Antibodies against PD-L1

Abstract

To provide novel classes of antibodies capable of binding human PD-L1, and to provide bispecific antibodies capable of binding human PD-L1 and capable of binding human CD3.SOLUTION: Provided is an antibody comprising an antigen-binding region capable of binding to human PD-L1, in which the antibody inhibits the binding of human PD-L1 to human PD-1 and competes for binding to human PD-L1 with an antibody comprising a VH sequence and VL sequence as set forth in particular amino acid sequences but does not compete for binding to human PD-L1 with an antibody comprising a VH sequence and VL sequence as set forth in particular amino acid sequences.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to novel antibodies and their uses in medicine. In particular, the present invention relates to bispecific antibodies capable of binding to human PD-L1 and human CD3. A novel class of antibodies capable of binding to human PD-L1 is also provided. The present invention further relates to uses of the antibodies of the present invention, as well as methods, nucleic acid constructs, and host cells for producing the antibodies of the present invention. [Background technology]

[0002] Background of the Invention Programmed death-ligand 1 (PD-L1, PDL1, CD274, B7H1, B7-H1) is a 33-kDa single-span type I membrane protein. Three PD-L1 isoforms have been described based on alternative splicing. PD-L1 belongs to the immunoglobulin (Ig) superfamily and contains one Ig-like C2-type domain and one Ig-like V-type domain. Freshly isolated T cells and B cells express minimal amounts of PD-L1, and only a small fraction (approximately 16%) of CD14+ monocytes constitutively express PD-L1 (Rietz and Chen, 2004 Am J Transplant 4: 8-14). Interferon-γ (IFN-γ) is known to upregulate PD-L1 on tumor cells (Abiko et al., 2015 Br J Cancer 112:1501-1509 (Non-Patent Document 2); Dong et al., 2002 Nature Medicine 8(8): 793-800 (Non-Patent Document 3)).

[0003] PD-L1 acts by (1) tolerizing tumor-reactive T cells by binding to its receptor PD-1 (CD279) on activated T cells (Dong et al., supra; Latchman et al., 2004 Proc Natl Acad Sci USA 101, 10691-6 (Non-Patent Document 4)), (2) rendering tumor cells resistant to CD8+ T cell- and Fas ligand-mediated lysis through PD-1 signaling via tumor cell-expressed PD-L1 (Azuma et al., 2008 Blood 111, 3635-43 (Non-Patent Document 5)); and (3) tolerizing T cells through reverse signaling via T cell-expressed CD80 (B7.1) (Butte et al., 2007 Immunity 27, 111-22 (Non-Patent Document 6); Park et al., 2010 Blood 116, 1291-8 (Non-Patent Document 7); and (4) interfere with antitumor immunity by promoting the development and maintenance of induced T regulatory cells (Francisco et al., 2009 J Exp Med 206, 3015-29 (Non-Patent Document 8)). PD-L1 is expressed in many human cancers, including melanoma, ovarian cancer, lung cancer, and colon cancer (Dong et al., supra).

[0004] PD-L1 blocking antibodies have shown clinical activity in several types of cancer (including melanoma and NSCLC) that are known to overexpress PD-L1. For example, atezolizumab is a humanized IgG1 monoclonal antibody against PD-L1. Atezolizumab is currently undergoing clinical trials as an immunotherapeutic agent for several indications, including various types of solid tumors (see, for example, Rittmeyer et al., 2017 Lancet 389:255-265). The PD-L1 antibody avelumab (Kaufman et al., Lancet Oncol. 2016;17(10):1374-1385) has been approved by the FDA for the treatment of adult and pediatric patients 12 years of age and older with metastatic Merkel cell carcinoma and is currently in clinical trials for several cancer indications, including bladder cancer, gastric cancer, head and neck cancer, mesothelioma, NSCLC, ovarian cancer, and kidney cancer. The PD-L1 antibody durvalumab has been approved for the treatment of locally advanced or metastatic urothelial carcinoma and is in clinical development for multiple solid tumors and hematological cancers (see, e.g., Massard et al., 2016 J Clin Oncol. 34(26):3119-25). Further anti-PD-L1 antibodies are described in WO2004004771 (Patent Document 1), WO2007005874 (Patent Document 2), WO2010036959 (Patent Document 3), WO2010077634 (Patent Document 4), WO2013079174 (Patent Document 5), WO2013164694 (Patent Document 6), WO2013173223 (Patent Document 7), and WO2014022758 (Patent Document 8).

[0005] Although significant progress has been made in eradicating cancer, further improvements in antibody-based cancer therapies are still needed. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2004004771

Patent document 2

Patent Document 3

Patent document 4

Patent document 5

Patent document 6

Patent document 7

Patent document 8

Non-licensed literature

[0007] [Non-licensed document 1] Rietz and Chen, 2004 Am J Transplant 4: 8-14 [Non-licensed document 2] Abiko et al., 2015 Br J Cancer 112:1501-1509 [Non-licensed document 3] Dong et al., 2002 Nature Medicine 8(8): 793-800

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

[0008] In a first aspect, the present invention provides novel anti-PD-L1 antibodies which comprise an antigen-binding region capable of binding to human PD-L1. The antibodies of this second aspect of the invention may be monospecific or multispecific, and if multispecific, the multispecific antibody may or may not comprise an antigen-binding region capable of binding to human CD3ε.

[0009] The present invention also provides bispecific antibodies comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD3ε (epsilon). Such bispecific antibodies have the following two effects:

[0010] First, the antibody binds to PD-L1-expressing tumor cells via its PD-L1-binding region. At the same time, the antibody binds to T cells via its CD3-binding region. Therefore, the antibody draws T cells into close proximity with tumor cells, thereby facilitating tumor cell killing by the T cells. Furthermore, without being limited to any particular theory, drawing PD-L1-expressing cells and effector T cells into close proximity with each other may initiate interferon-γ release, which may result in upregulation of PD-L1 on tumor cells, thereby recruiting more antibodies to the tumor and further enhancing its killing.

[0011] Second, the bispecific antibodies of the present invention inhibit the binding of human PD-L1 to human PD-1, thus preventing PD-L1 from interfering with anti-tumor immunity via PD-1.

[0012] CD3xPD-L1 bispecific antibodies are particularly useful in therapeutic settings where specific targeting and killing of PD-L1-expressing cells by T cells is desirable. CD3xPD-L1 bispecific antibodies are highly efficient at mediating killing of PD-L1-expressing cells, including, in some embodiments, cells with low PD-L1 copy numbers.

[0013] The antibodies of the invention can bind to PD-L1-expressing cells, such as MDA-MB-231 cells, PC3 cells, or HELA cells. Furthermore, the antibodies of the invention can inhibit the interaction between PD-L1 and PD-1 and mediate the killing of MDA-MB-231 cells, PC3 cells, and / or HELA cells by purified T cells or PBMCs.

[0014] In a further aspect, the present invention relates to the use of the antibodies of the invention in medicine, in particular the use of the antibodies of the invention for treating cancer.

[0015] [The present invention 1001] 1. An antibody comprising an antigen-binding region capable of binding to human PD-L1, Inhibits the binding of human PD-L1 to human PD-1, and (i) competes for binding to human PD-L1 with antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with antibody

[0547] comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with antibody

[0547] comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; The antibody. [The present invention 1002] An antibody of the present invention 1001 that competes with antibody

[0338] comprising the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:5 for binding to human PD-L1. [The present invention 1003] Any of the antibodies of the present invention, wherein the binding of the antibody to human PD-L1 is not displaced by antibody

[0476] comprising the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57. [The present invention 1004] Any of the antibodies of the present invention, wherein the binding of the antibody to human PD-L1 is not blocked by the binding of antibody

[0625] comprising the VH sequence set forth in SEQ ID NO:106 and the VL sequence set forth in SEQ ID NO:110. [The present invention 1005] Any of the antibodies of the present invention, wherein the binding of said antibody to human PD-L1 is blocked by antibody

[0547] comprising the VH sequence shown in SEQ ID NO:18 and the VL sequence shown in SEQ ID NO:22. [The present invention 1006] (i) capable of binding to the same human PD-L1 epitope as antibody

[0338] comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) capable of binding to the same human PD-L1 epitope as antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) It can bind to the same human PD-L1 epitope as antibody

[0547] , which comprises the VH sequence set forth in SEQ ID NO: 18 and the VL sequence set forth in SEQ ID NO: 22. The antibody of the present invention. [The present invention 1007] any one of antibodies 1001 to 1004 and 1006 of the invention, wherein the binding of said antibody to a mutant PD-L1 having one or more of the amino acid residues corresponding to position 113 (R113), 123 (Y123), and 125 (R125) in SEQ ID NO:94 substituted with alanine, compared to binding to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, is determined when the fold change in binding of the antibody is less than the average fold change in binding across all alanine mutants - 1.5 × SD, where SD is the standard deviation of all calculated fold changes of the antibody to mutant PD-L1, and the fold change in binding is calculated as set forth in Example 13

[0338] . [The present invention 1008] Any of the antibodies of the present inventions 1001 to 1002 and 1006, which bind to an epitope on PD-L1 (SEQ ID NO:94), wherein the epitope comprises the amino acid residue at position 113 (R113), the amino acid residue at position 123 (Y123), and / or the amino acid residue at position 125 (R125) of SEQ ID NO:94. [The present invention 1009] any one of antibodies 1001, 1003, 1004, and 1006 of the invention, which have reduced binding to mutant PD-L1 having one or more of the following substituted with alanine: amino acid residue corresponding to position 19 (F19), amino acid residue corresponding to position 42 (F42), amino acid residue corresponding to position 45 (E45), amino acid residue corresponding to position 46 (K46), amino acid residue corresponding to position 94 (L94), and amino acid residue corresponding to position 116 (I116) in SEQ ID NO:94, compared to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, and wherein the fold change in binding of the antibody is the average fold change in binding across all alanine mutants - Any of the antibodies 1001, 1003, 1004, and 1006 of the present invention, wherein reduced binding is determined if it is less than 1.5 x SD, where SD is the standard deviation of all calculated fold changes of the antibody against mutant PDL1, and the fold change of binding is calculated as shown in Example 13

[0511] . [The present invention 1010] Any of the antibodies 1001, 1003, 1004, and 1006 of the present invention, wherein the antibody binds to an epitope on PD-L1 (SEQ ID NO:94), wherein the epitope comprises one or more amino acid residues selected from the group consisting of the amino acid residue at position 45 (E45), the amino acid residue at position 46 (K46), and / or the amino acid residue at position 94 (L94) of SEQ ID NO:94. [The present invention 1011] any one of antibodies 1001, 1005, and 1006 of the invention, which exhibit reduced binding to a mutant PD-L1 comprising an alanine substitution at one or more of the amino acid residues corresponding to position 58 (E58) and 113 (R113) in SEQ ID NO:94, relative to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, wherein reduced binding is determined when the fold change in binding of the antibody is less than the average fold change in binding across all alanine mutants - 1.5 × SD, where SD is the standard deviation of all calculated fold changes of the antibody to mutant PD-L1, and the fold change in binding is calculated as set forth in Example 13

[0547] . [The present invention 1012] Any of the antibodies 1001, 1005, and 1006 of the present invention, wherein the antibody binds to an epitope on PD-L1 (SEQ ID NO:94), the epitope comprising the amino acid residue at position 58 (E58) and / or the amino acid residue at position 113 (R113) of SEQ ID NO:94. [The present invention 1013] Any of the aforementioned antibodies of the present invention, wherein the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, and the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:21. [The present invention 1014] the antigen-binding region capable of binding to human PD-L1 is (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7

[0338] ; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17

[0511] ; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21; and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:23, a CDR2 having the sequence DDN, and a CDR3 having the sequence set forth in SEQ ID NO:24. Any of the antibodies of the present invention, comprising: [The present invention 1015] Any of the antibodies of the invention, wherein the antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:18. [The present invention 1016] Any of the antibodies of the invention, wherein the antigen-binding region capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22. [The present invention 1017] the antigen-binding region capable of binding to human PD-L1 is (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO: 22. Any of the antibodies of the present invention, comprising: [The present invention 1018] 1017. The antibody of the present invention, wherein the VH sequence and the VL sequence each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, and wherein each combined FR1, FR2, FR3, and FR4 framework sequence of VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequence is not mutated, and each combined FR1, FR2, FR3, and FR4 framework sequence of VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequence is not mutated. [The present invention 1019] Any of the antibodies of the present invention that are capable of inducing dose-dependent lysis of adenocarcinoma epithelial cells, for example, dose-dependent lysis of MDA-MB-231, via antibody-dependent cellular cytotoxicity (ADCC). [The present invention 1020] The antibody of the invention 1019, which is capable of reducing the number of cells in a culture of said epithelial cells as a result of cell lysis by at least 5%, such as at least 6%, 7%, 8%, 9%, or at least 10%. [The present invention 1021] ADCC, 51 The antibody of the invention 1019 or 1020, as measured in vitro in a Cr release assay, for example, the assay disclosed in Example 14. [The present invention 1022] The antibody of the present invention 1019 or 1020, wherein ADCC is measured in vitro by incubating said epithelial cells with a composition comprising the antibody of the present invention 1019 or 1020 and effector cells, such as peripheral blood mononuclear cells (PBMCs), for 4 hours at 37°C, 5% CO2, wherein the amount of antibody in the composition is in the range of 0.1 to 1 μg / mL and the ratio of effector cells to epithelial cells is 100:1. [The present invention 1023] The antibody of the invention 1019 or 1020, wherein said lysis of epithelial cells is measured in vitro in a luciferase reporter assay as a surrogate for ADCC, for example, in the luminescence ADCC reporter bioassay disclosed in Example 14. [The present invention 1024] ADCC, (i) contacting the culture of epithelial cells with a composition comprising an antibody of the invention 1023 and Jurkat human T cells (effector cells) stably expressing FcγRIIIa (CD16) and firefly luciferase at an effector cell:epithelial cell ratio of 1:1; (ii) allowing the epithelial cell culture and the effector cells to equilibrate to room temperature for 15 minutes; (iii) incubating the culture of epithelial cells and the effector cells with a luciferase substrate; and (iv) measuring luciferase production in the cell culture It is measured in vitro by The antibody of the present invention 1023, wherein the amount of the antibody in the composition is within the range of 0.5 to 250 ng / mL, and the ratio of effector cells to epithelial cells is 1:1. [The present invention 1025] The ADCC of the epithelial cells is measured in a luciferase reporter assay, for example, the reporter assay defined in invention 1023 or 1024, and then the ADCC observed after incubating a culture of the epithelial cells with a test composition comprising any of antibodies of invention 1019, 1020, 1023, and 1024 is at least 1.5 times the ADCC observed after incubating a culture of the epithelial cells with a composition comprising a reference antibody, the ADCC being measured as relative light units (RLU), the antibody concentrations in the test composition and the composition comprising the reference antibody are the same and are in the range of 20 to 250 ng / ml, and the reference antibody is (a) an antibody comprising the VH sequence set forth in SEQ ID NO:74 and the VL sequence set forth in SEQ ID NO:78; and (b) an antibody comprising the VH sequence shown in SEQ ID NO:81 and the VL sequence shown in SEQ ID NO:85 Any of the antibodies 1019, 1020, 1023, and 1024 of the present invention selected from the group consisting of: [The present invention 1026] the antigen-binding region capable of binding to human PD-L1 is (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22

[0547] Any of the antibodies of the present invention, comprising: [The present invention 1027] 1. An antibody comprising an antigen-binding region capable of binding to human PD-L1, (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 33, the CDR2 sequence set forth in SEQ ID NO: 34, and the CDR3 sequence set forth in SEQ ID NO: 35, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 37, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO: 38

[0321] ; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 47, the CDR2 sequence set forth in SEQ ID NO: 48, and the CDR3 sequence set forth in SEQ ID NO: 49, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 51, a CDR2 having the sequence DVI, and a CDR3 having the sequence set forth in SEQ ID NO: 52

[0421] ; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 54, the CDR2 sequence set forth in SEQ ID NO: 55, and the CDR3 sequence set forth in SEQ ID NO: 56, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 58, a CDR2 having the sequence RDS, and a CDR3 having the sequence set forth in SEQ ID NO: 59

[0476] ; or (iv) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 61, the CDR2 sequence set forth in SEQ ID NO: 62, and the CDR3 sequence set forth in SEQ ID NO: 63, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 65, a CDR2 having the sequence DDS, and a CDR3 having the sequence set forth in SEQ ID NO: 66

[0516] ; or (v) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 107, the CDR2 sequence set forth in SEQ ID NO: 108, and the CDR3 sequence set forth in SEQ ID NO: 109; and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 111, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO: 113

[0625] ; (vi) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:68, the CDR2 sequence set forth in SEQ ID NO:69, and the CDR3 sequence set forth in SEQ ID NO:70; and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:72, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:73. The antibody comprising: [The present invention 1028] (i) the VH sequence set forth in SEQ ID NO: 32 and the VL sequence set forth in SEQ ID NO: 36

[0321] ; or (ii) the VH sequence set forth in SEQ ID NO: 46 and the VL sequence set forth in SEQ ID NO: 50

[0421] ; or (iii) the VH sequence set forth in SEQ ID NO: 53 and the VL sequence set forth in SEQ ID NO: 57

[0476] ; or (iv) the VH sequence set forth in SEQ ID NO: 60 and the VL sequence set forth in SEQ ID NO: 64

[0516] ; or The VH sequence shown in SEQ ID NO: 106 and the VL sequence shown in SEQ ID NO: 110

[0625] (v) the VH sequence shown in SEQ ID NO: 67 and the VL sequence shown in SEQ ID NO: 71

[0632] The antibody of the present invention, comprising: [The present invention 1029] Any of the antibodies of the present invention which is monovalent. [The present invention 1030] Any of the antibodies of the present invention is a bivalent antibody having two antigen-binding domains capable of binding to human PD-L1, and the two antigen-binding domains have the same variable region sequence. [The present invention 1031] Any of the aforementioned antibodies of the invention is a bivalent, bispecific antibody, and comprises, in addition to the (first) antigen-binding region capable of binding to human PD-L1, a (second) antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is not human CD3ε. [The present invention 1032] A bispecific antibody comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD3ε (epsilon), The bispecific antibody, wherein the antigen-binding region capable of binding to human PD-L1 has any of the characteristics set forth in the present invention. [The present invention 1033] 1032. A bispecific antibody of the present invention, wherein the antigen-binding region capable of binding to human CD3ε comprises: (a) a heavy chain variable region (VH) comprising CDR1 having the sequence shown in SEQ ID NO:26, CDR2 having the sequence shown in SEQ ID NO:27, and CDR3 having the sequence shown in SEQ ID NO:28, and a light chain variable region (VL) comprising CDR1 having the sequence shown in SEQ ID NO:30, CDR2 having the sequence GTN, and CDR3 having the sequence shown in SEQ ID NO:31. [The present invention 1034] (i) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7

[0338] ; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (ii) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17

[0338] ; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (iii) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21, and a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO:23, the CDR2 sequence set forth in the sequence DDN, and the CDR3 sequence set forth in SEQ ID NO:24

[0547] ; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:26, the CDR2 sequence set forth in SEQ ID NO:27, and the CDR3 sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO:30, the CDR2 having the sequence GTN, and the CDR3 sequence set forth in SEQ ID NO:31. The bispecific antibody of the present invention 1032 or 1033, comprising: [This invention 1035] The bispecific antibody of any of claims 1032 to 1034, wherein the antigen-binding region capable of binding to human CD3ε comprises the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29. [The present invention 1036] (i) the bispecific antibody has a lower affinity for human CD3ε binding compared to an antibody having an antigen-binding region that can comprise the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29, preferably the affinity is at least 2-fold lower, such as at least 5-fold lower, such as at least 10-fold lower, such as at least 25-fold lower, such as at least 50-fold lower; and (ii) the bispecific antibody is capable of mediating concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when PBMCs or purified T cells are used as effector cells, e.g., when assayed as described in Example 11 herein; The bispecific antibody of the present invention. [This invention 1037] the antigen-binding region capable of binding to human CD3ε is (i) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:99, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (ii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO: 100, a CDR2 having the sequence set forth in SEQ ID NO: 27, and a CDR3 having the sequence set forth in SEQ ID NO: 28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO: 31; or (iii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:101, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (iv) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:102, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (v) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:103, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (vi) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:104, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (vii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO: 26, a CDR2 having the sequence set forth in SEQ ID NO: 27, and a CDR3 having the sequence set forth in SEQ ID NO: 105, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO: 31. 1036. A bispecific antibody of the present invention comprising: [The present invention 1038] the antigen-binding region capable of binding to human CD3ε is (i) the VH sequence set forth in SEQ ID NO:39 and the VL sequence set forth in SEQ ID NO:29; or (ii) the VH sequence set forth in SEQ ID NO:40 and the VL sequence set forth in SEQ ID NO:29; or (iii) the VH sequence set forth in SEQ ID NO: 41 and the VL sequence set forth in SEQ ID NO: 29; or (iv) the VH sequence set forth in SEQ ID NO: 42 and the VL sequence set forth in SEQ ID NO: 29; or (v) the VH sequence set forth in SEQ ID NO: 43 and the VL sequence set forth in SEQ ID NO: 29; or (vi) the VH sequence set forth in SEQ ID NO: 44 and the VL sequence set forth in SEQ ID NO: 29; or (vii) the VH sequence shown in SEQ ID NO:45 and the VL sequence shown in SEQ ID NO:29 The bispecific antibody of the present invention 1036 or 1037, comprising: [This invention 1039] A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; The multispecific antibody, wherein the antigen-binding region capable of binding to human PD-L1 has the characteristics set forth in any one of claims 1001 to 1031 of the present invention. [The present invention 1040] A multispecific antibody of the present invention, wherein the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, and the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4

[0338] , SEQ ID NO:11

[0511] , and SEQ ID NO:21

[0547] . [This invention 1041] the first antigen-binding region capable of binding to human PD-L1 comprises: (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7

[0338] ; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17

[0511] ; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:23, a CDR2 having the sequence DDN, and a CDR3 having the sequence set forth in SEQ ID NO:24. 1040. A multispecific antibody of the present invention comprising: [The present invention 1042] The multispecific antibody of the present invention 1040 or 1041, wherein the first antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1

[0338] , SEQ ID NO:8

[0511] , and SEQ ID NO:18

[0547] . [This invention 1043] Any of the multispecific antibodies of the present inventions 1040 to 1042, wherein the first antigen-binding domain capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:5

[0338] , SEQ ID NO:15

[0511] , and SEQ ID NO:22

[0547] . [This invention 1044] the first antigen-binding region capable of binding to human PD-L1 comprises: (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO: 22. The multispecific antibody of any one of 1040 to 1043 of the present invention, comprising: [This invention 1045] any one of claims 1040 to 1044, wherein the VH sequence and the VL sequence each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, wherein each combined FR1, FR2, FR3, and FR4 framework sequence of VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequence is not mutated; and each combined FR1, FR2, FR3, and FR4 framework sequence of VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequence is not mutated. [The present invention 1046] the first antigen-binding region capable of binding to human PD-L1 comprises: (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22

[0547] The multispecific antibody of any one of 1040 to 1045 of the present invention, comprising: [This invention 1047] A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; (i) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; The multispecific antibody. [This invention 1048] 1047. A multispecific antibody of the invention that competes for binding to human PD-L1 with an antibody comprising the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:5. [This invention 1049] A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; The multispecific antibody, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57. [The present invention 1050] A multispecific antibody of the present invention 1049 that inhibits the binding of human PD-L1 to human PD-1. [This invention 1051] The multispecific antibody of the invention 1049 or 1050, which competes for binding to human PD-L1 with an antibody comprising the VH sequence shown in SEQ ID NO:18 and the VL sequence shown in SEQ ID NO:22. [This invention 1052] Any of the multispecific antibodies of the present inventions 1049 to 1051, wherein the binding of any of the multispecific antibodies of the present inventions 1049 to 1051 to human PD-L1 is blocked by an antibody comprising the VH sequence shown in SEQ ID NO:18 and the VL sequence shown in SEQ ID NO:22. [This invention 1053] A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; the first antigen-binding region comprises: (i) capable of binding to the same human PD-L1 epitope as antibody

[0338] comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) capable of binding to the same human PD-L1 epitope as antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) It can bind to the same human PD-L1 epitope as antibody

[0547] , which comprises the VH sequence set forth in SEQ ID NO: 18 and the VL sequence set forth in SEQ ID NO: 22. The multispecific antibody. [This invention 1054] The multispecific antibody of any one of 1040 to 1053 of the present invention, which is bispecific. [This invention 1055] A multispecific antibody of the present invention 1054, which is bivalent. [This invention 1056] The multispecific antibody of any of claims 1040 to 1055, wherein the multispecific antibody of any of claims 1040 to 1055 can bind to a second antigen, and the second antigen is not human CD3ε. [This invention 1057] Any of the antibodies of the present invention which are full-length antibodies. [This invention 1058] The antibody of the present invention 1057, which is a full-length IgG1 antibody. [This invention 1059] Any of the antibodies of the present invention, which are antibody fragments. [The present invention 1060] any one of the antibodies 1032 to 1059 of the present invention comprises two half molecules each comprising an antigen-binding region; (i) the half-molecule containing an antigen-binding region capable of binding to human PD-L1 is chimeric; and / or (ii) is chimeric, if present, with half molecules containing an antigen-binding region capable of binding to human CD3ε (epsilon); Any one of the antibodies 1032 to 1059 of the present invention. [This invention 1061] (i) the antigen-binding region capable of binding to human PD-L1 is humanized; and / or (ii) the antigen-binding region capable of binding to human CD3ε (epsilon), if present, is humanized; Any of the antibodies of the present invention. [This invention 1062] (i) the antigen-binding region capable of binding to human PD-L1 is human; and / or (ii) the antigen-binding region capable of binding to human CD3ε (epsilon), when present, is human; Any of the antibodies of the present invention. [This invention 1063] Any of the antibodies of the present invention, wherein each of the antigen-binding regions comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of the variable regions comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. [This invention 1064] The antibody of the present invention 1063, comprising two heavy chain constant regions (CH) and two chain constant regions (CL). [This invention 1065] Any of the antibodies of the present invention, wherein the antibody comprises a first heavy chain and a second heavy chain, each of which comprises at least a hinge region, CH2, and CH3 region, and wherein the first heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering), and the second heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering), and wherein the first heavy chain and the second heavy chain do not have substitutions at the same positions. [The present invention 1066] The antibody of the present invention, wherein (i) in the first heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L and in the second heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R, or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R and in the second heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L. [This invention 1067] any of the antibodies of the invention comprising a first heavy chain and a second heavy chain; and one or both heavy chains are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an otherwise identical antibody comprising an unmodified first and second heavy chains; Any of the antibodies of the present invention. [The present invention 1068] The antibody of the present invention 1067, wherein said Fc-mediated effector function is measured by determining Fc-mediated CD69 expression, by binding to an Fcγ receptor, by binding to C1q, or by inducing Fc-mediated FcR cross-linking. [This invention 1069] the antibody of the invention 1067 or 1068 has altered heavy and light chain constant sequences such that Fc-mediated CD69 expression is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% when compared to the wild-type antibody; The Fc-mediated CD69 expression is measured in a PBMC-based functional assay. The antibody of the present invention 1067 or 1068. [The present invention 1070] Any of the antibodies of the present invention, comprising a first heavy chain and a second heavy chain, wherein in at least one of the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. [This invention 1071] The antibody of the present invention 1070, wherein in said first heavy chain and said second heavy chain, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively. [This invention 1072] The antibody of the present invention 1071, wherein the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L. [This invention 1073] The antibody of the present invention 1070, wherein in said first heavy chain and said second heavy chain, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively. [This invention 1074] The antibody of the present invention 1073, wherein the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L. [This invention 1075] Any of the antibodies of the present invention which do not bind to human PD-L2. [This invention 1076] Approximately 10 -8 M or less, e.g., about 10-9 M or less, e.g., about 10 -10 M or less K D Any of the antibodies of the present invention which bind to human PD-L1 at [This invention 1077] Any of the antibodies of the present invention, which mediate concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when purified T cells are used as effector cells, when assayed as described in Example 11 herein. [This invention 1078] (i) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to human PD-L1 as defined in any one of claims 1001 to 1031; and / or (ii) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to human PD-L1 as defined in any one of claims 1001 to 1031 of the present invention; A nucleic acid construct comprising: [This invention 1079] (i) a nucleic acid sequence encoding a heavy chain sequence of an antibody comprising an antigen-binding region capable of binding to human CD3ε as defined in any one of claims 1033 to 1038 of the present invention; and (ii) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to human CD3ε as defined in any one of claims 1033 to 1038 of the present invention; The nucleic acid construct of the present invention 1073, further comprising: [The present invention 1080] 1078 or 1079. An expression vector comprising a nucleic acid construct as defined in the present invention. [This invention 1081] A host cell comprising a nucleic acid construct as defined in claim 1078 or 1079 of the present invention or an expression vector as defined in claim 1080 of the present invention. [This invention 1082] A host cell of the present invention 1081 that is a mammalian cell, such as a Chinese hamster ovary cell. [This invention 1083] A pharmaceutical composition comprising any one of the antibodies of the present inventions 1001 to 1077 and a pharmaceutically acceptable carrier. [This invention 1084] An antibody of any one of the antibodies of the present inventions 1001 to 1077 or a pharmaceutical composition of the present invention 1083 for use as a medicine. [This invention 1085] The antibody of any one of the antibodies of the present inventions 1001 to 1080 or the pharmaceutical composition of the present invention 1070 for use in treating cancer. [The present invention 1086] The antibody of any of claims 1001 to 1077 or the pharmaceutical composition of claim 1070 for use in treating a cancer disease characterized by the presence of a solid tumor. [This invention 1087] The antibody of any one of claims 1001 to 1078 or the pharmaceutical composition of claim 1070 for use in treating a cancer disease selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer. [This invention 1088] A method for treating a disease, comprising the step of administering to a subject in need thereof an antibody of any of the present inventions 1001 to 1075 or a pharmaceutical composition of the present invention 1083. [This invention 1089] Use of any of the antibodies of the present inventions 1001 to 1077 for the manufacture of a medicament, such as a medicament for treating cancer, e.g., a cancer disease characterized by the presence of a solid tumor, or a cancer disease selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer. [The present invention 1090] 108. The method or use of any of claims 1083 to 1089, including combination with one or more further therapeutic agents, for example combination with a chemotherapeutic agent. [This invention 1091] (a) culturing a host cell that produces a first antibody comprising an antigen-binding region capable of binding to human PD-L1 as defined in any one of claims 1001 to 1013, and purifying the first antibody from the culture; (b) culturing a host cell that produces a second antibody comprising an antigen-binding region capable of binding to a different PD-L1 epitope or a different antigen, such as a human CD3ε-binding region as defined in any one of claims 1014 to 1019 of the present invention, and purifying the second antibody from the culture; (c) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (d) Obtaining a bispecific antibody A method for producing any one of the antibodies of the present inventions 1001 to 1077, comprising: [This invention 1092] An anti-idiotype antibody that binds to an antigen-binding region capable of binding to human PD-L1 as defined in any one of claims 1001 to 1077 of the present invention. These and other aspects of the invention are described in further detail below. [Brief explanation of the drawings]

[0016] [Figure 1A] Binding of bispecific CD3xPD-L1 and b12xPD-L1 antibodies and their monospecific bivalent PD-L1 counterparts to MDA-MB-231 cells. (A) Binding of bsIgG1-huCD3-H1L1-FEALx338-FEAR and IgG1-338-FEAR, (B) Binding of bsIgG1-huCD3-H1L1-FEALx547-FEAR and IgG1-547-FEAR, (C) Binding of bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR, (D) Binding of bsIgG1-b12-FEALx338-FEAR, (E) Binding of bsIgG1-b12-FEALx547-FEAR, (F) Binding of bsIgG1-b12-FEALx511-LC33S-FEAR. Data shown are mean fluorescence intensities (MFI) of one representative experiment, measured by flow cytometry. [Figure 1B] See legend to Figure 1A. [Figure 1C]See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 1F] See legend to Figure 1A. [Figure 2A] Binding of bispecific CD3xPD-L1 and b12xPD-L1 antibodies and their monospecific bivalent PD-L1 counterparts to PC3 cells. (A) Binding of bsIgG1-huCD3-H1L1-FEALx338-FEAR and IgG1-338-FEAR, (B) Binding of bsIgG1-huCD3-H1L1-FEALx547-FEAR and IgG1-547-FEAR, (C) Binding of bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR, (D) Binding of bsIgG1-b12-FEALx338-FEAR, (E) Binding of bsIgG1-b12-FEALx547-FEAR, (F) Binding of bsIgG1-b12-FEALx511-LC33S-FEAR. Data shown are mean fluorescence intensities (MFI) of one representative experiment, measured by flow cytometry. [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Figure 2D] See legend to Figure 2A. [Figure 2E] See legend to Figure 2A. [Figure 2F] See legend to Figure 2A. [Figure 3]Binding of bispecific CD3xPD-L1 and b12xPD-L1 antibodies and their monospecific bivalent PD-L1 counterparts to HELA cells. (A) Binding of bsIgG1-huCD3-H1L1-FEALx338-FEAR and IgG1-338-FEAR, (B) Binding of bsIgG1-huCD3-H1L1-FEALx547-FEAR and IgG1-547-FEAR, (C) Binding of bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR, (D) Binding of bsIgG1-b12-FEALx338-FEAR, (E) Binding of bsIgG1-b12-FEALx547-FEAR, (F) Binding of bsIgG1-b12-FEALx511-LC33S-FEAR. Data shown are mean fluorescence intensities (MFI) of one representative experiment, measured by flow cytometry. [Figure 4] Binding of bispecific b12 x PD-L1 antibodies and their monospecific bivalent PD-L1 counterparts to SK-MES-1 cells. (A) Binding of bsIgG1-b12-FEALx338-FEAR and IgG1-338-FEAR, (B) Binding of bsIgG1-b12-FEALx547-FEAR and IgG1-547-FEAR, (C) Binding of bsIgG1-b12-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR. Data shown are mean fluorescence intensities (MFI) from one representative experiment, as measured by flow cytometry. [Figure 5A]Binding of bispecific CD3xPD-L1 and b12xPD-L1 antibodies and their monospecific bivalent PD-L1 counterparts to CHO cells transfected with cynomolgus PD-L1. (A) Binding of bsIgG1-huCD3-H1L1-FEALx338-FEAR and IgG1-338-FEAR, (B) Binding of bsIgG1-huCD3-H1L1-FEALx547-FEAR and IgG1-547-FEAR, (C) Binding of bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR, (D) Binding of bsIgG1-b12-FEALx338-FEAR, (E) Binding of bsIgG1-b12-FEALx547-FEAR, (F) Binding of bsIgG1-b12-FEALx511-LC33S-FEAR. Data shown are mean fluorescence intensities (MFI) from one representative experiment, measured by flow cytometry. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 5D] See legend to Figure 5A. [Figure 5E] See legend to Figure 5A. [Figure 5F] See legend to Figure 5A. [Figure 6A] Antibody cross-blocking. Antibody cross-blocking was determined using Biolayer Interferometry. All antibodies were produced with a FEAR IgG1 backbone. In this table, responses of 0.1 nm or greater were considered non-blocking antibody pairs (results shown as plain numbers in the table), and responses of less than 0.1 nm were considered blocking antibody pairs (results shown as bold numbers in the table). In contrast, some responses that were neither blocking nor non-blocking were indicative of antibodies that exhibited displacing behavior (results shown as an asterisk (*) in the table). MEDI=MEDI4736; MPDL=MPDL3280A. Representative figures are shown for (A) displacing, (B) blocking, and (C) non-blocking antibody pairs. [Figure 6B] See legend to Figure 6A. [Figure 6C] See legend to Figure 6A. [Figure 7] Effect of bispecific b12 x PD-L1 antibodies and their monospecific bivalent PD-L1 counterparts on PD-1 / PD-L1 interaction. The effects of (A) bsIgG1-b12-FEALx338-FEAR and IgG1-338-FEAR, (B) bsIgG1-b12-FEALx547-FEAR and IgG1-547-FEAR, and (C) bsIgG1-b12-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR were determined in a PD-1 / PD-L1 blockade bioassay. Data shown are fold induction compared to control (no antibody added) from one representative experiment. [Figure 8A] Induction of cytotoxicity by CD3×PD-L1 bispecific antibodies in MDA-MB-231 cells in vitro. MDA-MB-231 cells were incubated with bsIgG1-huCD3-H1L1-FEALx338-FEAR, bsIgG1-huCD3-H1L1-FEALx547-FEAR, and bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR. Purified T cells (A) or PBMCs (B) were used as effector cells. Data shown are the % viable cells from one representative experiment. [Figure 8B] See legend to Figure 8A. [Figure 9A] Induction of cytotoxicity by CD3xPD-L1 bispecific antibodies in PC-3 cells in vitro. PC-3 cells were incubated with bsIgG1-huCD3-H1L1-FEALx338-FEAR, bsIgG1-huCD3-H1L1-FEALx547-FEAR, and bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR. Purified T cells (A) or PBMCs (B) were used as effector cells. Data shown are % viable cells from one representative experiment. [Figure 9B] See legend to Figure 9A. [Figure 10A]Induction of cytotoxicity by CD3×PD-L1 bispecific antibodies in HELA cells in vitro. HELA cells were incubated with bsIgG1-huCD3-H1L1-FEALx338-FEAR, bsIgG1-huCD3-H1L1-FEALx547-FEAR, and bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR. Purified T cells (A) or PBMCs (B) were used as effector cells. Data shown are the % viable cells from one representative experiment. [Figure 10B] See legend to Figure 10A. [Figure 11] T cell proliferation and activation by CD3xPD-L1 bispecific antibodies. To measure T cell activation and proliferation, CD3xPD-L1 bispecific antibodies were tested in an in vitro assay using MDA-MB-231 cells as target cells and purified T cells as effector cells. (A) Total T cell count, (B) CD69+ T cell count, (C) CD25+ T cell count. Figure 12: Fold change in binding between PD-L1 antibodies and PD-L1 variants with alanine mutations at positions 42-131. Fold change was defined as Log10 (normalized gMFI [ala variant] / normalized gMFI [wt]). Residues with a fold change in binding less than the mean fold change - 1.5 × SD (shown by the dotted line) were considered "loss of binding variants." Residues with a positive fold change in binding are loss of binding residues (residues 75 and 86) relative to the IgG1-625-FEAR-A488 control antibody. Numbers on the x-axis refer to amino acid positions. [Figure 12] Fold change in binding between PD-L1 antibodies and PD-L1 variants with alanine mutations at positions 42-131. Fold change was defined as Log10(normalized gMFI [ala variant] / normalized gMFI [wt]). Residues with a fold change in binding less than the mean fold change - 1.5 × SD (indicated by the dotted line) were considered "loss of binding variants." Residues with a positive fold change in binding are loss of binding residues (residues 75 and 86) relative to the IgG1-625-FEAR-A488 control antibody. Numbers on the x-axis refer to amino acid positions. [Figure 13] Antibody-dependent cellular cytotoxicity of MDA-MB-231 cells by PD-L1 antibodies measured in a 51Cr release assay. ADCC of MDA-MB-231 cells was measured in an in vitro 51Cr release assay using freshly isolated PBMCs from healthy human donors at an E:T ratio of 100:1. For each data point, the mean and standard deviation of three replicate samples are shown. A representative example using PBMCs from one donor is shown. [Figure 14] Antibody-dependent cellular cytotoxicity of MDA-MB-231 cells induced by PD-L1 antibodies as measured in a luminescence ADCC reporter bioassay. ADCC of MDA-MB-231 cells induced by PD-L1 antibodies was quantified using FcγRIIIa-expressing Jurkat reporter cells, which express luciferase upon FcγRIIIa binding. Luciferase production is represented by relative light units (RLU). Each data point shows the mean and standard deviation of duplicate experiments. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description of the Invention definition The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of low molecular weight light (L) chains and one pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as VH or VH) and a heavy chain constant region (abbreviated herein as CH or CH). The heavy chain constant region typically consists of three domains: CH1, CH2, and CH3. The hinge region is located between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interaction between the two heavy chains in an IgG molecule. Each light chain typically consists of a light chain variable region (abbreviated herein as VL or VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region typically consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (or hypervariable regions that can vary significantly in sequence and / or take the form of loops with defined structures), also called complementarity-determining regions (CDRs), which are interspersed with regions that are more conserved than the hypervariable regions, called framework regions (FRs). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987). Unless otherwise specified or contradicted by context, CDR sequences herein are identified according to the IMGT rules using DomainGapAlign version 4.9.2 (2016-09-26) (Lefranc MP., Nucleic Acids Research 1999;27:209-212 and Ehrenmann F., Kaas Q. and Lefranc M.-P. Nucleic Acids Res., 38, D301-307 (2010); see also Internet http address http: / / www.imgt.org / (See also SEQ ID NO: 93, 1991, pp. 118-120, 2003). Unless otherwise specified or contradictory in context, the amino acid positions of the constant region in the present invention are described according to EU numbering (Edelman et al., Proc Natl Acad Sci US A. 1969 May;63(1):78-85; Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. 1991 NIH Publication No. 91-3242). For example, in the present specification, SEQ ID NO: 93 denotes amino acid positions 118-447 of the IgG1m(f) heavy chain constant region according to EU numbering.

[0018] As used herein, the term "amino acid corresponding to position ..." refers to the amino acid position number in the human IgG1 heavy chain. Corresponding amino acid positions in other immunoglobulins may be found by alignment with human IgG1. Thus, an amino acid or segment in one sequence that "corresponds to" an amino acid or segment in another sequence is an amino acid or segment that aligns with the other amino acid or segment using a standard sequence alignment program, e.g., ALIGN, ClustalW, or the like, typically using default settings, and that has at least 50%, at least 80%, at least 90%, or at least 95% identity with the human IgG1 heavy chain. Techniques for aligning sequences or segments within a sequence, thereby determining positions within a sequence that correspond to amino acid positions according to the invention, are considered well known in the art.

[0019] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions with a substantially long half-life, e.g., at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or more, about 48 hours or more, about 3 days, 4 days, 5 days, 6 days, 7 days, or more, or any other relevant, functionally defined period (e.g., a period sufficient to induce, promote, enhance, and / or modulate a physiological response associated with antibody-antigen binding and / or a period sufficient for the antibody to enhance effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigen. As used herein, the term "antibody binding region" refers to the region that interacts with an antigen and includes both the VH and VL regions. The term "antibody" as used herein includes not only monospecific antibodies but also multispecific antibodies that contain multiple, e.g., two or more, e.g., three or more, different antigen-binding regions. The constant region of an antibody (Ab) can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or host factors, including complement system components, such as C1q, the first component of the classical pathway of complement activation. As mentioned above, the term "antibody" as used herein includes antigen-binding fragments, i.e., antibody fragments that retain the ability to specifically bind to antigens, unless otherwise specified or clearly contradicted by the context. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.Examples of antigen-binding fragments encompassed within the term "antibody" include: (i) Fab' or Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains, or the monovalent antibodies described in WO2007059782 (Genmab); (ii) F(ab')2 fragments, bivalent fragments in which two Fab fragments are linked by a disulfide bridge at the hinge region; (iii) Fd fragments, consisting essentially of the VH and CH1 domains; (iv) Fv fragments, consisting essentially of the VL and VH domains of a single arm of an antibody; and (v) domain antibodies, consisting essentially of a VH domain (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11):484-90), also known as dAb fragments (Ward et al., Nature 341 , 544-546(1989)); (vi) camelids or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1):111-24), and (vii) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, the VL and VH regions pair to form a monovalent molecule (known as a single-chain antibody or single-chain Fv (scFv)). See, e.g., Bird et al., Science 242 , 423-426(1988) and Huston et al., PNAS USA 85The fragments may be joined using recombinant techniques by synthetic linkers that allow them to be produced as a single protein chain forming a single chain (see, e.g., J. Immunol., 5879-5883 (1988)). Such single-chain antibodies are included within the term antibody unless otherwise specified or clearly indicated by context. Although such fragments are generally included within the meaning of antibody, they are a unique feature of the present invention, collectively and individually exhibiting distinct biological properties and utilities. These and other useful antibody fragments, as well as bispecific forms of such fragments, in the context of the present invention are further discussed herein. The term antibody, unless otherwise specified, should also be understood to include polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, e.g., chimeric antibodies and humanized antibodies, as well as antibody fragments (antigen-binding fragments) that retain the ability to specifically bind to an antigen, provided by any known technique, e.g., enzymatic cleavage, peptide synthesis, and recombinant techniques. The antibodies produced may have any isotype. As used herein, the term "isotype" refers to the immunoglobulin class (e.g., IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by heavy chain constant region genes. When a particular isotype, e.g., IgG1, is referred to herein, the term is not limited to a particular isotype sequence, e.g., a particular IgG1 sequence, but is used to indicate that the sequence of the antibody resembles that isotype, e.g., IgG1, more than other isotypes. Thus, for example, an IgG1 antibody of the invention may be a sequence variant of a native IgG1 antibody, including altered constant regions.

[0020] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules with a single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody with a single binding specificity whose variable and constant regions are derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas in which B cells from transgenic or transchromosomal non-human animals, such as transgenic mice with genomes containing human heavy and light chain transgenes, are fused with immortalized cells.

[0021] In the context of the present invention, the term "bispecific antibody" or "bs" refers to an antibody having two different antigen-binding regions defined by different antibody sequences. In some embodiments, the different antigen-binding regions bind to different epitopes on the same antigen. However, in preferred embodiments, the different antigen-binding regions bind to different target antigens. The bispecific antibody may be any format of bispecific antibody, including any of the bispecific antibody formats described herein below.

[0022] As used herein, the terms "half molecule," "Fab arm," and "arm" refer to one heavy-light chain pair.

[0023] When a bispecific antibody is said to comprise a half-molecule antibody "derived from" a first antibody and a half-molecule antibody "derived from" a second antibody, the term "derived from" indicates that the bispecific antibody has been produced by recombining half molecules derived from each of the first and second antibodies into the resulting bispecific antibody by any known method. In this context, "recombining" is not intended to be limited by a particular recombination method, and thus includes all of the methods for producing bispecific antibodies described herein below, including, for example, recombination by half-molecule exchange, as well as recombination at the nucleic acid level and / or by co-expression of two half-molecules in the same cell.

[0024] The term "monovalent antibody" means in the context of the present invention that the antibody molecule can only bind to one type of antigen molecule and therefore cannot cross-link antigens or cells.

[0025] The term "full length," when used in the context of an antibody, indicates that the antibody is not a fragment, but contains all of the domains of a particular isotype that are normally found in nature for that particular isotype, e.g., the VH, CH1, CH2, CH3, hinge, VL, and CL domains in the case of an IgG1 antibody.

[0026] As used herein, unless the context indicates otherwise, the term "Fc region" refers to an antibody region consisting of two Fc sequences of an immunoglobulin heavy chain, said Fc sequences including at least a hinge region, a CH2 domain, and a CH3 domain.

[0027] As used herein, the term "heterodimeric interaction between a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and a second CH3 region in a first CH3 / second CH3 heterodimeric protein.

[0028] As used herein, the term "homodimeric interaction of a first CH3 region and a second CH3 region" refers to the interaction between a first CH3 region and another first CH3 region in a first CH3 / first CH3 homodimeric protein, and the interaction between a second CH3 region and another second CH3 region in a second CH3 / second CH3 homodimeric protein.

[0029] As used herein, the term "capable of binding" or "binding" with respect to the binding of an antibody to a given antigen or epitope typically refers to a binding activity of about 10, when measured using biolayer interferometry (BLI), e.g., as described in Example 8, or when measured using surface plasmon resonance (SPR) technology in a BIAcore 3000 instrument using the antigen as the ligand and the antibody as the analyte. -6 M or less, e.g., about 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less K D The antibody binds with an affinity corresponding to a K of at least 1 / 10, e.g., at least 1 / 100, e.g., at least 1 / 1,000, e.g., at least 1 / 10,000, e.g., at least 1 / 100,000, e.g., at least 1 / 100,000, of the affinity for binding to a non-specific antigen other than the given antigen or a closely related antigen (e.g., BSA, casein). D The amount by which affinity decreases is the antibody's K D As a result, the K D is very low (i.e., the antibody is highly specific), the affinity for the antigen may be at least 10,000-fold less than the affinity for a non-specific antigen.

[0030] As used herein, "k d" (sec -1 The term k ) refers to the dissociation rate constant of a particular antibody-antigen interaction. This value is k off or k dis Also called value.

[0031] As used herein, "K D The term "(M)" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. It is obtained by dividing kd by ka.

[0032] As used herein, "k a " (M -1 ×sec -1 The term k ) refers to the association rate constant of a particular antibody-antigen interaction. This value is k on Also called value or on-rate.

[0033] In a preferred embodiment, the antibodies of the present invention are isolated. As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities. In a preferred embodiment, an isolated bispecific antibody that specifically binds to PD-L1 and a second target, e.g., CD3, is substantially free of monospecific antibodies that specifically bind to PD-L1 or a second target, e.g., CD3. In another preferred embodiment, the antibody, or a pharmaceutical composition comprising the antibody, is substantially free of naturally occurring antibodies that are unable to bind to PD-L1. In a further preferred embodiment, the antibodies of the present invention have an altered amino acid sequence structure compared to the structure of a native anti-PD-L1 antibody, and this altered structure allows the antibody to exhibit altered functionality compared to the functionality exhibited by the native anti-PD-L1 antibody, the functionality being selected from the group consisting of (i) PD-L1 binding affinity, (ii) the ability to bind PD-L1 to PD-1, and (iii) the ability to induce Fc-mediated effector function.

[0034] The term "PD-L1," as used herein, refers to the programmed death-ligand 1 protein. PD-L1 is found in humans and other species, and thus, the term "PD-L1" is not limited to human PD-L1, unless the context indicates the contrary. Human PD-L1 sequences, macaque PD-L1 sequences, and mouse PD-L1 sequences can be found under Genbank accession numbers NP_054862.1, XP_005581836, and NP_068693, respectively.

[0035] The term "PD-L2," as used herein, refers to the human programmed death 1-ligand 2 protein (Genbank Accession No. NP_079515).

[0036] The term "PD-1," as used herein, refers to the human programmed death-1 protein, also known as CD279.

[0037] As used herein, the term "CD3" refers to the human cluster of differentiation 3 protein, which is part of the T-cell coreceptor protein complex and is composed of four distinct chains. CD3 is also found in other species, and therefore, the term "CD3" is not limited to human CD3, unless the context indicates otherwise. In mammals, this complex consists of one CD3γ (gamma) chain (human CD3γ chain UniProtKB / Swiss-Prot No. P09693, or cynomolgus monkey CD3γ UniProtKB / Swiss-Prot No. Q95LI7), one CD3δ (delta) chain (human CD3δ UniProtKB / Swiss-Prot No. P04234, or cynomolgus monkey CD3δ UniProtKB / Swiss-Prot No. Q95LI8), and two CD3ε (epsilon) chains (human CD3ε UniProtKB / Swiss-Prot No. P07766 (SEQ ID NO:95); cynomolgus monkey CD3ε UniProtKB / Swiss-Prot No. Q95LI5; or rhesus monkey CD3ε UniProtKB / Swiss-Prot No. G7NCB9), and one CD3ζ (zeta) chain (human CD3ζ UniProtKB / Swiss-Prot No P20963, cynomolgus monkey CD3ζ UniProtKB / Swiss-Prot No Q09TK0). These chains bind to a molecule known as the T cell receptor (TCR), generating an activation signal in T lymphocytes. The TCR and CD3 molecules together form the TCR complex.

[0038] A "PD-L1 antibody" or "anti-PD-L1 antibody" is an antibody, as described above, that specifically binds to the antigen PD-L1, in particular human PD-L1.

[0039] A "CD3 antibody" or "anti-CD3 antibody" is an antibody as described above that specifically binds to the antigen CD3, in particular human CD3ε (epsilon).

[0040] A "CD3xPD-L1 antibody," "anti-CD3xPD-L1 antibody," "PD-L1xCD3 antibody," or "anti-PD-L1xCD3 antibody" is a bispecific antibody that contains two different antigen-binding regions: one that specifically binds to the antigen PD-L1, and one that specifically binds to CD3.

[0041] The present invention also provides antibodies comprising functional variants of the VL region, VH region, or one or more CDRs of the example antibodies. Even if a functional variant of the VL, VH, or CDR is used in connection with an antibody, the antibody still retains at least a significant proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, or more) of the affinity and / or specificity / selectivity of the "reference" or "parent" antibody, and in some cases, such antibodies may be associated with greater affinity, selectivity, and / or specificity than the parent antibody.

[0042] Such functional variants typically retain significant sequence identity with the parent antibody. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., % homology = number of identical positions / total number of positions × 100). The percent identity between two nucleotide or amino acid sequences can be determined, for example, using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4, 11-17 (1988), incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch, J. Mol. Biol. 48, 444-453 (1970)).

[0043] Exemplary variants include those that differ from the VH and / or VL and / or CDR regions of a parent antibody sequence primarily by conservative substitutions, e.g., 10, e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the substitutions in the variant are conservative exchanges of amino acid residues.

[0044] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes reflected in the table below.

[0045] Amino acid residue classes for conservative substitutions TIFF0007640614000001.tif53139

[0046] In the context of the present invention, the following notation is used to describe mutations unless otherwise specified: (i) A substitution of an amino acid at a certain position is written, for example, K409R, which means that the lysine at position 409 is substituted with arginine. (ii) For specific variants, a specific three-letter or one-letter notation is used, with the symbols Xaa and X representing any amino acid residue. Thus, a substitution of lysine at position 409 with arginine is indicated by K409R, and a substitution of lysine at position 409 with any amino acid residue is indicated by K409X. In the case of a deletion of lysine at position 409, this is indicated by K409R. * It is shown as follows.

[0047] In the context of the present invention, "competition" refers to the significant reduction in the tendency of a particular molecule to bind to a particular binding partner in the presence of another molecule that binds to the binding partner. "Competition" can also be referred to as "blocking" or "displacement." That is, a competing molecule can be either a blocking molecule or a displacing molecule. "Displacement" refers to a state in which a second antibody displaces an antigen from a (previously formed) antigen-antibody complex, thereby allowing it to be exchanged for this antigen (Abdiche et al., 2017 Plos One 12(1): e0169535). Competition between two or more anti-PD-L1 antibodies for binding to PD-L1 can be measured by any suitable technique. In one embodiment, competition is measured as described in Example 9 herein.

[0048] Similarly, in the context of the present invention, "inhibition of binding of PD-L1 to PD-1" refers to a detectably significant reduction in the binding of PD-L1 to PD-1 in the presence of an antibody capable of binding to PD-L1. Typically, inhibition refers to at least about a 10% reduction in the binding between PD-L1 and PD-1 caused by the presence of an anti-PD-L1 antibody, such as at least about a 15%, such as at least about a 20%, such as at least a 40% reduction. Inhibition of binding of PD-L1 to PD-1 can be measured by any suitable technique. In one embodiment, inhibition is measured as described in Example 10 herein.

[0049] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, but the binding to the latter is not lost. Epitopes may include amino acid residues directly involved in binding as well as other amino acid residues not directly involved in binding, such as amino acid residues that are effectively blocked or covered by the specifically antigen-binding peptide (in other words, these amino acid residues are within the footprint of the specifically antigen-binding peptide).

[0050] As used herein, the term "chimeric antibody" refers to an antibody whose variable region is derived from a non-human species (e.g., from a rodent) and whose constant region is derived from a different species, such as human. To reduce antibody immunogenicity, chimeric monoclonal antibodies for therapeutic use have been developed. The term "variable region" or "variable domain" used in reference to a chimeric antibody refers to the region containing the CDRs and framework regions of both the heavy and light chains of an immunoglobulin. Chimeric antibodies can be produced using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Chapter 15. Chimeric antibodies can be genetically engineered or enzymatically engineered recombinant antibodies. Producing chimeric antibodies is within the knowledge of those skilled in the art, and therefore, chimeric antibodies according to the present invention can be produced by methods other than those described herein.

[0051] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting the six non-human antibody complementarity-determining regions (CDRs), which together form the antigen-binding site, to homologous human acceptor framework regions (FRs) (see WO92 / 22653 and EP0629240). To fully reconstitute the binding affinity and specificity of the parent antibody, it may be necessary to substitute (backmutate) framework residues from the parent antibody (i.e., non-human antibody) into a human framework. Structural homology modeling can help identify amino acid residues within the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody may comprise non-human CDR sequences, primarily human framework regions, optionally containing one or more amino acid backmutations to non-human amino acid sequences, and a fully human constant region. Optionally, further amino acid modifications, not necessarily back mutations, may be applied to obtain humanized antibodies with favorable characteristics, eg, affinity and biochemical properties.

[0052] As used herein, the term "human antibody" refers to an antibody having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Human monoclonal antibodies of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methods, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature 256:495 (1975). While somatic cell hybridization is generally preferred, other techniques for producing monoclonal antibodies, such as viral transformation or oncogenesis of B lymphocytes or phage display techniques using human antibody gene libraries, can also be used. A suitable animal system for preparing hybridomas secreting human monoclonal antibodies is the murine system. Hybridoma production in mice is a well-established procedure. Immunization protocols and methods for isolation of immunized splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known. Thus, human monoclonal antibodies can be generated, for example, using transgenic or transchromosomal mice or rats carrying parts of the human immune system in a mouse system but not in a rat system. Thus, in one embodiment, human antibodies are obtained from transgenic animals, such as mice or rats, that have human germline immunoglobulin sequences in place of the animal's immunoglobulin sequences. In such embodiments, the antibody originates from human germline immunoglobulin sequences introduced into the animal, but the final antibody sequence is the result of further modification of the human germline immunoglobulin sequences by somatic hypermutation and affinity maturation by the animal's endogenous antibody machinery.See, e.g., Mendez et al. 1997 Nat Genet. 15(2):146-56. The term "reducing conditions" or "reducing environment" refers to conditions or circumstances in which a substrate, here a cysteine ​​residue in the hinge region of an antibody, is more likely to be reduced than oxidized.

[0053] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which an expression vector, e.g., an expression vector encoding an antibody of the invention, has been introduced. Recombinant host cells include, for example, transfectomas such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NS0 cells, and lymphocytic cells.

[0054] The term "treatment" refers to the administration of a therapeutically active effective amount of an antibody of the invention with the intent to alleviate, ameliorate, arrest, or eradicate (cure) the symptoms or disease state.

[0055] The term "effective amount" or "therapeutically effective amount" refers to an amount that exhibits an effect effective to achieve a desired therapeutic result, at dosages and for periods of time necessary to achieve that result. A therapeutically effective amount of an antibody may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of an antibody or antibody portion are outweighed by the therapeutically beneficial effects.

[0056] The term "anti-idiotypic antibody" refers to an antibody that recognizes unique determinants generally associated with the antigen-binding site of an antibody.

[0057] Further aspects and embodiments of the present invention As mentioned above, in a first aspect, the present invention relates to a bispecific antibody comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD3ε (epsilon), wherein the bispecific antibody inhibits the binding of human PD-L1 to human PD-1. Thus, such a bispecific antibody comprises two different antigen-binding regions, one antigen-binding region with binding specificity for PD-L1 and one antigen-binding region with binding specificity for CD3.

[0058] In one embodiment, the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, wherein the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:21.

[0059] In a further embodiment, the antigen-binding region capable of binding to human PD-L1 comprises: (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO: 19, the CDR2 sequence shown in SEQ ID NO: 20, and the CDR3 sequence shown in SEQ ID NO: 21, and a light chain variable region (VL) comprising a CDR1 having the sequence shown in SEQ ID NO: 23, a CDR2 having the sequence DDN, and a CDR3 having the sequence shown in SEQ ID NO: 24. Includes:

[0060] In another embodiment, the antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:18.

[0061] In another embodiment, the antigen-binding region capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22.

[0062] In a further embodiment, the antigen-binding region capable of binding to human PD-L1 comprises: (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22. Includes:

[0063] Thus, for example, the antigen-binding region capable of binding to human PD-L1 may be: a VH sequence having at least 95% amino acid sequence identity with the VH sequence set forth in SEQ ID NO:1 and a VL sequence having at least 95% amino acid sequence identity with the VL sequence set forth in SEQ ID NO:5; or a VH sequence having at least 97% amino acid sequence identity with the VH sequence set forth in SEQ ID NO:1 and a VL sequence having at least 97% amino acid sequence identity with the VL sequence set forth in SEQ ID NO:5; or a VH sequence having at least 99% amino acid sequence identity with the VH sequence set forth in SEQ ID NO:1 and a VL sequence having at least 99% amino acid sequence identity with the VL sequence set forth in SEQ ID NO:5; or a VH sequence having at least 95% amino acid sequence identity with the VH sequence set forth in SEQ ID NO:8 and a VL sequence having at least 95% amino acid sequence identity with the VL sequence set forth in SEQ ID NO:15; or a VH sequence having at least 97% amino acid sequence identity with the VH sequence set forth in SEQ ID NO:8 and a VL sequence having at least 97% amino acid sequence identity with the VL sequence set forth in SEQ ID NO:15; or a VH sequence having at least 99% amino acid sequence identity with the VH sequence set forth in SEQ ID NO:8 and a VL sequence having at least 99% amino acid sequence identity with the VL sequence set forth in SEQ ID NO:15; or a VH sequence having at least 95% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18 and a VL sequence having at least 95% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22; or a VH sequence having at least 97% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18 and a VL sequence having at least 97% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22; or a VH sequence having at least 99% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 99% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22. Includes:

[0064] In further embodiments, the VH and VL sequences each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, and each combined FR1, FR2, FR3, and FR4 framework sequence of the VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequences are not mutated; and each combined FR1, FR2, FR3, and FR4 framework sequence of the VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequences are not mutated. For this embodiment, the percent identity refers to the percent identity that would result when the framework sequences were put together as one contiguous sequence, without the intervening CDR sequences.

[0065] In a preferred embodiment of the antibody of the invention, the antigen-binding region capable of binding to human PD-L1 comprises: (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5, or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22 Includes:

[0066] Different antibodies capable of binding to the same antigen, such as PD-L1, may bind to different regions of the antigen. In some cases, binding of one PD-L1 antibody to PD-L1 may still result in binding of a different PD-L1 antibody to PD-L1. However, in other cases, binding of one PD-L1 antibody to PD-L1 may compete with (block or replace) binding of a different PD-L1 antibody to PD-L1. Thus, competition experiments can provide information about where an antibody binds on a target antigen, which may affect the functional effect of antibody binding.

[0067] In one embodiment, the bispecific antibody of the invention comprises: (i) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15.

[0068] In a further embodiment herein, the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5.

[0069] Antibodies that compete for target antigen binding may bind to different epitopes on the antigen, and these epitopes may be so close to each other that a first antibody that binds to one epitope will block the binding of a second antibody to the other epitope. However, in other situations, two different antibodies may bind to the same epitope on an antigen.

[0070] Thus, in one embodiment, the antibody of the invention comprises: (i) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) it is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0071] In a further embodiment, the binding of the bispecific antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57.

[0072] In a further embodiment, the binding of the bispecific antibody to human PD-L1 is blocked by an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22. As used herein, "blocked" indicates that the antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22 competes with, but does not displace, the bispecific antibody.

[0073] As mentioned above, the bispecific antibody according to the first aspect of the present invention described above comprises an antigen-binding region capable of binding to human CD3ε.

[0074] In one embodiment, the antigen-binding region capable of binding to human CD3ε comprises a heavy chain variable (VH) region CDR1 having the sequence set forth in SEQ ID NO:26, a heavy chain variable (VH) region CDR2 having the sequence set forth in SEQ ID NO:27, and a heavy chain variable (VH) region CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising CDR1 having the sequence set forth in SEQ ID NO:30, CDR2 having the sequence GTN, and CDR3 having the sequence set forth in SEQ ID NO:31.

[0075] The six CDR sequences defined above are derived from a murine antibody designated SP34. A humanized version of this antibody has been produced, designated herein as huCD3, and further disclosed in WO2015001085 (Genmab).

[0076] In a preferred embodiment of the bispecific antibody of the present invention, the bispecific antibody comprises: (i) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (ii) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (iii) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:23, a CDR2 having the sequence DDN, and a CDR3 having the sequence set forth in SEQ ID NO:24; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31. Includes:

[0077] In one embodiment, the bispecific antibody comprises an antigen-binding region capable of binding to human CD3ε comprising a heavy chain variable region (VH), wherein said VH sequence has at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:25.

[0078] In another embodiment, the bispecific antibody comprises an antigen-binding region capable of binding to human CD3ε comprising a light chain variable region (VL), wherein said VL sequence has at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO:29.

[0079] In a preferred embodiment, the antigen-binding region capable of binding to human CD3ε comprises the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29.

[0080] In one aspect, bispecific antibodies according to the present invention may be modified to reduce the affinity of the antibody. This may be advantageous in some situations and may lead to increased efficacy. In particular, low affinity binding to human CD3ε may affect the motility of T cells in the circulation and at the tumor site, and thus may lead to better binding of T cells to tumor cells. See Molhoj et al., Molecular Immunology 44 (2007).

[0081] Thus, in a different embodiment of a bispecific antibody of the invention comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD3ε: (i) the bispecific antibody has a lower affinity for human CD3ε binding compared to an antibody having an antigen-binding region that can comprise the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29, preferably the affinity is at least 2-fold lower, such as at least 5-fold lower, such as at least 10-fold lower, such as at least 25-fold lower, such as at least 50-fold lower; and (ii) The bispecific antibody can mediate concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when PBMCs or purified T cells are used as effector cells.

[0082] Similarly, in one embodiment, an antibody of the invention comprises an antigen-binding region capable of binding to human CD3ε, (i) the antigen-binding region capable of binding to human CD3ε has a lower affinity for binding to human CD3ε compared to an antibody having an antigen-binding region that can comprise the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29, preferably the affinity is at least 2-fold lower, such as at least 5-fold lower, such as at least 10-fold lower, such as at least 25-fold lower, such as at least 50-fold lower; and (ii) The antigen-binding region capable of binding to human CD3ε can mediate concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when PBMCs or purified T cells are used as effector cells.

[0083] Affinity for human CD3ε may be measured, for example, using octet binding affinity measurements as described in Example 7 of WO2017009442.

[0084] The ability of an antibody to mediate cytotoxicity by PBMCs or purified T cells may be measured, for example, as described in Example 11 herein, i.e., MDA-MB-231, PC-3, or HELA cells are seeded and cultured in wells. Tumor cells, PBMCs, and serial dilutions of antibody are added, and after incubation, tumor cells are stained for viability.

[0085] As used herein, huCD3-H1L1 refers to an anti-CD3 antibody having the VH sequence shown in SEQ ID NO: 25 and the VL sequence shown in SEQ ID NO: 29. IgG1-huCD3-FEAL refers to a variant thereof that contains the substitutions L234F, L235E, D265A, and F405L (see elsewhere herein).

[0086] Examples of IgG1-huCD3-FEAL variants with reduced affinity for human CD3ε are described in WO2017009442 (Genmab). Example 7 (Table 6) of WO2017009442 discloses the affinities of IgG1-huCD3-FEAL and its seven variants measured using octet binding affinity measurements. TIFF0007640614000002.tif60128

[0087] In one embodiment of the bispecific antibody of the invention, the antigen-binding region capable of binding to human CD3ε comprises: (i) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:99, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (ii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO: 100, a CDR2 having the sequence set forth in SEQ ID NO: 27, and a CDR3 having the sequence set forth in SEQ ID NO: 28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO: 31; or (iii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:101, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (iv) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:102, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (v) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:103, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (vi) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:104, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (vii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO: 26, a CDR2 having the sequence set forth in SEQ ID NO: 27, and a CDR3 having the sequence set forth in SEQ ID NO: 105, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO: 31. Includes:

[0088] In another embodiment, the antigen-binding region capable of binding to human CD3ε is (i) the VH sequence set forth in SEQ ID NO:39 and the VL sequence set forth in SEQ ID NO:29; or (ii) the VH sequence set forth in SEQ ID NO:40 and the VL sequence set forth in SEQ ID NO:29; or (iii) the VH sequence set forth in SEQ ID NO: 41 and the VL sequence set forth in SEQ ID NO: 29; or (iv) the VH sequence set forth in SEQ ID NO: 42 and the VL sequence set forth in SEQ ID NO: 29; or (v) the VH sequence set forth in SEQ ID NO: 43 and the VL sequence set forth in SEQ ID NO: 29; or (vi) the VH sequence set forth in SEQ ID NO: 44 and the VL sequence set forth in SEQ ID NO: 29; or (vii) the VH sequence shown in SEQ ID NO:45 and the VL sequence shown in SEQ ID NO:29 Includes:

[0089] In a further preferred embodiment of the bispecific antibodies of the invention, each of the antigen-binding regions comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of said variable regions comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively.

[0090] In a further preferred embodiment of the bispecific antibody of the invention, said antibody comprises two heavy chain constant regions (CH) and two chain constant regions (CL).

[0091] In a preferred embodiment, the bispecific antibody comprises a first heavy chain and a second heavy chain, each of which comprises at least a hinge region, a CH2 region, and a CH3 region; wherein the first heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering); and the second heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering); and the first heavy chain and the second heavy chain do not have substitutions at the same positions.

[0092] Most preferably, (i) in the first heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L and in the second heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R, or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R and in the second heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L.

[0093] In a further particularly preferred embodiment, the antibody is a CD3xPD-L1 bispecific antibody comprising a first heavy chain and a second heavy chain, wherein both the first and second heavy chains have F and E at positions corresponding to L234 and L235 in the human IgG1 heavy chain according to EU numbering, respectively, and wherein (i) the first heavy chain has L at position corresponding to F405 in its human IgG1 heavy chain according to EU numbering and the second heavy chain has R at position corresponding to K409 in its human IgG1 heavy chain according to EU numbering, or (ii) the first heavy chain has R at position corresponding to K409 in its human IgG1 heavy chain according to EU numbering and the second heavy chain has L at position corresponding to F405 in its human IgG1 heavy chain according to EU numbering.

[0094] In a further particularly preferred embodiment, the antibody is a CD3xPD-L1 bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chains are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0095] A novel class of PD-L1 antibodies In a further aspect, the present invention provides novel anti-PD-L1 antibodies, which comprise an antigen-binding region capable of binding to human PD-L1. The antibodies of this aspect of the invention may be monospecific or multispecific, and if multispecific, the multispecific antibody may or may not comprise an antigen-binding region capable of binding to human CD3ε.

[0096] In one aspect, the present invention provides an antibody comprising an antigen-binding region capable of binding to human PD-L1, wherein said antigen-binding region capable of binding to human PD-L1 has the characteristics defined herein above.

[0097] In one aspect, the invention provides an antibody comprising an antigen-binding region capable of binding to human PD-L1, wherein the binding of the antibody to a mutant PD-L1 comprising one or more of the amino acid residues corresponding to position 113 (R113), 123 (Y123), and 125 (R125) in SEQ ID NO:94 substituted with alanine, is reduced compared to binding to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, wherein reduced binding is determined when the fold change in binding of the antibody is less than the mean fold change in binding across all alanine mutants - 1.5 x SD, where SD is the standard deviation of all calculated fold change values ​​of the antibody to mutant PD-L1, and the fold change in binding is calculated as described in Example 13.

[0098] The antibody may bind to an epitope on PD-L1 (SEQ ID NO:94), wherein the epitope includes the amino acid residue at position 113 (R113), the amino acid residue at position 123 (Y123), and / or the amino acid residue at position 125 (R125) of SEQ ID NO:94.

[0099] In one aspect, the invention provides an antibody comprising an antigen-binding region capable of binding to human PD-L1, wherein the antibody has reduced binding to a mutant PD-L1 having an alanine substitution at one or more of the following amino acid residues in SEQ ID NO:94: amino acid residue at a position corresponding to position 19 (F19), amino acid residue at a position corresponding to position 42 (F42), amino acid residue at a position corresponding to position 45 (E45), amino acid residue at a position corresponding to position 46 (K46), amino acid residue at a position corresponding to position 94 (L94), and amino acid residue at a position corresponding to position 116 (I116) in SEQ ID NO:94; wherein reduced binding is determined when the fold change in binding of the antibody is less than the mean fold change in binding across all alanine mutants - 1.5 × SD, where SD is the standard deviation of all calculated fold change values ​​of the antibody to mutant PD-L1, and the fold change in binding is calculated as described in Example 13.

[0100] The antibody may bind to an epitope on PD-L1 (SEQ ID NO:94), wherein the epitope comprises one or more amino acid residues selected from the group consisting of the amino acid residue at position 45 (E45), the amino acid residue at position 46 (K46); and / or the amino acid residue at position 94 (L94) of SEQ ID NO:94.

[0101] In one aspect, the invention provides an antibody comprising an antigen-binding region capable of binding to human PD-L1, wherein the antibody has reduced binding to a mutant PD-L1 comprising an alanine substitution at one or more of the amino acid residues corresponding to position 58 (E58) and 113 (R113) in SEQ ID NO:94, relative to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, wherein reduced binding is determined when the fold change in binding of the antibody is less than the mean fold change in binding across all alanine mutants - 1.5 x SD, where SD is the standard deviation of all calculated fold change values ​​of the antibody to mutant PD-L1, and the fold change in binding is calculated as described in Example 13.

[0102] The antibody may bind to an epitope on PD-L1 (SEQ ID NO:94), wherein the epitope includes the amino acid residue at position 58 (E58) and / or the amino acid residue at position 113 (R113) of SEQ ID NO:94.

[0103] In a further embodiment, the antibody according to the invention is capable of inducing dose-dependent lysis of adenocarcinoma epithelial cells, for example MDA-MB-231, via antibody-dependent cellular cytotoxicity (ADCC).

[0104] In a further embodiment, the antibody according to the invention is capable of reducing the number of cells in a culture of said epithelial cells as a result of cell lysis by at least 5%, such as at least 6%, 7%, 8%, 9%, or at least 10%.

[0105] ADCC is 51ADCC may also be measured in vitro in a Cr release assay, such as the assay disclosed in Example 14. In particular, ADCC is measured in vitro by incubating the epithelial cells with a composition comprising the antibody and effector cells, such as peripheral blood mononuclear cells (PBMCs), for 4 hours at 37°C, 5% CO2, where the amount of antibody in the composition is in the range of 0.1-1 μg / mL and the ratio of effector cells to epithelial cells is 100:1.

[0106] Epithelial cell lysis may be measured in vitro in a luciferase reporter assay as a surrogate for ADCC, for example, the luminescence ADCC reporter bioassay disclosed in Example 14.

[0107] ADCC, in particular, (i) contacting the culture of epithelial cells with a composition comprising the antibody and Jurkat human T cells stably expressing FcγRIIIa (CD16) and firefly luciferase (effector cells) at a 1:1 effector cell:epithelial cell ratio; (ii) allowing the epithelial cell cultures and effector cells to equilibrate to room temperature for 15 minutes; (iii) incubating the epithelial cell culture and the effector cells with a luciferase substrate; and (iv) measuring luciferase production in the cell culture The amount of antibody in the composition may be in the range of 0.5 to 250 ng / mL, and the ratio of effector cells to epithelial cells is 1:1.

[0108] The ADCC of epithelial cells may be measured in a luciferase reporter assay, such as a reporter assay as defined in claim 23 or 24, and then the ADCC observed after incubating a culture of epithelial cells with a test composition comprising the antibody is at least 1.5 times the ADCC observed after incubating a culture of epithelial cells with a composition comprising a reference antibody, where ADCC is measured as relative light units (RLU), and the antibody concentrations in the test composition and the composition comprising the reference antibody are the same and are in the range of 20 to 250 ng / ml, and the reference antibody is (a) an antibody comprising the VH sequence set forth in SEQ ID NO:74 and the VL sequence set forth in SEQ ID NO:78; and (b) an antibody comprising the VH sequence shown in SEQ ID NO:81 and the VL sequence shown in SEQ ID NO:85 It may be selected from the following.

[0109] In one embodiment, an antibody is provided, which comprises an antigen-binding region capable of binding to human PD-L1, wherein the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, and a light chain variable region (VL) comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, wherein the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:21.

[0110] In a further aspect, an antibody is provided comprising an antigen-binding region capable of binding to human PD-L1, wherein said antigen-binding region capable of binding to human PD-L1 is: (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO: 19, the CDR2 sequence shown in SEQ ID NO: 20, and the CDR3 sequence shown in SEQ ID NO: 21, and a light chain variable region (VL) comprising a CDR1 having the sequence shown in SEQ ID NO: 23, a CDR2 having the sequence DDN, and a CDR3 having the sequence shown in SEQ ID NO: 24. Includes:

[0111] In a further embodiment, an antibody is provided, comprising an antigen-binding region capable of binding to human PD-L1, wherein said antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:18.

[0112] In a further embodiment, an antibody is provided, comprising an antigen-binding region capable of binding to human PD-L1, wherein said antigen-binding region capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:52, SEQ ID NO:15, and SEQ ID NO:22.

[0113] In a further aspect, an antibody is provided comprising an antigen-binding region capable of binding to human PD-L1, wherein said antigen-binding region capable of binding to human PD-L1 is: (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22. Includes:

[0114] In a further embodiment, an antibody is provided, comprising an antigen-binding region capable of binding to human PD-L1, wherein the antigen-binding region capable of binding to human PD-L1 comprises a VH sequence and a VL sequence, wherein the VH sequence and the VL sequence each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, and wherein each combined FR1, FR2, FR3, and FR4 framework sequence of the VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and The CDR sequences are not mutated, and each combined FR1, FR2, FR3, and FR4 framework sequence of the VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequences are not mutated.

[0115] In a further aspect, an antibody is provided comprising an antigen-binding region capable of binding to human PD-L1, wherein said antigen-binding region capable of binding to human PD-L1 is: (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5, or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22 Includes:

[0116] In a further aspect, an antibody is provided comprising an antigen-binding region capable of binding to human PD-L1, said antibody comprising: (i) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15.

[0117] Preferably, the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:5.

[0118] In a further embodiment, an antibody is provided that comprises an antigen-binding region that is capable of binding to human PD-L1, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence set forth in SEQ ID NO:53 and the VL sequence set forth in SEQ ID NO:57.

[0119] In a further embodiment, an antibody is provided that comprises an antigen-binding region that is capable of binding to human PD-L1, wherein the binding of the antibody to human PD-L1 is not blocked by the binding of an antibody comprising the VH sequence set forth in SEQ ID NO:106 and the VL sequence set forth in SEQ ID NO:110.

[0120] In a further embodiment, an antibody is provided that comprises an antigen-binding region that is capable of binding to human PD-L1, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence set forth in SEQ ID NO:53 and the VL sequence set forth in SEQ ID NO:57, and that inhibits the binding of human PD-L1 to human PD-1.

[0121] In a further embodiment, an antibody is provided that comprises an antigen-binding region capable of binding to human PD-L1, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence set forth in SEQ ID NO:53 and the VL sequence set forth in SEQ ID NO:57, and the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0122] In a further embodiment, an antibody is provided that comprises an antigen-binding region capable of binding to human PD-L1, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence set forth in SEQ ID NO:53 and the VL sequence set forth in SEQ ID NO:57, and the binding of the antibody to human PD-L1 is blocked by an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22. As used herein, "blocked" indicates that an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22 competes with, but does not displace, the bispecific antibody.

[0123] In a further aspect, an antibody is provided comprising an antigen-binding region capable of binding to human PD-L1, said antibody comprising: (i) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) it is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0124] In a further aspect, the invention relates to an antibody comprising an antigen-binding region capable of binding to human PD-L1, said antibody comprising: (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:33, the CDR2 sequence set forth in SEQ ID NO:34, and the CDR3 sequence set forth in SEQ ID NO:35, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:37, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:38; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:47, the CDR2 sequence set forth in SEQ ID NO:48, and the CDR3 sequence set forth in SEQ ID NO:49, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:51, a CDR2 having the sequence DVI, and a CDR3 having the sequence set forth in SEQ ID NO:52; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:54, the CDR2 sequence set forth in SEQ ID NO:55, and the CDR3 sequence set forth in SEQ ID NO:56, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:58, a CDR2 having the sequence RDS, and a CDR3 having the sequence set forth in SEQ ID NO:59; or (iv) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:61, the CDR2 sequence set forth in SEQ ID NO:62, and the CDR3 sequence set forth in SEQ ID NO:63, and a light chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:65, a CDR2 having the sequence DDS, and a CDR3 having the sequence set forth in SEQ ID NO:66; or (v) a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO: 107, the CDR2 sequence shown in SEQ ID NO: 108, and the CDR3 sequence shown in SEQ ID NO: 109, and a light chain variable region (VL) comprising a CDR1 having the sequence shown in SEQ ID NO: 111, a CDR2 having the sequence EDS, and a CDR3 having the sequence shown in SEQ ID NO: 113; (vi) a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO: 68, the CDR2 sequence shown in SEQ ID NO: 69, and the CDR3 sequence shown in SEQ ID NO: 70, and a light chain variable region (VL) comprising a CDR1 having the sequence shown in SEQ ID NO: 72, a CDR2 having the sequence EDS, and a CDR3 having the sequence shown in SEQ ID NO: 73. Includes:

[0125] In a further embodiment, the antibody (i) the VH sequence set forth in SEQ ID NO: 32 and the VL sequence set forth in SEQ ID NO: 36, or (ii) the VH sequence set forth in SEQ ID NO:46 and the VL sequence set forth in SEQ ID NO:50; or (iii) the VH sequence set forth in SEQ ID NO: 53 and the VL sequence set forth in SEQ ID NO: 57; or (iv) the VH sequence set forth in SEQ ID NO: 60 and the VL sequence set forth in SEQ ID NO: 64; or (v) the VH sequence set forth in SEQ ID NO: 106 and the VL sequence set forth in SEQ ID NO: 110; or (v) the VH sequence shown in SEQ ID NO:67 and the VL sequence shown in SEQ ID NO:71 Includes:

[0126] In one embodiment, the antibody comprising an antigen-binding region capable of binding to human PD-L1 is monovalent.

[0127] In another embodiment, the antibody comprising an antigen-binding region capable of binding to human PD-L1 is a monospecific antibody comprising two or more identical antigen-binding regions.

[0128] In a further embodiment, the antibody comprising an antigen-binding region capable of binding to human PD-L1 is a bivalent antibody having two antigen-binding regions capable of binding to human PD-L1, and the two antigen-binding regions have identical variable region sequences.

[0129] In a different embodiment, the antibody comprising an antigen-binding region capable of binding to human PD-L1 is a bivalent, bispecific antibody, which comprises a (first) antigen-binding region capable of binding to said human PD-L1 and a (second) antigen-binding region capable of binding to a second antigen, wherein said second antigen is human CD3ε.

[0130] In a different embodiment, the antibody comprising an antigen-binding region capable of binding to human PD-L1 is a bivalent, bispecific antibody, which comprises, in addition to said (first) antigen-binding region capable of binding to human PD-L1, a (second) antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein said second antigen is not human CD3ε.

[0131] In a further aspect, the invention relates to a multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is optionally not human CD3ε; and the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, wherein the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4, SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:49, SEQ ID NO:56, SEQ ID NO:63, and SEQ ID NO:70. As used herein, "different epitopes" means that the epitope to which the second antigen-binding region binds is different from the epitope to which the first antigen-binding region binds.

[0132] In one embodiment of the multispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises: (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO: 19, the CDR2 sequence shown in SEQ ID NO: 20, and the CDR3 sequence shown in SEQ ID NO: 21, and a light chain variable region (VL) comprising a CDR1 having the sequence shown in SEQ ID NO: 23, a CDR2 having the sequence DDN, and a CDR3 having the sequence shown in SEQ ID NO: 24; (iv) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 33, the CDR2 sequence set forth in SEQ ID NO: 34, and the CDR3 sequence set forth in SEQ ID NO: 35, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 37, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO: 38; or (v) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:47, the CDR2 sequence set forth in SEQ ID NO:48, and the CDR3 sequence set forth in SEQ ID NO:49, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:51, a CDR2 having the sequence DVI, and a CDR3 having the sequence set forth in SEQ ID NO:52; or (vi) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 54, the CDR2 sequence set forth in SEQ ID NO: 55, and the CDR3 sequence set forth in SEQ ID NO: 56, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 58, a CDR2 having the sequence RDS, and a CDR3 having the sequence set forth in SEQ ID NO: 59; or (vii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:61, the CDR2 sequence set forth in SEQ ID NO:62, and the CDR3 sequence set forth in SEQ ID NO:63, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:65, a CDR2 having the sequence DDS, and a CDR3 having the sequence set forth in SEQ ID NO:66; or (viii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 68, the CDR2 sequence set forth in SEQ ID NO: 69, and the CDR3 sequence set forth in SEQ ID NO: 70, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 72, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO: 73. Includes:

[0133] In another embodiment of the multispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:18.

[0134] In another embodiment of the multispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22.

[0135] In another embodiment of the multispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises: (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22. Includes:

[0136] In another embodiment of the multispecific antibody, the VH and VL sequences each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, and each combined FR1, FR2, FR3, and FR4 framework sequence of the VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequences are not mutated; and each combined FR1, FR2, FR3, and FR4 framework sequence of the VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequences are not mutated.

[0137] In another embodiment of the multispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises: (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5, or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) the VH sequence set forth in SEQ ID NO: 18 and the VL sequence set forth in SEQ ID NO: 22; or (iv) the VH sequence set forth in SEQ ID NO: 32 and the VL sequence set forth in SEQ ID NO: 36; or (v) the VH sequence set forth in SEQ ID NO: 46 and the VL sequence set forth in SEQ ID NO: 50; or (vi) the VH sequence set forth in SEQ ID NO: 53 and the VL sequence set forth in SEQ ID NO: 57; or (v) the VH sequence set forth in SEQ ID NO: 106 and the VL sequence set forth in SEQ ID NO: 110; or (vii) the VH sequence set forth in SEQ ID NO: 60 and the VL sequence set forth in SEQ ID NO: 64; or (viii) the VH sequence shown in SEQ ID NO: 67 and the VL sequence shown in SEQ ID NO: 71 Includes:

[0138] In a further aspect, the invention relates to a multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is optionally not human CD3ε, and wherein the antibody is (i) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15.

[0139] In one embodiment of the multispecific antibody, the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5.

[0140] In a further aspect, the invention relates to a multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different epitope on human PD-L1, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57.

[0141] In one embodiment herein, the antibody inhibits the binding of human PD-L1 to human PD-1.

[0142] In a further embodiment, the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0143] In a further embodiment, the binding of the antibody to human PD-L1 is blocked by an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22. As used herein, "blocked" indicates that the antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22 competes with, but does not displace, the bispecific antibody.

[0144] In a further aspect, the present invention relates to a multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is optionally not human CD3ε, and the first antigen-binding region is (i) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) it is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0145] In a further aspect, the invention relates to a bivalent, bispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is optionally not human CD3ε; and wherein the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, and the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:21.

[0146] In one embodiment of the bivalent, bispecific antibody, the antigen-binding region capable of binding to human PD-L1 is (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence shown in SEQ ID NO: 19, the CDR2 sequence shown in SEQ ID NO: 20, and the CDR3 sequence shown in SEQ ID NO: 21, and a light chain variable region (VL) comprising a CDR1 having the sequence shown in SEQ ID NO: 23, a CDR2 having the sequence DDN, and a CDR3 having the sequence shown in SEQ ID NO: 24. Includes:

[0147] In another embodiment of the bivalent, bispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:18.

[0148] In another embodiment of the bivalent, bispecific antibody, the antigen-binding region capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22.

[0149] In another embodiment of the bivalent, bispecific antibody, the antigen-binding region capable of binding to human PD-L1 is (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity with the VL sequence set forth in SEQ ID NO: 22. Includes:

[0150] In another embodiment of the bivalent, bispecific antibody, the VH and VL sequences each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively, and each combined FR1, FR2, FR3, and FR4 framework sequence of the VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequences are not mutated; and each combined FR1, FR2, FR3, and FR4 framework sequence of the VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequences are not mutated.

[0151] In another embodiment of the bivalent, bispecific antibody, the antigen-binding region capable of binding to human PD-L1 is (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5, or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22 Includes:

[0152] In a further aspect, the invention relates to a bivalent, bispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is optionally not human CD3ε, and wherein the antibody: (i) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15.

[0153] In one embodiment of the bivalent, bispecific antibody, the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5.

[0154] In a further aspect, the invention relates to a bivalent, bispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different epitope on human PD-L1, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57.

[0155] In one embodiment herein, the antibody inhibits the binding of human PD-L1 to human PD-1.

[0156] In a further embodiment, the antibody competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0157] In a further embodiment, the binding of the antibody to human PD-L1 is blocked by an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22. As used herein, "blocked" indicates that the antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22 competes with, but does not displace, the bispecific antibody.

[0158] In a further aspect, the present invention relates to a bivalent, bispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is optionally not human CD3ε, and the first antigen-binding region is (i) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) it is capable of binding to the same human PD-L1 epitope as an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22.

[0159] Further aspects of the antibodies of the invention In one embodiment, the antibody according to the invention has a cytotoxicity of about 10 when measured as described in Example 8 herein. -8 M or less, e.g., about 10 -9 M or less, e.g., about 10 -10 Binds to human PD-L1 with a KD of M or less.

[0160] In a further embodiment, the antibodies of the invention mediate concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when purified T cells are used as effector cells, when assayed as described in Example 11 herein.

[0161] In a preferred embodiment, the antibodies of the invention do not bind to human PD-L2.

[0162] antibody format As mentioned above, various antibody formats have been described in the art. The antibodies of the present invention can be of any isotype in principle. The choice of isotype is typically driven by the desired Fc-mediated effector function, e.g., ADCC induction, or the requirements for an antibody lacking Fc-mediated effector function (an "inert" antibody). Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4. Either kappa or lambda human light chain constant regions can be used. The effector functions of the antibodies of the present invention can be altered by isotype switching, e.g., to an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody for various therapeutic uses. In one embodiment, both heavy chains of the antibody of the present invention are of the IgG1 isotype, e.g., IgG1,κ. Optionally, the heavy chains can be modified in the hinge and / or CH3 region as described elsewhere herein.

[0163] Preferably, each antigen-binding region comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of the variable regions comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. Furthermore, preferably, the antibody comprises two heavy chain constant regions (CH) and two light chain constant regions (CL).

[0164] In one embodiment, the antibody is a full-length antibody, for example, a full-length IgG1 antibody. In another embodiment, the antibody is a full-length IgG4 antibody, preferably a full-length IgG4 antibody with a stabilized hinge region. Modifications that stabilize the IgG4 hinge region, for example, the S228P mutation in the core hinge, have been described in the art. See, for example, Labrijn et al., 2009 Nat Biotechnol.27(8):767-71.

[0165] In other embodiments, the antibody of the invention is an antibody fragment, such as an Fab' or Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains, a monovalent antibody described in WO2007059782 (Genmab), an F(ab')2 fragment, an Fd fragment, an Fv fragment, a dAb fragment, a camelid or nanobody, or an isolated complementarity determining region (CDR).

[0166] Antibodies of the invention are preferably human, humanized, or chimeric. In embodiments where the antibody is a bispecific antibody, both half molecules may be human, humanized, or chimeric, or the half molecules may differ in terms of the origin of their sequences.

[0167] For example, in one embodiment, a bispecific antibody comprises two half molecules, each comprising an antigen-binding region: (i) the half-molecule containing an antigen-binding region capable of binding to human PD-L1 is chimeric; and / or (ii) Half-molecules containing antigen-binding regions capable of binding to human CD3ε (epsilon), if present, are chimeric.

[0168] For example, in another embodiment, a bispecific antibody comprises two half molecules, each comprising an antigen-binding region: (i) the half molecule containing the antigen-binding region capable of binding to human PD-L1 is humanized; and / or (ii) The half molecules containing the antigen-binding region capable of binding to human CD3ε (epsilon), if present, are humanized.

[0169] For example, in further embodiments, a bispecific antibody comprises two half molecules, each comprising an antigen-binding region: (i) the half-molecule containing the antigen-binding region capable of binding to human PD-L1 is human; and / or (ii) Half-molecules containing antigen-binding regions capable of binding to human CD3ε (epsilon), if present, are human.

[0170] Thus, for example, in one embodiment, the antigen-binding region capable of binding to human PD-L1 is humanized, and the antigen-binding region capable of binding to human CD3ε (epsilon), if present, is humanized.

[0171] In different embodiments, the antigen-binding region capable of binding to human PD-L1 is human, and the antigen-binding region capable of binding to human CD3ε (epsilon), if present, is human.

[0172] In a further embodiment, the antibody is a bispecific antibody comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD3ε (epsilon), wherein the half-molecule comprising the antigen-binding region capable of binding to human PD-L1 is human, humanized, or chimeric, and the half-molecule comprising the antigen-binding region capable of binding to human CD3ε (epsilon) is humanized.

[0173] Preferably, the half molecule comprising the antigen-binding region capable of binding to human PD-L1 is human, and the half molecule comprising the antigen-binding region capable of binding to human CD3ε (epsilon) is humanized.

[0174] Bispecific antibody format Many different formats and uses of bispecific antibodies are known in the art and have been reviewed by Kontermann; Drug Discovery Today, 2015 Jul;20(7):838-47 and MAbs, 2012 Mar-Apr;4(2):182-97.

[0175] Bispecific antibodies according to the present invention are not limited to any particular bispecific format or method of producing the bispecific format.

[0176] Examples of bispecific antibody molecules that can be used in the present invention include: (i) a single antibody with two arms comprising different antigen-binding regions; (ii) a single-chain antibody with specificity for two different epitopes, e.g., via two scFvs linked in tandem by an extra peptide linker; and (iii) a dual-variable-domain antibody (DVD-Ig), in which each light and heavy chain comprises two variable domains in tandem via a short peptide bond (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig™) Molecule, In: Antibody Engineering, Springer Berlin). Heidelberg (2010); (iv) chemically linked bispecific (Fab') fragments; (v) Tandabs, in which two single-chain diabodies are fused to form tetravalent bispecific antibodies with two binding sites for each target antigen; (vi) Flexibodies, in which scFvs and diabodies are combined to form multivalent molecules; (vii) so-called "dock and lock" molecules, based on the "dimerization and docking domain" of protein kinase A. When applied to Fabs, this can result in trivalent bispecific binding proteins in which two identical Fab fragments are linked to a single different Fab fragment; (viii) so-called scorpion molecules, in which, for example, two scFvs are fused to both ends of a human Fab arm; and (ix) diabodies.

[0177] In one embodiment, the bispecific antibody of the invention is a diabody, a crossbody, or a bispecific antibody obtained via directed Fab arm exchange (e.g., as described in WO2011131746 (Genmab)).

[0178] Examples of different classes of bispecific antibodies include, but are not limited to, (i) IgG-like molecules with complementary CH3 domain molecules that force heterodimerization; (ii) recombinant IgG-like dual targeting molecules, each side of which contains an Fab fragment or portion of an Fab fragment of at least two different antibodies; (iii) IgG fusion molecules, in which a full-length IgG antibody is fused to an extra Fab fragment or portion of an Fab fragment; (iv) Fc fusion molecules, in which a single-chain Fv molecule or stabilized diabody is fused to a heavy-chain constant domain, Fc region, or portion thereof; (v) Fab fusion molecules, in which different Fab fragments are fused together and fused to a heavy-chain constant domain, Fc region, or portion thereof; and (vi) ScFv- and diabody-based antibodies and heavy-chain antibodies (e.g., domain antibodies, nanobodies) in which different single-chain Fv molecules or different diabodies or different heavy-chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule fused to a heavy-chain constant domain, Fc region, or portion thereof.

[0179] Examples of IgG-like molecules with complementary CH3 domain molecules include Triomab / Quadroma molecules (Trion Pharma / Fresenius Biotech; Roche, WO2011069104), so-called Knob-into-Hole molecules (Genentech, WO9850431), CrossMAb (Roche, WO2011117329) and electrostatically matched molecules (Amgen, EP1870459 and WO2009089004; Chugai, US201000155133; Oncomed, WO2010129304), LUZ-Y molecules (Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9, doi: 10.1074 / jbc.M112.397869. Epub 2012 Nov 1), DIG body and PIG body molecules (Pharmabcine, WO2010134666, WO2014081202), Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD Serono, WO2007110205), Biclonics molecules (Merus, WO2013157953), FcΔAdp molecules (Regeneron, WO201015792), bispecific IgG1 and IgG2 molecules (Pfizer / Rinat, WO11143545), Azymetric scaffold molecules (Zymeworks / Merck, WO2012058768), mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific antibodies (WO2009080254), and DuoBody® molecules (Genmab, WO2011131746).

[0180] Examples of recombinant IgG-like dual targeting molecules include Dual Targeting (DT)-Ig molecules (WO2009058383), Two-in-one Antibodies (Genentech; Bostrom, et al 2009. Science 323, 1610-1614.), Cross-linked Mabs (Karmanos Cancer Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia; LaFleur et al. MAbs. 2013 Mar-Apr;5(2):208-18), common light chain approaches (Crucell / Merus, US7,262,028), Kappa Lambda bodies (NovImmune, WO2012023053), and CovX bodies (CovX / Pfizer; Doppalapudi, VR, et al 2007. Bioorg. Med. Chem. Lett. 17,501-506.)

[0181] Examples of IgG fusion molecules include Dual Variable Domain (DVD)-Ig molecules (Abbott, US 7,612,181), Dual domain double head antibodies (Unilever; Sanofi Aventis, WO20100226923), IgG-like bispecific molecules (ImClone / Eli Lilly, Lewis et al. Nat Biotechnol. 2014 Feb;32(2):191-8), Ts2Ab (MedImmune / AZ; Dimasi et al. J Mol Biol. 2009 Oct 30;393(3):672-92), and BsAb molecules (Zymogenetics, WO2010111625), HERCULE molecules (Biogen Idec, US007951918), scFv fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam Biotech Inc, CN 102250246), and TvAb molecules (Roche, WO2012025525, WO2012025530).

[0182] Examples of Fc fusion molecules include, but are not limited to, ScFv / Fc fusions (Pearce et al., Biochem Mol Biol Int. 1997 Sep;42(6):1179-88), Scorpion molecules (Emergent BioSolutions / Trubion, Blankenship JW, et al. AACR 100th Annual meeting 2009 (Abstract # 5465); Zymogenetics / BMS, WO2010111625), Dual Affinity Retargeting Technology (Fc-DART) molecules (MacroGenics, WO2008157379, WO2010080538), and dual (ScFv)2-Fab molecules (National Research Center for Antibody Medicine-China).

[0183] Examples of Fab-fused bispecific antibodies include, but are not limited to, F(ab)2 molecules (Medarex / AMGEN; Deo et al. J. Immunol. 1998 Feb. 15;160(4):1677-86), Dual-Action or Bis-Fab molecules (Genentech, Bostrom, et al. 2009. Science 323, 1610-1614), Dock-and-Lock (DNL) molecules (ImmunoMedics, WO2003074569, WO2005004809), bivalent bispecific molecules (Biotechnol, Schoonjans, J. Immunol. 2000 Dec. 15;165(12):7050-7), and Fab-Fv molecules (UCB-Celltech, WO2009040562A1).

[0184] Examples of ScFv antibodies, diabody-based antibodies, and domain antibodies include Bispecific T Cell Engager (BiTE) molecules (Micromet, WO2005061547), Tandem Diabody molecules (TandAb) (Affimed, Le Gall et al., Protein Eng Des Sel. 2004 Apr;17(4):357-66.), Dual Affinity Retargeting Technology (DART) molecules (MacroGenics, WO2008157379, WO2010080538), Single Chain Diabody molecules (Lawrence, FEBS Lett. 1998 Apr 3;425(3):479-84), TCR-like antibodies (AIT, ReceptorLogics), Human Serum Albumin ScFv Fusion (Merrimack, WO2010059315), and COMBODY molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010 Aug;88(6):667-75.), dual targeting molecules (Ablynx, Hmila et al., FASEB J. 2010), and dual targeting heavy chain only domain antibodies.

[0185] In one aspect, the bispecific antibody of the present invention comprises a first Fc sequence comprising a first CH3 region and a second Fc sequence comprising a second CH3 region, wherein the sequences of the first CH3 region and the second CH3 region are different and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region, respectively. Further details about these interactions and how they can be achieved are provided in WO2011131746 and WO2013060867 (Genmab), which are incorporated herein by reference.

[0186] As further described herein, stable bispecific antibodies, e.g., bispecific CD3xPD-L1 antibodies, contain only a small number of conservative asymmetric mutations in the CH3 regions and can be obtained in high yield using certain methods based on one homodimeric starting PD-L1 antibody and one homodimeric starting antibody (e.g., a homodimeric starting CD3 antibody) that can bind to a different PD-L1 epitope or a different antigen. Asymmetric mutations mean that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at positions that are not identical.

[0187] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, 407, and 409; and the second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, 407, and 409; and the first CH3 region and the second CH3 region are not substituted at the same positions.

[0188] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 366, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 368, 370, 399, 405, 407, and 409. In one embodiment, the amino acid at position 366 is selected from Ala, Asp, Glu, His, Asn, Val, or Gln.

[0189] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 368, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 370, 399, 405, 407, and 409.

[0190] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 370, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 399, 405, 407, and 409.

[0191] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 399, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 405, 407, and 409.

[0192] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 405, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 407, and 409.

[0193] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 407, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, and 409.

[0194] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid substitution at position 409, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, and 407.

[0195] Thus, in one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the sequences of said first and second CH3 regions contain asymmetric mutations, i.e. mutations at different positions in the two CH3 regions, for example a mutation at position 405 in one of the CH3 regions and a mutation at position 409 in the other CH3 region.

[0196] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second CH3 region has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, and 407. In one such embodiment, the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Cys, Lys, or Leu. In a further embodiment thereof, the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has an amino acid other than Phe, Arg, or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys.

[0197]

[0042] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region comprises a Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys; and said second CH3 region comprises an amino acid other than Phe at position 405, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, Leu, Met, or Cys; and a Lys at position 409. In a further embodiment thereof, said first CH3 region comprises a Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second CH3 region comprises an amino acid other than Phe, Arg, or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Met, Lys, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and comprises a Lys at position 409.

[0198] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region comprises a Phe at position 405 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second CH3 region comprises a Leu at position 405 and a Lys at position 409. In a further embodiment thereof, said first CH3 region comprises a Phe at position 405 and an Arg at position 409, and said second CH3 region comprises an amino acid other than Phe, Arg, or Gly at position 405, e.g., Leu, Ala, Val, Ile, Ser, Thr, Lys, Met, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and a Lys at position 409. In another embodiment, said first CH3 region comprises a Phe at position 405 and an Arg at position 409, and said second CH3 region comprises a Leu at position 405 and a Lys at position 409.

[0199] In a further embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region comprises an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region comprises a Lys at position 409, a Thr at position 370, and a Leu at position 405. In a further embodiment, the first CH3 region comprises an Arg at position 409, and the second CH3 region comprises a Lys at position 409, a Thr at position 370, and a Leu at position 405.

[0200] In still further embodiments of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region comprises a Lys at position 370, a Phe at position 405, and an Arg at position 409, and said second CH3 region comprises a Lys at position 409, a Thr at position 370, and a Leu at position 405.

[0201] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region comprises an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region comprises a Lys at position 409, (a) an Ile at position 350 and a Leu at position 405, or (b) a Thr at position 370 and a Leu at position 405.

[0202] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region comprises an Arg at position 409 and said second CH3 region comprises a Lys at position 409, (a) an Ile at position 350 and a Leu at position 405, or (b) a Thr at position 370 and a Leu at position 405.

[0203] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405, and an Arg at position 409, and the second CH3 region comprises a Lys at position 409, and (a) an Ile at position 350 and a Leu at position 405, or (b) a Thr at position 370 and a Leu at position 405.

[0204] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region comprises a Thr at position 350, a Lys at position 370, a Phe at position 405, and an Arg at position 409; and said second CH3 region comprises an Ile at position 350, a Thr at position 370, a Leu at position 405, and a Lys at position 409.

[0205] In one embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has an amino acid other than Lys, Leu, or Met at position 409 and said second CH3 region has an amino acid other than Phe at position 405, such as an amino acid other than Phe, Arg, or Gly at position 405, or said first CH3 region has an amino acid other than Lys, Leu, or Met at position 409 and said second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0206] In one embodiment, a bispecific antibody defined in any embodiment disclosed herein comprises a first CH3 region having an amino acid other than Lys, Leu, or Met at position 409 and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407.

[0207] In one embodiment, a bispecific antibody defined in any embodiment disclosed herein comprises a first CH3 region having a Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and Lys at position 409.

[0208] In one embodiment, a bispecific antibody defined in any embodiment disclosed herein comprises a first CH3 region having a Tyr at position 407 and an Arg at position 409, and a second CH3 region having an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407 and a Lys at position 409.

[0209] In another embodiment, the first CH3 region has an amino acid at position 409 other than Lys, Leu, or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has an amino acid at position 407 other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys. In another embodiment, the first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407.

[0210] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second CH3 region has Gly, Leu, Met, Asn, or Trp at position 407.

[0211]

[0039] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has a Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys; and said second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys; and

[0212] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has a Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and said second CH3 region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and a Lys at position 409.

[0213]

[0039] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has a Tyr at position 407 and an amino acid other than Lys, Leu, or Met at position 409, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys; and said second CH3 region has a Gly, Leu, Met, Asn, or Trp at position 407 and a Lys at 409.

[0214] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has a Tyr at position 407 and an Arg at position 409, and said second CH3 region has an amino acid other than Tyr, Asp, Glu, Phe, Lys, Gln, Arg, Ser, or Thr at position 407, e.g., Leu, Met, Gly, Ala, Val, Ile, His, Asn, Pro, Trp, or Cys, and has a Lys at position 409.

[0215] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has a Tyr at position 407 and an Arg at position 409, and said second CH3 region has an Ala, Gly, His, Ile, Leu, Met, Asn, Val, or Trp at position 407 and a Lys at position 409.

[0216] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, said first CH3 region has a Tyr at position 407 and an Arg at position 409, and said second CH3 region has a Gly, Leu, Met, Asn, or Trp at position 407 and a Lys at position 409.

[0217] In another embodiment of the bispecific antibody defined in any embodiment disclosed herein, the first CH3 region has an amino acid at position 409 other than Lys, Leu, or Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Phe, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys, and the second CH3 region has (i) at position 368, an amino acid other than Phe, Leu, and Met, e.g., Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asp, Asn, Glu, Gln, Pro, Trp, Tyr, or Cys; or (ii) has a Trp at position 370, or (iii) has an amino acid other than Asp, Cys, Pro, Glu, or Gln at position 399, e.g., Phe, Leu, Met, Gly, Ala, Val, Ile, Ser, Thr, Lys, Arg, His, Asn, Trp, Tyr, or Cys; or (iv) At position 366, it has an amino acid other than Lys, Arg, Ser, Thr, or Trp, e.g., Phe, Leu, Met, Ala, Val, Gly, Ile, Asn, His, Asp, Glu, Gln, Pro, Tyr, or Cys.

[0218] In one embodiment, the first CH3 region has an Arg, Ala, His, or Gly at position 409 and the second CH3 region has (i) at position 368, has Lys, Gln, Ala, Asp, Glu, Gly, His, Ile, Asn, Arg, Ser, Thr, Val, or Trp; or (ii) has a Trp at position 370, or (iii) at position 399, Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, Trp, Phe, His, Lys, Arg, or Tyr; or (iv) At position 366, it has Ala, Asp, Glu, His, Asn, Val, Gln, Phe, Gly, Ile, Leu, Met, or Tyr.

[0219] In one embodiment, the first CH3 region has an Arg at position 409 and the second CH3 region has (i) at position 368, has Asp, Glu, Gly, Asn, Arg, Ser, Thr, Val, or Trp; or (ii) has a Trp at position 370, or (iii) at position 399, Phe, His, Lys, Arg, or Tyr; or (iv) At position 366, it has Ala, Asp, Glu, His, Asn, Val, or Gln.

[0220] In a preferred embodiment, the bispecific antibody comprises a first heavy chain and a second heavy chain, each of which comprises at least a hinge region, a CH2, and a CH3 region, and wherein (i) in the first heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L and in the second heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R, or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R and in the second heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L.

[0221] In addition to the amino acid substitutions described above, the first and second heavy chains may contain further amino acid substitutions, deletions, or insertions compared to the wild-type heavy chain sequence.

[0222] In a further embodiment, the first and second Fab arms (or heavy chain constant domains) comprise, but for the specified mutations, a CH3 sequence independently selected from (IgG1m(a)) (SEQ ID NO:96), (IgG1m(f)) (SEQ ID NO:97), and (IgG1m(ax) (SEQ ID NO:98).

[0223] In one embodiment, neither said first nor said second Fc sequence comprises a Cys-Pro-Ser-Cys sequence in the (core) hinge region.

[0224] In a further embodiment, said first Fc sequence and said second Fc sequence both comprise a Cys-Pro-Pro-Cys sequence in the (core) hinge region.

[0225] Methods for preparing bispecific antibodies Conventional methods, such as hybrid hybridoma and chemical conjugation techniques (Marvin and Zhu (2005) Acta Pharmacol Sin 26:649), can be used to prepare bispecific antibodies of the present invention. Co-expression of two antibodies consisting of different heavy and light chains in a host cell results in a mixture of potential antibody products in addition to the desired bispecific antibody. The desired bispecific antibody can then be isolated, for example, by affinity chromatography or similar methods.

[0226] Strategies that favor the formation of functional bispecific products when different antibody constructs are coexpressed, such as those described by Lindhofer et al. (1995 J Immunol 155:219), can also be used. Fusion of rat and mouse hybridomas producing different antibodies results in a limited number of heterodimeric proteins due to species-restricted preferential heavy / light chain pairing. Another strategy to promote heterodimer formation over homodimers is the "knob-into-hole" strategy. In this strategy, a protrusion is introduced into a first heavy chain polypeptide and a corresponding cavity into a second heavy chain polypeptide, allowing the protrusion to be positioned within the cavity at the interface between the two heavy chains, in order to promote heterodimer formation and prevent homodimer formation. The "knob" is constructed by exchanging a small amino acid side chain from the interface of the first polypeptide with a larger side chain. A compensating "cavity" of the same or similar size as the protrusion is created at the interface of the second polypeptide by exchanging large amino acid side chains for small ones (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and WO2009089004 (Amgen) describe other strategies to favor heterodimer formation when different antibody domains are co-expressed in host cells. In these methods, one or more residues constituting the CH3-CH3 interface in both CH3 domains are exchanged with charged amino acids so that homodimer formation is electrostatically unfavorable and heterodimerization is electrostatically favored. WO2007110205 (Merck) describes yet another strategy in which differences between IgA and IgG CH3 domains are exploited to promote heterodimerization.

[0227] Another in vitro method for producing bispecific antibodies is described in WO2008119353 (Genmab), in which bispecific antibodies are formed by "Fab arm" or "half molecule" exchange (swapping of heavy chains and attached light chains) between two monospecific IgG4 antibodies or IgG4-like antibodies when incubated under reducing conditions. The resulting product is a bispecific antibody with two Fab arms that may contain different sequences.

[0228] A preferred method for preparing a bispecific antibody of the invention, such as a bispecific CD3xPD-L1 antibody, comprises: (a) providing a first antibody comprising an Fc region, wherein the Fc region comprises a first CH3 region; (b) providing a second antibody comprising a second Fc region, wherein said Fc region comprises a second CH3 region, and wherein the first antibody is a PD-L1 antibody according to the invention and the second antibody is an antibody capable of binding to a different PD-L1 epitope or a different antigen, such as human CD3, or vice versa, and wherein the sequences of the first CH3 region and the second CH3 region are different and are such that the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region, respectively; (c) incubating the first antibody with the second antibody under reducing conditions; and (d) obtaining the bispecific antibody, e.g., a bispecific PD-L1xCD3 antibody. The methods described in WO2011131746 and WO2013060867 (Genmab) include:

[0229] Similarly, (a) culturing a host cell that produces a first antibody comprising an antigen-binding region capable of binding to human PD-L1 as defined herein, and purifying the first antibody from the culture; (b) culturing a host cell that produces a second antibody that comprises an antigen-binding region capable of binding to a different PD-L1 epitope or to a different antigen, e.g., a human CD3ε-binding region as defined herein, and purifying the second antibody from the culture; (c) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (d) obtaining the bispecific antibody A method for producing an antibody according to the invention is provided, comprising:

[0230] In one embodiment, said first antibody and said second antibody are incubated under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization, and the heterodimeric interaction between said first antibody and second antibody in the resulting heterodimeric antibody is such that no Fab arm exchange occurs after 24 hours at 37°C with 0.5 mM GSH.

[0231] Without being limited by theory, in step (c), heavy chain disulfide bonds in the hinge region of the parent antibody are reduced, and the resulting cysteine ​​can then form an inter-heavy chain disulfide bond with a cysteine ​​residue of another parent antibody molecule (originally with different specificity). In one embodiment of this method, the reducing conditions in step (c) comprise the addition of a reducing agent, e.g., a reducing agent selected from the group consisting of 2-mercaptoethylamine (2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione, tris(2-carboxyethyl)phosphine (TCEP), L-cysteine, and β-mercaptoethanol, preferably a reducing agent selected from the group consisting of 2-mercaptoethylamine, dithiothreitol, and tris(2-carboxyethyl)phosphine. In a further embodiment, step (c) comprises restoring conditions to non-reducing or less reducing conditions, e.g., by removing the reducing agent, e.g., by desalting.

[0232] For this method, any of the aforementioned antibodies can be used, e.g., the CD3 and PD-L1 antibodies described above, including first and second CD3 and PD-L1 antibodies, respectively, comprising a first Fc region and / or a second Fc region. Examples of such first and second Fc regions can include any of those described above, including combinations of such first and second Fc regions. In certain embodiments, the first and second antibodies, e.g., the CD3 and PD-L1 antibodies, respectively, can be selected to obtain a bispecific antibody as described herein.

[0233] In one embodiment of this method, said first antibody and / or second antibody is a full-length antibody.

[0234] The Fc regions of the first and second antibodies may be of any isotype, including, but not limited to, IgG1, IgG2, IgG3, or IgG4. In one embodiment of this method, the Fc regions of both the first and second antibodies are of the IgG1 isotype. In another embodiment, one of the antibody Fc regions is of the IgG1 isotype and the other is of the IgG4 isotype. In the latter embodiment, the resulting bispecific antibody contains IgG1 and IgG4 Fc sequences and may therefore have interesting intermediate properties with respect to activation of effector function.

[0235] In a further embodiment, one of the antibody starting proteins is engineered not to bind to Protein A, thus allowing separation of the heterodimeric protein from the homodimeric starting protein by passing the product over a Protein A column.

[0236] As described above, the sequences of the first CH3 region and the second CH3 region of the homodimeric starting antibody are different, and the heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than the homodimeric interaction between the first CH3 region and the second CH3 region, respectively. Further details about these interactions and how to achieve them are provided in WO2011131746 and WO2013060867 (Genmab). WO2011131746 and WO2013060867 (Genmab) are incorporated herein by reference in their entirety.

[0237] In particular, stable bispecific antibodies, for example, bispecific CD3xPD-L1 antibodies, can be obtained in high yield using the above-described methods of the present invention based on two homodimeric starting antibodies, one that binds to PD-L1 and the other that binds to a different antigen or a different PD-L1 epitope, for example, CD3, and contain only a small number of conservative asymmetric mutations in the CH3 region. Asymmetric mutations mean that the sequences of the first CH3 region and the second CH3 region contain amino acid substitutions at positions that are not identical.

[0238] The bispecific antibody of the present invention may also be obtained by co-expressing constructs encoding the first and second polypeptides in a single cell. (a) providing a first nucleic acid construct encoding a first polypeptide comprising a first Fc sequence and a first antigen-binding region of a first antibody heavy chain, wherein the first Fc sequence comprises a first CH3 region; (b) providing a second nucleic acid construct encoding a second polypeptide comprising a second Fc sequence and a second antigen-binding region of a second antibody heavy chain, wherein the second Fc sequence comprises a second CH3 region; the sequences of the first CH3 region and the second CH3 region are different and are such that a heterodimeric interaction between the first CH3 region and the second CH3 region is stronger than a homodimeric interaction between the first CH3 region and the second CH3 region, respectively; the first homodimeric protein has an amino acid other than Lys, Leu, or Met at position 409; and the second homodimeric protein has an amino acid substitution at a position selected from the group consisting of positions 366, 368, 370, 399, 405, and 407; optionally, said first and second nucleic acid constructs encode light chain sequences of said first and second antibodies; (c) co-expressing the first and second nucleic acid constructs in a host cell; and (d) obtaining the heterodimeric protein from the cell culture. The present invention relates to a method for producing a bispecific antibody, comprising:

[0239] Materials and methods for producing antibodies of the invention In a further aspect, the present invention relates to materials and methods for the recombinant production of antibodies according to the invention. Suitable expression vectors comprising promoters, enhancers etc., and suitable host cells for producing antibodies are well known in the art.

[0240] Thus, in one aspect, (i) a nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen-binding region capable of binding to human PD-L1, as defined herein; and / or (ii) a nucleic acid sequence encoding the light chain sequence of an antibody comprising an antigen-binding region capable of binding to human PD-L1, as defined herein; A nucleic acid construct is provided, comprising:

[0241] In one embodiment, the nucleic acid construct comprises: (i) a nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen-binding region capable of binding to a different PD-L1 epitope or a different antigen, e.g., human CD3ε, as defined herein; and (ii) a nucleic acid sequence encoding an antibody light chain sequence comprising an antigen-binding region capable of binding to a different PD-L1 epitope or a different antigen, e.g., human CD3ε, as defined herein; Further includes:

[0242] In a still further aspect, the present invention relates to an expression vector comprising a nucleic acid construct as defined herein above.

[0243] In the context of the present invention, an expression vector can be any suitable vector, including chromosomal nucleic acid vectors, non-chromosomal nucleic acid vectors, and synthetic nucleic acid vectors (nucleic acid sequences comprising an appropriate set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the antibody-encoding nucleic acid, e.g., a PD-L1-encoding nucleic acid or a CD3 antibody-encoding nucleic acid, is expressed in a naked DNA or RNA vector, e.g., comprising a linear expression element (e.g., as described in Sykes and Johnston, Nat Biotech 17, 355-59 (1997)), a compacted nucleic acid vector (e.g., as described in US 6,077,835 and / or WO 00 / 70087), a plasmid vector, e.g., pBR322, pUC19 / 18, or pUC118 / 119, a "midge" minimal size nucleic acid vector (e.g., as described in Schakowski et al., Mol Ther 3, 793-800 (2001)), or a precipitated nucleic acid vector construct, e.g., a Ca3(P04)2-precipitated construct (e.g., as described in WO 200046147; Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7, 603 (1981). Such nucleic acid vectors and their uses are well known in the art (see, for example, US Pat. No. 5,589,466 and US Pat. No. 5,973,972).

[0244] In one embodiment, the vector is suitable for expressing the antibody, e.g., a PD-L1 antibody and / or a CD3 antibody, in bacterial cells. Examples of such vectors include expression vectors, e.g., BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem264 , 5503-5509 (1989), pET vectors (Novagen, Madison WI), etc.

[0245] The expression vector may also or alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system can be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as α-factor, alcohol oxidase, and PGH (F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987) and Grant et al., Methods in Enzymol. 153 , 516-544 (1987).

[0246] The expression vector may also or alternatively be a vector suitable for expression in mammalian cells, such as a vector containing glutamine synthetase as a selectable marker, such as the vector described in Bebbington (1992) Biotechnology (NY) 10:169-175.

[0247] The nucleic acid and / or vector may also include a nucleic acid sequence encoding a secretion / localization sequence. Secretion / localization sequences can target a polypeptide, e.g., a nascent polypeptide chain, to the periplasmic space or into the cell medium. Such sequences are known in the art and include secretion leaders or signal peptides.

[0248] The expression vector may contain or be linked to any suitable promoter, enhancer, and other expression-promoting elements. Examples of such elements include a strong expression promoter (e.g., the human CMV IE promoter / enhancer and the RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), an effective poly(A) termination sequence, an origin of replication for the plasmid product in E. coli, an antibiotic resistance gene as a selectable marker, and / or a convenient cloning site (e.g., a polylinker). The nucleic acid may also contain an inducible promoter, e.g., CMV IE, as opposed to a constitutive promoter.

[0249] In one embodiment, the antibody-encoding expression vector, such as a PD-L1 antibody-encoding expression vector and / or a CD3 antibody-encoding expression vector, may be placed in and / or delivered to a host cell or animal via a viral vector.

[0250] In a still further aspect, the present invention relates to a host cell comprising one or more of the nucleic acid constructs or expression vectors specified herein above.

[0251] Thus, the present invention also relates to recombinant eukaryotic or prokaryotic host cells, such as transfectomas, which produce the antibodies of the invention.

[0252] Examples of host cells include yeast, bacterial cells, plant cells, and mammalian cells, such as CHO, CHO-S, HEK, HEK293, HEK-293F, Expi293F, PER.C6, or NS0 cells, or lymphocytic cells. A preferred host cell is a CHO-K1 cell.

[0253] For example, in one embodiment, the host cell may comprise a first nucleic acid construct and a second nucleic acid construct stably integrated into the cellular genome. In another embodiment, the invention provides a cell comprising a non-integrated nucleic acid, e.g., a plasmid, cosmid, phagemid, or linear expression element, comprising the above-specified first and second nucleic acid constructs.

[0254] In a further aspect, the invention relates to a hybridoma producing a PD-L1 antibody as defined herein.

[0255] Fc area In some embodiments, an antibody according to the invention comprises, in addition to the antigen-binding region, an Fc region consisting of the Fc sequences of two heavy chains.

[0256] The first Fc sequence and the second Fc sequence may each be of any isotype, including, but not limited to, IgG1, IgG2, IgG3, and IgG4, and may contain one or more mutations or modifications. In one embodiment, the first Fc sequence and the second Fc sequence may each be of or derived from the IgG4 isotype, optionally with one or more mutations or modifications. In another embodiment, the first Fc sequence and the second Fc sequence may each be of or derived from the IgG1 isotype, optionally with one or more mutations or modifications. In another embodiment, one of the Fc sequences is an Fc sequence of the IgG1 isotype and the other is an Fc sequence of the IgG4 isotype, or is derived from each of such isotypes, optionally with one or more mutations or modifications.

[0257] In one embodiment, one or both Fc sequences are effector function-deficient. For example, the Fc sequences may be Fc sequences of an IgG4 isotype or of a non-IgG4 type, such as IgG1, IgG2, or IgG3, that have been mutated to reduce or even eliminate effector function, e.g., the ability to mediate ADCC. Such mutations are described, for example, in Dall'Acqua WF et al., J Immunol. 177 (2):1129-1138 (2006) and Hezareh M, J Virol.; 75 (24):12161-12168 (2001). In another embodiment, one or both Fc sequences comprise an IgG1 wild-type sequence.

[0258] The antibodies of the present invention may contain modifications in the Fc region. When an antibody contains such modifications, it may become an inactive or non-activated antibody. As used herein, the terms "inactive," "inactive," or "non-activating" refer to an Fc region that is at least incapable of binding to any Fcγ receptor, incapable of inducing Fc-mediated FcR cross-linking, incapable of inducing FcR-mediated cross-linking of target antigens via the two Fc regions of an individual antibody, or incapable of binding to C1q. The inactivity of the Fc region of an antibody, for example, a humanized or chimeric CD3 antibody, is advantageously tested using the antibody in a monospecific format.

[0259] For therapeutic antibody development, several variants of the Fc region of an antibody can be constructed to render it inert to interactions with Fcγ (gamma) receptors and C1q. Examples of such variants are described herein.

[0260] Thus, in one embodiment of the antibody of the invention, the antibody comprises a first heavy chain and a second heavy chain, wherein one or both heavy chains are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an otherwise identical antibody comprising the unmodified first and second heavy chains, and the Fc-mediated effector function may be measured by determining Fc-mediated CD69 expression, binding to Fcγ receptors, binding to C1q, or induction of Fc-mediated FcR cross-linking.

[0261] In one such embodiment, the heavy chain constant sequence is modified such that the antibody reduces Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% when compared to a wild-type (unmodified) antibody, as determined, for example, in a PBMC-based functional assay as described in Example 3 of WO2015001085.

[0262] In another such embodiment, the heavy chain constant sequence and the light chain constant sequence are modified such that C1q binding to said antibody is reduced by at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% compared to an unmodified antibody, and C1q binding is measured by ELISA.

[0263] In another embodiment, the antibody comprises an Fc region that has been modified to mediate Fc-mediated T-cell proliferation that is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to an unmodified antibody, wherein said T-cell proliferation is measured in a peripheral blood mononuclear cell (PBMC)-based functional assay.

[0264] Thus, amino acids within the Fc region that play a central role in interactions with C1q and Fcγ receptors can be altered.

[0265] For example, examples of amino acid positions that can be modified in an IgG1 isotype antibody include positions L234, L235, and P331, the combination of which, for example, L234F / L235E / P331S, can significantly reduce binding to human CD64, CD32, CD16, and C1q.

[0266] Thus, in one embodiment, the amino acids at at least one position corresponding to L234, L235, and P331 may be A, A, and S, respectively (Xu et al., 2000, Cell Immunol. 200(1):16-26; Oganesyan et al., 2008, Acta Cryst.(D64):700-4). Furthermore, L234F and L235E amino acid substitutions can result in an Fc region with reduced interaction with Fcγ receptors and C1q (Canfield et al., 1991, J. Exp. Med. (173):1483-91; Duncan et al., 1988, Nature (332):738-40). Thus, in one embodiment, the amino acids at positions corresponding to L234 and L235 may be F and E, respectively. The D265A amino acid substitution can reduce binding to all Fcγ receptors and inhibit ADCC (Shields et al., 2001, J. Biol. Chem. (276):6591-604). Thus, in one embodiment, the amino acid at the position corresponding to D265 can be A. Binding to C1q can be inhibited by mutating positions D270, K322, P329, and P331. Mutation of these positions to D270A, K322A, P329A, or P331A can render the antibody deficient in CDC activity (Idusogie EE, et al., 2000, J. Immunol. 164: 4178-84). Thus, in one embodiment, the amino acids at at least one position corresponding to D270, K322, P329, and P331 can be A, A, A, and A, respectively.

[0267] An alternative approach to minimize the interaction of the Fc region with Fcγ receptors and C1q is to remove the glycosylation site of the antibody. Mutating position N297 to, for example, Q, A, or E removes a glycosylation site critical for IgG-Fcγ receptor interaction. Thus, in one embodiment, the amino acid at the position corresponding to N297 can be G, Q, A, or E. (Leabman et al., 2013, MAbs; 5(6):896-903). Another alternative approach to minimize the interaction of the Fc region with Fcγ receptors can be achieved by the following mutations: P238A, A327Q, P329A, or E233P / L234V / L235A / G236del (Shields et al., 2001, J. Biol. Chem. (276):6591-604).

[0268] Alternatively, human IgG2 and IgG4 subclasses are thought to be naturally impaired in their interactions with C1q and Fcγ receptors, but interactions with Fcγ receptors have been reported (Parren et al., 1992, J. Clin Invest. 90: 1537-1546; Bruhns et al., 2009, Blood 113: 3716-3725). In both isotypes, adding mutations that suppress these remaining interactions can reduce undesirable side effects associated with FcR binding. For IgG2, these include L234A and G237A, and for IgG4, they include L235E. Thus, in one embodiment, the amino acids at positions corresponding to L234 and G237 in a human IgG2 heavy chain may be A and A, respectively. In one embodiment, the amino acid at the position corresponding to L235 in a human IgG4 heavy chain may be E.

[0269] Other approaches to further minimize interactions with Fcγ receptors and C1q in IgG2 antibodies include those described in WO2011066501 and Lightle, S., et al., 2010, Protein Science (19):753-62.

[0270] The hinge region of the antibody may also be important for interactions with Fcγ receptors and complement (Brekke et al., 2006, J Immunol 177:1129-1138; Dall'Acqua WF, et al., 2006, J Immunol 177:1129-1138). Therefore, mutations in or deletions of the hinge region may affect the effector functions of the antibody.

[0271] Thus, in one embodiment, the antibody comprises a first immunoglobulin heavy chain and a second immunoglobulin heavy chain, wherein in at least one of the first immunoglobulin heavy chain and the second immunoglobulin heavy chain, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0272] In one embodiment, in both the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain are not L, L, D, N, and P, respectively.

[0273] In one embodiment, in both said first and second heavy chains the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is not D.

[0274] Thus, in one embodiment, the amino acid at the position corresponding to position D265 in a human IgG1 heavy chain is selected from the group consisting of A and E in both the first and second heavy chains.

[0275] In a further embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are not L and L, respectively.

[0276] In a particular embodiment, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0277] In one embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234 and L235 in a human IgG1 heavy chain are F and E, respectively.

[0278] In certain embodiments, in at least one of said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0279] In a particularly preferred embodiment, in both said first and second heavy chains, the amino acids at positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain are F, E, and A, respectively.

[0280] In a further particularly preferred embodiment, the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, in which the positions corresponding to positions L234 and L235 in the human IgG1 heavy chain according to EU numbering in both the first and second heavy chains are F and E, respectively, and (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering in the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering in the second heavy chain is L.

[0281] In a further particularly preferred embodiment, the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, wherein the positions in both the first and second heavy chains corresponding to positions L234, L235, and D265 in the human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L.

[0282] Antibody variants having a combination of the three amino acid substitutions L234F, L235E, and D265A, and further having a K409R or F405L mutation, are designated herein with the suffix "FEAR" or "FEAL," respectively.

[0283] As used herein, huCD3-H1L1 refers to the humanized SP34 anti-CD3 antibody having the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29.

[0284] In a preferred embodiment, the bispecific antibody of the present invention comprises: (i) half-molecular antibodies derived from IgG1-huCD3-H1L1-FEAL, and half-molecular antibodies derived from IgG1-PDL1-338-FEAR, IgG1-PDL1-511-FEAR, or IgG1-PDL1-547-FEAR; or (ii) Half-molecular antibodies derived from IgG1-huCD3-H1L1-FEAR, and half-molecular antibodies and half-molecular antibodies derived from IgG1-PDL1-338-FEAL, IgG1-PDL1-511-FEAL, or IgG1-PDL1-547-FEAL. Includes:

[0285] In a further embodiment, the first heavy chain or half molecule comprises the sequence set forth in SEQ ID NO:90 and the second heavy chain comprises the sequence set forth in SEQ ID NO:89.

[0286] In a further embodiment of the present invention, to manipulate the serum half-life of a bispecific antibody, one or both antibody-forming portions of the bispecific antibody are engineered to reduce or increase binding to fetal Fc receptors (FcRn). Techniques for increasing or decreasing serum half-life are well known in the art. See, for example, Dall'Acqua et al. 2006, J. Biol. Chem., 281:23514-24; Hinton et al. 2006, J. Immunol., 176:346-56; and Zalevsky et al. 2010 Nat. Biotechnol., 28:157-9.

[0287] combination In a further aspect, the present invention provides antibodies linked or conjugated to one or more therapeutic moieties, such as cytokines, immunosuppressants, immunostimulatory molecules, and / or radioisotopes. Such conjugates are referred to herein as "immunoconjugates" or "drug conjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins."

[0288] In one embodiment, the first Fc sequence and / or the second Fc sequence is conjugated to a drug or prodrug or contains a drug or prodrug acceptor group, which may be, for example, an unnatural amino acid.

[0289] composition In a further aspect, the invention relates to a pharmaceutical composition comprising an antibody according to any one of the embodiments disclosed herein and a pharmaceutically acceptable carrier.

[0290] The pharmaceutical composition of the present invention may contain one antibody of the present invention or a combination of different antibodies of the present invention.

[0291] The pharmaceutical compositions can be formulated according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. Pharmaceutical compositions of the present invention may include, for example, diluents, fillers, salts, buffers, surfactants (e.g., non-ionic surfactants such as Tween-20 or Tween-80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions.

[0292] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, antioxidants, and absorption delaying agents that are physiologically compatible with the antibodies of the present invention. Examples of suitable aqueous and nonaqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, saline, phosphate-buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils, carboxymethylcellulose colloidal solutions, tragacanth gum, and injectable organic esters such as ethyl oleate, and / or various buffers. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0293] The pharmaceutical compositions of the present invention may also contain pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, e.g., ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, e.g., ascorbic acid palmitate, butylhydroxyanisole, butylhydroxytoluene, lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, e.g., citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0294] The pharmaceutical compositions of the present invention may also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol, or sodium chloride in the composition.

[0295] The pharmaceutical compositions of the present invention may also contain one or more auxiliary agents suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersing agents, antiseptics, or buffers, which can enhance the shelf life or effectiveness of the pharmaceutical composition. The antibodies of the present invention may also be prepared using carriers that protect the antibodies from rapid release, such as sustained-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid alone or with wax, or other materials well known in the art. Methods for preparing such formulations are generally known to those skilled in the art.

[0296] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent, for example, with one or combination of the components listed above as needed, and then carry out sterilization microfiltration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components that are required, for example, those listed above.For the sterile powder that is used to prepare sterile injectable solution, the example of preparation method is vacuum drying and freeze-drying (lyophilization), which produces the powder of active ingredient and any other desired components from the solution of active ingredient and any other desired components that has been previously sterilized by filtration.

[0297] The actual dosage level of the active ingredient in the pharmaceutical composition can be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and method of administration without toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the invention or its amide employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0298] The pharmaceutical composition can be administered by any suitable route and method.In one embodiment, the pharmaceutical composition of the present invention is administered parenterally.As used herein, "administered parenterally" refers to an administration method other than enteral administration and topical administration, usually by injection, including epidermal, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0299] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0300] use In one aspect, the present invention relates to an antibody according to any one of the embodiments disclosed herein or a pharmaceutical composition disclosed herein, for use as a medicament.

[0301] In a further aspect, the present invention relates to an antibody according to any one of the embodiments disclosed herein or a pharmaceutical composition disclosed herein for use in the treatment of a disease such as cancer.

[0302] In a further aspect, the present invention relates to a method for treating a disease, comprising administering to a subject in need thereof an effective amount of an antibody according to any one of the embodiments disclosed herein or a pharmaceutical composition disclosed herein.

[0303] In particular, bispecific antibodies according to the invention may be useful in therapeutic settings where specific targeting and killing of PD-L1 expressing cells by T cells is desirable, and may be more efficient than conventional anti-PD-L1 antibodies in certain such indications and situations.

[0304] The antibodies of the invention also have additional utility in the therapy and diagnosis of various PD-L1-associated diseases. For example, the antibodies can be used to induce one or more of the following biological activities in vivo or in vitro: inhibiting the proliferation and / or differentiation of PD-L1-expressing cells; killing PD-L1-expressing cells; mediating phagocytosis or ADCC of PD-L1-expressing cells in the presence of human effector cells; mediating CDC of PD-L1-expressing cells in the presence of complement; mediating apoptosis of PD-L1-expressing cells; and / or inducing translocation to lipid rafts upon PD-L1 binding.

[0305] In one aspect, the invention relates to an antibody according to any one of the embodiments disclosed herein or a pharmaceutical composition disclosed herein for use in the treatment of cancer.

[0306] In a further aspect, the present invention relates to an antibody according to any one of the embodiments disclosed herein or a pharmaceutical composition disclosed herein for use in the treatment of a cancer disease characterized by the presence of a solid tumor.

[0307] In a further aspect, the present invention relates to the antibody according to any one of the embodiments disclosed herein or the pharmaceutical composition disclosed herein for use in treating a cancer disease selected from the group consisting of melanoma, ovarian cancer, lung cancer, colorectal cancer, head and neck cancer, gastric cancer, breast cancer, kidney cancer, bladder cancer, esophageal cancer, pancreatic cancer, liver cancer, thymoma and thymic cancer, brain cancer, glioma, adrenocortical carcinoma, thyroid cancer, other skin cancers, sarcoma, multiple myeloma, leukemia, lymphoma, myelodysplastic syndrome, ovarian cancer, endometriosis cancer, prostate cancer, penile cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, Merkel cell carcinoma, and mesothelioma.

[0308] In a further aspect, the invention relates to the use of an antibody according to any one of the embodiments disclosed herein for the manufacture of a medicament, such as a medicament for treating cancer, e.g., a cancer disease characterized by the presence of a solid tumor, or a cancer disease selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer.

[0309] The present invention also relates to methods for inhibiting the growth and / or proliferation of one or more PD-L1-expressing tumor cells comprising the administration of an antibody of the invention to an individual in need thereof.

[0310] The present invention also provides (a) selecting a subject suffering from a cancer comprising PD-L1-expressing tumor cells; and (b) administering the antibody of the present invention or the pharmaceutical composition of the present invention to a subject. The present invention relates to a method for treating cancer, comprising:

[0311] The dosage regimen in the above-mentioned treatment methods and uses is adjusted to achieve the optimum desired response (e.g., therapeutic response).For example, a single bolus may be administered, or several divided doses may be administered over a period of time, and the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.Parenteral compositions may be formulated in unit dosage form for ease of administration and uniformity of dosage.

[0312] The effective dosage and administration regimen of the antibody depend on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range of a therapeutically effective amount of the compound of the present invention is about 0.001 to 10 mg / kg, for example, about 0.001 to 5 mg / kg, for example, about 0.001 to 2 mg / kg, for example, about 0.001 to 1 mg / kg, for example, about 0.001 mg / kg, about 0.01 mg / kg, about 0.1 mg / kg, about 1 mg / kg, or about 10 mg / kg. Another exemplary, non-limiting range of a therapeutically effective amount of an antibody of the present invention is about 0.1 to 100 mg / kg, for example, about 0.1 to 50 mg / kg, for example, about 0.1 to 20 mg / kg, for example, about 0.1 to 10 mg / kg, for example, about 0.5 mg / kg, for example, about 0.3 mg / kg, about 1 mg / kg, about 3 mg / kg, about 5 mg / kg, or about 8 mg / kg.

[0313] A physician or veterinarian skilled in the art can easily determine and prescribe the effective amount of pharmaceutical composition required. For example, a physician or veterinarian can start the dosage of the antibody used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Generally, a suitable daily dose of the antibody of the present invention is the amount of the compound that is the minimum dose effective to produce a therapeutic effect. Administration can be, for example, parenteral, e.g., intravenous, intramuscular, or subcutaneous. In one embodiment, the antibody is administered in an amount of 1 mg / m 2The antibody may be administered by infusion in a weekly dose calculated in units. Such doses may be based on the mg / kg doses set forth above, for example, according to dose (mg / kg) x 70:1.8. Such administration may be repeated, for example, 1 to 8 times, for example, 3 to 5 times. Administration may be by continuous infusion over a period of 2 to 24 hours, for example, 2 to 12 hours. In one embodiment, to reduce toxic side effects, the antibody may be administered by slow continuous infusion over an extended period, for example, more than 24 hours.

[0314] In one embodiment, the antibody may be administered in a weekly dose calculated as a fixed dose of up to eight doses, e.g., four to six doses, when given once a week. Such a regimen may be repeated one or more times as needed, e.g., after six or twelve months. Such fixed doses may be based on the mg / kg doses indicated above, for an estimated body weight of 70 kg. Dosage may be determined or adjusted by measuring the blood level of the antibody of the invention at the time of administration, e.g., by collecting a biological sample and using an anti-idiotypic antibody targeting the PD-L1 antigen-binding region of the antibody of the invention.

[0315] In one embodiment, the antibody may be administered as a maintenance therapy, for example, once a week for six months or more.

[0316] The antibody may also be administered prophylactically to reduce the risk of developing cancer, to delay the onset of events in cancer progression, and / or to reduce the risk of recurrence if the cancer is in remission.

[0317] The antibodies of the invention may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition to be treated. Thus, in one embodiment, the medicament containing the antibody is for use in combination with one or more additional therapeutic agents, for example, a cytotoxic agent, a chemotherapeutic agent, or an anti-angiogenic agent.

[0318] In a further aspect, the invention relates to an anti-idiotypic antibody that binds to the PD-L1 binding region defined in any one of the embodiments disclosed herein.

[0319] Further items of this disclosure: 1. An antibody comprising an antigen-binding region capable of binding to human PD-L1, Inhibits the binding of human PD-L1 to human PD-1, and (i) competes for binding to human PD-L1 with antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with antibody

[0547] comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with antibody

[0547] comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; The antibody. 2. The antibody described in item 1, which competes with antibody

[0338] comprising the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:5 for binding to human PD-L1. 3. The antibody of any preceding item, wherein the binding of the antibody to human PD-L1 is not displaced by antibody

[0476] comprising the VH sequence set forth in SEQ ID NO:53 and the VL sequence set forth in SEQ ID NO:57. 4. The antibody of any preceding item, wherein the binding of the antibody to human PD-L1 is not blocked by the binding of antibody

[0625] comprising the VH sequence set forth in SEQ ID NO:106 and the VL sequence set forth in SEQ ID NO:110. 5. The antibody of any preceding item, wherein the binding of the antibody of any preceding item to human PD-L1 is blocked by antibody

[0547] comprising the VH sequence of SEQ ID NO:18 and the VL sequence of SEQ ID NO:22. 6. (i) It is capable of binding to the same human PD-L1 epitope as antibody

[0338] comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) capable of binding to the same human PD-L1 epitope as antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) It can bind to the same human PD-L1 epitope as antibody

[0547] , which comprises the VH sequence set forth in SEQ ID NO: 18 and the VL sequence set forth in SEQ ID NO: 22. The antibody according to item 1. 7. The antibody of any of Items 1 to 4 and 6, wherein the binding of the antibody to a mutant PD-L1 having one or more of the amino acid residues corresponding to position 113 (R113), 123 (Y123), and 125 (R125) in SEQ ID NO:94 substituted with alanine compared to binding to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, wherein reduced binding is determined when the fold change in binding of the antibody is less than the mean fold change in binding across all alanine mutants - 1.5 × SD, where SD is the standard deviation of all calculated fold changes of the antibody to mutant PD-L1, and the fold change in binding is calculated as set forth in Example 13

[0338] . 8. The antibody of any of items 1 to 2 and 6, wherein the antibody binds to an epitope on PD-L1 (SEQ ID NO:94), the epitope comprising the amino acid residue at position 113 (R113), the amino acid residue at position 123 (Y123), and / or the amino acid residue at position 125 (R125) of SEQ ID NO:94. 9. The antibody of any of paragraphs 1, 3, 4, and 6, wherein the antibody exhibits reduced binding to a mutant PD-L1 having an alanine substitution at one or more of the following amino acid residues in SEQ ID NO:94: amino acid residue at a position corresponding to position 19 (F19), amino acid residue at a position corresponding to position 42 (F42), amino acid residue at a position corresponding to position 45 (E45), amino acid residue at a position corresponding to position 46 (K46), amino acid residue at a position corresponding to position 94 (L94), and amino acid residue at a position corresponding to position 116 (I116) in SEQ ID NO:94, relative to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, wherein reduced binding is determined when the fold change in binding of the antibody is less than the mean fold change in binding across all alanine mutants - 1.5 × SD, where SD is the standard deviation of all calculated fold change values ​​of the antibody to mutant PD-L1, and the fold change in binding is calculated as set forth in Example 13. 10. The antibody of any of paragraphs 1, 3, 4, and 6, wherein the antibody binds to an epitope on PD-L1 (SEQ ID NO:94), and the epitope comprises one or more amino acid residues selected from the group consisting of the amino acid residue at position 45 (E45), the amino acid residue at position 46 (K46), and / or the amino acid residue at position 94 (L94) of SEQ ID NO:94. 11. The antibody of any of paragraphs 1, 5, and 6, wherein the antibody exhibits reduced binding to a mutant PD-L1 comprising an alanine substitution at one or more of the amino acid residues corresponding to position 58 (E58) and 113 (R113) in SEQ ID NO:94, relative to wild-type PD-L1 having the amino acid sequence set forth in SEQ ID NO:94, wherein reduced binding is determined when the fold change in binding of the antibody is less than the mean fold change in binding across all alanine mutants - 1.5 × SD, where SD is the standard deviation of all calculated fold changes of the antibody to mutant PD-L1, and the fold change in binding is calculated as set forth in Example 13. 12. The antibody of any of items 1, 5, and 6, wherein the antibody binds to an epitope on PD-L1 (SEQ ID NO:94), the epitope comprising the amino acid residue at position 58 (E58) and / or the amino acid residue at position 113 (R113) of SEQ ID NO:94. 13. The antibody of any preceding item, wherein the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, and the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4, SEQ ID NO:11, and SEQ ID NO:21. 14. The antigen-binding region capable of binding to human PD-L1 is (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7

[0338] ; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17

[0511] ; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21; and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:23, a CDR2 having the sequence DDN, and a CDR3 having the sequence set forth in SEQ ID NO:24. The antibody of any preceding item, comprising: 15. The antibody of any preceding item, wherein the antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1, SEQ ID NO:8, and SEQ ID NO:18. 16. The antibody of any preceding item, wherein the antigen-binding region capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO:5, SEQ ID NO:15, and SEQ ID NO:22. 17. The antigen-binding region capable of binding to human PD-L1 is (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO: 22. The antibody of any preceding item, comprising: 18. The antibody of item 17, wherein the VH sequence and the VL sequence each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively; wherein each combined FR1, FR2, FR3, and FR4 framework sequence of the VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequences are not mutated; and wherein each combined FR1, FR2, FR3, and FR4 framework sequence of the VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequences are not mutated. 19. The antibody of any preceding item, which is capable of inducing dose-dependent lysis of adenocarcinoma epithelial cells, e.g., dose-dependent lysis of MDA-MB-231, via antibody-dependent cellular cytotoxicity (ADCC). 20. The antibody described in item 19, which is capable of reducing the number of cells in a culture of said epithelial cells by at least 5%, for example, at least 6%, 7%, 8%, 9%, or at least 10% as a result of cell lysis. 21. ADCC 51 21. The antibody of item 19 or 20, as measured in vitro in a Cr release assay, e.g., the assay disclosed in Example 14. 22. The antibody of item 19 or 20, wherein ADCC is measured in vitro by incubating the epithelial cells with a composition comprising the antibody of item 19 or 20 and effector cells, e.g., peripheral blood mononuclear cells (PBMCs), for 4 hours at 37°C and 5% CO2, wherein the amount of antibody in the composition is in the range of 0.1 to 1 μg / mL and the ratio of effector cells to epithelial cells is 100:1. 23. The antibody of item 19 or 20, wherein the lysis of epithelial cells is measured in vitro in a luciferase reporter assay as a surrogate for ADCC, e.g., a luminescence ADCC reporter bioassay disclosed in Example 14. 24. ADCC (i) contacting the culture of epithelial cells with a composition comprising the antibody of paragraph 23 and Jurkat human T cells stably expressing FcγRIIIa (CD16) and firefly luciferase (effector cells) at an effector cell:epithelial cell ratio of 1:1; (ii) allowing the epithelial cell culture and the effector cells to equilibrate to room temperature for 15 minutes; (iii) incubating the culture of epithelial cells and the effector cells with a luciferase substrate; and (iv) measuring luciferase production in the cell culture It is measured in vitro by 24. The antibody according to item 23, wherein the amount of the antibody in the composition is within the range of 0.5 to 250 ng / mL and the ratio of effector cells to epithelial cells is 1:1. 25. The ADCC of the epithelial cells is measured in a luciferase reporter assay, for example, the reporter assay defined in item 23 or 24, and then the ADCC observed after incubating a culture of the epithelial cells with a test composition comprising an antibody according to any one of items 19, 20, 23, and 24 is at least 1.5 times the ADCC observed after incubating a culture of the epithelial cells with a composition comprising a reference antibody, the ADCC being measured as relative light units (RLU), the antibody concentrations in the test composition and the composition comprising the reference antibody are the same and are in the range of 20 to 250 ng / ml, and the reference antibody is (a) an antibody comprising the VH sequence set forth in SEQ ID NO:74 and the VL sequence set forth in SEQ ID NO:78; and (b) an antibody comprising the VH sequence shown in SEQ ID NO:81 and the VL sequence shown in SEQ ID NO:85 25. The antibody of any of items 19, 20, 23, and 24, selected from: 26. The antigen-binding region capable of binding to human PD-L1 is (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22

[0547] The antibody of any preceding item, comprising: 27. An antibody comprising an antigen-binding region capable of binding to human PD-L1, (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 33, the CDR2 sequence set forth in SEQ ID NO: 34, and the CDR3 sequence set forth in SEQ ID NO: 35, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 37, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO: 38

[0321] ; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 47, the CDR2 sequence set forth in SEQ ID NO: 48, and the CDR3 sequence set forth in SEQ ID NO: 49, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 51, a CDR2 having the sequence DVI, and a CDR3 having the sequence set forth in SEQ ID NO: 52

[0421] ; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 54, the CDR2 sequence set forth in SEQ ID NO: 55, and the CDR3 sequence set forth in SEQ ID NO: 56, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 58, a CDR2 having the sequence RDS, and a CDR3 having the sequence set forth in SEQ ID NO: 59

[0476] ; or (iv) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 61, the CDR2 sequence set forth in SEQ ID NO: 62, and the CDR3 sequence set forth in SEQ ID NO: 63, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 65, a CDR2 having the sequence DDS, and a CDR3 having the sequence set forth in SEQ ID NO: 66

[0516] ; or (v) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO: 107, the CDR2 sequence set forth in SEQ ID NO: 108, and the CDR3 sequence set forth in SEQ ID NO: 109, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 111, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO: 113

[0625] ; or (vi) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:68, the CDR2 sequence set forth in SEQ ID NO:69, and the CDR3 sequence set forth in SEQ ID NO:70; and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:72, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:73. The antibody comprising: 28. (i) the VH sequence set forth in SEQ ID NO: 32 and the VL sequence set forth in SEQ ID NO: 36

[0321] ; or (ii) the VH sequence set forth in SEQ ID NO: 46 and the VL sequence set forth in SEQ ID NO: 50

[0421] ; or (iii) the VH sequence set forth in SEQ ID NO: 53 and the VL sequence set forth in SEQ ID NO: 57

[0476] ; or (iv) the VH sequence set forth in SEQ ID NO: 60 and the VL sequence set forth in SEQ ID NO: 64

[0516] ; or (v) the VH sequence set forth in SEQ ID NO: 106 and the VL sequence set forth in SEQ ID NO: 110

[0625] ; or (vi) the VH sequence shown in SEQ ID NO: 67 and the VL sequence shown in SEQ ID NO: 71

[0632] 28. The antibody according to Item 27, comprising: 29. The antibody of any preceding item, which is monovalent. 30. The antibody according to any of the preceding items, wherein the antibody is a bivalent antibody having two antigen-binding regions capable of binding to human PD-L1, and the two antigen-binding regions have the same variable region sequence. 31. The antibody of any preceding item, wherein the antibody is a bivalent, bispecific antibody and comprises, in addition to the (first) antigen-binding region capable of binding to human PD-L1, a (second) antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope, wherein the second antigen is not human CD3ε. 32. A bispecific antibody comprising an antigen-binding region capable of binding to human PD-L1 and an antigen-binding region capable of binding to human CD3ε (epsilon), The bispecific antibody, wherein the antigen-binding region capable of binding to human PD-L1 has the characteristic described in any of the preceding items. 33. The bispecific antibody of item 32, wherein the antigen-binding region capable of binding to human CD3ε comprises: (a) a heavy chain variable region (VH) comprising CDR1 having the sequence set forth in SEQ ID NO: 26, CDR2 having the sequence set forth in SEQ ID NO: 27, and CDR3 having the sequence set forth in SEQ ID NO: 28, and a light chain variable region (VL) comprising CDR1 having the sequence set forth in SEQ ID NO: 30, CDR2 having the sequence GTN, and CDR3 having the sequence set forth in SEQ ID NO: 31. 34. (i) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (ii) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17

[0338] ; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (iii) an antigen-binding region capable of binding to human PD-L1, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21, and a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO:23, the CDR2 sequence set forth in the sequence DDN, and the CDR3 sequence set forth in SEQ ID NO:24

[0547] ; and (a) an antigen-binding region capable of binding to human CD3ε, comprising a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:26, the CDR2 sequence set forth in SEQ ID NO:27, and the CDR3 sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising the CDR1 sequence set forth in SEQ ID NO:30, the CDR2 having the sequence GTN, and the CDR3 sequence set forth in SEQ ID NO:31. 34. The bispecific antibody of item 32 or 33, comprising: 35. The bispecific antibody according to any of items 32 to 34, wherein the antigen-binding region capable of binding to human CD3ε comprises the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29. 36. (i) the bispecific antibody has a lower affinity for human CD3ε binding compared to an antibody having an antigen-binding region that may comprise the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29, preferably the affinity is at least 2-fold lower, such as at least 5-fold lower, such as at least 10-fold lower, such as at least 25-fold lower, such as at least 50-fold lower; and (ii) the bispecific antibody is capable of mediating concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when PBMCs or purified T cells are used as effector cells, e.g., when assayed as described in Example 11 herein; 33. The bispecific antibody of item 32. 37. The antigen-binding region capable of binding to human CD3ε is (i) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:99, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (ii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO: 100, a CDR2 having the sequence set forth in SEQ ID NO: 27, and a CDR3 having the sequence set forth in SEQ ID NO: 28, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO: 31; or (iii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:101, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (iv) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:102, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (v) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:103, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (vi) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO:26, a CDR2 having the sequence set forth in SEQ ID NO:27, and a CDR3 having the sequence set forth in SEQ ID NO:104, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO:31; or (vii) a heavy chain variable region (VH) comprising a CDR1 having the sequence set forth in SEQ ID NO: 26, a CDR2 having the sequence set forth in SEQ ID NO: 27, and a CDR3 having the sequence set forth in SEQ ID NO: 105, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO: 30, a CDR2 having the sequence GTN, and a CDR3 having the sequence set forth in SEQ ID NO: 31. 37. The bispecific antibody of item 36, comprising: 38. The antigen-binding region capable of binding to human CD3ε is (i) the VH sequence set forth in SEQ ID NO:39 and the VL sequence set forth in SEQ ID NO:29; or (ii) the VH sequence set forth in SEQ ID NO:40 and the VL sequence set forth in SEQ ID NO:29; or (iii) the VH sequence set forth in SEQ ID NO: 41 and the VL sequence set forth in SEQ ID NO: 29; or (iv) the VH sequence set forth in SEQ ID NO: 42 and the VL sequence set forth in SEQ ID NO: 29; or (v) the VH sequence set forth in SEQ ID NO: 43 and the VL sequence set forth in SEQ ID NO: 29; or (vi) the VH sequence set forth in SEQ ID NO: 44 and the VL sequence set forth in SEQ ID NO: 29; or (vii) the VH sequence shown in SEQ ID NO:45 and the VL sequence shown in SEQ ID NO:29 38. The bispecific antibody of item 36 or 37, comprising: 39. A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; 32. The multispecific antibody, wherein the antigen-binding region capable of binding to human PD-L1 has the characteristics as set forth in any of items 1 to 31. 40. The multispecific antibody of Item 39, wherein the antigen-binding region capable of binding to human PD-L1 comprises a heavy chain variable region (VH) comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region (VL) comprising CDR1, CDR2, and CDR3 sequences, and the VH CDR3 sequence is selected from the group consisting of the sequences set forth in SEQ ID NO:4

[0338] , SEQ ID NO:11

[0511] , and SEQ ID NO:21

[0547] . 41. The first antigen-binding region capable of binding to human PD-L1 is (i) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:2, the CDR2 sequence set forth in SEQ ID NO:3, and the CDR3 sequence set forth in SEQ ID NO:4, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:6, a CDR2 having the sequence KAS, and a CDR3 having the sequence set forth in SEQ ID NO:7

[0338] ; or (ii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:9, the CDR2 sequence set forth in SEQ ID NO:10, and the CDR3 sequence set forth in SEQ ID NO:11, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:16, a CDR2 having the sequence EDS, and a CDR3 having the sequence set forth in SEQ ID NO:17

[0511] ; or (iii) a heavy chain variable region (VH) comprising the CDR1 sequence set forth in SEQ ID NO:19, the CDR2 sequence set forth in SEQ ID NO:20, and the CDR3 sequence set forth in SEQ ID NO:21, and a light chain variable region (VL) comprising a CDR1 having the sequence set forth in SEQ ID NO:23, a CDR2 having the sequence DDN, and a CDR3 having the sequence set forth in SEQ ID NO:24. 41. The multispecific antibody of item 40, comprising: 42. The multispecific antibody of Aspect 40 or 41, wherein the first antigen-binding region capable of binding to human PD-L1 comprises a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VH sequence selected from the group consisting of the sequences set forth in SEQ ID NO:1

[0338] , SEQ ID NO:8

[0511] , and SEQ ID NO:18

[0547] . 43. The multispecific antibody of any of Items 40 to 42, wherein the first antigen-binding domain capable of binding to human PD-L1 comprises a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to a VL sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 5

[0338] , SEQ ID NO: 15

[0511] , and SEQ ID NO: 22

[0547] . 44. The first antigen-binding region capable of binding to human PD-L1 is (i) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:1, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO:8, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) a VH sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VH sequence set forth in SEQ ID NO: 18, and a VL sequence having at least 90%, at least 95%, at least 97%, at least 99%, or 100% amino acid sequence identity to the VL sequence set forth in SEQ ID NO: 22. 44. The multispecific antibody according to any of Items 40 to 43, comprising: 45. The multispecific antibody according to any of items 40 to 44, wherein the VH sequence and the VL sequence each comprise three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively; wherein each combined FR1, FR2, FR3, and FR4 framework sequence of VH has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VH sequence, and the VH CDR sequences are not mutated; and wherein each combined FR1, FR2, FR3, and FR4 framework sequence of VL has at least 90%, at least 95%, at least 97%, or at least 99% amino acid sequence identity with each combined FR1, FR2, FR3, and FR4 framework sequence of the VL sequence, and the VL CDR sequences are not mutated. 46. ​​The first antigen-binding region capable of binding to human PD-L1 is (i) the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5

[0338] ; or (ii) the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15

[0511] ; or (iii) the VH sequence shown in SEQ ID NO: 18 and the VL sequence shown in SEQ ID NO: 22

[0547] 46. ​​The multispecific antibody according to any of Items 40 to 45, comprising: 47. A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; (i) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; The multispecific antibody. 48. The multispecific antibody of item 47, which competes for binding to human PD-L1 with an antibody comprising the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:5. 49. A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; The multispecific antibody, wherein the binding of the antibody to human PD-L1 is not displaced by an antibody comprising the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57. 50. The multispecific antibody described in item 49, which inhibits the binding of human PD-L1 to human PD-1. 51. The multispecific antibody of item 49 or 50, which competes for binding to human PD-L1 with an antibody comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22. 52. The multispecific antibody of any of items 49 to 51, wherein the binding of the multispecific antibody of any of items 49 to 51 to human PD-L1 is blocked by an antibody comprising the VH sequence shown in SEQ ID NO:18 and the VL sequence shown in SEQ ID NO:22. 53. A multispecific antibody comprising a first antigen-binding region capable of binding to human PD-L1 and a second antigen-binding region capable of binding to a second antigen or to a different human PD-L1 epitope; the first antigen-binding region comprises: (i) capable of binding to the same human PD-L1 epitope as antibody

[0338] comprising the VH sequence set forth in SEQ ID NO:1 and the VL sequence set forth in SEQ ID NO:5; or (ii) capable of binding to the same human PD-L1 epitope as antibody

[0511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; or (iii) It can bind to the same human PD-L1 epitope as antibody

[0547] , which comprises the VH sequence set forth in SEQ ID NO: 18 and the VL sequence set forth in SEQ ID NO: 22. The multispecific antibody. 54. The multispecific antibody according to any one of items 40 to 53, which is bispecific. 55. The multispecific antibody described in item 54, which is bivalent. 56. The multispecific antibody according to any of items 40 to 55, which can bind to a second antigen, and the second antigen is not human CD3ε. 57. The antibody of any preceding item, which is a full-length antibody. 58. The antibody described in item 57, which is a full-length IgG1 antibody. 59. The antibody of any preceding item, which is an antibody fragment. 60. The antibody according to any one of items 32 to 59, comprising two half molecules each comprising an antigen-binding region; (i) the half-molecule containing an antigen-binding region capable of binding to human PD-L1 is chimeric; and / or (ii) is chimeric, if present, with half molecules containing an antigen-binding region capable of binding to human CD3ε (epsilon); 59. The antibody according to any one of items 32 to 59. 61. (i) the antigen-binding region capable of binding to human PD-L1 is humanized; and / or (ii) the antigen-binding region capable of binding to human CD3ε (epsilon), if present, is humanized; The antibody according to any of the preceding items. 62. (i) the antigen-binding region capable of binding to human PD-L1 is human, and / or (ii) the antigen-binding region capable of binding to human CD3ε (epsilon), when present, is human; The antibody according to any of the preceding items. 63. The antibody of any of the preceding items, wherein each of the antigen-binding regions comprises a heavy chain variable region (VH) and a light chain variable region (VL), and each of the variable regions comprises three CDR sequences, CDR1, CDR2, and CDR3, respectively, and four framework sequences, FR1, FR2, FR3, and FR4, respectively. 64. The antibody described in item 63, comprising two heavy chain constant regions (CH) and two chain constant regions (CL). 65. The antibody of any preceding item, wherein the antibody comprises a first heavy chain and a second heavy chain, each of which comprises at least a hinge region, CH2, and CH3 region; wherein the first heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering); and the second heavy chain has at least one amino acid substitution at a position corresponding to a position selected from the group consisting of T366, L368, K370, D399, F405, Y407, and K409 (according to EU numbering); and wherein the first heavy chain and the second heavy chain do not have substitutions at the same positions. 66. The antibody of item 65, wherein (i) in the first heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L and in the second heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R, or (ii) in the first heavy chain, the amino acid at the position corresponding to K409 (according to EU numbering) is R and in the second heavy chain, the amino acid at the position corresponding to F405 (according to EU numbering) is L. 67. The antibody of any of the preceding items, comprising a first heavy chain and a second heavy chain; and one or both heavy chains are modified such that the antibody induces Fc-mediated effector function to a lesser extent than an otherwise identical antibody comprising an unmodified first and second heavy chains; The antibody according to any of the preceding items. 68. The antibody of item 67, wherein the Fc-mediated effector function is measured by determining Fc-mediated CD69 expression, by binding to an Fcγ receptor, by binding to C1q, or by induction of Fc-mediated FcR cross-linking. 69. The antibody according to item 67 or 68, wherein the heavy chain constant sequence and the light chain constant sequence are modified to reduce Fc-mediated CD69 expression by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, or 100% compared to the wild-type antibody; The Fc-mediated CD69 expression is measured in a PBMC-based functional assay. 69. The antibody of item 67 or 68. 70. The antibody of any preceding item, comprising a first heavy chain and a second heavy chain, wherein in at least one of the first heavy chain and the second heavy chain, one or more amino acids at positions corresponding to positions L234, L235, D265, N297, and P331 in a human IgG1 heavy chain according to EU numbering are not L, L, D, N, and P, respectively. 71. The antibody of item 70, wherein in the first heavy chain and the second heavy chain, the positions corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively. 72. The antibody of Item 71, wherein the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234 and L235 in a human IgG1 heavy chain according to EU numbering are F and E, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L. 73. The antibody of item 70, wherein in the first heavy chain and the second heavy chain, the positions corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively. 74. The antibody of Item 73, wherein the antibody is a bispecific antibody comprising a first heavy chain and a second heavy chain, and wherein the positions in both the first heavy chain and the second heavy chain corresponding to positions L234, L235, and D265 in a human IgG1 heavy chain according to EU numbering are F, E, and A, respectively, and wherein (i) the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is L and the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is R, or (ii) the position corresponding to K409 in the human IgG1 heavy chain according to EU numbering of the first heavy chain is R and the position corresponding to F405 in the human IgG1 heavy chain according to EU numbering of the second heavy chain is L. 75. The antibody of any preceding item, which does not bind to human PD-L2. 76. Approximately 10 -8 M or less, e.g., about 10 -9 M or less, e.g., about 10 -10 M or less K D2. The antibody of any preceding item, which binds to human PD-L1 at 77. The antibody of any preceding paragraph, which mediates concentration-dependent cytotoxicity of MDA-MB-231 cells, PC-3 cells, and / or HELA cells when purified T cells are used as effector cells, when assayed as described in Example 11 herein. 78. (i) a nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen-binding region capable of binding to human PD-L1 as defined in any one of items 1 to 31; and / or (ii) a nucleic acid sequence encoding an antibody light chain sequence comprising an antigen-binding region capable of binding to human PD-L1 as defined in any one of items 1 to 31; A nucleic acid construct comprising: 79. (i) A nucleic acid sequence encoding an antibody heavy chain sequence comprising an antigen-binding region capable of binding to human CD3ε as defined in any of items 33 to 38; and (ii) a nucleic acid sequence encoding a light chain sequence of an antibody comprising an antigen-binding region capable of binding to human CD3ε as defined in any one of items 33 to 38; 74. The nucleic acid construct of item 73, further comprising: 80. An expression vector comprising the nucleic acid construct defined in item 78 or 79. 81. A host cell comprising the nucleic acid construct defined in item 78 or 79 or the expression vector defined in item 80. 82. The host cell according to item 81, which is a mammalian cell such as a Chinese hamster ovary cell. 83. A pharmaceutical composition comprising the antibody according to any one of items 1 to 77 and a pharmaceutically acceptable carrier. 84. The antibody according to any one of items 1 to 77 or the pharmaceutical composition according to item 83 for use as a medicament. 85. The antibody according to any one of items 1 to 80 or the pharmaceutical composition according to item 70 for use in treating cancer. 86. The antibody according to any one of items 1 to 77 or the pharmaceutical composition according to item 70 for use in the treatment of a cancer disease characterized by the presence of a solid tumor. 87. The antibody according to any one of items 1 to 78 or the pharmaceutical composition according to item 70 for use in treating a cancer disease selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer. 88. A method for treating a disease, comprising administering to a subject in need thereof the antibody according to any one of items 1 to 75 or the pharmaceutical composition according to item 83. 89. Use of the antibody according to any of items 1 to 77 for the manufacture of a medicament, such as a medicament for treating cancer, e.g., a cancer disease characterized by the presence of a solid tumor, or a cancer disease selected from the group consisting of melanoma, ovarian cancer, lung cancer, colon cancer, and head and neck cancer. 90. The method or use according to any of items 83 to 89, comprising combination with one or more further therapeutic substances, for example combination with a chemotherapeutic agent. 91. (a) culturing a host cell that produces a first antibody comprising an antigen-binding region capable of binding to human PD-L1 as defined in any one of items 1 to 13, and purifying the first antibody from the culture; (b) culturing a host cell that produces a second antibody that comprises an antigen-binding region capable of binding to a different PD-L1 epitope or a different antigen, for example, a human CD3ε-binding region as defined in any of items 14 to 19, and purifying the second antibody from the culture; (c) incubating the first antibody with the second antibody under reducing conditions sufficient to allow cysteines in the hinge region to undergo disulfide bond isomerization; and (d) Obtaining a bispecific antibody 78. A method for producing the antibody according to any one of items 1 to 77, comprising: 92. An anti-idiotypic antibody that binds to an antigen-binding region capable of binding to human PD-L1 as defined in any of items 1 to 77.

[0320] The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention. [Example]

[0321] Example 1: Generation of PD-L1 antibodies Immunization of OmniRat animals and hybridoma generation Immunization and hybridoma generation were performed at Aldevron GmbH (Freiburg, Germany). cDNA encoding amino acids 19-238 of human PD-L1 was cloned into an expression plasmid proprietary to Aldevron. OmniRat animals (transgenic rats expressing a diverse antibody repertoire with fully human idiotypes; Ligand Pharmaceuticals Inc., San Diego, USA) were immunized by intradermal application of DNA-coated gold particles using a handheld device for particle bombardment ("gene gun"). Cell surface expression on transiently transfected HEK cells was confirmed using Genentech's MPDL3280A-based anti-PD-L1 antibody. Serum samples were collected after each immunization series and tested by flow cytometry against HEK cells transiently transfected with the aforementioned expression plasmid. Antibody-producing cells were isolated and fused with mouse myeloma cells (Ag8) according to standard procedures. Hybridomas producing PD-L1-specific antibodies were identified by screening using the same assay described above. Cell pellets of positive hybridoma cells were prepared using an RNA protecting agent (RNAlater, ThermoFisher Scientific, Cat. No. AM7020) and further processed to sequence the variable domains of the antibodies.

[0322] Sequence analysis of PD-L1 antibody variable domains and cloning into expression vectors 0.2 to 5 × 10 6Total RNA was prepared from hybridoma cells, and 5'-RACE-complementary DNA (cDNA) was prepared from the total RNA using the SMART RACE cDNA Amplification kit (Clontech) according to the manufacturer's instructions. The VH and VL coding regions were PCR amplified and cloned directly and in-frame into the pOMTG1f-FEAR-LIC (human IgG1) and pEFC33D-κ (human κ) or pOMTL-LIC (human λ) expression vectors by ligation-independent cloning (Aslanidis, C. and PJ de Jong, Nucleic Acids Res 1990;18(20):6069-74). In these plasmids, antibody sequences are expressed using a CMV promoter. For each antibody, eight VL clones and eight VH clones were sequenced. CDR sequences were defined according to the IMGT definition [Lefranc MP. et al., Nucleic Acids Research, 27, 209-212, 1999; Brochet X. Nucl. Acids Res. 36, W503-508 (2008)]. Clones with the correct open reading frame (ORF) were selected for further study and expression. LEE PCR products of all combinations of heavy and light chains discovered for each hybridoma culture were transiently co-expressed in Expi293F cells using ExpiFectamine. For each hybridoma, the HC / LC pair that showed the best binding in a homogeneous dose-response screen was selected as a lead candidate.

[0323] Three PD-L1 antibodies, numbered 338, 511, and 547, were selected for further experiments, and their variable region sequences are shown in the sequence listing herein.

[0324] For the antibody IgG1-PDL1-511-FEAR, a variant with a point mutation in the variable domain to remove cysteine ​​residues that could potentially form undesired disulfide bridges was generated: IgG1-PDL1-511-FEAR-LC33S. This mutant was generated by gene synthesis (Geneart).

[0325] LEE PCR Linear expression elements (LEEs) were generated by amplifying fragments containing the CMV promoter and either the HC or LC coding region and poly(A) signal containing elements from the expression plasmid using Accuprime Taq DNA polymerase (Life Technologies) and primers CMVPf(BsaI)2 and TkpA(BsaI)r, with 35 cycles of 45 s at 94°C, 30 s at 55°C, and 2 min (LC) or 3 min (HC) at 68°C, using 50x diluted plasmid miniprep material as DNA template.

[0326] Transient expression of LEE fragments in Expi293F cells For LEE expression of Ab, 1.11 μl of HC LEE PCR reaction mixture and 1.11 μl of LC PCR reaction mixture were mixed and transfected in Expi293F cells in a total volume of 125 μl using ExpiFectamin 293 as the transfection reagent according to the manufacturer's instructions (Thermo Fisher Scientific, USA) using a 96-well plate as the vessel.

[0327] Antibody expression Antibodies were expressed as IgG1,κ (for 338) or IgG1,λ (for 511 and 547). Plasmid DNA mixtures encoding both the heavy and light chains of the antibodies were transiently expressed using the Expi293F expression platform (Thermo Fisher Scientific, USA) essentially as described by the manufacturer.

[0328] Homogeneous Binding Assay Antibodies were tested for binding in a homogeneous dose-response screen using CHO cells transfected with PDL1, PDL1mm, or PDL1Mf (see also Example 2). Untransfected CHO cells were used as a negative control.

[0329] cells (cells 2.5×10 5 100 cells / ml) was mixed with goat anti-human IgG Alexa647, Fcγ fragment specific (0.2 μg / ml; Jackson ImmunoResearch Laboratories, 109-605-098). Serial dilutions of the test and control antibodies (ranging from 0.001 to 3 μg / mL in two-fold dilution steps) were prepared, and 2 μl of the antibody dilution was added to 5 μl of the cell / conjugate mixture in a 1536-well plate (Greiner, 789866). The plate was incubated at room temperature for 9 hours, after which the fluorescence intensity was measured using an ImageXpress Velos Laser Scanning Cytometer (Molecular Devices).

[0330] Antibody purification Culture supernatants were filtered through a 0.2 μm dead-end filter, loaded onto a 5 mL MabSelect SuRe column (GE Healthcare), and eluted with 0.1 M sodium citrate-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9, and dialyzed overnight against 8.7 mM NaHPO, 1.8 mM NaHPO, 140.3 mM NaCl, pH 7.4 (B. Braun or GE Healthcare). Alternatively, after purification, the eluate was loaded onto a HiPrep Desalting column, and the antibody was exchanged into 8.7 mM NaHPO, 1.8 mM NaHPO, 140.3 mM NaCl, pH 7.4 (B. Braun or GE Healthcare) buffer. After dialysis or buffer exchange, the sample was sterile filtered through a 0.2 μm dead-end filter. Purity was determined by CE-SDS using a LabChip GXII (Caliper Life Sciences, MA), and IgG concentration was measured using a Nanodrop ND-1000 spectrophotometer (Isogen Life Science, Maarssen, The Netherlands). Purified antibodies were stored at 4°C.

[0331] Example 2: Preparation of screening materials Expression constructs for PD-L1 The following codon-optimized constructs were generated to express full-length PD-L1: human (Homo sapiens) PD-L1 (Genbank accession number NP_054862), Macaca fascicularis PD-L1 (Genbank accession number XP_005581836), and Mus musculus PD-L1 (Genbank accession number NP_068693).

[0332] Additionally, the following codon-optimized constructs were generated for PD-L1 ECD: the extracellular domain (ECD) of human PD-L1 (aa 1-238) with a C-terminal His-tag and C-tag (PDLoneECDHisC-tag).

[0333] The construct contained suitable restriction sites for cloning and an optimal Kozak (GCCGCCACC) sequence [Kozak et al. (1999) Gene 234: 187-208]. The construct was cloned into the mammalian expression vector pMA (Geneart).

[0334] Expression construct for PD-L2 Similarly, the following codon-optimized construct was generated to express full-length human PD-L2: human PD-L2 (Genbank accession number NP_079515).

[0335] Expression in CHO-S cells CHO-S cells were transiently transfected with pMA vectors containing the complete human PD-L1 coding sequence, the complete cynomolgus monkey coding sequence, and the complete mouse coding sequence.

[0336] Purification of His-tagged PD-L1 PDLoneECDHisC-tagged was expressed in HEK-293F cells. The His-tag allows for purification by immobilized metal affinity chromatography. In this process, the chelator immobilized on the chromatography resin is Co. 2+ The supernatant containing the His-tagged protein is incubated with the resin in a batch format (i.e., in solution). The His-tagged protein binds strongly to the resin beads, whereas other proteins present in the culture supernatant do not bind or bind weakly compared to the His-tagged protein. After incubation, the beads are recovered from the supernatant and packed into a column. The column is washed to remove weakly bound proteins. The strongly bound His-tagged protein is then separated by a His- and Co-cleavage reaction. 2+ The protein is eluted with a buffer containing imidazole, which competes with binding to the ATP. The eluant is removed from the protein by buffer exchange on a desalting column.

[0337] Example 3: Humanized CD3 antibody to generate CD3xPDL1 bispecific antibody The production of the humanized antibody IgG1-huCD3-H1L1 is described in Example 1 of WO2015001085. The antibody huCD3-H1L1-FEAL is a variant thereof with the following substitutions as described herein above: L234F, L235E, D265A, and F405L.

[0338] Example 4: Generation of bispecific antibodies by 2-MEA-induced Fab arm exchange Bispecific IgG1 antibodies were generated by Fab arm exchange under controlled reducing conditions. This method is based on the use of complementary CH3 domains to promote heterodimer formation under specific assay conditions, as described in WO2011 / 131746. To generate antibody pairs with complementary CH3 domains, F405L and K409R (EU numbering) mutations were introduced into the relevant antibodies.

[0339] To generate bispecific antibodies, two parental complementary antibodies at a final concentration of 0.5 mg / mL of each antibody were incubated with 75 mM 2-mercaptoethylamine-HCl (2-MEA) in a total volume of 100 μL TE for 5 h at 31° C. The reduction reaction was stopped by removing the reducing agent 2-MEA using a spin column (Microcon centrifugal filter, 30k, Millipore) according to the manufacturer's protocol.

[0340] The following antibodies were used in the examples:

[0341] CD3 antibody IgG1-huCD3-H1L1-FEAL (having the VH sequence shown in SEQ ID NO:25 and the VL sequence shown in SEQ ID NO:29) bsIgG1-huCD3-H1L1-FEALxb12-FEAR is a bispecific antibody that uses the gp120-specific antibody b12 (Barbas, C. F. J. Mol. Biol. 1993 Apr. 5;230(3):812-23) as the second arm.

[0342] PDL1 antibody and CD3×PDL1 bispecific antibody IgG1-338-FEAR (having the VH sequence shown in SEQ ID NO:1 and the VL sequence shown in SEQ ID NO:5) IgG1-338-F405L bsIgG1-huCD3-H1L1-FEALx338-FEAR bsIgG1-b12-FEALx338-FEAR

[0343] IgG1-511-LC33S-FEAR (having the VH sequence shown in SEQ ID NO:8 and the VL sequence shown in SEQ ID NO:15) IgG1-511-F405L-LC33S bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR bsIgG1-b12-FEALx511-LC33S-FEAR

[0344] IgG1-547-FEAR (having the VH sequence shown in SEQ ID NO:18 and the VL sequence shown in SEQ ID NO:22) IgG1-547-F405L bsIgG1-huCD3-H1L1-FEALx547-FEAR bsIgG1-b12-FEALx547-FEAR

[0345] IgG1-321-FEAR (having the VH sequence shown in SEQ ID NO:32 and the VL sequence shown in SEQ ID NO:36)

[0346] IgG1-421-LC91S-FEAR (having the VH sequence shown in SEQ ID NO:46 and the VL sequence shown in SEQ ID NO:50)

[0347] IgG1-476-N101Q-LC33S-FEAR (having the VH sequence shown in SEQ ID NO:53 and the VL sequence shown in SEQ ID NO:57)

[0348] IgG1-625-FEAR (having the VH sequence shown in SEQ ID NO:106 and the VL sequence shown in SEQ ID NO:110)

[0349] IgG1-632-FEAR (having the VH sequence shown in SEQ ID NO:67 and the VL sequence shown in SEQ ID NO:71) IgG1-516-FEAR (having the VH sequence shown in SEQ ID NO:60 and the VL sequence shown in SEQ ID NO:64)

[0350] IgG1-MPDL3280A-FEAR (based on Genentech's PDL1 antibody MPDL3280A; has the VH sequence shown in SEQ ID NO:74 and the VL sequence shown in SEQ ID NO:78) IgG1-MPDL3280A-K409R

[0351] IgG1-MEDI4736-FEAR (based on MediImmune's PDL1 antibody MEDI4736; has the VH sequence shown in SEQ ID NO:81 and the VL sequence shown in SEQ ID NO:85) IgG1-MEDI4736-F405L

[0352] Example 5: Binding of PD-L1 antibodies or CD3xPD-L1 or b12xPD-L1 bispecific antibodies to tumor cells The binding of PD-L1 antibodies and CD3×PD-L1 and b12×PD-L1 bispecific antibodies to the human tumor cell lines SK-MES-1 (lung squamous cell carcinoma; ATCC; Catalog No. HTB-58), MDA-MB-231 (breast adenocarcinoma; ATCC; Catalog No. HTB-26), PC-3 (prostate adenocarcinoma; ATCC; Catalog No. CRL-1435), and HELA (cervical adenocarcinoma; ATCC; Catalog No. CCL-2) was analyzed by flow cytometry.

[0353] cells (cells 3~5 x 10 4 The cells (cells / well) were incubated in a polystyrene 96-well round-bottom plate (Greiner bio-one, catalog no. 650101) with serial dilutions of antibody (ranging from 0.0001 to 10 μg / mL in 5-fold dilution steps) dissolved in 50 μL of PBS / 0.1% BSA / 0.02% azide (staining buffer) at 4°C for 30 minutes.

[0354] After washing twice with staining buffer, cells were incubated with 50 μL of secondary antibody at 4°C for 30 minutes. R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab')2 (catalog number 109-116-098, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) diluted 1:500 in staining buffer was used as the secondary antibody in all experiments. Cells were then washed twice with staining buffer, resuspended in 20 μL of staining buffer, and analyzed using an iQue screener (Intellicyt Corporation, USA). Binding curves were analyzed using nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V75.04 software (GraphPad Software, San Diego, CA, USA).

[0355] To quantify target expression on the plasma membrane of MDA-MB-231, PC-3, and HELA cells and to determine the number of bound PDL1 molecules, quantitative flow cytometry (QIFIKIT®, Dako; catalog number K0078) was performed as described (Poncelet and Carayon, 1985, J. Immunol. Meth. 85: 65-74). The cell lines had the following PD-L1 antigen densities (ABC, antibody binding capacity): SK-MES-1: approximately 30,000 ABCs / cell, MDA-MB-231: approximately 21,000 ABCs / cell, PC-3: Approximately 6,000 ABCs / cell, HELA cells: approximately 2,000 ABCs / cell It was determined that he had

[0356] Binding to MDA-MB-231 cells Figure 1 shows that bsIgG1-huCD3-H1L1-FEALx338-FEAR (A), bsIgG1-b12-FEALx338-FEAR (D), bsIgG1-huCD3-H1L1-FEALx547-FEAR (B), and bsIgG1-b12-FEALx547-FEAR (E) showed dose-dependent binding to MDA-MB-231 cells, with higher maximum binding than the monospecific bivalent PD-L1 antibodies IgG1-338-FEAR and IgG1-547-FEAR. The maximum binding of bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR (C) and bsIgG1-b12-FEALx511-LC33S-FEAR (F) was lower than the bivalent monospecific PD-L1 antibody IgG1-511-LC33S-FEAR.

[0357] Binding to PC-3 cells Figure 2 shows that bsIgG1-huCD3-H1L1-FEALx338-FEAR (A), bsIgG1-b12-FEALx338-FEAR (D), bsIgG1-huCD3-H1L1-FEALx547-FEAR (B), and bsIgG1-b12-FEALx547-FEAR (E) showed dose-dependent binding to PC3 cells. The maximum binding of bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR (C) and bsIgG1-b12-FEALx511-LC33S-FEAR (F) was lower than that of the bivalent monospecific PD-L1 antibody IgG1-511-LC33S-FEAR.

[0358] Binding to HELA cells Figure 3 shows that bsIgG1-huCD3-H1L1-FEALx338-FEAR (A) and bsIgG1-huCD3-H1L1-FEALx547-FEAR (B) exhibited dose-dependent binding to HELA cells. Maximum binding of the monospecific bivalent PD-L1 antibodies IgG1-338-FEAR and IgG1-547-FEAR could not be determined within the concentration range used. (C) bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR did not bind to HELA cells.

[0359] Binding to SK-MES-1 cells Figure 4 shows that bsIgG1-b12-FEALx338-FEAR (A) and bsIgG1-b12-FEALx547-FEAR (B) showed dose-dependent binding to SK-MES-1 cells, with higher maximum binding than the monospecific bivalent PD-L1 antibodies IgG1-338-FEAR and IgG1-547-FEAR. The maximum binding of bsIgG1-b12-FEALx511-LC33S-FEAR (C) was lower than the bivalent monospecific PD-L1 antibody IgG1-511-LC33S-FEAR.

[0360] Example 6: Binding to human PD-L2 To demonstrate specific binding to PD-L1 but not to human PD-L2, the binding of bsIgG1-huCD3-H1L1-FEALx338-FEAR and IgG1-338-FEAR, bsIgG1-huCD3-H1L1-FEALx547-FEAR and IgG1-547-FEAR, and bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR and IgG1-511-LC33S-FEAR to human PD-L2-expressing CHO cells was measured by flow cytometry using the methods described above. A PE-conjugated PD-L2-specific antibody (Mylteni, clone MIH18; catalog no. 130-098-651) was used as a positive control. None of the antibodies tested bound to CHO-PD-L2 cells.

[0361] Example 7: Binding of PD-L1 antibodies or CD3xPD-L1 or b12xPD-L1 bispecific antibodies to cynomolgus monkey PD-L1 Binding to cynomolgus monkey PD-L1-expressing CHO cells was measured by flow cytometry using the methods described above. Figure 5 shows that bsIgG1-huCD3-H1L1-FEALx338-FEAR (A), bsIgG1-huCD3-H1L1-FEALx547-FEAR (B), bsIgG1-huCD3-H1L1-FEALx511-LC33S-FEAR (C), bsIgG1-b12-FEALx338-FEAR (D), bsIgG1-b12-FEALx547-FEAR (E), and bsIgG1-b12-FEALx511-LC33S-FEAR (F) showed dose-dependent binding to cynomolgus PD-L1-expressing CHO cells, with higher maximum binding than the monospecific bivalent PD-L1 antibodies IgG1-338-FEAR, IgG1-547-FEAR, and IgG1-511-LC33S-FEAR.

[0362] Example 8: Measurement of human PD-L1 affinity and cynomolgus monkey PD-L1 affinity using biolayer interferometry In the first set of experiments, affinity for recombinantly expressed human PD-L1 protein was measured using biolayer interferometry (BLI) on an Octet HTX in...

Claims

[Claim 1] An antibody comprising an antigen-binding region capable of binding to human PD-L1, Inhibits the binding of human PD-L1 to human PD-1, and (i) competes for binding to human PD-L1 with antibody [511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15, but does not compete for binding to human PD-L1 with antibody [547] comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22; or (ii) competes for binding to human PD-L1 with antibody [547] comprising the VH sequence set forth in SEQ ID NO:18 and the VL sequence set forth in SEQ ID NO:22, but does not compete for binding to human PD-L1 with antibody [511] comprising the VH sequence set forth in SEQ ID NO:8 and the VL sequence set forth in SEQ ID NO:15; The antibody.

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