different epitope than the reference anti-αvβ8 antibody. However, if the anti-αvβ8 antibody in question fails to bind to αvβ8 after saturation binding with a reference anti-αvβ8 antibody, the anti-αvβ8 antibody in question may bind to the same epitope as the reference anti-αvβ8 antibody. To confirm whether the antibody in question binds to the same epitope or is blocked due to steric hindrance, standard experiments can be used (e.g., peptide mutation and binding assays using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art). This assay should be performed in two settings, i.e., both antibodies are saturated antibodies. If, in both settings, only the first (saturated) antibody is able to bind to αvβ8, it can be concluded that the anti-αvβ8 antibody in question and the reference anti-αvβ8 antibody compete for αvβ8 binding.

[0059] In some respects, such as those measured in competitive binding assays, if one antibody inhibits the binding of another antibody by 1, 5, 10, 20, or 100 times at least 50%, at least 75%, at least 90%, or even 99% or higher, the two antibodies are considered to bind to the same or overlapping epitopes (see, for example, Junghans et al., Cancer Res. 50 (1990) 1495-1502).

[0060] In some respects, if virtually all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of another antibody, then the two antibodies are considered to bind to the same epitope. If only a subset of amino acid mutations that reduce or eliminate the binding of one antibody reduces or eliminates the binding of another antibody, then the two antibodies are considered to have “overlapping epitopes.”

[0061] The term "cancer" refers to a disease caused by the uncontrolled division of abnormal cells in a part of the body. In some embodiments, cancer is GBM, low-grade glioma, pheochromocytoma, adrenal carcinoma, mesothelioma, uveal melanoma, sarcoma, melanoma, cholangiocarcinoma, clear cell renal cell carcinoma (ccRCC), thymoma, papillary RCC, germ cell carcinoma, ovarian cancer, diffuse large B-cell lymphoma (DLBCL), or endometrial cancer. In a specific embodiment, cancer is ovarian cancer. In a specific embodiment, cancer is ccRCC. In an embodiment, cancer is cancer that exhibits increased expression of αvβ8 and decreased expression of αvβ6 compared to normal tissue. In an embodiment, cancers with a higher αvβ8 to αvβ6 expression ratio than that of comparable normal tissue, such as ovarian cancer, are selected for treatment. Because av integrins are components of both avb8 and avb6, the expression of b8 and / or b6 integrins can be used as a substitute for avb8 and / or avb6 expression. Cancer may be locally advanced or metastatic. In some cases, cancer is locally advanced. In others, cancer is metastatic. In some cases, cancer may be unresectable (e.g., unresectable locally advanced or metastatic cancer).

[0062] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a specific source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0063] An antibody's "class" refers to the type of constant structural domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. In some respects, the antibody belongs to the IgG1 isotype. In some respects, the antibody belongs to the IgG1 isotype but has P329G, L234A, and L235A mutations to reduce Fc region effector function. In other respects, the antibody is the IgG2 isotype. In some respects, the antibody is the IgG4 isotype with an S228P mutation in the hinge region to improve the stability of the IgG4 antibody. For example, a disclosed anti-avb8 antibody may be an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 isotype. Specifically, anti-avb8 antibodies belong to the IgG1 isotype and possess P329G, L234A, and L235A mutations to reduce Fc region effector function. Specifically, anti-avb8 antibodies belong to the IgG2 isotype. Specifically, anti-avb8 antibodies belong to the IgG4 isotype and possess the S228P mutation in the hinge region to improve the stability of IgG4 antibodies. The constant heavy chain domains corresponding to different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively. The light chain of an antibody, based on the amino acid sequence of its constant domain, can be classified into one of two types: kappa (κ) and lamuda (λ).

[0064] As used in this application, the term "constant region derived from human origin" or "human constant region" refers to the constant heavy chain region and / or constant light chain region κ or λ region of a human antibody belonging to the subclass IgG1, IgG2, IgG3, or IgG4. Such constant regions are well known in the art and are described, for example, by the following: Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991) (see also, for example, Johnson, G. and Wu, TT, Nucleic Acids Res. 28 (2000) 214-218; Kabat, EA et al., Proc. Natl. Acad. Sci. USA 72 (1975) 2785-2788). Unless otherwise specified herein, the amino acid residues in the constant region are numbered according to the EU numbering system, also known as Kabat's EU index, as described in Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0065] "Effective functions" refer to those biological activities attributable to the Fc region of an antibody that vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

[0066] The “effective amount” of a drug (such as a pharmaceutical composition) refers to the amount that effectively achieves the desired therapeutic or preventative outcome within the required dose and time period.

[0067] The term "Fc region" used herein is used to define the C-terminal region of an immunoglobulin heavy chain that comprises at least a portion of the constant region. This term includes both native sequence Fc regions and variant Fc regions. In one aspect, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the C-terminus of the heavy chain. However, antibodies produced by host cells can undergo post-translational cleavage of one or more (particularly one or two) amino acids from the C-terminus of the heavy chain. Therefore, antibodies produced by host cells by expressing a specific nucleic acid molecule encoding the full-length heavy chain can comprise the full-length heavy chain, or the antibody can comprise a cleaved variant of the full-length heavy chain. This could be the case where the last two C-terminal amino acids of the heavy chain are glycine (G446) and lysine (K447, EU numbering system). Therefore, the C-terminal lysine (Lys447) or the C-terminal glycine (Gly446) and lysine (Lys447) of the Fc region may or may not be present. In one aspect, a heavy chain comprising an Fc region as specified herein is included in an antibody according to the invention, said heavy chain comprising an additional C-terminal glycine-lysine dipeptide (G446 and K447, EU numbering system). In another aspect, a heavy chain comprising an Fc region as specified herein is included in an antibody according to the invention, said heavy chain comprising an additional C-terminal glycine residue (G446, according to EU index number). Unless otherwise specified herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In some embodiments, the antibody described herein comprises the Fc region of SEQ ID NO: 201. In some embodiments, the antibody described herein comprises the Fc region of SEQ ID NO: 220. In some embodiments, the antibody described herein comprises the Fc region of SEQ ID NO: 203. In some embodiments, the antibody described herein comprises the Fc region of SEQ ID NO:221. In some embodiments, the antibody described herein comprises the Fc region according to SEQ ID NO:204.

[0068] "Frame" or "FR" refers to the variable domain residues other than the complementarity-determining region (CDR). A variable domain FR typically consists of four FR domains: FR1, FR2, FR3, and FR4. Therefore, the CDR and FR sequences usually appear in the VH (or VL) as follows: FR1-CDR-H1(CDR-L1)-FR2-CDR-H2(CDR-L2)-FR3-CDR-H3(CDR-L3)-FR4.

[0069] The terms “full-length antibody,” “intact antibody,” and “all antibody” are used interchangeably herein to refer to antibodies having a structure substantially similar to that of natural antibodies or having a heavy chain containing an Fc region as defined herein.

[0070] The terms “host cell,” “host cell line,” and “host cell culture” are used interchangeably and refer to cells in which exogenous nucleic acids have been introduced, including progeny cells. Host cells include “transformations” and “transformed cells,” which include primary transformed cells and progeny derived from those primary transformed cells, regardless of passage number. Progeny cells may not have completely identical nucleic acid contents to the parent cells and may contain mutations. This article includes mutant progeny with the same function or biological activity as those screened or selected from the original transformed cells.

[0071] A "human antibody" is an antibody whose amino acid sequence corresponds to that of an antibody produced by a human or human cell, or to a non-human antibody derived from a complete library of human antibodies or other antibody-encoding sequences. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues.

[0072] "Humanized" antibodies refer to chimeric antibodies that contain amino acid residues from a non-human CDR and amino acid residues from a human FR. In some respects, humanized antibodies will contain substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDRs correspond to the CDRs of the non-human antibody, and all or substantially all of the FRs correspond to the FRs of the human antibody. Humanized antibodies may optionally contain at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0073] As used herein, the term "hypervariant region" or "HVR" refers to the regions within the variable domain of an antibody that are hypervariable in sequence and determine antigen-binding specificity, such as the "complementarity-determining region" ("CDR").

[0074] Typically, an antibody contains six CDRs; three in the VH region (CDR-H1, CDR-H2, CDR-H3) and three in the VL region (CDR-L1, CDR-L2, CDR-L3). In this document, exemplary CDRs include:

[0075] (a) Hypervariable rings present at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917(1987));

[0076] (b) CDRs appearing at the following amino acid residues: 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)); and

[0077] (c) Antigen contact sites appearing at the following amino acid residues: 27c to 36 (L1), 46 to 55 (L2), 89 to 96 (L3), 30 to 35b (H1), 47 to 58 (H2), and 93 to 101 (H3) (MacCallum et al., J. Mol. Biol. 262: 732-745 (1996)).

[0078] Unless otherwise specified, the CDR is determined according to the method described by Kabat et al., citing above. Those skilled in the art will understand that the CDR name may also be determined according to Chothia, McCallum, or any other scientifically accepted naming system, citing above.

[0079] The “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (such as cows, sheep, cats, dogs, and horses), primates (such as humans and non-human primates, such as monkeys), rabbits, and rodents (such as mice and rats). In some respects, the individual or subject is a human.

[0080] "Isolated" antibodies are antibodies that have been separated from components of their natural environment. In some respects, antibodies are purified to a purity greater than 95% or 99%, as determined by methods such as electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods used to assess antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0081] The terms "nucleic acid molecule" or "polynucleotide" include any compound and / or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e., deoxyribose or ribose), and a phosphate group. Typically, nucleic acid molecules are described by a base sequence, where the bases represent the primary structure (linear structure) of the nucleic acid molecule. Base sequences are typically represented from 5' to 3'. In this document, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA) (including, for example, complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA) (particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and mixed polymers containing two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Additionally, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone bonds or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct in vitro and / or in vivo (e.g., in a host or patient) expression of antibodies used in this invention. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors can be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and / or the expression of the encoding molecule, enabling the mRNA to be injected into a subject to generate in vivo antibodies (see, for example, Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi:10.1038 / nm.4356 or EP 2 101823 B1).

[0082] "Isolated" nucleic acids refer to nucleic acid molecules that have been separated from components of their natural environment. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain nucleic acid molecules, but which are located outside the chromosome or at a chromosomal location different from their natural chromosomal location.

[0083] "Isolated nucleic acid encoding anti-αvβ8 antibody" refers to one or more nucleic acid molecules encoding the heavy and light chains (or fragments thereof) of anti-αvβ8 antibody, including such nucleic acid molecules in a single vector or in separate vectors, and such nucleic acid molecules present at one or more locations in the host cell.

[0084] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al., Sequences of Immunological Interest. 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is typically used (e.g., the EU index reported by Kabat et al. above). The “EU index described by Kabat” refers to the residue numbering of human IgG1 EU antibodies.

[0085] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, i.e., individual antibodies comprising this group are identical and / or bind to the same epitopes, except for possible variant antibodies (e.g., those containing naturally occurring mutations or generated during the production of the monoclonal antibody formulation, such variants typically exist in small quantities). In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier "monoclonal" indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies according to the invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0086] "Naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. Naked antibodies may be present in pharmaceutical compositions.

[0087] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 Daltons, composed of two identical light chains and two identical heavy chains bonded by disulfides. Each heavy chain has a variable domain (VH), also called a variable heavy chain domain or heavy chain variable region, from the N-terminus to the C-terminus, followed by three constant heavy chain domains (CH1, CH2, and CH3). Similarly, each light chain has a variable domain (VL), also called a variable light chain domain or light chain variable region, from the N-terminus to the C-terminus, followed by a constant light chain (CL) domain.

[0088] The term "packaging insert" is used to refer to the instruction leaflet typically included in the commercial packaging of a therapeutic product, which contains information concerning the indications, usage, dosage, administration, combination therapy, contraindications, and / or warnings related to the use of such therapeutic products.

[0089] The "percentage of amino acid sequence identity (%)" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to those in the reference polypeptide sequence after aligning the candidate sequence with the reference polypeptide sequence and introducing vacancies (if necessary) to achieve the maximum percentage of sequence identity, and for alignment purposes without considering any conserved substitutions as part of sequence identity. Alignment used to determine the percentage of amino acid sequence identity can be performed in various ways within the scope of the art, such as using publicly available computer software, such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. Alternatively, the sequence comparison computer program ALIGN-2 can be used to generate the percentage of identity values. The ALIGN-2 sequence comparison computer program was written by Genentech, and the source code has been submitted with the user documentation to the US Copyright Office, Washington DC, 20559, registered under US Copyright Registry No. TXU510087 and described in WO 2001 / 007611.

[0090] Unless otherwise indicated, for the purposes of this article, the ggsearch program of FASTA package version 36.3.8c or later was used to generate the percentage of amino acid sequence identity using the BLOSUM50 comparison matrix. The FASTA package was created by WR Pearson and DJ Lipman (1988), “Improved Tools for Biological Sequence Analysis”, PNAS 85:2444-2448; WR Pearson (1996) “Effective proteins sequence comparison” Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36 and is publicly available at www.fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml or www.ebi.ac.uk / Tools / sss / fasta. Alternatively, sequences can be compared using a public server accessible at fasta.bioch.virginia.edu / fasta_www2 / index.cgi, using the ggsearch (global protein: protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup = 2) to ensure global rather than local alignment is performed. The percentage of amino acid identity is given in the output alignment header.

[0091] The terms “pharmaceutical composition” or “pharmaceutical formulation” refer to a formulation in which the active ingredient contained therein is in a biologically effective form and does not contain any additional components that would have unacceptable toxicity to a subject to whom the pharmaceutical composition will be administered.

[0092] "Pharmaceutical carrier" refers to a component in a pharmaceutical composition or preparation other than the active ingredient, which is non-toxic to the subject. Pharmaceutical carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0093] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction between a PD-1 axis binding partner and one or more of its binding partners to eliminate T cell dysfunction caused by signal transduction along the PD-1 signaling axis, resulting in the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, and / or target cell killing). As used herein, PD-1 axis binding antagonists include PD-L1 binding antagonists, PD-1 binding antagonists, and PD-L2 binding antagonists. In some cases, PD-1 axis binding antagonists include either PD-L1 binding antagonists or PD-1 binding antagonists. In a preferred aspect, the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0094] The term "PD-L1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 and / or B7-1). In some cases, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 with its binding partners. In one specific aspect, a PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some cases, PD-L1 binding antagonists include anti-PD-L1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signal transduction resulting from the interaction of PD-L1 with one or more of its binding partners (such as PD-1 and / or B7-1). In one scenario, a PD-L1 binding antagonist reduces negative co-stimulatory signals mediated by PD-L1 signaling, either mediated by cell surface proteins expressed on T lymphocytes or by signal transduction mediated by PD-L1, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some cases, the PD-L1 binding antagonist binds to PD-L1. In some cases, the PD-L1 binding antagonist is an anti-PD-L1 antibody (e.g., an anti-PD-L1 antagonist antibody). Exemplary anti-PD-L1 Antagonist antibodies include atezolizumab, MDX-1105, MEDI4736 (durvalumab), MSB0010718C (avelumab), SHR-1316, CS1001, envafolimab, TQB2450, ZKAB001, LP-002, CX-072, IMC-001, KL-A167, APL-502, cosibelimab, lodapolimab, FAZ053, TG-1501, BGB-A333, BCD-135, AK-106, LDP, GR1405, HLX20, MSB2311, RC98, PDL-GEX, KD036, KY1003, and YBL-007. And HS-636. In some respects, the anti-PD-L1 antibody is atezolizumab, MDX-1105, MEDI4736 (dvorumab), or MSB0010718C (averimumab). In one specific respect, the PD-L1 binding antagonist is MDX-1105. In another specific respect, the PD-L1 binding antagonist is MEDI4736 (dvorumab). In yet another specific respect, the PD-L1 binding antagonist is MSB0010718C (averimumab).In other respects, the PD-L1 binding antagonist can be a small molecule, such as GS-4224, INCB086550, MAX-10181, INCB090244, CA-170, or ABSK041, which in some cases can be administered orally. Other exemplary PD-L1 binding antagonists include AVA-004, MT-6035, VXM10, LYN192, GB7003, and JS-003. In a preferred aspect, the PD-L1 binding antagonist is atezolizumab.

[0095] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling generated by the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. PD-1 (programmed cell death 1) is also referred to in the art as "programmed cell death 1," "PDCD1," "CD279," and "SLEB2." Exemplary human PD-1 is shown in UniProtKB / Swiss-Prot accession number Q15116. In some cases, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 with one or more of its binding partners. In one specific aspect, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies and their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling generated by the interaction of PD-1 with PD-L1 and / or PD-L2. In one case, a PD-1 binding antagonist reduces negative co-stimulatory signals mediated by PD-1 signaling, either mediated by or through cell surface proteins expressed on T lymphocytes, thereby reducing dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some cases, the PD-1 binding antagonist binds to PD-1. In some cases, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., an anti-PD-1 antagonist antibody). Exemplary anti-PD-1 antagonist antibodies include nivolumab, pembrolizumab, MEDI-0680, PDR001 (spartazolizumab), REGN2810 (cimiprizumab), BGB-108, palolizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dotalimab, rivanlimab, sazasanlimab, penamprizumab, CS1003, HLX10, SCT-I10A, cepalimumab, batitilimab, genolimab, and BI. 754091, nivolumab, YBL-006, BAT1306, HX008, bucrolimus, AMG404, CX-188, JTX-4014, 609A, Sym021, LZM009, F520, SG001, AM0001, ENUM 244C8, ENUM388D4, STI-1110, AK-103, and hAb21. In one specific aspect, the PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, the PD-1 binding antagonist is MK-3475 (pembrolizumab). In yet another specific aspect, the PD-1 binding antagonist is a PD-L2 fusion protein, such as AMP-224.In another specific aspect, the PD-1 binding antagonist is MED1-0680. In another specific aspect, the PD-1 binding antagonist is PDR001 (spartazolizumab). In another specific aspect, the PD-1 binding antagonist is REGN2810 (cimiprizumab). In another specific aspect, the PD-1 binding antagonist is BGB-108. In another specific aspect, the PD-1 binding antagonist is palolizumab. In another specific aspect, the PD-1 binding antagonist is camrelizumab. In another specific aspect, the PD-1 binding antagonist is sintilimab. In another specific aspect, the PD-1 binding antagonist is tislelizumab. In another specific aspect, the PD-1 binding antagonist is toripalimab. Other exemplary PD-1 binding antagonists include BION-004, CB201, AUNP-012, ADG104, and LBL-006.

[0096] The term "PD-L2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signaling generated by the interaction of PD-L2 with one or more binding partners, such as PD-1. PD-L2 (programmed cell death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2," "PDCD1LG2," "CD273," "B7-DC," "Btdc," and "PDL2." Exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot accession number Q9BQ51. In some cases, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 with one or more of its binding partners. In one specific aspect, a PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. Exemplary PD-L2 antagonists include anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, eliminate, or interfere with signaling generated by the interaction of PD-L2 with one or more of its binding partners (such as PD-1). In one aspect, the PD-L2 binding antagonist reduces negative co-stimulatory signaling mediated by or expressed by cell surface proteins on T lymphocytes via PD-L2 signaling, thereby alleviating dysfunction of dysfunctional T cells (e.g., enhancing effector responses to antigen recognition). In some aspects, the PD-L2 binding antagonist binds to PD-L2. In some aspects, the PD-L2 binding antagonist is an immunoadhesin. In other aspects, the PD-L2 binding antagonist is an anti-PD-L2 antagonist antibody.

[0097] The terms “programmed cell death ligand 1” and “PD-L1” refer herein to the natural sequence human PD-L1 polypeptide. The natural sequence PD-L1 polypeptide is provided under Uniprotocol accession number Q9NZQ7. For example, the natural sequence PD-L1 may have the amino acid sequence described in Uniprotocol accession number Q9NZQ7-1 (isomer 1). In another example, the natural sequence PD-L1 may have the amino acid sequence described in Uniprotocol accession number Q9NZQ7-2 (isomer 2). In yet another example, the natural sequence PD-L1 may have the amino acid sequence described in Uniprotocol accession number Q9NZQ7-3 (isomer 3). PD-L1 is also referred to in the art as “programmed cell death 1 ligand 1”, “PDCD1LG1”, “CD274”, “B7-H”, and “PDL1”.

[0098]

[0099] As used herein, “treatment” (and its grammatical variations such as treat or treating) refers to an attempt to alter the natural course of a disease in the treated individual and can be performed for prevention or may be performed during a clinicopathological process. The desired effects of treatment include, but are not limited to, preventing the onset or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the disease state, and alleviating or improving prognosis. In some aspects, the antibodies of this invention are used to delay the development of disease or slow its progression.

[0100] The term "patient" refers to a person of any age. In some embodiments, a patient is an adult.

[0101] The term "variable region" or "variable domain" refers to a domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable domains (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FRs) and three complementarity-determining regions (CDRs). (See, for example, Kindt et al., Kuby Immunology, p. 6) (WH Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen binding specificity. Furthermore, antibodies binding to a specific antigen can be separated using either the VH or VL domain from the antibody binding that antigen, to screen libraries containing complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0102] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures, as well as vectors incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of the nucleic acid to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0103] II. Compositions and Methods

[0104] In one aspect, this document provides antibodies or antigen-binding moieties thereof that specifically bind to αvβ8 (such as human αvβ8, mouse αvβ8, cynomolgus monkey αvβ8, and / or rabbit αvβ8) (i.e., anti-αvβ8 antibodies, synonymously referred to as αvβ8 antibodies). In some embodiments, the antibody or antigen-binding moieties thereof do not significantly (e.g., nonspecifically) bind to αvβ6 (e.g., in some embodiments, the antibody or antigen-binding moieties thereof bind to αvβ8 with an affinity at least 10 times higher than αvβ6 (such as an affinity for αvβ8 at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 1000 times, or higher than αvβ6). The antibodies and their antigen-binding portions described herein can be used to diagnose or treat cancers, such as cancers expressing αvβ8 (including cancers expressing high levels of αvβ8 and low levels of αvβ6, cancers expressing normal levels of αvβ8 and low levels of αvβ6, and cancers expressing normal levels of αvβ8 and normal levels of αvβ6). In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is renal papillary carcinoma. In one embodiment, the cancer is bladder cancer. The antibodies described herein can be specifically used to treat cancers expressing normal or higher levels of αvβ8 but lower levels of αvβ6. In some embodiments, cancer is defined as expressing an αvβ8 to αvβ6 ratio greater than the normal value for that tissue (such as approximately 1.1 times, approximately 1.2 times, approximately 1.3 times, approximately 1.4 times, approximately 1.5 times, approximately 1.6 times, approximately 1.7 times, approximately 1.8 times, approximately 1.9 times, approximately 2.0 times, approximately 2.5 times, approximately 3.0 times, approximately 3.5 times, approximately 4.0 times, approximately 5.0 times, approximately 10 times, approximately 15 times, approximately 20 times, approximately 25 times, approximately 30 times, approximately 35 times, approximately 40 times, approximately 45 times, approximately 50 times, approximately 60 times, approximately 70 times, approximately 80 times, approximately 90 times, approximately 100 times, approximately 200 times, approximately 300 times, approximately 400 times, approximately 500 times, approximately 600 times). Cancer at rates of approximately 700, 800, 900, 1000, or greater.Antibodies and their antigen-binding portions can also be used in combination with therapies containing PD-1 axis antagonists (such as PD-1 binding antagonists or PD-L1 binding antagonists, such as anti-PD-1 or anti-PD-L1 antibodies) to treat the cancer. In a preferred embodiment, the anti-PD-L1 antibody is atezolizumab.

[0105] A. Exemplary anti-αvβ8 antibody

[0106] In one aspect, the present invention provides an antibody or antigen-binding moiety thereof that binds to αvβ8. In one aspect, a separate antibody that binds to αvβ8 is provided. In one aspect, the present invention provides an antibody that specifically binds to αvβ8. In some aspects, the anti-αvβ8 antibody or its antigen-binding moiety exhibits at least one of the following properties: (a) at 1 nM or less K D Binds to human αvβ8 with 1 nM or less K D Binding to mouse αvβ8, and / or with 1 nM or less K D (a) binding to cynomolgus monkey αvβ8; (b) inhibiting αvβ8-mediated activation of human leucine-rich repeat protein 32 (LRRC32), LRRC33 and / or latent TGFβ-binding protein (LTBP) and / or its associated latent TGFβ1 (LTGFβ1) and TGFβ3; (c) blocking the binding of TGFβ peptide to αvβ8; and / or (d) binding to αvβ8 in the absence of divalent cations. In some embodiments, the K of the antibody... D Evaluation is performed by surface plasmon resonance. In some embodiments, the anti-αvβ8 antibody described herein or its antigen-binding moiety binds to αvβ8 with an affinity at least 10 times greater than that of αvβ6 (such as an affinity at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 750, 1000 or more times greater than that of αvβ6).

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-65.H1L1, as defined in Tables 1, 2, or 3. In another aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-65.H15L2, as defined in Tables 1, 2, or 3. In yet another aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-65.H15L2.QS, as defined in Tables 1, 2, or 3. In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-65.H15L2.NA, as defined in Tables 1, 2, or 3. In another aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-65.H15L2.NT, as defined in Tables 1, 2, or 3. In yet another aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-92.H1L1, as defined in Tables 1, 2, or 3. In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs of an antibody named Hu.aVb8-92.H13L1, which is defined according to CDRs in Table 1, Table 2, or Table 3.

[0113] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[0114] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:10; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:11; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:12.

[0115] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:13; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:14; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:15; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:16; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:17; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18.

[0116] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:19; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:20; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:21; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:22; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:23; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:24.

[0117] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:26; (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:27; (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO:28; (e) CDR-H2 comprising the amino acid sequence of SEQ ID NO:29; and (f) CDR-H3 comprising the amino acid sequence of SEQ ID NO:30.

[0118] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:31; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:32; (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:33; (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO:34; (e) CDR-H2 comprising the amino acid sequence of SEQ ID NO:35; and (f) CDR-H3 comprising the amino acid sequence of SEQ ID NO:36.

[0119] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:42.

[0120] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:43; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:44; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:45; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:47; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:48.

[0121] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54.

[0122] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:55; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:56; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:57; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:58; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:60.

[0123] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:61; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:62; (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:63; (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO:64; (e) CDR-H2 comprising the amino acid sequence of SEQ ID NO:65; and (f) CDR-H3 comprising the amino acid sequence of SEQ ID NO:66.

[0124] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:68; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:69; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:70; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:71; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:72.

[0125] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:73; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:74; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:75; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:76; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:77; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78.

[0126] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:79; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:80; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:81; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:82; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:83; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84.

[0127] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:85; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:86; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:87; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:88; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:89; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:90.

[0128] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:91; (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:92; (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:93; (d) CDR-H1 comprising the amino acid sequence of SEQ ID NO:94; (e) CDR-H2 comprising the amino acid sequence of SEQ ID NO:95; and (f) CDR-H3 comprising the amino acid sequence of SEQ ID NO:96.

[0129] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:98; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:99; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:100; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:101; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:102.

[0130] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:103; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:105; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:106; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:107; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:108.

[0131] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:109; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:110; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:111; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:112; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:113; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:114.

[0132] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:115; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:117; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:118; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:119; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:120.

[0133] In one aspect, the present invention provides an anti-αvβ8 antibody comprising at least one, at least two, at least three, at least four, at least five, or all six CDRs selected from: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:121; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:122; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:123; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:124; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:125; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:126.

[0134] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:150. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:152. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:154. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:156. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:158. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:160. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:162. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:164. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-L1, CDR-L2, and CDR-L3 from the variable light chain sequence shown in SEQ ID NO:166.

[0135] In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:151. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:153. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:155. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:157. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:159. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:161. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:163. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:165. In some embodiments, the antibody or its antigen-binding fragment comprises CDR-H1, CDR-H2, and CDR-H3 from the heavy chain variable region shown in SEQ ID NO:167.

[0136] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6.

[0137] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:10; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:11; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:12.

[0138] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:13; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:14; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:15; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:16; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:17; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18.

[0139] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:19; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:20; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:21; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:22; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:23; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:24.

[0140] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:26; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:27; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:28; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:29; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:30.

[0141] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:31; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:32; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:33; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:34; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:35; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:36.

[0142] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:42.

[0143] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:43; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:44; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:45; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:47; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:48.

[0144] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54.

[0145] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:55; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:56; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:57; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:58; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:60.

[0146] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:61; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:62; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:63; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:64; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:66.

[0147] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:68; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:69; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:70; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:71; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:72.

[0148] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:73; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:74; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:75; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:76; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:77; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78.

[0149] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:79; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:80; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:81; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:82; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:83; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84.

[0150] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:85; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:86; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:87; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:88; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:89; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:90.

[0151] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:92; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:93; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:94; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:95; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:96.

[0152] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:98; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:99; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:100; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:101; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:102.

[0153] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:103; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:105; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:106; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:107; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:108.

[0154] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:109; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:110; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:111; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:112; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:113; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:114.

[0155] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:115; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:117; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:118; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:119; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:120.

[0156] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:121; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:122; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:123; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:124; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:125; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:126.

[0157] In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:150 and the CDR sequence of VH of SEQ ID NO:151. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:152 and the CDR sequence of VH of SEQ ID NO:153. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:154 and the CDR sequence of VH of SEQ ID NO:155. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:156 and the CDR sequence of VH of SEQ ID NO:157. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:158 and the CDR sequence of VH of SEQ ID NO:159. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:160 and the CDR sequence of VH of SEQ ID NO:161. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:162 and the CDR sequence of VH of SEQ ID NO:163. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:164 and the CDR sequence of VH of SEQ ID NO:165. In some embodiments, the anti-αvβ8 antibody or its antigen-binding fragment comprises the CDR sequence of VL of SEQ ID NO:166 and the CDR sequence of VH of SEQ ID NO:167.

[0158] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:1; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:2; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:3; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:4; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:5; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:6, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:151, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:150. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 151. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 150.

[0159] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:7; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:8; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:9; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:10; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:11; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:12, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:153, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:152. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 153. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 152.

[0160] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:13; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:14; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:15; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:16; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:17; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:18, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:155, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:154. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 155. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 154.

[0161] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:19; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:20; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:21; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:22; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:23; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:24, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:157, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:156. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 157. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 156.

[0162] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:25; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:26; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:27; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:28; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:29; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:30, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:159, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:158. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 159. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 158.

[0163] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:31; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:32; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:33; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:34; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:35; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:36, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:161, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:160. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 161. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 160.

[0164] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:42, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:163, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:162. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 163. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 162.

[0165] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:42, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:165, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:164. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 165. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 164.

[0166] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:38; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:39; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:40; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:41; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:42, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:167, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:166. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 167. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 166.

[0167] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:43; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:44; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:45; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:46; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:47; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:48, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:151, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:150. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 151. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 150.

[0168] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:49; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:50; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:51; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:52; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:53; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:54, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:153, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:152. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 153. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 152.

[0169] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:55; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:56; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:57; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:58; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:59; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:60, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:155, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:154. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 155. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 154.

[0170] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:61; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:62; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:63; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:64; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:65; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:66, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:157, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:156. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 157. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 156.

[0171] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:67; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:68; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:69; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:70; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:71; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:72, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:159, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:158. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 159. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 158.

[0172] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:73; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:74; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:75; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:76; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:77; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:78, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:161, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:160. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 161. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 160.

[0173] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:79; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:80; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:81; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:82; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:83; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:163, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:162. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 163. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 162.

[0174] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:79; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:80; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:81; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:82; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:83; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:165, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:164. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 165. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 164.

[0175] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:79; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:80; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:81; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:82; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:83; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:84, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:167, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:166. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 167. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 166.

[0176] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:85; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:86; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:87; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:88; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:89; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:90, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:151, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:150. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 151. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 150.

[0177] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:91; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:92; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:93; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:94; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:95; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:96, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:153, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:152. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 153. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 152.

[0178] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:97; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:98; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:99; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:100; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:101; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:102, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:155, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:154. The amino acid sequence of the VL domain has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 155. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 154.

[0179] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:103; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:104; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:105; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:106; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:107; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:108, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:157, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:156. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 157. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 156.

[0180] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:109; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:110; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:111; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:112; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:113; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:114, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:159, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:158. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 159. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 158.

[0181] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:115; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:116; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:117; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:118; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:119; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:120, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:161, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:160. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 161. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 160.

[0182] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:121; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:122; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:123; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:124; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:125; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:126, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:163, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:162. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 163. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 162.

[0183] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:121; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:122; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:123; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:124; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:125; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:126, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:165, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:164. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 165. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 164.

[0184] In one aspect, the present invention provides an anti-αvβ8 antibody comprising: (a) a CDR-L1 comprising the amino acid sequence of SEQ ID NO:121; (b) a CDR-L2 comprising the amino acid sequence of SEQ ID NO:122; (c) a CDR-L3 comprising the amino acid sequence of SEQ ID NO:123; (d) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:124; (e) a CDR-H2 comprising the amino acid sequence of SEQ ID NO:125; and (f) a CDR-H3 comprising the amino acid sequence of SEQ ID NO:126, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:167, and a VH domain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:166. The amino acid sequence of the [specific amino acid] has a VL domain with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In one aspect, the VH domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 167. In another aspect, the VL domain has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 166.

[0185] In some embodiments, the anti-αvβ8 antibody comprises a light chain variable region (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:150. In some embodiments, the anti-αvβ8 antibody comprises a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:150. In some embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence, but the anti-αvβ8 antibody containing this sequence retains its ability to bind to αvβ8. In some aspects, in SEQ ID NO:150, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In some aspects, the substitution, insertion, or deletion occurs in a region outside the CDR (i.e., in the FR). Optionally, the anti-αvβ8 antibody comprises the VL sequence shown in SEQ ID NO:150, including post-translational modifications of that sequence. In some embodiments, the anti-αvβ8 antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:151. In one aspect, the anti-αvβ8 antibody comprises a heavy chain variable domain (VH) sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:151. In some aspects, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to the reference sequence, but the anti-αvβ8 antibody comprising this sequence retains its ability to bind to αvβ8. In some respects, in SEQ ID NO:151, a total of 1 to 10 amino acids have been substituted, inserted, and / or deleted. In some respects, the substitution, insertion, or deletion occurs in regions outside the CDR (i.e., in the FR). Optionally, the anti-αvβ8 antibody comprises the VH sequence shown in SEQ ID NO:151, including post-translational modifications of that sequence.

[0186] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:151 and SEQ ID NO:150, respectively, including post-translational modifications of those sequences.

[0187] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:153 and SEQ ID NO:152, respectively, including post-translational modifications of those sequences.

[0188] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:155 and SEQ ID NO:154, respectively, including post-translational modifications of those sequences.

[0189] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:157 and SEQ ID NO:156, respectively, including post-translational modifications of those sequences.

[0190] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:159 and SEQ ID NO:158, respectively, including post-translational modifications of those sequences.

[0191] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:161 and SEQ ID NO:160, respectively, including post-translational modifications of those sequences.

[0192] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:163 and SEQ ID NO:162, respectively, including post-translational modifications of those sequences.

[0193] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:165 and SEQ ID NO:164, respectively, including post-translational modifications of those sequences.

[0194] In another aspect, an anti-αvβ8 antibody is provided, wherein the antibody comprises a VH sequence as provided in any of the aspects above and a VL sequence as provided in any of the aspects above. In one aspect, the antibody comprises the VH sequence and VL sequence as in SEQ ID NO:167 and SEQ ID NO:166, respectively, including post-translational modifications of those sequences.

[0195] In another aspect of the invention, the anti-αvβ8 antibody according to any of the foregoing aspects is a monoclonal antibody, including chimeric, humanized, or human antibodies. In one aspect, the anti-αvβ8 antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, biantibody, or F(ab')2 fragment.

[0196] In the other case, the antibody is a full-length antibody, such as a full-length IgG1 antibody or other antibody class or isotype as defined herein.

[0197] In some respects, the antibodies described herein belong to the IgG1 isotype / subclass. In some embodiments, the antibodies described herein belong to the IgG1 isotype / subclass and are modified to reduce Fc region effector function. In some embodiments, the antibodies described herein belong to the IgG1 isotype / subclass and contain amino acid substitutions L234A and L235A, numbered according to the Kabat EU index. In some embodiments, the antibodies described herein belong to the IgG1 isotype / subclass and contain amino acid substitution P329G, numbered according to the Kabat EU index. In some embodiments, the antibodies described herein contain the Fc region according to SEQ ID NO:204. In some embodiments, the antibodies described herein contain an Fc region exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:204. In some embodiments, the antibodies described herein comprise C-terminal glycine (Gly446). In some embodiments, the antibodies described herein comprise C-terminal glycine (Gly446) and C-terminal lysine (Lys447).

[0198] In some embodiments, the antibody described herein comprises the light chain sequence of SEQ ID NO:200 and the heavy chain sequence of SEQ ID NO:201. In some embodiments, the antibody described herein comprises the light chain sequence of SEQ ID NO:200 and the heavy chain sequence of SEQ ID NO:220. In some embodiments, the antibody described herein comprises: a light chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:200, and a heavy chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:201. In some embodiments, the antibody described herein comprises: a light chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:200; and a heavy chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:220.

[0199] In some embodiments, the antibody described herein comprises the light chain sequence of SEQ ID NO:202 and the heavy chain sequence of SEQ ID NO:203. In some embodiments, the antibody described herein comprises the light chain sequence of SEQ ID NO:202 and the heavy chain sequence of SEQ ID NO:221. In some embodiments, the antibody described herein comprises: a light chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:202; and a heavy chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:203. In some embodiments, the antibody described herein comprises: a light chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:202; and a heavy chain sequence exhibiting at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO:221.

[0200] In another respect, as described in the following sections 1-8 below, anti-αvβ8 antibodies according to any of the above aspects may combine any feature, alone or in combination:

[0201] 1. Antibody affinity

[0202] In some respects, this paper provides an antibody with a dissociation constant (K) D ) is ≤ 1 μM, ≤ 100 nM, ≤ 10 nM, ≤ 1 nM, ≤ 0.1 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g., 10 -8 M or smaller, such as 10 -8 M to 10-13 M, for example, 10 -9 M to 10 -13 M). In some embodiments, the K between the antibody and human αvβ8 D Less than about 5 nM, such as less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM. In some embodiments, the K value between the antibody and mouse αvβ8 is... D Less than about 5 nM, such as less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM. In some embodiments, the K-value between the antibody and cynomolgus monkey αvβ8 is... D Less than about 5 nM, such as less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM.

[0203] On the one hand, using BIACORE ® Surface plasmon resonance method for measuring K D For example, using BIACORE® T200, BIACORE ® -2000, BIACORE ® Assays at -3000 or BIACORE® 8K (BIAcore, Inc., Piscataway, NJ) were performed at 25°C using an immobilized antigen CM5 chip at approximately 10 response units (RU). In one aspect, the carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen was diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8, and then injected at a flow rate of 5 μl / min to obtain approximately 10 response units (RU) of conjugate protein. Following antigen injection, 1 M ethanolamine was injected to block unreacted groups. Regarding kinetic measurements, at 25°C and a flow rate of approximately 25 μl / min, it was injected into a solution containing 0.05% polysorbate 20 (TWEEN-20). TM The surfactant (PBST) in PBS was serially diluted twice (0.78 nM to 500 nM). A simple one-to-one Langmuir binding model (BIACORE) was used.® Evaluation Software version 3.2 calculates the association rate (k) by simultaneously fitting association and dissociation sensor maps. on ) and dissociation rate (k off Equilibrium dissociation constant (K) D ) Calculated as ratio k off / k on For example, Chen et al., J.Mol. Biol. 293:865-881 (1999). If the association rate obtained by the above surface plasmon resonance determination exceeds 10... 6 M -1 s -1 The association rate can then be determined using fluorescence quenching techniques, such as those found in spectrometers equipped with flow-stopping devices (e.g., Aviv Instruments) or the 8000 series SLM-AMINCO. TM The fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS pH 7.2 at 25°C was measured in a stirred cuvette using a spectrophotometer (ThermoSpectronic) in the presence of gradually increasing antigen concentration.

[0204] In an alternative method, K is measured by radiolabeled antigen binding assay (RIA). D In one aspect, RIA is performed using the Fab form of the target antibody and its antigen. For example, by titrating a series of unlabeled antigens in the presence of the minimum concentration ( 125 I) The labeled antigen was equilibrated with Fab, and the bound antigen was then captured using a plate coated with anti-Fab antibody to measure the solution-binding affinity of Fab to the antigen (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions used for the assay, MICROTITER was coated with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6). ® Multi-well plates (ThermoScientific) were incubated overnight, followed by blockade with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorbent plates (Nunc #269620), 100 pM or 26 pM [ 125I] The antigen is mixed with a serially diluted target Fab (e.g., following the evaluation of anti-VEGF antibody (Fab-12) in Presta et al., Cancer Res. 57:4593-4599 (1997)). The target Fab is then incubated overnight; however, incubation may be prolonged (e.g., approximately 65 hours) to ensure equilibration. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution is then removed and mixed with 0.1% polysorbate 20 (TWEEN-20) in PBS. ® Wash the plate eight times. When the plate is dry, add 150 μl / well of scintillator (MICROSCINT-20). TM Packard), and in TOPCOUNT TM Use a γ counter (Packard) to count the filaments for several tens of minutes. Select the concentration of each Fab that gives a maximum binding of less than or equal to 20% for use in competitive binding assays.

[0205] 2. Antibody fragments

[0206] In some respects, the antibodies presented herein are antibody fragments that specifically bind to αvβ8 (such as human αvβ8, cynomolgus monkey αvβ8, and / or mouse αvβ8). As used herein, αvβ8 antibody fragments are αvβ8 antibodies that retain specific binding to αvβ8 to any part.

[0207] In one respect, antibody fragments are Fab, Fab', Fab'-SH, or F(ab')2 fragments, particularly Fab fragments. Papain digestion of an intact antibody produces two identical antigen-binding fragments called "Fab" fragments, each containing a heavy chain variable domain and a light chain variable domain (VH and VL, respectively), as well as a constant domain (CL) of the light chain and a first constant domain (CH1) of the heavy chain. Thus, the term "Fab fragment" refers to an antibody fragment comprising a light chain containing the VL and CL domains and a heavy chain containing the VH and CH1 domains. Fab' fragments differ from Fab fragments in that Fab' fragments have residues added to the carboxyl terminus of the CH1 domain, including one or more cysteine ​​residues from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residues of the constant domain have free thiol groups. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and a portion of the Fc region. For a discussion of the Fab and F(ab')2 fragments containing salvage receptor-binding epitope residues and having an increased in vivo half-life, see U.S. Patent No. 5,869,046.

[0208] In another respect, antibody fragments are bisomatic, trisomatic, or tetrasomatic antibodies. A “bisomatic antibody” is an antibody fragment having two antigen-binding sites, which can be bivalent or bispecific. See, for example, EP 404,097; WO1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Trisomatic and tetrasomatic antibodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0209] On the other hand, the antibody fragment is a single-chain Fab fragment. A “single-chain Fab fragment” or “scFab” is a polypeptide composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), and a linker, wherein the antibody domains and linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1, or d) VL-CH1-linker-VH-CL. Specifically, the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single-chain Fab fragment is stabilized via a native disulfide bond between the CL domain and the CH1 domain. Furthermore, these single-chain Fab fragments can be further stabilized by generating interchain disulfide bonds via the insertion of cysteine ​​residues (e.g., position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0210] On the other hand, the antibody fragment is a single-chain variable fragment (scFv). A "single-chain variable fragment" or "scFv" is a fusion protein of the antibody's heavy chain variable domain (VH) and light chain variable domain (VL), linked by a linker. Specifically, the linker is a short polypeptide of approximately 10 to 25 amino acids, typically rich in glycine for flexibility and serine or threonine for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, the protein retains the specificity of the original antibody. For reviews of scFv fragments, see, for example, Plückthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore (Springer-Verlag, New York), pp. 269–315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458.

[0211] On the other hand, antibody fragments are single-domain antibodies. A "single-domain antibody" is an antibody fragment containing all or part of the variable heavy chain domain or all or part of the variable light chain domain of an antibody. In some respects, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, for example, U.S. Patent No. 6,248,516 B1).

[0212] Antibody fragments can be prepared using various techniques, including but not limited to the proteolytic digestion of intact antibodies and recombinant production from recombinant host cells (e.g., E. coli), as described herein.

[0213] 3. Chimeric antibodies and humanized antibodies

[0214] In some respects, the antibodies described herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one instance, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In another instance, a chimeric antibody is a “class-switching” antibody in which the class or subclass has been altered from that of the parent antibody. Chimeric antibodies include their antigen-binding fragment.

[0215] In some respects, chimeric antibodies are humanized antibodies. Typically, nonhuman antibodies are humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent nonhuman antibody. Humanized antibodies typically contain one or more variable domains, wherein the CDR (or a portion thereof) is derived from the nonhuman antibody, and the FR (or a portion thereof) is derived from the human antibody sequence. Humanized antibodies may also optionally contain at least a portion of the human constant region. In some respects, some FR residues in the humanized antibody are replaced by corresponding residues from the nonhuman antibody (e.g., the antibody from which the CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0216] Humanized antibodies and their preparation methods have been reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l. Acad. Sci. USA 86:10029-10033 (1989); US patents 5,821,337, 7,527,791, 6,982,321 and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) transplantation); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “surface reshaping”); Dall'Acqua Methods 36:43-60 (2005) (describes “FR shuffling”); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describes a “guided selection” approach for FR shuffling).

[0217] Human frame regions that can be used for humanization include, but are not limited to: frame regions selected using a "best fit" method (see, for example, Sims et al., J. Immunol. 151:2296 (1993)); frame regions derived from the common sequences of human antibodies from specific subgroups of the light or heavy chain variable regions (see, for example, Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) frame regions or human germline frame regions (see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frame regions derived from screening FR libraries (see, for example, Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0218] 4. Human antibodies

[0219] In some respects, the antibodies provided herein are human antibodies. Human antibodies can be generated using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0220] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce a complete human antibody or a complete antibody with a human variable region in response to antigen stimulation. Such animals typically contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus, or is present extrachromosomally or randomly integrated into the animal's chromosome. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, for example, a description of XENOMOUSE. TM The technologies described are described in U.S. Patent Nos. 6,075,181 and 6,150,584; U.S. Patent No. 5,770,429 describing HuMab® technology; U.S. Patent No. 7,041,870 describing KM MOUSE® technology; and U.S. Patent Application Publication No. US 2007 / 0061900 describing VELOCIMOUSE® technology. The human variable region derived from intact antibodies produced by such animals can be further modified, for example, by combining it with different human constant regions.

[0221] Human antibodies can also be prepared using hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines used to produce human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Human antibodies produced via human B-cell hybridoma technology are also described by Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include, for example, those described in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridoma). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0222] Human antibodies can also be generated by isolating variable domain sequences selected from human phage display libraries. These variable domain sequences can then be bound to the desired human constant domain. The technique for selecting human antibodies from antibody libraries is described below.

[0223] 5. Antibodies derived from a library

[0224] In some respects, the antibodies provided herein are derived from libraries. The antibodies of the present invention can be isolated by screening combinatorial libraries for antibodies having one or more desired activities. Methods for screening combinatorial libraries are reviewed, for example, in Lerner et al., Nature Reviews 16:498-508 (2016). For example, various methods are known in the art for generating phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed in, for example, Frenzel et al., mAbs 8:1177-1194 (2016); Bazan et al., Human Vaccines and Immunotherapeutics 8:1817-1828 (2012); and Zhao et al., Critical Reviews in Biotechnology 36:276-289 (2016), as well as Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., editors, Human Press, Totowa, NJ, 2001) and Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, editors, Human Press, Totowa, NJ, 2003).

[0225] In some phage display methods, the entire set of VH and VL genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library. Antigen-binding phages can then be screened from said phage library, as described in Winter et al., Annual Review of Immunology 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, all natural components (e.g., all natural components from humans) can be cloned to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization, as described by Griffiths et al., EMBO Journal 12: 725-734 (1993). In addition, natural libraries are synthesized by cloning an unrearranged V gene segment from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 region and perform in vitro rearrangement, as described by Hoogenboom and Winter in Journal of Molecular Biology 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent Nos. 5,750,373; 7,985,840; 7,785,903 and 8,679,490, and U.S. Patent Publications Nos. 2005 / 0079574, 2007 / 0117126, 2007 / 0237764 and 2007 / 0292936.

[0226] Other examples of methods known in the art for screening combinatorial libraries of antibodies with one or more desired activities include ribosome and mRNA display, as well as methods for displaying and selecting antibodies on bacterial, mammalian, insect, or yeast cells. Methods for yeast surface display are reviewed, for example, in Scholler et al., Methods in Molecular Biology 503:135-56 (2012), Cherf et al., Methods in Molecular Biology 1319:155-175 (2015), and Zhao et al., Methods in Molecular Biology 889:73-84 (2012). Methods for ribosome display are described, for example, in He et al., Nucleic Acids Research 25:5132-5134 (1997), and Hanes et al., PNAS 94:4937-4942 (1997).

[0227] In this paper, antibodies or antibody fragments isolated from human antibody libraries are considered to be human antibodies or human antibody fragments.

[0228] 6. Multispecific antibodies

[0229] In some respects, the antibodies presented herein are multispecific antibodies, particularly bispecific antibodies. A “multispecific antibody” is a monoclonal antibody that has binding specificity to at least two distinct sites (i.e., different epitopes on different antigens or different epitopes on the same antigen). In some respects, multispecific antibodies have three or more binding specificities. In some respects, one of the binding specificities is against αvβ8, and another specificity is against any other antigen. In some respects, bispecific antibodies can bind to two (or more) distinct epitopes of αvβ8. Multispecific (e.g., bispecific) antibodies can also be used to target cytotoxic agents or cells to cells expressing αvβ8. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0230] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of heavy-light chain pairs of two immunoglobulins with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)) and engineered “mortar and pestle structures” (see, for example, U.S. Patent 5,731,168 and Atwell et al., J. Mol. Biol. 270:26 (1997)). Multispecific antibodies can also be prepared by: engineering electrostatic manipulation effects for the preparation of antibody Fc-heterodimer molecules (see, for example, WO 2009 / 089004); crosslinking two or more antibodies or fragments (see, for example, U.S. Patent No. 4,676,980, and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to generate bispecific antibodies (see, for example, Kostelny et al., J. Immunol., 148(5):1547-1553 (1992) and WO 2011 / 034605); using common light chain techniques to avoid light chain mismatch problems (see, for example, WO 98 / 50431); using “dibody antibody” techniques for the preparation of bispecific antibody fragments (see, for example, Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448). (1993)); and the use of single-chain Fv (sFv) dimers (see, for example, Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies as described in Tutt et al., J. Immunol. 147:60 (1991).

[0231] This document also includes engineered antibodies having three or more antigen-binding sites, including, for example, “octopus antibodies” or DVD-Ig (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies having three or more antigen-binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792 and WO 2013 / 026831. Bispecific antibodies or their antigen-binding fragments also include “dual-acting FAbs” or “DAFs” containing antigen-binding sites that bind to αvβ8 and another different antigen or to two different epitopes of αvβ8 (see, for example, US 2008 / 0069820 and WO 2015 / 095539).

[0232] Multispecific antibodies can also be provided in an asymmetric form, wherein there is domain interchange in one or more binding arms having the same antigen specificity, i.e., by exchanging the VH / VL domain (see, for example, WO 2009 / 080252 and WO2015 / 150447), the CH1 / CL domain (see, for example, WO 2009 / 080253), or the complete Fab arm (see, for example, WO 2009 / 080251, WO 2016 / 016299, and also Schaefer et al., PNAS, 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-20). In one aspect, multispecific antibodies contain cross-Fab fragments. The terms “cross-Fab fragment” or “xFab fragment” or “exchangeable Fab fragment” refer to Fab fragments in which variable or constant regions of the heavy and light chains are exchanged. Cross-Fab fragments comprise polypeptide chains consisting of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), or a heavy chain variable region (VH) and a light chain constant region (CL). Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations into the domain interfaces to guide correct Fab pairing. See, for example, WO 2016 / 172485.

[0233] Various other molecular forms of multispecific antibodies are known in the art and are included herein (see, for example, Spiess et al., Mol Immunol 67 (2015) 95-106).

[0234] This article also includes a specific type of multispecific antibody, which is a bispecific antibody designed to simultaneously bind to a surface antigen on a target cell (e.g., a tumor cell) and an activation-invariant component of the T cell receptor (TCR) complex (such as CD3) for retargeting T cells to kill the target cell. Therefore, in some respects, the antibodies provided herein are multispecific antibodies, particularly bispecific antibodies, wherein one binding specificity targets αvβ8 while the other targets CD3.

[0235] Examples of bispecific antibody forms that can be used for this purpose include, but are not limited to: so-called “BiTE” (bispecific T cell recruiter) molecules in which two scFv molecules are fused by a flexible linker (see, for example, WO 2004 / 106381; WO2005 / 061547; WO 2007 / 042261; WO 2008 / 119567; Nagorsen and Bäuerle, Exp Cell Res 317, 1255-1260 (2011)); bispecific antibodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and their derivatives, such as tandem bispecific antibodies (“TandAb”; Kipriyanov et al., J Mol Biol 293, 41-56 (1999)); “DART” (biaffinity retargeting) molecules, which are based on bispecific antibody forms but have a C-terminal disulfide bond for additional stabilization (Johnson et al., J Mol Biol). 399, 436-449 (2010)); and so-called trifunctional antibodies, which are complete hybrid mouse / rat IgG molecules (as reviewed by Seimetz et al.: Cancer Treat Rev 36, 458-467 (2010)). The specific T-cell bispecific antibody forms included in this article are described in the following references: WO 2013 / 026833; WO 2013 / 026839; WO 2016 / 020309; Bacac et al., Oncoimmunology 5(8) (2016) e1203498.

[0236] 7. Antibody variants

[0237] In some respects, amino acid sequence variants of the antibodies presented herein are envisioned. For example, it may be desirable to alter the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to achieve the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0238] a) Substitution, insertion, and deletion variants

[0239] In some respects, antibody variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include CDR and FR. Conserved substitutions are shown under the heading “Preferred Substitutions” in Table 6. Further substantial variations are provided under the heading “Exemplary Substitutions” in Table 6, and are described further below with reference to the amino acid side chain categories. Amino acid substitutions can be introduced into the target antibody, and the product can be screened for desired activities (e.g., preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0240] Table 6

[0241]

[0242] Amino acids can be grouped based on their common side-chain characteristics:

[0243] (1) Hydrophobicity: Leucine, Met, Ala, Val, Leu, Ile;

[0244] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0245] (3) Acidic: Asp, Glu;

[0246] (4) Alkaline: His, Lys, Arg;

[0247] (5) Residues affecting chain orientation: Gly, Pro;

[0248] (6) Fang ethnic group: Trp, Tyr, Phe.

[0249] Non-conservative substitution would require swapping members of one of these categories for members of another category.

[0250] One type of substitution variant involves replacing one or more highly variable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, one or more resulting variants selected for further research will alter (e.g., improve) certain biological properties (e.g., increased affinity, decreased immunogenicity) and / or will substantially retain certain biological properties of the parent antibody, relative to the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated and the variant antibody is displayed on a phage and screened for specific biological activities (e.g., binding affinity).

[0251] For example, changes (e.g., substitutions) can be made in the CDR to improve antibody affinity. Such alterations can occur in CDR “hotspots,” which are residues encoded by codons that undergo high-frequency mutations during somatic maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or residues in contact with the antigen (detecting the binding affinity of the resulting variant VH or VL). Affinity maturation achieved by constructing and reselecting from a secondary library has been described, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, (2001)). In some aspects of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is subsequently screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves CDR directed approaches, in which several CDRs are... Residues (e.g., 4 to 6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutations or modeling. Specifically, CDR-H3 and CDR-L3 are often targeted.

[0252] In some respects, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions as provided herein) can be made within the CDR. Such changes can, for example, be external to the antigen-contacting residues in the CDR. In some variant VH and VL sequences provided above, each CDR either remains unchanged or contains no more than one, two, or three amino acid substitutions.

[0253] A method used to identify antibody residues or regions that can be targeted for mutagenesis is called "alanine scan mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or a group of target residues (e.g., charged residues such as arg, asp, his, lys, and glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine if the antibody-antigen interaction is affected. Additional substitutions can be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or additionally, the contact points between the antibody and antigen can be identified using the crystal structure of the antigen-antibody complex. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine if they possess the desired properties.

[0254] Amino acid sequence insertions include the fusion of amino and / or carboxyl terms of peptides ranging in length from one residue to one hundred or more residues, as well as intra-sequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionine residue. Other insertion variants of antibody molecules include the fusion of the N-terminus or C-terminus of the antibody with an enzyme (e.g., for ADEPT (antibody-directed enzyme prodrug therapy)) or peptide that increases the antibody's serum half-life.

[0255] b) Glycosylation variants

[0256] In some respects, the antibodies presented herein can be modified to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to antibodies can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0257] When an antibody contains an Fc region, the oligosaccharide associated with it can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example, Wright et al., TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose of GlcNAc attached to the “backbone” of the biantennary oligosaccharide structure. In some aspects, the oligosaccharides in the antibodies of the present invention can be modified to produce antibody variants with certain improved properties.

[0258] On the one hand, antibody variants with unfucosylated oligosaccharides are provided, i.e., oligosaccharide structures lacking (directly or indirectly) fucose linked to the Fc region. Such unfucosylated oligosaccharides (also known as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack the fucose residues linking the first GlcNAc in the stem of the biantennary oligosaccharide structure. On the other hand, antibody variants with an increased proportion of unfucosylated oligosaccharides in the Fc region compared to natural or parental antibodies are provided. For example, the proportion of unfucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., no fucose oligosaccharides present). The percentage of non-fucosylated oligosaccharides, as described, for example, in WO 2006 / 082515, and measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking fucosylated residues relative to the sum of all oligosaccharides (e.g., complex, heterozygous, and high-mannose structures) linked to Asn 297. Asn 297 refers to the asparagine residue (EU number of Fc region residues) located at approximately position 297 in the Fc region; however, due to minor sequence variations in antibodies, Asn 297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies with an increased proportion of non-fucosylated oligosaccharides in the Fc region may exhibit improved FcγRIIIa receptor binding and / or improved effector function, particularly improved ADCC function. See, for example, US 2003 / 0157108 and US 2004 / 0093621.

[0259] Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US 2003 / 0157108; and WO 2004 / 056312, especially in Example 11), and knockout cell lines, such as those with the α-1,6-fucosylation gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107), or cells with reduced or eliminated GDP-fucose synthesis or transporter activity (see, for example, US2004259150, US2005031613, US2004132140, US2004110282).

[0260] On the other hand, antibody variants provide bipartite oligosaccharides, for example, in which biantennary oligosaccharides linked to the Fc region of the antibody are bipartitely divided by GlcNAc. As mentioned above, such antibody variants can have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99 / 54342; WO 2004 / 065540, WO 2003 / 011878.

[0261] Antibody variants having at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087, WO 1998 / 58964 and WO 1999 / 22764.

[0262] c) Fc region variant

[0263] In some respects, one or more amino acid modifications may be introduced into the Fc region of the antibody provided herein, thereby generating an Fc region variant. The Fc region variant may contain a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0264] In some respects, the present invention considers antibody variants possessing some, but not all, effector functions, making them ideal candidates for applications where the in vivo half-life of the antibody is important, but certain effector functions, such as complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC), are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding capacity. Primary NK cells, which mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of target molecules are described in U.S. Patent Nos. 5,500,362 (see, for example, Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be used (see, for example, the ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA); and CytoTox 96). ®Non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest can be assessed in vivo in animal models such as those disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind C1q and therefore lacks CDC activity. See, for example, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays can also be performed using methods known in the art (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0265] Antibodies with reduced effector function include those with substitutions of one or more of the Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at two or more of the amino acids at positions 265, 269, 270, 297, and 327, including the so-called “DANA” Fc mutant, in which residues 265 and 297 are substituted with alanine (US Patent No. 7,332,581).

[0266] Certain antibody variants with improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604(2001).)

[0267] In some respects, antibody variants contain an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 of the Fc region (EU numbers of the residues).

[0268] In some aspects, the antibody variant includes an Fc region with one or more amino acid substitutions that reduce FcγR binding, such as substitutions at positions 234 and 235 of the Fc region (EU numbers of the residues). In one aspect, the substitutions are L234A and L235A (LALA). In some aspects, the antibody variant further includes D265A and / or P329G in an Fc region derived from the human IgG1 Fc region. In one aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and P329G (LALA-PG). (See, for example, WO 2012 / 130831). In another aspect, in the Fc region derived from the human IgG1 Fc region, the substitutions are L234A, L235A, and D265A (LALA-DA).

[0269] In some respects, alterations are made in the Fc region that result in changes (i.e., improvements or reductions) in C1q binding and / or complement-dependent cytotoxicity (CDC), for example, as described in U.S. Patent Nos. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0270] Antibodies with prolonged half-lives and improved neonatal Fc receptor (FcRn) binding, responsible for transferring maternal IgG to the fetus (Guyer, RL et al., J. Immunol. 117:587 (1976), and Kim, JK et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include Fc variants with substitutions at one or more of the following Fc region residues: 238, 252, 254, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, a substitution of Fc region residue 434 (see, for example, U.S. Patent No. 7,371,826; Dall'Acqua, WF et al. J. Biol. Chem. 281 (2006) 23514-23524).

[0271] Fc region residues crucial for the mouse Fc-mouse FcRn interaction have been identified through site-directed mutagenesis (see, for example, Dall'Acqua, WF et al. J. Immunol 169 (2002) 5171-5180). The interaction involves residues I253, H310, H433, N434, and H435 (EU numbers of the residues) (Medesan, C. et al. Eur. J. Immunol. 26 (1996) 2533; Firan, M. et al. Int. Immunol. 13 (2001) 993; Kim, JK et al. Eur. J. Immunol. 24 (1994) 542). Residues I253, H310, and H435 were found to be crucial for the interaction between human Fc and mouse FcRn (Kim, JK et al., Eur. J. Immunol. 29 (1999) 2819). Studies on the human Fc-human FcRn complex showed that residues I253, S254, H435, and Y436 were crucial for the interaction (Firan, M. et al., Int. Immunol. 13 (2001) 993; Shields, RL et al., J. Biol. Chem. 276 (2001) 6591-6604). Various mutants of residues 248 to 259 and 301 to 317 and 376 to 382 and 424 to 437 have been reported and examined in Yeung, YA et al. (J. Immunol. 182 (2009) 7667-7671).

[0272] In some respects, antibody variants comprise Fc regions with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 253, and / or 310, and / or 435 of the Fc region (EU numbers of the residues). In some respects, antibody variants comprise Fc regions with amino acid substitutions at positions 253, 310, and 435. In one instance, in the Fc region derived from the human IgG1 Fc region, the substitutions are I253A, H310A, and H435A. See, for example, Grevys, A. et al., J. Immunol. 194 (2015) 5497-5508.

[0273] In some respects, antibody variants comprise Fc regions with one or more amino acid substitutions that reduce FcRn binding, for example, substitutions at positions 310, and / or 433, and / or 436 of the Fc region (EU numbers of the residues). In some respects, antibody variants comprise Fc regions with amino acid substitutions at positions 310, 433, and 436. In one instance, in the Fc region derived from the human IgG1 Fc region, the substitutions are H310A, H433A, and Y436A. (See, for example, WO 2014 / 177460A1).

[0274] In some respects, antibody variants comprise an Fc region having one or more amino acid substitutions that increase FcRn binding, such as substitutions at positions 252, and / or 254, and / or 256 of the Fc region (EU numbers of the residues). In some respects, antibody variants comprise an Fc region having amino acid substitutions at positions 252, 254, and 256. In one respect, in the Fc region derived from the human IgG1 Fc region, the substitutions are M252Y, S254T, and T256E. For other examples of Fc region variants, see also: Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351.

[0275] The C-terminus of the heavy chain of the antibody reported herein may be a full C-terminus ending with the amino acid residue PGK. The C-terminus of the heavy chain may be a shortened C-terminus in which one or two C-terminal amino acid residues have been removed. In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending with PG. In one aspect of all aspects reported herein, as specified herein, an antibody comprising a heavy chain including a C-terminal CH3 domain comprises a C-terminal glycine-lysine dipeptide (G446 and K447, EU index numbers of amino acid positions). In one aspect of all aspects reported herein, as specified herein, an antibody comprising a heavy chain including a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, EU index number of amino acid position).

[0276] d) Cysteine-engineered antibody variants

[0277] In some respects, it may be necessary to produce cysteine-engineered antibodies, such as THIOMAB. TMAn antibody in which one or more residues of the antibody are replaced by cysteine ​​residues. In a particular embodiment, the substituted residues are located at an accessible site of the antibody. As further described herein, by replacing those residues with cysteine, a reactive thiol group is thereby positioned at an accessible site of the antibody and can be used to conjugate the antibody to other parts (such as a pharmaceutical part or a linker-pharmaceutical part) to produce an immunoconjugate. Cysteine-engineered antibodies can be produced, for example, as described in U.S. Patent Nos. 7,521,541, 8,30,930, 7,855,275, 9,000,130, or WO 2016040856.

[0278] e) Antibody derivatives

[0279] In some respects, the antibodies provided herein can be further modified to include additional non-protein moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers) and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. PEG-propionaldehyde may be advantageous in manufacturing due to its stability in water. Polymers can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on the following considerations, including but not limited to the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for a limited therapy.

[0280] 8. Immunoconjugates

[0281] The present invention also provides an immunoconjugate comprising the anti-αvβ8 antibody described herein, which is conjugated (chemically bonded) to one or more therapeutic agents, such as cytotoxic agents, chemotherapeutic agents, pharmaceuticals, growth inhibitors, toxins (e.g., protein toxins, bacterial, fungal, plant or animal-derived enzyme-active toxins or fragments thereof) or radioisotopes.

[0282] On the one hand, immunoconjugates are antibody-drug conjugates (ADCs), in which an antibody is conjugated to one or more therapeutic agents. A linker is typically used to connect the antibody to one or more therapeutic agents. An overview of ADC technology is provided in Pharmacol Review 68:3-19 (2016), which includes examples of therapeutic agents, drugs, and linkers.

[0283] In another aspect, the immunoconjugate comprises an antibody conjugated herein to an enzyme-active toxin or a fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, absinthecin A chain, senna root toxin A chain, α-arbusculin, tung oil protein, caryophyllein protein, pokeweed antiviral proteins (PAPI, PAPII, and PAP-S), bitter melon inhibitor, curcumin, crotonin, soapwort inhibitor, gelatin, mitochondriin, localized aspergillin, phenolmycin, enoxacin, and trichosporine.

[0284] In another aspect, immunoconjugates include antibodies described herein that are conjugated with radioactive atoms to form radioconjugates. A variety of radioisotopes can be used to prepare radioconjugates. Examples include At... 211 I 131 I 125 Y 90 Re 186 Re 188 、Sm 153 Bi 212 P 32 Pb 212 Radioactive isotopes of Lu. When radioactive conjugates are used for detection, they may contain radioactive atoms used for scintillation studies, such as tc99m or I123, or spin-labeled elements used for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0285] A variety of bifunctional protein conjugates, such as N-succinimino-3-(2-pyridyldithio)propionate (SPDP), 4-(N-maleiminomethyl)cyclohexane-1-carboxylic acid succinimide ester (SMCC), iminothiacyclopentane (It), bifunctional derivatives of imino esters (such as dimethyl adipate HCl), active esters (such as disuccinimide octanoate), aldehydes (such as glutaraldehyde), diazid compounds (such as bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (such as bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and difluorinated compounds (such as 1,5-difluoro-2,4-dinitrobenzene), can be used to prepare conjugates of antibodies and cytotoxic agents. For example, ricin immunotoxin can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides to antibodies. See WO 94 / 11026. The adapter can be a “cleavable adapter” that promotes the release of cytotoxic drugs from cells. For example, acid-labile adapters, peptidase-sensitive adapters, light-labile adapters, dimethyl adapters, or disulfide-containing adapters can be used (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0286] The immunoconjugates or ADCs discussed in this article are explicitly considered, but not limited to, such conjugates prepared with cross-linking agents, including but not limited to commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA) BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfon-EMCS, sulfon-GMBS, sulfon-KMUS, sulfon-MBS, sulfon-SIAB, sulfon-SMCC, sulfon-SMPB, and SVSB (succinimino-(4-vinyl sulfone)benzoate).

[0287] B. Exemplary anti-PD-L1 antibody

[0288] In some embodiments, an anti-αvβ8 antibody or its antigen-binding fragment is administered in combination with an effective amount of one or more other therapeutic agents. In a preferred embodiment, the anti-αvβ8 antibody is administered in combination with a PD-1 axis antagonist, such as a PD-1 binding antagonist or a PD-L1 binding antagonist, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. In another preferred embodiment, the anti-αvβ8 antibody is administered in combination with an anti-PD-L1 antibody, wherein the anti-PD-L1 antibody is atezolizumab. In some embodiments, anti-PD-L1 antibody examples comprise HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2, and HVR-H3 as defined in Table 7 below. In some aspects, the anti-PD-L1 antibody comprises the heavy chain variable region (VH) sequence of SEQ ID NO:216 and the light chain variable region (VL) sequence of SEQ ID NO:217. In some embodiments, the anti-PD-L1 antibody comprises the heavy chain sequence of SEQ ID NO:218 and the light chain sequence of SEQ ID NO:219. Treatment methods using the anti-αvβ8 antibody described herein in combination with effective amounts of one or more other therapeutic agents are discussed in Part II(G) of this specification.

[0289]

[0290] C. Recombination methods and compositions

[0291] Antibodies can be generated using recombinant methods and compositions, such as those described in US 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0292] In the case of natural antibodies or fragments of natural antibodies, two nucleic acids are required: one for the light chain or a fragment thereof, and one for the heavy chain or a fragment thereof. These nucleic acids encode the amino acid sequence of the VL containing the antibody and / or the amino acid sequence of the VH containing the antibody (e.g., the light chain and / or heavy chain of the antibody). These nucleic acids can be expressed on the same expression vector or on different expression vectors.

[0293] In the case of certain bispecific antibodies containing heterodimeric heavy chains, four nucleic acids are required: one for the first light chain, one for the first heavy chain containing the Fc region of the first heteromonomer, one for the second light chain, and one for the second heavy chain containing the Fc region of the second heteromonomer. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors. These nucleic acids encode the amino acid sequence constituting the first VL of the antibody and / or the amino acid sequence constituting the first VH containing the Fc region of the first heteromonomer and / or the amino acid sequence constituting the second VL of the antibody and / or the amino acid sequence constituting the second VH containing the Fc region of the second heteromonomer (e.g., the first light chain and / or the second light chain and / or the first heavy chain and / or the second heavy chain of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors; typically, these nucleic acids are located on two or three expression vectors, meaning that a single vector can contain more than one of these nucleic acids. Examples of these bispecific antibodies are cross-Mabs (see, for example, Schaefer, W. et al., PNAS, 108 (2011) 11187-1191). For instance, one of the heteromonads in the heavy chain contains a so-called "hole mutation" (T366W, and optionally one of S354C or Y349C), and the other contains a so-called "hole mutation" (T366S, L368A, and Y407V, and optionally Y349C or S354C) (see, for example, Carter, P. et al., Immunotechnol. 2 (1996) 73), according to EU index number.

[0294] In one aspect, isolated nucleic acids encoding antibodies used in the methods reported herein are provided.

[0295] In one aspect, a method for preparing an anti-αvβ8 antibody is provided, wherein the method includes culturing a host cell comprising a nucleic acid encoding an antibody as provided above under conditions suitable for antibody expression, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0296] For the recombinant production of anti-αvβ8 antibodies, the nucleic acid encoding the antibody (e.g., as described above) is isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using routine procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody), or obtained through recombinant methods or chemical synthesis.

[0297] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. Antibodies can be generated in bacteria, for example, especially when glycosylation and Fc effector function are not required. For information on the expression of antibody fragments and peptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523. (See also Charlton, KA, in: Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2003), pp. 245-254, describing the expression of antibody fragments in *E. coli*.) Antibodies can be separated from the bacterial cell paste in a soluble fraction after expression and can be further purified.

[0298] Besides prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for antibody-encoding vectors. These eukaryotic microorganisms, including fungal and yeast strains, have "humanized" glycosylation pathways, resulting in antibodies with partial or complete human glycosylation patterns. See Gerngross, TU, Nat. Biotech. 22 (2004) 1409-1414; and Li, H. et al., Nat. Biotech. 24 (2006) 210-215.

[0299] Suitable host cells for expressing glycosylated antibodies also originate from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0300] Plant cell cultures can also be used as hosts. See, for example, US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978 and US 6,417,429 (which describe PLATNIBODIES™ technology for producing antibodies in transgenic plants).

[0301] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted for growth in suspension may be useful. Other examples of useful mammalian host cell lines include the monkey kidney CV1 line (COS-7) transformed with SV40; human embryonic kidney cell lines (such as 293 or 293T cells as described, for example, in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells described, for example, in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); Buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumors (MMT 060562); and TRI cells (such as those described, for example, in Mather, JP et al., Annals NY Acad.). (As described in Sci. 383 (1982) 44-68); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220); and myeloma cell lines such as Y0, NSO, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0302] On the one hand, the host cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., Y0, NSO, Sp20 cells).

[0303] D. Measurement

[0304] The physical / chemical properties and / or biological activity of the anti-αvβ8 antibody provided herein can be identified, screened, or characterized by various assays known in the art.

[0305] 1. Combining measurements with other measurements

[0306] In one aspect, the antibodies of the present invention are tested for their antigen-binding activity by known methods such as ELISA and Western blotting.

[0307] In another aspect, competitive assays can be used to identify antibodies that compete with any of the antibodies described herein for binding to αvβ8. In some aspects, such competitive antibodies bind to the same epitope (e.g., linear or conformational epitope) that is bound by other antibodies specific for αvβ8. Detailed exemplary methods for locating the epitope to which the antibody binds are provided in: Morris (1996), “Epitope Mapping Protocols”, in Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

[0308] In an exemplary competitive assay, immobilized αvβ8 is incubated in a solution containing a first labeled antibody (such as the antibody described herein) that binds to αvβ8 and a second unlabeled antibody (being tested for its ability to competitively bind to αvβ8 against the first antibody). This second antibody may be present in hybridoma supernatant. As a control, the immobilized αvβ8 is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions allowing the first antibody to bind to αvβ8, excess unbound antibody is removed, and the amount of label associated with the immobilized αvβ8 is measured. If the amount of label associated with the immobilized αvβ8 is substantially reduced relative to the control sample in the test sample, it indicates that the second antibody competitively binds to αvβ8 against the first antibody. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0309] In another aspect, in some embodiments, the anti-αvβ8 antibody described herein does not depend on the presence of a divalent cation to bind to αvβ8. In exemplary methods, the binding of anti-αvβ8 to αvβ8 (e.g., human αvβ8 or mouse αvβ8) is assessed by SPR (such as on a BIACORE® system) in the presence and absence of a divalent cation (e.g., MgCl2 or CaCl2).

[0310] 2. Activity Assay

[0311] In one aspect, an assay is provided for identifying the biological activity of anti-αvβ8 antibodies. Biological activity may include, for example, antitumor activity. Antibodies exhibiting such biological activity in vivo and / or in vitro are also provided.

[0312] In some respects, such biological activities of the antibodies of the present invention are tested.

[0313] In some embodiments, the anti-αvβ8 antibody described herein inhibits αvβ8-mediated activation of L-TGFβ1 and / or latent TGFβ-binding protein (LTBP) via human leucine-rich repeat protein 33 (LRRC33). Pro-TGFβ1 dimers in a large latent complex and forms disulfide bonds with LTBP or GARP. αvβ8 binding to the motif in the arm domain of pro-TGFβ1 is required for in vivo TGFβ1 activation.

[0314] E. Methods and compositions for diagnosis and detection

[0315] In some respects, any of the anti-αvβ8 antibodies provided herein can be used to detect the presence of αvβ8 in biological samples. As used herein, the term "detection" encompasses both quantitative and qualitative detection. In some respects, the biological sample comprises cells or tissues.

[0316] In one aspect, an anti-αvβ8 antibody is provided for use in a diagnostic or detection method. In another aspect, a method for detecting the presence of αvβ8 in a biological sample is provided. In some aspects, the method includes contacting the biological sample with the anti-αvβ8 antibody under conditions that allow both the anti-αvβ8 antibody and αvβ8 to bind, and detecting whether a complex has formed between the anti-αvβ8 antibody and αvβ8. Such methods can be in vitro or in vivo. In one aspect, the anti-αvβ8 antibody is used to select subjects eligible for treatment with the anti-αvβ8 antibody, for example, where αvβ8 is a biomarker used for patient selection.

[0317] In some respects, labeled anti-αvβ8 antibodies are provided. Labeling includes, but is not limited to, labels or portions for direct detection (such as fluorescence, chromogenicity, electron density, chemiluminescence, and radioactive labeling) and portions for indirect detection (e.g., via enzymatic reactions or molecular interactions) (such as enzymes or ligands). Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C 125 I, 3 H and 131I; fluorophores, such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone; luciferases, such as firefly luciferase and bacterial luciferase (US Patent No. 4,737,456); luciferin; dihydrophthalazinedione; horseradish peroxidase (HRP); alkaline phosphatase; β-galactosidase; glucosylamylase; lysozyme; sugar oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase; heterocyclic oxidases, such as urate oxidase and xanthine oxidase; conjugated with enzymes that use hydrogen peroxide to oxidize dye precursors (such as HRP, lactoperoxidase, or microperoxidase); biotin / antibiotin protein; spinning labeling; phage labeling; stable free radicals, etc.

[0318] F. Pharmaceutical Composition

[0319] In another aspect, pharmaceutical compositions comprising any of the antibodies provided herein are provided, for example, in any of the following treatment methods. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutical carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent, such as those described below.

[0320] The pharmaceutical compositions (formulations) of the anti-αvβ8 antibody described herein can be prepared by combining the antibody with a pharmaceutically acceptable carrier or excipient known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed., 1980), Shire S., Monoclonal Antibodies: Meeting the Challenges in Manufacturing, Formulation, Delivery and Stability of Final Drug Product, 1st edition. See Woodhead Publishing (2015), §4 and Falconer RJ, Biotechnology Advances (2019), 37, 107412. Exemplary pharmaceutical compositions of anti-αvβ8 antibodies as described herein are lyophilized, aqueous, frozen, etc.

[0321] Pharmaceutical carriers are generally non-toxic to recipients at the doses and concentrations used, including but not limited to: buffers such as histidine, phosphates, citrates, acetates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethyl diammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; m-cresol); and low molecular weight (less than about 10). (1 residue) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0322] The pharmaceutical compositions described herein may also contain more than one active ingredient essential for the specific indication being treated, preferably active ingredients having complementary activities that do not adversely affect each other. Such active ingredients are appropriately combined in amounts effective for the intended purpose.

[0323] Pharmaceutical compositions intended for internal administration are typically sterile. For example, sterility can be readily achieved through filtration using a sterile filter membrane.

[0324] G. Treatment methods and routes of administration

[0325] Any of the anti-αvβ8 antibodies provided in this article can be used in treatment methods.

[0326] In one aspect, an anti-αvβ8 antibody is provided for use as a medicament. In another aspect, an anti-αvβ8 antibody is provided for the treatment of cancers, including but not limited to glioblastoma multiforme (GBM), low-grade glioma, pheochromocytoma, adrenal carcinoma, ovarian cancer, melanoma, uveal melanoma, sarcoma, mesothelioma, clear cell renal cell carcinoma (ccRCC), thymoma, papillary RCC, germ cell carcinoma, diffuse large B-cell lymphoma (DLBCL), breast cancer (such as triple-negative breast cancer (TNBC)), non-small cell lung cancer (NSCLC), colorectal cancer, cholangiocarcinoma, endometrial cancer, papillary renal carcinoma, or bladder cancer. In some embodiments, the cancer is a cancer exhibiting increased expression of αvβ8 and decreased expression of αvβ6, such as compared to normal tissue or compared to cancers of the same type. For example, in some embodiments, after comparing two cancers of the same type (such as two breast cancers), the cancer with higher expression of αvβ8, lower expression of αvβ6, and / or a higher ratio of αvβ8 to αvβ6 is selected for treatment with an anti-αvβ8 antibody.

[0327] In some aspects, an anti-αvβ8 antibody is provided for use in treatment methods. In some aspects, the invention provides an anti-αvβ8 antibody for use in a method of treating an individual with cancer, including but not limited to ovarian cancer, triple-negative breast cancer (TNBC), non-small cell lung cancer (NSCLC), colorectal cancer, bile duct cancer, endometrial cancer, renal papillary carcinoma, or bladder cancer, the method comprising administering an effective amount of the anti-αvβ8 antibody to the individual. In one such aspect, as described below, the method further comprises administering an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents) to the individual. The "individual" according to any of the foregoing aspects is preferably a human being.

[0328] In another aspect, the present invention provides the use of anti-αvβ8 antibodies in the preparation or manufacture of pharmaceuticals. In one aspect, the pharmaceutical medicament is used to treat cancers such as glioblastoma multiforme (GBM), low-grade glioma, pheochromocytoma, adrenal carcinoma, ovarian cancer, melanoma, uveal melanoma, sarcoma, mesothelioma, clear cell renal cell carcinoma (ccRCC), thymoma, papillary RCC, germ cell carcinoma, diffuse large B-cell lymphoma (DLBCL), breast cancer (such as triple-negative breast cancer (TNBC)), non-small cell lung cancer (NSCLC), colorectal cancer, bile duct cancer, endometrial cancer, papillary renal carcinoma, or bladder cancer, the method comprising administering an effective amount of the pharmaceutical medicament to an individual suffering from cancer. In one such aspect, as described below, the method further comprises administering an effective amount of at least one other therapeutic agent to the individual. The “individual” according to any of the foregoing aspects can be a human being.

[0329] In another aspect, the present invention provides a method for treating cancers such as glioblastoma multiforme (GBM), low-grade glioma, pheochromocytoma, adrenal carcinoma, ovarian cancer, melanoma, uveal melanoma, sarcoma, mesothelioma, clear cell renal cell carcinoma (ccRCC), thymoma, papillary RCC, germ cell carcinoma, diffuse large B-cell lymphoma (DLBCL), breast cancer (such as triple-negative breast cancer (TNBC)), non-small cell lung cancer (NSCLC), colorectal cancer, cholangiocarcinoma, endometrial cancer, renal papillary carcinoma, or bladder cancer. In one aspect, the method includes administering an effective amount of anti-αvβ8 antibody to an individual suffering from cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC). In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is cholangiocarcinoma. In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is renal papillary carcinoma. In one embodiment, the cancer is bladder cancer. In one aspect, as described below, the method further includes administering an effective amount of at least one additional therapeutic agent to the individual.

[0330] The “individual” in any of the above aspects can be a person of any age, such as an adult.

[0331] In another aspect, the present invention provides a pharmaceutical composition comprising any of the anti-αvβ8 antibodies provided herein, for example, in any of the above-described treatment methods. In one aspect, a pharmaceutical composition comprising any of the anti-αvβ8 antibodies provided herein, and a pharmaceutical carrier. In another aspect, the pharmaceutical composition comprises any of the anti-αvβ8 antibodies provided herein and at least one additional therapeutic agent, for example, as described below.

[0332] The antibodies of the present invention can be administered alone or in combination therapy. For example, such combination therapy includes administering the antibody of the present invention and at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). In some aspects, the combination therapy includes administering the antibody of the present invention and at least one additional therapeutic agent, such as a PD-1 axis antagonist, such as a PD-1 binding antagonist or a PD-L1 binding antagonist, such as an anti-PD-1 antibody or an anti-PD-L1 antibody. In a preferred embodiment, the PD-1 axis antagonist is an anti-PD-L1 antibody, preferably atezolizumab.

[0333] Other therapeutic agents, such as PD-1 axis antagonists, may be administered before, concurrently with, or after the administration of the anti-αvβ8 antibody. As used herein, "concurrently" does not necessarily mean that the anti-αvβ8 antibody and the other therapeutic agent are present in the same composition. It should be understood that administration of the anti-αvβ8 antibody and the other therapeutic agent at similar times (such as on the same day) will be concurrent. "Before or after" administration of the anti-αvβ8 antibody may be at least 1 day before or after administration, such as at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, or longer before or after administration.

[0334] It should be understood that the anti-αvβ8 antibody and the one or more additional therapeutic agents in combination therapy may have different dosing regimens. Such combination therapies mentioned above encompass combined administration (where two or more therapeutic agents are contained in the same or separate pharmaceutical composition) and single administration, in which case the administration of the antibody of the present invention may occur before, simultaneously with, and / or after administration of the other one or more therapeutic agents (such as PD-1 axis antagonists, such as atezolizumab). In one aspect, administration of the anti-αvβ8 antibody and administration of the other therapeutic agent (such as a PD-1 axis antagonist) occur within about one month, or within about one week, two weeks, or three weeks, or within about one day, two days, three days, four days, five days, or six days. In one aspect, the antibody and the other therapeutic agent are administered to the patient on day 1 of treatment. The antibody of the present invention can also be used in combination with radiotherapy.

[0335] The antibody (anti-αvβ8 antibody) and any other therapeutic agents of the present invention may be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, as well as intralesional administration (if local treatment is required). Parenteral infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Administration may be carried out by any suitable route, such as by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is transient or long-term. Various dosing regimens are considered herein, including but not limited to single or multiple administrations at various time points, bolus administration, and pulsatile infusion.

[0336] The antibodies of this invention will be formulated, administered, and applied in accordance with good medical practice. In this context, factors considered include the specific disease to be treated, the specific mammal to be treated, the individual patient's clinical condition, the cause of the disease, the site of delivery of the agent, the method of administration, the administration regimen, and other factors known to the medical practitioner. The antibody is not mandatory but may optionally be formulated in conjunction with one or more formulations currently used for the prevention or treatment of the disease in question. The effective amount of these other formulations depends on the amount of antibody present in the pharmaceutical composition, the type of disease or treatment, and other factors discussed above. These are typically used at the same dosage and route of administration as described herein, or at about 1% to 99% of the dosage described herein, or at any dosage and via any route determined empirically / clinically to be appropriate.

[0337] For the prevention or treatment of disease, the appropriate dosage of the antibody of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, the patient's medical history and response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient once or in a series of treatments. For repeated administration over several days or longer, treatment will generally continue until the desired suppression of disease symptoms is achieved, depending on the condition. However, other dosage regimens may be available. The progress of this therapy can be easily monitored using conventional techniques and assays.

[0338] H. Products

[0339] In another aspect of the invention, an article is provided containing a substance that can be used to treat, prevent, and / or diagnose the aforementioned conditions. The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that, alone or in combination with another composition, is effective in treating, preventing, and / or diagnosing the condition, and the container may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. Furthermore, the article may include (a) a first container containing a composition comprising the antibody of the present invention; and (b) a second container containing a composition comprising additional cytotoxic agents or other therapeutic agents. The article of this aspect of the invention may also include a package insert indicating that the composition is used to treat a specific condition. Alternatively or additionally, the article may further include a second (or third) container comprising a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and glucose solution. The article may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0340] III. Examples

[0341] The following are examples of the methods and compositions of the present invention. It should be understood that various other embodiments may be practiced given the general description provided above.

[0342] Example 1: Generation of rabbit anti-αvβ8 monoclonal antibody

[0343] The workflow for screening αvβ8 antibodies is illustrated in Figure 1. New Zealand white (NZW) rabbits were co-immunized with recombinant human αvβ8 protein and a self-made mouse αvβ8 (PUR1BX42109). Single B cells were isolated using a protocol relevant to published literature. See, for example, Lin et al., Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology, PLoS ONE 15(12), 2020. This workflow involved direct FACS sorting of IgG+, human and / or mouse αvβ8+ B cells (a total of 9788 B cells) into single wells. The B cells were cultured for 7 days, and the binding of the supernatant to both human and mouse αvβ8 was determined by ELISA, FACS, and SPR. For ELISA screening, the supernatant was measured for binding to both human and mouse αvβ8, and reverse screening was performed for other human integrins (including αvβ1, 3, 5, and 6). In addition to 45 human-specific B cells and 162 mouse-specific B cells, 805 B cells also bound to both human and mouse αvβ8 proteins. ELISA-positive B cells were lysed and immediately cryopreserved at -80°C until molecular cloning. Subsequently, the ELISA hu / mu double-positive B cell supernatant was screened by FACS analysis for binding to LN229 (human cells endogenously expressing αvβ8) and TRAMPC2-H1 (mouse cells endogenously expressing αvβ8). FACS analysis further narrowed down to 215 B cell clones.

[0344] Using a BIACORE® 8K instrument, the supernatant of those 215 B cells was further screened for SPR binding affinity. In short, rabbit B cell supernatant was first captured on a protein A sensor chip. Then, both human and mouse αvβ8 solutions were injected through a flow cell at a concentration of 100 nM. Phenylephrine was selected for its affinity for human and mouse cells at 5 nM or less. D Equally bound clones were used for molecular cloning. As previously described, variable regions (VH and VL) of various monoclonal antibodies derived from rabbit B cells were cloned into expression vectors derived from extracted mRNA. Single recombinant rabbit antibodies were expressed in Expi293 cells and subsequently purified with protein A.

[0345] Sprague Dawley rats (Charles River, Hollister, CA) were separately immunized at multiple sites with recombinant human and mouse αvβ8 protein dissolved in a detergent containing CFA (Sigma-Aldrich, St. Louis, MO) or a mixture of MPL+TDM adjuvant (Sigma-Aldrich, St. Louis, MO) or a combination of the following TLR agonists: 50 µg MPL (Sigma-Aldrich), 20 µg R848 (Invivogen, San Diego, CA), 10 µg PolyI:C (Invivogen), and 10 µg CpG (Invivogen). Additional 6- to 8-fold boosters were performed every two weeks. Following immunization, enriched B cells from lymph nodes were FACS-sorted and cultured as single avb8-positive cells as previously described (Marei H et al., Nature 610 (7930):182-9 (2022)). A total of 10,752 individual B cells were sorted, and the supernatant was analyzed for ELISA binding against human and mouse avb8 recombinant protein (154 clone-positive) or cell lines expressing human or mouse avb8 (75 clone-positive). RNA was extracted from B cells showing FACS binding for molecular cloning and recombinant expression.

[0346] For immunization, the full-length extracellular domains (head and legs) are used for both αV (residues M1 to V992) and β8 (residues M1 to R684). Heterodimerization is induced in the absence of the transmembrane domains of αV and β8 by fusing acidic / basic loops to the C-terminus of the αV and β8 ECDs, respectively. αvβ8-positive (αvβ8+) immunoglobulin G-positive (IgG+) monocellular B cells are isolated. Hu αvβ8+ / IgG+ B cells are fused with LN229 cells, and mu αvβ8+ / IgG+ B cells are fused with TRAMPC2-H1 cells. The B cell supernatant is then collected and antigen-specific (αvβ8+, αVβ1-, αVβ3-, αVβ5-, αVβ6-) antibodies are screened by ELISA, FAC, and BIACORE®.

[0347] Purified anti-αvβ8 antibodies were screened based on their binding affinity to αvβ8, selectivity for αvβ8, epitope characterization, and cell-based functional activity.

[0348] αvβ1 binding assay using rabbit αvβ8 antibody

[0349] The binding affinity of the antibodies was determined using a BIAcore™ T200 instrument. For kinetic measurements, antibodies were captured on a research-grade Protein A chip (Cytiva, USA) to achieve approximately 60 RU. A 10-fold serial dilution of human and cynomolgus aVb8 was injected at 100 μL / min into the same running buffer as described above at 37°C. Binding (ka) and dissociation (kd) were calculated using a 1:1 Langmuir binding model (BIAcore™ T200 evaluation software version 2.0). The equilibrium dissociation constant (KD) was calculated as the ratio kd / ka. No binding with human αVβ1, αVβ3, αVβ5, or αVβ6 was observed, confirming the specificity of those antibodies.

[0350] To determine the ability of rabbit αvβ8 antibody to block αvβ8-dependent L-TGFβ1 activation. LN-229 cells expressing αvβ1 were co-cultured with 3T3-Nano luciferase TGFβ reporter cells expressing the cell surface molecule GARP (which binds to TGFβ1) and human TGFβ1.

[0351] Figures 2A and 2B show the IC50 (nM) values ​​of the rabbit αvβ8 antibody. Compared with mAb C6D4, the rabbit αvβ8 antibody has a lower IC50 value.

[0352] Cross-reactivity of rabbit αvβ8 antibody with cynomolgus monkey, human, or mouse αvβ8

[0353] An array-based SPR imaging system (Carterra USA) was used for epitope binning of 20 of the most potent rabbit monoclonal antibodies, including ADWA11-2.4 and C6D4. Purified antibodies were diluted to 10 µg / ml in 10 mM sodium acetate buffer at pH 4.5. Using amine conjugation, antibodies were directly immobilized onto an SPR sensorprism CMD 200M chip (XanTec Bioanalytics, Germany) using a Continuous Flow Microspotter (Carterra, USA). For analysis, the binding of analytes to immobilized ligands was evaluated using an IBIS MX96 SPRi (Carterra USA). Human αvβ8 was first injected at 50 nM for 4 minutes, followed by a second 4-minute injection of individual monoclonal antibodies at 10 µg / ml. Between cycles, surface regeneration was performed with 10 mM glycine at pH 1.5. The experiment was conducted at 25°C in a running buffer consisting of 0.01M HEPES (pH 7.4), 0.15M NaCl, 0.05% surfactant P20, and 0.5mM CaCl2. Tabletop binning data were processed using Carterra binning software.

[0354] The relative binding of rabbit αvβ8 antibody to cynomolgus monkey, human, or mouse αvβ8 was determined. Binding assays were performed by SPR. The results of three separate binding assays are shown in Table 8 and Figures 3A to 3C. The relative binding of commercial human C6D4 mIgG2aLALALG control antibody and rabbit αvβ8 antibody was evaluated in the presence of divalent cations (Figure 3D).

[0355] Table 8: Rabbit αvβ8 antibody binding assay

[0356]

[0357] Rabbit αvβ8 antibodies rb.αvβ8-65 and rb.αvβ8-92 bind to human and mouse αvβ8 and commercial αvβ8 antibody huC6D4 (Figures 3A to 3C).

[0358] Example 2: Humanization of rabbit αvβ8 antibody

[0359] After screening as described in Example 1, monoclonal antibodies with inhibitory activity were selected for further characterization. Antibodies with the strongest binding affinity, inhibitory activity, and fewest manufacturing problems, named αvβ8-65 and αvβ8-92, were selected for humanization.

[0360] The rabbit monoclonal antibodies αvβ8-65 and αvβ8-92 were humanized as described below. The residue numbers are based on Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991).

[0361] The variants constructed during the humanization of rb.αvβ8-65 and rb.αvβ8-92 were evaluated as human IgG. Hypervariable regions of each rabbit antibody (i.e., positions 24–34 (L1), 50–56 (L2), and 89–97 (L3) in the VL domain and positions 26–35 (H1), 50–65 (H2), and 95–102 (H3) in the VH domain) were transplanted into their respective receptor frames. Specifically, for αvβ8-65, the VL CDR was transplanted into KV1-12*01, and the VH CDR was transplanted into HV3-23*01. Additionally, all VL and VH cursor positions from the rabbit antibodies were transplanted into their corresponding human germline frames. A graft containing all rabbit amino acids at the cursor position is called H1L1 (hu.αvβ8-65.H1L1).

[0362] The binding affinity of the hu.αvβ8.H1L1 antibody was compared with that of their chimeric parent clones. Rabbit cursor positions of the H1L1 antibody were converted back to human residues to assess the contribution of each rabbit cursor position to the binding affinity of human αvβ8. An additional light chain variant L2 (CDR graft) and ten additional heavy chain variants H2-H11 were prepared. Based on the binding affinity assessment of the above variant antibodies, Gly49 on the heavy chain was identified as a key rabbit cursor residue (data not shown). G49, along with the CDR residue, was also transplanted into the human germline HV3-23*01 as H15. The final humanized sequence of αvβ8-65 is hu.αvβ8-65.H15L2.

[0363] For rb.αvβ8-92, the VL CDR was transplanted into KV4-1*01, and the VH CDR was transplanted into both HV3-33*02 and HV3-23*02. Additionally, all VL and VH cursor positions from the rabbit antibody were transplanted into their corresponding human germline frames. The graft with all rabbit amino acids at the cursor position was designated H1L1 (hu.αvβ8-92.H1L1). Similar to the above description for αvβ8-65, all VL and VH cursor positions from the rabbit antibody were transplanted into their corresponding human germline frames. Nine additional heavy chain variants, H2-H10, were prepared for HV3-33:H2-10. For the light chain, based on binding affinity assessments of the aforementioned variant antibodies, all three cursor residues, Ala2, Leu4, and Arg68, were identified as key rabbit residues (data not shown). For the heavy chain, based on the binding affinity assessment of the aforementioned variant antibodies, Gln2, Ile48, Gly49, Ser73, Phe91, and Pro105 were identified as key rabbit residues (data not shown). These residues, along with the CDR residues, were also transplanted into the human line HV3-23*02 as H13. The final humanized sequence of αvβ8-92 is hu.αvβ8-92.H13L1.

[0364] The sequences of the humanized and rabbit αvβ8 antibodies are provided in Tables 1 to 5 of this specification, and sequence alignments of the light chain variable region and heavy chain variable region sequences between the humanized rabbit αvβ8 antibodies are provided in Figures 4A and 4B.

[0365] Structure and interaction of rabbit anti-αvβ8-65 and αvβ8 The αvβ8 FAB was subjected to cryo-EM structural determination to assess the antibody structure. The FAB fragment comprising both the heavy and light chains of FAB 68 (Cormier NSMB 2018PMID 30061598) was expressed in CHO cells. The protein was purified using Protein G chromatography resin followed by size exclusion chromatography and dialysis. The final sample concentration was 5 mg / mL in 20 mM histidine acetate, 0.15 M NaCl, pH 5.5.

[0366] The FAB fragment (Cormier NSMB 2018 PMID30061598), including both the heavy and light chains of FAB 8B8, was expressed in CHO cells. The protein was purified using Protein G chromatography resin followed by size exclusion chromatography and dialysis. The final sample concentration was 5.6 mg / mL in 20 mM histidine acetate, 0.15 M NaCl, pH 5.5.

[0367] FAB fragments, including both the heavy and light chains of FAB 65 (anti-ITGB8.αvβ8-65), were expressed in Expi293 cells. The protein was purified using Protein G chromatography resin followed by size exclusion chromatography and dialysis. The final sample concentration was 3.47 mg / mL in 20 mM sodium acetate, 150 mM NaCl, pH 4.5.

[0368] Recombinant human αvβ8 was obtained by co-expressing residues M1-V992 of human integrin subunit α (αV) and residues M1-R684 of human integrin subunit β8 (b8) in HEK293 cells. To promote the formation and purification of αvβ8 heterodimers, acidic rings and Strep tags were used. ® The protein was fused to the C-terminus of the αV domain, and the basic loop and hexahistine tag were fused to the C-terminus of the b8 domain. The protein was purified using immobilized metal affinity chromatography and size exclusion chromatography. The final protein concentration was 2.6 mg / mL in 20 mM histidine acetate, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 5.5.

[0369] The purified αvβ8 + FAB 65 + FAB 68 + FAB 8B8 complex (4 µL) was applied to a glow discharge grid (UltraAuFoil) with a porous gold film using a Vitrobot™ (Thermo Fisher Scientific) vitrification robot. ® The images were frozen by immersion in liquid ethane. A stack of 16,093 images was collected using a Titan Krios equipped with a Falcon4 detector (0.731 Å pixel size; Thermo Fisher Scientific; Waltham, MA). The images were processed using the software packages cryoSPARC™ Live, cryoSPARC™ (Structura Biotechnology; Toronto, ON), and cisTEM (Grant et al. 2018) to obtain the final 3D map with an estimated resolution of 2.45 Å.

[0370] The coordinates of the published structure of αvβ8 (PDB code 6UJB) from the Protein Database (PDB) have been mapped to the map, as have the initial Fab model generated using the Swiss model. The coordinates were then refined through iterative interactive reconstruction using a crystal-oriented toolkit (PMID 20383002) and real-space refinement (PMID 31588918).

[0371] FAB 68 and FAB 8B8 (Cormier NSMB 2018 PMID 30061598) were included in the sample formulation to act as structural escort proteins and facilitate high-resolution structural determination using cryo-EM. Neither FAB 68 nor FAB 8B8 alters the function of αvβ8 (Cormier NSMB 2018 PMID 30061598). The αvβ8 + FAB 65 + FAB 68 + FAB 8B8 complex was synthesized by mixing 60 μL of αvβ8 with 30 μL of FAB 65, 20 μL of FAB 68, and 20 μL of FAB 8B8 and incubating on ice for 2 h. The stoichiometric complex was then separated by pooling appropriate peak fractions from size exclusion chromatography in 25 mM Hepes, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, pH 7.2 (data not shown).

[0372] Cryo-EM analysis of the αvβ8 + FAB 65 + FAB 68 + FAB 8B8 sample allowed for three-dimensional reconstruction of the αvβ8 + FAB 65 + FAB 68 + FAB 8B8 complex at 2.4 Å resolution, thus clearly defining the Fab65 epitope on human αvβ8 (data not shown). Comparison of the obtained αvβ8 + FAB 65 + FAB 68 + FAB 8B8 complex structure with the published structure of the human αvβ8 complex with porcine latent transforming growth factor β1 (L-TGFb1) (PDB ID 6UJA, PMID 31955848 – Campbell et al. 2020 Cell 180: 490) confirmed that FAB 65 blocks the entry of L-TGFb1, thereby preventing the binding of L-TGFb1 to αvβ8 (data not shown).

[0373] Analysis of the interaction interface between FAB 65 and αvβ8 indicates that the buried surface area on αVβ8 is approximately 1200 Å. 2Both the variable heavy chain and the variable light chain of FAB 65 contribute to this. The CDRH2, CDRH3, CDRL1 and CDRL3 rings of FAB 65 bind at the ligand-binding gap between αV and β8, where the RGDLXXI / L co-epitome of L-TGFb1 (also known as the integrin-binding epitome) also binds (data not shown).

[0374] Specifically, residues on the CDRH2 ring of FAB 65 bind to the specificity-determining ring 1 (SDL1) of β8 and the α head region of αV, thereby interacting with the αVβ8 region, similar to the L-TGFb1 integrin-binding motif (data not shown). Residues from the CDRL1 and CDRL3 rings of FAB 65 form a broad network of polar interactions with the specificity-determining ring 2 (SDL2) of β8 and the α head region of αV (data not shown). In summary, the interactions formed between FAB 65 and αVβ8 further rationalize how the binding of FAB 65 to αVβ8 prevents the binding of L-TGFb1 or L-TGFβ3 to αVβ8.

[0375] Furthermore, Figures 5A through 5D illustrate the results of cryo-EM, highlighting the interaction between rb.αVβ8-65 (anti-αVβ8 integrin antibody) and αVβ8 integrin (Figures 5A and 5B, rotated 90° relative to Figure 5A) and the interaction between latent TGFβ1 (L-TGFβ1) and αVβ8 integrin (Figures 5C and 5D, rotated 90° relative to Figure 5A)). Figures 5A through 5D demonstrate that Fab65 binding blocks the interaction between latent TGFβ1 and αVβ8. Figures 5C and 5D show the location of the L-TGFb1 RGDLXXI / L motif inserted into the interface between the αV and β8 subunits. Figures 5A and 5B show that FAB65 occupies a similar position to L-TGFb1 relative to αVβ8, thus effectively blocking the L-TGFb1 binding site.

[0376] Figures 5E to 5H are enlarged views of the interface between hu.αVβ8-65 (anti-αVβ8 integrin antibody) and αVβ8 integrin (Figures 5E and 5G), and the interface between L-TGFβ1 and αVβ8 integrin (Figures 5F and 5H). Several residues in αVβ8 integrin that interact with hu.αVβ8-65 or L-TGFβ1 are highlighted. Figures 5E and 5G show that residues F177 and D218 of αV specifically contact CDRH2; K119, Q120, E121, and D148 of αV specifically contact CDRL1; N219 of β8 specifically contacts CDRH2; and R164 of β8 specifically contacts CDRL1.

[0377] Figures 5I to 5K are enlarged views of the interface between hu.αVβ8-65 (anti-αVβ8 integrin antibody) and αVβ8 integrin, showing the salt bridge formed between hu.αVβ8-65 (anti-αVβ8 integrin antibody) and αVβ8 integrin. Specific residues are highlighted.

[0378] Figure 5L shows the sequences of the αV and β8 subunits of αVβ8 and the EM structure of the interface between hu.αVβ8-65 (anti-αVβ8 integrin antibody) and αVβ8 integrin. Residues located within 5 Å of hu.αVβ8-65 are highlighted in the sequences of the αV subunits of αVβ8 (i.e., R115, 118M, 119K, 120Q, 121E, 123E, 147I, 148D, 149A, 150D, 154F, 177F, 178Y, 180Q, 212T, 213A, 214Q, 215A, and 218D) and the β8 subunits (i.e., 118H, 119N, 122E, 158I, 159S, 160I, 164R, 166H, 169C, 170S, 171D, 172Y, 206G, 207N, and 208I) and depicted as putative αVβ8 epitopes bound by Fab65.

[0379] Figure 5M shows the subtype specificity assessment of the binding between Fab65 and αV compared to other αV integrins.

[0380] Figure 5N shows the isotype specificity assessment of the binding between Fab65 and β8 compared to other β8 integrins.

[0381] The aim of this study was to define the interaction site between human αVβ8 and the FAB 65 antibody fragment that blocks TGFb1. Size exclusion chromatography revealed that αVb8 and FAB 65 form a 1:1 stoichiometric complex. Cryo-EM structural analysis of the αVβ8-FAB65 complex indicated that FAB 65 binds directly to αVb8, thereby spatially blocking the binding of TGFb1 to αVβ8.

[0382] Baculovirus (BV) conjugation assays were used to screen rabbits and humans for αVβ8-65 and αVβ8-92. One approach to reducing the number of antibodies with rapid nonspecific clearance (CL) is to screen for general nonspecific binding of antibodies using a baculovirus (BV) binding assay (Hötzel, I. et al., (2012) mAbs 4(6):753–760; Yadav, DB et al., (2015) J. Biol. Chem. 290:29732-29741, WO 2013 / 177470). In short, antibodies can be screened in an ELISA for binding to baculovirus particles, lysates, or antigens. The result is a baculovirus score (BV score). In some embodiments, an antibody is selected if the uptake of the antibody by a macrophage population is equal to or less than a predetermined threshold and the BV score is less than any of about 1, 2, 3, 4, or 5. Antibodies that bind nonspecifically to baculovirus may have a shorter half-life in vivo. Rabbit and humanized αVβ8-65 and αVβ8-92 passed the BV ELISA test. The BV test scores can be found in Table 9 below.

[0383] Table 9: BV ELISA Test Scoring

[0384]

[0385] Screening of humanized αVβ8-65 and αVβ8-92 cells using TGFβ reporter cellsThe blocking activity of the anti-αVβ8 antibody against the activation of latent TGF-β1 was evaluated using a co-culture of LN 229 cells with TGFβ reporter cells overexpressing human GARP and latent TGF-β1. LN 229 cells derived from human glioblastoma were maintained in high-glucose Durbeco modified Igor medium (DMEM) containing 10% fetal bovine serum (FBS; VWR; Brisbane, CA), 2 mM L glutamine, 100 IU / mL penicillin, and 100 µg / mL streptomycin at 37 ± 0.5 °C and 5% CO2. TGF-β reporter cells derived from the 3T3 fibroblast cell line were transfected with the SMAD-inducible NanoLuc® luciferase reporter gene and a constitutively expressed firefly luciferase gene. Further stabilization transfection was performed using human GARP and latent TGF-β1. Cells were maintained in high-glucose DMEM supplemented with 10% FBS and 2 mM glutamine at 37 ± 0.5 °C and 5% CO2, and incubated with penicillin (100 IU / mL), streptomycin (100 µg / mL), genomicamycin (500 µg / mL), and hygromycin (200 µg / mL).

[0386] Endogenous expression of integrin αvβ8 on the surface of LN 229 cells binds to and activates latent TGF-β1 presented on the surface of TGFβ reporter cells by GARP. This activation causes TGF-β1 to bind to its cell surface receptor on the reporter cell, which signals via pSMAD and leads to the production of NanoLuc® luciferase. Constitutively expressed firefly luciferase was used for data normalization. The quantities of NanoLuc® luciferase and firefly luciferase were assessed using the NanoGlo® Dual Luciferase® reporter assay system (Promega; Madison, WI).

[0387] On the day of assay, LN 229 cells were seeded at a density of 40,000 cells / well into 96-well, flat, clear-bottomed, white polystyrene, tissue culture-treated microplates (Corning; New York, NY) by adding 42 µL of cells diluted in test medium (high glucose DMEM, supplemented with 10% heat-inactivated FBS, 100 units / mL penicillin, 100 µg / mL streptomycin, and 2 mM glutamine). Anti-αvβ8 antibody was diluted to 100 µg / mL (333.3 nM) for antibody screening, using a single dose or serially diluted 4-fold up to 11 times in test medium. Subsequently, 8 µL of diluted antibody was added to LN229 cells and incubated for 30 minutes (the first 10 minutes in a biosafety cabinet, followed by incubation at 37°C in a 5% CO2 atmosphere). TGFβ reporter cells were then seeded into plates at a density of 20,000 cells / well by adding 30 µL of cells (667,000 cells / mL). The plates were incubated at 37°C in a 5% CO2 atmosphere for 18 to 20 hours.

[0388] To measure the levels of NanoLuc® and firefly luciferase, ONE Glo™ EX reagent (Promega) was preheated to room temperature and 80 µL was added to each well of the cells. After incubation at room temperature for 20 minutes with vigorous stirring, firefly luciferase luminescence was measured using an EnSight® multimode plate reader (PerkinElmer; Waltham, MA). NanoDLR™ Stop & Glo® reagent (80 µL; Promega) was then added to each well. After incubation at room temperature for 20 minutes with vigorous stirring, NanoLuc® luciferase luminescence was measured using an EnSight® multimode plate reader. The NanoLuc® luciferase signal was normalized to the firefly signal and multiplied by 1000. The half-maximum inhibitory concentration (IC50) was calculated from the titration curve using the Prism [inhibitor] with response-variable slope (four parameters) model (GraphPad Software; San Diego, CA). The percentage of inhibition was calculated using the following equation: %inhibition = 100 x [1 - (X - MIN) / (MAX - MIN)], where MIN and MAX are the normalized NanoLuc® luciferase signals from individual reporter cells and reporter cells co-cultured with LN 229 cells in test medium, respectively.

[0389] Humanized αVβ8-65 (aVb8-65.H15L2.hIgG1.LALAPG) retains high binding affinity for human, cynomolgus monkey, and mouse αVβ8. See Table 10 and Figures 6A to 6B.

[0390] Table 10: Humanized αVβ8-65 combined with measurement results

[0391]

[0392] Humanized αVβ8-92 (PARS-20463-65 (IH) aVb8-92.H13L1.hIgG1.LALAPG) retains high affinity for human, cynomolgus monkey and mouse αVβ8, as shown in Table 11 and Figures 7A to 7B.

[0393] Table 11: Humanized αVβ8-92 combined with measurement results

[0394]

[0395] Evaluation of the binding of humanized αVβ8-65 antibody to recombinant human, cynomolgus monkey, mouse, and rat αVβ8 protein.Humanized αVβ8-65 yielded a 10.29 mg / mL solution in 20 mM histidine acetate (pH 5.5) and 150 mM NaCl. Recombinant human (PARS-17102), cynomolgus monkey (PARS-19393), mouse (PARS-17103), and rat (PARS-22180) αVβ8 proteins were prepared and stored at -80°C.

[0396] The ability of αVβ8-65 to bind to different αVβ8 proteins was evaluated using SPR measurements (Karlsson et al. 1991) on a Biacore™ T200 (Cytiva Life Sciences; Marlborough, MA) instrument (Säfsten et al. 2006). Anti-αVβ8-65 was first captured on a Biacore™ Protein A biosensor chip (Cytiva Life Sciences) to achieve approximately 60 reaction units. Binding measurements were performed using a run buffer consisting of 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, and 0.005% surfactant P20 (Cytiva Life Sciences). A series of 3-fold dilutions of the analyte protein (range, 0 to 100 nM in the run buffer) were injected into the Biacore™ T200. All infusions were performed over 180 seconds, with a dissociation time of 1200 seconds, at a flow rate of 100 µL / min and a temperature of 37°C. Between sample infusions, 10 mM glycine (pH 1.5) was injected twice to regenerate the sensor chip (each injection was performed at 10 µL / min over 30 seconds). To determine the binding kinetics and affinity constants of anti-αVβ8-65 with various αVβ8 proteins, the signal from the reference flow cell (FC1, which appeared to contain only protein A) was "double-referenced" by subtracting the signal observed after sample infusion into FC1, and then subtracting the signal observed after infusion of the run buffer alone. The kinetic constants of anti-αVβ8-65 binding to recombinant human, cynomolgus monkey, mouse, and rat αVβ8 proteins were calculated using nonlinear regression fitting of the data based on the 1:1 Langmuir binding model using Biacore™ evaluation software (Cytiva Life Sciences).

[0397] The anti-αVβ8-65 antibody binds to recombinant human, cynomolgus monkey, mouse and rat αVβ8 protein with high affinity, and the mean KD values ​​determined using a 1:1 binding model are 0.50, 0.7, 0.94 and 1.0 nM, respectively, as shown in Table 12 and Figure 8.

[0398] Table 12: Humanized αVβ8-65 combined with measurement results

[0399]

[0400] Example 3: Affinity of anti-αVβ8 monoclonal antibody to human αVβ8

[0401] The ability of humanized αvβ8-65, hC6D4, and hADWA11-2.4 to bind to human aVb8 integrin protein was evaluated using SPR measurements (Karlsson et al. 1991) on a Biacore™ T200 (Cytiva Life Sciences; Marlborough, MA) instrument (Säfsten et al. 2006). All antibodies tested in the assay were internally generated. Materials were stored in a refrigerator set to maintain a temperature range of 2°C to 8°C prior to use in the study.

[0402] In SPR-based biosensors, changes in the refractive index angle near a surface are monitored and converted into a measured response signal. If a protein target (“ligand”) is covalently immobilized on the sensor chip surface, SPR can be used to monitor the non-covalent interactions of binding partners (“analytes”) injected onto the surface. Those “real-time” measurements of analyte binding can be used to determine both the kinetics and affinity of the interactions.

[0403] First, the antibody was captured on a Biacore™ Protein A Biosensor Chip (Cytiva LifeSciences) or an anti-mouse Fc chip to achieve approximately 60 reaction units. Binding measurements were performed using a run buffer consisting of 10 mM HEPES (pH 7.4), 150 mM NaCl, 0.5 mM CaCl2, and 0.005% surfactant P20 (Cytiva Life Sciences). For comparisons between αvβ8-65 and hC6D4, a 5-fold series of human αvβ8 dilutions (range, 0 to 100 nM in run buffer) were injected into the Biacore™ T200. For comparisons between αvβ8-65 and hADWA11-2.4, a 5-fold series of human αvβ8 dilutions (range, 0 to 100 nM in run buffer) were injected. To compare all three antibodies in the mouse Fc form, a 5-fold dilution series of human αvβ8 (range, 0 to 50 nM in running buffer) was injected. All injections were performed over 180 seconds, with a dissociation time of 1200 seconds, a flow rate of 100 µL / min, and a temperature of 37°C. Between sample injections, regeneration reagents (10 mM glycine pH 1.5 for protein A chips; 10 mM glycine pH 1.7 for anti-mouse Fc chips) were injected to regenerate the sensor chips (twice, 10 µL / min for 30 seconds each time). To determine the binding kinetics and affinity constants of the antibody to the human αvβ8 protein, the signal from the reference flow cell (FC1, which appears to contain only protein A) was "double-referenced" by subtracting the signal observed after sample injection into FC1 and then subtracting the signal observed after injection of running buffer alone. Based on the 1:1 Langmuir combination model, the kinetic constants were calculated using nonlinear regression fitting of the data with Biacore™ evaluation software (Cytiva Life Sciences).

[0404] The kinetic constants kon, koff, and KD for the binding of αvβ8-65, hC6D4, and ADWA11-2.4 to various αvβ8 proteins are summarized in Table 13. The KD value was determined using a 1:1 Langmuir binding model. The observed sensing patterns for the binding of various αvβ8 proteins to the captured antibodies are illustrated in Figures 9A, 9B, and 10. The solid lines superimposed on the experimental curves in the figures represent the results of analyses performed using the 1:1 binding model, indicating that the 1:1 binding model is sufficient to describe the interaction.

[0405] Table 13: Kinetics and affinity constants of the binding of recombinant human αvβ8 protein to captured αvβ8-65, hC6D4, and hADWA11-2.4, as determined by surface plasmon resonance at 37 °C.

[0406]

[0407] Example 4: The efficacy of anti-αVβ8 monoclonal antibodies against human αVβ8

[0408] All antibodies were of the mIgG2a.LALAPG isotype. All antibodies tested in the assay were stored in a refrigerator set to maintain a temperature range of 2°C to 8°C.

[0409] LN-229 cells, derived from human glioblastoma and endogenously expressing αvβ8, were obtained from the Genentech Cell Bank (gCell). Cells were maintained in high-glucose Durbeco-modified Igor medium (DMEM) containing 10% fetal bovine serum, 2 mM L glutamine, 100 IU / mL penicillin, and 100 μg / mL streptomycin at 37 ± 0.5 °C under a 5% CO2 atmosphere.

[0410] 3T3-Nano cells are a TGFβ reporter cell line generated through stable transfection of 3T3 cells obtained from gCell. These cells are derived from a mouse fibroblast cell line and possess SMAD-inducible NanoLuc cells. ® Luciferase reporter gene and constitutively expressed firefly luciferase gene. Cells were maintained in high-glucose DMEM supplemented with 100 IU / mL penicillin, 100 µg / mL streptomycin, and 500 µg / mL genomicamycin, containing 10% FBS and 2 mM glutamine, and incubated at 37 ± 0.5 °C and 5% CO2.

[0411] 3T3-Nano-human GARP / LTGF-β1 (WT) reporter cells were used to determine the potency of antibodies that block integrin αvβ8-mediated TGF-β1 (WT) activation and were generated by stable transfection of 3T3-Nano cells with human GARP and latent TGF-β1 (WT).

[0412] 3T3-Nano-human GARP / LTGF-β1 (non-releasable, NR) reporter cells were generated by transiently transfecting 3T3-Nano cells with human GARP and latent TGF-β1 (NR) to determine the potency of antibodies that block integrin avb8-mediated LTGF-β1 (NR) activation.

[0413] 3T3-Nano-human GARP / LTGF-β3 (WT) reporter cells were used to determine the potency of antibodies that block integrin avb8-mediated activation of latent TGF-β3 (WT) and were generated by transiently transfecting 3T3-Nano cells with human GARP and latent TGF-β3 (WT).

[0414] LN-229 cells endogenously expressing integrin αvβ8, along with co-cultures of TGFβ reporter cells, 3T3-Nano-human GARP / LTGF-β1 (WT), 3T3-Nano-human GARP / LTGF-β1 (NR), or 3T3-Nano-human GARP / LTGF-β3 (WT), were used to evaluate the blocking activity of antibodies against integrin αvβ8-mediated activation of LTGF-β1 (WT), LTGF-β1 (NR), or LTGF-β3 (WT)β. Expression of integrin αvβ8 on the surface of LN-229 cells can bind to and activate latent TGF-β1 (WT or NR) and latent TGF-β presented on the surface of TGFβ reporter cells by GARP. This activation causes TGF β1 (WT or NR) or LTGF β3 (WT) to bind to its cell surface receptor on the reporter cell, which signals via pSMAD and leads to NanoLuc... ® Production of luciferase. Constitutively expressed firefly luciferase is used for data normalization. NanoLuc ® The amounts of luciferase and firefly luciferase were measured using Nano Glo ® Dual Luciferase ® The assessment was conducted using the Report Measurement System (Promega; Madison, WI).

[0415] On day 1 of the assay, 6 x 10⁶ g of the medium (high glucose DMEM containing 10% FBS, 2 mM L glutamine and 500 µg / mL gemcitabine) was added to 12 mL of culture medium. 63T3-Nano cells were seeded into T75 cell culture flasks (Corning; New York, NY) and incubated at 37°C in an incubator with a 5% CO2 atmosphere for 20 to 24 hours.

[0416] On day 2 of the assay, 3T3-Nano cells were washed with 12 ml of transfection medium (high glucose DMEM containing 10% FBS and 2 mM glutamine) and then transfected with human GARP and latent TGF β1 (NR) expression vector (3T3-Nano-human GARP / LTGF β1(NR)) or human GARP and latent TGF β3 (WT) (3T3-Nano-human GARP / LTGF β3(WT)) using Lipofectamine 3000 (Thermo Fisher Scientific; Waltham, MA) according to the manufacturer's instructions. The flasks were incubated at 37°C in a 5% CO2 atmosphere for 22 to 24 hours.

[0417] On day 3 of the assay, LN-229 cells were seeded at a density of 40,000 cells / well into 96-well, flat, clear-bottomed, white polystyrene, tissue culture-treated microplates (Corning; New York, NY) by adding 42 µL of cells diluted in the test medium (high glucose DMEM, supplemented with 10% heat-inactivated FBS, 100 units / mL penicillin, 100 µg / mL streptomycin, and 2 mM glutamine). The test material was diluted to a concentration of 500 µg / mL (3333.3 nM) and serially diluted 4-fold in phosphate-buffered saline (PBS) to 11 dilution points. Subsequently, 8 µL of diluted antibody or PBS was added to LN-229 cells and incubated for 30 minutes (the first 10 minutes in a biosafety cabinet, followed by incubation at 37°C in a 5% CO2 atmosphere). Then, reporter cells, 3T3-Nano-human GARP / LTGF β1(WT), 3T3-Nano-human GARP / LTGF β1(NR), or 3T3-Nano-human GARP / LTGF β3(WT) cells were seeded into assay plates at a density of 20,000 cells / well by adding 30 µL of cells (667,000 cells / mL). The plates were incubated at 37°C in a 5% CO2 atmosphere for 18 to 20 hours.

[0418] On day 4 of the assay, NanoLuc was measured separately. ® The levels of latent TGF-β1 (WT or NR) or latent TGF-β3 (WT) activation and TGF-β reporter cell normalization were determined by the amount of firefly luciferase. ONE Glo™ EX reagent (Promega) was preheated to room temperature, and 80 µL was added to each well. After incubation at room temperature for 20 minutes with vigorous stirring, the cells were analyzed using EnSight. ® A multi-mode plate reader (PerkinElmer; Waltham, MA) was used to measure the luminescence of firefly luciferase. Then, NanoDLR™ Stop & Glo... ® Reagent (80 µL; Promega) was added to each well. After incubation at room temperature for 20 minutes with vigorous stirring, the solution was analyzed using EnSight. ® Multi-mode plate reader measures NanoLuc ® Luciferase emits light. NanoLuc ®The luciferase signal was normalized to the firefly signal and multiplied by 1000. The half-maximum inhibitory concentration (IC50) was calculated from the titration curve using the Prism [inhibitor] and response-variable slope (four parameters) model (GraphPad Software; SanDiego, CA).

[0419] In vitro cell-based power assays in cocultures of LN-229 and 3T3-Nano-human GARP / LTGFβ1 (WT) are shown in Figures 11A (Experiment #1) and 11B (Experiment #2). In vitro cell-based power assays in cocultures of LN-229 and 3T3-Nano-human GARP / LTGFβ1 (non-releasable-(NR)) are shown in Figure 11C (Experiment #3). In vitro cell-based power assays in cocultures of LN-229 and 3T3-Nano-human GARP / LTGFβ3 (WT) are shown in Figure 11D (Experiment #4). A summary of IC50 (nM) is depicted in Table 14.

[0420] Table 14 - Summary of IC50 (nM)

[0421]

[0422] Example 5: Competitive assay of anti-αVβ8 monoclonal antibody

[0423] All tested antibodies were maintained within a temperature range of 2°C to 8°C.

[0424] EMT6 mouse breast cancer and HCC1159 human ovarian cancer cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in RPMI (Gibco, USA) supplemented with 10% FBS (Gibco, USA) under standard conditions (37°C, in a humid atmosphere containing 5% CO2).

[0425] Cells were isolated from the culture using 2.5 mM EDTA and then mixed with Mouse BD FC block. TMCells were incubated on ice for 30 minutes with 5 µg / ml (catalog number 553142, BD Biosciences, San Jose, CA). Cells were then stained on ice for 1 hour in 96 wells with different antibody combinations (PE-clone 65 with AF647-hC6D4 or PE-clone 65 with AF647-hADWA11) at different concentrations (0 µg / ml to 40 µg / ml). Finally, cells were incubated on ice for 30 minutes with LIVE / DEAD® Aqua to fix dead cells (catalog number L34957; Thermo Fisher Scientific; Waltham, MA) and then fixed with 1% PFA. Flow cytometry data were collected using BD FACS symphony (BD Biosciences). Flow cytometry data were analyzed in FlowJo (version 10.8.1). The half-maximum inhibitory concentration (IC50) value was calculated from the titration curve using the Prism [inhibitor] and response-variable slope (four parameters) model (GraphPad Software; San Diego, CA).

[0426] The competition assay between anti-αVβ8-65 and ADWA11 in the EMT6 cell line is shown in Figure 12A (1 µg / mL) and Figure 12B (40 µg / mL). The competition assay between anti-αVβ8-65 and C6D4 in the EMT6 cell line is shown in Figure 12C (1 µg / mL) and Figure 12D (40 µg / mL). The competition assay between anti-αVβ8-65 and ADWA11 in the HCC1159 cell line is shown in Figure 12E (1 µg / mL) and Figure 12F (10 µg / mL). The competition assay between anti-αVβ8-65 and C6D4 in the HCC1159 cell line is shown in Figure 12G (1 µg / mL) and Figure 12H (10 µg / mL). A summary of IC50 (mg / mL) is shown in Table 15.

[0427] Table 15 – Summary of IC50 (mg / mL)

[0428]

[0429] Example 6: Pharmacokinetic (PK) Characteristics of Humanized αVβ8 Antibody in Various Animal Models

[0430] SCID mice.SCID mice were IV-administered with 10 mg / kg of hu.αVβ8-65 or hu.αVβ8-92, and serum concentrations of the two antibodies were measured over 21 days, as shown in Figure 13. The pharmacokinetic characteristics of αVβ8-65 or hu.αVβ8-92 are provided in Table 16.

[0431] Table 16: Pharmacokinetic characteristics of humanized αVβ8 antibody in SCID mice.

[0432]

[0433] Crab-eating macaques. Cynomolgus monkeys were administered 10 mg / kg of hu.aVb8-92 or hu.aVb8-65, and serum concentrations of both antibodies were measured over 35 days, as shown in Figure 14. The observed Cmax was higher (approximately 1.5-fold) than expected based on cynomolgus monkey serum volume (Table 17). Lower-than-typical Vss (approximately 80 ml / kg) was also observed in cynomolgus monkeys (Table 17).

[0434] Table 17: Pharmacokinetic characteristics of humanized αVβ8 antibody in cynomolgus monkeys.

[0435]

[0436] CD-1 mice. The safety of the hu.aVb8-65 antibody was measured in a 56-day study in CD-1 mice at two separate doses (10 mg / kg or 50 mg / kg) (Figure 15). The study design is shown in Table 18. Mice were administered the two separate doses IV three times a week for four weeks. Only one animal died on day 30 (50 mg / kg hu.aVb8-65). All other animals survived to the scheduled necropsy. There were no clinical observations, effects on body weight, or toxicological significance in CD-1 mice administered at either dose.

[0437] Table 18: Pharmacokinetic characteristics of humanized αVβ8 antibody in CD-1 mice.

[0438]

[0439] Example 7: In vivo antitumor efficacy of humanized αvβ8 antibody

[0440] All anti-αVβ8 antibodies were against the mouse IgG2a.LALAPG isotype. The mouse (Mu) IgG1 anti-PD-L16E11 monoclonal antibody (Mu anti-PD-L1) was generated by immunizing PD-L1 knockout mice with the PF-L1-Fc fusion protein and cloned onto the mouse IgG1 isotype antibody. The Mu IgG1 anti-glycoprotein 120 (Mu IgG1 anti-gp120) served as a control for Mu anti-PD-L1. The Mu IgG2a LALAPG anti-gp120 (Mu IgG2a anti-gp120) served as a control for the anti-αVβ8 antibody. All antibodies tested in the assay were internally generated. Materials were stored in a refrigerator set to maintain a temperature range of 2°C to 8°C prior to use in the study.

[0441] EMT6 mouse breast cancer cell lines obtained from the American Type Culture Collection (Manassas, VA) were cultured in RPMI 1640 medium containing 1% L-glutamine and 10% fetal bovine serum (FBS; catalog number F2442; Sigma-Aldrich, St. Louis, MO). Cells were isolated using 0.5% trypsin in phosphate-buffered saline containing EDTA and collected in RPMI 1640 medium containing 1% L-glutamine and 10% FBS. The collected cells were centrifuged, washed once with Hanks balanced salt solution (HBSS), counted, and divided into 1x10⁻¹⁰ samples. 6 Cells were resuspended at a density of 1 cells / mL in a 1:1 solution of HBSS and matrix gel (Corning; Bedford, MA) and then inoculated into animals.

[0442] Female Balb / c mice (8 to 9 weeks old, approximately 20 g at the start of the study) were obtained from Charles River Laboratories (Hollister, CA). Mice were housed in standard rodent miniature isolation cages and acclimatized to study conditions for at least 3 days prior to tumor cell implantation. Only animals that appeared healthy and without obvious abnormalities were used in the study.

[0443] 1 x 10⁻⁶ cells suspended in 100 µL of HBSS:Matrigel 5One syngeneic EMT6 cell line was inoculated into the left mammary fat pad #5 of mice. Tumors were monitored until they reached a volume of approximately 180 mm³ (7 days post-inoculation). On day 0 of the study, mice were randomly assigned to 6 groups (n=9 per group) based on tumor volume. Antibodies were administered twice weekly for 3 weeks (first dose intravenously, followed by intraperitoneal administration). Mice were treated with an isotype control, aPD-L1, or a combination of aPD-L1 and anti-αvβ8 antibody. All antibodies were administered at a dose of 10 mg / kg. All concentrations were calculated based on the mean body weight of 18.5 g in the Balb / c mouse strain used in this study. Antibody stock solutions were diluted using histidine buffer (20 mM histidine acetate, 240 mM sucrose, 0.02% polysorbate-20, pH 5.5). Test materials were diluted to a concentration permissible for administration of 10 mg / kg. Throughout the research process, tumor measurements, body weight, and general clinical observations were recorded twice a week.

[0444] Tumors were measured using calipers, and tumor volume was calculated using the modified elliptic formula. Mice were euthanized if their tumor volume exceeded 1500 mm³, their tumors ulcerated, or their weight loss was equal to or greater than 20% of their starting weight. All animal studies were approved by the Genentech Animal Care and Use Committee. The length and width of the tumors were measured using UltraCal-IV calipers (model 54-10-111; Fred V. Fowler Co; Newton, MA). Tumor volume was calculated in Excel (version 11.5.6; Microsoft; Redmond, WA) using the following formula: Tumor volume (mm³) = (length x width) 2 ) x 0.5.

[0445] Tumor growth was analyzed and compared using a custom function package in R (version 4.1.0; R Foundation for Statistical Computing; Vienna, Austria), which integrates software from open-source packages (e.g., lme4, mgcv, gamm4, multcomp, settings, and plyr) and several packages from the tidyverse (e.g., magrittr, dplyr, tidyr, and ggplot2) (Forrest et al., Generalized additive mixed modeling of longitudinal tumor growth reduces bias and improves decision making in translational oncology, Cancer Res 2020;80(22):5089-97). In short, since tumors typically grow exponentially, a natural logarithmic transformation of tumor volume was performed before analysis. All original tumor volume measurements less than 8 mm³ were considered to reflect the complete absence of tumor and were converted to 8 mm³ before the natural logarithmic transformation. Furthermore, tumors smaller than 16 mm³ were considered too small to be accurately measured as microtumors and were converted to 16 mm³ before natural logarithmic transformation. A generalized additive mixture model (GAMM) was then applied to fit the time curves of logarithmically transformed tumor volumes across all study groups using regression arcs and automatically generated arc bases. This approach addresses both repeated measurements on the same subjects and the issue of appropriate exit before the study concludes.

[0446] Strong antitumor responses leading to a reduction in tumor size were tracked as partial response (PR, defined as animals whose original tumor volume measurement decreased by more than 50% at any time point during the study, but whose final original tumor volume measurement was still greater than 8 mm3) and complete response (CR, defined as animals whose final original unconverted tumor volume measurement was less than 8 mm3. For animals counted as CR, their final tumor volume measurement did not indicate the presence of a tumor).

[0447] Several studies were performed following the procedure described above. For each study, the percentage of complete remission (CR) above anti-PD-L1 was calculated as follows: %CR = number of CRs / number of mice in the treatment group; %CR above anti-PD-L1 = %CR of the combination group - %CR of the anti-PD-L1 group.

[0448] rb.avb8-65 and hu.avb8-65 showed similar inhibition of pSMAD2 / 3 in tumors and lymph nodes (Figs. 16A and 16B). EMT6 Thy1.1 WT tumor-bearing mice were treated with aPD-L1 and GP120 (ctr), or mice were treated with aPD-L1 and Galunisertib (1 hour before removal; SMI of TGFbR2), anti-TGFb1 21D1, anti-hu.avb8.65.m2a.LALAPG, or anti-rb.avb8.65.m2a.LALAPG. Approximately 55% to 56% of pSMAD2 / 3 was inhibited with administration of rb.avb8-65 and hu.avb8-65.

[0449] Next, the antitumor efficacy of the humanized antibody and the rabbit antibody was investigated by co-injecting mice with the antibody and aPD-L1. Tumor volume was measured for several consecutive days. The humanized and rabbit αVβ8 antibodies were superior to Mu.C6D4 (Figure 17).

[0450] Hu.avb8-65 and -92, when used in combination with anti-PD-L1, also showed better complete response (CR) than the baseline (ADWA11) in the EMT6 tumor model (Figures 18A and 18B). Hu.avb8-65, when used in combination with anti-PD-L1, also showed better CR than ADWA11 in the EMT6 tumor model (Figures 19A and 19B).

[0451] In the EMT6 model, Balb / c mice were treated with 0.1 x 10⁻⁶ mol / L ... 6 10 EMT6 cells were injected. No significant weight loss was observed, and complete remission (CR) was achieved in all patients up to day 51 of the study. Similarly, hu.avb8-65 and -92 demonstrated tumor-killing efficacy in the MC38 tumor model when used in combination with anti-PD-L1 (Figs. 20A and 20B). In the MC38 model, C57BL6 mice were injected with 1x10 EMT6 cells. 6 MC38 cells were injected. No significant weight loss was observed in mice.

[0452] Although the invention has been described in considerable detail above by way of example and illustration for the purpose of clarity, such description and examples should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly incorporated herein by reference in their entirety.

[0453] Example 8: In vivo antitumor efficacy of αvβ8 antibody.

[0454] The same EMT6 mouse breast cancer cell line and female BALB / c mice from the same source as in Example 7 were used. In this example, the following antibodies were used: (1) mouse anti-gp120-IgG1 (isotype control), (2) mouse anti-gp120-IgG2a-LALAPG (isotype control), (3) mouse anti-PD-L1 / IgG1 (clone 6E11, as discussed in Example 7), and (4) mouse Ch-anti-αvβ8 IgG2a.LALAPG (“Ch-anti-αvβ8-65”) and mouse anti-αvβ8-ADWA11 IgG2a.LALAPG (see the preceding examples).

[0455] EMT6 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2 mM L-glutamine and 10% fetal bovine serum (FBS; HyClone, Waltham, MA). Cells in logarithmic growth phase were centrifuged, washed once with Hank balanced salt solution (HBSS), counted, and cultured at 1 x 10⁻⁶ cells / mL. 6 A concentration of 1 x 10 cells / mL was resuspended in 50% HBSS and 50% Matrigel (Corning; Bedford, MA) for injection into mice. 1 x 10 cells / mL was used in 100 μL of HBSS:Matrigel (1:1). 5 EMT6 cells were inoculated into mice. The cells were seeded into the left mammary fat pad #5 of mice. When the tumor volume reached 130 to 230 mm... 3 Animals were assigned to treatment groups based on tumor volume and treated with isotype control antibodies (mouse IgG1 anti-gp120, 10 mg / kg; mouse IgG2aLALAPG anti-gp120, 10 mg / kg), anti-PD-L1 (mouse IgG1 clone 6E11, 10 mg / kg), Ch-anti-αvβ8-65 (10 mg / kg), anti-αvβ8-ADWA11 (10 mg / kg), or a combination of anti-PD-L1 and Ch-anti-αvβ8-65 or anti-αvβ8-ADWA11 antibodies. Antibodies were administered twice weekly for 21 days, with the first dose administered intravenously and subsequent doses administered intraperitoneally. Mice were euthanized if the tumor volume exceeded 1500 mm3. Weight changes in each individual animal were tracked during the study. All animal studies were approved by the Institutional Animal Care and Use Committee.

[0456] Tumor growth was analyzed and compared using a custom function package in R (version 4.1.0 (2021-05-18); R Foundation for Statistical Computing; Vienna, Austria), which integrates software from open-source packages (e.g., lme4, mgcv, gamm4, multcomp, settings, and plyr) and several packages from the tidyverse (e.g., magrittr, dplyr, tidyr, and ggplot2) (Forrest et al., Generalized additive mixed modeling of longitudinal tumor growth reduces bias and improves decision making in translationaloncology, Cancer Res 2020;80(22):5089-97). In short, since tumors typically grow exponentially, the tumor volume was transformed using the natural logarithm before analysis. All raw tumor volume measurements from 0 to 8 mm³ were considered to reflect the complete absence of tumor and were converted to 8 mm³ before the natural logarithmic transformation. Then, a generalized additive mixed model was applied to describe the transformation of tumor volume over time using regression splines with automatically generated spline bases. This approach addresses both repeated measurements on the same subjects and the issue of prudent exit before the end of the study. Tumor growth was analyzed and compared using a custom function package in R (version: 4.1.0; R Foundation for Statistical Computing; Vienna, Austria), which integrates software from open-source packages (e.g., lme4, mgcv, gamm4, multcomp, settings, and plyr) and several packages from tidyverse (e.g., magrittr, dplyr, tidyr, and ggplot2) (Forrest et al., Generalizedadditive mixed modeling of longitudinal tumor growth reduces bias and improves decision making in translational oncology, Cancer Res 2020;80(22):5089-97).Furthermore, all original tumor volume measurements smaller than 16 mm3 were considered too small. All original tumor volume measurements smaller than 8 mm3 were judged to reflect the complete absence of tumor and converted to "no tumor upper limit".

[0457] Strong antitumor responses leading to tumor size reduction were tracked as partial response (PR, defined as a reduction of >50% in tumor volume from the initial tumor size) and complete response (CR, defined as a reduction of 100% in tumor volume). The %CR for each treatment group was calculated using a series of formulas:

[0458] (Number of CRs / Number of mice in the group) * 100

[0459] Tumor growth rate is calculated using Dunnett's comparison of endpoint gain integrated in time (eGaIT) estimates. This type of eGaIT comparison represents the difference in growth rate between the treatment and reference groups, derived from the area under the curve (AUC) fitted on a natural logarithmic scale over a common time period. To obtain the growth rate from the fitted AUC on this scale over that time period, the AUC is corrected for the initial tumor burden and then undergoes a slope-equivalent "normalization". Mathematically, this normalization is achieved by dividing the estimated baseline-corrected AUC value by half the square of the common study period, resulting in natural logarithmic units per day. When the tumor exhibits log-linear growth (i.e., fitted to a line on a natural logarithmic scale), the slope-equivalent "normalization" of the AUC causes the calculation of the fitted slope. In cases where the tumor exhibits non-log-linear growth (i.e., the fit curves on the natural logarithmic scale), slope-equivalent "normalization" results in the calculation of a constant log-linear growth rate required to produce the AUC corrected for the baseline observed in the fit. A more negative contrast value indicates a greater anti-tumor effect; a more positive contrast value indicates a greater growth-promoting effect.

[0460] The comparison between anti-αvβ8 clone 65 and ADWA11 was made by calculating the following: (1) the percentage of complete response (CR) of the two antibodies in combination with anti-PD-L1 (combo ADWA11 or combo clone 65) relative to anti-PD-L1 treatment alone in each experiment, using the following formula: % CR relative to anti-PD-L1 = % combo CR - anti-PDL1 CR; and (2) the percentage of mice with tumor regression (eGaIT <= 0.05) on day 14.

[0461] resultFigure 21 shows the antitumor activity of Ch anti-αvβ8-65 and anti-αvβ8-ADWA11 in combination with anti-PD-L1 compared to the allotype control and anti-PD-L1 alone. Anti-PD-L1 alone did not result in a significant complete response rate, while the combination with clone 65 resulted in a significant 70% CR rate, which was significantly higher than the combination with anti-αvβ8-ADWA11. %EGaIT at day 14 was calculated as 20% for the combination with ADWA11 and 30% for the combination with clone 65. The mean eGaIT at day 14 was calculated as 0.0966 for the combination with ADWA11 and 0.0354 for the combination with clone 65. Figures 22A, 22B, and 22C illustrate the comparison of the antitumor activity of Ch anti-αvβ8-65 and anti-αvβ8-ADWA11 by the following: (Figure 22A) the percentage of CR in the combination group compared to the anti-PD-L1 group alone across several studies; (Figure 22B) a direct comparison (i.e., using both molecules in the same study) of the percentage of CR in the combination group compared to the anti-PD-L1 group alone; and (Figure 22C) the percentage of mice with tumor regression at day 14, measured in studies testing both molecules.

Claims

1. An antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety thereof exhibits at least one of the following properties: (a) K with 1 nM or less D It binds to human αvβ8; (b) With K of 1 nM or less D Binds to mouse αvβ8; (c) With K of 1 nM or less D Binding with cynomolgus monkey αvβ8; (d) Inhibition of αvβ8-mediated activation of human leucine-rich repeat protein 32 (LRRC32), LRRC32 and / or latent TGFβ-binding protein (LTBP) and / or its associated LTGFβ1 and LTGFβ3; and / or (e) Block the binding of TGFβ peptide to αvβ8.

2. The antibody or its antigen-binding portion according to claim 1, comprising: a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3, and a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, wherein: (a) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:152, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:153; (b) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:154, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:155; (c) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:156, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:157; (d) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:158, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:159; (e) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:160, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:161; (f) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:164, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:165; (g) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:166, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:167; (h) CDR-L1 according to SEQ ID NO:7, CDR-L2 according to SEQ ID NO:8, CDR-L3 according to SEQ ID NO:9, CDR-H1 according to SEQ ID NO:10, CDR-H2 according to SEQ ID NO:11, and CDR-H3 according to SEQ ID NO:12; (i) CDR-L1 according to SEQ ID NO:13, CDR-L2 according to SEQ ID NO:14, CDR-L3 according to SEQ ID NO:15, CDR-H1 according to SEQ ID NO:16, CDR-H2 according to SEQ ID NO:17, and CDR-H3 according to SEQ ID NO:18; (j) CDR-L1 according to SEQ ID NO:19, CDR-L2 according to SEQ ID NO:20, CDR-L3 according to SEQ ID NO:21, CDR-H1 according to SEQ ID NO:22, CDR-H2 according to SEQ ID NO:23, and CDR-H3 according to SEQ ID NO:24; (k) CDR-L1 according to SEQ ID NO:25, CDR-L2 according to SEQ ID NO:26, CDR-L3 according to SEQ ID NO:27, CDR-H1 according to SEQ ID NO:28, CDR-H2 according to SEQ ID NO:29, and CDR-H3 according to SEQ ID NO:30; (l) CDR-L1 according to SEQ ID NO:31, CDR-L2 according to SEQ ID NO:32, CDR-L3 according to SEQ ID NO:33, CDR-H1 according to SEQ ID NO:34, CDR-H2 according to SEQ ID NO:35, and CDR-H3 according to SEQ ID NO:36; (m) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:42; (n) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:150, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:151; (o) CDR-L1 according to SEQ ID NO:1, CDR-L2 according to SEQ ID NO:2, CDR-L3 according to SEQ ID NO:3, CDR-H1 according to SEQ ID NO:4, CDR-H2 according to SEQ ID NO:5, and CDR-H3 according to SEQ ID NO:6; (p) The CDR-L1, CDR-L2, and CDR-L3 sequences originate from the VL domain of SEQ ID NO:162, and the CDR-H1, CDR-H2, and CDR-H3 sequences originate from the VH domain of SEQ ID NO:163; or (q) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:

42.

3. The antibody or its antigen-binding portion according to claim 1 or claim 2, comprising: a VL containing CDR-L1, CDR-L2 and CDR-L3, and a VH containing CDR-H1, CDR-H2 and CDR-H3, wherein the sequences of CDR-L1, CDR-L2 and CDR-L2 are derived from the VL domain of SEQ ID NO:166, and the sequences of CDR-H1, CDR-H2 and CDR-H3 are derived from the VH domain of SEQ ID NO:

167.

4. The antibody or antigen-binding portion thereof according to any one of claims 1 to 3, comprising: a VL comprising CDR-L1, CDR-L2 and CDR-L3, and a VH comprising CDR-H1, CDR-H2 and CDR-H3, wherein CDR-L1 is according to SEQ ID NO:37, CDR-L2 is according to SEQ ID NO:38, CDR-L3 is according to SEQ ID NO:39, CDR-H1 is according to SEQ ID NO:40, CDR-H2 is according to SEQ ID NO:41, and CDR-H3 is according to SEQ ID NO:

42.

5. The antibody or antigen-binding portion thereof according to claim 1 or claim 2, comprising: a VL containing CDR-L1, CDR-L2 and CDR-L3, and a VH containing CDR-H1, CDR-H2 and CDR-H3, wherein the sequences of CDR-L1, CDR-L2 and CDR-L2 are derived from the VL domain of SEQ ID NO:154, and the sequences of CDR-H1, CDR-H2 and CDR-H3 are derived from the VH domain of SEQ ID NO:

155.

6. The antibody or antigen-binding portion thereof according to any one of claims 1, 2, and 5, comprising: a VL comprising CDR-L1, CDR-L2, and CDR-L3, and a VH comprising CDR-H1, CDR-H2, and CDR-H3, wherein CDR-L1 is according to SEQ ID NO:13, CDR-L2 is according to SEQ ID NO:14, CDR-L3 is according to SEQ ID NO:15, CDR-H1 is according to SEQ ID NO:16, CDR-H2 is according to SEQ ID NO:17, and CDR-H3 is according to SEQ ID NO:

18.

7. An antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety comprises: a heavy chain variable domain (VH) comprising CDR-H1, CDR-H2, and CDR-H3, and a light chain variable domain (VL) comprising CDR-L1, CDR-L2, and CDR-L3, wherein: (a) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:152, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:153; (b) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:154, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:155; (c) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:156, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:157; (d) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:158, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:159; (e) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:160, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:161; (f) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:164, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:165; (g) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:166, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:167; (h) CDR-L1 according to SEQ ID NO:7, CDR-L2 according to SEQ ID NO:8, CDR-L3 according to SEQ ID NO:9, CDR-H1 according to SEQ ID NO:10, CDR-H2 according to SEQ ID NO:11, and CDR-H3 according to SEQ ID NO:12; (i) CDR-L1 according to SEQ ID NO:13, CDR-L2 according to SEQ ID NO:14, CDR-L3 according to SEQ ID NO:15, CDR-H1 according to SEQ ID NO:16, CDR-H2 according to SEQ ID NO:17, and CDR-H3 according to SEQ ID NO:18; (j) CDR-L1 according to SEQ ID NO:19, CDR-L2 according to SEQ ID NO:20, CDR-L3 according to SEQ ID NO:21, CDR-H1 according to SEQ ID NO:22, CDR-H2 according to SEQ ID NO:23, and CDR-H3 according to SEQ ID NO:24; (k) CDR-L1 according to SEQ ID NO:25, CDR-L2 according to SEQ ID NO:26, CDR-L3 according to SEQ ID NO:27, CDR-H1 according to SEQ ID NO:28, CDR-H2 according to SEQ ID NO:29, and CDR-H3 according to SEQ ID NO:30; (l) CDR-L1 according to SEQ ID NO:31, CDR-L2 according to SEQ ID NO:32, CDR-L3 according to SEQ ID NO:33, CDR-H1 according to SEQ ID NO:34, CDR-H2 according to SEQ ID NO:35, and CDR-H3 according to SEQ ID NO:36; (m) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:42; (n) The CDR-L1, CDR-L2 and CDR-L3 sequences are derived from the VL domain of SEQ ID NO:150, and the CDR-H1, CDR-H2 and CDR-H3 sequences are derived from the VH domain of SEQ ID NO:151; (o) CDR-L1 according to SEQ ID NO:1, CDR-L2 according to SEQ ID NO:2, CDR-L3 according to SEQ ID NO:3, CDR-H1 according to SEQ ID NO:4, CDR-H2 according to SEQ ID NO:5, and CDR-H3 according to SEQ ID NO:6; (p) The CDR-L1, CDR-L2, and CDR-L3 sequences originate from the VL domain of SEQ ID NO:162, and the CDR-H1, CDR-H2, and CDR-H3 sequences originate from the VH domain of SEQ ID NO:163; or (q) CDR-L1 according to SEQ ID NO:37, CDR-L2 according to SEQ ID NO:38, CDR-L3 according to SEQ ID NO:39, CDR-H1 according to SEQ ID NO:40, CDR-H2 according to SEQ ID NO:41, and CDR-H3 according to SEQ ID NO:

42.

8. An antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety comprises: a VH containing CDR-H1, CDR-H2 and CDR-H3, and a VL containing CDR-L1, CDR-L2 and CDR-L3, wherein the sequences of CDR-L1, CDR-L2 and CDR-L2 are derived from the VL domain of SEQ ID NO:166, and the sequences of CDR-H1, CDR-H2 and CDR-H3 are derived from the VH domain of SEQ ID NO:

167.

9. An antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety comprises: a VH comprising CDR-H1, CDR-H2 and CDR-H3, and a VL comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 is according to SEQ ID NO:37, CDR-L2 is according to SEQ ID NO:38, CDR-L3 is according to SEQ ID NO:39, CDR-H1 is according to SEQ ID NO:40, CDR-H2 is according to SEQ ID NO:41, and CDR-H3 is according to SEQ ID NO:

42.

10. An antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety comprises: a VH containing CDR-H1, CDR-H2 and CDR-H3, and a VL containing CDR-L1, CDR-L2 and CDR-L3, wherein the sequences of CDR-L1, CDR-L2 and CDR-L2 are derived from the VL domain of SEQ ID NO:154, and the sequences of CDR-H1, CDR-H2 and CDR-H3 are derived from the VH domain of SEQ ID NO:

155.

11. An antibody or antigen-binding moiety thereof that specifically binds to αvβ8, wherein the antibody or antigen-binding moiety comprises: a VH comprising CDR-H1, CDR-H2 and CDR-H3, and a VL comprising CDR-L1, CDR-L2 and CDR-L3, wherein CDR-L1 is according to SEQ ID NO:13, CDR-L2 is according to SEQ ID NO:14, CDR-L3 is according to SEQ ID NO:15, CDR-H1 is according to SEQ ID NO:16, CDR-H2 is according to SEQ ID NO:17, and CDR-H3 is according to SEQ ID NO:

18.

12. The antibody or its antigen-binding portion according to any one of claims 1 to 11, wherein it is a monoclonal antibody.

13. The antibody or its antigen-binding portion according to any one of claims 1 to 12, wherein it is a humanized antibody or a chimeric antibody.

14. The antibody or its antigen-binding portion according to any one of claims 1 to 13, wherein it is an antibody fragment that specifically binds to human αvβ8.

15. The antibody or its antigen-binding portion according to any one of claims 1 to 14, comprising a sequence selected from the group consisting of: (a) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:152 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:153; (b) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:154 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:155; (c) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:156 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:157; (d) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:158 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:159; (e) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:160 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:161; (f) A VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:164 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:165; (g) The VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:166 and the VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:167; and (h) The VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:162 and the VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

163.

16. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, 7 to 9 and 12 to 15, comprising a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:166 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

167.

17. The antibody or antigen-binding portion thereof according to any one of claims 1 to 2, 5 to 7 and 10 to 15, comprising a VL sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:154 and a VH sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

155.

18. The antibody or antigen-binding portion thereof according to any one of claims 1 to 17, comprising a sequence selected from the group consisting of: (a) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:152 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:153; (b) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:154 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:155; (c) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:156 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:157; (d) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:158 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:159; (e) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:160 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:161; (f) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:164 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:165; (g) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:166 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:167; (h) A VL sequence comprising the amino acid sequence shown in SEQ ID NO:150 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:151; and (i) A VL sequence containing the amino acid sequence shown in SEQ ID NO:162 and a VH sequence containing the amino acid sequence shown in SEQ ID NO:

163.

19. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, 7 to 9, 12 to 15, 16 and 18, comprising: a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 166 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:

167.

20. The antibody or antigen-binding portion thereof according to any one of claims 1 to 2, 5 to 7, 10 to 15, 17 and 18, comprising: a VL sequence comprising the amino acid sequence shown in SEQ ID NO: 154 and a VH sequence comprising the amino acid sequence shown in SEQ ID NO:

155.

21. The antibody or its antigen-binding portion according to any one of claims 1 to 20, wherein it is a full-length antibody of the IgG1 isotype.

22. The antibody or antigen-binding portion thereof according to any one of claims 1 to 21, comprising a variant IgG1 Fc region having reduced effector function.

23. The antibody or antigen-binding portion thereof according to claim 21 or claim 22, wherein the Fc region comprises amino acid substitutions L234A / L235A according to the EU index number of Kabat.

24. The antibody or antigen-binding portion thereof according to any one of claims 21 to 23, wherein the Fc region comprises amino acid substitution P329G according to the EU index number of Kabat.

25. The antibody or antigen-binding portion thereof according to any one of claims 1 to 24, wherein the antibody binds to human αvβ8 at a KD of 1 nM or less as measured by surface plasmon resonance.

26. The antibody or its antigen-binding portion according to any one of claims 1 to 25, comprising: (a) A heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:201 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:200; (b) A heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:203 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:202; (c) a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:220 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:200; or (d) Heavy chains exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:221 and light chains exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

202.

27. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, 7 to 9, 12 to 15, 16, 18, 19 and 21 to 26, comprising: a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 203 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

202.

28. The antibody or antigen-binding portion thereof according to any one of claims 1 to 2, 5 to 7, 10 to 15, 17, 18 and 20 to 26, comprising: a heavy chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:201 and a light chain exhibiting at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO:

200.

29. The antibody or its antigen-binding portion according to any one of claims 1 to 28, comprising: (a) The heavy chain of SEQ ID NO:201 and the light chain of SEQ ID NO:200; (b) The heavy chain of SEQ ID NO:203 and the light chain of SEQ ID NO:202; (c) The heavy chain of SEQ ID NO:220 and the light chain of SEQ ID NO:200; or (d) The heavy chain of SEQ ID NO:221 and the light chain of SEQ ID NO:

202.

30. The antibody or antigen-binding portion thereof according to any one of claims 1 to 4, 7 to 9, 12 to 15, 16, 18, 19, 21 to 26 and 29, comprising the heavy chain of SEQ ID NO:203 and the light chain of SEQ ID NO:202 or the heavy chain of SEQ ID NO:221 and the light chain of SEQ ID NO:

202.

31. The antibody or antigen-binding portion thereof according to any one of claims 1 to 2, 5 to 7, 10 to 15, 17, 18, 20 to 26 and 29, comprising the heavy chain of SEQ ID NO:201 and the light chain of SEQ ID NO:200 or the heavy chain of SEQ ID NO:220 and the light chain of SEQ ID NO:

200.

32. An isolated nucleic acid encoding an antibody according to any one of claims 1 to 31.

33. A vector comprising the nucleic acid according to claim 32.

34. A host cell comprising the nucleic acid according to claim 32 or the vector according to claim 33.

35. A method for generating an antibody that binds to αvβ8, the method comprising culturing a host cell according to claim 34 under conditions suitable for the expression of the antibody.

36. The method of claim 35, further comprising recovering the antibody from the host cell.

37. An antibody produced by the method according to claim 36.

38. A pharmaceutical composition comprising: an antibody or an antigen-binding portion thereof according to any one of claims 1 to 31, and a pharmaceutical carrier.

39. A method of treating cancer in an individual with this need, the method comprising administering to the individual an effective amount of an antibody or antigen-binding portion thereof according to any one of claims 1 to 31 or a pharmaceutical composition according to claim 38.

40. A method of treating cancer in an individual with this need, said method comprising administering: (a) An effective amount of the antibody or its antigen-binding fragment according to any one of claims 1 to 31, or the pharmaceutical composition according to claim 38, and (b) Effective amount of PD-1 axis antagonist.

41. The method of claim 40, wherein the PD-1 axis antagonist is an anti-PD-L1 antibody.

42. The method of claim 40 or claim 41, wherein the PD-1 axis antagonist is atezolizumab.

43. The method according to any one of claims 39 to 42, the method further comprising assessing the level of αvβ6 expressed in the cancer.

44. The method according to any one of claims 39 to 43, wherein the cancer is selected from the list of ovarian cancer, triple-negative breast cancer, non-small cell lung cancer, colorectal cancer, bile duct cancer, endometrial cancer, renal papillary carcinoma, and bladder cancer.

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