Human monoclonal antibodies to programmed death ligand 1 (PD-l1)

Abstract

To provide an isolated monoclonal antibody, particularly human monoclonal antibody, that specifically binds to PD-L1 with high affinity.SOLUTION: A method for preparing an anti-PD-L1 antibody comprises: (a) providing a heavy chain variable region antibody sequence including a specific CDR3 sequence, and / or a light chain variable region antibody sequence including a specific CDR3 sequence; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to create at least one altered antibody sequence; and (c) expressing the altered antibody sequence as a protein.SELECTED DRAWING: Figure 47

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 60 / 696,426, filed July 1, 2005, which is incorporated herein by reference in its entirety. [Background technology]

[0002] background Programmed death 1 (PD-1) is a member of the CD28 family of receptors, which also includes CD28, CTLA-4, ICOS, PD-1, and BTLA. The first members of the family, CD28 and ICOS, were discovered by their functional effects on T cell proliferation, which increased after the addition of monoclonal antibodies (Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980) Immunogenics 10:247-260). Two cell surface glycoprotein ligands for PD-1, PD-L1 and PD-L2, have been identified and shown to downregulate T cell activation and cytokine secretion upon binding to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). Both PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind to PD-1 but not other CD28 family members (Blank et al. (2004)). PD-L1 expression on the cell surface has also been shown to be upregulated by IFN-γ stimulation.

[0003] PD-L1 expression has been found in several murine and human cancers, including human lung, ovarian, and colon cancers, as well as various myelomas (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). PD-L1 has been suggested to play a role in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8:793-800). It has also been suggested that PD-L1 may be involved in intestinal mucosal inflammation, and that inhibiting PD-L1 suppresses the debilitating disease associated with colitis (Kanai et al. (2003) J Immunol 171:4156-63). Summary of the Invention

[0004] overview The present invention provides isolated monoclonal antibodies, particularly human monoclonal antibodies, that bind to PD-L1 and exhibit a number of desirable properties, including high affinity binding to human PD-L1. Furthermore, the antibodies of the invention have also been shown to increase T cell proliferation, IFN-γ secretion, and IL-2 secretion in a mixed lymphocyte reaction.

[0005] In one aspect, the present invention relates to an isolated monoclonal antibody, or antigen-binding portion thereof, that exhibits at least one of the following properties: (a) 1×10 -7 M or lower K D binds to human PD-L1; (b) increase T-cell proliferation in mixed lymphocyte reaction (MLR) assays; (c) Increases interferon-γ production in the MLR assay; (d) increases IL-2 secretion in the MLR assay; (e) stimulating an antibody response; or (f) Reversing the effects of regulatory T cells on T cell effector cells and / or dendritic cells.

[0006] Preferably, the antibody is a human antibody, although in alternative embodiments, the antibody may be, for example, a murine antibody, a chimeric antibody, or a humanized antibody.

[0007] In certain embodiments, the antibody is 5×10 -8 M or lower K D binds to human PD-L1 at 1 × 10 -8 M or lower K D binds to human PD-L1 at 5 × 10 -9 M or lower K D binds to human PD-L1 at 5 × 10 -9 M or lower K D bind to human PD-L1 at 1 × 10 -8 M~1×10 -10 K between M D It binds to human PD-L1.

[0008] In another embodiment, the invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, that cross-competes for binding to PD-L1 with a reference antibody comprising: (a) a human heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and (b) a human light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0009] In various embodiments, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:12; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:13; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:14; or Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:15; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:16; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:17; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:18; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:19; Alternatively, the reference antibody is (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:20.

[0010] In another aspect, the present invention provides a method for the preparation of a human V H 1-18 gene product or human V H The present invention further relates to an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-18 gene. H 1-69 gene product or human V H The present invention also provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-69 gene. H 1-3 gene product or human V H The present invention also provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the human V gene. H 3-9 gene product or human V H The present invention also provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 3-9 gene. K It is the product of the L6 gene or human V K The present invention also provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the L6 gene. K L15 gene product or human V KThe present invention also provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the L15 gene. K A27 gene product or human V K The present invention also provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the A27 gene. K It is the product of the L18 gene or human V K Also provided is an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the L18 gene.

[0011] In particularly preferred embodiments, the present invention provides isolated monoclonal antibodies, or antigen-binding portions thereof, that specifically bind to PD-L1, comprising: (a) Human V H The heavy chain variable region of the 1-18 gene; and (b) Human V K Light chain variable region of the L6 gene.

[0012] In another preferred embodiment, the present invention provides an isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising: (a) Human V H the heavy chain variable region of the 1-69 gene; and (b) Human V K Light chain variable region of the L6 gene.

[0013] In another preferred embodiment, the present invention provides an isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising: (a) Human V H The heavy chain variable region of the 1-3 gene; and (b) Human V K Light chain variable region of the L15 gene.

[0014] In another preferred embodiment, the present invention provides an isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising: (a) Human V H the heavy chain variable region of the 1-69 gene; and (b) Human V K Light chain variable region of the A27 gene.

[0015] In another preferred embodiment, the present invention provides an isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising: (a) Human V H The heavy chain variable region of the 3-9 gene; and (b) Human V K Light chain variable region of the L15 gene.

[0016] In another preferred embodiment, the present invention provides an isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising: (a) Human V H The heavy chain variable region of the 3-9 gene; and (b) Human V K Light chain variable region of the L18 gene.

[0017] In another aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising: a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; and a light chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; (a) the CDR3 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, and conservative variants thereof; (b) the CDR3 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, and conservative variants thereof; and (c) the antibody specifically binds to human PD-L1; An isolated monoclonal antibody or antigen-binding portion thereof is provided.

[0018] Preferably, the CDR2 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and conservative variants thereof, and the CDR2 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, and conservative variants thereof. Preferably, the CDR1 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, and conservative variants thereof, and the CDR1 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, and conservative variants thereof.

[0019] In yet another aspect, the present invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region; (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; (b) the light chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and (c) Antibody is 1 × 10 -7 M or lower K D binds to human PD-L1 with An isolated monoclonal antibody or antigen-binding portion thereof is provided.

[0020] In preferred embodiments, the antibody further comprises at least one of the following properties: (a) the antibody increases T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; or (b) the antibody increases interferon-γ production in an MLR assay; (c) The antibody increases IL-2 secretion in an MLR assay.

[0021] In a preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising: (a) a CDR1 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; (b) a CDR2 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; (c) a CDR3 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; (d) a CDR1 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60; (e) a CDR2 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70; and (f) a CDR3 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80.

[0022] Preferred combinations include: (a) heavy chain variable region CDR1 comprising SEQ ID NO:21; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:31; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:41; (d) a light chain variable region CDR1 comprising SEQ ID NO:51; (e) a light chain variable region CDR2 comprising SEQ ID NO:61; and (f) Light chain variable region CDR3 comprising SEQ ID NO:71.

[0023] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:22; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:32; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:42; (d) a light chain variable region CDR1 comprising SEQ ID NO:52; (e) a light chain variable region CDR2 comprising SEQ ID NO:62; and (f) Light chain variable region CDR3 comprising SEQ ID NO:72.

[0024] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:23; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:33; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:43; (d) a light chain variable region CDR1 comprising SEQ ID NO:53; (e) a light chain variable region CDR2 comprising SEQ ID NO:63; and (f) Light chain variable region CDR3 comprising SEQ ID NO:73.

[0025] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:24; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:34; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:44; (d) a light chain variable region CDR1 comprising SEQ ID NO:54; (e) a light chain variable region CDR2 comprising SEQ ID NO:64; and (f) Light chain variable region CDR3 comprising SEQ ID NO:74.

[0026] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:25; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:35; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:45; (d) a light chain variable region CDR1 comprising SEQ ID NO:55; (e) a light chain variable region CDR2 comprising SEQ ID NO:65; and (f) Light chain variable region CDR3 comprising SEQ ID NO:75.

[0027] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:26; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:36; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:46; (d) a light chain variable region CDR1 comprising SEQ ID NO:56; (e) a light chain variable region CDR2 comprising SEQ ID NO:66; and (f) Light chain variable region CDR3 comprising SEQ ID NO:76.

[0028] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:27; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:37; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:47; (d) a light chain variable region CDR1 comprising SEQ ID NO:57; (e) a light chain variable region CDR2 comprising SEQ ID NO:67; and (f) Light chain variable region CDR3 comprising SEQ ID NO:77.

[0029] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:28; (b) heavy chain variable region CDR2 comprising SEQ ID NO:38; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:48; (d) a light chain variable region CDR1 comprising SEQ ID NO:58; (e) a light chain variable region CDR2 comprising SEQ ID NO:68; and (f) Light chain variable region CDR3 comprising SEQ ID NO:78.

[0030] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:29; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:39; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:49; (d) a light chain variable region CDR1 comprising SEQ ID NO:59; (e) a light chain variable region CDR2 comprising SEQ ID NO:69; and (f) Light chain variable region CDR3 comprising SEQ ID NO:79.

[0031] Another preferred combination includes: (a) heavy chain variable region CDR1 comprising SEQ ID NO:30; (b) heavy chain variable region CDR2 comprising SEQ ID NO:40; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:50; (d) a light chain variable region CDR1 comprising SEQ ID NO:60; (e) a light chain variable region CDR2 comprising SEQ ID NO:70; and (f) Light chain variable region CDR3 comprising SEQ ID NO:80.

[0032] Other preferred antibodies of the invention that specifically bind to PD-L1, or antigen-binding portions thereof, include: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0033] Preferred combinations include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11.

[0034] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:12.

[0035] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:13.

[0036] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:14.

[0037] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:15.

[0038] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:16.

[0039] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:17.

[0040] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:18.

[0041] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:19.

[0042] Another preferred combination includes: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:20.

[0043] In another aspect of the disclosure, there is provided an antibody, or antigen-binding portion thereof, that competes with any of the aforementioned antibodies for binding to PD-L1.

[0044] The antibodies of the disclosure can be, for example, full-length antibodies, e.g., of the isotype IgG1 or IgG4, or they can be antibody fragments, such as Fab or Fab'2 fragments, or single chain antibodies.

[0045] The present disclosure also provides immunoconjugates comprising an antibody of the invention, or an antigen-binding portion thereof, linked to a therapeutic agent, such as a cytotoxin or a radioisotope. The present invention also provides bispecific molecules comprising an antibody of the invention, or an antigen-binding portion thereof, linked to a second functional moiety having a binding specificity different from the antibody of the invention, or an antigen-binding portion thereof.

[0046] Compositions comprising an antibody or antigen-binding portion thereof, or immunoconjugate or bispecific molecule of this disclosure and a pharmaceutically acceptable carrier are also provided.

[0047] Nucleic acid molecules encoding the antibodies of the invention or antigen-binding portions thereof, as well as expression vectors containing such nucleic acids and host cells containing such expression vectors, are also encompassed by the invention. Additionally, the invention provides transgenic mice containing human immunoglobulin heavy and light chain transgenes that express the antibodies of the invention, and hybridomas prepared from such mice that produce the antibodies of the invention.

[0048] In yet another aspect, the invention provides a method of modulating the immune response of a subject, comprising administering to the subject an antibody of the invention, or antigen-binding portion thereof, such that the subject's immune response is modulated. Preferably, the antibody of the invention enhances, stimulates, or increases the subject's immune response.

[0049] In another aspect, the present invention provides a method of inhibiting tumor cell growth in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody, or antigen-binding portion thereof. Although an antibody of the present invention is preferably used in this method, other anti-PD-L1 antibodies may be used instead (or in combination with an anti-PD-L1 antibody of the present invention). For example, a chimeric anti-PD-L1 antibody, a humanized anti-PD-L1 antibody, or a fully human anti-PD-L1 antibody may be used in the method of inhibiting tumor growth.

[0050] In another aspect, the present invention provides a method of treating an infectious disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody, or antigen-binding portion thereof. Although an antibody of the present invention is preferably used in this method, other anti-PD-L1 antibodies may be used instead (or in combination with an anti-PD-L1 antibody of the present invention). For example, a chimeric anti-PD-L1 antibody, a humanized anti-PD-L1 antibody, or a fully human anti-PD-L1 antibody may be used in the method of treating an infectious disease.

[0051] Furthermore, the present invention provides a method for enhancing an immune response to an antigen in a subject, the method comprising the step of administering to the subject (i) the antigen and (ii) an anti-PD-L1 antibody, or antigen-binding portion thereof, such that the immune response to the antigen in the subject is enhanced. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Although an antibody of the present invention is preferably used in this method, other anti-PD-L1 antibodies may be used instead (or in combination with an anti-PD-L1 antibody of the present invention). For example, a chimeric anti-PD-L1 antibody, a humanized anti-PD-L1 antibody, or a fully human anti-PD-L1 antibody may be used in the method for enhancing an immune response to an antigen in a subject.

[0052] The present invention also provides methods for making "second generation" anti-PD-L1 antibodies based on the sequences of the anti-PD-L1 antibodies provided herein. For example, the present invention provides a method for preparing an anti-PD-L1 antibody, comprising the steps of: (a)(i) a CDR1 sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, a CDR2 sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and a CDR2 sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. or (ii) a heavy chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, a CDR2 sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, and a CDR3 sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80. providing a light chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80; (b) modifying at least one amino acid residue in at least one variable region antibody sequence selected from a heavy chain variable region antibody sequence and a light chain variable region antibody sequence to generate at least one modified antibody sequence; and (c) expressing the modified antibody sequence as a protein.

[0053] Other features and advantages of the present invention will become apparent from the following detailed description and examples, which should not be construed as limiting. The contents of all references, Genbank accession numbers, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. [Brief explanation of the drawings]

[0054] [Figure 1A] The nucleotide sequence (SEQ ID NO:81) and amino acid sequence (SEQ ID NO:1) of the heavy chain variable region of the 3G10 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:21), CDR2 (SEQ ID NO:31), and CDR3 (SEQ ID NO:41) regions are underlined, and the germline V, D, and J are indicated. [Figure 1B] The nucleotide sequence (SEQ ID NO:91) and amino acid sequence (SEQ ID NO:11) of the light chain variable region of the 3G10 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:51), CDR2 (SEQ ID NO:61), and CDR3 (SEQ ID NO:71) regions are underlined and the germline V and J are indicated. [Figure 2A] The nucleotide sequence (SEQ ID NO:82) and amino acid sequence (SEQ ID NO:2) of the heavy chain variable region of the 12A4 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:22), CDR2 (SEQ ID NO:32), and CDR3 (SEQ ID NO:42) regions are underlined and the germline V and J are indicated. [Figure 2B]The nucleotide sequence (SEQ ID NO:92) and amino acid sequence (SEQ ID NO:12) of the light chain variable region of the 12A4 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:52), CDR2 (SEQ ID NO:62), and CDR3 (SEQ ID NO:72) regions are underlined and the germline V and J are indicated. [Figure 3A] The nucleotide sequence (SEQ ID NO:83) and amino acid sequence (SEQ ID NO:3) of the heavy chain variable region of the 10A5 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:23), CDR2 (SEQ ID NO:33), and CDR3 (SEQ ID NO:43) regions are underlined and the germline V and J are indicated. [Figure 3B] The nucleotide sequence (SEQ ID NO:93) and amino acid sequence (SEQ ID NO:13) of the light chain variable region of the 10A5 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:53), CDR2 (SEQ ID NO:63), and CDR3 (SEQ ID NO:73) regions are underlined and the germline V and J are indicated. [Figure 4A] The nucleotide sequence (SEQ ID NO:84) and amino acid sequence (SEQ ID NO:4) of the heavy chain variable region of the 5F8 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:24), CDR2 (SEQ ID NO:34), and CDR3 (SEQ ID NO:44) regions are underlined and the germline V and J are indicated. [Figure 4B] The nucleotide sequence (SEQ ID NO:94) and amino acid sequence (SEQ ID NO:14) of the light chain variable region of the 5F8 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:54), CDR2 (SEQ ID NO:64), and CDR3 (SEQ ID NO:74) regions are underlined and the germline V and J are indicated. [Figure 5A]The nucleotide sequence (SEQ ID NO:85) and amino acid sequence (SEQ ID NO:5) of the heavy chain variable region of the 10H10 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:35), and CDR3 (SEQ ID NO:45) regions are underlined and the germline V and J are indicated. [Figure 5B] The nucleotide sequence (SEQ ID NO:95) and amino acid sequence (SEQ ID NO:15) of the light chain variable region of the 10H10 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:55), CDR2 (SEQ ID NO:65), and CDR3 (SEQ ID NO:75) regions are underlined and the germline V and J are indicated. [Figure 6A] The nucleotide sequence (SEQ ID NO:86) and amino acid sequence (SEQ ID NO:6) of the heavy chain variable region of the 1B12 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:26), CDR2 (SEQ ID NO:36), and CDR3 (SEQ ID NO:46) regions are underlined and the germline V and J are indicated. [Figure 6B] The nucleotide sequence (SEQ ID NO:96) and amino acid sequence (SEQ ID NO:16) of the light chain variable region of the 1B12 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:56), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:76) regions are underlined and the germline V and J are indicated. [Figure 7A] The nucleotide sequence (SEQ ID NO:87) and amino acid sequence (SEQ ID NO:7) of the heavy chain variable region of the 7H1 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:27), CDR2 (SEQ ID NO:37), and CDR3 (SEQ ID NO:47) regions are underlined and the germline V and J are indicated. [Figure 7B]The nucleotide sequence (SEQ ID NO:97) and amino acid sequence (SEQ ID NO:17) of the light chain variable region of the 7H1 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:57), CDR2 (SEQ ID NO:67), and CDR3 (SEQ ID NO:77) regions are underlined and the germline V and J are indicated. [Figure 8A] The nucleotide sequence (SEQ ID NO:88) and amino acid sequence (SEQ ID NO:8) of the heavy chain variable region of the 11E6 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:28), CDR2 (SEQ ID NO:38), and CDR3 (SEQ ID NO:48) regions are underlined, and the germline V and J are indicated. [Figure 8B] The nucleotide sequence (SEQ ID NO:98) and amino acid sequence (SEQ ID NO:18) of the light chain variable region of the 11E6 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:58), CDR2 (SEQ ID NO:68), and CDR3 (SEQ ID NO:78) regions are underlined and the germline V and J are indicated. [Figure 9A] The nucleotide sequence (SEQ ID NO:89) and amino acid sequence (SEQ ID NO:9) of the heavy chain variable region of the 12B7 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:29), CDR2 (SEQ ID NO:39), and CDR3 (SEQ ID NO:49) regions are underlined and the germline V and J are indicated. [Figure 9B] The nucleotide sequence (SEQ ID NO:99) and amino acid sequence (SEQ ID NO:19) of the light chain variable region of the 12B7 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:59), CDR2 (SEQ ID NO:69), and CDR3 (SEQ ID NO:79) regions are underlined and the germline V and J are indicated. [Figure 10A]The nucleotide sequence (SEQ ID NO:90) and amino acid sequence (SEQ ID NO:10) of the heavy chain variable region of the 13G4 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:30), CDR2 (SEQ ID NO:40), and CDR3 (SEQ ID NO:50) regions are underlined and the germline V and J are indicated. [Figure 10B] The nucleotide sequence (SEQ ID NO:100) and amino acid sequence (SEQ ID NO:20) of the light chain variable region of the 13G4 human monoclonal antibody are shown. The CDR1 (SEQ ID NO:60), CDR2 (SEQ ID NO:70), and CDR3 (SEQ ID NO:80) regions are underlined and the germline V and J are indicated. [Figure 11] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 3G10 (SEQ ID NO:1) with the human germline VH1-18 amino acid sequence (SEQ ID NO:101). [Figure 12] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 12A4 (SEQ ID NO:2) with the human germline VH1-69 amino acid sequence (SEQ ID NO:102). The JH6b germline sequence is disclosed as SEQ ID NO:110. [Figure 13] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 10A5 (SEQ ID NO:3) with the human germline VH1-3 amino acid sequence (SEQ ID NO:103). The JH4b germline sequence is disclosed as SEQ ID NO:111. [Figure 14] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 5F8 (SEQ ID NO:4) with the human germline VH1-69 amino acid sequence (SEQ ID NO:102). The JH4b germline sequence is disclosed as SEQ ID NO:111. [Figure 15]1 shows an alignment of the amino acid sequence of the heavy chain variable region of 10H10 (SEQ ID NO:5) with the human germline VH3-9 amino acid sequence (SEQ ID NO:104). The JH4b germline sequence is disclosed as SEQ ID NO:111. [Figure 16] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 1B12 (SEQ ID NO:6) with the human germline VH1-69 amino acid sequence (SEQ ID NO:102). The JH6b germline sequence is disclosed as SEQ ID NO:110. [Figure 17] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 7H1 (SEQ ID NO:7) with the human germline VH1-69 amino acid sequence (SEQ ID NO:102). The JH6b germline sequence is disclosed as SEQ ID NO:110. [Figure 18] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 11E6 (SEQ ID NO:8) with the human germline VH1-69 amino acid sequence (SEQ ID NO:102). The JH6c germline sequence is disclosed as SEQ ID NO:112. [Figure 19] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 12B7 (SEQ ID NO:9) with the human germline VH1-69 amino acid sequence (SEQ ID NO:102). The JH6b germline sequence is disclosed as SEQ ID NO:110. [Figure 20] 1 shows an alignment of the amino acid sequence of the heavy chain variable region of 13G4 (SEQ ID NO:10) with the human germline VH3-9 amino acid sequence (SEQ ID NO:104). The JH4b germline sequence is disclosed as SEQ ID NO:113. [Figure 21] 1 shows an alignment of the amino acid sequence of the light chain variable region of 3G10 (SEQ ID NO:11) with the human germline VkL6 amino acid sequence (SEQ ID NO:105). The JK1 germline sequence is disclosed as SEQ ID NO:114. [Figure 22]1 shows an alignment of the amino acid sequence of the light chain variable region of 12A4 (SEQ ID NO:12) with the human germline VkL6 amino acid sequence (SEQ ID NO:105). The JK1 germline sequence is disclosed as SEQ ID NO:115. [Figure 23] 1 shows an alignment of the amino acid sequence of the light chain variable region of 10A5 (SEQ ID NO:13) with the human germline VkL15 amino acid sequence (SEQ ID NO:106). The JK2 germline sequence is disclosed as SEQ ID NO:116. [Figure 24] 1 shows an alignment of the amino acid sequence of the light chain variable region of 5F8 (SEQ ID NO:14) with the human germline VkA27 amino acid sequence (SEQ ID NO:107). The JK1 germline sequence is disclosed as SEQ ID NO:114. [Figure 25] 1 shows an alignment of the amino acid sequence of the light chain variable region of 10H10 (SEQ ID NO:15) with the human germline VkL15 amino acid sequence (SEQ ID NO:106). The JK2 germline sequence is disclosed as SEQ ID NO:116. [Figure 26] 1 shows an alignment of the amino acid sequence of the light chain variable region of 1B12 (SEQ ID NO:16) with the human germline VkL6 amino acid sequence (SEQ ID NO:105). The JK1 germline sequence is disclosed as SEQ ID NO:115. [Figure 27] 1 shows an alignment of the amino acid sequence of the light chain variable region of 7H1 (SEQ ID NO:17) with the human germline VkL6 amino acid sequence (SEQ ID NO:105). The JK1 germline sequence is disclosed as SEQ ID NO:115. [Figure 28] 1 shows an alignment of the amino acid sequence of the light chain variable region of 11E6 (SEQ ID NO:18) with the human germline VkA27 amino acid sequence (SEQ ID NO:107). The JK4 germline sequence is disclosed as SEQ ID NO:117. [Figure 29]1 shows an alignment of the amino acid sequence of the light chain variable region of 11E6a (SEQ ID NO:109) with the human germline VkA27 amino acid sequence (SEQ ID NO:107). The JK4 germline sequence is disclosed as SEQ ID NO:118. [Figure 30] 1 shows an alignment of the amino acid sequence of the light chain variable region of 12B7 (SEQ ID NO:19) with the human germline VkL6 amino acid sequence (SEQ ID NO:105). The JK5 germline sequence is disclosed as SEQ ID NO:119. [Figure 31] 1 shows an alignment of the amino acid sequence of the light chain variable region of 13G4 (SEQ ID NO:20) with the human germline VkL18 amino acid sequence (SEQ ID NO:108). The JK3 germline sequence is disclosed as SEQ ID NO:120. [Figure 32] Figure 1 shows the results of a flow cytometry experiment demonstrating that human monoclonal antibodies 3G10, 10A5, and 12A4 against human PD-L1 bind to the cell surface of CHO cells transfected with full-length human PD-L1. (A) Flow cytometry plot for 3G10. (B) Flow cytometry plot for 10A5. (C) Flow cytometry plot for 12A4. [Figure 33] Figure 1 shows the results of flow cytometry experiments demonstrating that the human monoclonal antibodies 3G10, 10A5, and 12A4 against human PD-L1 bind to the cell surface of CHO cells transfected with full-length human PD-L1 in a concentration-dependent manner. [Figure 34] Figure 1 shows the results of an ELISA experiment demonstrating that the human monoclonal antibodies 3G10, 10A5, and 12A4 against human PD-L1 bind to a PD-L1-Fc fusion protein. [Figure 35] 1 shows the results of an experiment demonstrating HuMab titration on stimulated human CD4+ T cells. [Figure 36] 1 shows the results of an experiment demonstrating HuMab titration on stimulated cynomolgus monkey PBMCs. [Figure 37]Figure 1 shows the results of a flow cytometry experiment demonstrating that human monoclonal antibodies 3G10, 10A5, and 12A4 against human PD-L1 bind to PD-L1 on the cell surface of activated T cells. (A) Flow cytometry plot for 3G10. (B) Flow cytometry plot for 10A5. (C) Flow cytometry plot for 12A4. [Figure 38] Binding of HuMabs to ES-2 cells is demonstrated. [Figure 39A] Figure 39A shows the results of experiments demonstrating that human monoclonal antibodies against human PD-L1 promote T cell proliferation, IFN-γ secretion, and IL-2 secretion in a mixed lymphocyte reaction assay. Figure 39B is a bar graph showing concentration-dependent T cell proliferation with HuMAb 10A5. [Figure 39B] Figure 39B shows the results of experiments demonstrating that human monoclonal antibodies against human PD-L1 promote T cell proliferation, IFN-γ secretion, and IL-2 secretion in a mixed lymphocyte reaction assay. Figure 39B is a bar graph showing concentration-dependent IFN-γ secretion with HuMAb 10A5. [Figure 39C] Figure 39C shows the results of experiments demonstrating that human monoclonal antibodies against human PD-L1 promote T cell proliferation, IFN-γ secretion, and IL-2 secretion in a mixed lymphocyte reaction assay. Figure 39D is a bar graph showing IFN-γ secretion with HuMAb 3G10 and HuMAb 12A4. [Figure 39D] Figure 39D shows the results of experiments demonstrating that human monoclonal antibodies against human PD-L1 promote T cell proliferation, IFN-γ secretion, and IL-2 secretion in a mixed lymphocyte reaction assay. Figure 39C is a bar graph showing the concentration-dependent IL-2 secretion with HuMAb 10A5. [Figure 40] Figure 1 shows the effect of human anti-PD-L1 antibodies on proliferation and IFN-γ secretion in an MLR with allogeneic dendritic cells and T cells (CD4+ effector T cells). [Figure 41A]Figure 41A shows the results of experiments demonstrating that a human monoclonal antibody against human PD-L1 promotes T cell proliferation and IFN-γ secretion in an MLR containing regulatory T cells. Figure 41A is a bar graph showing concentration-dependent T cell proliferation with HuMAb 10A5. [Figure 41B] Figure 41B shows the results of experiments demonstrating that a human monoclonal antibody against human PD-L1 promotes T cell proliferation and IFN-γ secretion in an MLR containing regulatory T cells. Figure 41B is a bar graph showing concentration-dependent IFN-γ secretion with HuMAb 10A5. [Figure 42] Demonstrates the effect of anti-PD-L1 antibodies on cell proliferation in a mixed lymphocyte reaction in the presence of regulatory T cells. [Figure 43] Demonstrates the effect of anti-PD-L1 antibodies on cytokine production in a mixed lymphocyte reaction in the presence of regulatory T cells. [Figure 44] Demonstrates the results of anti-PD-L1 antibodies on IFN-γ secretion in human PBMCs stimulated with CMV lysate. [Figure 45] Figure 1 shows the results of flow cytometry experiments demonstrating that human monoclonal antibodies against human PD-L1 block the binding of PD-L1 to transfected CHO cells expressing PD-1. [Figure 46] Figure 1 shows that anti-PD-L1 antibodies block PD-1 binding to IFNγ-treated ES-2 cells. [Figure 47] Figure 1 shows the effect of anti-PD-L1 antibodies on tumor growth in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0055] Detailed Description In one aspect, the present disclosure relates to isolated monoclonal antibodies, particularly human monoclonal antibodies, that specifically bind to PD-L1. In particular embodiments, the antibodies of the invention exhibit one or more desirable functional properties, such as high affinity binding to PD-L1, the ability to increase T cell proliferation, IFN-γ and / or IL-2 secretion in a mixed lymphocyte reaction, the ability to inhibit PD-L1 binding to the PD-1 receptor, the ability to stimulate an antibody response, and / or the ability to reverse the suppressive function of regulatory T cells. Additionally or alternatively, the antibodies of the invention are derived from particular heavy chain and light chain germline sequences and / or comprise particular structural features, such as CDR regions comprising particular amino acid sequences.

[0056] The disclosure provides, for example, isolated antibodies, methods of making such antibodies, immunoconjugates and bispecific molecules comprising such antibodies, and pharmaceutical compositions comprising the antibodies, immunoconjugates, or bispecific molecules of the invention.

[0057] In another aspect, the disclosure relates to methods of inhibiting the growth of tumor cells in a subject using anti-PD-L1 antibodies. The invention also relates to methods of using antibodies to alter immune responses and to treat diseases such as cancer or infectious diseases, or to stimulate a protective autoimmune response, or to stimulate an antigen-specific immune response (e.g., by co-administering anti-PD-L1 with an antigen of interest).

[0058] In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are provided throughout the detailed description.

[0059] The term "immune response" refers to the action of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the foregoing cells or the liver that selectively damage, destroy, or remove from the human body invading pathogens, pathogen-infected cells or tissues, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0060] "Signal transduction pathway" refers to the biochemical relationship between various signal transduction molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. As used herein, the phrase "cell surface receptor" includes, for example, molecules and complexes of molecules that can receive a signal and transmit such a signal across the plasma membrane of a cell. An example of a "cell surface receptor" of the present invention is the PD-L1 receptor.

[0061] The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (referred to herein as V H The heavy chain constant region is composed of three domains: C H1 , C H2 , and C H3 Each light chain is composed of a light chain variable region (referred to herein as V L The light chain constant region consists of one domain, C L It consists of: V H Area and V L The region can be further subdivided into regions of hypervariability, called complementarity-determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). H and VL is composed 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. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0062] The term "antigen-binding portion" of an antibody (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., PD-L1). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, i.e., a V L Domain, V H Domain, C L domain, and C H1 (ii) a monovalent fragment consisting of the V(ab')2 fragment, i.e., a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a V(ab')2 fragment, i.e., a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; H Domain and C H1 (iv) the V of the short arm of the antibody; L Domains and V H Fv fragment consisting of domains; (v) V H (v) isolated complementarity-determining regions (CDRs); and (vi) isolated complementarity-determining regions (CDRs). In addition, two domains of the Fv fragment, V, and VD, are included. L and V H are encoded by separate genes, but V L Area and V HThese regions can be linked, using recombinant methods, by a synthetic linker that allows them to be produced as a single protein chain in which they pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0063] An "isolated antibody," as used herein, is intended to mean an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-L1). However, an isolated antibody that specifically binds PD-L1 may have cross-reactivity to other antigens, such as PD-L1 molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0064] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0065] The term "human antibody," as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include 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.

[0066] The term "human monoclonal antibody" refers to antibodies displaying a single binding specificity having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy chain and light chain transgenes, fused to an immortalized cell.

[0067] The term "recombinant human antibody," as used herein, includes all human antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes, or hybridomas prepared therefrom (described in more detail below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., from transfectomas; (c) antibodies isolated from recombinant human antibody combinatorial libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means, including splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), thus modifying the V and CDR regions of the recombinant antibody. H Area and V L The amino acid sequence of the region is similar to that of the human germline V H Array and V L These are sequences that are derived from and related to sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0068] As used herein, "isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by heavy chain constant region genes.

[0069] The phrases "antibody that recognizes an antigen" and "antibody specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen."

[0070] The term "human antibody derivative" refers to any modified form of a human antibody, for example, a conjugate of the antibody with another agent or antibody.

[0071] The term "humanized antibody" is intended to mean an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications may be made within the human framework sequences.

[0072] The term "chimeric antibody" is intended to mean an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0073] As used herein, an antibody that "specifically binds to human PD-L1" refers to an antibody that specifically binds to human PD-L1 in an amount of 1×10 -7 M or less, preferably 5×10 -8 M or less, more preferably 1×10 -8 M or less, preferably 5×10 -9 M or less, and even more preferably 1×10 -8 M~1×10 -10 K between or below M D "antibody" is intended to mean an antibody that binds to human PD-L1 at

[0074] "K assoc " or "K a " as used herein is intended to mean the binding rate of a particular antibody-antigen interaction, whereas "K dis " or "K d The term "K", as used herein, is intended to mean the off-rate of a particular antibody-antigen interaction. D " as used herein means K a K d The ratio of (i.e., K d / K a ) and expressed as a molar concentration (M). D The K value can be determined using methods well established in the art. DA preferred method for determining is by using surface plasmon resonance, preferably using a biosensor system such as a Biacore® system.

[0075] As used herein, the term "high affinity" for an IgG antibody refers to a K D is 10 -8 M or less, more preferably 10 -9 M or less, and even more preferably 10 -10 However, "high affinity" binding may be different for other antibody isotypes. For example, "high affinity" binding for an IgM isotype refers to an antibody with a K D is 10 -7 M or less, more preferably 10 -8 M or less, even more preferably 10 -9 This refers to antibodies that are M or less.

[0076] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0077] Various aspects of the disclosure are described in more detail in the following subsections.

[0078] Anti-PD-L1 antibody The antibodies of the present invention are characterized by particular functional characteristics or properties of the antibody. For example, these antibodies specifically bind to human PD-L1. Preferably, the antibodies of the present invention bind with high affinity, e.g., 1×10 -7 M or lower K D The anti-PD-L1 antibodies of the invention preferably exhibit one or more of the following characteristics: (a) 1×10 -7 M or lower K Dbinds to human PD-L1; (b) increase T-cell proliferation in mixed lymphocyte reaction (MLR) assays; (c) Increases interferon-γ production in the MLR assay; (d) increases IL-2 secretion in the MLR assay; (e) stimulating an antibody response; and / or (f) Reversing the effects of regulatory T cells on T cell effector cells and / or dendritic cells.

[0079] Preferably, the antibody is 5×10 -8 M or lower K D binds to human PD-L1 at 1 × 10 -8 M or lower K D binds to human PD-L1 at 5 × 10 -9 M or lower K D binds to human PD-L1 at 4 × 10 -9 M or lower K D binds to human PD-L1 at 2 × 10 -9 M or lower K D bind to human PD-L1 at 1 × 10 -9 M~1×10 -10 K between or below M D It binds to human PD-L1.

[0080] Standard assays for assessing the binding ability of an antibody to PD-L1 are known in the art, including, for example, ELISA, Western blot, and RIA. Suitable assays are described in detail in the Examples. The binding kinetics (e.g., binding affinity) of an antibody can also be assessed by standard assays known in the art, such as Biacore® analysis.

[0081] Monoclonal antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 Preferred antibodies of the invention are human monoclonal antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, which were isolated and structurally characterized as described in Examples 1 and 2. The V H The amino acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively. L The amino acid sequences are shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20, respectively.

[0082] Each of these antibodies binds to PD-L1, allowing V H Array and V L Sequences can be "mixed and matched" to create other anti-PD-L1 binding molecules of the invention. PD-L1 binding of such "mixed and matched" antibodies can be tested using the binding assays (e.g., ELISAs) described above and in the Examples. Preferably, V H Chain and V L When strands are mixed and matched, individual V H / V L V comes from a pair H The sequence is structurally similar to V H Similarly, each V H / V L V comes from a pair L The sequence is structurally similar to V LIt is replaced by an array.

[0083] Accordingly, in one aspect, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, preferably human PD-L1, comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

[0084] Preferred heavy and light chain combinations include the following: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:11; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:12; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:13; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:4; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:14; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:5; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:15; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:6; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:16; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:7; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:17; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:8; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:18; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:9; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:19; or (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:10; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:20.

[0085] In another aspect, the present invention provides antibodies comprising the heavy and light chain CDR1, CDR2, and CDR3 of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4, or a combination thereof. H The amino acid sequences of CDR1 are shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively. HThe amino acid sequences of CDR2 are shown in SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, respectively. H The amino acid sequences of CDR3 are shown in SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively. k The amino acid sequences of CDR1 are shown in SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, respectively. k The amino acid sequences of CDR2 are shown in SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, respectively. kThe amino acid sequences of CDR3 are shown in SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, respectively. The CDR regions are delineated using the Kabat system (Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242).

[0086] Each of these antibodies can bind to PD-L1, and the antigen-binding specificity is primarily provided by the CDR1, CDR2, and CDR3 regions. H and the CDR1, CDR2, and CDR3 sequences of V k The CDR1, CDR2, and CDR3 sequences of the V H CDR1, CDR2, and CDR3 of V k Other anti-PD-L1 binding molecules of the invention can be generated based on the CDR1, CDR2, and CDR3 of V. The PD-L1 binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples (e.g., ELISA, Biacore analysis). Preferably, V H When CDR sequences are mixed and matched, individual V H The CDR1, CDR2, and / or CDR3 sequences derived from the V sequence are replaced with structurally similar CDR sequences. k When CDR sequences are mixed and matched, individual V kThe CDR1, CDR2, and / or CDR3 sequences derived from the V sequence are preferably replaced with structurally similar CDR sequences. H CDR region sequences and / or V L Novel V sequences were created by replacing the CDR region sequences with structurally similar sequences derived from the CDR sequences disclosed herein for monoclonal antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. H Sequence and / or V L It will be readily apparent to one skilled in the art that sequences can be made.

[0087] Accordingly, in another aspect, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, preferably human PD-L1, comprising: (a) a CDR1 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; (b) a CDR2 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; (c) a CDR3 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; (d) a CDR1 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60; (e) a CDR2 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70; and (f) a CDR3 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80.

[0088] In a preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:21; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:31; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:41; (d) a light chain variable region CDR1 comprising SEQ ID NO:51; (e) a light chain variable region CDR2 comprising SEQ ID NO:61; and (f) Light chain variable region CDR3 comprising SEQ ID NO:71.

[0089] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:22; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:32; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:42; (d) a light chain variable region CDR1 comprising SEQ ID NO:52; (e) a light chain variable region CDR2 comprising SEQ ID NO:62; and (f) Light chain variable region CDR3 comprising SEQ ID NO:72.

[0090] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:23; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:33; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:43; (d) a light chain variable region CDR1 comprising SEQ ID NO:53; (e) a light chain variable region CDR2 comprising SEQ ID NO:63; and (f) Light chain variable region CDR3 comprising SEQ ID NO:73.

[0091] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:24; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:34; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:44; (d) a light chain variable region CDR1 comprising SEQ ID NO:54; (e) a light chain variable region CDR2 comprising SEQ ID NO:64; and (f) Light chain variable region CDR3 comprising SEQ ID NO:74.

[0092] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:25; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:35; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:45; (d) a light chain variable region CDR1 comprising SEQ ID NO:55; (e) a light chain variable region CDR2 comprising SEQ ID NO:65; and (f) Light chain variable region CDR3 comprising SEQ ID NO:75.

[0093] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:26; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:36; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:46; (d) a light chain variable region CDR1 comprising SEQ ID NO:56; (e) a light chain variable region CDR2 comprising SEQ ID NO:66; and (f) Light chain variable region CDR3 comprising SEQ ID NO:76.

[0094] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:27; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:37; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:47; (d) a light chain variable region CDR1 comprising SEQ ID NO:57; (e) a light chain variable region CDR2 comprising SEQ ID NO:67; and (f) Light chain variable region CDR3 comprising SEQ ID NO:77.

[0095] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:28; (b) heavy chain variable region CDR2 comprising SEQ ID NO:38; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:48; (d) a light chain variable region CDR1 comprising SEQ ID NO:58; (e) a light chain variable region CDR2 comprising SEQ ID NO:68; and (f) Light chain variable region CDR3 comprising SEQ ID NO:78.

[0096] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:29; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:39; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:49; (d) a light chain variable region CDR1 comprising SEQ ID NO:59; (e) a light chain variable region CDR2 comprising SEQ ID NO:69; and (f) Light chain variable region CDR3 comprising SEQ ID NO:79.

[0097] In another preferred embodiment, the antibody comprises: (a) heavy chain variable region CDR1 comprising SEQ ID NO:30; (b) heavy chain variable region CDR2 comprising SEQ ID NO:40; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:50; (d) a light chain variable region CDR1 comprising SEQ ID NO:60; (e) a light chain variable region CDR2 comprising SEQ ID NO:70; and (f) Light chain variable region CDR3 comprising SEQ ID NO:80.

[0098] It is well known in the art that the CDR3 domain can solely determine the binding specificity of an antibody to its cognate antigen, independent of the CDR1 and / or CDR2 domains, and that multiple antibodies with the same binding specificity can be predictably generated based on a common CDR3 sequence. For example, Klimka et al., British J. of Cancer 83(2):252-260(2000) (describes the generation of a humanized anti-CD30 antibody using only the heavy chain variable domain CDR3 of the murine anti-CD30 antibody Ki-4); Beiboer et al., J. Mol. Biol. 296:833-849(2000) (describes the generation of an epithelial glycoprotein-2 (EGP-2) recombinant antibody using only the heavy chain CDR3 sequence of the murine MOC-31 anti-EGP-2 parent antibody); Rader et al., Proc. Natl. Acad. Sci. USA 95:8910-8915(1998) (describes the generation of a murine anti-integrin α v Humanized anti-integrin α using variable CDR3 domains of the heavy and light chains of the β3 antibody LM609 vdescribes a panel of β3 antibodies, each antibody member containing a unique sequence outside the CDR3 domain and capable of binding to the same epitope as the murine parent antibody with affinity as high as or higher than that of the murine parent antibody; Barbas et al., J. Am. Chem. Soc. 116:2161-2162(1994) (disclosing that the CDR3 domain contributes most significantly to antigen binding); Barbas et al., Proc. Natl. Acad. Sci. USA 92:2529-2533(1995) (describes grafting the heavy chain CDR3 sequences of three Fabs (SI-1, SI-40, and SI-32) against human placental DNA onto the heavy chain of an anti-tetanus toxoid Fab, thereby replacing the existing heavy chain CDR3 and demonstrating that the CDR3 domain alone conferred binding specificity); and Ditzel et al., J. Immunol. 157:739-749 (1996) (describing grafting studies in which introducing only the heavy chain CDR3 of the polyspecific parental Fab LNA3 into the heavy chain of the monospecific IgG tetanus toxoid-binding Fab p313 antibody was sufficient to retain the binding specificity of the parental Fab). Each of these references is incorporated herein by reference in its entirety.

[0099] Thus, in certain aspects, the invention provides monoclonal antibodies that comprise one or more heavy chain and / or light chain CDR3 domains derived from a non-human antibody, such as a mouse or rat antibody, and that are capable of specifically binding to PD-L1. In some embodiments, such antibodies of the invention that comprise one or more heavy chain and / or light chain CDR3 domains derived from a non-human antibody (a) can compete for binding, (b) retain functional characteristics, (c) bind to the same epitope, and / or (d) have similar binding affinity as the corresponding non-human parent antibody.

[0100] In other aspects, the invention provides monoclonal antibodies that comprise one or more heavy chain CDR3 domains and / or light chain CDR3 domains derived from a first human antibody, such as a human antibody obtained from a non-human animal, where the first human antibody is capable of specific binding to PD-L1, and the CDR3 domains from the first human antibody replace CDR3 domains in a human antibody that lacks binding specificity for PD-L1 to generate a second human antibody that is capable of specific binding to PD-L1. In some embodiments, antibodies of the present disclosure that comprise one or more heavy chain CDR3 domains and / or light chain CDR3 domains derived from a first human antibody (a) can compete for binding, (b) retain functional characteristics, (c) bind to the same epitope, and / or (d) have a similar binding affinity as the corresponding first parent human antibody.

[0101] Antibodies with specific germline sequences In certain embodiments, antibodies of the invention comprise a heavy chain variable region derived from a particular germline heavy chain immunoglobulin gene and / or a light chain variable region derived from a particular germline light chain immunoglobulin gene.

[0102] For example, in a preferred embodiment, the present invention provides a method for the production of human V H 1-18 gene product or human V H In another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-18 gene. H 1-69 gene product or human V H In another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-69 gene. H 1-3 gene product or human V HIn another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the human V gene. H 3-9 gene product or human V H In yet another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a heavy chain variable region derived from the human V3-9 gene. K It is the product of the L6 gene or human V K In yet another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the L6 gene. K L15 gene product or human V K In yet another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the L15 gene. K A27 gene product or human V K In yet another preferred embodiment, the present invention provides an isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the A27 gene. K It is the product of the L18 gene or human V K In yet another preferred embodiment, the present invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, that specifically binds to PD-L1, comprising a light chain variable region derived from the L18 gene. (a) Human V (encoding the amino acid sequences shown in SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104, respectively) H 1-18 gene, human V H 1-69 gene, human V H 1-3 genes, or human V H3-9 gene product or a heavy chain variable region derived therefrom, (b) Human V (encoding the amino acid sequences shown in SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, and SEQ ID NO:108, respectively) K L6 gene, human V K L15 gene, human V K A27 gene, or human V K comprising a light chain variable region that is the product of or derived from an L18 gene; and (c) specifically binds to PD-L1, preferably human PD-L1.

[0103] V H 1-18 and V K L6 V H and V K An example of an antibody having each of the following is 3G10. H 1-69 and V K L6 V H and V K Examples of antibodies with the VH and VH sequences are 12A4, 1B12, 7H1, and 12B7. H 1-3 and V K L15 V H and V K An example of an antibody having each of the following is 10A5. H 1-69 and V K A27 V H and V K Examples of antibodies with the V H 3-9 and V K L15 V H and V K An example of an antibody having each of the following amino acids is 10H10. H 1-3 and V K L15 V H and V K An example of an antibody having each of the following is 10A5. H 3-9 and V K L18 V H and V KAn example of an antibody having each of the following is 13G4.

[0104] As used herein, when the antibody variable region is obtained from a system that uses human germline immunoglobulin genes, the human antibody contains a heavy chain variable region or a light chain variable region that is the "product" or "derived" from a particular germline sequence. Such systems involve immunizing transgenic mice carrying human immunoglobulin genes with the antigen of interest, or screening a human immunoglobulin gene library displayed on phage with the antigen of interest. A human antibody that is the "product" or "derived" from a human germline immunoglobulin sequence can be identified by comparing the amino acid sequence of the human antibody with that of a human germline immunoglobulin and selecting the human germline immunoglobulin sequence that is most similar in sequence to the human antibody (i.e., has the highest percent identity). A human antibody that is the "product" or "derived" from a particular human germline immunoglobulin sequence may contain amino acid differences compared to the germline sequence, for example, due to natural somatic mutations or the intentional introduction of site-specific mutations. However, the selected human antibody will generally be at least 90% identical in amino acid sequence to the amino acid sequence encoded by a human germline immunoglobulin gene and contain amino acids that identify the human antibody as of human origin when compared to the germline immunoglobulin amino acid sequence of another species (e.g., a murine germline sequence). In certain cases, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical in amino acid sequence to the amino acid sequence encoded by a germline immunoglobulin gene. In certain embodiments, a human antibody derived from a particular human germline sequence will exhibit no more than 10 amino acid differences compared to the amino acid sequence encoded by a human germline immunoglobulin gene. In other specific embodiments, a human antibody may exhibit no more than 5, or even no more than 4, 3, 2, or 1 amino acid differences compared to the amino acid sequence encoded by a germline immunoglobulin gene.

[0105] Homologous antibodies In yet another embodiment, the antibodies of the invention comprise heavy chain and light chain variable regions that comprise amino acid sequences that are homologous to the amino acid sequences of the preferred antibodies described herein that retain the desired functional properties of the anti-PD-L1 antibodies of the invention.

[0106] For example, the present invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region and a light chain variable region, (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% homologous to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; (b) the light chain variable region comprises an amino acid sequence at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; (c) Antibody: 1 × 10 -7 M or lower K D binds to human PD-L1; (d) the antibody increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) the antibody increases interferon-γ production in an MLR assay; (f) the antibody increases IL-2 secretion in an MLR assay; (g) the antibody stimulates an antibody response; and (h) Reversing the effects of regulatory T cells on T cell effector cells and / or dendritic cells.

[0107] In other embodiments, V H Amino acid sequence and / or V L The amino acid sequence may be 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the aforementioned sequences. H Area and V L V with high (i.e., 80% or more) homology to the region H Area and V L Antibodies having the regions can be obtained by mutating nucleic acid molecules encoding SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30 (e.g., by site-directed mutagenesis or PCR-based mutagenesis), and then testing the encoded modified antibodies for retained function (i.e., the functions set forth in (c) through (h) above) using the functional assays described herein.

[0108] As used herein, the percentage of homology between two amino acid sequences is equal to the percentage of identity between the two sequences. The percentage of identity between two sequences is a function of the number of identical positions shared by these sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap (i.e., percentage of homology = number of identical positions / total number of positions x 100). Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0109] The percent identity between two amino acid sequences can be determined 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 residue weight table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can also be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) incorporated into the GAP program of the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6.

[0110] In certain cases, the protein sequences of the present disclosure can also be used as a "query sequence" to perform a search against public databases, for example, to identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed using the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0111] Conservatively modified antibodies In certain embodiments, antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprise a designated amino acid sequence based on a preferred antibody described herein (e.g., 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4) or a conservative variant thereof, and wherein these antibodies retain the desired functional properties of the anti-PD-L1 antibodies of the invention. Accordingly, the invention provides an isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences, and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences; (a) the CDR3 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, and conservative variants thereof; (b) the CDR3 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, and conservative variants thereof; (c) Antibody is 1 × 10 -7 M or lower K D binds to human PD-L1; (d) the antibody increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (e) the antibody increases interferon-γ production in an MLR assay; (f) the antibody increases IL-2 secretion in an MLR assay; (g) the antibody stimulates an antibody response; and (h) reversing the effects of regulatory T cells on T cell effector cells and / or dendritic cells; An isolated monoclonal antibody or antigen-binding portion thereof is provided.

[0112] In a preferred embodiment, the CDR2 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and conservative variants thereof, and the CDR2 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, and conservative variants thereof. In another preferred embodiment, the CDR1 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, and conservative variants thereof, and the CDR1 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, and conservative variants thereof.

[0113] As used herein, the term "conservative sequence modifications" is intended to mean amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-based mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acids with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention can be substituted with other amino acid residues from the same side chain family, and the modified antibody can be tested for retained function (i.e., the functions set forth in (c) through (h) above) using the functional assays described herein.

[0114] Antibodies that bind to the same epitope as the anti-PD-L1 antibodies of the present invention In another embodiment, the invention provides antibodies that bind to the same epitope on human PD-L1 as any of the PD-L1 monoclonal antibodies of the invention (i.e., antibodies that have the ability to cross-compete with any of the monoclonal antibodies of the invention for binding to PD-L1). In a preferred embodiment, the reference antibody for cross-competition studies is monoclonal antibody 3G10 (V as shown in SEQ ID NO:1 and SEQ ID NO:11, respectively). H Array and V L ), or monoclonal antibody 12A4 (V shown in SEQ ID NO:2 and SEQ ID NO:12, respectively). H Array and V L ), or monoclonal antibody 10A5 (V shown in SEQ ID NO:3 and SEQ ID NO:13, respectively). H Array and V L ), or monoclonal antibody 10A5 (V shown in SEQ ID NO:3 and SEQ ID NO:13, respectively). H Array and V L ), or monoclonal antibody 5F8 (V shown in SEQ ID NO:4 and SEQ ID NO:14, respectively). H Array and V L ), or monoclonal antibody 10H10 (V shown in SEQ ID NO:5 and SEQ ID NO:15, respectively). H Array and V L ), or monoclonal antibody 1B12 (V shown in SEQ ID NO:6 and SEQ ID NO:16, respectively). H Array and V L ), or monoclonal antibody 7H1 (V shown in SEQ ID NO:7 and SEQ ID NO:17, respectively). H Array and V L sequence), or monoclonal antibody 11E6 (V shown in SEQ ID NO:8 and SEQ ID NO:18, respectively). H Array and V L), or monoclonal antibody 12B7 (V shown in SEQ ID NO:9 and SEQ ID NO:19, respectively). H Array and V L ), or monoclonal antibody 13G4 (V shown in SEQ ID NO:10 and SEQ ID NO:20, respectively). H Array and V L Such cross-competing antibodies can be identified based on their ability to cross-compete with 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4 in standard PD-L1 binding assays. For example, BIAcore analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with the antibodies of the invention. If a test antibody is able to inhibit the binding of, for example, 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4 to human PD-L1, it demonstrates that the test antibody can compete with 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4 for binding to human PD-L1, and therefore binds to the same epitope on human PD-L1 as 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4. In a preferred embodiment, the antibody that binds to the same epitope on human PD-L1 as 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4 is a human monoclonal antibody. Such human monoclonal antibodies can be prepared and isolated as described in the Examples.

[0115] Engineered and modified antibodies The antibodies of the present invention may further comprise one or more V H Sequence and / or V LAn antibody having the sequence can also be prepared by using it as a starting material to engineer modified antibodies, which may have altered properties from the starting antibody. H and / or V L ), for example, by modifying one or more residues in one or more CDR regions and / or one or more framework regions. Additionally or alternatively, antibodies can be engineered by modifying residues in the constant region(s), for example, to alter the effector functions of the antibody.

[0116] One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with target antigens primarily through amino acid residues located in the six complementarity-determining regions (CDRs) of the heavy and light chains. For this reason, the amino acid sequences within the CDRs are more diverse between individual antibodies than the sequences outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular natural antibody by constructing expression vectors containing CDR sequences from that particular natural antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann, L. et al. (1998) Nature 332:323-327; Jones, P. et al. (1986) Nature 321:522-525; Queen, C. et al. (1989) Proc. Natl. Acad. Sci. USA 86:10029-10033; U.S. Pat. No. 5,225,539 to Winter and U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.).

[0117] Accordingly, another aspect of the present invention is an isolated monoclonal antibody or antigen-binding portion thereof, comprising: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; and SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, respectively. a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, and a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, and a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ IDThe present invention relates to an isolated monoclonal antibody or antigen-binding portion thereof, comprising a light chain variable region comprising CDR1, CDR2, and CDR3 sequences comprising amino acid sequences selected from the group consisting of NO:80, NO:10, NO:11, NO:12, NO:13, NO:14, NO:15, NO:16, NO:17, NO:18, NO:19, NO:20, NO:21, NO:22, NO:23, NO:24, NO:25, NO:26, NO:27, NO:28, NO:29 ... H CDR sequences and V L It contains CDR sequences but may contain framework sequences that differ from these antibodies.

[0118] Such framework sequences can be obtained from public DNA databases or published references that contain germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes are available in the "VBase" human germline sequence database (available on the Internet at www.mrc-cpe.cam.ac.uk / vbase), as well as in Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, IM, et al. (1992) "The Repertoire of Human Germline V H Sequences Reveals about Fifty Groups of V H Segments with Different Hypervariable Loops" J. Mol. Biol. 227:776-798; and Cox, JPL et al. (1994) "A Directory of Human Germ-line V H"Segments Reveal a Strong Bias in Their Usage," Eur. J. Immunol. 24:827-836 (the contents of each of which are expressly incorporated herein by reference).

[0119] Antibody protein sequences are compared against compiled protein sequence databases using a sequence similarity search method known as Gapped BLAST (Altschul et al. (1997) Nucleic Acids Research 25:3389-3402), which is well known to those skilled in the art. BLAST is a heuristic algorithm in that a statistically significant alignment between an antibody sequence and a database sequence is likely to contain high-scoring segment pairs (HSPs) of aligned words. Segment pairs whose scores cannot be improved by extension or trimming are called hits. Briefly, nucleotide sequences from VBASE (vbase.mrc-cpe.cam.ac.uk / vbase1 / list2.php) are translated, and the regions between and including the FR1 to FR3 framework regions are retained. The average length of database sequences is 98 residues. Identical sequences with perfect matches over the entire length of the protein are excluded. A BLAST search of proteins using the blastp program with default standard parameters except for the low complexity region filter turned off and the BLOSUM62 substitution matrix selects the top five hits showing sequence matches. The nucleotide sequence is translated in all six frames, and frames without a stop codon in the matched segment of the database sequence are considered potential hits. This is further confirmed using the BLAST program tblastx. In this case, the antibody sequence is translated in all six frames and these translations are compared to the dynamically translated VBASE nucleotide sequence in all six frames.

[0120] A match is a perfect amino acid match between the antibody sequence and the protein database over the entire length of the sequence. A positive (match + substitution match) is not identical, but has amino acid substitutions guided by the BLOSUM62 substitution matrix. If the antibody sequence matches two of the database sequences with the same identity, the strongest positive hit is determined to be the matching sequence hit.

[0121] Preferred framework sequences for use in the antibodies of the invention are those that are structurally similar to the framework sequences used by the selected antibodies of the invention, e.g., the V H 1-18 framework sequence (SEQ ID NO: 101) and / or V H 1-69 framework sequence (SEQ ID NO: 102) and / or V H 1-3 framework sequence (SEQ ID NO: 103) and / or V H 3-9 framework sequence (SEQ ID NO: 104), and / or V K L6 framework sequence (SEQ ID NO:105) and / or V K L15 framework sequence (SEQ ID NO:106) and / or V K A27 framework sequence (SEQ ID NO:107) and / or V K It is similar to the L18 framework sequence (SEQ ID NO: 107). H and the CDR1, CDR2, and CDR3 sequences of V KThe CDR1, CDR2, and CDR3 sequences of the above can be grafted onto framework regions having the same sequences as those present in the germline immunoglobulin gene from which the framework sequences are derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations compared to the germline sequences. For example, in some cases, it has been found to be beneficial to mutate residues within the framework regions to maintain or enhance the antigen-binding ability of the antibody (see, e.g., U.S. Patent Nos. 5,530,101, 585,089, 5,693,762, and 6,180,370 by Queen et al.).

[0122] Another type of variable region modification is the V H and / or V K Amino acid residues within the CDR1, CDR2, and / or CDR3 regions of an antibody of interest are mutated to thereby improve one or more binding characteristics (e.g., affinity) of the antibody of interest. Mutations can be introduced using site-directed mutagenesis or PCR-based mutagenesis, and the effect on antibody binding or other functional properties of interest can be assessed in in vitro or in vivo assays described herein and provided in the Examples. Preferably, conservative modifications (as described above) are introduced. Mutations can be amino acid substitutions, additions, or deletions, but are preferably substitutions. Furthermore, typically, no more than one, two, three, four, or five residues within the CDR regions are altered.

[0123] Accordingly, in another aspect, the invention provides an isolated anti-PD-L1 monoclonal antibody, or an antigen-binding portion thereof, comprising a heavy chain variable region comprising: (a) an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; H CDR1 region; (b) an amino acid sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; H CDR2 region; (c) V comprising an amino acid sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50. H CDR3 region; (d) an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60; K CDR1 region; (e) an amino acid sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70; K the CDR2 region; and (f) V comprising an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, or an amino acid sequence having one, two, three, four, or five amino acid substitutions, deletions, or additions compared to SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80. K CDR3 region.

[0124] The engineered antibodies of the invention may include, for example, V H and / or V KThese framework modifications include those in which framework residues within the framework region are modified. Typically, such framework modifications are performed to reduce the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, antibodies that have undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody is derived. For example, as described below, several amino acids have been changed in the framework regions of anti-PD-L1 antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 that differ from the germline parent sequence. To return these framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence, for example, by site-directed mutagenesis or PCR-based mutagenesis. Germline V H V of 3G10 relative to the 1-18 parent sequence H The alignment of the regions is shown in Figure 11. Germline V H V of 12A4 relative to the 1-69 parent sequence H The alignment of the regions is shown in Figure 12. Germline V H V of 10A5 relative to the 1-3 parent sequence H The alignment of the regions is shown in Figure 13. Germline V H V of 5F8 relative to the 1-69 parent sequence H The alignment of the regions is shown in Figure 14. Germline V H V of 10H10 relative to the 3-9 parent sequence H The alignment of the regions is shown in Figure 15. Germline V H V of 1B12 relative to the 1-69 parent sequence H The alignment of the regions is shown in Figure 16. Germline V H V of 7H1 relative to the 1-69 parent sequence H The alignment of the regions is shown in Figure 17. Germline V H V of 11E6 relative to the 1-69 parent sequence H The alignment of the regions is shown in Figure 18. Germline V HV of 12B7 relative to the 1-69 parent sequence H The alignment of the regions is shown in Figure 19. Germline V H V of 13G4 relative to the 3-9 parent sequence H The alignment of the regions is shown in FIG.

[0125] For example, in the case of 3G10, V H The amino acid residue 79 (in FR3) of is valine, whereas the corresponding V H This residue in the 1-18 germline sequence is alanine. To return the framework region sequences to their germline configuration, somatic mutations can be "backmutated" to the germline sequence by, for example, site-directed mutagenesis or PCR-based mutagenesis (e.g., V of 3G10). H Residue 79 of (residue 13 of FR3) can be "backmutated" from valine to alanine).

[0126] As another example, for 12A4, V H The amino acid residue 24 (in FR1) of is threonine, whereas the corresponding V H This residue in the 1-69 germline sequence is alanine. To return the framework region sequences to their germline configuration, e.g., V of 12A4 H Residue 24 of the antibody can be "backmutated" from threonine to alanine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0127] As another example, for 12A4, V H The amino acid residue 27 (in FR1) of is aspartic acid, whereas the corresponding V H This residue in the 1-69 germline sequence is a glycine. To return the framework region sequences to their germline configuration, e.g., V of 12A4 H Residue 27 of the antibody can be "backmutated" from aspartic acid to glycine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0128] As another example, for 12A4, V HThe amino acid residue 95 (in FR3) of is phenylalanine, whereas the corresponding V H This residue in the 1-69 germline sequence is a tyrosine. To return the framework region sequences to their germline configuration, e.g., V of 12A4 H Residue 95 of (residue 29 of FR3) can be "backmutated" from phenylalanine to tyrosine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0129] As another example, in the case of 5F8, the amino acid residue at position 24 (in FR1) is a valine, whereas the corresponding V H This residue in the 1-69 germline sequence is alanine. To restore the framework region sequences to their germline configuration, e.g., V of 5F8 H Residue 24 of the antibody can be "backmutated" from valine to alanine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0130] As another example, in the case of 5F8, the amino acid residue at position 28 (in FR1) is isoleucine, whereas the corresponding V H This residue in the 1-69 germline sequence is threonine. To restore the framework region sequences to their germline configuration, e.g., V of 5F8 H Residue 28 of the antibody can be "backmutated" from isoleucine to threonine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0131] As another example, in the case of 10H10, the amino acid residue at position 24 (in FR1) is a valine, whereas the corresponding V H This residue in the 3-9 germline sequence is alanine. To return the framework region sequences to their germline configuration, e.g., V of 10H10 is H Residue 24 of the antibody can be "backmutated" from valine to alanine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0132] As another example, in the case of 10H10, one amino acid can be inserted after amino acid residue 97 (within FR3). This amino acid is valine. To return the framework region sequences to their germline configuration, for example, V in 10H10 can be inserted. H The inserted amino acid after residue 97 of can be "backmutated" to remove this valine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0133] As another example, in the case of 1B12, amino acid residue 24 (in FR1) is a threonine, whereas the corresponding V H This residue in the 1-69 germline sequence is alanine. To return the framework region sequences to their germline configuration, e.g., V of 1B12 H Residue 24 of the antibody can be "backmutated" from threonine to alanine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0134] As another example, in the case of 1B12, amino acid residue 27 (in FR1) is aspartic acid, whereas the corresponding V H This residue in the 1-69 germline sequence is a glycine. To return the framework region sequences to their germline configuration, e.g., V of 1B12 H Residue 27 of the antibody can be "backmutated" from aspartic acid to glycine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0135] As another example, in the case of 1B12, amino acid residue 95 (in FR3) is phenylalanine, while the corresponding V H This residue in the 1-69 germline sequence is a tyrosine. To restore the framework region sequences to their germline configuration, e.g., V of 1B12 H Residue 95 of (residue 29 of FR3) can be "backmutated" from phenylalanine to tyrosine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0136] As another example, in the case of 7H1, amino acid residue 24 (in FR1) is a threonine, whereas the corresponding V H This residue in the 1-69 germline sequence is alanine. To return the framework region sequences to their germline configuration, e.g., V of 7H1 H Residue 24 of the antibody can be "backmutated" from threonine to alanine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0137] As another example, in the case of 7H1, amino acid residue 77 (within FR3) is a threonine, whereas the corresponding V H This residue in the 1-69 germline sequence is serine. To return the framework region sequences to their germline configuration, e.g., V of 7H1 H Residue 72 of (residue 11 of FR3) can be "backmutated" from threonine to serine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0138] As another example, in the case of 11E6, amino acid residue 78 (in FR3) is alanine, whereas the corresponding V H This residue in the 1-69 germline sequence is threonine. To return the framework region sequences to their germline configuration, e.g., V of 11E6 H Residue 78 of (residue 12 of FR3) can be "backmutated" from alanine to threonine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0139] As another example, in the case of 12B7, amino acid residue 13 (in FR1) is glutamic acid, whereas the corresponding V H This residue in the 1-69 germline sequence is a lysine. To return the framework region sequences to their germline configuration, e.g., V of 12B7 H Residue 13 of the formula (I) can be "backmutated" from glutamic acid to lysine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0140] As another example, in the case of 12B7, amino acid residue 30 (within FR1) is an asparagine, while the corresponding V H This residue in the 1-69 germline sequence is serine. To restore the framework region sequences to their germline configuration, e.g., V of 12B7 H Residue 30 of the antibody can be "backmutated" from asparagine to serine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0141] As another example, in the case of 12B7, amino acid residue 77 (within FR3) is an asparagine, while the corresponding V H This residue in the 1-69 germline sequence is serine. To restore the framework region sequences to their germline configuration, e.g., V of 12B7 H Residue 377 (residue 11 of FR3) can be "backmutated" from asparagine to serine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0142] As another example, in the case of 12B7, amino acid residue 82 (within FR3) is aspartic acid, whereas the corresponding V H This residue in the 1-69 germline sequence is glutamic acid. To restore the framework region sequences to their germline configuration, e.g., V of 12B7 H Residue 82 of (residue 16 of FR3) can be "backmutated" from aspartic acid to glutamic acid. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0143] As another example, in the case of 13G4, the amino acid residue at position 27 (in FR1) is isoleucine, whereas the corresponding V H This residue in the 1-69 germline sequence is phenylalanine. To return the framework region sequences to their germline configuration, e.g., V of 12B7 HResidue 27 of the antibody can be "backmutated" from isoleucine to phenylalanine. Such "backmutated" antibodies are also intended to be encompassed by the present invention.

[0144] Another type of framework modification involves mutating one or more residues within the framework regions, or even within one or more CDR regions, to remove T-cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Patent Application Publication No. 20030153043 by Carr et al.

[0145] In addition to, or instead of, modifications made in the framework or CDR regions, antibodies of the invention can be engineered to contain modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. Furthermore, antibodies of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter their glycosylation, also to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. The numbers of residues in the Fc region are those of the EU index of Kabat.

[0146] In one embodiment, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light chain and heavy chain, or to increase or decrease the stability of the antibody.

[0147] In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the Fc hinge fragment, which is the interface region of the CH2-CH3 domain, so that the antibody has impaired Staphylococcus protein A (SpA) binding compared to the SpA binding of the native Fc hinge domain. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0148] In another embodiment, the antibody is modified to extend its biological half-life. Various approaches are possible. For example, as described in U.S. Patent No. 6,277,375 by Ward, one or more of the following mutations can be introduced: T252L, T254S, T256F. Alternatively, to extend biological half-life, the CH1 or CL region of the antibody can be modified to include a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patent Nos. 5,869,046 and 6,121,022 by Presta et al.

[0149] In yet another embodiment, the Fc region is modified by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be substituted with a different amino acid residue so that the antibody retains the antigen-binding ability of the parent antibody while altering its affinity for the effector ligand. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0150] In another example, one or more amino acids selected from amino acid residues 329, 331, and 322 can be substituted with a different amino acid residue such that the antibody's C1q binding is altered and / or complement-dependent cytotoxicity (CDC) is reduced or abolished. This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0151] In another example, one or more amino acid residues within amino acid positions 231 to 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in PCT Publication WO 94 / 29351 by Bodmer et al.

[0152] In yet another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcγ receptor by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283 , 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in PCT Publication WO 00 / 42072 by Presta. Furthermore, the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. Furthermore, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0153] In yet another embodiment, the glycosylation of the antibody is modified. For example, a glycosylated antibody can be generated (i.e., the antibody lacks glycosylation). The glycosylation can be altered to, for example, increase the affinity of the antibody for the antigen. Such carbohydrate modifications can be achieved, for example, by altering the glycosylation at one or more sites within the antibody sequence. For example, one or more amino acid substitutions can be made that eliminate one or more glycosylation sites in the variable region framework, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0154] In other specific embodiments, antibodies can be generated with altered glycosylation types, such as hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express the recombinant antibodies of the invention, thereby producing antibodies with altered glycosylation. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α(1,6) fucosyltransferase), and as a result, antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lack fucose on the carbohydrate. Ms704, Ms705, and Ms709 FUT8 - / -A cell line was created by targeting and disrupting the FUT8 gene in CHO / DG44 cells using two replacement vectors (see U.S. Patent Application Publication No. 20040110704 by Yamane et al. and Yamane-Ohnuki et al. (2004) Biotechnol Bioeng 87:614-22). As another example, EP 1,176,195 by Hanai et al. describes a cell line in which the FUT8 gene encoding fucosyltransferase is functionally disrupted, thereby reducing or eliminating the enzyme associated with α1,6 linkage, so that antibodies expressed in such cell lines exhibit low fucosylation. Hanai et al. also describe cell lines with low or no enzymatic activity for adding fucose to N-acetylglucosamine linked to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication WO 03 / 035835 by Presta describes a variant CHO cell line, Lec13 cells, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrates and results in hypofucosylation of antibodies expressed in the host cells (see also Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycosyltransferases (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) that modify glycoproteins such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures, resulting in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17:176-180). Alternatively, fucose residues on antibodies can be cleaved off using a fucosidase enzyme. For example, the fucosidase α-L-fucosidase removes fucosyl residues from antibodies (Tarentino, AL et al. (1975) Biochem. 14:5516-23).

[0155] Another modification of the antibodies herein contemplated by the present invention is pegylation. For example, an antibody can be pegylated to extend the antibody's biological (e.g., serum) half-life. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in one or more PEG groups being attached to the antibody or antibody fragment. Preferably, pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG used to derivatize other proteins, such as mono(C1-C10)alkoxypolyethyleneglycol or aryloxypolyethyleneglycol, or polyethyleneglycolmaleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present invention. See, for example, EP 0154316 by Nishimura et al. and EP 0401384 by Ishikawa et al.

[0156] Methods for engineering antibodies As mentioned above, the V disclosed herein H Array and V K Using an anti-PD-L1 antibody having the sequence, VH and / or V KBy modifying the sequence or the constant regions associated therewith, new anti-PD-L1 antibodies can be generated. Thus, in another aspect of the invention, the structural features of the anti-PD-L1 antibodies of the invention, such as 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4, are used to generate structurally related anti-PD-L1 antibodies that retain at least one functional property of the antibodies of the invention, such as binding to human PD-L1. For example, one or more CDR regions of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, or 13G4, or variants thereof, can be recombinantly combined with known framework regions and / or other CDRs, as described above, to generate additional recombinantly engineered anti-PD-L1 antibodies of the invention. Other types of modifications include those described in the previous sections. The starting material for the method of operation may be one or more of the V H Sequence and / or V K To generate an engineered antibody, one or more V sequences provided herein may be used. H Sequence and / or V K It is not necessary to actually prepare (i.e., express as a protein) an antibody having the sequence, or one or more CDR regions thereof. Rather, the information contained in the sequence is used as starting material to create a "second generation" sequence derived from the original sequence, which is then prepared and expressed as a protein.

[0157] Accordingly, in another aspect, the present invention provides a method for preparing an anti-PD-L1 antibody, comprising the steps of: (a)(i) a CDR1 sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, a CDR2 sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and / or a CDR2 sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, and SEQ ID NO:50; and / or (ii) a heavy chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, a CDR2 sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, and / or ...71, SEQ ID NO:72, SEQ ID NO: providing a light chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80; (b) altering at least one amino acid residue within the heavy chain variable region antibody sequence and / or the light chain variable region antibody sequence to generate at least one modified antibody sequence; and (c) expressing the modified antibody sequence as a protein.

[0158] Standard molecular biology techniques can be used to prepare and express the altered antibody sequence.

[0159] Preferably, the antibody encoded by the modified antibody sequence retains one, some, or all of the functional properties of the anti-PD-L1 antibodies described herein, including, but not limited to, the following: (i) 1 × 10 -7 M or lower K D binds to human PD-L1; (ii) increase T-cell proliferation in mixed lymphocyte reaction (MLR) assays; (iii) increase interferon-γ production in the MLR assay; (iv) increases IL-2 secretion in the MLR assay; (v) stimulating an antibody response; and / or (vi) Reverse the effects of regulatory T cells on T cell effector cells and / or dendritic cells.

[0160] The functional properties of the modified antibodies can be assessed using standard assays available in the art and / or described herein, such as those described in the Examples (e.g., flow cytometry, binding assays).

[0161] In certain embodiments of the methods for engineering antibodies of the invention, mutations can be introduced randomly or selectively along all or part of the anti-PD-L1 antibody coding sequence, and the resulting modified anti-PD-L1 antibodies can be screened for binding activity and / or other functional properties as described herein. Mutation methods have been described in the art. For example, PCT Publication WO 02 / 092780 by Short describes methods for generating and screening antibody variants using saturation mutagenesis, synthetic ligation assembly, or a combination thereof. Alternatively, PCT Publication WO 03 / 074679 by Lazar et al. describes methods for using computational screening methods to optimize the physiochemical properties of antibodies.

[0162] Nucleic acid molecules encoding antibodies of the present disclosure Another aspect of the present disclosure relates to nucleic acid molecules encoding the antibodies of the present invention. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. A nucleic acid is "isolated" or "substantially purified" when it has been purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See F. Ausubel, et al., eds. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

[0163] Nucleic acids of the invention can be obtained using standard molecular biology techniques. In the case of antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes, as described further below), cDNAs encoding the light and heavy chains of the antibodies produced by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. In the case of antibodies obtained from an immunoglobulin gene library (e.g., using phage display technology), nucleic acids encoding the antibodies can be recovered from the library.

[0164] Preferred nucleic acid molecules of the invention encode the VH and VL sequences of monoclonal antibodies 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4. DNA sequences encoding the VH sequences of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 are set forth in SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, and SEQ ID NO:90, respectively. The DNA sequences encoding the VL sequences of 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 are shown in SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, and SEQ ID NO:100, respectively.

[0165] After obtaining the DNA fragments encoding the VH and VL segments, these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the VL- or VH-encoding DNA fragment is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

[0166] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operably linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be the constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD, but is most preferably the constant region of IgG1 or IgG4. In the case of a Fab fragment heavy chain gene, the VH-encoding DNA can be operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0167] The isolated DNA encoding the VL region can be converted into a full-length light chain gene (as well as a Fab light chain gene) by operably linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (see, for example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa constant region or a lambda constant region, but is most preferably a kappa constant region.

[0168] To generate an scFv gene, a DNA fragment encoding the VH and VL is operably linked to another fragment encoding a flexible linker, such as a fragment encoding the amino acid sequence (Gly4-Ser)3, so that the VH and VL sequences can be expressed as a continuous single-chain protein with the VL and VH regions connected by a flexible linker (see, e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., (1990) Nature 348:552-554).

[0169] Production of the monoclonal antibodies of the present invention The monoclonal antibodies (mAbs) of the invention can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein (1975) Nature 256:495. Although somatic cell hybridization procedures are preferred, in principle, other techniques for producing monoclonal antibodies can be used, e.g., viral or oncogenic transformation of B lymphocytes.

[0170] The preferred animal system for preparing hybridomas is the mouse system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immune splenocytes for fusion are known in the art. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known.

[0171] Chimeric or humanized antibodies of the present invention can be prepared based on the sequences of mouse monoclonal antibodies prepared as described above. DNA encoding heavy and light chain immunoglobulins can be obtained from the mouse hybridoma of interest using standard molecular biology techniques and engineered to include non-mouse (e.g., human) immunoglobulin sequences. For example, to create chimeric antibodies, mouse variable regions can be linked to human constant regions using methods known in the art (see, e.g., U.S. Patent No. 4,816,567 by Cabilly et al.). To create humanized antibodies, mouse CDR regions can be inserted into a human framework using methods known in the art (see, e.g., U.S. Patent No. 5,225,539 by Winter, and U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,762, and 6,180,370 by Queen et al.).

[0172] In a preferred embodiment, the antibodies of the invention are human monoclonal antibodies. Such human monoclonal antibodies against PD-L1 can be generated using transgenic or transchromosomic mice carrying parts of the human immune system, instead of the mouse system. These transgenic and transchromosomic mice include those referred to herein as HuMAb mice and KM mice™, respectively, and are collectively referred to herein as "human Ig mice."

[0173] HuMAb Mice® (Medarex, Inc.) contain human immunoglobulin gene minilocuses encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474): 856-859). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high-affinity human IgGκ monoclonals (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immumol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-546).The preparation and use of HuMab mice, and the genomic modifications they carry, are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-3724; Choi et al. (1993) Nature Genetics 4:117-123; Chen, J. et al. (1993) EMBO J. 12:821-830; Tuaillon et al. (1994) J. Immunol. 152:2912-2920; Taylor, L. et al. (1994) International Immunology 6:579-591; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, the contents of all of which are specifically incorporated herein by reference in their entireties. Further, U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429 (all to Lonberg and Kay); U.S. Patent No. 5,545,807 (to Surani et al.); PCT Publications WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO See further PCT Publication No. 99 / 45962 (all by Lonberg and Kay); and PCT Publication No. WO 01 / 14424 (by Korman et al.).

[0174] In another embodiment, human antibodies of the invention can be produced using mice carrying human immunoglobulin sequences on a transgene and transchromosome, such as mice carrying a human heavy chain transgene and a human light chain transchromosome. Such mice, referred to herein as "KM Mice™," are described in detail in PCT Publication WO 02 / 43478 by Ishida et al.

[0175] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the anti-PD-L1 antibodies of the invention. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used. Such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963 by Kucherlapati et al.

[0176] Furthermore, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the anti-PD-L1 antibodies of the invention. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, referred to as "TC mice," can be used. Such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad Sci. USA 97:722-727. Furthermore, cows carrying a human heavy chain transchromosome and a human light chain transchromosome have been described in the art (Kuroiwa et al. (2002) Nature Biotechnology, 20:889-894) and can be used to raise the anti-PD-L1 antibodies of the invention.

[0177] The human monoclonal antibody of the present invention can also be prepared by using phage display method to screen the library of human immunoglobulin genes.Such phage display method for isolating human antibody has been established in the art.For example, see U.S. Patent Nos. 5,223,409, 5,403,484 and 5,571,698 by Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 by Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 by McCafferty et al.; and U.S. Patent Nos. 5,885,793, 6,521,404, 6,544,731, 6,555,313, 6,582,915 and 6,593,081 by Griffiths et al.

[0178] Human monoclonal antibodies of the invention can also be prepared using SCID mice reconstituted with human immune cells so that a human antibody response can be generated upon immunization. Such mice are described, for example, in U.S. Patent Nos. 5,476,996 and 5,698,767 by Wilson et al.

[0179] Immunization of human Ig mice When human Ig mice are used to generate the human antibodies of the invention, such mice can be immunized with a purified or concentrated preparation of PD-L1 antigen and / or recombinant PD-L1, or a PD-L1 fusion protein, as described in Lonberg, N. et al. (1994) Nature 368(6474):856-859, Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, and PCT publications WO 98 / 24884 and WO 01 / 14424. Preferably, the mice are 6-16 weeks old at the time of the first injection. For example, a purified or recombinant preparation of PD-L1 antigen (5 μg-50 μg) can be used to immunize the human Ig mice intraperitoneally.

[0180] A detailed procedure for generating fully human monoclonal antibodies against PD-L1 is described in Example 1 below. Cumulative experience with various antigens has shown that transgenic mice respond to an initial intraperitoneal (IP) immunization with antigen in complete Freund's adjuvant, followed by weekly IP immunizations with antigen in incomplete Freund's adjuvant (for a total of up to six immunizations). However, adjuvants other than Freund's adjuvant have also proven effective. Furthermore, whole cells in the absence of adjuvant have been shown to be highly immunogenic. The immune response can be monitored throughout the immunization protocol using plasma samples obtained by retroorbital bleeds. Plasma can be screened by ELISA (as described below), and mice with sufficient titers of anti-PD-L1 human immunoglobulin can be used for fusions. Mice can be boosted with antigen intravenously three days before sacrifice and pancreatectomy. It is expected that two to three fusions may need to be performed for each immunization. Typically, between 6 and 24 mice are immunized for each antigen. Usually, both HCo7 and HCo12 strains are used. Furthermore, both HCo7 and HCo12 transgenes can be introduced together to breed a single mouse with two different human heavy chain transgenes (HCo7 / HCo12). Alternatively, or in addition, the KM Mouse™ strain can be used, as described in Example 1.

[0181] Generation of hybridomas producing human monoclonal antibodies of the present disclosure To generate hybridomas producing the human monoclonal antibodies of the present invention, spleen cells and / or lymph node cells from immunized mice can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. The resulting hybridomas can be screened for the production of antigen-specific antibodies. For example, a single-cell suspension of splenic lymphocytes from immunized mice can be fused with one-sixth the number of P3X63-Ag8.653 non-secreting mouse myeloma cells (ATCC, CRL 1580) using 50% PEG. Approximately 2 × 10 5 Cells are seeded into flat-bottom microtiter plates and then incubated for 2 weeks in selection medium containing 20% ​​fetal clonal serum, 18% "653" conditioned medium, 5% origen (IGEN), 4 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units / ml penicillin, 50 mg / ml streptomycin, 50 mg / ml gentamicin, and 1x HAT (Sigma; HAT is added 24 hours postfusion). After approximately 2 weeks, cells may be cultured in medium in which the HAT is replaced with HT. Individual wells can then be screened for human monoclonal IgM and IgG antibodies by ELISA. Once extensive hybridoma growth has occurred, medium may be observed, usually after 10-14 days. The antibody-secreting hybridomas can be replated, screened again, and if still positive for human IgG, the monoclonal antibodies can be subcloned at least twice by limiting dilution. The stable subclones can then be cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.

[0182] To purify human monoclonal antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated, then subjected to affinity chromatography using Protein A Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS, and the concentration can be determined by OD280 using an extinction coefficient of 1.43. The monoclonal antibodies can be aliquoted and stored at -80°C.

[0183] Generation of transfectomas producing human monoclonal antibodies of the present disclosure The antibodies of the present invention can also be produced in host cell transfectomas, for example, using a combination of recombinant DNA technology and gene transfection methods well known in the art (e.g., Morrison, S. (1985) Science 229:1202).

[0184] For example, to express an antibody or antibody fragment thereof, DNA encoding a portion or the entire length of the light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest), and the DNA can be inserted into an expression vector such that the genes are operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. Expression vectors and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present). The light chain variable region and heavy chain variable region of the antibodies described herein are V H The segment is C in the vector H operably linked to the segment and V K The segment is C in the vector L The antibody chain segments can be used to generate full-length antibody genes of any antibody isotype by inserting them operably linked into an expression vector already encoding the heavy and light chain constant regions of the desired isotype. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0185] In addition to the antibody chain genes, the recombinant expression vectors of the invention contain regulatory sequences that control the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, for example, in Goeddel (Gene Expression Technology. Methods in Enzymology 185, Academic Press, San Diego, CA (1990)). Those skilled in the art will understand that the design of the expression vector, including the selection of regulatory sequences, can depend on factors such as the choice of the host cell to be transformed, the desired expression level of protein, and the like. Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma virus. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or the β-globin promoter, can be used. Furthermore, regulatory elements have been constructed from sequences derived from different sources, such as the SRα promoter system, which contains sequences derived from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

[0186] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, the selectable marker gene typically confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cells into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0187] To express the light and heavy chains, expression vectors encoding the light and heavy chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. While it is theoretically possible to express the antibodies of the present invention in either prokaryotic or eukaryotic host cells, expression of the antibodies in eukaryotic cells, and most preferably mammalian host cells, is most preferred. This is because eukaryotic cells, and particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete correctly folded and immunologically active antibodies. Prokaryotic expression of antibody genes has been reported to be ineffective for producing high yields of active antibody (Boss, MA and Wood, CR (1985) Immunology Today 6:12-13).

[0188] Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese hamster ovary (CHO) cells (e.g., as described by RJ Kaufman and PA Sharp (1982) Mol. Biol. 159:601-621, including the dhfr-CHO cells described by Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with the DHFR selection marker), NSO myeloma cells, COS cells, and SP2 cells. Another preferred expression system, particularly for use with NSO myeloma cells, is the GS gene expression system disclosed in WO 87 / 04462, WO 89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing those host cells for a period of time sufficient to allow for expression of the antibody in the host cells, or more preferably, secretion of the antibody into the medium that the host cells are grown in. The antibody can be recovered from the medium using standard protein purification methods.

[0189] Characterization of antibody binding to antigen Antibodies of the invention can be tested for binding to PD-L1, for example, by standard ELISA. Briefly, microtiter plates are coated with 0.25 μg / ml of purified PD-L1 in PBS, then blocked with 5% bovine serum albumin in PBS. Dilutions of antibody (e.g., dilutions of plasma from mice immunized with PD-L1) are added to each well and incubated for 1-2 hours at 37°C. The plates are washed with PBS / Tween and then incubated with a secondary reactant conjugated to alkaline phosphatase (e.g., for human antibodies, a goat anti-human IgG Fc-specific polyclonal reactant) for 1 hour at 37°C. After washing, the plates are developed with pNPP substrate (1 mg / ml) and analyzed at an OD of 405-650. Preferably, mice with the highest titers are used for fusions.

[0190] The ELISA assay described above can also be used to screen for hybridomas that show positive reactivity to PD-L1 immunogens. Hybridomas that bind to PD-L1 with high avidity are subcloned and further characterized. One clone from each hybridoma that retains the reactivity of the parental cells (by EILSA) can be selected to generate a cell bank of 5-10 vials stored at -140°C and to purify the antibody.

[0191] To purify anti-PD-L1 antibodies, selected hybridomas can be grown in 2-liter spinner flasks for monoclonal antibody purification. The supernatant can be filtered and concentrated, then subjected to affinity chromatography using Protein A Sepharose (Pharmacia, Piscataway, NJ). The eluted IgG can be checked by gel electrophoresis and high-performance liquid chromatography to ensure purity. The buffer can be exchanged into PBS, and the concentration can be determined by measuring the OD using an extinction coefficient of 1.43. 280 The monoclonal antibodies can be aliquoted and stored at -80°C.

[0192] To determine whether selected anti-PD-L1 monoclonal antibodies bind to unique epitopes, each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, IL). Competition studies using unlabeled and biotinylated monoclonal antibodies can be performed using PD-L1-coated ELISA plates as described above. Binding of biotinylated mAbs can be detected using a streptavidin-alkaline phosphatase probe.

[0193] To determine the isotype of purified antibodies, isotype ELISA can be performed using reagents specific for antibodies of a particular isotype. For example, to determine the isotype of a human monoclonal antibody, the wells of a microtiter plate can be coated with 1 μg / ml of anti-human immunoglobulin overnight at 4°C. After blocking with 1% BSA, the plates are reacted with 1 μg / ml or lower concentrations of the test monoclonal antibody or purified isotype control for 1 to 2 hours at ambient temperature. The wells can then be reacted with either an alkaline phosphatase-conjugated probe specific for human IgG1 or human IgM. The plates are developed and analyzed as described above.

[0194] Anti-PD-L1 human IgG can be further tested for reactivity with the PD-L1 antigen by Western blotting. Briefly, PD-L1 can be prepared and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. After electrophoresis, the separated antigens are transferred to a nitrocellulose membrane, blocked with 10% fetal bovine serum, and probed with the monoclonal antibody to be tested. Human IgG binding can be detected using anti-human IgG alkaline phosphatase and developed using BCIP / NBT substrate tablets (Sigma Chem. Co., St. Louis, Mo.).

[0195] Antibody physical properties The antibodies of the invention may be further characterized by various physical properties of the anti-PD-L1 antibodies, and various assays may be used to detect and / or distinguish between different classes of antibodies based on these physical properties.

[0196] In some embodiments, antibodies of the invention may contain one or more glycosylation sites in either the light chain variable region or the heavy chain variable region. The presence of one or more glycosylation sites in the variable region may alter antigen binding, thereby increasing the immunogenicity of the antibody or changing the antibody's pK (Marshall et al (1972) Annu Rev Biochem 41:673-702; Gala FA and Morrison SL (2004) J Immunol 172:5489-94; Wallick et al (1988) J Exp Med 168:1099-109; Spiro RG (2002) Glycobiology 12:43R-56R; Parekh et al (1985) Nature 316:452-7; Mimura et al. (2000) Mol Immunol 37:697-706). Glycosylation is known to occur in motifs containing NXS / T sequences. Glycosylation of the variable region can be tested using a Glycoblot assay, in which an antibody is cleaved to generate Fab fragments, which are then tested for glycosylation using an assay that measures periodate oxidation and Schiff base formation. Alternatively, variable region glycosylation can be tested using Dionex light chromatography (Dionex-LC), in which sugars from the Fab are cleaved into monosaccharides and analyzed for individual sugar content. In some cases, it may be preferable to have an anti-PD-L1 antibody that does not contain variable region glycosylation. This can be achieved by selecting an antibody that does not contain a glycosylation motif in the variable region, or by mutating residues within the glycosylation motif using standard techniques known in the art.

[0197] In a preferred embodiment, the antibody of the present invention does not contain an asparagine isomerism site. Deamidation or isoaspartic acid formation can occur at the NG or DG sequence, respectively. Deamidation or isoaspartic acid formation results in the production of isoaspartic acid, which reduces the stability of the antibody by creating a twisted structure away from the carboxyl end of the side chain rather than the main chain. The production of isoaspartic acid can be measured using an isoquant assay, which tests for isoaspartic acid using reverse-phase HPLC.

[0198] Each antibody is believed to have a unique isoelectric point (pI), but antibodies are generally believed to fall within a pH range between 6 and 9.5. The pI of IgG1 antibodies typically falls within a pH range of 7 to 9.5, and the pI of IgG4 antibodies typically falls within a pH range of 6 to 8. Antibodies can have pIs outside this range. While these effects are generally unknown, there is speculation that antibodies with pIs outside the normal range may exhibit some unfolding and instability under in vivo conditions. Isoelectric point can be tested using a capillary isoelectric focusing assay, which creates a pH gradient and can utilize laser focusing for increased accuracy (Janini et al. (2002) Electrophoresis 23:1605-11; Ma et al. (2001) Chromatographia 53:S75-89; Hunt et al. (1998) J Chromatogr A 800:355-67). In some cases, it may be preferable to have an anti-PD-L1 antibody with a pI value that falls within the normal range. This can be achieved by selecting an antibody with a pI in the normal range, or by mutating charged surface residues using standard techniques well known in the art.

[0199] Each antibody has a melting temperature that indicates its thermal stability (Krishnamurthy R and Manning MC (2002) Curr Pharm Biotechnol 3:361-71). Higher thermal stability indicates greater overall antibody stability in vivo. The melting point of an antibody can be measured using techniques such as differential scanning calorimetry (Chen et al (2003) Pharm Res 20:1952-60; Ghirlando et al (1999) Immunol Lett 68:47-52). M1 indicates the temperature at which the antibody first unfolds. T M2 indicates the temperature at which the antibody completely unfolds. Generally, the T M1 is preferably greater than 60°C, preferably greater than 65°C, and even more preferably greater than 70°C. Alternatively, antibody thermal stability can be measured using circular dichroism (Murray et al. (2002) J. Chromatogr Sci 40:343-9). The thermal stabilities of the anti-PD-L1 antibodies disclosed herein are summarized in Table 1.

[0200] (Table 1) TIFF2024174925000002.tif88128

[0201] In preferred embodiments, antibodies are selected that do not rapidly degrade. Fragmentation of anti-PD-L1 antibodies can be measured using capillary electrophoresis (CE) and MALDI-MS, as is well understood in the art (Alexander AJ and Hughes DE (1995) Anal Chem 67:3626-32).

[0202] In another preferred embodiment, an antibody with minimal aggregation effect is selected. Aggregation can induce unwanted immune responses and / or result in altered or unfavorable pharmacokinetic properties. Generally, antibodies with aggregation of 25% or less, preferably 20% or less, even more preferably 15% or less, even more preferably 10% or less, and even more preferably 5% or less are acceptable. Aggregation can be measured by several techniques well known in the art, including size exclusion column (SEC) high performance liquid chromatography (HPLC) to identify monomers, dimers, trimers, or multimers, and light scattering.

[0203] immune complex In another aspect, the invention features an anti-PD-L1 antibody, or fragment thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, as well as analogs or homologs thereof. Therapeutic agents include, for example, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil), Also included are chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum(II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and antimitotics (e.g., vincristine and vinblastine).

[0204] Other preferred examples of therapeutic cytotoxins that can be conjugated to the antibodies of the invention include duocarmycins, calicheamicins, maytansines, and auristatins, and their derivatives. Examples of calicheamicin-antibody conjugates are commercially available (Mylotarg™, Wyeth-Ayerst).

[0205] Cytotoxin can be attached to the antibody of the present invention using linker technology available in the art. Examples of linker types used to attach cytotoxin to antibody include, but are not limited to, hydrazone, thioether, ester, disulfide, and peptide-containing linkers. For example, a linker can be selected that is susceptible to cleavage by the low pH in the lysosomal compartment, or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, D).

[0206] For further discussion of cytotoxin types, linkers, and methods for attaching therapeutic agents to antibodies, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55:199-215; Trail, PA et al. (2003) Cancer Immunol. Immunother. 52:328-337; Payne, G. (2003) Cancer Cell 3:207-212; Allen, TM (2002) Nat. Rev. Cancer 2:750-763; Pastan, I. and Kreitman, RJ (2002) Curr. Opin. Investig. Drugs 3:1089-1091; Senter, PD and Springer, CJ (2001) Adv. Drug Deliv. Rev. 53:247-264.

[0207] The antibodies of the present invention can also be conjugated to radioisotopes to create cytotoxic radiopharmaceuticals, also called radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine 131 ,indium 111 ,yttrium 90 , and lutetium 177 Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates, including Zevalin™ (IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), are commercially available, and similar methods can be used to prepare radioimmunoconjugates using the antibodies of the invention.

[0208] The antibody conjugates of the present invention can be used to modify a given biological response, and the drug moiety should not be construed as being limited to conventional chemical therapeutic agents. For example, the drug moiety can be a protein or polypeptide possessing a desired biological activity. Such proteins can include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor or interferon-γ; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF"), granulocyte-colony-stimulating factor ("G-CSF"), or other growth factors.

[0209] Techniques for conjugating such therapeutic moieties to antibodies are well known and can be found, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery (2nd ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospects Of The Therapeutic Use See, "Of Radiolabeled Antibodies In Cancer Therapy," Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev., 62:119-58 (1982).

[0210] bispecific molecules In another aspect, the present invention features bispecific molecules comprising an anti-PD-L1 antibody of the invention, or a fragment thereof. An antibody of the invention, or its antigen-binding portion thereof, can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., a ligand for another antibody or receptor), to create a bispecific molecule that binds to at least two different binding sites or target molecules. An antibody of the invention can actually be derivatized or linked to multiple other functional molecules to create a multispecific molecule that binds to more than two different binding sites and / or target molecules. Such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be operatively linked (e.g., by chemical bond, genetic fusion, noncovalent bonding, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, such that a bispecific molecule results.

[0211] Thus, the present invention includes bispecific molecules comprising at least one first binding specificity for PD-L1 and a second binding specificity for a second target epitope. In certain embodiments of the invention, the second target epitope is an Fc receptor, e.g., human FcγRI (CD64) or human Fcα receptor (CD89). Thus, the present invention includes bispecific molecules that can bind to both effector cells expressing FcγR or FcαR (e.g., monocytes, macrophages, or polymorphonuclear cells (PMNs)) and target cells expressing PD-L1. These bispecific molecules target PD-L1-expressing cells to effector cells and induce Fc receptor-mediated effector cell activity, such as phagocytosis of PD-L1-expressing cells, antibody-dependent cell-mediated cytotoxicity (ADCC), cytokine release, or superoxide anion generation.

[0212] In embodiments of the present invention in which the bispecific molecule is multispecific, the molecule may further comprise a third binding specificity in addition to the anti-Fc binding specificity and the anti-PD-L1 binding specificity. In one embodiment, the third binding specificity is an anti-enhancement factor (EF) moiety, e.g., a molecule that binds to a surface protein involved in cytotoxic activity, thereby enhancing the immune response against the target cell. The "anti-enhancement factor moiety" can be an antibody, functional antibody fragment, or ligand that binds to a given molecule, e.g., an antigen or receptor, thereby enhancing the action of the binding determinant on the Fc receptor or target cell antigen. The "anti-enhancement factor moiety" can bind to an Fc receptor or target cell antigen. Alternatively, the anti-enhancement factor moiety can bind to an entity different from the entity bound by the first binding specificity and the second binding specificity. For example, the anti-enhancement factor moiety can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, ICAM-1, or other immune cells, resulting in an enhanced immune response against the target cell).

[0213] In one embodiment, the bispecific molecules of the invention comprise as binding specificities at least one antibody or antibody fragment thereof, including, for example, Fab, Fab', F(ab'), Fv, or single-chain Fv. The antibody may also be a light-chain or heavy-chain dimer, or any small fragment thereof, such as an Fv or single-chain construct, as described in U.S. Patent No. 4,946,778 by Ladner et al., the contents of which are expressly incorporated by reference.

[0214] In one embodiment, the binding specificity for an Fcγ receptor is provided by a monoclonal antibody, the binding of which is not blocked by human immunoglobulin G (IgG). As used herein, the term "IgG receptor" refers to any of the eight gamma chain genes located on chromosome 1. These genes encode a total of 12 transmembrane or soluble receptor isoforms, which are divided into three Fcγ receptor classes: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). In one preferred embodiment, the Fcγ receptor is a human high-affinity FcγRI. Human FcγRI is a 72 kDa molecule that exhibits high affinity for monomeric IgG (10 8 M -1 ~10 9 M -1 ).

[0215] The production and characterization of several preferred anti-Fcγ monoclonal antibodies are described by Fanger et al. in PCT Publication WO 88 / 00052 and U.S. Pat. No. 4,954,617, the teachings of which are incorporated herein by reference in their entireties. These antibodies bind to epitopes of FcγRI, FcγRII, or FcγRIII at sites distinct from the Fcγ binding site of the receptor, and therefore, their binding is not substantially blocked by physiological levels of IgG. Specific anti-FcγRI antibodies useful in the present invention are mAb22, mAb32, mAb44, mAb62, and mAb197. The hybridoma producing mAb32 is available from the American Type Culture Collection (ATCC) Accession No. HB9469. In other embodiments, the anti-Fcγ receptor antibody is a humanized version of monoclonal antibody 22 (H22). The production and characterization of the H22 antibody are described in Graziano, RF et al. (1995) J. Immunol 155(10):4996-5002 and PCT publication WO 94 / 10332. The cell line producing the H22 antibody has been deposited with the American Type Culture Collection under the designation HA022CL1 and has the accession number CRL 11177.

[0216] In yet another preferred embodiment, the binding specificity for an Fc receptor is provided by an antibody that binds to a human IgA receptor, e.g., the Fcα receptor (FcαRI (CD89)), the binding of which is preferably not blocked by human immunoglobulin A (IgA). The term "IgA receptor" is intended to include the gene product of one α gene (FcαRI) located on chromosome 19. This gene is known to encode several alternatively spliced ​​55-110 kDa transmembrane isoforms. FcαRI (CD89) is constitutively expressed on monocytes / macrophages, eosinophilic and neutrophilic granulocytes, but not on cell populations that are not effector cells. FcαRI has a moderate affinity (approximately 5×10) for both IgA1 and IgA2. 7 M -1 ), which increases upon exposure to cytokines such as G-CSF or GM-CSF (Morton, HC et al. (1996) Critical Reviews in Immunology 16:423-440). Four FcαRI-specific monoclonal antibodies, identified as A3, A59, A62, and A77, which bind to FcαRI outside the IgA ligand-binding domain, have been described (Monteiro, RC et al. (1992) J. Immunol. 148:1764).

[0217] FcαRI and FcγRI are preferred trigger receptors for use in the bispecific molecules of the invention because (1) they are primarily expressed on immune effector cells, e.g., monocytes, PMNs, macrophages, and dendritic cells; (2) they are expressed at high levels (e.g., 5,000-100,000 per cell); (3) they are mediators of cytotoxic activity (e.g., ADCC, phagocytosis); and (4) they mediate enhanced antigen presentation of antigens, including self-antigens, that are targeted to them.

[0218] Although human monoclonal antibodies are preferred, other antibodies that can be used in the bispecific molecules of the invention are murine, chimeric, and humanized monoclonal antibodies.

[0219] Bispecific molecules of the invention can be prepared by conjugating the component binding specificities, e.g., an anti-FcR binding specificity and an anti-PD-L1 binding specificity, using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohaxane-1-carboxylate (sulfo-SMCC) (see, e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83 and Glennie et al. (1987) J. Immunol. 139:2367-2375. Preferred linking agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[0220] When the binding specificities are antibodies, they can be attached via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to attachment.

[0221] Alternatively, both binding specificities can be encoded in the same vector and expressed in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. Bispecific molecules of the invention can be single-chain molecules containing one single-chain antibody and one binding determinant, or single-chain bispecific molecules containing two binding determinants. Bispecific molecules can contain at least two single-chain molecules. Methods for preparing bispecific molecules are described, for example, in U.S. Patent Nos. 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498, and 5,482,858.

[0222] The binding of bispecific molecules to specific targets can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growth inhibition), or Western blot assay. Each of these assays generally detects the presence of a specific protein-antibody complex of interest by using a labeled reactant (e.g., an antibody) specific to the complex of interest. For example, FcR-antibody complexes can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody-FcR complex. Alternatively, these complexes can be detected using any of a variety of other immunoassay methods. For example, antibodies can be radiolabeled and used in radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated herein by reference). The radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by autoradiography.

[0223] Pharmaceutical Compositions In another aspect, the present invention provides compositions, e.g., pharmaceutical compositions, comprising one or a combination of the monoclonal antibodies or antigen-binding portions thereof of the present invention, formulated together with a pharmaceutically acceptable carrier. Such compositions can contain one or a combination (e.g., two or more different) antibodies, or immunoconjugates or bispecific molecules of the present invention. For example, pharmaceutical compositions of the present invention can contain a combination of antibodies (or immunoconjugates or bispecifics) that bind to different epitopes on the target antigen or have complementary activities.

[0224] Pharmaceutical compositions of the invention can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy may include an anti-PD-L1 antibody of the invention combined with at least one other anti-inflammatory or immunosuppressive agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on uses of the antibodies of the invention.

[0225] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and isotonic and absorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immunoconjugate, or bispecific molecule, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0226] The pharmaceutical compounds of the present invention may include one or more pharmaceutically acceptable salts. The term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, for example, Berge, SM, et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as those derived from non-toxic organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, and calcium, and those derived from non-toxic organic amines, such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0227] The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0228] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.Proper fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants.

[0229] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by both the above-mentioned sterilization procedures and the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, and phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0230] Pharmaceutically acceptable carriers include sterile aqueous solution or aqueous dispersion and sterile powder, which can be used to prepare sterile injectable solution or injectable dispersion immediately.The use of such media and active substances for pharmaceutically active substances is known in the art.Except when any conventional media or active substance is incompatible with active compound, it is contemplated to use it in the pharmaceutical compositions of the present invention.Auxiliary active compounds can also be mixed into the composition.

[0231] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be prepared as solutions, microemulsions, liposomes, or other ordered structures suitable to high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. 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. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0232] Sterile injectable solution can be prepared by mixing the active compound in the required amount in suitable solvent with one or combination of the above-mentioned components as needed, and then sterilizing by filtration.Generally, dispersion is prepared by mixing the active compound in the sterile vehicle that contains the basic dispersion medium and the other components that are required from the above-mentioned components.For the sterile powder that is used to prepare sterile injectable solution, the preferred method is vacuum drying and freeze-drying (lyophilization), which produces the powder of active ingredient and any additional desired components from the solution that has been previously sterilized and filtered.

[0233] The amount of active ingredient that can be combined with carrier material to make a single dosage form will vary depending on the subject being treated and the individual mode of administration.The amount of active ingredient that can be combined with carrier material to make a single dosage form will generally be the amount of the composition that produces a therapeutic effect.Generally, out of 100 percent, this amount will be in the range of about 0.01% to about 99% of active ingredient, preferably about 0.1% to about 70%, most preferably about 1% to about 30% of active ingredient combined with pharmaceutically acceptable carrier.

[0234] Dosage regimens are adjusted to provide the optimum desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as required by the exigencies of the therapeutic situation. It is particularly advantageous to prepare parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. As used herein, unit dosage form refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated. Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are determined by and directly depend on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the constraints inherent in the technology of formulating such active compounds to treat individual susceptibility.

[0235] For administration of antibodies, dosages range from about 0.0001 mg / kg to 100 mg / kg of the recipient's body weight, and more usually 0.01 mg / kg to 5 mg / kg of the recipient's body weight. For example, dosages can be 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 5 mg / kg, or 10 mg / kg of body weight, or within the range of 1 mg / kg to 10 mg / kg. Exemplary treatment regimens involve administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every three months, or once every three to six months. Preferred dosing regimens for the anti-PD-L1 antibodies of the invention include 1 mg / kg or 3 mg / kg body weight via intravenous administration, where the antibody is given using one of the following dosing schedules: (i) six doses every four weeks, then every three months; (ii) every three weeks; (iii) one dose of 3 mg / kg body weight, then 1 mg / kg body weight every three weeks.

[0236] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, with the dosage of each antibody administered falling within the indicated range. Antibodies are typically administered on multiple occasions. The interval between doses can be, for example, weekly, monthly, quarterly, or yearly. The intervals can also be irregular, as indicated by measuring the patient's blood levels of antibody against the target antigen. In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1 μg / ml to 1000 μg / ml, and in some methods, to achieve a plasma antibody concentration of about 25 μg / ml to 300 μg / ml.

[0237] Alternatively, the antibody can be administered as a sustained-release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, human antibodies exhibit the longest half-life, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency can vary depending on whether the treatment is preventive or therapeutic. In preventive applications, relatively low dosages are administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages at relatively short intervals may be required until the progression of the disease is alleviated or terminated, and preferably until the patient shows partial or complete improvement in the symptoms of the disease. Thereafter, a preventive treatment regimen can be administered to the patient.

[0238] The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied for a particular patient, composition, and mode of administration to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response without being toxic to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present invention or its ester, salt, or amide used, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health, and previous medical history of the patient being treated, and similar factors well known in the pharmaceutical arts.

[0239] A "therapeutically effective dose" of an anti-PD-L1 antibody of the invention preferably results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or prevention of functional impairment or disability due to disease affliction. For example, in the case of treating PD-L1+ tumors, a "therapeutically effective dose" preferably inhibits cell proliferation or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, relative to untreated subjects. The ability of a compound to inhibit tumor growth can be assessed in an animal model system that is predictive of efficacy in human tumors. Alternatively, this property of a composition can be assessed by examining the compound's ability to inhibit such inhibition in vitro using assays known to those of skill in the art. A therapeutically effective amount of a therapeutic compound may reduce tumor size or otherwise ameliorate the subject's symptoms. One of skill in the art would be able to determine such amounts based on factors such as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[0240] The compositions of the present invention can be administered via one or more administration routes using one or more of a variety of methods known in the art. As will be understood by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. Preferred administration routes for the antibodies of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral administration routes, for example, by injection or infusion. As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0241] Alternatively, antibodies of the invention can be administered via non-parenteral routes, such as topical, epidermal, or mucosal routes of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0242] Active compound can be prepared with carriers that are considered to protect compound from rapid release, such as controlled release preparations, including implants, transdermal patches and microencapsulated delivery systems.Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid can be used.Many methods for preparing such preparations are patented or generally known to those skilled in the art.For example, see Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0243] The therapeutic composition can be administered using medical devices known in the art.For example, in a preferred embodiment, the therapeutic composition of the present invention can be administered using a needleless subcutaneous administration device, such as the device disclosed in U.S. Patent No. 5,399,163, U.S. Patent No. 5,383,851, U.S. Patent No. 5,312,335, U.S. Patent No. 5,064,413, U.S. Patent No. 4,941,880, U.S. Patent No. 4,790,824 or U.S. Patent No. 4,596,556. Examples of well-known implants and modules useful in the present invention include U.S. Pat. No. 4,487,603, which discloses an implantable microinfusion pump for administering medication at a controlled rate; U.S. Pat. No. 4,486,194, which discloses a therapeutic device for administering medical agents through the skin; U.S. Pat. No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Pat. No. 4,447,224, which discloses an implantable variable flow rate infusion device for sustained drug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drug delivery system having multiple chamber compartments; and U.S. Pat. No. 4,475,196, which discloses an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[0244] In certain embodiments, the human monoclonal antibodies of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., U.S. Patent Nos. 4,522,811, 5,374,548, and 5,399,331. Liposomes may contain one or more moieties that selectively transport into specific cells or organs, thereby improving targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin. Pharmacol. 29:685). Exemplary targeting moieties include folic acid or biotin (see, e.g., U.S. Patent No. 5,416,016 to Low et al.), mannosides (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038), antibodies (PG Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39:180), surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233:134), p120 (Schreier et al. (1994) J. Biol. Chem. 269:9090). See also K. Keinanen; ML Laukkanen (1994) FEBS Lett. 346:123; JJ Killion; IJ Fidler (1994) Immunomethods 4:273.

[0245] Uses and methods of the present invention The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo utilities, including, for example, enhancing immune responses by detecting PD-L1 or blocking PD-L1. In preferred embodiments, the antibodies of the present invention are human antibodies. For example, these molecules can be administered to cultured cells, in vivo or ex vivo, or to human subjects, e.g., in vivo, to enhance immunity in a variety of situations. Thus, in one aspect, the present invention provides a method of altering an immune response in a subject, the method comprising administering to the subject an antibody of the present invention, or an antigen-binding portion thereof, such that the immune response in the subject is altered. Preferably, the response is enhanced, stimulated, or upregulated.

[0246] As used herein, the term "subject" is intended to include humans and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, with mammals such as non-human primates, sheep, dogs, cats, cows, and horses being preferred. Preferred subjects include human patients in need of an enhanced immune response. These methods are particularly suitable for treating human patients with diseases that can be treated by enhancing T cell-mediated immune responses. In certain embodiments, these methods are particularly suitable for treating cancer cells in vivo. To achieve antigen-specific enhancement of immunity, anti-PD-L1 antibodies can be administered with an antigen of interest. When an antibody against PD-L1 is administered with another agent, the two can be administered sequentially or simultaneously.

[0247] The present invention also provides a method for detecting the presence or measuring the amount of human PD-L1 antigen in a sample, comprising contacting the sample and a control sample with a human monoclonal antibody, or antigen-binding portion thereof, that specifically binds human PD-L1 under conditions that allow the formation of a complex between the antibody or portion thereof and human PD-L1. The formation of the complex is then detected, whereby a difference in complex formation with the sample compared to the control sample indicates the presence of human PD-L1 antigen in the sample.

[0248] cancer Blocking PD-L1 with antibodies can enhance a patient's immune response to cancer cells. PD-L1 is not expressed on normal human cells, but is abundant in various human cancers (Dong et al. (2002) Nat Med 8:787-9). PD-1 and PD-L1 interaction results in a reduction in tumor-infiltrating lymphocytes, reduced T cell receptor-mediated proliferation, and immune evasion by cancer cells (Dong et al. (2003) J Mol Med 81:281-7; Blank et al. (2004) Cancer Immunol. Immunother. [Electronic Publication]; Konishi et al. (2004) Clin. Cancer Res. 10:5094-100). Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L2, and the effect is additive if the interaction of PD-L1 with PD-L2 is similarly disrupted (Iwai et al. (2002) PNAS 99:12293-7; Brown et al. (2003) J. Immunol. 170:1257-66). Anti-PD-L1 antibodies can be used alone to inhibit the growth of cancerous tumors. Alternatively, anti-PD-L1 antibodies can be used in combination with other immunogenic substances, standard cancer therapeutics, or other antibodies, as described below.

[0249] Accordingly, in one embodiment, the invention provides a method of inhibiting the growth of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody, or antigen-binding portion thereof. Preferably, the antibody is a human anti-PD-L1 antibody (such as any of the human anti-human PD-L1 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric anti-PD-L1 antibody or a humanized anti-PD-L1 antibody.

[0250] Preferred cancers whose growth can be inhibited using the antibodies of the invention include cancers that typically respond to immunotherapy. Non-limiting examples of cancers that are preferred for treatment include melanoma (e.g., metastatic malignant melanoma), kidney cancer, prostate cancer, breast cancer, colon cancer, and lung cancer. Other examples of cancers that can be treated using the methods of the invention include bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, and chronic myeloid leukemia. leukemia, acute or acute leukemia including acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumors, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of the foregoing cancers. The present invention is also useful for treating metastatic cancers, particularly metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 17:133-144).

[0251] Optionally, antibodies against PD-L1 may be combined with immunogenic materials such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and carbohydrate molecules), cells, and cells transfected with genes encoding immune-stimulating cytokines (He et al (2004) J. Immunol. 173:4919-28). Non-limiting examples of tumor vaccines that can be used include peptides of melanoma antigens, such as peptides of gp100, MAGE antigens, Trp-2, MART1, and / or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF (discussed further below).

[0252] In humans, some tumors, such as melanoma, have been shown to be immunogenic. By raising the threshold for T cell activation by PD-L1 blockade, we anticipate activating tumor responses in the host.

[0253] PD-L1 blockade is likely to be most effective when combined with vaccination protocols. Many experimental strategies for tumor vaccination have been devised (see Rosenberg, S., 2000, Development of Cancer Vaccines, ASCO Educational Book Spring:60-62; Logothetis, C., 2000, ASCO Educational Book Spring:300-302; Khayat, D. 2000, ASCO Educational Book Spring:414-428; Foon, K. 2000, ASCO Educational Book Spring:730-738. See also Restifo, N. and Sznol, M., Cancer Vaccines, Ch. 61, pp.3023-3043; DeVita, V. et al. (eds.), 1997, Cancer: Principles and Practice of Oncology. 5th ed.). In one of these strategies, vaccines are prepared using autologous or allogeneic tumor cells. These cellular vaccines have been shown to be most effective when the tumor cells are transduced to express GM-CSF. GM-CSF has been shown to be a potent activator of antigen presentation for tumor vaccines (Dranoff et al. (1993) Proc. Natl. Acad. Sci. USA 90:3539-43).

[0254] Studies of gene expression and large-scale gene expression patterns in various tumors have defined so-called tumor-specific antigens (Rosenberg, SA (1999) Immunity 10:281-7). These tumor-specific antigens are often differentiation antigens expressed in tumors and in the cells from which tumors originate, such as the melanocyte antigen gp100, MAGE antigens, and Trp-2. More importantly, many of these antigens can be shown to be targets for tumor-specific T cells present in the host. PD-L1 blockade can be used in combination with a group of recombinant proteins and / or peptides expressed in tumors to generate an immune response against these proteins. These proteins are usually perceived by the immune system as self-antigens and are therefore tolerated. Tumor antigens can also include the protein telomerase, which is required for the synthesis of chromosomal telomeres and is expressed in more than 85% of human cancers and in a limited number of somatic tissues (Kim, N et al. (1994) Science 266:2011-2013). (These somatic tissues can be protected from immune attack by various means.) Tumor antigens can also be "neoantigens" or idiotypes from B-cell tumors that are expressed in cancer cells due to somatic mutations that alter the protein sequence or create fusion proteins between two unrelated sequences (i.e., bcr-abl in the Philadelphia chromosome).

[0255] Other tumor vaccines may include proteins derived from viruses associated with human cancers, such as human papillomavirus (HPV), hepatitis viruses (HBV and HCV), and Kaposi's herpes sarcoma virus (KHSV). Another form of tumor-specific antigen that may be used in combination with PD-L1 blockade is purified heat shock proteins (HSPs) isolated from the tumor tissue itself. These heat shock proteins contain fragments of proteins derived from tumor cells, and these HSPs are remarkably efficient at being delivered to antigen-presenting cells to induce tumor immunity (Suot, R and Srivastava, P (1995) Science 269:1585-1588; Tamura, Y. et al. (1997) Science 278:117-120).

[0256] Dendritic cells (DCs) are potent antigen-presenting cells that can be used to prime antigen-specific responses. DCs can be produced ex vivo and loaded with various protein and peptide antigens and tumor cell extracts (Nestle, F. et al. (1998) Nature Medicine 4:328-332). DCs can also be transduced by genetic means to similarly express these tumor antigens. DCs have also been directly fused to tumor cells for immunization (Kugler, A. et al. (2000) Nature Medicine 6:332-336). As a vaccination method, DC immunization can be effectively combined with PD-L1 blockade to activate stronger anti-tumor responses.

[0257] PD-L1 blockade can also be combined with standard cancer treatments. PD-L1 blockade can be effectively combined with chemotherapy regimens. In these cases, it may be possible to reduce the dose of chemotherapy drugs administered (Mokyr, M. et al. (1998) Cancer Research 58:5301-5304). An example of such a combination is an anti-PD-L1 antibody combined with dacarbazine for the treatment of melanoma. Another example is an anti-PD-L1 antibody combined with interleukin-2 (IL-2) for the treatment of melanoma. The scientific rationale supporting the combination of PD-L1 blockade and chemotherapy is that cell death, which results from the cytotoxic action of most chemotherapeutic compounds, should result in increased levels of tumor antigens in the antigen presentation pathway. Other combination therapies that may synergize with PD-L1 blockade through cell death are radiation therapy, surgery, and hormone suppression. Each of these protocols creates a source of tumor antigens in the host. Angiogenesis inhibitors may also be combined with PD-L1 blockade. Inhibition of angiogenesis can lead to tumor cell death, thereby delivering tumor antigens into the host's antigen presentation pathway.

[0258] PD-L1 blocking antibodies can also be used in combination with bispecific antibodies that target effector cells expressing Fcα or Fcγ receptors to tumor cells (see, e.g., U.S. Patent Nos. 5,922,845 and 5,837,243). Bispecific antibodies can be used to target two separate antigens. For example, anti-Fc receptor / anti-tumor antigen (e.g., Her-2 / neu) bispecific antibodies have been used to target macrophages to tumor sites. This targeting can more effectively activate tumor-specific responses. These responses of T cell arms are likely to be enhanced by the use of PD-L1 blockade. Alternatively, antigens can be delivered directly to DCs using bispecific antibodies that bind to tumor antigens and cell surface markers specific to dendritic cells.

[0259] Tumors evade host immune surveillance through a wide variety of mechanisms. Many of these mechanisms can be overcome by inactivating proteins expressed by tumors that are immunosuppressive. These include, among others, TGF-β (Kehrl, J. et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard, M. and O'Garra, A. (1992) Immunology Today 13:198-200), and Fas ligand (Hahne, M. et al. (1996) Science 274:1363-1365). Antibodies against each of these entities can be used in combination with anti-PD-L1 to counteract the effects of immunosuppressants and promote host immune responses to tumors.

[0260] Other antibodies that can be used to activate host immune responsiveness can be used in combination with anti-PD-L1. These include molecules on the surface of dendritic cells that activate DC function and antigen presentation. Anti-CD40 antibodies can effectively substitute for T cell helper activity (Ridge, J. et al. (1998) Nature 393:474-478) and can be used in combination with PD-L1 antibodies (Ito, N. et al. (2000) Immunobiology 201(5)527-40). Increased levels of T cell activation can also be achieved by activating antibodies against T cell costimulatory molecules such as OX-40 (Weinberg, A. et al. (2000) Immunol 164:2160-2169), 4-1BB (Melero, I. et al. (1997) Nature Medicine 3:682-685(1997), and ICOS (Hutloff, A. et al. (1999) Nature 397:262-266), as well as antibodies that block the activity of negative costimulatory molecules such as CTLA-4 (e.g., U.S. Pat. No. 5,811,097) or BTLA (Watanabe, N. et al. (2003) Nat Immunol 4:670-9), B7-H4 (Sica, GL et al. (2003) Immunity 18:849-61).

[0261] Bone marrow transplantation is currently used to treat a variety of tumors derived from the hematopoietic system. Although graft-versus-host disease is a consequence of this treatment, therapeutic benefit can be obtained from a graft-versus-tumor response. PD-L1 blockade can be used to enhance the efficacy of transplanted tumor-specific T cells from the donor.

[0262] For antigen-specific T cells against tumors, several experimental therapeutic protocols also involve ex vivo activation and expansion of antigen-specific T cells and adoptive transfer of these cells into recipients (Greenberg, R. and Riddell, S. (1999) Science 285:546-51). These methods can also be used to activate T cell responses to infectious agents such as CMV. Ex vivo activation in the presence of anti-PD-L1 antibodies can be expected to enhance the frequency and activity of adoptively transferred T cells.

[0263] infectious disease Other methods of the invention are used to treat patients who have been exposed to a particular toxin or pathogen. Accordingly, another aspect of the invention provides a method of treating an infectious disease in a subject, comprising administering to the subject an anti-PD-L1 antibody, or an antigen-binding portion thereof, such that the infectious disease in the subject is treated. Preferably, the antibody is a human anti-human PD-L1 antibody (such as any of the human anti-PD-L1 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric or humanized antibody.

[0264] Similar to the tumor application described above, antibody-mediated PD-L1 blockade can be used as an adjuvant, alone or in combination with vaccines, to stimulate immune responses against pathogens, toxins, and self-antigens. Examples of pathogens for which this therapeutic approach may be particularly useful include those for which there are currently no effective vaccines or for which conventional vaccines are not sufficiently effective. These include, but are not limited to, HTV, hepatitis (A, B, and C), influenza, herpes, giardia, malaria, leishmania, Staphylococcus aureus, and Pseudomonas aeruginosa. PD-L1 blockade is particularly useful against established infections caused by pathogens such as HIV, which exhibit antigenic shifts during the course of the infection. These novel epitopes are recognized as foreign upon administration of anti-human PD-L1, thus eliciting robust T cell responses that are not suppressed by PD-L1-mediated negative signaling.

[0265] Some examples of pathogenic viruses that cause infections treatable by the methods of the present invention include HIV, hepatitis (A, B, or C), herpesviruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus), adenovirus, influenza virus, flavivirus, echovirus, rhinovirus, coxsackievirus, cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, molluscum contagiosum virus, poliovirus, rabies virus, JC virus, and arboviral encephalitis virus.

[0266] Some examples of pathogenic bacteria that cause infections treatable by the methods of the present invention include chlamydia, rickettsia, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthracis, Yersinia pestis, Leptospira, and Bordetella burgdorferi.

[0267] Some examples of pathogenic fungi that cause infections treatable by the methods of the present invention include Candida (e.g., albicans, krusei, glabrata, tropicalis), Cryptococcus neoformans, Aspergillus (e.g., fumigatus, niger), Mucorales (e.g., mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, and the like. brasiliensis, Coccidioides immitis, and Histoplasma capsulatum.

[0268] Some examples of pathogenic parasites that cause infections treatable by the methods of the present invention include Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia lamblia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, Nippostrongylus brasiliensis, and the like. brasiliensis).

[0269] In all of the above methods, PD-L1 blockade can be combined with other forms of immunotherapy, such as cytokine therapy (e.g., interferon, GM-CSF, G-CSF, IL-2), or bispecific antibody therapy to enhance tumor antigen presentation (see, e.g., Holliger (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123).

[0270] Autoimmune reactions Anti-PD-L1 antibodies can provoke and amplify autoimmune responses. Indeed, induction of anti-tumor responses using tumor cell and peptide vaccines has revealed that many anti-tumor responses are accompanied by anti-self reactivity (such as the hypopigmentation observed with anti-CTLA-4+GM-CSF-modified B16 melanoma by van Elsas et al., supra; hypopigmentation in mice vaccinated with Trp-2 (Overwijk, W. et al. (1999) Proc. Natl. Acad. Sci. USA 96:2982-2987); TRAMP tumor cell vaccine (Hurwitz, A. (2000) supra), autoimmune prostatitis induced by melanoma peptide antigen vaccination, and vitiligo observed in human clinical trials (Rosenberg, SA and White, DE (1996) J. Immunother Emphasis Tumor Immunol 19(1):81-4)).

[0271] Therefore, it is possible to consider using anti-PD-L1 blockade with various self-proteins to devise vaccination protocols that efficiently generate immune responses against these self-proteins for disease treatment. For example, Alzheimer's disease is associated with the inappropriate accumulation of Aβ peptides in amyloid deposits in the brain; antibody responses against amyloid can clear these amyloid deposits (Schenk et al., (1999) Nature 400:173-177).

[0272] Other self-proteins, such as IgE for the treatment of allergies and asthma and TNFα for rheumatoid arthritis, can also be used as targets. Finally, antibody responses against various hormones can be induced by the use of anti-PD-L1 antibodies. Neutralizing antibody responses against reproductive hormones can be used for contraception. Neutralizing antibody responses against hormones and other soluble factors required for the growth of certain tumors can also be considered as possible vaccination targets.

[0273] Similar methods described above for using anti-PD-L1 antibodies can be used to induce therapeutic autoimmune responses to treat patients with inappropriate accumulation of amyloid deposits, including Aβ in Alzheimer's disease, cytokines such as TNFα, and other autoantigens, such as IgE.

[0274] vaccine Anti-PD-L1 antibodies can be used to stimulate antigen-specific immune responses by co-administration of the anti-PD-L1 antibody with an antigen of interest (e.g., a vaccine). Accordingly, in another aspect, the present invention provides a method for enhancing an immune response to an antigen in a subject, comprising administering to the subject (i) the antigen and (ii) an anti-PD-L1 antibody, or an antigen-binding portion thereof, such that the immune response to the antigen in the subject is enhanced. Preferably, the antibody is a human anti-human PD-L1 antibody (such as any of the human anti-PD-L1 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric or humanized antibody. The antigen may be, for example, a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen. Non-limiting examples of such antigens include those discussed in the sections above, such as the tumor antigens (or tumor vaccines) described above, or antigens derived from the aforementioned viruses, bacteria, or other pathogens.

[0275] Anti-PD-L1 antibodies may also be used to inhibit secondary effects associated with the disease, such as the T cell-mediated wasting syndrome associated with colitis (Kanai et al. (2003) J. Immunol. 171:4156-63). Thus, in another aspect, the invention provides a method of inhibiting leukocyte infiltration and reducing the production of IFN-γ, IL-2, and IFN-α by T cells. Preferably, the antibody is a human anti-human PD-L1 antibody (such as any of the human anti-PD-L1 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric or humanized antibody.

[0276] Anti-PD-L1 antibodies may also be used to treat diseases such as chronic inflammatory diseases such as lichen planus, a T cell-mediated chronic inflammatory mucocutaneous disease (Youngnak-Piboonratanakit et al. (2004) Immunol Letters 94:215-22). Thus, in another aspect, the invention provides a method of inhibiting T cell-mediated chronic inflammatory diseases. Preferably, the antibody is a human anti-human PD-L1 antibody (such as any of the human anti-PD-L1 antibodies described herein). Additionally, or alternatively, the antibody may be a chimeric or humanized antibody.

[0277] Suitable routes for administering the antibody compositions of the invention (e.g., human monoclonal antibodies, multispecific and bispecific molecules, and immunoconjugates) in vivo and in vitro are well known in the art and can be selected by one of ordinary skill in the art. For example, antibody compositions can be administered by injection (e.g., intravenously or subcutaneously). The appropriate dosage of the molecule used depends on the age and weight of the subject, as well as the concentration and / or formulation of the antibody composition.

[0278] As described above, the human anti-PD-L1 antibodies of the invention may be co-administered with one or more other therapeutic agents, such as cytotoxic agents, radiotoxic agents, or immunosuppressive agents. The antibody may be linked to the agent (as an immunocomplex) or administered separately from the agent. In the latter case (separate administration), the antibody may be administered before, after, or simultaneously with the agent, or may be administered simultaneously with other known therapies, such as anti-cancer therapies, such as radiation therapy. Such therapeutic agents include, among others, anti-tumor agents such as doxorubicin (adriamycin), cisplatin bleomycin sulfate, carmustine, chlorambucil, and cyclophosphamide hydroxyurea, which, by themselves, are only effective at toxic or subtoxic levels in patients. Cisplatin is administered intravenously at 100 mg / dose once every four weeks, and adriamycin is administered intravenously at a dose of 60 mg / ml to 75 mg / ml once every 21 days. Co-administration of the human anti-PD-L1 antibodies or antigen-binding fragments thereof of the invention with chemotherapeutic agents provides two anti-cancer agents that function via different mechanisms to cause cytotoxicity in human tumor cells, which can overcome problems resulting from the development of drug resistance or antigenic changes in tumor cells that may reduce their responsiveness to antibodies.

[0279] Also within the scope of the present invention are kits comprising the antibody compositions of the invention (e.g., human antibodies, bispecific or multispecific molecules, or immunoconjugates) and instructions for use. The kits may further comprise at least one additional reagent, or one or more additional human antibodies of the invention (e.g., human antibodies with complementary activity that bind to a different epitope in the PD-L1 antigen than the first human antibody). The kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing or recorded material on or supplied with the kit, or which otherwise accompanies the kit.

[0280] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all figures and all references, patents, and published patent applications cited throughout this application are hereby expressly incorporated by reference. [Example]

[0281] Example Example 1: Generation of human monoclonal antibodies against PD-L1 antigen The immunization protocol used both (i) a recombinant fusion protein containing the extracellular portion of PD-L1 and (ii) membrane-bound full-length PD-L1 as antigens, both of which were produced by recombinant transfection in a CHO cell line.

[0282] Transgenic mice (KM-Mouse® colony) Fully human monoclonal antibodies against PD-L1 were prepared using the KM strain of transgenic mice expressing human antibody genes. In this mouse strain, the endogenous mouse kappa light chain gene is homozygously disrupted as described in Chen et al. (1993) EMBO J. 12:811-820, and the endogenous mouse heavy chain gene is homozygously disrupted as described in Example 1 of PCT Publication WO 01 / 09187. In addition, this mouse strain harbors the human kappa light chain transgene KCo5, described in Fishwild et al. (1996) Nature Biotechnology 14:845-851, and the SC20 transchromosome, described in PCT Publication WO 02 / 43478.

[0283] Immunization of KM Mouse® To generate fully human monoclonal antibodies against PD-L1, cohorts of KM Mouse® strain mice were immunized with purified recombinant PD-L1-Ig as antigen and PD-L1-transfected CHO cells. The general immunization scheme for HuMab mice is described in Lonberg, N. et al. (1994) Nature 368(6474):856-859; Fishwild, D. et al. (1996) Nature Biotechnology 14:845-851, and PCT Publication WO 98 / 24884. Mice were 6-16 weeks old at the time of the first injection of antigen. Purified recombinant preparations of PD-L1-Ig antigen (5 μg-50 μg) and 5-10 × 10 6 The cells were used to immunize HuMab mice intraperitoneally (IP), subcutaneously (Sc), or by footpad injection.

[0284] Transgenic mice were immunized intraperitoneally twice with antigen in complete Freund's or Ribi adjuvant, followed by intraperitoneal immunizations on days 3 to 21 (maximum total of 11 immunizations) with antigen in incomplete Freund's or Ribi adjuvant. Immune responses were monitored by retroorbital bleeding. Plasma was screened by ELISA (as described below), and mice with sufficient titers of anti-PD-L1 human immunoglobulin were used for fusions. Mice were boosted intravenously with antigen 3 days before sacrifice and pancreatectomy. Typically, 10 to 35 fusions were performed for each antigen. Several dozen mice were immunized for each antigen.

[0285] Selection of KM Mice® producing anti-PD-L1 antibodies To select HuMab mice producing antibodies that bound to PD-L1, sera from immunized mice were tested by ELISA as described by Fishwild, D. et al. (1996). Briefly, microtiter plates were coated with purified recombinant PD-L1 fusion protein from transfected CHO cells (100 μl / well) at 1 μg / ml to 2 μg / ml in PBS, incubated overnight at 4°C, and then blocked with 200 μl / well of 5% fetal bovine serum in PBS / Tween (0.05%). Dilutions of sera from PD-L1-immunized mice were added to each well and incubated for 1 to 2 hours at ambient temperature. The plates were washed with PBS / Tween and then incubated with a goat anti-human IgG polyclonal antibody conjugated to horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with ABTS substrate (Sigma, A-1888, 0.22 mg / ml) and analyzed spectrophotometrically at OD 415-495. Mice that developed the highest titers of anti-PD-L1 antibodies were used for fusions. Fusions were performed as described below, and hybridoma supernatants were tested for anti-PD-L1 activity by ELISA.

[0286] Generation of hybridomas producing human monoclonal antibodies against PD-L1 Mouse splenocytes isolated from KM mice were fused to a mouse myeloma cell line using PEG according to standard protocols. The resulting hybridomas were then screened for the production of antigen-specific antibodies. Single-cell suspensions of splenocytes from immunized mice were fused to one-quarter the number of SP2 / 0 non-secretory mouse myeloma cells (ATCC, CRL 1581) using 50% PEG (Sigma). Approximately 1 × 10 cells were fused. 5Hybridomas were seeded at 1000kJ / well in flat-bottom microtiter plates and subsequently incubated for approximately 2 weeks in selection medium containing 10% fetal bovine serum, 10% P388D1 (ATCC, CRL TIB-63) conditioned medium, 3%-5% origen (IGEN), 0.055 mM 2-mercaptoethanol, 50 mg / ml gentamicin, and 1x HAT (Sigma, CRL P-7185) in DMEM (Mediatech, CRL 10013, containing high glucose, L-glutamine, and sodium pyruvate) and 5 mM HEPES. After 1-2 weeks, cells were cultured in medium in which the HAT was replaced with HT. Individual wells were then screened for human anti-PD-L1 monoclonal IgG antibodies by ELISA (as described above). Once extensive hybridoma growth occurred, medium was monitored, usually after 10-14 days. The antibody-secreting hybridomas were replated, screened again, and if still positive for human IgG, the anti-PD-L1 monoclonal antibodies were subcloned at least twice by limiting dilution. The stable subclones were then cultured in vitro to generate small amounts of antibody in tissue culture medium for further characterization.

[0287] Hybridoma clones 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 were selected for further analysis.

[0288] Example 2: Structural characterization of human monoclonal antibodies 3G10, 12A4, and 10A5 cDNA sequences encoding the heavy and light chain variable regions of the 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 monoclonal antibodies were obtained from the 3G10, 12A4, 10A5, 5F8, 10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 hybridomas, respectively, using standard PCR techniques and sequenced using standard DNA sequencing techniques.

[0289] The nucleotide and amino acid sequences of the heavy chain variable region of 3G10 are shown in Figure 1A and in SEQ ID NO:81 and SEQ ID NO:1, respectively.

[0290] The nucleotide and amino acid sequences of the light chain variable region of 3G10 are shown in Figure 1B and in SEQ ID NO:91 and SEQ ID NO:11, respectively.

[0291] Comparison of the 3G10 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 3G10 heavy chain uses a VH segment derived from human germline VH1-18, an undetermined D segment, and a JH segment derived from human germline JH6b. An alignment of the 3G10 VH sequence to the germline VH1-18 sequence is shown in Figure 11. The 3G10 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figure 1A and Figure 11, and SEQ ID NO: 21, SEQ ID NO: 31, and SEQ ID NO: 41, respectively.

[0292] Comparison of the 3G10 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 3G10 light chain uses a VL segment derived from human germline VKL6 and a JK segment derived from human germline JK1. An alignment of the 3G10 VL sequence to the germline VKL6 sequence is shown in Figure 21. The 3G10 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 1B and Figure 21, and in SEQ ID NO: 51, SEQ ID NO: 61, and SEQ ID NO: 71, respectively.

[0293] The nucleotide and amino acid sequences of the heavy chain variable region of 12A4 are shown in Figure 2A and in SEQ ID NO:82 and SEQ ID NO:2, respectively.

[0294] The nucleotide and amino acid sequences of the light chain variable region of 12A4 are shown in Figure 2B and in SEQ ID NO:92 and SEQ ID NO:12, respectively.

[0295] Comparison of the 12A4 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 12A4 heavy chain uses a VH segment derived from human germline VH1-69, a D segment derived from human germline 3-10, and a JH segment derived from human germline JH6b. An alignment of the 12A4 VH sequence to the germline VH1-69 sequence is shown in Figure 12. The 12A4 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figures 2A and 12, and in SEQ ID NOs: 22, 32, and 42, respectively.

[0296] Comparison of the 12A4 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 12A4 light chain uses a VL segment derived from human germline VKL6 and a JK segment derived from human germline JK1. An alignment of the 12A4 VL sequence to the germline VKL6 sequence is shown in Figure 22. The 12A4 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 2B and Figure 22, and in SEQ ID NO: 52, SEQ ID NO: 62, and SEQ ID NO: 72, respectively.

[0297] The nucleotide and amino acid sequences of the heavy chain variable region of 10A5 are shown in Figure 3A and in SEQ ID NO:83 and SEQ ID NO:3, respectively.

[0298] The nucleotide and amino acid sequences of the light chain variable region of 10A5 are shown in Figure 3B and in SEQ ID NO:93 and SEQ ID NO:13, respectively.

[0299] Comparison of the 10A5 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 10A5 heavy chain uses a VH segment derived from human germline VH1-3, a D segment derived from human germline 5-5, and a JH segment derived from human germline JH4b. An alignment of the 10A5 VH sequence to the germline VH1-3 sequence is shown in Figure 13. The 10A5 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figures 3A and 13, and in SEQ ID NOs: 23, 33, and 43, respectively.

[0300] Comparison of the 10A5 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 10A5 light chain uses a VL segment derived from human germline VK L15 and a JK segment derived from human germline JK2. An alignment of the 10A5 VL sequence to the germline VK L15 sequence is shown in Figure 23. The 10A5 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 3B and Figure 23, and in SEQ ID NO:53, SEQ ID NO:63, and SEQ ID NO:73, respectively.

[0301] The nucleotide and amino acid sequences of the heavy chain variable region of 5F8 are shown in Figure 4A and in SEQ ID NO:84 and SEQ ID NO:4, respectively.

[0302] The nucleotide and amino acid sequences of the light chain variable region of 5F8 are shown in Figure 4B and in SEQ ID NO:94 and SEQ ID NO:14, respectively.

[0303] Comparison of the 5F8 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 5F8 heavy chain uses a VH segment derived from human germline VH1-69, a D segment derived from human germline 6-13, and a JH segment derived from human germline JH4b. An alignment of the 5F8 VH sequence to the germline VH1-69 sequence is shown in Figure 14. The 5F8 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figure 4A and Figure 14, and in SEQ ID NO:24, SEQ ID NO:34, and SEQ ID NO:44, respectively.

[0304] Comparison of the 5F8 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 5F8 light chain uses a VL segment derived from human germline VK A27 and a JK segment derived from human germline JK1. An alignment of the 5F8 VL sequence to the germline VK A27 sequence is shown in Figure 24. The 5F8 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 4B and Figure 24, and in SEQ ID NO:54, SEQ ID NO:64, and SEQ ID NO:74, respectively.

[0305] The nucleotide and amino acid sequences of the heavy chain variable region of 10H10 are shown in Figure 5A and in SEQ ID NO:85 and SEQ ID NO:5, respectively.

[0306] The nucleotide and amino acid sequences of the light chain variable region of 10H10 are shown in Figure 5B and in SEQ ID NO:95 and SEQ ID NO:15, respectively.

[0307] Comparison of the 10H10 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 10H10 heavy chain uses a VH segment derived from human germline VH3-9, a D segment derived from human germline 4-17, and a JH segment derived from human germline JH4b. An alignment of the 10H10 VH sequence to the germline VH 3-9 sequence is shown in Figure 15. The 10H10 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figure 5A and Figure 15, and in SEQ ID NO:25, SEQ ID NO:35, and SEQ ID NO:45, respectively.

[0308] Comparison of the 10H10 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 10H10 light chain uses a VL segment derived from human germline VK L15 and a JK segment derived from human germline JK 2. An alignment of the 10H10 VL sequence to the germline VK L15 sequence is shown in Figure 25. The 10H10 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 5B and Figure 25, and in SEQ ID NO:55, SEQ ID NO:65, and SEQ ID NO:75, respectively.

[0309] The nucleotide and amino acid sequences of the heavy chain variable region of 1B12 are shown in Figure 6A and in SEQ ID NO:86 and SEQ ID NO:6, respectively.

[0310] The nucleotide and amino acid sequences of the light chain variable region of 1B12 are shown in Figure 6B and in SEQ ID NO:96 and SEQ ID NO:16, respectively.

[0311] Comparison of the 1B12 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 1B12 heavy chain uses a VH segment derived from human germline VH1-69, a D segment derived from human germline 3-10, and a JH segment derived from human germline JH6b. An alignment of the 1B12 VH sequence to the germline VH1-69 sequence is shown in Figure 16. The 1B12 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figures 6A and 16, and in SEQ ID NOs: 26, 36, and 46, respectively.

[0312] Comparison of the 1B12 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 1B12 light chain uses a VL segment derived from human germline VK L6 and a JK segment derived from human germline JK1. An alignment of the 1B12 VL sequence to the germline VK L6 sequence is shown in Figure 26. The 1B12 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 6B and Figure 26, and in SEQ ID NO:56, SEQ ID NO:66, and SEQ ID NO:76, respectively.

[0313] The nucleotide and amino acid sequences of the heavy chain variable region of 7H1 are shown in Figure 7A and in SEQ ID NO:87 and SEQ ID NO:7, respectively.

[0314] The nucleotide and amino acid sequences of the light chain variable region of 7H1 are shown in Figure 7B and in SEQ ID NO:97 and SEQ ID NO:17, respectively.

[0315] Comparison of the 7H1 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 7H1 heavy chain uses a VH segment derived from human germline VH1-69, a D segment derived from human germline 3-10, and a JH segment derived from human germline JH6b. An alignment of the 7H1 VH sequence to the germline VH1-69 sequence is shown in Figure 17. The 7H1 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figures 7A and 17, and in SEQ ID NOs:27, 37, and 47, respectively.

[0316] Comparison of the 7H1 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 7H1 light chain uses a VL segment derived from human germline VK L6 and a JK segment derived from human germline JK1. An alignment of the 7H1 VL sequence to the germline VK L6 sequence is shown in Figure 27. The 7H1 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 7B and Figure 27, and in SEQ ID NO:57, SEQ ID NO:67, and SEQ ID NO:77, respectively.

[0317] The nucleotide and amino acid sequences of the heavy chain variable region of 11E6 are shown in Figure 4A and in SEQ ID NO:84 and SEQ ID NO:4, respectively.

[0318] The nucleotide and amino acid sequences of the light chain variable region of 11E6 are shown in Figure 4B and in SEQ ID NO:94 and SEQ ID NO:14, respectively.

[0319] Comparison of the 11E6 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 11E6 heavy chain uses a VH segment derived from human germline VH1-69, a D segment derived from human germline 6-19, and a JH segment derived from human germline JH6c. An alignment of the 11E6 VH sequence to the germline VH1-69 sequence is shown in Figure 18. The 11E6 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figures 8A and 18, and in SEQ ID NOs: 28, 38, and 48, respectively.

[0320] Comparison of the 11E6 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 11E6 light chain uses a VL segment derived from human germline VK A27 and a JK segment derived from human germline JK4. An alignment of the 11E6 VL sequence to the germline VK A27 sequence is shown in Figure 27. The 11E6 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figures 8B and 28, and in SEQ ID NOs: 58, 68, and 78, respectively. Additionally, a second related clone contained the VK sequence shown in SEQ ID NO: 109. This antibody is referred to herein as 11E6a.

[0321] The nucleotide and amino acid sequences of the heavy chain variable region of 12B7 are shown in Figure 9A and in SEQ ID NO:89 and SEQ ID NO:9, respectively.

[0322] The nucleotide and amino acid sequences of the light chain variable region of 12B7 are shown in Figure 9B and in SEQ ID NO:99 and SEQ ID NO:19, respectively.

[0323] Comparison of the 12B7 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 12B7 heavy chain uses a VH segment derived from human germline VH1-69, a D segment derived from human germline 3-10, and a JH segment derived from human germline JH6b. An alignment of the 12B7 VH sequence to the germline VH1-69 sequence is shown in Figure 19. The 12B7 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figures 9A and 19, and in SEQ ID NOs: 29, 39, and 49, respectively.

[0324] Comparison of the 12B7 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 12B7 light chain uses a VL segment derived from human germline VK L6 and a JK segment derived from human germline JK5. An alignment of the 12B7 VL sequence to the germline VK L6 sequence is shown in Figure 29. The 12B7 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 9B and Figure 29, and in SEQ ID NO: 59, SEQ ID NO: 69, and SEQ ID NO: 79, respectively.

[0325] The nucleotide and amino acid sequences of the heavy chain variable region of 13G4 are shown in Figure 10A and in SEQ ID NO:90 and SEQ ID NO:10, respectively.

[0326] The nucleotide and amino acid sequences of the light chain variable region of 13G4 are shown in Figure 10B and in SEQ ID NO:100 and SEQ ID NO:20, respectively.

[0327] Comparison of the 13G4 heavy chain immunoglobulin sequence with known human germline immunoglobulin heavy chain sequences demonstrated that the 13G4 heavy chain uses a VH segment derived from human germline VH3-9, a D segment derived from human germline 3-9, and a JH segment derived from human germline JH4b. An alignment of the 13G4 VH sequence to the germline VH3-9 sequence is shown in Figure 20. The 13G4 VH sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the heavy chain were delineated as shown in Figure 10A and Figure 20, and in SEQ ID NO: 30, SEQ ID NO: 40, and SEQ ID NO: 50, respectively.

[0328] Comparison of the 13G4 light chain immunoglobulin sequence with known human germline immunoglobulin light chain sequences demonstrated that the 13G4 light chain uses a VL segment derived from human germline VK L18 and a JK segment derived from human germline JK3. An alignment of the 13G4 VL sequence to the germline VK L18 sequence is shown in Figure 30. The 13G4 VL sequence was further analyzed using the Kabat system for determining CDR regions, and the CDR1, CDR2, and CD3 regions of the light chain were delineated as shown in Figure 10B and Figure 30, and in SEQ ID NO: 60, SEQ ID NO: 70, and SEQ ID NO: 80, respectively.

[0329] Example 3 Characterization of the binding specificity and binding kinetics of anti-PD-L1 human monoclonal antibodies In this example, the binding affinity and binding kinetics of anti-PD-L1 antibodies were examined by Biacore analysis, and the binding specificity and cross-competition were examined by flow cytometry.

[0330] Binding affinity and binding kinetics Anti-PD-L1 antibodies were characterized for affinity and binding kinetics by Biacore analysis (Biacore AB, Uppsala, Sweden). Purified recombinant human PD-L1 fusion protein was covalently coupled to a CM5 chip (a chip coated with carboxymethyldextran) via primary amines to a density of 562 RU using standard amine coupling chemistry and a kit provided by Biacore. Binding was measured by flowing 133 nM antibody in HBS EP buffer (provided by Biacore AB) at a flow rate of 50 μl / min. Antigen-antibody binding kinetics was followed for 1 minute, and dissociation kinetics was followed for 1 minute. Binding and dissociation curves were fitted to a 1:1 Langmuir binding model using BIA evaluation software (Biacore AB). To minimize the effect of binding activity on estimating the binding constant, only the initial portions of the data corresponding to the association and dissociation phases were used for fitting. The determined K D , k on , and k off The values ​​are shown in Table 2.

[0331] Table 2. Biacore binding data for PD-L1 human monoclonal antibodies TIFF2024174925000003.tif21150

[0332] Additional binding data obtained by equilibrium binding methods and analyzed with GraphPad Prizm are shown in Table 3.

[0333] Table 3. Biacore equilibrium binding data for PD-L1 human monoclonal antibodies TIFF2024174925000004.tif61128

[0334] Binding specificity by flow cytometry A Chinese hamster ovary (CHO) cell line expressing recombinant human PD-L1 on the cell surface was developed and used to determine the specificity of PD-L1 human monoclonal antibodies by flow cytometry. CHO cells were transfected with an expression plasmid containing a full-length cDNA encoding transmembrane PD-L1. Binding of the 3G10, 10A5, and 12A4 anti-PD-L1 human monoclonal antibodies was assessed by incubating the transfected cells with anti-PD-L1 human monoclonal antibodies. The cells were washed, and binding was detected using an FITC-labeled anti-human IgG Ab. Flow cytometry analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Binding was compared to the parental CHO cell line. The results are shown in Figure 32A (HuMAb 3G10), Figure 32B (HuMAb 10A5), and Figure 32C (HuMAb 12A4). Binding was also tested using various concentrations of anti-PD-L1 antibodies. The results are shown in Figure 33. The anti-PD-L1 human monoclonal antibodies 3G10, 10A5, and 12A4 bound to PD-L1-transfected CHO cells in a concentration-dependent manner. These data demonstrate that the anti-PD-L1 human monoclonal antibodies specifically bind to cell-surface PD-L1.

[0335] Binding specificity by ELISA The specificity of the anti-PD-L1 monoclonal antibodies was determined for binding to human PD-L1 fusions to the immunoglobulin Fc region using a standard ELISA assay.

[0336] Human PD-L1 Fc fusion proteins were tested for binding to the anti-PD-L1 human monoclonal antibodies 3G10, 10A5, and 12A4. Standard ELISA procedures were performed. Various concentrations of anti-PD-L1 human monoclonal antibodies were added. A goat anti-human IgG (κ chain specific) polyclonal antibody conjugated with horseradish peroxidase (HRP) was used as the secondary antibody. The results are shown in Figure 34. The anti-PD-L1 human monoclonal antibodies 3G10, 12A4, and 10A5 each bound to PD-L1 with high specificity.

[0337] Example 4: Characterization of anti-PD-L1 antibodies that bind to PD-L1 expressed on the cell surface of human and monkey T cells Anti-PD-L1 antibodies were tested by flow cytometry for binding to activated human or cynomolgus monkey T cells expressing PD-L1 on their surface.

[0338] Human T cells or monkey T cells were activated with anti-CD3 antibody to induce PD-L1 expression and then bound to human anti-PD-L1 monoclonal antibodies. The binding of the 3G10, 1B12, 13G4, and 12A4 anti-PD-L1 human monoclonal antibodies was assessed by incubating the activated cells with serial dilutions of the anti-PD-L1 human monoclonal antibodies. An isotype control antibody was used as a negative control. The cells were washed, and binding was detected using an FITC-labeled anti-human Igκ light chain Ab. Flow cytometry analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figure 35 and Figure 36. The anti-PD-L1 human monoclonal antibodies 3G10, 1B12, 13G4, and 12A4 bound to activated human T cells and monkey T cells. These data demonstrate that the anti-PD-L1 human monoclonal antibodies bind to human and cynomolgus monkey cell surface PD-L1.

[0339] Example 5:Characterization of anti-PD-L1 antibodies that bind to PD-L1 expressed on the cell surface of human T cells Anti-PD-L1 antibodies were tested by flow cytometry for binding to activated human T cells expressing PD-L1 on the cell surface.

[0340] Human T cells were activated with anti-CD3 antibody to induce PD-L1 expression on the T cells, followed by binding with human anti-PD-L1 monoclonal antibodies. Activated T cells were incubated with anti-PD-L1 human monoclonal antibodies at a concentration of 20 μg / ml to assess binding of the 3G10, 10A5, and 12A4 anti-PD-L1 human monoclonal antibodies. An isotype control antibody was used as a negative control. The cells were washed, and binding was detected using an FITC-labeled anti-human IgG Ab. Flow cytometry analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figure 37A (HuMAb 3G10), Figure 37B (HuMAb 10A5), and Figure 37C (HuMAb 12A4). The anti-PD-L1 monoclonal antibodies 3G10, 10A5, and 12A4 bound to activated human T cells (thick line) as shown in the histogram plots compared to the control (thin line). These data demonstrate that the anti-PD-L1 human monoclonal antibodies bind to human cell-surface PD-L1.

[0341] Example 6: Binding specificity by flow cytometry The specificity of PD-L1 human monoclonal antibodies was determined by flow cytometry using the ES-2 human ovarian cancer cell line, which expresses human PD-L1 on the cell surface. ES-2 cells were treated overnight with 500 IU / mL recombinant hIFN-γ to increase PD-L1 expression above basal levels. Binding of the 12A4, 1B12, 3G10, 10A5, 12B7, 13G4, 11E6, and 5F8 anti-PD-L1 human monoclonal antibodies was assessed by incubating the induced cells with serial dilutions of the anti-PD-L1 human monoclonal antibodies. The cells were washed, and binding was detected using a PE-labeled anti-human IgG Ab. Flow cytometry analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). Binding was compared to an isotype control antibody. The results are shown in Figure 38. The anti-PD-L1 human monoclonal antibodies 12A4, 1B12, 3G10, 10A5, 12B7, 13G4, 11E6, and 5F8 bound to hIFN-γ-induced ES-2 cells in a concentration-dependent manner. These data demonstrate that the anti-PD-L1 human monoclonal antibodies specifically bind to cell-surface PD-L1.

[0342] Example 7: Effects of human anti-PD-L1 antibodies on cell proliferation and cytokine production in mixed lymphocyte reactions A mixed lymphocyte reaction was used to demonstrate the effect of PD-L1 / PD-1 pathway blockade on lymphocyte effector cells. T cells were tested in assays for proliferation, IFN-γ secretion, and IL-2 secretion in the presence or absence of an anti-PD-L1 human monoclonal antibody.

[0343] Human CD4+ T cells were purified from PBMCs using a CD4+ positive selection kit (Dynal Biotech). Dendritic cells were derived from purified monocytes cultured for 7 days with 1000 U / ml IL-4 and 500 U / ml GM-CSF (R&D Biosystems). Monocytes were prepared using a monocyte negative selection kit (Mitenyi Biotech). Each culture contained 10 cells in a total volume of 200 μl. 5 10 purified T cells and 10 4 Each culture contained 100 μl of allogeneic dendritic cells. Various antibody concentrations of anti-PD-L1 monoclonal antibodies 10A5, 12A4, or 3G10 were added to each culture. Either no antibody or an isotype control antibody was used as a negative control. These cells were cultured at 37°C for 5 days. After 5 days, 100 μl of medium was collected from each culture for cytokine measurement. IFN-γ and IL-2 levels were measured using an OptEIA ELISA kit (BD Biosciences). These cells 3 The cells were labeled with 3H-thymidine, cultured for an additional 18 hours, and analyzed for cell proliferation. The results are shown in Figure 39A (T cell proliferation), Figure 39B (IFN-γ secretion with HuMAb 10A5), Figure 39C (IFN-γ secretion with HuMAb 12A4 or HuMAb 3G10), and Figure 39D (IL-2 secretion). The anti-PD-L1 human monoclonal antibody 10A5 promoted T cell proliferation, IFN-γ secretion, and IL-2 secretion in a concentration-dependent manner. The anti-PD-L1 human monoclonal antibodies 12A4 and 3G10 also showed increased IFN-γ secretion. In contrast, cultures containing control antibodies did not show increased T cell proliferation, IFN-γ secretion, or IL-2 secretion.

[0344] In another experiment, an allogeneic mixed lymphocyte reaction (MLR) was used to demonstrate the effect of PD-L1 / PD-1 pathway blockade on lymphocyte effector cells. T cells were tested in the assay for proliferation and IFN-γ secretion in the presence or absence of an anti-PD-L1 human monoclonal antibody or an isotype control antibody.

[0345] Human CD4+ T cells were purified from PBMCs using a CD4+ negative selection kit (Miltenyi). Monocytes were prepared using a monocyte negative selection kit (Mitenyi Biotech). Dendritic cells were derived from purified monocytes cultured for 7 days with 1000 U / ml IL-4 and 500 U / ml GM-CSF (R&D Biosystems). Each MLR culture contained 10 cells in a total volume of 200 μl. 5 10 purified T cells and 10 4 Each culture contained 1000 allogeneic dendritic cells. Various antibody concentrations of the anti-PD-L1 monoclonal antibodies 12A4, 11E6, 3G10, 13G4, 1B12, 10A5, and 12B7 were added to each culture. Either no antibody or an isotype control antibody was used as a negative control. The cells were cultured at 37°C for 5 days. On day 5, 50μl of medium was collected from each culture for cytokine measurement, and 1μCi of 3 The medium was replaced with an equal volume of medium containing 3H-thymidine. The cells were cultured for an additional 18 hours, harvested, and analyzed for cell proliferation. IFN-γ levels in the culture medium were measured using an OptEIA hIFN-γ ELISA kit (BD Biosciences). The results are shown in Figure 40. Anti-PD-L1 human monoclonal antibodies promote T cell proliferation and IFN-γ secretion in a concentration-dependent manner. In contrast, cultures containing a control antibody showed no increase in T cell proliferation or IFN-γ secretion.

[0346] Example 8: The effect of human anti-PD-L1 antibodies on regulatory T cell function Regulatory T cells (CD4+, CD25+) are lymphocytes that suppress immune responses. We examined the effect of adding regulatory T cells on proliferation and IFN-γ secretion in MLRs of allogeneic dendritic cells and T cells in the presence or absence of an anti-PD-L1 human monoclonal antibody.

[0347] Regulatory T cells were purified from PBMCs using a CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec). Regulatory T cells were added to a mixed lymphocyte reaction (MLL) containing purified CD4+CD25- T cells and allogeneic dendritic cells at a 2:1 ratio of CD4+CD25- cells to regulatory T cells (see above). The anti-PD-L1 monoclonal antibody 10A5 was added to each culture at a concentration of 10 μg / mL. Either no antibody or an isotype control antibody was used as a negative control. The cells were cultured at 37°C for 5 days, at which time the supernatants were analyzed for IFN-γ secretion using the Beadlyte cytokine detection system (Upstate). The cells were cultured at 37°C for 5 days, at which time the supernatants were analyzed for IFN-γ secretion using the Beadlyte cytokine detection system (Upstate). 3 The cells were labeled with H-thymidine, cultured for an additional 18 hours, and analyzed for cell proliferation. The results are shown in Figure 41A (T cell proliferation) and Figure 41B (IFN-γ secretion). Addition of the anti-PD-L1 human monoclonal antibody 10A5 promoted both T cell proliferation and IFN-γ secretion in cell cultures of allogeneic dendritic cells, T cells, and regulatory T cells, indicating that anti-PD-L1 antibodies can reverse the effects of regulatory T cells in allogeneic DC-T cell-MLR.

[0348] In separate experiments, the human anti-PD-L1 antibodies 12A4 and 13G4, as well as the control antibody 1D12, were tested in an MLR assay using regulatory T cells. The results are shown in Figure 42 (T cell proliferation) and Figure 43 (IFN-γ secretion). The addition of anti-PD-L1 human monoclonal antibodies 12A4 or 13G4 partially reversed the inhibition of both T cell proliferation and IFN-γ secretion in cell cultures of allogeneic dendritic cells and T cells containing regulatory T cells, indicating that anti-PD-L1 antibodies can act on regulatory T cells.

[0349] Example 9: Effect of anti-PD-1 antibody on cytokine secretion by viral antigen-stimulated PBMC cells from positive CMV-responsive donors CMV antigen-responsive human PBMCs (Astarte Biologics, Redmond, WA) were cultured at 2e5 cells / well in TC-treated flat-bottom 96-well plates in the presence of 0.5µg / ml CMV lysate (Astarte Biologics) + / - titrated anti-PD-L1 antibodies. AIM-V medium (Invitrogen) supplemented with heat-inactivated FBS (final concentration 10%) was used in a total volume of 200µl / well. The cells were cultured at 37°C and 5% CO2 for 4 days, at which time culture supernatants were harvested for measurement of secreted interferon-γ by ELISA (OptEIA hIFN-γ ELISA kit, BD Biosciences). The results are shown in Figure 44. Anti-PD-L1 human monoclonal antibodies stimulate IFN-γ secretion by CMV-specific T cells in a dose-dependent manner. The strongest responses compared to the isotype control were generated by antibodies 13G4, 1B12, and 12A4. These results indicate that anti-PD-L1 HuMAbs can stimulate IFN-γ release in memory T cell responses from PBMC cells pre-stimulated against an antigen.

[0350] Example 10: Blockage of PD-L1 ligand binding to PD-1 by human anti-PD-L1 antibodies Anti-PD-L1 human monoclonal antibodies were tested for their ability to block the binding of the ligand PD-L1 to PD-1 expressed on transfected CHO cells by using a cytometric assay.

[0351] PD-1-expressing CHO cells were suspended in FACS buffer (PBS containing 4% fetal bovine serum). Various concentrations of anti-PD-L1 HuMAbs 3G10, 10A5, or 12A4 were added to the cell suspension tubes for 30 minutes at 4°C, followed by the addition of FITC-labeled PD-L1 fused to an immunoglobulin Fc region. Flow cytometry analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figure 45. The anti-PD-L1 monoclonal antibodies 3G10, 10A5, and 12A4 blocked the binding of PD-L1 to CHO cells transfected with human PD-1, as measured by the mean fluorescent intensity (MFI) of staining. These data demonstrate that anti-PD-L1 HuMAbs block the binding of the PD-L1 ligand to cell-surface PD-1.

[0352] Example 11: Inhibition of soluble PD-1 binding to cell surface PD-L1 by human anti-PD-L1 antibodies Anti-PD-L1 human monoclonal antibodies were tested for their ability to block the binding of a soluble dimeric form of the PD-1 receptor (PD-1-hFc) to PD-L1 expressed on hIFN-γ-induced ES-2 human ovarian cancer cells using a flow cytometry assay, and blockade was compared to an isotype control antibody.

[0353] ES-2 cells were induced with 500 IU / mL hIFN-γ overnight to upregulate cell surface expression of hPD-L1. Induced cells were suspended in FACS buffer. Serial dilutions of anti-PD-L1 HuMAbs 12A4, 1B12, 3G10, 10A5, 12B7, 13G4, 11E6, and 5F8 were added to the cell suspension tubes for 30 minutes at 4°C, followed by two washes to remove unbound antibody. PD-1-hFc protein was then added to all wells at a constant concentration of 2 μg / mL for 30 minutes at 4°C, followed by two washes to remove unbound PD-1-hFc. Next, biotinylated, non-blocking anti-PD-1 HuMab 26D5, which binds to PD-1 when bound to PD-L1, was added for 30 minutes at 4°C, followed by two washes to remove unbound antibody, to detect bound PD-1-Fc on ES-2 cells. Finally, streptavidin-PE conjugate was added for 30 minutes at 4°C, followed by two washes to remove unbound conjugate, to detect bound 26D5 antibody. Flow cytometry analysis was performed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA). The results are shown in Figure 46. The anti-PD-L1 monoclonal antibodies 12A4, 1B12, 3G10, 10A5, 12B7, 13G4, 11E6, and 5F8 interfered with PD-L1 binding to ES-2 cells expressing human PD-1, as measured by the geometric mean fluorescence intensity (GMFI) of staining. These data demonstrate that the anti-PD-L1 HuMAbs interfere with the binding of soluble PD-L1 receptor to cell-surface PD-1.

[0354] Example 12: Treatment of in vivo tumor models with anti-PD-L1 antibodies Mice bearing cancerous tumors were treated in vivo with anti-PD-L1 antibodies to examine the in vivo effects of the antibodies on tumor growth. For tumor studies, female AJ mice (Harlan Laboratories) between 6 and 8 weeks of age were randomly divided into six groups based on body weight. On day 0, 2 x 10 SA1 / N fibrosarcoma cells dissolved in 200 μl of DMEM medium were injected into the mice. 6The mice are subcutaneously implanted with 100 μl of antibody-containing PBS or vehicle via intraperitoneal injection on days 1, 4, 8, and 11. Each group contains 10 animals and consists of (i) a vehicle group, (ii) control mouse IgG, and (iii) anti-PD-L1 antibody. The mice are monitored twice weekly for tumor growth for approximately 6 weeks. Using electronic calipers, the three dimensions of the tumor are measured (height x width x length), and tumor volume is calculated. When the tumor reaches the tumor endpoint (1500 mm), the tumor volume is calculated. 3 Euthanize mice when they reach 0.05 mg / kg body weight (BMI) or show more than 15% weight loss.

[0355] Example 13: In vivo efficacy of combination therapy (anti-CTLA-4 antibody and anti-PD-L1 antibody) on tumor establishment and growth MC38 colorectal cancer cells (available from Dr. N. Restifo, National Cancer Institute, Bethesda, MD; or Jeffrey Schlom, National Institutes of Health, Bethesda, MD) were implanted into C57BL / 6 mice (2 × 10 6 cells / mouse) and tumor size is 100 mm 3 ~200mm 3Mice were selected for treatment when they reached a median age of 10. On day 0 (i.e., the first day of treatment), four groups of 10 mice each received an intraperitoneal (IP) injection of one of the following: (1) 10 mg / kg mouse IgG and 10 mg / kg rat IgG (control), (2) 10 mg / kg anti-CTLA-4 monoclonal antibody 9D9 (mouse anti-mouse CTLA-4, obtained from J. Allison, Memorial Sloan-Kettering Cancer Center, New York, NY) and 10 mg / kg rat IgG, (3) anti-PD-L1 monoclonal antibody MIH5 (rat anti-mouse PD-L1, eBioscience) and 10 mg / kg mouse IgG, or (4) 10 mg / kg anti-CTLA-4 antibody 9D9 and 10 mg / kg anti-PD-L1 antibody MIH5. Further antibody injections were then administered on days 3 and 6. Using electronic calipers, three dimensions of the tumor were measured (height x width x length), and tumor volume was calculated. Mice were euthanized when tumors reached the designated tumor endpoints. The results are shown in Figure 47.

[0356] This study shows that in the MC38 mouse tumor model, anti-PD-L1 antibody treatment alone has a modest effect on tumor growth, resulting in tumor growth delay, whereas anti-CTLA-4 has little effect in this model. However, combined treatment with CTLA-4 and PD-L1 antibodies has a significantly greater effect on tumor growth, resulting in tumor-free mice.

[0357] Example 14: Immunohistochemistry using anti-PD-L1 antibody To evaluate the tissue binding profiles of HuMabs anti-PD-L1, unmodified 12A4, 13G4, 3G10, and 12B7 were tested in a panel of normal (non-tumorous) human tissues (one sample per tissue), including spleen, tonsils, cerebrum, cerebellum, heart, liver, lung, kidney, pancreas, pituitary gland, skin, epidermis, and small intestine, as well as lung cancer tissues. ES-2 cells were used as a positive control. Hu-IgG1 and Hu-IgG4 were used as isotype control antibodies.

[0358] Snap-frozen, OCT-embedded normal and tumor tissues were purchased from the Cooperative Human Tissue Network (Philadelphia, PA) or the National Disease Research Institute (Philadelphia, PA). Five-micron cryostat sections were fixed in acetone at room temperature for 10 minutes and stored at -80°C until use. An immunohistochemistry protocol developed by Medarex was performed using unmodified HuMab anti-PD-L1, with primary antibodies (12A4, 13G4, 3G10, and 12B7) and secondary antibodies (FITC-conjugated Fab fragment of goat anti-Hu-IgG, Jackson ImmunoResearch Laboratories, West Grove, PA) pre-complexed prior to application to the sections. Briefly, 1 μg / ml or 5 μg / ml of unconjugated primary antibody was mixed with a threefold excess of secondary antibody and incubated at room temperature for 30 minutes. An excess of human gamma globulin was then added for another 30 minutes to block unbound secondary antibody. In parallel, the isotype control antibodies Hu-IgG1 or Hu-IgG4 were similarly pre-complexed. The slides were washed twice with PBS (Sigma, St. Louis, MO) and then incubated for 10 minutes with the peroxidase block provided in the Dako EnVision+ System (Dako, Carpinteria, CA). After two PBS washes, the slides were incubated with Dako protein block to block nonspecific binding sites. Subsequently, the pre-complexed primary antibody or isotype control was added to the sections and incubated for 1 hour. After three PBS washes, the slides were incubated with mouse anti-FITC antibody (20 μg / ml, Sigma) for 30 minutes. After three additional washes with PBS, the slides were incubated for 30 minutes with the peroxidase-conjugated anti-mouse IgG polymer provided in the Dako EnVision+ System. Finally, the slides were washed as above and reacted with the DAB substrate-chromogen solution provided in the Dako EnVision+ System for 6 minutes.Slides were then washed in deionized water, counterstained with Mayer's hematoxylin (Dako), dehydrated, cleared, and coverslipped with Permount (Fischer Scientific, Fair Lawn, NJ) according to routine histological procedures.

[0359] Weak to moderate staining was observed in ES-2 cells and tumor cells in lung cancer tissue. In tonsil sections, strong staining was observed in the crypt epithelium, which was heavily infiltrated with lymphocytes, but not in the mucosal stratified squamous epithelial cells. Moderate staining was observed in some cells in the interfollicular region, and very weak staining was observed in large cells (dendritic reticular cells) scattered in the germinal centers. In the lung, weak staining was found in alveolar macrophages. The staining pattern in tonsil and lung tissue was similar in immunohistochemistry sections using a commercially available anti-PD-L1 mAb (eBiosciences, San Diego, CA). HuMab staining was generally less intense, particularly in germinal centers. In the spleen, widespread, faint immunoreactivity in the red pulp barely exceeded background staining. In addition, weak to moderate staining was demonstrated in Kupffer-like cells in the liver and scattered cells in Peyer's patches, as well as in macrophage-like cells and fibroblasts scattered primarily in focal areas of the muscularis externa of the small intestine.

[0360] No significant staining was observed in cerebellum, cerebrum, heart, kidney, pancreas, pituitary, and skin tissues when stained with all four anti-PD-L1 HuMabs. No obvious differences in staining were observed among these four antibodies, except that 12B7 and / or 3G10 showed slightly stronger staining in liver cells and ES-2 cells.

[0361] PD-L1 Antibody Summary TIFF2024174925000005.tif191144TIFF2024174925000006.tif221144TIFF2024174925000007.tif45144

[0362] Array information SEQUENCE LISTING <110> E. R. SQUIBB & SONS L.L.C. <120> HUMAN MONOCLONAL ANTIBODIES TO PROGRAMMED DEATH LIGAND 1 (PD-L1) <150> US 60 / 696,426 <151> 2005-07-01 <160> 120 <170> PatentIn Ver. 3.3 <210> 1[[ID=2,2]] <211> 117 <212> PRT <213> Homo sapiens <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 2,0 25 30 Gly Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 2 <211> 123 <212> PRT <213> Homo sapiens <400> 2 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 3 <211> 118 <212> PRT <213> Homo sapiens <400> 3 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asp Val His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Leu His Ala Asp Thr Gly Ile Thr Lys Phe Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Arg Ile Gln Leu Trp Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 4 <211> 120 <212> PRT <213> Homo sapiens <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Val Ser Gly Gly Ile Phe Ser Thr Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn His Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gln Gly Ile Ala Ala Ala Leu Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 113 <212> PRT <213> Homo sapiens <400> 5 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Val Val His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Asn Ser Gly Asn Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Val Pro Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser <210> 6 <211> 123 <212> PRT <213> Homo sapiens <400> 6 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Arg Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 7 <211> 123 <212> PRT <213> Homo sapiens <400> 7 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Lys Tyr Asp Tyr Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 8 <211> 121 <212> PRT <213> Homo sapiens <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Ser Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Ala Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Ser Gly Trp Ser Arg Tyr Tyr Met Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 9 <211> 123 <212> PRT <213> Homo sapiens <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Glu Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Asn Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Leu Phe Gly Ile Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Asp Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Lys Tyr Ser Tyr Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 10 <211> 121 <212> PRT <213> Homo sapiens <400> 10 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Thr Phe Asp Asp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Arg Gly Arg Ile Glu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Gly Arg Phe Arg Tyr Phe Asp Trp Phe Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 11 <211> 107 <212> PRT <213> Homo sapiens <400> 11 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Val Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Arg 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 106 <212> PRT <213> Homo sapiens <400> 12 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 107 <212> PRT <213> Homo sapiens <400> 13 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 14 <211> 108 <212> PRT <213> Homo sapiens <400> 14 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 15 <211> 107 <212> PRT <213> Homo sapiens <400> 15 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 16 <211> 106 <212> PRT <213> Homo sapiens <400> 16 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 17 <211> 106 <212> PRT <213> Homo sapiens <400> 17 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 18 <211> 106 <212> PRT <213> Homo sapiens <400> 18 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 19 <211> 106 <212> PRT <213> Homo sapiens <400> 19 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Homo sapiens <400> 20 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 <210> 21 <211> 5 <212> PRT <213> Homo sapiens <400> 21 Asp Tyr Gly Phe Ser 1 5 <210> 22 <211> 5 <212> PRT <213> Homo sapiens <400> 22 Thr Tyr Ala Ile Ser 1 5 <210> 23 <211> 5 <212> PRT <213> Homo sapiens <400> 23 Ser Tyr Asp Val His 1 5 <210> 24 <211> 5 <212> PRT <213> Homo sapiens <400> 24 Thr Tyr Ala Ile Asn 1 5 <210> 25 <211> 5 <212> PRT <213> Homo sapiens <400> 25 Asp Tyr Val Val His 1 5 <210> 26 <211> 5 <212> PRT <213> Homo sapiens <400> 26 Watch Tyr Ala Ile Watch 1 5 <210> 27 <211> 5 <212> PRT <213> Homo sapiens <400> 27 Watch Tyr Ala Ile Watch 1 5 <210> 28 <211> 5 <212> PRT <213> Homo sapiens <400> 28 Ser Tyr Ala Ile Asn 1 5 <210> 29 <211> 5 <212> PRT <213> Homo sapiens <400> 29 Watch Tyr Ala Ile Watch 1 5 <210> 30 <211> 5 <212> PRT <213> Homo sapiens <400> 30 Asp Tyr Gly Met His 1 5 <210> 31 <211> 17 <212> PRT <213> Homo sapiens <400> 31 Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu Gln 1 5 10 15 Gly <210> 32 <211> 17 <212> PRT <213> Homo sapiens <400> 32 Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 33 <211> 17 <212> PRT <213> Homo sapiens <400> 33 Trp Leu His Ala Asp Thr Gly Ile Thr Lys Phe Ser Gln Lys Phe Gln 1 5 10 15 Gly <210> 34 <211> 17 <212> PRT <213> Homo sapiens <400> 34 Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn His Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 35 <211> 17 <212> PRT <213> Homo sapiens <400> 35 Gly Ile Ser Gly Asn Ser Gly Asn Ile Gly Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 36 <211> 17 <212> PRT <213> Homo sapiens <400> 36 Gly Ile Ile Pro Ile Phe Gly Arg Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 37 <211> 17 <212> PRT <213> Homo sapiens <400> 37 Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 38 <211> 17 <212> PRT <213> Homo sapiens <400> 38 Gly Ile Ile Pro Ile Phe Gly Ser Ala Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Asp <210> 39 <211> 17 <212> PRT <213> Homo sapiens <400> 39 Gly Ile Ile Pro Leu Phe Gly Ile Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 40 <211> 17 <212> PRT <213> Homo sapiens <400> 40 Gly Ile Ser Trp Asn Arg Gly Arg Ile Glu Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 41 <211> 8 <212> PRT <213> Homo sapiens <400> 41 Asp Tyr Phe Tyr Gly Met Asp Val 1 5 <210> 42 <211> 14 <212> PRT <213> Homo sapiens <400> 42 Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 43 <211> 9 <212> PRT <213> Homo sapiens <400> 43 Glu Arg Ile Gln Leu Trp Phe Asp Tyr 1 5 <210> 44 <211> 11 <212> PRT <213> Homo sapiens <400> 44 Asp Gln Gly Ile Ala Ala Ala Leu Phe Asp Tyr 1 5 10 <210> 45 <211> 4 <212> PRT <213> Homo sapiens <400> 45 Pro Phe Asp Tyr 1 <210> 46 <211> 14 <212> PRT <213> Homo sapiens <400> 46 Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 47 <211> 14 <212> PRT <213> Homo sapiens <400> 47 Lys Tyr Asp Tyr Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 48 <211> 12 <212> PRT <213> Homo sapiens <400> 48 Asp Ser Ser Gly Trp Ser Arg Tyr Tyr Met Asp Val 1 5 10 <210> 49 <211> 14 <212> PRT <213> Homo sapiens <400> 49 Lys Tyr Ser Tyr Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 50 <211> 12 <212> PRT <213> Homo sapiens <400> 50 Gly Arg Phe Arg Tyr Phe Asp Trp Phe Leu Asp Tyr 1 5 10 <210> 51 <211> 11 <212> PRT <213> Homo sapiens <400> 51 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Val 1 5 10 <210> 52 <211> 11 <212> PRT <213> Homo sapiens <400> 52 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 53 <211> 11 <212> PRT <213> Homo sapiens <400> 53 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 54 <211> 12 <212> PRT <213> Homo sapiens <400> 54 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 55 <211> 11 <212> PRT <213> Homo sapiens <400> 55 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210> 56 <211> 11 <212> PRT <213> Homo sapiens <400> 56 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 57 <211> 11 <212> PRT <213> Homo sapiens <400> 57 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 58 <211> 12 <212> PRT <213> Homo sapiens <400> 58 Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala 1 5 10 <210> 59 <211> 11 <212> PRT <213> Homo sapiens <400> 59 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 60 <211> 11 <212> PRT <213> Homo sapiens <400> 60 Arg Ala Ser Gln Gly Ile Ser Ser Ala Leu Ala 1 5 10 <210> 61 <211> 7 <212> PRT <213> Homo sapiens <400> 61 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 62 <211> 7 <212> PRT <213> Homo sapiens <400> 62 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 63 <211> 7 <212> PRT <213> Homo sapiens <400> 63 Wing Wing Ser Ser Leu Gln Ser 1 5 <210> 64 <211> 7 <212> PRT <213> Homo sapiens <400> 64 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 65 <211> 7 <212> PRT <213> Homo sapiens <400> 65 Wing Wing Ser Ser Leu Gln Ser 1 5 <210> 66 <211> 7 <212> PRT <213> Homo sapiens <400> 66 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 67 <211> 7 <212> PRT <213> Homo sapiens <400> 67 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 68 <211> 7 <212> PRT <213> Homo sapiens <400> 68 Gly Ala Ser Ser Arg Ala Thr 1 5 <210> 69 <211> 7 <212> PRT <213> Homo sapiens <400> 69 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 70 <211> 7 <212> PRT <213> Homo sapiens <400> 70 Asp Ala Ser Ser Leu Glu Ser 1 5 <210> 71 <211> 9 <212> PRT <213> Homo sapiens <400> 71 Gln Gln Arg Ser Asn Trp Pro Arg Thr 1 5 <210> 72 <211> 8 <212> PRT <213> Homo sapiens <400> 72 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 73 <211> 9 <212> PRT <213> Homo sapiens <400> 73 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 74 <211> 9 <212> PRT <213> Homo sapiens <400> 74 Gln Gln Tyr Gly Ser Ser Pro Trp Thr 1 5 <210> 75 <211> 9 <212> PRT <213> Homo sapiens <400> 75 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 76 <211> 8 <212> PRT <213> Homo sapiens <400> 76 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 77 <211> 8 <212> PRT <213> Homo sapiens <400> 77 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 78 <211> 7 <212> PRT <213> Homo sapiens <400> 78 Gln Gln Tyr Gly Ser Ser Pro 1 5 <210> 79 <211> 8 <212> PRT <213> Homo sapiens <400> 79 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 80 <211> 9 <212> PRT <213> Homo sapiens <400> 80 Gln Gln Phe Asn Ser Tyr Pro Phe Thr 1 5 <210> 81 <211> 351 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(351) <400> 81 cag gtt cag ctg gtg cag tct gga gct gag gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtc tcc tgc aag gct tct ggt tac acc ttt acc gac tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 ggt ttc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144 Gly Phe Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga tgg atc acc gct tac aat ggt aac aca aac tat gca cag aag ctc 192 Gly Trp Ile Thr Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 cag ggc aga gtc acc atg acc aca gac aca tcc acg agc aca gtc tac 240 Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 atg gag ctg agg agc ctg aga tct gac gac acg gcc gtg tat tac tgt 288 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 gcg aga gac tac ttc tac ggt atg gac gtc tgg ggc caa ggg acc acg 336 Ala Arg Asp Tyr Phe Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 gtc acc gtc tcc tca 351 Val Thr Val Ser Ser 115 <210> 82 <211> 369 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(369) <400> 82 cag gtc cag ctg gtg cag tct ggg gct gag gtg aag aag cct ggg tcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 tcg gtg aag gtc tcc tgc aag act tct gga gac acc ttc agc acc tat 96 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 gct atc agc tgg gtg cga cag gcc cct gga caa ggg ctt gag tgg atg 144 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 gga ggg atc atc cct ata ttt ggt aaa gca cac tac gca cag aag ttc 192 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 cag ggc aga gtc acg att acc gcg gac gaa tcc acg agc aca gcc tac 240 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 atg gag ctg agc agc ctg aga tct gag gac acg gcc gtg tat ttt tgt 288 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 gcg aga aag ttt cac ttt gtt tcg ggg agc ccc ttc ggt atg gac gtc 336 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 tgg ggc caa ggg acc acg gtc acc gtc tcc tca 369 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 83 <211> 354 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(354) <400> 83 cag gtc caa ctt gtg cag tct ggg gct gag gtg aag aag cct ggg gcc 48 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 tca gtg aag gtt tcc tgc aag gct tct gga tac acc ttc act agc tat 96 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 gat gta cat tgg gtg cgc cag gcc ccc gga caa agg ctt gag tgg atg 144 Asp Val His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 gga tgg ctc cac gct gac act ggt atc aca aaa ttt tca cag aag ttc 192 Gly Trp Leu His Ala Asp Thr Gly Ile Thr Lys Phe Ser Gln Lys Phe 50 55 60 cag ggc aga gtc acc att acc agg gac aca tcc gcg agc aca gcc tac 240 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 atg g...

Claims

1. An isolated human monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to human PD-L1 and exhibits at least one of the following properties: (a) 1×10 -7 M or lower K D binds to human PD-L1; (b) increasing T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (c) increasing interferon-γ production in an MLR assay; or (d) Increases interleukin-2 (IL-2) secretion in an MLR assay.

2. The antibody of claim 1, which is a full-length antibody of isotype IgG1, IgG2, or IgG4.

3. The antibody of claim 1 which is an antibody fragment or a single chain antibody.

4. 5×10 -9 M or lower K D The antibody of claim 1, which binds to human PD-L1 at

5. 2×10 -9 M or lower K D The antibody of claim 1, which binds to human PD-L1 at

6. An isolated human monoclonal antibody, or an antigen-binding portion thereof, that cross-competes for binding to PD-L1 with a reference antibody, including: (a) a human heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and (b) a human light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:

20.

7. The antibody of claim 6, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1, and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:

11.

8. The antibody of claim 6, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 2 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:

12.

9. The antibody of claim 6, wherein the human heavy chain variable region comprises the amino acid sequence of SEQ ID NO:3 and the human light chain variable region comprises the amino acid sequence of SEQ ID NO:

13.

10. Human V H 1-18 gene product or human V H An isolated monoclonal antibody that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-18 gene, or an antigen-binding portion thereof.

11. Human V H 1-69 gene product or human V H An isolated monoclonal antibody that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-69 gene, or an antigen-binding portion thereof.

12. Human V H 1-3 gene product or human V H An isolated monoclonal antibody that specifically binds to PD-L1, comprising a heavy chain variable region derived from the 1-3 gene, or an antigen-binding portion thereof.

13. Human V KL6 gene product or human V K An isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising a light chain variable region derived from the L6 gene.

14. Human V K L15 gene product or human V K An isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising a light chain variable region derived from the L15 gene.

15. An isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, including: (a) Human V H The heavy chain variable region of the 1-18 gene; and (b) The light chain variable region of the human VkL6 gene.

16. An isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, including: (a) Human V H The heavy chain variable region of the 1-69 gene; and (b) The light chain variable region of the human VkL6 gene.

17. An isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, including: (a) Human V H The heavy chain variable region of the 1-3 gene; and (b) The light chain variable region of the human VkL15 gene.

18. a heavy chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence; and a light chain variable region comprising a CDR1 sequence, a CDR2 sequence, and a CDR3 sequence, (a) the CDR3 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, and conservative variants thereof; (b) the CDR3 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80, and conservative variants thereof; and (c) the antibody specifically binds to human PD-L1; An isolated monoclonal antibody or an antigen-binding portion thereof.

19. The antibody of claim 18, wherein the CDR2 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40, and conservative variants thereof, and the CDR2 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70, and conservative variants thereof.

20. The antibody of claim 19, wherein the CDR1 sequence of the heavy chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, and conservative variants thereof, and the CDR1 sequence of the light chain variable region comprises an amino acid sequence selected from the group consisting of the amino acid sequences of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, and conservative variants thereof.

21. comprising a heavy chain variable region and a light chain variable region, (a) the heavy chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; (b) the light chain variable region comprises an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; and Antibody, 1×10 -7 M or lower K D binds to human PD-L1 with An isolated monoclonal antibody or an antigen-binding portion thereof.

22. The antibody of claim 21, further comprising one or more properties selected from the following: (a) the antibody increases T-cell proliferation in a mixed lymphocyte reaction (MLR) assay; (b) the antibody increases interferon-γ production in an MLR assay; and (c) The antibody increases IL-2 secretion in an MLR assay.

23. An isolated monoclonal antibody, or an antigen-binding portion thereof, that specifically binds to PD-L1, including: (a) a CDR1 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30; (b) a CDR2 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, and SEQ ID NO: 40; (c) a CDR3 of a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50; (d) a CDR1 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60; (e) a CDR2 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, and SEQ ID NO: 70; and (f) a CDR3 of a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO:

80.

24. The antibody of claim 23, comprising: (a) a heavy chain variable region CDR1 comprising SEQ ID NO:21; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:31; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:41; (d) a light chain variable region CDR1 comprising SEQ ID NO:51; (e) a light chain variable region CDR2 comprising SEQ ID NO: 61; and (f) light chain variable region CDR3 comprising SEQ ID NO:

71.

25. The antibody of claim 23, comprising: (a) heavy chain variable region CDR1 comprising SEQ ID NO:22; (b) a heavy chain variable region CDR2 comprising SEQ ID NO:32; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:42; (d) a light chain variable region CDR1 comprising SEQ ID NO:52; (e) a light chain variable region CDR2 comprising SEQ ID NO: 62; and (f) light chain variable region CDR3 comprising SEQ ID NO:

72.

26. The antibody of claim 23, comprising: (a) heavy chain variable region CDR1 comprising SEQ ID NO:23; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 33; (c) a heavy chain variable region CDR3 comprising SEQ ID NO:43; (d) a light chain variable region CDR1 comprising SEQ ID NO:53; (e) a light chain variable region CDR2 comprising SEQ ID NO: 63; and (f) light chain variable region CDR3 comprising SEQ ID NO:

73.

27. An isolated monoclonal antibody that specifically binds to PD-L1, or an antigen-binding portion thereof, comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:

20.

28. 28. The antibody of claim 27, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

11.

29. 28. The antibody of claim 27, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2; and (b) A light chain variable region comprising the amino acid sequence of SEQ ID NO:

12.

30. 28. The antibody of claim 27, comprising: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:3; and (b) a light chain variable region comprising the amino acid sequence of SEQ ID NO:

13.

31. A composition comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 30 and a pharma- ceutically acceptable carrier.

32. 31. An immunoconjugate comprising the antibody or antigen-binding portion thereof of any one of claims 1 to 30 linked to a therapeutic agent.

33. 33. A composition comprising the immunoconjugate of claim 32 and a pharma- ceutically acceptable carrier.

34. 33. The immunoconjugate of claim 32, wherein the therapeutic agent is a cytotoxin.

35. 35. A composition comprising the immunoconjugate of claim 34 and a pharma- ceutically acceptable carrier.

36. 33. The immunoconjugate of claim 32, wherein the therapeutic agent is a radioisotope.

37. 35. A composition comprising the immunoconjugate of claim 34 and a pharma- ceutically acceptable carrier.

38. A bispecific molecule comprising the antibody or antigen-binding portion thereof according to any one of claims 1 to 30, linked to a second functional moiety having a binding specificity different from the antibody or antigen-binding portion thereof.

39. 40. A composition comprising the bispecific molecule of claim 38 and a pharma- ceutically acceptable carrier.

40. 31. An isolated nucleic acid molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1 to 30.

41. 41. An expression vector comprising the nucleic acid molecule of claim 40.

42. 42. A host cell comprising the expression vector of claim 41.

43. A transgenic mouse comprising a human immunoglobulin heavy chain transgene and a light chain transgene expressing the antibody of any one of claims 1 to 30.

44. A hybridoma prepared from the mouse of claim 43, which produces an antibody.

45. 31. A method for modulating an immune response in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof according to any one of claims 1 to 30, such that the immune response in the subject is modulated.

46. A method of inhibiting the proliferation of tumor cells in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody, or an antigen-binding portion thereof.

47. 47. The method of claim 46, wherein the antibody is a chimeric antibody.

48. 47. The method of claim 46, wherein the antibody is a humanized antibody.

49. 47. The method of claim 46, wherein the antibody is a fully human antibody.

50. 47. The method of claim 46, wherein the tumor cells are from a cancer selected from the group consisting of melanoma, renal cancer, prostate cancer, breast cancer, colon cancer, and lung cancer.

51. Tumor cells may be classified as: bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia 47. The method of claim 46, wherein the cancer is selected from the list consisting of chronic or acute leukemia, including solid tumors of childhood, lymphocytic lymphoma, bladder cancer, cancer of the kidney or ureter, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, and combinations of said cancers.

52. 31. A method for inhibiting tumor cell proliferation in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof described in any one of claims 1 to 30 in an amount effective to inhibit tumor cell proliferation.

53. 31. A method for treating an infectious disease in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof according to any one of claims 1 to 30, such that the infectious disease in the subject is treated.

54. 54. The method of claim 53, wherein the infectious disease is selected from the list consisting of: HIV, Influenza, Herpes, Giardia, Malaria, Leishmania; Hepatitis viruses (A, B, and C), Herpes viruses (e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein-Barr virus, Adenovirus, Influenza virus, Flavivirus, Echovirus, Rhinovirus, Coxsackievirus, Cornovirus, Respiratory syncytial virus, Mumps virus, Rotavirus, Measles virus, Rubella virus, Parvovirus, Vaccinia virus, HTLV virus, Dengue virus, Papilloma virus, Molluscum contagiosum virus, Polio virus, Pathogenic infections with rabies virus, JC virus, and arboviral encephalitis virus; pathogenic infections with chlamydia, rickettsia bacteria, mycobacteria, staphylococci, streptococci, pneumonococci, meningococci, and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, Vibrio cholerae, Clostridium tetani, Clostridium botulinum, Bacillus anthracis, Yersinia pestis, Leptospira, and Borrelia burgdorferi;Fungi such as Candida (albicans, krusei, glabrata, tropicalis, etc.), Cryptococcus neoformans, Aspergillus (fumigatus, niger, etc.), Mucorales (mucor, absidia, rhizophus), Sporothrix schenkii, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis immitis and Histoplasma capsulatum; as well as the parasites Entamoeba histolytica, Balantidium coli, Naegleria fowleri, Acanthamoeba spp., Giardia lambia, Cryptosporidium spp., Pneumocystis carinii, Plasmodium vivax, Babesia microti, Trypanosoma brucei, Trypanosoma cruzi, Leishmania donovani, Toxoplasma gondii, and ... gondii), a pathogenic infection caused by the Brazilian hookworm (Nippostrongylus brasiliensis);

55. 31. A method for enhancing an immune response to an antigen in a subject, comprising administering to the subject (i) an antigen, and (ii) an antibody or antigen-binding portion thereof described in any one of claims 1 to 30, such that the immune response to the antigen in the subject is enhanced.

56. 56. The method of claim 55, wherein the antigen is a tumor antigen, a viral antigen, a bacterial antigen, or an antigen derived from a pathogen.

57. A method for treating or preventing an inflammatory disease in a subject, comprising administering to the subject an antibody or antigen-binding portion thereof described in any one of claims 1 to 30, such that the inflammatory disease in the subject is treated.

58. 58. The method of claim 57, wherein the inflammatory disease is lichen planus (LP).

59. A method for preparing an anti-PD-L1 antibody, comprising the steps of: (a)(i) a CDR1 sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, a CDR2 sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40, and a CDR2 sequence selected from the group consisting of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO: or (ii) a heavy chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, a CDR2 sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, and SEQ ID NO:70, and a heavy chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, and SEQ ID NO:60, a CDR2 sequence selected from the group consisting of SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO: providing a light chain variable region antibody sequence comprising a CDR3 sequence selected from the group consisting of SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, and SEQ ID NO:80; (b) altering at least one amino acid residue in at least one variable region antibody sequence selected from a heavy chain variable region antibody sequence and a light chain variable region antibody sequence to generate at least one modified antibody sequence; and (c) expressing the modified antibody sequence as a protein.

60. An anti-PD-L1 antibody, or an antigen-binding portion thereof, for use in a method of inhibiting the proliferation of tumor cells.

61. Use of an anti-PD-L1 antibody, or an antigen-binding portion thereof, for the preparation of a medicament for inhibiting the proliferation of tumor cells.

62. 31. The antibody or antigen-binding portion thereof of any one of claims 1 to 30 for use in a method for inhibiting the proliferation of tumor cells.

63. 31. Use of an antibody or antigen-binding portion thereof according to any one of claims 1 to 30 for the preparation of a medicament for inhibiting the proliferation of tumor cells.

64. 31. The antibody or antigen-binding portion thereof of any one of claims 1 to 30 for use in a method for treating an infectious disease.

65. 31. Use of an antibody or antigen-binding portion thereof according to any one of claims 1 to 30 for the preparation of a medicament for the treatment of an infectious disease.

66. 31. The antibody or antigen-binding portion thereof of any one of claims 1 to 30 for use in a method for treating an inflammatory disease.

67. 31. Use of an antibody or antigen-binding portion thereof according to any one of claims 1 to 30 for the preparation of a medicament for treating an inflammatory disease.

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