Anti-pd-l1 antibodies and anti-pd-l1 / il10 fusion proteins

Abstract

The present disclosure provides antibodies, including antibody fusions, that specifically bind to human PD-L1 protein (huPD-L1) and are capable of reducing, inhibiting, and / or completely blocking the immunoregulatory effects mediated by PD-L1, such as binding to the immune checkpoint molecule PD-1 in the tumor microenvironment. In addition, the antibodies include fusions with the cytokine inhibitory factor IL10 that are capable of supplementing and / or activating the cytotoxic effects of CD8+ T cells in the tumor microenvironment. The present disclosure also provides methods of using the antibodies (and compositions thereof) to treat diseases and conditions that are responsive to reducing, inhibiting, and / or blocking the immunoregulatory functions or activities mediated by the binding of PD1 to PD-L1.

Description

[0001] Cross-Reference to Related Applications

[0002] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application Serial No. 63 / 024,855, filed May 14, 2020, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to antibodies and fusion proteins that bind to PD-L1 protein, and methods of using such antibodies and fusion proteins.

[0004] Reference to Sequence Listing

[0005] The official copy of the sequence listing is submitted concurrently with the specification as an ASCII formatted text file via EFS-Web, with a file name of “09793-006WO1_SeqList_ST25.txt”, a creation date of May 12, 2021, and a size of 234,722 bytes. The sequence listing submitted via EFS-Web is part of the specification and is hereby incorporated in its entirety by this reference. BACKGROUND

[0006] Cancer represents a large class of diseases that involve abnormal cell growth, the potential to invade or spread to other parts of the body, and constitute a major cause of death. Because cancer cells are transformed from normal cells (carcinogenesis), the antigenic surface proteins and / or glycoproteins presented by cancer cells are identical or highly similar to those present on normal, non-tumor cells in the host organism. Thus, the immune system of the host organism can have difficulty detecting and distinguishing cancer cells from normal cells. Moreover, cancer cells can employ another mechanism to avoid detection by the host immune system.

[0007] Programmed death ligand (PD-L1) is a transmembrane protein that binds to the inhibitory checkpoint molecule PD1, thereby inhibiting adaptive immune responses during pregnancy, autoimmune diseases, and other disease states such as hepatitis. In addition, PD-L1 is highly expressed in cancerous tissues, and its expression level has been found to be closely related to tumor invasiveness. Overexpression of PD-L1 and its binding to its receptor protein PD1 are believed to be important mechanisms for cancer cells to avoid being destroyed by the immune system of the host organism.

[0008] Interleukin 10 or "IL10" (also known as cytokine synthesis inhibitory factor, CSIF, IL-10, IL10A, GVHDS, or TGIF) is a cytokine with multiple roles in immune regulation and inflammation. IL10 is known to downregulate the expression of Th1 cytokines, MHC class II antigens, and costimulatory molecules on macrophages. IL10 is also known to enhance B cell survival, proliferation, and antibody production. IL10 can block NF-κΒ activity and is involved in the regulation of the JAK-STAT signaling pathway. IL10 is capable of inhibiting the synthesis of proinflammatory cytokines such as IFN-γ, IL-2, IL-3, TNFα, and GM-CSF produced by cells such as macrophages and Th1 T cells. It also shows a strong ability to inhibit the antigen-presenting capacity of antigen-presenting cells; however, it also stimulates certain T cells (Th2) and mast cells, and stimulates B cell maturation and antibody production.

[0009] IL10 has been recognized as a potential inhibitor of tumor metastasis and an immunostimulatory agent that can be used in immunooncology therapy. In transgenic mice, expression of IL10 or administration of IL10 has been observed to control primary tumor growth and reduce metastatic burden. A pegylated form of recombinant mouse IL10 has been shown to induce IFNγ and CD8+ T cell-dependent anti-tumor immunity in mouse models. Pegylated recombinant human IL10 has been shown to enhance CD8+ T cell secretion of the cytotoxic molecules granzyme B and perforin, and to enhance T cell receptor-dependent IFNγ secretion. In clinical trials, pegylated recombinant human IL10 (PEG-rHuIL-10, AM0010) has been found to have significant anti-tumor effectiveness, eliciting dose-titratable induction of the immunostimulatory cytokines IFNγ, IL-18, IL-7, GM-CSF, and IL-4. Patients receiving the treatment also showed increases in peripheral CD8+ T cells expressing activation markers, such as PD1, lymphocyte-activation gene 3 (LAG3)+, and increased Fas ligand (FasL), as well as reductions in serum TGFβ. These findings suggest that IL10 therapy produces a predominantly immunostimulatory effect in humans. SUMMARY

[0010] The present disclosure provides anti-PD-Ll antibodies that bind specifically to human PD-L1 with high affinity. The antibodies are capable of reducing, inhibiting, and / or completely blocking the immunomodulatory effects mediated by the binding of PD-L1 to the immune checkpoint molecule PD1. The present disclosure also provides fusions of anti-PD-Ll antibodies with one or two IL10 polypeptides. These anti-PD-Ll / IL10 fusion proteins of the present disclosure are capable of providing a combined therapeutic effect of blocking the immunomodulatory effects mediated by the binding of PD-L1 to PD1 and providing the immunostimulatory effects mediated by IL10.

[0011] In at least one embodiment, the present disclosure provides an anti-PD-L1 antibody comprising (i) a first light chain complementarity determining region (CDR-L1), a second light chain complementarity determining region (CDR-L2), and a third light chain complementarity determining region (CDR-L3), and / or (ii) a first heavy chain complementarity determining region (CDR-H1), a second heavy chain complementarity determining region (CDR-H2), and a third heavy chain complementarity determining region (CDR-H3), wherein:

[0012] (a) the CDR-H1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 57, 65, 87, 93, 99, 105, 119, and 124;

[0013] (b) the CDR-H2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 50, 58, 66, 88, 94, 100, 106, 110, 115, 120, and 125;

[0014] (c) the CDR-H3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 51, 59, 67, 89, 95, 101, and 111;

[0015] (d) the CDR-L1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 53, 61, and 69;

[0016] (e) the CDR-L2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 54, 62, and 70;

[0017] (f) the CDR-L3 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55, 63, 71, 91, 97, 103, 108, 113, 117, and 122.

[0018] In at least one embodiment, the present disclosure provides an anti-PD-L1 antibody, wherein:

[0019] (a) the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 49, the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 50, and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 51;

[0020] (b) the CDR-H1 comprises an amino acid sequence of SEQ ID NO: 57, the CDR-H2 comprises an amino acid sequence of SEQ ID NO: 58, and the CDR-H3 comprises an amino acid sequence of SEQ ID NO: 59;

[0021] (c) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 65, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 66 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 67;

[0022] (d) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 87, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 88 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 89;

[0023] (e) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 94 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95;

[0024] (f) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 99, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 100 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 101 ;

[0025] (g) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 105, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 106 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95;

[0026] (h) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 110 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 111 ;

[0027] (i) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 115 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95;

[0028] (j) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 119, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 120 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95; or

[0029] (k) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 124, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 125 and CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95.

[0030] In at least one embodiment, the present disclosure provides an anti-PD-L1 antibody, wherein:

[0031] (a) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 55;

[0032] (b) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 61, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 62, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 63;

[0033] (c) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 69, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 70, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 71;

[0034] (d) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 91;

[0035] (e) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 97;

[0036] (f) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 103;

[0037] (g) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 108;

[0038] (h) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 113;

[0039] (i) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 117; or

[0040] (j) CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 122.

[0041] In at least one embodiment, the present disclosure provides an anti-PD-L1 antibody, wherein:

[0042] (a) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 49, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 50, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 51, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 55;

[0043] (b) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 57, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 58, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 59, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 61, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 62, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 63;

[0044] (c) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 65, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 66, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 67, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 69, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 70, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 71;

[0045] (d) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 87, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 88, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 89, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 91 ;

[0046] (e) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 94, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 97;

[0047] (f) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 99, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 100, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 101, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 103;

[0048] (g) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 105, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 106, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 108;

[0049] (h) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 110, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 111, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 113;

[0050] (i) CDR-H1 contains the amino acid sequence of SEQ ID NO: 93, CDR-H2 contains the amino acid sequence of SEQ ID NO: 115, CDR-H3 contains the amino acid sequence of SEQ ID NO: 95, CDR-L1 contains the amino acid sequence of SEQ ID NO: 53, CDR-L2 contains the amino acid sequence of SEQ ID NO: 54 and CDR-L3 contains the amino acid sequence of SEQ ID NO: 117;

[0051] (j) CDR-H1 contains the amino acid sequence of SEQ ID NO: 119, CDR-H2 contains the amino acid sequence of SEQ ID NO: 120, CDR-H3 contains the amino acid sequence of SEQ ID NO: 95, CDR-L1 contains the amino acid sequence of SEQ ID NO: 53, CDR-L2 contains the amino acid sequence of SEQ ID NO: 54, and CDR-L3 contains the amino acid sequence of SEQ ID NO: 122; or

[0052] (k) CDR-H1 contains the amino acid sequence of SEQ ID NO: 124, CDR-H2 contains the amino acid sequence of SEQ ID NO: 125, CDR-H3 contains the amino acid sequence of SEQ ID NO: 95, CDR-L1 contains the amino acid sequence of SEQ ID NO: 53, CDR-L2 contains the amino acid sequence of SEQ ID NO: 54 and CDR-L3 contains the amino acid sequence of SEQ ID NO: 55.

[0053] In at least one embodiment, this disclosure provides an anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable domain (V H The amino acid sequence having at least 90% identity with the sequences selected from SEQ ID NO: 52, 60, 68, 90, 96, 102, 107, 112, 116, 121, and 126; and / or a light chain variable domain (V L The amino acid sequence having at least 90% identity with the sequence selected from SEQ ID NO: 56, 64, 72, 92, 98, 104, 109, 114, 118 and 123; optionally, wherein:

[0054] (a) The antibody contains V that has at least 90% identity with SEQ ID NO: 52. H Amino acid sequence; and V that has at least 90% identity with SEQ ID NO: 56 L amino acid sequence;

[0055] (b) The antibody contains V that has at least 90% identity with SEQ ID NO: 60.H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 64 L amino acid sequence;

[0056] (c) the antibody comprises V having at least 90% identity to SEQ ID NO: 68 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 72 L amino acid sequence;

[0057] (d) the antibody comprises V having at least 90% identity to SEQ ID NO: 90 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 92 L amino acid sequence;

[0058] (e) the antibody comprises V having at least 90% identity to SEQ ID NO: 96 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 98 L amino acid sequence;

[0059] (f) the antibody comprises V having at least 90% identity to SEQ ID NO: 102 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 104 L amino acid sequence;

[0060] (g) the antibody comprises V having at least 90% identity to SEQ ID NO: 107 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 109 L amino acid sequence;

[0061] (h) the antibody comprises V having at least 90% identity to SEQ ID NO: 112 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 114 L amino acid sequence;

[0062] (i) the antibody comprises V having at least 90% identity to SEQ ID NO: 116 H amino acid sequence; and V having at least 90% identity to SEQ ID NO: 118 L amino acid sequence;

[0063] (j) the antibody comprises V having at least 90% identity to SEQ ID NO: 121H an amino acid sequence; and V L an amino acid sequence; or

[0064] (k) the antibody comprises a V H an amino acid sequence; and V L an amino acid sequence.

[0065] In at least one embodiment, the present disclosure provides an anti-PD-L1 antibody, wherein the antibody comprises a heavy chain (HC) amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 149, 150, 152, 154, 155, 156, 157, 158, 159, 160, and 161, and / or a light chain (LC) amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, 142, 151, and 153; optionally, wherein the antibody comprises:

[0066] (a) the HC amino acid sequence of SEQ ID NO: 149, and the LC amino acid sequence of SEQ ID NO: 142;

[0067] (b) the HC amino acid sequence of SEQ ID NO: 150, and the LC amino acid sequence of SEQ ID NO: 151;

[0068] (c) the HC amino acid sequence of SEQ ID NO: 152, and the LC amino acid sequence of SEQ ID NO: 153;

[0069] (d) the HC amino acid sequence of SEQ ID NO: 154, and the LC amino acid sequence of SEQ ID NO: 128;

[0070] (e) the HC amino acid sequence of SEQ ID NO: 155, and the LC amino acid sequence of SEQ ID NO: 130;

[0071] (f) the HC amino acid sequence of SEQ ID NO: 156, and the LC amino acid sequence of SEQ ID NO: 132;

[0072] (g) the HC amino acid sequence of SEQ ID NO: 157, and the LC amino acid sequence of SEQ ID NO: 134;

[0073] (h) the HC amino acid sequence of SEQ ID NO: 158, and the LC amino acid sequence of SEQ ID NO: 136;

[0074] (i) the HC amino acid sequence of SEQ ID NO: 159, and the LC amino acid sequence of SEQ ID NO: 138;

[0075] (j) the HC amino acid sequence of SEQ ID NO: 160, and the LC amino acid sequence of SEQ ID NO: 140;

[0076] (k) the HC amino acid sequence of SEQ ID NO: 161, and the LC amino acid sequence of SEQ ID NO: 142.

[0077] In at least one embodiment, the present disclosure provides an anti-PD-Ll antibody, wherein the antibody comprises a heavy chain (HC) fused to a cytokine selected from IL2, IL7, IL10, IL12, IL15, IL21, or IFN-alpha via a linker; optionally, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 74, 75, 76, 77, 78, and 79.

[0078] In at least one embodiment, the anti-PD-Ll antibody comprises a HC fused to a cytokine via a linker, the cytokine being IL10; optionally, wherein:

[0079] (a) the HC fused to the IL10 polypeptide comprises a HC-IL10 fusion amino acid sequence that is at least 90% identical to a sequence selected from SEQ ID NOs: 83, 84, 85, 127, 129, 131, 133, 135, 137, 139, and 141;

[0080] (b) the IL10 comprises an amino acid sequence of SEQ ID NO: 73; and / or

[0081] (c) the IL10 is an engineered variant of IL10 that is naturally occurring or retains its cytokine activity;

[0082] (d) the IL10 is a synthetically modified form of IL10 that retains its cytokine activity; and / or

[0083] (e) the IL10 comprises one, two, or four IL10 polypeptides.

[0084] In at least one embodiment, the present disclosure provides an anti-PD-Ll antibody comprising a HC fused to an IL10 polypeptide via a linker, wherein the antibody comprises (i) a first light chain complementarity determining region (CDR-L1), a second light chain complementarity determining region (CDR-L2), and a third light chain complementarity determining region (CDR-L3), and (ii) a first heavy chain complementarity determining region (CDR-H1), a second heavy chain complementarity determining region (CDR-H2), and a third heavy chain complementarity determining region (CDR-H3), wherein:

[0085] (a) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 1, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 3, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 5, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 6, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 7;

[0086] (b) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 9, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 10, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 11, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 13, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 14, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 15;

[0087] (c) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 17, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 18, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 19, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 21, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 22, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 23;

[0088] (d) the CDR-H1 comprises the amino acid sequence of SEQ ID NO: 25, the CDR-H2 comprises the amino acid sequence of SEQ ID NO: 26, the CDR-H3 comprises the amino acid sequence of SEQ ID NO: 27, the CDR-L1 comprises the amino acid sequence of SEQ ID NO: 29, the CDR-L2 comprises the amino acid sequence of SEQ ID NO: 30, and the CDR-L3 comprises the amino acid sequence of SEQ ID NO: 31;

[0089] (e) CDR-H1 comprises the amino acid sequence of SEQ ID NO:33, CDR-H2 comprises the amino acid sequence of SEQ ID NO:34, CDR-H3 comprises the amino acid sequence of SEQ ID NO:35, CDR-L1 comprises the amino acid sequence of SEQ ID NO:37, CDR-L2 comprises the amino acid sequence of SEQ ID NO:38, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:39;

[0090] (f) CDR-H1 comprises the amino acid sequence of SEQ ID NO:41, CDR-H2 comprises the amino acid sequence of SEQ ID NO:42, CDR-H3 comprises the amino acid sequence of SEQ ID NO:43, CDR-L1 comprises the amino acid sequence of SEQ ID NO:45, CDR-L2 comprises the amino acid sequence of SEQ ID NO:46, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:47;

[0091] (g) CDR-H1 comprises the amino acid sequence of SEQ ID NO:49, CDR-H2 comprises the amino acid sequence of SEQ ID NO:50, CDR-H3 comprises the amino acid sequence of SEQ ID NO:51, CDR-L1 comprises the amino acid sequence of SEQ ID NO:53, CDR-L2 comprises the amino acid sequence of SEQ ID NO:54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:55;

[0092] (h) CDR-H1 comprises the amino acid sequence of SEQ ID NO:57, CDR-H2 comprises the amino acid sequence of SEQ ID NO:58, CDR-H3 comprises the amino acid sequence of SEQ ID NO:59, CDR-L1 comprises the amino acid sequence of SEQ ID NO:61, CDR-L2 comprises the amino acid sequence of SEQ ID NO:62, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:63;

[0093] (i) CDR-H1 comprises the amino acid sequence of SEQ ID NO:65, CDR-H2 comprises the amino acid sequence of SEQ ID NO:66, CDR-H3 comprises the amino acid sequence of SEQ ID NO:67, CDR-L1 comprises the amino acid sequence of SEQ ID NO:69, CDR-L2 comprises the amino acid sequence of SEQ ID NO:70, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:71;

[0094] (j) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 87, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 88, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 89, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 91 ;

[0095] (k) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 94, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 97;

[0096] (l) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 99, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 100, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 101, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 103;

[0097] (m) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 105, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 106, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 95, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 108;

[0098] (n) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 110, CDR-H3 comprises the amino acid sequence of SEQ ID NO: 111, CDR-L1 comprises the amino acid sequence of SEQ ID NO: 53, CDR-L2 comprises the amino acid sequence of SEQ ID NO: 54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 113;

[0099] (o) CDR-H1 comprises the amino acid sequence of SEQ ID NO:93, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 115, CDR-H3 comprises the amino acid sequence of SEQ ID NO:95, CDR-L1 comprises the amino acid sequence of SEQ ID NO:53, CDR-L2 comprises the amino acid sequence of SEQ ID NO:54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 117;

[0100] (p) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 119, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 120, CDR-H3 comprises the amino acid sequence of SEQ ID NO:95, CDR-L1 comprises the amino acid sequence of SEQ ID NO:53, CDR-L2 comprises the amino acid sequence of SEQ ID NO:54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO: 122; or

[0101] (q) CDR-H1 comprises the amino acid sequence of SEQ ID NO: 124, CDR-H2 comprises the amino acid sequence of SEQ ID NO: 125, CDR-H3 comprises the amino acid sequence of SEQ ID NO:95, CDR-L1 comprises the amino acid sequence of SEQ ID NO:53, CDR-L2 comprises the amino acid sequence of SEQ ID NO:54, and CDR-L3 comprises the amino acid sequence of SEQ ID NO:55.

[0102] In at least one embodiment, the present disclosure provides an anti-PD-Ll antibody comprising a HC fused to an IL10 polypeptide by a linker, wherein the antibody comprises a heavy chain variable domain (VH) amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 90, 96, 102, 107, 112, 116, 121, and 126; and / or a light chain variable domain (VL) amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 92, 98, 104, 109, 114, 118, and 123; optionally, wherein: H ) an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 90, 96, 102, 107, 112, 116, 121, and 126; and / or a light chain variable domain (VL) amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 92, 98, 104, 109, 114, 118, and 123; optionally, wherein: L ) an amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 4, 12, 20, 28, 36, 44, 52, 60, 68, 90, 96, 102, 107, 112, 116, 121, and 126; and / or a light chain variable domain (VL) amino acid sequence having at least 90% identity to a sequence selected from the group consisting of SEQ ID NOs: 8, 16, 24, 32, 40, 48, 56, 64, 72, 92, 98, 104, 109, 114, 118, and 123; optionally, wherein:

[0103] (a) the antibody comprises a V Hamino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 8 L amino acid sequence;

[0104] (b) the antibody comprises V that is at least 90% identical to SEQ ID NO: 12 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 16 L amino acid sequence;

[0105] (c) the antibody comprises V that is at least 90% identical to SEQ ID NO: 20 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 24 L amino acid sequence;

[0106] (d) the antibody comprises V that is at least 90% identical to SEQ ID NO: 28 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 32 L amino acid sequence;

[0107] (e) the antibody comprises V that is at least 90% identical to SEQ ID NO: 36 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 40 L amino acid sequence;

[0108] (f) the antibody comprises V that is at least 90% identical to SEQ ID NO: 44 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 48 L amino acid sequence;

[0109] (g) the antibody comprises V that is at least 90% identical to SEQ ID NO: 52 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 56 L amino acid sequence;

[0110] (h) the antibody comprises V that is at least 90% identical to SEQ ID NO: 60 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 64 L amino acid sequence;

[0111] (i) the antibody comprises V that is at least 90% identical to SEQ ID NO: 68H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 72 L amino acid sequence;

[0112] (j) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 90 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 92 L amino acid sequence;

[0113] (k) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 96 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 98 L amino acid sequence;

[0114] (l) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 102 H amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 104 L amino acid sequence;

[0115] (m) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 107 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 109 L amino acid sequence;

[0116] (n) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 112 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 114 L amino acid sequence;

[0117] (o) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 116 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 118 L amino acid sequence;

[0118] (p) the antibody comprises a V that is at least 90% identical to SEQ ID NO: 121 H amino acid sequence; and / or V that is at least 90% identical to SEQ ID NO: 123 L amino acid sequence; or

[0119] (q) the antibody comprises a V H amino acid sequence that is at least 90% identical to SEQ ID NO: 56; and / or a V L amino acid sequence that is at least 90% identical to SEQ ID NO: 56.

[0120] In at least one embodiment, the present disclosure provides an anti-PD-Ll antibody comprising a HC fused to an IL10 polypeptide by a linker, wherein the antibody comprises a HC-IL10 fusion amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 80, 81, 82, 83, 84, 85, 127, 129, 131, 133, 135, 137, 139, and 141, and a light chain (LC) amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, and 148; optionally, wherein

[0121] (a) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 80 and the LC amino acid sequence of SEQ ID NO: 144;

[0122] (b) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 81 and the LC amino acid sequence of SEQ ID NO: 146;

[0123] (c) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 82 and the LC amino acid sequence of SEQ ID NO: 148;

[0124] (d) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 83 and the LC amino acid sequence of SEQ ID NO: 142;

[0125] (e) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 84 and the LC amino acid sequence of SEQ ID NO: 151;

[0126] (f) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 85 and the LC amino acid sequence of SEQ ID NO: 153;

[0127] (g) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 127 and the LC amino acid sequence of SEQ ID NO: 128;

[0128] (h) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 129 and the LC amino acid sequence of SEQ ID NO: 130;

[0129] (i) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 131 and the LC amino acid sequence of SEQ ID NO: 132;

[0130] (j) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 133 and the LC amino acid sequence of SEQ ID NO: 134;

[0131] (k) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 135 and the LC amino acid sequence of SEQ ID NO: 136;

[0132] (l) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 137 and the LC amino acid sequence of SEQ ID NO: 138;

[0133] (m) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 139 and the LC amino acid sequence of SEQ ID NO: 140; or

[0134] (n) the HC-IL10 fusion amino acid sequence of SEQ ID NO: 141 and the LC amino acid sequence of SEQ ID NO: 142.

[0135] In at least one embodiment, the present disclosure provides an anti-PD-L1 antibody, wherein:

[0136] (a) the antibody binds to human PD-L1 with a binding affinity of 1 x 10 -8 M or lower, 1 x 10 -9 M or lower, 1 x 10 -10 M or lower; optionally, wherein the binding affinity is measured by the equilibrium dissociation constant (KD) to the huPD-L1 polypeptide of SEQ ID NO: 174;

[0137] (b) the antibody binds to cynomolgus monkey PD-L1 with a binding affinity of 1 x 10 -8 M or lower, 1 x 10 -9 M or lower, 1 x 10 -10 M or lower; optionally, wherein the binding affinity is measured by the equilibrium dissociation constant (KD) to the cynoPD-L1 polypeptide of SEQ ID NO: 176;

[0138] (c) the protein increases MC / 9 cell proliferation by at least 25%, at least 50%, at least 100%, at least 150%, at least 200% or more;

[0139] (d) the protein increases IFNy and granzyme B production by activated CD8 T cells by at least 25%, at least 50%, at least 100% or more; and / or

[0140] (e) the antibody reduces tumor volume measured in a syngeneic mouse tumor model at 28 days by at least 25%, at least 50%, at least 75% or more, wherein the mouse tumor model is selected from the group consisting of: CT26 colon carcinoma, EMT6 breast carcinoma.

[0141] The present disclosure also provides embodiments of the anti-PD-Ll antibodies disclosed herein, including embodiments wherein: (i) the antibody is a human, humanized or chimeric antibody; (ii) the antibody comprises a fusion to a recombinant protein; optionally, a fusion to an IL10 polypeptide; (iii) the antibody is a full-length antibody of the IgG class, optionally, the IgG class antibody has an isotype selected from the group consisting of: IgGl, IgG2, IgG3 and IgG4; (iv) the antibody comprises an Fc region variant, optionally, an Fc region variant that alters effector function and / or a variant that alters antibody half-life; (v) the antibody is an antibody fragment, optionally selected from the group consisting of: F(ab')2, Fab', Fab, Fv, single domain antibody (VHH) and scFv; (vi) the antibody comprises an immunoconjugate, optionally, wherein the immunoconjugate comprises a therapeutic agent for treating a PD-Ll -mediated disease or condition; or (vii) the antibody is a multispecific antibody, optionally, a bispecific antibody.

[0142] In at least one embodiment, the present disclosure provides an isolated polynucleotide or vector encoding an anti-PD-Ll antibody of the present disclosure. In at least one embodiment, the present disclosure provides an isolated host cell comprising a polynucleotide or vector encoding an anti-PD-Ll antibody of the present disclosure. In at least one embodiment, the present disclosure also provides a method of producing an anti-PD-Ll antibody of the present disclosure, comprising culturing a host cell comprising a polynucleotide or vector encoding the anti-PD-Ll antibody, thereby producing the antibody.

[0143] In at least one embodiment, the present disclosure provides a pharmaceutical composition comprising an anti-PD-Ll antibody of the present disclosure and a pharmaceutically acceptable carrier; optionally, wherein the composition further comprises an IL10 polypeptide, a chemotherapeutic agent and / or an antibody specific for an immune checkpoint molecule.

[0144] In at least one embodiment, the present disclosure provides a method of treating a PD-L1 mediated disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody of the present disclosure, or administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure; optionally, wherein the disease is a cancer; optionally, wherein the cancer is selected from colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, gastric cancer, head and neck cancer, and oral cancer.

[0145] In at least one embodiment, the present disclosure provides a method for treating a cancer in a subject, comprising administering to the subject a PD-L1 antagonist and an IL10 agonist; optionally, wherein the PD-L1 antagonist comprises an anti-PD-L1 antibody, an shRNA, an siRNA, an miRNA, a small molecule inhibitor of PD-L1, or a combination thereof; optionally, wherein the IL10 agonist is IL-10, an IL10 receptor binding protein, or a combination thereof; optionally, wherein the PD-L1 antagonist is an anti-PD-L1 antibody of the present disclosure; optionally, wherein the PD-L1 antagonist and the IL10 agonist comprise an anti-PD-L1 antibody having a HC fused to an IL10 polypeptide by a linker; optionally, wherein the method further comprises administering to the subject a T cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0146] Figure 1A 、 Figure 1B and Figure 1C SDS-PAGE gel images of exemplary anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion proteins in full length IgG format produced, cloned, expressed, and purified as described in Example 1 are described. Figure 1A : Avdorubuzumab, SDS-PAGE images of Avdorubuzumab / IL10. Figure 1B : SDS-PAGE images of Durvalumab and Durvalumab / IL-10. Figure 1C : SDS-PAGE images of anti-PD-L1 antibodies: PHS102, PHS206, PHS219 and anti-PD-L1 / IL10 fusions: PHS102 / IL10, PHS206 / IL10 and PHS219 / IL10. N: non-reducing, R: reducing.

[0147] Figure 2A 、 Figure 2B and Figure 2CGraphs depicting the results of a competitive ELISA study showing the ability of exemplary anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusion proteins to block the specific binding of human PD-Ll to human PD1. Recombinant human PD-Ll (1 pg / mL) was immobilized on microtiter wells, biotin-conjugated human PD1 was added, and detected by streptavidin using an ELISA as described in Example 2. To detect competitive activity, serial dilutions of anti-PD-Ll or anti-PD-Ll / IL10 fusion proteins were added. Figure 2A : Blocking of PD1 binding to PD-L1 exhibited by exemplary anti-PD-Ll antibodies atezolizumab, PHS102, PHS206, and PHS219. Figure 2B : Blocking of PD1 binding to PD-L1 exhibited by exemplary anti-PD-Ll / IL10 fusions, atezolizumab / IL10, PHS102 / IL10, PHS206 / IL10, and PHS219 / IL10. Figure 2C : Blocking of PD1 binding to PD-L1 exhibited by exemplary anti-PD-Ll / IL10 fusions, avelumab / IL10, and durvalumab / IL10.

[0148] Figure 3A and Figure 3B Graphs depicting the results of a flow cytometry study of the binding of exemplary anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusion proteins to stable F293 cells overexpressing human PD-Ll ("F293 / hPDLl"), which were generated by transfecting a full-length human PD-Ll expression construct into F293 cells and then selecting for resistance to a selection drug as described in Example 3. Figure 3A Results are shown for cells incubated with serial dilutions of anti-PD-Ll antibodies prepared as in Example 1. Figure 3B Results are shown for cells incubated with serial dilutions of the corresponding anti-PD-Ll / IL10 fusion proteins as prepared in Example 1. Cells were analyzed for cell surface binding by flow cytometry and expressed as geometric MFI.

[0149] Figure 3C and Figure 3D Graphs depicting the results of a flow cytometry study showing the ability of exemplary anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusion proteins to block the specific binding of human PD1 to stable F293 cells overexpressing human PD-Ll ("F293 / hPDLl"). F293 / hPDLl expressing cells were generated by transfecting a full-length human PD-Ll expression construct into F293 cells and then selecting for resistance to a selection drug as described in Example 3. F293 / hPDLl cells were incubated with biotin-conjugated hPD1 (20 pg / mL) and exemplary anti-PD-Ll antibodies (Figure 3C ) or anti-PD-L1 / IL10 fusion protein ( Figure 3D The serial dilutions of PD1 were incubated on ice for 1 hour. Cell surface binding of PD1 was detected by streptavidin and analyzed by flow cytometry.

[0150] Figure 4A and Figure 4B The results of the exemplary anti-PD-L1 antibody and anti-PD-L1 / IL10 fusion protein inhibiting PD1 signal transduction as described in Example 4 are illustrated. The ability of the exemplary anti-PD-L1 antibody and anti-PD-L1 / IL10 fusion protein to block PD1 activation mediated by the U2OS PD-L1 cell line co-culture was determined. U2OS PD-L1 cells were subjected to serially diluted anti-PD-L1 antibody (… Figure 4A ) or anti-PD-L1 / IL10 fusion protein ( Figure 4B The cells were treated for 1 hour, and then stimulated with Jurkat PD1 signaling at room temperature for 2 hours.

[0151] Figure 5A and Figure 5B The figure depicts the results of a study demonstrating the ability of exemplary anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion proteins to enhance T cell activation in CD4 T cell-DC-mixed lymphocyte response (MLR). Figure 5A CD4 T cells were co-cultured with allogeneic mature dendritic cells (DCs) in the presence of 0.67 μg / mL anti-PD-L1 antibody or IgG control. Figure 5B CD4 T cells were co-cultured with allogeneic mature dendritic cells (DCs) in the presence of various anti-PDL1 / IL10 fusion proteins (or IL10-Fc controls) at 0.2 μg / mL. After 2 days, the supernatant was used to analyze IL-2 production by ELISA. Results are shown as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0152] Figure 6A , Figure 6B , Figure 6C , Figure 6D , Figure 6E and Figure 6F The data obtained in the study are illustrated in graphs, demonstrating that the exemplary anti-PD-L1 / IL10 fusion protein as described in Example 6 induces MC / 9 cell proliferation. MC / 9 cells were co-cultured with IL10-Fc and the exemplary anti-PD-L1 / IL10 fusion protein of this disclosure for 3 days. Cell proliferation was measured using a CellTiter-Glo assay.

[0153] Figure 7A andFigure 7B The data obtained in the study are illustrated in the figure, which shows exemplary enhancement of IFNγ and granzyme B production from activated CD8 T cells using the anti-PD-L1 / IL10 fusion protein, as described in Example 7. CD8 T cells isolated using anti-CD3 and anti-CD28 activation were used for 3 days. Activated CD8 T cells were treated with IL10-Fc or the anti-PD-L1 / IL10 fusion protein for 3 days and triggered with anti-CD3 for 4 hours. IFNγ was measured by ELISA. Figure 7A ) and cytotoxic protein granzyme B ( Figure 7B ) level.

[0154] Figure 8A A graph depicting data obtained in a study (described in Example 8) shows that the exemplary anti-PD-L1 / IL10 fusion protein of this disclosure plays a role in controlling tumor burden in a homologous mouse CT26 tumor model. Once the CT26 tumor reaches 50-100 mm... 3 Mice were randomly assigned to groups and subsequently administered PBS control, IL10-Fc (3 mg / kg), anti-PD-L1 / TGFβR (5.8 mg / kg), anti-PD-L1 (4.9 mg / kg), anti-CSF1R / IL10 (36 mg / kg), or anti-PD-L1 / IL10 (6 mg / kg) twice weekly for 3 weeks. Tumor volume was measured over time from day 0 after tumor cell implantation. n = 7 mice per group. Results are shown as mean ± SEM. ***p<0.001, ****p<0.0001.

[0155] Figure 8B A graph depicting data obtained in a study (described in Example 8) shows that the exemplary anti-PD-L1 / IL10 fusion protein of the disclosed method plays a role in controlling tumor burden in a homologous mouse EMT6 tumor model. Once the EMT6 tumor reaches 50-100 mm... 3 Mice were randomly assigned to groups and subsequently administered PBS control, anti-PDL1 (5 mg / kg), IL10-Fc (3 mg / kg), anti-PDL1 / TGFβR (6 mg / kg), or anti-PDL1 / IL10 (6 mg / kg) twice weekly for 3 weeks. Tumor volume was measured over time after tumor cell implantation in mice from day 0. n = 7 mice per group. Results are shown as mean ± SEM. *p<0.05, ***p<0.001, ****p<0.0001. Detailed Implementation

[0156] The present disclosure provides antibodies, including humanized antibodies, that specifically bind PD-L1 with high affinity, thereby inhibiting, reducing, and / or completely blocking the function of PD-L1 as a protein ligand involved in immune regulation, particularly the function of PD-L1 as a ligand for the immune checkpoint molecule PD1. It is believed that inhibiting PD-L1 / PD1 immune checkpoint signaling can enhance anti-tumor T cell responses. In clinical trials, PD-L1 inhibitors have limited anti-tumor effects when administered as a single agent to patients, and it is believed that these inhibitors need to be used in combination with other anti-tumor treatment methods. IL10 is a cytokine with anti-inflammatory and CD8+ T cell activating properties. Strong IL-10 signals can promote tumor-specific CD8+ T cell proliferation, rejuvenate exhausted T cells, and thereby increase T cell cytotoxicity. The present disclosure relates to the use of anti-PD-L1 antibodies in combination with IL10 agonists, including as anti-PD-L1 antibodies fused to human IL10 polypeptides. As disclosed herein, the combined inhibition of PD-L1 to reduce PD-L1 / PD1 signaling immunosuppression, and a bolus dose of IL10 to enhance CD8+ T cell cytotoxicity in the TME, can provide an improved treatment method for cancer treatment.

[0157] Accordingly, it is contemplated that any of the compositions or formulations comprising the anti-PD-L1 antibodies of the present disclosure, including the anti-PD-L1 antibodies fused to IL10 polypeptides, can serve as a therapeutic agent for treating a disease mediated by the function of PD-L1 or its target receptor protein PD1, such as cancer. Further, it is contemplated that the anti-PD-L1 antibodies of the present disclosure can serve as a therapeutic agent in combination with other therapeutic agents, such as antibodies that activate CD8+ T cells, and / or other target immune checkpoint molecules, including but not limited to, PD1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, and ICOS.

[0158] SUMMARY OF TERMS AND TECHNIQUES

[0159] For the description herein and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" includes more than one compound. It should also be noted that the claims can be drafted to exclude any optional element. Thus, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "only," "solely," and the like, in connection with the recitation of claim elements, or the use of a "negative" limitation. The use of "including," "comprising," "having" and "containing" are interchangeable and do not appear to be intended to exclude any elements not specifically recited. It is further understood that where the description indicates certain implementations "include," "comprise," "have" or "contain" certain elements, it is contemplated that these implementations can, in some specific cases, but not necessarily, include, comprise, have, or contain those elements without commensurate

[0160] In the event that a range of values is provided, unless otherwise stated the recited range of values can be understood as a disclosure of each and every value and sub-range encompassed therein. The upper value and lower value of a range of values can be independently included in the range. In the event that a range of values is provided, unless otherwise stated, the range is intended to include the beginning and end values of the range as well as any intervening value unless the context clearly indicates otherwise. These smaller ranges can be independently combined with other smaller ranges. For example, "1 to 50" includes "2 to 25," "5 to 20," "25 to 50," "1 to 10," etc.

[0161] In general, the nomenclature used herein and the techniques and procedures described herein include those conventionally used by those of skill in the art, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter "Sambrook"); Molecular Cloning - A Laboratory Manual Current Protocols in Molecular Biology (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989 (hereinafter "Sambrook"); Antibody Engineering Monoclonal Antibodies: Methods and Protocols Therapeutic Antibodies: From Bench to Clinic Ausubel, F. M. et al., eds., Current Protocols, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. joint venture (supplemented through 2011) (hereinafter "Ausubel"); Phage Display DETAILED DESCRIPTION OF VARIOUS EMBODIMENTSTable 1: PD-L1 and PD1 Sequences Kontermann and S. Dubel, Springer-Verlag, Berlin and Heidelberg (2010); Table 2: Table 3 Therapeutic Antibodies: Methods and Protocols, V. Ossipow and N. Fischer, eds., 2ndEdition, Humana Press (2014); 1. Anti-PD-L1 antibody binding affinities and functional characteristics 2. Anti-PD-L1 antibody fragments Antibodies: Methods and Protocols, Z. An, ed., J. Wiley & Sons, Hoboken, N.J. (2009); and 3. Chimeric, humanized and human anti-PD-L1 antibodies Antibodies: Methods and Protocols, Z. An, ed., J. Wiley & Sons, Hoboken, N.J. (2009); and

[0162] All publications, patents, patent applications and other documents cited in this disclosure are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes.

[0163] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs. It is to be understood that the terms used herein are for the purpose of describing particular embodiments and the terminology is not intended to be limiting. For the purposes of interpreting this disclosure, the following descriptions of terms will apply and where appropriate, terms used in the singular will also include the plural and vice versa.

[0164] As used herein, "PD-L1" (or "PDL1") refers to the transmembrane protein, Programmed Death-Ligand 1, and as used herein encompasses the PD-L1 protein in humans, cynomolgus monkeys, mice, and any isoforms of these proteins. The amino acid sequences of various exemplary PD-L1 proteins are well known in the art and are provided in Table 1 below and the attached sequence listing.

[0165] As used herein, a "PD-L1 -mediated condition" or "PD-L1 -mediated disease" includes any medical condition associated with the specific binding of PD-L1 to a receptor and the immune checkpoint molecule PD1 (or "PD-1"). For example, the specific binding of PD-L1 to PD1 on a T cell can inhibit its activation as part of an immune response. Thus, a PD-L1 -mediated disease can include, but is not limited to, any disease or condition mediated and / or responsive to an antagonist or inhibitor of PD-L1 and / or PD1, including but not limited to cancer.

[0166] ​As used herein, “IL10” or “IL-10” refers to the cytokine interleukin 10, which is also known as cytokine synthesis inhibitory factor (CSIF), and is intended to also include natural variants, engineered variants, and / or synthetically modified forms of interleukin 10 that retain its cytokine function. Amino acid sequences of various exemplary IL10 polypeptides and recombinant IL10 fusion constructs are provided in Table 2 below and the accompanying sequence listing. Other exemplary engineered and / or modified IL10 polypeptides that retain cytokine function are well known in the art (see, e.g., US 7,749,490 B2; US 2017 / 0015747 Al; Naing, A. et al., “PEGylated IL-10 (Pegilodecakin) Induces Systemic Immune Activation, CD8+ T Cell Invigoration and Polyclonal T Cell Expansion in Cancer Patients.” Cancer Cell 34, 775-791. e3 (2018); Gorby, C. et al., “Engineered IL-10 variants elicit potent immunomodulatory effects at low ligand doses.” Sci Signal 13, (2020); Yoon, S.I. et al., “Epstein-Barr virus IL-10 engages IL-10R1 by a two-step mechanism leading to altered signaling properties.” J Biol Chem 287, 26586-26595 (2012).

[0167] As used herein, “fusion protein” refers to two or more protein and / or polypeptide molecules that are linked (or “fused”) together in a non-naturally occurring configuration. Exemplary fusion proteins of the present disclosure include “IL10-Fc” fusion proteins comprising an IL10 polypeptide covalently linked to an immunoglobulin Fc region polypeptide via a polypeptide linker sequence at its C-terminus. Fusion proteins of the present disclosure also include “antibody fusions” comprising a full-length IgG antibody (with heavy and light chain polypeptides) covalently linked to an IL10 polypeptide via a polypeptide linker sequence at the C-terminus of its heavy chain.

[0168] As used herein, a "polypeptide linker" or "linker sequence" refers to a chain of two or more amino acids that is covalently attached at each end to a different polypeptide molecule, thereby serving to conjugate or fuse different polypeptides. Typically, a polypeptide linker comprises a polypeptide chain of 5 to 30 amino acids. A wide variety of polypeptide linkers are well known in the art and can be used in the compositions and methods of the present disclosure. Exemplary polypeptide linkers included in the compositions and methods of the present disclosure include (GGGGS) n , (SSSSG) n , (GGGG)(SGGGG) n , (EAAAK) n , (XP) n , ENLYFQ(-G / S), typically where n is 2 to 6, and other specific linker sequences as disclosed elsewhere herein.

[0169] As used herein, an "antibody" refers to a molecule comprising one or more polypeptide chains that specifically binds to or is immunologically reactive with a particular antigen. Exemplary antibodies of the present disclosure include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, multi-specific antibodies (e.g., bi-specific antibodies), monovalent antibodies (e.g., single-arm antibodies), multivalent antibodies, antigen binding fragments (e.g., Fab', F(ab')2, Fab, Fv, rIgG, and scFv fragments), and synthetic antibodies (or antibody mimetics).

[0170] An "anti-PD-Ll antibody" or "antibody that binds PD-Ll" refers to an antibody that binds PD-Ll with sufficient affinity to be useful as a therapeutic and / or diagnostic agent for targeting PD-Ll. In some embodiments, the extent of binding of an anti-PD-Ll specific antibody to an unrelated, non-PD-Ll antigen is less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the binding of the antibody to PD-Ll as measured, e.g., by radioimmunoassay (RIA) or surface plasmon resonance (SPR). In some embodiments, an anti-PD-Ll antibody of the present disclosure has a dissociation constant (KD) of <1 μΜ, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <1 pM (e.g., 10 -8 M or less, e.g., 10 -8 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0171] "Full-length antibody," "intact antibody," or "whole antibody" are used interchangeably herein to refer to an antibody with a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region as defined herein.

[0172] “Antibody fusions” refer to antibodies covalently conjugated (or fused) to a polypeptide or protein, typically via a linker at the end of an antibody light chain (LC) or heavy chain (HC). Exemplary antibody fusions of the present disclosure include an anti-PD-L1 antibody fused to a recombinant IL10 polypeptide by a 15 amino acid polypeptide linker from the C-terminus of the antibody heavy chain to the N-terminus of the IL10 polypeptide (e.g., SEQ ID NO: 74). Antibody fusions are denoted herein with the “antibody / polypeptide” nomenclature to indicate the fusion components, such as “Ab / IL10” or “anti-PD-L1 / IL10”. As described elsewhere herein, antibody fusions of the present disclosure can comprise a full-length IgG antibody comprising a dimeric complex of heavy chain-light chain pairs, wherein each heavy chain C-terminus is linked to an IL10 polypeptide by a polypeptide linker sequence.

[0173] “Antibody fragments” refer to a portion of a full-length antibody, that is capable of binding the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, monovalent or single-arm antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments.

[0174] The “class” of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, d, e, g, and m, respectively.

[0175] A “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt et al., Kuby Immunology, 6th Ed., W.H. Freeman and Co., page 91). A single VH or VL domain can be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen can be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0176] As used herein, "hypervariable region" or "HVR" refers to each of the regions which are hyper-variable in sequence and / or form structurally defined loops ("hypervariable loops") in the variable domain of an antibody. Generally, native antibodies comprise four chains which have six HVRs; three in the variable domain of the heavy chain, V H (HVR-H1, HVR-H2, HVR-H3), and three in the variable domain of the light chain, V L (HVR-L1, HVR-L2, HVR-L3). HVRs typically comprise amino acid residues from the hypervariable loops and / or from the "complementarity determining regions" (CDRs). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. The following table shows the hypervariable region residue ranges defined under these systems.

[0177]

[0178]

[0179] In addition to the above systems, HVRs and CDRs can be identified using the international ImMunoGeneTics information system, known as IMGT / V-Quest, described in Brochet, X. et al., Nucl. Acids Res. 36, W503-508 (2008), PMID: 18503082; and available online at www.imgt.org / IMGT_vquest / input. IMGT / V-Quest uses IMGT unique numbering analysis of alignment to the closest germline V gene variable region nucleotide sequence to identify HVRs and CDRs.

[0180] As used herein, hypervariable region (HVR) can include extended or alternative hypervariable regions as follows: 27-32, 27-36, 24-34, or 24-38 in the VH domain (HVR-L1); 50-52, 54-56, 50-56, or 54-60 (HVR-L2); 89-97 or 93-101 (HVR-L3); 26-33, 26-35, or 31-35 (HVR-H1); 51-58, 50-61, or 50-66 (H2); and 97-110, 97-112, 99-110, or 99-112 (H3). For each of these definitions, variable domain residues are numbered according to Kabat et al. (supra).

[0181] As used herein, "complementarity determining region" or "CDR" refers to a region within an HVR of a variable domain that has the highest sequence variability and / or is involved in antigen recognition. Generally, a native antibody comprises four chains with six CDRs; three in the heavy chain variable domain, V H (CDR-H1, CDR-H2, CDR-H3), and three in the light chain variable domain, V L (CDR-L1, CDR-L2, CDR-L3). Exemplary CDRs occur at variable domain amino acid residue positions: 24-34, 27-32, 27-36, 24-38 (CDR-L1); 50-56, 50-52, 54-56, or 54-60 (CDR-L2); 89-97 or 93-101 (CDR-L3); 31-35 or 26-33 (CDR-H1); 50-66 or 51-58 (CDR-H2); and 99-112, 99-110, 97-112, or 97-110 (CDR-H3).

[0182] "Framework region" or "FR" refers to variable domain residues other than HVR residues. The FR of a variable domain generally consists of four domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following order, from N- to C-terminus: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4. H (or V L ).

[0183] Unless otherwise indicated, residue positions in HVRs, CDRs, FRs, and other residues in variable domains are numbered herein according to Kabat et al. (supra).

[0184] "Naturally occurring antibody" refers to an immunoglobulin molecule that exists in nature. For example, a naturally occurring IgG antibody is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are linked together by disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (V H), also called variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, each light chain has a variable region (VL L ), also called variable light domain or light chain variable domain, followed by a constant light (CL) domain. The antibody's light chains can be assigned to one of two types, called kappa (K) and lambda (l), based on the amino acid sequences of their constant domains.

[0185] "Monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can arise during production of the monoclonal antibody, such variants being present in minor amounts. The term "monoclonal" refers to the characteristic that the antibody is obtained from a single clone, and does not refer to a particular method for making the antibody. For example, the monoclonal antibodies to be used in accordance with the present application can be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and transgenic animals containing all or part of the human immunoglobulin loci, as described herein and as exemplified by the following reference descriptions.

[0186] "Chimeric antibody" refers to an antibody in which a portion of the heavy and / or light chain is derived from a specific source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0187] "Humanized antibody" refers to a chimeric antibody that contains amino acid sequences from non-human HVRs and amino acid sequences from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, such as a non-human antibody, refers to an antibody that has undergone humanization.

[0188] "Human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoires or other human antibody-encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0189] "Human consensus framework" is a framework which represents the consensus sequence of human immunoglobulin V L or VH The selection of the framework sequence is based on the most common amino acid residues in the framework. Typically, human immunoglobulin V... L or V H The sequence is selected from a subgroup of variable-domain sequences. Typically, this sequence subgroup is one such subgroup as those in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, NIHP Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one implementation, for V... L This subgroup is subgroup κI, as described by Kabat et al. (ibid.). In one implementation, for V H This subgroup is subgroup III, as in Kabat et al. (ibid.).

[0190] As used in this article, “recipient human framework” refers to a light chain variable domain (V) containing a light chain variable domain derived from the human immunoglobulin framework or the human common framework. L )frame or heavy-chain variable field (V H A frame of amino acid sequences. Receptor frames “derived from” the human immunoglobulin frame or the human common frame may contain the same amino acid sequence or may contain amino acid sequence variations. In some embodiments, the number of amino acid variations is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, V L The recipient human framework is sequence-related to V L The human immunoglobulin framework sequence or the human common framework sequence is the same.

[0191] The “Fc region” refers to a dimer complex containing the C-terminal polypeptide sequence of the immunoglobulin heavy chain, where the C-terminal polypeptide sequence is a sequence obtainable by digesting an intact antibody with papain. The Fc region may contain native or variant Fc sequences. Although the boundaries of the Fc sequence of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc sequence is generally defined as extending from approximately amino acid residue Cys226 or from approximately Pro230 to the C-terminus of the Fc sequence. However, the C-terminal lysine (Lys447) of the Fc sequence may or may not be present. The Fc sequence of immunoglobulins typically contains two constant domains, namely the CH2 domain and the CH3 domain, and optionally includes a CH4 domain.

[0192] "Fc receptor" or "FcR" refers to a receptor which binds the Fc region of an antibody. In some embodiments, the FcR is a native sequence human FcR. In some embodiments, the FcR is one which binds an IgG antibody (a gamma receptor) and includes FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences, except in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). As used herein, FcR also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgGs to fetuses (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and for the homeostatic regulation of immunoglobulin levels. FcRs are reviewed in, e.g., Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995).

[0193] A "multispecific antibody" is an antibody that has at least two different binding sites, each site having a different binding specificity. The multispecific antibody can be a full-length antibody or an antibody fragment, and the different binding sites can each bind to a different antigen, or the different binding sites can bind to two different epitopes of the same antigen.

[0194] An "Fv" fragment is a fragment of an antibody that contains the complete antigen- recognition and -binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight association with each other. The tight association between the two domains of the Fv fragment gives rise to a structure that is stabile in that the variable domains of each subunit are far less mobile than in a Fab fragment. In this configuration, three HVRs from each of the variable domains contribute to the antigen binding site. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a significantly lower affinity than the entire binding site. H -V L The antigen binding site is defined by six HVRs or a subset thereof. These six HVRs confer antigen binding specificity to the antibody. However, a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a significantly lower affinity than the entire binding site.

[0195] "Fab fragment" refers to an antibody fragment that contains the variable and constant domains of the light chain and the variable domain and the first constant domain (CH1) of the heavy chain. "F(ab')2 fragments" comprise a pair of Fab fragments that are generally covalently linked near their carboxy termini by their interchain disulfide cysteines. Other chemical couplings of antibody fragments are also known in the art.

[0196] As used herein, "antigen binding arm" refers to an antibody component that has the ability to specifically bind a target molecule of interest. Typically, an antigen binding arm is a complex of immunoglobulin polypeptide sequences (e.g., HVR and / or variable domain sequences of immunoglobulin light and heavy chains).

[0197] "Single-chain Fv" or "scFv" refers to an antibody fragment that comprises the V H and V L domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the V H and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0198] "Affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). "Binding affinity" refers to the intrinsic binding affinity, which reflects the 1 : 1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (K D ). Affinity can be measured by common methods known in the art, including those described herein. Particular illustrative and exemplary embodiments for measuring binding affinity are described below.

[0199] "Specifically binds" or "specific binding" refers to the binding of an antibody to an antigen with an affinity value of no more than about 1 x 10 -7 M. In some embodiments, an antibody can have a secondary affinity for an antigen other than the one to which it specifically binds, where "secondary affinity" generally refers to the binding of an antibody to a secondary antigen with an affinity value greater than about 10 nM, as otherwise described herein. Where an antibody can have a secondary affinity for a secondary antigen, the antibody still specifically binds to the primary antigen.

[0200] "Isolated antibody" refers to an antibody that is separated from a component of its natural environment. In some embodiments, an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) methods. For a review on methods for antibody purity assessment, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87.

[0201] "Effector function" refers to a biological activity attributed to an antibody Fc region that varies with antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0202] "Immunoconjugate" refers to an antibody conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

[0203] "Treatment" (noun), "treat" (verb), or "treating" (verb) refer to clinical intervention made with the expectation of altering the natural course of the individual's condition. Desirable effects of treatment can include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. For example, treatment can include administering to an individual a therapeutically effective amount of a pharmaceutical formulation comprising an anti-PD-Ll antibody to delay the onset or slow the progression of a disease or condition mediated by PD-Ll and / or its binding to PD1 or other ligands or in which PD-Ll can play a role in pathogenesis and / or progression.

[0204] "Pharmaceutical formulation" refers to a preparation which releases a biologically active agent into a biological milieu of a subject and does not itself toxic to the subject receiving the formulation. A pharmaceutical formulation can include one or more active agents. For example, a pharmaceutical formulation can include an anti-PD-Ll antibody as the only active agent of the formulation, or can include an anti-PD-Ll antibody and one or more additional active agents, immune activators (such as IL10) or inhibitors of immune checkpoint molecules.

[0205] As used herein, "only active agent" refers to an active agent in a pharmaceutical formulation that is the only active agent present in that pharmaceutical formulation that provides or is intended to provide the relevant pharmacological effect to treat a condition in a subject. A pharmaceutical formulation that includes an only active agent does not exclude the presence of one or more non-active agents in that formulation, e.g., a pharmaceutically acceptable carrier. A "non-active agent" is an agent that is not intended to provide or otherwise significantly contribute to the relevant pharmacological effect intended to treat a condition in a subject.

[0206] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical formulation other than the active ingredients that is not toxic to the subject to which the formulation is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0207] As used herein, "immune checkpoint molecule" refers to a molecule that functions to regulate immune system pathways and thereby prevent it from attacking cells unnecessarily. Many immune checkpoint molecules (inhibitory and co-stimulatory) are targets for immunotherapy (e.g., to block antibodies to block immune inhibition or to agonists to promote immune stimulation) for the treatment of cancer and viral infections. Exemplary immune checkpoint molecules targeted by cancer immunotherapy include, but are not limited to, PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS.

[0208] "Therapeutically effective amount" refers to the amount of an active ingredient or agent (e.g., a pharmaceutical formulation) that will elicit the desired therapeutic or prophylactic result (e.g., treatment or prevention of a disease, disorder, or condition in a subject). In the case of a PD-L1 mediated disease or condition, a therapeutically effective amount of a therapeutic agent is an amount that alleviates, prevents, inhibits, and / or ameliorates to some extent one or more symptoms associated with the disease, disorder, or condition. For cancer treatment, in vivo efficacy can be measured, for example, by assessing primary tumor growth, secondary tumor occurrence and / or growth, metastasis occurrence and / or number, duration, severity, and / or recurrence of symptoms, response rate (RR), duration of response, and / or quality of life.

[0209] As used herein, "simultaneously" refers to administration of two or more therapeutic agents where at least a portion of the administration overlaps in time. Thus, simultaneous administration includes dosing regimens where administration of one or more agents is continued after administration of one or more other agents has stopped.

[0210] "Individual" or "subject" refers to a mammal, including, but not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0211] 4. Anti-PD-L1 antibody variants from libraries

[0212] I. PD-L1 and PD1

[0213] The sequence and annotation for human PD-L1 (also referred to herein as "huPD-L1" or "hPD-L1) can be found in UniProt entry Q9NZQ7, the full-length 290 amino acid isoform 1 sequence of hPD-L1 is set forth in SEQ ID NO: 173. In the examples described elsewhere herein, a shorter recombinant human PD-L1.ECD segment of SEQ ID NO: 174 was used.

[0214] The sequence and annotation of cynomolgus monkey PD-L1 (also referred to herein as“cynoPD-L1”) can be found at NCBI Reference Sequence XP_015292694.1 / XP_014973151.1. A shorter recombinant cyno PD-L1.ECD segment of SEQ ID NO: 176 was used in the binding assays of the examples described elsewhere herein.

[0215] The sequence and annotation of human PD1, which is a cognate receptor for human PD-1, can be found at UniProt entry Q8IX89. A shorter recombinant human PD1.ECD segment of SEQ ID NO: 175 was used in the examples described elsewhere herein.

[0216] Table 1 below provides an overview of the sequences of the PD-L1 and PD1 proteins and recombinant constructs used in the present disclosure, as well as their sequence identifiers. The sequences are also contained in the attached sequence listing.

[0217] 5. Multi-specific antibodies and antibody fusions

[0218]

[0219] II. IL10

[0220] The human IL10 cytokine is a homodimeric protein of two 178 amino acid polypeptide subunits. IL10 signals through a receptor complex composed of two IL10 receptor 1 (IL-10Ra subunit) and two IL10 receptor 2 (IL-10Rb subunit) proteins. Thus, a functional receptor is composed of four IL10 receptor molecules. Binding of IL10 to IL-10Ra induces STAT3 signaling through phosphorylation of the cytoplasmic tail of the IL10 receptor by JAK1 and Tyk2. IL10 is produced primarily by monocytes, to a lesser extent by lymphocytes, i.e. Type II T helper (T H 2), mast cells, CD4 + CD25 + Foxp3 + regulatory T cells as well as certain activated T and B cell subsets. IL10 can be produced by monocytes upon PD1 triggering. Table 2 below provides an overview of the amino sequences of the human IL10 polypeptides and recombinant IL10-Fc fusion constructs used in the examples of the present disclosure, as well as their sequence identifiers. The sequences are also contained in the attached sequence listing.

[0221] 6. Variants of anti-PD-L1 antibodies Recombinant IL10 polypeptides and polypeptide linkers

[0222]

[0223] In addition to naturally occurring human IL10, a variety of engineered and / or synthetically modified IL10 polypeptides that retain the cytokine function of IL10 are known in the art. Pegylated IL10, Pegilodecakin, has been shown to retain the anti-tumor immune surveillance function of naturally occurring human IL10. See, Naing, A. et al., “PEGylated IL-10 (Pegilodecakin) Induces Systemic Immune Activation, CD8+ T Cell Invigoration and Polyclonal T Cell Expansion in Cancer Patients.” Cancer Cell 34, 775-791. (2018). The engineered IL-10 variant R5A11 has been shown to have higher affinity for IL10R2, exhibit enhanced signaling activity in human CD8+ T cells, and enhance anti-tumor function of CAR-T cells. See, Gorby, C. et al., “Engineered IL-10 variants elicit potent immunomodulatory effects at low ligand doses.” Sci Signal 13, (2020). IL-10 from Epstein-Barr virus binds IL-10R1 less strongly but retains the immunosuppressive cytokine activity of human IL10 while losing the ability to induce immune stimulatory activity in certain cells. See, Yoon, S.I. et al., “Epstein-Barr virus IL-10 engages IL-10R1 by a two-step mechanism leading to altered signaling properties.” J Biol Chem 287, 26586-26595 (2012). US 7,749,490 B2 and US 2017 / 0015747 Al describe engineered IL10 mutants (e.g., F129S-IL10) that exhibit lower immune stimulatory activity in MC / 9 cell proliferation assays. In general, any engineered or modified form of IL10 polypeptide that retains some IL10 cytokine function is contemplated for use in any of the anti-PD-L1 / IL10 fusion protein compositions and methods of the present disclosure.

[0224] III. Anti-PD-L1 Antibodies

[0225] In some embodiments, the present disclosure provides structures of anti-PD-L1 antibodies, including various well-known immunoglobulin features (e.g., CDRs, FRs, VH , V L Amino acid and encoding nucleotide sequences of the variable domains and full length heavy and light chains). Table 3 below provides an overview of the anti-PD-L1 antibody sequences of the present disclosure, including antibody fusions, and their sequence identifiers. The sequences are also contained in the attached Sequence Listing.

[0226] A. Substitution, insertion and deletion variants : Anti-PD-L1 antibody (including antibody fusion) sequences

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244] B. Glycosylation variants

[0245] In some embodiments, the anti-PD-L1 antibodies provided herein have a KDof < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10 -8 M or less, 10 -8 M to 10-13 M, e.g., 10 -9 M to 10 -13 M) of the equilibrium dissociation constant (K D ) binds PD-L1.

[0246] It is contemplated that various anti-PD-L1 antibodies produced as disclosed herein include antibodies that are capable of binding huPD-L1, cynoPD-L1, and both huPD-L1 and cynoPD-L1 with high affinity. More specifically, in some embodiments, anti-PD-L1 antibodies of the present disclosure bind huPD-L1 with a binding affinity of 1 x 10 -8 M or less, 1 x 10 -9 M or less, 1 x 10 -10 M or less, or 1 x 10 -11 M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant (K D ) for binding to a huPD-L1 polypeptide of SEQ ID NO: 174. In some embodiments, anti-PD-L1 antibodies of the present disclosure bind huPD-L1 with a binding affinity of 1 x 10 -8 M or less, 1 x 10 -9 M or less, 1 x 10 -10 M or less, or 1 x 10 -11 M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant (K D ) for binding to a cynoPD-L1 polypeptide of SEQ ID NO: 176. In some embodiments, anti-PD-L1 antibodies of the present disclosure bind cynoPD-L1 with a binding affinity of 1 x 10 -8 M or less, 1 x 10 -9 M or less, 1 x 10 -10 M or less, or 1 x 10 -11 M or less. In some embodiments, the binding affinity is measured as the equilibrium dissociation constant (K D ) for binding to both a huPD-L1 polypeptide of SEQ ID NO: 174 and a cynoPD-L1 polypeptide of SEQ ID NO: 176.

[0247] In general, the binding affinity of a ligand for its receptor can be determined using any of a variety of assay methods and expressed in a variety of quantitative values. Particular PD-L1 binding assays useful for determining antibody affinity are disclosed in the Examples herein. In addition, antigen binding assays are known in the art and can be used in the present application, including but not limited to any direct or competitive binding assay using techniques such as Western Blot, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassays, surface plasmon resonance-based assays (e.g., BIAcore assay as described in WO 2005 / 012359), immunoprecipitation assays, fluorescence immunoassays, protein A immunoassays, flow cytometry, and fluorescence activated cell sorting (FACS) assays.

[0248] Thus, in some embodiments, the binding affinity is expressed as a K D value and reflects the intrinsic binding affinity (e.g., with minimized avidity effects). Anti-PD-L1 antibodies of the present disclosure exhibit strong binding affinity for the huPD-L1 polypeptide of SEQ ID NO: 174, e.g., exhibit a K D value of between 10 nM and 1 pM. Thus, anti-PD-L1 antibodies of the present disclosure can compete with antibodies having lower affinity for the same or overlapping epitope of PD-L1.

[0249] In some embodiments, the anti-PD-L1 antibodies provided herein reduce, inhibit, and / or completely block the binding of PD1 to PD-L1, as well as the immunomodulatory and / or immunosignaling mediated by the binding of PD1 to PD-L1, including the inhibition of T cell activation in the tumor microenvironment (TME). The ability of the antibodies to inhibit these immunomodulatory and immunosignaling pathways mediated by the binding of PD1 to PD-L1 can be determined in vitro using known cell-based assays, including those assays described in the Examples of the present disclosure.

[0250] In addition, the anti-PD-L1 antibodies provided herein comprise an antibody fusion with IL10 and thus can provide effects mediated by IL10 agonist activity, including the activation of CD8+ T cells in the tumor microenvironment. The ability of the anti-PD-L1 antibody fusion with IL10 to provide IL10 agonist effects can be determined in vitro using known cell-based assays, including those cell-based assays described in the Examples of the present disclosure.

[0251] Thus, in some embodiments, the anti-PD-L1 antibodies of the present disclosure are characterized by one or more of the following functional properties based on the ability to reduce, inhibit, and / or completely block intracellular signaling mediated by PD-L1.

[0252] In at least one embodiment, the anti-PD-Ll antibody binds human PD-L1 with a binding affinity of 1 x 10 -8 M or less, 1 x 10 -9 M or less, 1 x 10 -10 M or less; optionally wherein the binding affinity is measured by the equilibrium dissociation constant (K D ) for the huPD-Ll polypeptide of SEQ ID NO: 174.

[0253] In at least one embodiment, the anti-PD-Ll antibody binds cynomolgus monkey PD-L1 with a binding affinity of 1 x 10 -8 M or less, 1 x 10 -9 M or less, 1 x 10 -10 M or less; optionally wherein the binding affinity is measured by the equilibrium dissociation constant (K D ) for the cynoPD-Ll polypeptide of SEQ ID NO: 176.

[0254] In at least one embodiment, the anti-PD-Ll / IL10 fusion protein increases MC / 9 cell proliferation by at least 25%, at least 50%, at least 100%, at least 150%, at least 200% or more.

[0255] In at least one embodiment, the anti-PD-Ll / IL10 fusion protein increases production of IFNy and granzyme B in activated CD8 T cells by at least 25%, at least 50%, at least 100% or more.

[0256] In at least one embodiment, the anti-PD-Ll antibody reduces tumor volume in a syngeneic mouse tumor model selected from the group consisting of: CT26 colon carcinoma and EMT6 breast carcinoma by at least 25%, at least 50%, at least 75% or more measured at day 28.

[0257] C. Fc region variants

[0258] In some embodiments, the anti-PD-Ll antibody of the present disclosure can be an antibody fragment. Antibody fragments of the present disclosure that can be used to bind a determinant include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, scFv fragments, monovalent, single-domain antibodies, one-armed or single-armed antibodies, and other fragments described herein and known in the art. Thus, in some embodiments of the anti-PD-Ll antibody of the present disclosure, the antibody is an antibody fragment selected from the group consisting of: F(ab')2, Fab', Fab, Fv, single-domain antibody (VHH), one-armed antibody, and scFv.

[0259] For a review of various antibody fragments, see, e.g., Hudson et al., Nat. Med. 9: 129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a description of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-lives, see U.S. Patent No. 5,869,046. Other monovalent antibody forms are described, e.g., in WO 2007 / 048037, WO 2008 / 145137, WO 2008 / 145138, and WO 2007 / 059782. Monospecific, single-arm antibodies are described, e.g., in WO 2005 / 063816. Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific (see, e.g., EP 0 404 097; WO 93 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).

[0260] In some embodiments, an antibody fragment is a single-domain antibody comprising all or a portion of a heavy chain variable domain or all or a portion of a light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516).

[0261] Antibody fragments can be produced by various techniques, including but not limited to proteolytic digestion of whole antibodies and production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0262] D. Non-protein antibody derivatives - immunoconjugates

[0263] In some embodiments, an anti-PD-L1 antibody of the present disclosure is a chimeric antibody. (See, e.g., chimeric antibodies described in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). In one embodiment, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In some embodiments, a chimeric antibody is a "class switched" antibody in which the class or subclass is changed from that of the parent antibody. It is contemplated that a chimeric antibody can include an antigen-binding fragment thereof.

[0264] In some embodiments, an anti-PD-L1 antibody of the present disclosure is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, CDRs (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived) to restore or improve antibody specificity or affinity.

[0265] Humanized antibodies and methods of making them are reviewed in, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described in, e.g., Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-HVR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface reshaping"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling). Acad. Sci. USA 86:10029-10033 (1989); US Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-HVR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "surface reshaping"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

[0266] Human framework regions that can be used in humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from an consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol, 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271 :2261 1-22618 (1996)).

[0267] In some embodiments, an anti-PD-L1 antibody of the present disclosure can be a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies can be prepared by administering the immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to an antigenic challenge. Such animals are typically TM techniques; the K-M techniques; the K-M techniques; and the K-M techniques. The human variable regions from intact antibodies generated by such animals can be further modified, such as by combining with different human constant regions.

[0268] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies. See, e.g., Kozbor J. Immunol, 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol, 147:86 (1991). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described in, e.g., U.S. Patent No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005).

[0269] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from phage display libraries of human origin. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0270] EXAMPLES

[0271] In at least one embodiment, variants of improved anti-PD-Ll antibodies can be isolated by screening combinatorial libraries for antibodies with the desired improved functional properties, e.g., binding affinity or cross-reactivity. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for variant antibodies with improved binding properties. Other methods for generating such libraries of antibodies of interest can be found, e.g., in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Humana Press, Totowa, NJ, 2001); McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, m Methods in Molecular Biology 248:161-175 (Lo, ed., Humana Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0272] scFv reformatting and cloning:

[0273] In at least one embodiment, the anti-PD-Ll antibodies contemplated by the present disclosure can be multispecific antibodies, e.g., bispecific antibodies. In some embodiments, the multispecific antibodies have at least two different binding sites, each with a binding specificity for a different antigen, at least one of which specifically binds PD-L1. In at least one embodiment, the multispecific antibodies contemplated are bispecific antibodies comprising a specificity for PD-L1 and a specificity for another antigen that mediates immune regulation, immune signaling, and / or is expressed on a cancer or tumor cell. For example, the other specificity can be for an immune checkpoint molecule, e.g., PD1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, or ICOS.

[0274] Techniques for making multispecific antibodies include, but are not limited to, recombinant co- expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see, e.g., Milstein and Cuello, Nature 305: 537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)). “Knob-in-hole” engineering can also be used to generate bispecific antibodies of the anti-PD-L1 antibodies useful in the present disclosure. Techniques for knob-in-hole engineering are known in the art and described in, e.g., U.S. Patent No. 5,731,168.

[0275] Multispecific antibodies can also be produced by engineering an “electrostatic steering” effect that favors formation of Fc heterodimeric antibody molecules rather than homodimers (WO 2009 / 089004 Al); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol, 148(5): 1547-1553 (1992)); using the “diabody” technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993)); using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol, 152: 5368 (1994)); or trispecific antibodies (see, e.g., Tutt et al., J. Immunol. 147: 60 (1991)) to prepare.

[0276] In at least one embodiment, the anti-PD-L1 antibodies provided herein can comprise an antibody fusion with a protein. Methods of making and using antibody fusions or fusion proteins are well known in the art and described elsewhere herein, including the Examples. Typically, the antibody is covalently conjugated (or fused) to the protein via a polypeptide linker comprising a chain of 5-30 amino acids. Typically, the linker is conjugated to the C-terminus of the antibody heavy chain (HC) constant region, but it can also be conjugated via the N-terminus, or to either end of the antibody light chain (LC). Antibody fusions can also be made with various antibody fragments, where these fragments comprise the CDRs required for specific binding to an antigen.

[0277] In at least one embodiment, the antibody fusions of the present disclosure can include a full-length anti-PD-Ll antibody conjugated at the end of a light chain or heavy chain to a polypeptide linker sequence conjugated at its other end to a T cell activation or immunostimulatory cytokine. The cytokine can include, but is not limited to, IL2, IL7, IL10, IL12, IL15, IL21, or IFN-alpha. Such anti-PD-Ll antibody fusions can block PD-L1 / PD1 signal-mediated activity and provide an immunostimulatory cytokine effect. The ability of such anti-PD-Ll / cytokine antibody fusions to provide an immunostimulatory cytokine effect can be determined in vitro using known cell-based assays related to the cytokine, including those described in the Examples.

[0278] As described elsewhere herein, the antibody fusions of the present disclosure can include a full-length IgG antibody comprising a dimeric complex of heavy chain-light chain pairs, wherein the C-terminus of each heavy chain is linked by a polypeptide linker sequence to an IL10 polypeptide. In one exemplary embodiment, the antibody fusions of the present disclosure can include an anti-PD-Ll antibody fused to a recombinant IL10 polypeptide by a 15 amino acid polypeptide linker from the C-terminus of the antibody heavy chain to the N-terminus of the IL10 polypeptide (e.g., SEQ ID NO: 74). The PD-L1 binding and immunostimulatory IL10 effects of a number of such exemplary anti-PD-Ll / IL10 antibody fusions are further described and characterized in the Examples.

[0279] Production of anti-PD-L1 / IL10 fusion proteins:

[0280] In some embodiments, variants of the anti-PD-Ll antibodies of the present disclosure are contemplated that have improved properties, such as binding affinity and / or other biological properties of the antibody. Variants can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired qualities of PD-L1 antigen binding.

[0281] Expression of full-length antibodies and fusion proteins:

[0282] In some embodiments, in addition to those described herein, variants of the anti-PD-Ll antibody having one or more amino acid substitutions are provided. Sites for mutagenesis can include HVRs and FRs. Typical "conservative" amino acid substitutions and / or those that are based on common side-chain classes or properties can be made and are known in the art, and can be made in embodiments of the present disclosure. The present disclosure also contemplates variants based on non- conserved amino acid substitutions, where a member of one of the amino acid side chain classes is substituted for another. Amino acid side chains are typically grouped according to the following classes or common properties: (1) hydrophobic: Met, Ala, Val, Leu, He, norleucine; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) big al: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Techniques for making amino acid substitutions to antibodies and subsequently screening for desired function, e.g., maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC, are known in the art.

[0283] Amino acid substitution variants can also include variants having one or more substitutions in the hypervariable region of a parent antibody. Typically, the resulting variant(s) selected for further study will have alterations in certain biological properties relative to the parent antibody, such as increased affinity, reduced immunogenicity, and / or retention of a specific biological property of the parent antibody. Exemplary substitution variants are affinity matured antibodies, which can be conveniently generated using phage display based affinity maturation techniques. Briefly, one or more HVR residues are mutated and variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0284] One useful method for identifying antibody residues or regions that can be targeted for mutagenesis is "alanine scanning mutagenesis" (see, e.g., Cunningham and Wells (1989) Science, 244: 1081-1085). In this method, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu are commonly targeted for substitution) and replaced by a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interactions of the antibody with antigen are affected. Further substitutions can be introduced at amino acids locations demonstrating functional sensitivity to the initial substitution. Alternatively, or additionally, the crystal structure of the antigen-antibody complex can be determined to identify contact points between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated to be candidates for substitution. Variants can be screened for desired properties.

[0285] Amino acid sequence insertions including amino- and / or carboxyl-terminal fusions, ranging in length from one residue to polypeptides of one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues, can be prepared by standard methods. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of the antibody molecule can include a fusion with an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0286] Other residue substitutions can be made in HVRs to improve antibody affinity. Such alterations can be made in "hot spot" residues, i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), where the binding affinity of the resulting variant V H or V L HVRs are tested. In one embodiment, affinity maturation is performed by constructing and reselecting from secondary libraries. (See, e.g., Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ (2001)). Another method to introduce diversity involves HVR-directed approaches, in which a few HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, e.g., by alanine scanning mutagenesis or modeling. In particular, HVR-H3 and HVR-L3 are often targeted. Usually, substitutions, insertions or deletions are made in one or more HVRs, so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (e.g., conservative substitutions) as provided herein can be made in the HVRs, without substantially reducing the ability of the antibody to bind antigen. Such alterations can be outside of HVR "hot spots."

[0287] In some embodiments, it is contemplated that the anti-PD-Ll antibodies described herein can be substituted at particular non-HVR positions with cysteine residues to generate reactive thiol groups. Such engineered "thioMAbs" can be used to conjugate the antibody to e.g., drug moieties or linker-drug moieties, to generate immunoconjugates, as described elsewhere herein. The generation of cysteine engineered antibodies can be performed as described in, e.g., U.S. Patent No. 7,521,541. In some embodiments, any one or more of the following antibody residues can be substituted with cysteine: V205 of the light chain (Kabat numbering); Al 18 of the heavy chain (EU numbering); and S400 of the heavy chain Fc region (EU numbering).

[0288] Purification and SDS-PAGE characterization of full-length antibodies and fusion proteins:

[0289] In some embodiments, an anti-PD-L1 antibody of the present disclosure is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can be made by altering the amino acid sequence such that one or more glycosylation sites is created or removed. In embodiments where the antibody comprises an Fc region, the carbohydrate attached to the Fc region can be altered. Typically, a native antibody produced by a mammalian cell comprises a branched, biantennary oligosaccharide that is attached by an N-linkage to asparagine ("N297") at about position 297 of the CH2 domain of the Fc region (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharide can include various carbohydrates, such as mannose, N-acetylglucose amine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modification of the oligosaccharide of an antibody Fc region can yield variants with certain improved properties.

[0290] In some embodiments, an anti-PD-L1 antibody of the present disclosure can be a variant that comprises a sugar structure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be from about 1% to about 80%, from about 1% to about 65%, from about 5% to about 65%, or from about 20% to about 40%. The amount of fucose can be determined by calculating the average amount of fucose in the sugar chain attached to residue N297, relative to the sum of all sugar-structures attached at N297 (e.g., complex, hybrid, and high mannose structures), as measured by MALDI-TOF mass spectrometry (see, e.g., WO 2008 / 077546).

[0291] In some embodiments, fucosylation variants can provide improved ADCC function of the variant antibody. See, e.g., U.S. Patent Publication Nos. US 2003 / 0157108 or US 2004 / 0093621. Examples of "defucosylated" or "afucosylated" antibodies and related methods of making the same are disclosed, e.g., in US 2003 / 0157108; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US 2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2000 / 61739; WO 2001 / 29246; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004). Cell lines useful for producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (see, e.g., Ripka et al., Arch. Biochem. Biophys. 249: 533-545 (1986); US 2003 / 0157108 and WO 2004 / 056312), and knockout cell lines such as CHO cells knocked out for the alpha-1,6-fucosyltransferase gene (FUT8) (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0292] SDS-PAGE results:

[0293] In some embodiments, the anti-PD-L1 antibodies of the present disclosure can comprise one or more amino acid modifications in the Fc region (i.e., Fc region variants). Fc region variants can comprise a human Fc region sequence (e.g., a human IgGl, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid substitution at one or more amino acid residue positions. A wide variety of Fc region variants known in the art that can be used in the anti-PD-L1 antibodies of the present disclosure are described below.

[0294] In some embodiments, the anti-PD-Ll antibody is an Fc region variant with altered effector function. In some embodiments, the antibody with altered effector function has some (but not all) of the effector functions, has reduced effector function, or lacks any one of the effector functions of the parent antibody (e.g., an effectorless). Effectorless Fc region variants are more desirable for certain applications in which effector functions such as ADCC are not required or deleterious and / or the half-life of the antibody in vivo is important. Fc region variant antibodies with reduced or no effector function can result from amino acid substitutions at one or more of the following Fc region positions: 238, 265, 269, 270, 297, 327, and 329 (see, e.g., U.S. Patent No. 6,737,056). Such Fc region variants can include amino acid substitutions at two or more of positions 265, 269, 270, 297, and 327. Such Fc region variants can also include substitutions of residues 265 and 297 to alanine (see, e.g., U.S. Patent No. 7,332,581).

[0295] Some Fc region variants are capable of providing improved or diminished binding to FcRs (see, e.g., U.S. Patent No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)). Some Fc region variants that provide improved ADCC comprise one or more amino acid substitutions at positions 298, 333, and / or 334 of the Fc region (based on EU numbering). Fc region variants with altered (i.e., improved or diminished) Clq binding and / or complement dependent cytotoxicity (CDC) are described, e.g., in U.S. Patent No. 6,194,551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0296] Some Fc region variants provide increased half-lives and improved binding to the neonatal Fc receptor (FcRn), disclosed, for example, in US 2005 / 0014934 Al (Hinton et al.). Such Fc region variants include amino acid substitutions at one or more of positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, and 434. Other Fc region variants with increased half-lives include the YTE mutation set at positions 252, 254, and 256 (i.e., M252Y / S254T / T256E), described, for example, in US 7658921 B2 (Dall’Acqua et al.). Additional examples of Fc region variants can be found in, for example, US Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351.

[0297] In general, in vitro and / or in vivo cytotoxicity assays can be conducted to confirm reduction / depletion of CDC and / or ADCC activity in the Fc region variants. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence can lack ADCC activity) but retains the ability to bind FcRn. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII, and FcyRIII. Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)); and Hellstrom et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166: 1351-1361 (1987)). Alternatively, non-radioactive assays methods can be used (see, e.g., ACTI TM non-radioactive cytotoxicity assays (see, e.g., ACTI Nonradioactive cytotoxicity assays (Promega, Madison, Wl). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). Clq-dosing assays can also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. See, e.g., Clq and C3c binding ELISA in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al. J. Immunol. Methods 202: 163 (1996); Cragg, M.S. et al. Blood 101 : 1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, SW 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can be performed using methods known in the art (see, e.g., Petkova et al. Intl. Immunol. 18(12): 1759-1769 (2006)).

[0298] Figure 1A

[0299] In some embodiments, the anti-PD-Ll antibodies of the present disclosure can be further modified (i.e., derivatized) with nonproteinaceous moieties. Nonproteinaceous moieties suitable for derivatizing antibodies include, but are not limited to, the following: water-soluble polymers of the type known as "polyethylene glycol" (PEG), copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyethylene glycol homopolymers and copolymers, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxpane, ethylene / maleic acid copolymers, polyamino acids other than gelatin, and dextran or poly(n-vinyl pyrrolidone) copolymers, polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohols, and mixtures thereof. In some embodiments, the modification of the antibody with methoxy- polyethylene glycol propionaldehyde can be utilized. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers conjugated to the antibody can vary, and if more than one polymer is conjugated, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody, e.g., whether the antibody derivative is to be used for therapy under specified conditions.

[0300] In some embodiments, the anti-PD-Ll antibodies of the present disclosure can also be immunoconjugates, wherein the immunoconjugate comprises an anti-PD-Ll antibody conjugated to one or more cytotoxic agents. Suitable cytotoxic agents contemplated by the present disclosure include chemotherapeutic agents, drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), wherein an anti-PD-Ll antibody described herein is conjugated to one or more drugs. In some embodiments, the immunoconjugate of the present disclosure comprises an anti-PD-Ll antibody described herein conjugated to a drug or therapeutic agent for treating a PD-Ll -mediated disease or disorder.

[0301] In some embodiments, the anti-PD-Ll antibodies described herein can be conjugated to an enzymatically active toxin or fragments thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, phytolacaemia A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, enomycin, and the

[0302] In some embodiments, the immunoconjugates of the present disclosure comprise an anti-PD-Ll antibody described herein conjugated to a radioisotope (i.e., a radioconjugate). A number of radioisotopes can be used to produce such radioconjugates. Examples include 211 At、 131 I、 125 I、 90 Y、 186 Re、 188 Re、 153 Sm、 212 Bi、 32 P、 212 Pb and Lu. In some embodiments, the immunoconjugate can comprise a radioisotope for scintigraphic detection, or a spin label for NMR detection or MRI. Suitable radioisotopes or spin labels can include, for example 123 I, 131 I, 111 In, 13 C, 19 F, 15 N,17 O, various isotopes of Gd, Mn, and Fe.

[0303] Immunoconjugates of an anti-PD-Ll antibody and a cytotoxic agent can be made using a variety of bifunctional crosslinkers known to those skilled in the art. Such agents include, but are not limited to: N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC), iminothiolate (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HQ), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), azino compounds (such as bis-(p-azidophenyl)- hexanediamine), bis-diazonium biosuccinmidyl ester, and bis-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). Reagents for making immunoconjugates of the present disclosure can also include commercially available "cross-linking" reagents such as BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (see, e.g., Pierce Biotechnology, Inc., Rockford, IL., U.S.A).

[0304] IV. Recombinant Methods and Compositions

[0305] Anti-PD-Ll antibodies of the present disclosure can be produced using recombinant methods and materials well known in the art of antibody production. In some embodiments, the present disclosure provides an isolated nucleic acid encoding an anti-PD-Ll antibody. The nucleic acid can encode an amino acid sequence comprising a V L of an antibody and / or an amino acid sequence comprising a V H of an antibody (e.g., a light chain and / or a heavy chain of an antibody). In some embodiments, one or more vectors (e.g., expression vectors) comprising nucleic acid sequences encoding an anti-PD-Ll antibody of the present disclosure are provided. In some embodiments, a host cell comprising nucleic acid sequences encoding an anti-PD-Ll antibody of the present disclosure is provided. In one embodiment, a host cell is transformed with a vector comprising nucleic acid sequences encoding an amino acid sequence comprising a V L of an antibody and an amino acid sequence comprising a V H of an antibody. In another embodiment, a host cell is transformed with a vector comprising nucleic acid sequences encoding an amino acid sequence comprising a VL a first vector comprising nucleic acid encoding an antibody comprising a V H a second vector comprising nucleic acid encoding an antibody comprising a V

[0306] In some embodiments of the recombinant methods, the host cells used are eukaryotic cells, such as Chinese Hamster Ovary (CHO) cells or lymphoid cells (e.g., Y0, NS0, Sp20). In one embodiment, a method of making an anti-PD-Ll antibody is provided, wherein the method comprises culturing a host cell as previously provided containing nucleic acid encoding an antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0307] Briefly, recombinant production of an anti-PD-Ll antibody is performed by isolating nucleic acid encoding an antibody (e.g., as described herein) and inserting this nucleic acid into one or more vectors for further cloning (e.g., in Ma cells) and / or expression in a host cell. Such nucleic acid is readily isolated and sequenced using conventional procedures (e.g., by employing oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the desired antibody). Suitable host cells and culture methods for cloning or expression of antibody-encoding vectors are well known in the art and include prokaryotic or eukaryotic cells. In general, after expression, the antibody can be isolated from the cell paste in a soluble fraction and further purified. In addition to prokaryotes, eukaryotic microbes are suitable

[0308] Host cells suitable for expression of the glycosylated anti-PD-Ll antibodies of the present disclosure can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures can also be utilized as hosts (see, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, and 7,125,978).

[0309] Examples of mammalian host cell lines useful for production of the anti-PD-Ll antibodies of the present disclosure include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells (see e.g., Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); myeloma cell lines such as Y0, NS0 and Sp2 / 0; monkey kidney CVl cell line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described e.g., in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells as described e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TR1 cells (see e.g., Mather et al., Annals N Y. Acad. Sci. 383:44-68 (1982) and US 6,235,498); Medical Research Council 5 (MRC 5) cells (e.g., those available from ATCC, also known as CCL-171); and Foreskin 4 (FS-4) cells (see e.g., Vilcek et al., Ann. N. Y. Acad. Sci. 284:703-710 (1977), Gardner & Vilcek, J. Gen. Virol. 44:161-168 (1979), and Pang et al., Proc. Natl. Acad. Sci. U.S.A. 77:5341-5345 (1980)). For a review of general recombinant expression methods suitable for production of antibodies see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0310] V. Pharmaceutical compositions and formulations of anti-PD-Ll antibodies

[0311] The present disclosure also provides pharmaceutical compositions and pharmaceutical formulations comprising an anti-PD-Ll antibody. In some embodiments, the present disclosure provides a pharmaceutical formulation comprising an anti-PD-Ll antibody described herein and a pharmaceutically acceptable carrier. In some embodiments, the anti-PD-Ll antibody is the only active agent of the pharmaceutical composition. Such pharmaceutical formulations can be prepared by mixing an anti-PD-Ll antibody having the desired degree of purity with one or more pharmaceutically acceptable carriers. In general, such antibody formulations can be aqueous (see, e.g., U.S. Pat. No. 6,171,586 and WO 2006 / 044908) or non-aqueous (see, e.g., U.S. Pat. No. 6,267,958) suspensions.

[0312] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. A wide range of such pharmaceutically acceptable carriers are known in the art (see, e.g., Remington's Pharmaceutical Sciences 16thEdition, Osol, A. Ed., (1980)). Exemplary pharmaceutically acceptable carriers that can be used in formulations of the present disclosure can include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents (e.g., EDTA); sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0313] Pharmaceutically acceptable carriers that can be used in formulations of the present disclosure can also include interstitial drug dispersing agents such as a soluble, neutral-active hyaluronidase glycoprotein (sHASEGP) (see, e.g., U.S. Pat. Pub. Nos. 2005 / 0260186 and 2006 / 0104968), such as a human soluble PH-20 hyaluronidase glycoprotein (e.g., rHuPH20 or Vyzalta®, and HYLENEX®, from Baxter International, Inc.).

[0314] It is also contemplated that the formulations disclosed herein can contain active ingredients other than anti-PD-Ll, as necessary for the particular indication being treated in the subject to which the formulation is administered. Preferably, any additional active ingredients have activity that is complementary to that of the anti-PD-Ll antibody, and the activities of the agents do not adversely affect one another.

[0315] As disclosed elsewhere herein, including the Examples, it has been demonstrated that the anti-PD-Ll antibodies of the present disclosure can be used in conjunction with IL10 polypeptides to provide improved therapeutic effects in the treatment of cancer. Accordingly, in some embodiments, the present disclosure provides a pharmaceutical composition or formulation useful for the treatment of cancer comprising a PD-L1 antagonist (e.g., an anti-PD-Ll) and an IL10 agonist (e.g., IL10). Further, while the anti-PD-Ll antibodies of the present disclosure are used as the PD-L1 antagonist in such pharmaceutical formulations or compositions, it is also contemplated that other antagonists can be used, including but not limited to shRNA, siRNA, miRNA, small molecule inhibitors of PD-L1, or combinations thereof. Small molecule inhibitors of PD-L1 that can be used in such pharmaceutical compositions or formulations can include known compounds in clinical development, including but not limited to, AUNP12 (Aurigene), CA-170 (Aurigene / Curis), and BMS-986189 (Bristol-Myers Squibb). In addition to the anti-PD-Ll antibodies of the present disclosure, other known anti-PD-Ll antibodies that can be used in such combination pharmaceutical compositions or formulations with IL10 can include any known antibody that binds PD-L1, including those in clinical development for the treatment of cancer, such as atezolizumab, avelumab, durvalumab, lirilumab, FAZ053 (BAP058-huml3), and MDX-1105, which are described elsewhere herein.

[0316] As described elsewhere herein, in some embodiments, the present disclosure provides a pharmaceutical composition or formulation for use in combination therapy comprising a PD-L1 antagonist and an IL10 agonist. In some embodiments, such a combination can be provided as a single pharmaceutical composition or formulation comprising an anti-PD-Ll antibody fusion having an anti-PD-Ll antibody covalently fused to IL10 via a polypeptide linker (e.g., the linker sequence of SEQ ID NO: 74, 75, 76, 77, 78, or 79). Examples demonstrating such anti-PD-Ll antibody fusions (e.g., PHS102 / IL10) and their use in pharmaceutical compositions to reduce tumor volume in a variety of syngeneic mouse cancer models are provided elsewhere herein.

[0317] In some embodiments, the pharmaceutical composition comprises an anti-PD-Ll antibody and an additional active agent for cancer treatment, such as an immune checkpoint inhibitor. Immune checkpoint inhibitors useful in such embodiments include, but are not limited to, a second antibody comprising specificity for an antigen that is an immune checkpoint molecule. In some embodiments, the second antibody comprises specificity for an immune checkpoint molecule selected from the group consisting of PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS. In at least one embodiment, the pharmaceutical composition comprises an anti-PD-Ll antibody and an additional active agent, wherein the additional active agent is an antibody comprising specificity for the immune checkpoint molecule PD1. Exemplary antibodies comprising specificity for PD1 useful in the pharmaceutical composition embodiments disclosed herein include, but are not limited to, pembrolizumab, nivolumab, cemiplimab, pidilizumab, dostarlimab, and HX008.

[0318] In colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules or in a coarse suspension, the active ingredient can be present together with at least one excipient that is designated for the preparation of these pharmaceutical dosage forms, such as, for example, glycerol, sorbitol or mannitol, or together with at least one polymer, such as, for example, polyvinylpyrrolidone or hypromellose. Other suitable excipients are disclosed in Remington's Pharmaceutical Sciences, 16thEdition, Osol, A. Ed. (1980).

[0319] In some embodiments, the formulation can be a sustained-release formulation of the antibody and / or other active ingredients. Suitable examples of sustained-release formulations include a semipermeable matrix of solid hydrophobic polymer containing the antibody, which is in the form of shaped articles, such as films or microcapsules.

[0320] Generally, the formulations within the present disclosure to be administered to a subject are sterile. Sterile formulations can be readily prepared using well-known techniques, such as by filtration through a sterile filtration membrane.

[0321] VI. Uses and methods of treatment

[0322] It is contemplated that any composition or formulation containing an anti-PD-L1 antibody of the present disclosure can be used in any method or use, e.g., a therapeutic method that utilizes the ability of the anti-PD-L1 antibody to specifically bind to a PD-L1 protein, thereby inhibiting, reducing, and / or completely blocking the function of PD-L1 as a participant in immune modulation or signaling, in particular, the function of PD-L1 to specifically bind to the immune checkpoint molecule PD1, thereby inhibiting anti-tumor immune responses (e.g., T cell activation) in the tumor microenvironment (TME) and promoting tumor growth and progression.

[0323] By inhibiting, reducing, and / or completely blocking the immune modulatory and / or immune signaling activity of PD-L1, in particular, the effect of PD-L1 on tumor progression, a range of diseases, disorders, and conditions can potentially be treated. The diseases, disorders, and conditions include, but are not limited to, cancer, including, but not limited to, colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, gastric cancer, head and neck cancer, or oral cancer. It is contemplated that any composition or formulation containing an anti-PD-L1 antibody of the present disclosure, including an anti-PD-L1 antibody fusion with an IL10 polypeptide, can be used in a method or use for treating any of the above-listed cancers. In some embodiments, the cancer is selected from colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, gastric cancer, head and neck cancer, or oral cancer. In some embodiments, the present disclosure provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-PD-L1 antibody of the present disclosure or administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an anti-PD-L1 antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0324] As disclosed herein, including in the Examples below, the anti-PD-L1 antibodies of the present disclosure have the ability to reduce, inhibit, and / or block the binding of PD1 to PD-L1, thereby altering the interaction of PD1 with the immune signaling pathway mediated by PD-L1. Accordingly, in some embodiments, the present disclosure provides a method of treating a PD-L1 mediated disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody of the present disclosure or administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-PD-L1 antibody of the present disclosure and a pharmaceutically acceptable carrier. Similarly, in some embodiments, the present disclosure provides a method of treating a disease mediated by binding to PD-L1 expressed on a cell in a subject, the method comprising administering to the subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody of the present disclosure or administering to the subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-PD-L1 antibody of the present disclosure and a pharmaceutically acceptable carrier.

[0325] Administration of an anti-PD-Ll antibody, composition, or pharmaceutical preparation according to the methods of treatment provides an antibody-induced therapeutic effect that protects a subject from PD-Ll -mediated disease progression and / or treats PD-Ll -mediated disease progression in a subject. In some embodiments, the methods of treatment can further comprise administration of one or more additional therapeutic agents or treatments known to those of skill in the art to prevent and / or treat a PD-Ll -mediated disease or condition. Such methods that include administration of one or more additional agents can include combination administration (where two or more therapeutic agents are included in the same or separate formulations) and separate administration, in which case administration of the antibody composition or formulation can precede, coincide with, and / or follow administration of the additional therapeutic agent(s).

[0326] The cytokine IL10 exhibits anti-inflammatory and CD8+ T cell activation properties. Strong IL-10 signals can promote tumor-specific CD8+ T cell proliferation, rejuvenate exhausted T cells, and thereby increase T cell cytotoxicity. Accordingly, in at least one embodiment, the present disclosure contemplates a method of treatment using a PD-L1 antagonist (e.g., an anti-PD-Ll antibody) in combination with an IL10 agonist. In at least one embodiment, such combination treatment can be performed using an anti-PD-Ll antibody fusion with an IL10 polypeptide. As disclosed herein, the inhibition of PD-L1 binding to reduce its immunosuppressive effects with a concentrated IL10 signal near the TME to enhance T cell cytotoxicity can provide an improved cancer treatment. Accordingly, in any embodiment of a method of treating a PD-L1 -mediated disease (e.g., a cancer) using an anti-PD-Ll antibody of the present disclosure, it is contemplated that the anti-PD-Ll antibody can be an antibody fusion (or fusion protein) with an IL10 polypeptide as disclosed elsewhere herein.

[0327] Other PD-L1 or PD1 antagonists are also contemplated for use in this combination treatment with IL10, including but not limited to, shRNA, siRNA, miRNA, or small molecule inhibitors of PD-L1, or combinations thereof. Small molecule inhibitors of PD-L1 that can be used in such methods can include known PD-L1 inhibitor compounds in clinical development, such as AUNP12 (Aurigene), CA-170 (Aurigene / Curis), and BMS-986189 (Bristol-Myers Squibb). In addition, other known PD-L1 antagonist antibodies can be used in this combination treatment with IL10, including known antibodies that block PD-L1, including those in clinical development for cancer treatment, such as atezolizumab, avelumab, durvalumab, lodecabtagene, FAZ053 (BAP058-hum13), and MDX-1105.

[0328] In some embodiments of the methods of treatment of the disclosure, the anti-PD-Ll antibody or pharmaceutical formulation comprising the anti-PD-Ll antibody is administered to the subject, or to the desired target tissue, by any mode of administration that systemically delivers the agent. Systemic administration generally refers to any mode of administration that results in the antibody being administered to the subject at a site other than directly into the desired target site, tissue, or organ, such that the antibody or formulation thereof enters the circulatory system of the subject and is thus subject to metabolism and other similar processes.

[0329] Accordingly, modes of administration that can be used in the methods of treatment of the disclosure can include, but are not limited to, injection, infusion, instillation, and inhalation. Injection administration can include intravenous, intramuscular, intraarterial, intrathecal, intracerebroventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0330] In some embodiments, the pharmaceutical formulation of the anti-PD-Ll antibody is formulated such that the antibody is protected from inactivation in the intestinal tract. Accordingly, the methods of treatment can include oral administration of the formulation.

[0331] In some embodiments, the use of a composition or formulation comprising the anti-PD-Ll antibody of the disclosure as a medicament is also provided. Further, in some embodiments, the use of a composition or formulation comprising the anti-PD-Ll antibody in the manufacture or preparation of a medicament, particularly a medicament for treating, preventing, or inhibiting a PD-Ll -mediated disease, is also provided. In further embodiments, the medicament is for use in a method of treating, preventing, or inhibiting a PD-Ll -mediated disease, comprising administering to an individual having a PD-Ll -mediated disease an effective amount of the medicament. In particular embodiments, the medicament further comprises an effective amount of at least one additional therapeutic agent or treatment. In at least one embodiment, the additional therapeutic agent or treatment is an IL10 agonist, such as an IL10 polypeptide, which is administered in combination with the anti-PD-Ll antibody (rather than as an antibody fusion).

[0332] As disclosed elsewhere herein, it is also contemplated that additional therapeutic agents or treatments that can be used in such medicaments having an anti-PD-Ll antibody of the present disclosure can include, but are not limited to, therapeutic antibodies comprising specificity for immune checkpoint molecules such as PD1, PD-L1, LAG3, CTLA-4, A2AR, TIM-3, BTLA, CD276, CD328, VTCN1, IDO, KIR, NOX2, VISTA, OX40, CD27, CD28, CD40, CD122, CD137, GITR, ICOS. Exemplary antibodies comprising specificity for immune checkpoint molecules include, but are not limited to, anti-PD1 antibodies selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, pidilizumab, dostarlimab, and HX008.

[0333] In further embodiments, the medicament is for treating, inhibiting, or preventing a PD-L1 mediated disease, such as a cancer, in a subject, comprising administering to the subject an effective amount of the medicament to treat, inhibit, or prevent the PD-L1 mediated disease.

[0334] Suitable dosages of the anti-PD-Ll antibodies included in the compositions and formulations of the present disclosure, when used alone or in conjunction with one or more additional therapeutic agents, depend upon the particular disease or condition being treated, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the treating physician. The anti-PD-Ll antibodies included in the compositions described herein are suitably administered to the patient at one time or over a series of treatments. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at various time points, bolus administration, and pulse infusion.

[0335] Depending on the type and severity of the disease, from about 1 pg / kg to 15 mg / kg of the anti-PD-Ll antibody in the formulations of the present disclosure is an initial candidate dosage for administration to a human subject, whether, for example, by one or more separate administrations, or by continuous infusion. Typically, the dosage of the antibody ranges from about 0.05 mg / kg to about 10 mg / kg. In some embodiments, one or more dosages of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to the patient.

[0336] Dose administration can be maintained for several days or longer, depending on the condition of the subject, e.g., administration can continue until the PD-L1 mediated disease is adequately treated, as determined by methods known in the art. In some embodiments, an initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimes can be useful. The progress of the therapeutic effects of dose administration can be monitored by routine techniques and assays.

[0337] Thus, in some embodiments of the methods of the disclosure, administration of the anti-PD-Ll antibody comprises a daily dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the dose of the anti-PD-Ll antibody comprises a daily dose of at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg, or at least about 30 mg / kg.

[0338] Figure 1B

[0339] The various features and embodiments of the disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. Those skilled in the art will readily understand that the specific examples merely illustrate the application as described more fully in the claims that follow. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained within this application.

[0340] Example 1: Generation and binding analysis of anti-PD-Ll antibodies and IL10 fusions

[0341] This example illustrates the use of phage display antibody library technology to generate exemplary anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusion proteins of the disclosure that specifically bind to human PD-L1 and / or IL10R and the ability to block PD-L1 binding to PD-1.

[0342] A. Selection of anti-PD-Ll scFv binders from phage display antibody libraries

[0343] The panning procedure is outlined as follows. First, human PD-L1 / ECD antigen (5 μg per well, SinoBiological) was coated in PBS buffer (pH 7.4) in 96-well plates (NUNC Maxisorb Immuno Plates) at 4°C overnight, then blocked with 5% nonfat milk in PBST [0.1% (v / v) Tween 20] for 1 h. After blocking, 100 μL of concentrated phage library (10 13CFU) for 1 h, then 50 mL of 2X YT medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin was added and incubated at 37°C with vigorous shaking overnight. The rescued phage library was precipitated with 20% polyethylene glycol / NaCl and resuspended in PBS. The concentrated phage solution was used for the next round of panning. 11 CFU) for 1 h, then 50 mL of 2X YT medium containing 50 μg / mL kanamycin and 100 μg / mL ampicillin was added and incubated at 37°C with vigorous shaking overnight. The rescued phage library was precipitated with 20% polyethylene glycol / NaCl and resuspended in PBS. The concentrated phage solution was used for the next round of panning.

[0344] After 3-4 rounds of selection-expansion cycles, individual colonies were randomly picked into 96-deep well plates (Plate A: secreted scFv); each well contained 950 μL of 2YT (100 μg / mL ampicillin). After 3 h of incubation at 37°C with shaking, 50 μL of bacterial culture was transferred to the corresponding well of a fresh 96-deep well plate (Plate B: phage form); each well contained 0.8 mL of 2YT and 100 μg / mL ampicillin. Simultaneously, 50 μL of M13KO7 (a total of ~5 x 10 10CFU) was added to each well of plate B. After 1 h of incubation, 100 μL of 2YT containing IPTG (10 mM) was added to each well of plate A; 100 μL of 2YT containing kanamycin (500 μg / mL) was added to each well of plate B. After overnight incubation at 37 °C with vigorous shaking, the cultures were centrifuged at 3000 g for 10 min at 4 °C. Plate B was stored for further sequencing assay. For secreted scFv culture plates (plate A), 100 μL of media and 100 μL of 5% PBST milk were added to pre-coated with protein L (0.1 μg / well), human CD36 (0.5-1 μg / well), and bovine serum albumin (BSA) (2 μg / well) with 5% PBST milk blocking, respectively. After 1 h of incubation at room temperature, the plates were washed 3 times with PBST. 100 μL of protein A-HRP (Thermo Scientific) was added to each well of protein L-coated immunoplates; 100 μL of anti-E-tag-HRP (ICL Inc.) was added to each well of human PD-L1.ECD antigen-coated plates and BSA-coated plates. After 1 h of incubation, the plates were washed 3 times with PBST buffer, 2 times with PBS, and developed with 3,3',5,5'-tetramethyl-benzidine peroxidase substrate (Kirkegaard & Perry Laboratories) for 3 min, quenched with 1.0 M HC1, and read spectrophotometrically at 450 nm.

[0345] Positive clones were selected by the following criteria: OD450>0.2 for human PD-L1.ECD antigen-coated wells (antigen binding positive); OD450<0.05 for BSA-coated wells (non-specific binding negative); OD450>0.5 for protein L-coated wells (soluble scFv binds to both protein L and protein A to ensure proper folding in solution), followed by DNA sequencing.

[0346] The polynucleotide sequences of exemplary scFv of anti-PD-L1 antibodies PHS102 (SEQ ID NO: 162), PHS206 (SEQ ID NO: 163), and PHS219 (SEQ ID NO: 164) obtained from phage display panning are provided in Table 3 and the accompanying sequence listing.

[0347] To further increase the affinity of the anti-PDLl antibody PHS102, six phage display libraries were created against individual CDRs of PHS102. After the first round of panning, 38 unique CDRs were selected from CDR-L3, CDR-H1, CDR-H2, and CDR-H3 and assembled as new libraries for off-rate screening. To select phage with improved off-rate of scFv, off-rate screening was performed by co-culturing with 10, 100, or 1000-fold of hPDLl.ECD protein during panning. Further panning of these phage display libraries consisting of variant CDR sequences derived from the anti-PD-Ll antibody PHS102 resulted in the following eight exemplary anti-PD-Ll antibodies (scFv polynucleotide sequences): YT6D (SEQ ID NO: 165), YP11F (SEQ ID NO: 166), YT10H (SEQ ID NO: 167), YT7A (SEQ ID NO: 170), YT7H (SEQ ID NO: 171), YP7G (SEQ ID NO: 172), HSYPP31F (SEQ ID NO: 168), and HSYPP411C (SEQ ID NO: 169). The CDRs, V H , V L The amino acid sequences of the heavy and light chains are also listed in Table 3 and the accompanying sequence listing.

[0348] B. Production of full-length anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusions

[0349] Results The PD-L1 binding determinants of the scFv selected by phage display panning were reconstituted into full-length IgG antibodies by cloning the V H and V L domains of the fragments into a human IgGl-N297A heavy chain vector and a human kappa light chain vector using restriction enzyme sites MluI / NheI and BsiWI / DraIII, respectively. The V H and V LDomain: (1) PhageLib_VL_Fw: 5'-AATCACgATgTgATATTCAAATgACCCAgAgCCCgAgC-3' (SEQ ID NO: 177), (2) PhageLib_VL_Rv: 5'-AATCgTACgTTTgATTTCCACTTTggTgCCTTg-3' (SEQ ID NO: 178), (3) PhageLib_VH_Fw: 5'-AATACgCgTgTCCTgTCCgAAgTgCAgCTggTggAATCg-3' (SEQ ID NO: 179), and (4) PhageLib_VH_Rv: 5'-AATgCTAgCCgAgCTCACggTAACAAg-3' (SEQ ID NO: 180).

[0350] Table 4 The design of the recombinant-Fc fusion protein (SEQ ID NO: 86) was to genetically fuse IL-10 (SEQ ID NO: 73) to the N-terminus of human IgGl-Fc, separated by a 15 amino acid linker sequence -GGGGSGGGGSGGGGS- (SEQ ID NO: 76). The design of the recombinant anti-PD-Ll / IL10 fusion protein was to genetically fuse IL-10 to the C-terminus of the antibody heavy chain, separated by an amino acid linker sequence -LGGGGSGGGGSGGGG- (SEQ ID NO: 74). Other useful linker sequences are provided in Table 2. The desired gene fragments, signature the IL-2 secretion sequence required for secretion of the recombinant protein, were obtained from Thermo Gene Synthesis Services and cloned in mammalian expression vectors for transfection and expression in ExpiCHO-S cells.

[0351] Full length anti-PD-Ll antibodies, atezolizumab, avelumab, durvalumab, lodecabtagene, FAZ053, and MDX-1105 were obtained using Thermo Gene Synthesis Services and cloned into mammalian expression vectors for transfection and expression in ExpiCHO-S cells. Polypeptide sequences are provided in Table 3 and the accompanying sequence listing.

[0352] Anti-PD-L1 antibodies Vectors cloned with rearranged anti-PD-Ll antibodies or anti-PD-Ll / IL10 fusion genes were transiently expressed in ExpiCHO-S cells (Thermo Scientific). At the exponential growth phase, 6xl05cells were transiently transfected with 20 pg of vector using ExpiFectamine CHO transfection reagent kit (Thermo Scientific). The cells were cultured for 10 days in ExpiCHO-S medium (Thermo Scientific) with 8 mM L-glutamine, 4 mM sodium pyruvate, and 10% FBS. The supernatants were collected and filtered using 0.22 pm filter units. The filtered supernatants were analyzed by ELISA for the presence of the recombinant proteins. 6ExpiCHO-S cells. 18-22 hours post transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added to the flasks. Cells were cultured for 8 days and the supernatant from each culture was centrifuged and then filtered through a 0.45 pm filter.

[0353] PHS102 Antibodies and Ab / IL10 fusion proteins were purified from transfected cell supernatants using Protein A Sepharose Fast Flow beads (GE Healthcare). Antibody loaded columns were washed with 20 column volumes of PBS and then eluted directly into 1 / 10 volume of 1 M Tris buffer (pH 9.0) with 3 bead volumes of 0.1 M glycine (pH 2.5). Antibody containing fractions were pooled and dialyzed against PBS. The quality of purified anti-PD-L1 antibodies and fusion proteins was determined using SDS-PAGE in the presence or absence of reducing agent.

[0354] PHS206 A. ELISA for antigen-specific binding of full-length antibodies and fusion proteins PHS219 B. BLI for antigen-specific binding of full-length antibodies and fusion proteins Anti-PD-L1 / IL10 fusions Inspection of the SDS-PAGE images described in Example 1 indicated that the clones, expression and purification produced purified anti-PD-L1 antibodies in full-length IgG format, as well as anti-PD-L1 fused to IL10 polypeptides.

[0355] Example 2: Specific binding assays for anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion proteins

[0356] This example demonstrates that ELISA and BLI studies showed specific antigen binding and blocking function of anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusions.

[0357] A. ELISA for antigen-specific binding of full-length antibodies and fusion proteins

[0358] Recombinant human PD-L1-mFc fusion protein (1 pg / mL), recombinant cynomolgus PD-L1 (1 pg / mL; Sino Biological), or IL10R a-Fc fusion protein (1 pg / mL, R&D Systems) were immobilized on 96-well microtiter plates at a concentration of 1 pg / ml in coating solution (SeraCare) overnight at 4°C. Wells were washed with wash solution (Imidazole-buffered saline with 0.05% Tween 20) and blocked with 1% BSA. Serial dilutions of anti-PD-L1 or anti-PD-L1 / IL10 fusion proteins were added to the wells. After incubation at 37°C for 1 hr, the wells were washed using wash solution. Peroxidase-conjugated goat anti-human kappa light chain antibody (Sigma) was applied to each well and incubated at 37°C for 1 h. For PD-1 / PD-L1 competitive ELISA, biotin-conjugated PD-1-Fc protein (30 pg / mL, Biolegend) was added. Binding of PD-1 was detected by streptavidin-HRP. After washing, the wells were developed with TMB substrate at room temperature for 5-10 min, and then stopped with 1 N HC1. Thereafter, absorbance was measured at 450 nm and 650 nm. EC50 and IC50 values were calculated using GraphPad Prism 7.

[0359] atezolizumab / IL10

[0360] EC50 values from ELISA assays show the specific binding activity to human PD-L1 exhibited by exemplary anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion of the present disclosure, as shown in Table 4 below.

[0361] avelumab / IL10 : Specific binding activity to human PD-L1 protein

[0362] avelumab / TGFpR EC 50 (M)]]> durvalumab / IL10 0.784E-10 PHS102 / IL10 0.525E-10 PHS206 / IL10 2.043E-10 PHS219 / IL10 Table 5 0.865E-10 Anti-PD-L1 antibodies 1.835E-10 atezolizumab 1.067E-10 PHS102 1.207E-10 PHS206 0.798E-10 Table 6 0.629E-10 Anti-PD-L1 / IL10 fusions 1.643E-10

[0363] EC50 values from ELISA assays show the specific binding activity to cynomolgus PD-L1 exhibited by exemplary anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion of the present disclosure, as shown in Table 5 below.

[0364] EC50 (M) : Specific binding activity to cynomolgus PD-L1 protein

[0365] atezolizumab / IL10 EC 50 (M)]]> avelumab / IL10 0.140E-09 durvalumab / IL10 0.216E-09 PHS102 / IL10 0.187E-09

[0366] EC50 values from ELISA assays show the specific binding activity to IL10R exhibited by exemplary anti-PD-L1 / IL10 fusion of the present disclosure, as shown in Table 6 below.

[0367] PHS206 / IL10 Specific binding activity to human IL10Rα

[0368] PHS219 / IL10 Figure 2A Figure 2B 8.725E-08 Figure 2C 2.591E-08 Table 7 3.755E-08 IC50 (M) 9.426E-08 Anti-PD-L1 antibodies 6.194E-08 atezolizumab 6.626E-08

[0369] Binding data graphs obtained by competitive ELISA are shown in PHS102 , PHS206 and PHS219 . IC50 values show the specific activity exhibited by the exemplary anti-PD-L1 / IL10 fusion of the disclosure for blocking the binding of PD-1 to PD-L1, as shown in Table 7 below.

[0370] Anti-PD-L1 / IL10 fusions Specific activity for blocking interaction of hPD-1 and hPD-L1

[0371] IC50 (M) atezolizumab / IL10 avelumab / IL10 0.239E-09 durvalumab / IL10 0.182E-09 PHS102 / IL10 0.083E-09 PHS206 / IL10 3.8900E-05 PHS219 / IL10 Figure 2A Figure 2B 0.124E-09 Figure 2C 0.559E-09 Table 7 0.188E-09 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizumab PHS102 PHS206 PHS219 Anti-PD-L1 / IL10 fusions IC50 (M) atezolizumab / IL10 avelumab / IL10 durvalumab / IL10 PHS102 / IL10 PHS206 / IL10 PHS219 / IL10 Figure 2A Figure 2B Figure 2C Table 7 IC50 (M) Anti-PD-L1 antibodies atezolizum 0.061E-09 PHS206 / IL10 0.116E-09 PHS219 / IL10 1.204E-09

[0372] B. BLI analysis of anti-PD-L1 binding kinetics

[0373] Bio-layer interferometry (BLI) (ForteBio Octet RED96) assays were performed using AHC (anti-hIgG Fc capture) biosensors (ForteBio) to capture each anti-PD-L1 antibody (5 pg / mL) to achieve a 0.5 nm shift, then the biosensors were immersed in different concentrations (i.e., 0, 1.5625, 3.125, 6.25, 4.94, 12.5, 25, 50, and 100 nM) of recombinant human PD-L1-His in running buffer containing PBS-Tween 20 (0.1%), BSA (0.1%). Rate constants were calculated by curve fitting analysis (1:1 Langmuir model) of binding reactions with 2.5 min association and 5 min dissociation interaction times.

[0374] Results: Dissociation constants K D and kinetic rate constants k a and k d for the specific binding of exemplary anti-PD-L1 antibodies to the antigen PD-L1 were determined using BLI assays, as shown in Table 8 below

[0375] Table 8 Specific binding kinetics of anti-PD-L1 antibodies to hPD-L1

[0376] Anti-PD-L1 Ab K D (M)]]> k a (1 / Ms)]]> k d (1 / s) PHS102 1.44E-08 4.73E+05 6.79E-03 YT6D 2.16E-09 3.30E+05 7.13E-04 YP11F 1.84E-09 3.34E+05 6.15E-04 YT10H 1.92E-09 3.50E+05 6.70E-04 HSYPP31F 1.88E-09 2.88E+05 5.42E-04 HSYPP411C 2.03E-09 3.21E+05 6.53E-04 YT7A 1.99E-09 3.03E+05 6.03E-04 YT7H 6.16E-09 2.03E+05 1.25E-03 YP7G 5.51E-09 2.47E+05 1.36E-03

[0377] Example 3: Cell binding assays for anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion proteins

[0378] This example illustrates flow cytometry studies showing specific binding of exemplary anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusion proteins of the present disclosure to F293 cells expressing PD-Ll.

[0379] Materials and Methods

[0380] A. Preparation of F293 cells expressing PD-L1: A gene fragment encoding full-length human PD-Ll was obtained using Thermo Gene Synthesis Service and cloned into mammalian expression vector pCDNA3.4. Freestyle 293-F cells (Thermo Scientific) were transfected with PD-Ll expression vector by polyethyleneimine (PEI) method and selected with Geneticin (Thermo Scientific) to establish a stable F293 cell line expressing PD-Ll.

[0381] B. Flow cytometry: F293 cells or F293 cells overexpressing PD-Ll were incubated with anti-PD-Ll antibodies or anti-PD-Ll / IL10 fusion proteins at 4°C for 30 min. After washing with FACS buffer (PBS with 2% FBS), cells were stained with anti-human IgG-Alexa Fluor 647 and analyzed by Attune NxT flow cytometer (Thermo Scientific), and cell surface binding was expressed as geometric MFI.

[0382] C. PD-1 blockade assay: F293 / hPD-Ll cells were incubated with serial dilutions of anti-PD-Ll antibodies or anti-PD-Ll / IL10 fusion proteins on ice for 30 min. 20 pg / ml biotin-conjugated human PD1.ECD protein (SEQ ID NO: 175) was added and incubated on ice for 60 min. After washing with FACS buffer (PBS with 2% FBS), cells were stained with streptavidin-Alexa Fluor 647 and analyzed by Attune NxT flow cytometer (Thermo Scientific).

[0383] Results

[0384] As Figure 3A and Figure 3BExemplary anti-PD-L1 antibodies (PHS102, PHS206, PHS219, and atezolizumab) and corresponding anti-PD-L1 / IL10 fusion proteins (PHS102 / IL10, PHS206 / IL10, PHS219 / IL10, and atezolizumab / IL10) showed specific binding activity to human PD-L1 expressed on the surface of F293 cells, as shown in the flow cytometry data plots in FIGS. 1A-1D.

[0385] As shown in the flow cytometry data plots in FIGS. 2A-2D, exemplary anti-PD-L1 antibodies (PHS102, PHS206, PHS219, and atezolizumab) and corresponding anti-PD-L1 / IL10 fusion proteins (PHS102 / IL10, PHS206 / IL10, PHS219 / IL10, and atezolizumab / IL10) showed specific blocking activity to the binding of human PD-1 to human PD-L1 expressed on the surface of F293 cells. Figure 3C Figure 3D As shown in the flow cytometry data plots in FIGS. 2A-2D, exemplary anti-PD-L1 antibodies (PHS102, PHS206, PHS219, and atezolizumab) and corresponding anti-PD-L1 / IL10 fusion proteins (PHS102 / IL10, PHS206 / IL10, PHS219 / IL10, and atezolizumab / IL10) showed specific blocking activity to the binding of human PD-1 to human PD-L1 expressed on the surface of F293 cells.

[0386] Example 4: Assay for PD-1 / PD-L1 cell signaling blockade

[0387] This example illustrates a study of the ability of exemplary anti-PD-L1 antibodies and anti-PD-L1 / IL10 fusion proteins of the disclosure to block PD-1 / PD-L1 cell signaling.

[0388] Materials and Methods

[0389] Using A PD-1 / PD-L1 blockade bioassay was performed using a PD-1 signaling bioassay kit (Eurofins). Serial dilutions of exemplary anti-PD-L1 antibodies or anti-PDL1 / IL10 fusion proteins were pre-incubated with U2OS bioassay cells (1 x 105cells / well) in the presence of PD-L1 at 37 °C for 1 hour. Jurkat PD-1 signaling cells (2 x 105cells / well) were added to the PD-L1 -presenting cells and incubated at room temperature for 2 hours, followed by the addition of detection reagents. 4 4

[0390] Results

[0391] As shown in the flow cytometry data plots in FIGS. 2A-2D, exemplary anti-PD-L1 antibodies (PHS102, PHS206, PHS219, and atezolizumab) and corresponding anti-PD-L1 / IL10 fusion proteins (PHS102 / IL10, PHS206 / IL10, PHS219 / IL10, and atezolizumab / IL10) showed specific blocking activity to the binding of human PD-1 to human PD-L1 expressed on the surface of F293 cells. Figure 4A Figure 4B ​​​​Exemplary anti-PD-Ll antibodies (PHS102, HSYPP31F, HSYPP411C, YT7A, YT7H, YP10H) and anti-PD-Ll / IL10 fusion proteins (PHS102 / IL10, YT7A / IL10, YT7H / IL10, YP10H / IL10) were able to block cell signaling transduction mediated by PD-1 binding to PD-L1, as shown in the blocking assay data plotted and the IC50 values listed in Table 9.

[0392] Table 9 Specific activity of blocking PD-1 / PD-L1 signaling

[0393] IC50 (nM) Anti-PD-L1 antibody PHS102 3.178 HSYPP31F 3.563 HSYPP411C 3.741 YT7A 6.07 YT7H 3.478 YP10H 3.381 Anti-PD-L1 / IL10 fusion PHS102 / IL10 4.221 YT7A / IL10 4.867 YT7H / IL10 2.471 YP10H / IL10 2.492

[0394] Example 5: Anti-PD-Ll antibodies and fusion proteins enhance T cell activation

[0395] This example illustrates a study of the ability of exemplary anti-PD-Ll antibodies and anti-PD-Ll / IL10 fusion proteins of the disclosure to enhance T cell activation in a mixed lymphocyte reaction (MLR).

[0396] Materials and Methods

[0397] Human peripheral blood was taken from healthy donors. Peripheral blood mononuclear cells (PBMCs) were immediately isolated by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare). To serve as allogeneic antigen presenting cells (APCs), CD14+ monocytes were first isolated from donor A using anti-human CD14 conjugated magnetic beads (Miltenyi Biotec). For differentiation of immature dendritic cells (DCs), monocytes were cultured with GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) in RPMI1640 supplemented with 10% FBS for 6 days. For generation of mature DCs, immature DCs were treated with LPS (500 ng / mL) for 24 hr. Mature DCs were treated with mitomycin C at 40 μg / mL for 30 min at 37°C before co-culturing with T cells.

[0398] CD4+ T cells were isolated from donor B using anti-human CD4 conjugated magnetic beads (Miltenyi Biotec). Responder CD4 T cells were resuspended in culture medium at 4 x 10 6 cells / mL, and 50 μL of T cells were added to all wells except the DC only wells. Stimulator DCs were added to the wells at 4 x 10 6Cells / mL were resuspended in culture medium and 50 μΐ^DC were added to all wells except the CD4 T-only wells. An additional 100 μΐ^of culture medium containing 0.1-2 μg IL10-Fc or anti-PD-L1 / IL10 fusion proteins were added to the CD4 T-DC cultures in 96-well U-bottom plates. The co-cultures were incubated at 37°C. The concentration of IL-2 in the cell culture medium after 2 days of co-culture was determined by ELISA (Biolegend) according to the manufacturer's instructions.

[0399] Results

[0400] As shown in the assay data plotted in Figure 5A and Figure 5B , exemplary anti-PD-L1 antibodies (PHS102, PHS206, atezolizumab, avelumab, durvalumab) and anti-PD-L1 / IL10 fusion proteins (avelumab / IL10, PHS102 / IL10, YT7A / IL10, YT10H / IL10, HSYPP31F / IL10, HSYPP411C / IL10, YT7H / IL10, YP7G / IL10, YP11F / IL10) were able to enhance T cell activation in the MLR relative to hlgG or IL10-Fc controls.

[0401] Example 6: Anti-PD-L1 / IL10 fusion proteins stimulate MC / 9 cell proliferation

[0402] This example illustrates a study of the ability of IL10 polypeptides linked to exemplary anti-PD-L1 / IL10 fusion proteins of the disclosure to stimulate MC / 9 cell proliferation.

[0403] Materials and Methods

[0404] The biological activity of IL10 was determined by using a proliferation assay. MC / 9 (ATCC, CRL-8306) mouse mast cells were cultured in DMEM (GIBCO) supplemented with 2 mM L-glutamine, 0.05 nM 2-mercaptoethanol, 10% Rat T-STIM (Becton Dickenson), and 10% FBS. In the proliferation assay, MC / 9 cells were plated at 1 x 105per well in the presence of IL10-Fc or exemplary anti-PD-L1 / IL10 fusion proteins. 4 Seeded in 96-well plates in 200 μΐ assay medium (DMEM containing 10% FBS). MC / 9 cell proliferation was measured using the CellTiter-Glo assay after 72 hours of stimulation.

[0405] Results

[0406] As shown in the assay data plotted in Figure 6A , Figure 6B , Figure 6C , Figure 6D , Figure 6E and Figure 6F , the exemplary anti-PD-L1 / IL10 fusion proteins (atezolizumab / IL10, avelumab / IL10, durvalumab / IL10, PHS102 / IL10, PHS206 / IL10, YT7A / IL10, YT10H / IL10, HSYPP31F / IL10, HSYPP411C / IL10, YT6D / IL10, YT7H / IL10, YP7G / IL10, YP11F / IL10) were able to stimulate MC / 9 proliferation at enhanced levels relative to the IL10-Fc protein, as shown by the assay data plotted in

[0407] Table 10 Specific activity in stimulating MC / 9 proliferation

[0408] EC50 (nM) IL10-Fc 0.4781 Anti-PD-L1 / IL10 fusion Atezolizumab / IL10 0.0191 Avelumab / IL10 0.0197 Durvalumab / IL10 0.1309 PHS102 / IL10 0.0689 PHS206 / IL10 0.0626 PHS102 / IL10 0.04972 YT7A / IL10 0.04662 YT10H / IL10 0.05056 PHS102 / IL10 0.0515 HSYPP31F / IL10 0.02683 HSYPP411C / IL10 0.04183 PHS102 / IL10 0.05331 YT6D / IL10 0.008582 YT7H / IL10 0.02209 PHS102 / IL10 0.04052 YP7G / IL10 0.0561 YP11F / IL10 0.009712

[0409] Example 7: Anti-PD-L1 / IL10 fusion proteins activate CD8 T cells

[0410] This example illustrates a study of IL10 polypeptides linked to exemplary anti-PD-L1 / IL10 fusion proteins of the disclosure to activate CD8 T cells.

[0411] Materials and Methods

[0412] Human CD8 T cells were isolated from PBMCs by using CD8 magnetic beads (Miltenyi Biotec). Isolated CD8 T cells (1 x 10 7 cells / 3 mL / well in a 6-well plate) were cultured in AIM-V medium (Thermo Scientific) and activated for 3 days using T Cell TransAct (Miltenyi Biotec). After activation, the CD8 T cells were then washed and plated at 4 x 10 5 cells per well in a 96-well plate and treated with anti-PD-L1 / IL10 fusion proteins for 3 days. After treatment with anti-PD-L1 / IL10 fusion proteins, the cells were restimulated with 1 pg / mL soluble anti-CD3 (Biolegend) for 4 h. The concentration of IFN-g and granzyme B in the cell culture medium was determined by ELISA (Biolegend) according to the manufacturer’s instructions.

[0413] Results

[0414] As shown by the assays plotted in Figure 7A and Figure 7B and the EC50 values listed in Table 11 below, the exemplary anti-PD-L1 / IL10 fusion protein (avelumab / IL10, PHS102 / IL10) was able to activate CD8 T cells (as determined by IFNy and granzyme B levels) at enhanced levels relative to the IL10-Fc protein.

[0415] Table 11 : Activation of CD8 T cells

[0416]

[0417] Example 8: Anti-tumor activity of anti-PD-L1 / IL10 fusion proteins in syngeneic tumor models

[0418] This example illustrates the study of the anti-tumor activity of exemplary anti-PD-L1 / IL10 fusion proteins of the disclosure in two syngeneic tumor models, CT26 and EMT6.

[0419] Materials and Methods

[0420] BALB / c mice (6-8 weeks old, female) were subcutaneously implanted with 5x10 5 CT26 cells (ATCC CRL-2638) or 5x10 5 EMT6 cells (ATCC CRL-2755). After 8 days, when the tumor volume reached 50-100 mm 3 , the mice were randomly assigned to treatment groups. The mice were then intraperitoneally injected with PBS control, 3 mg / kg IL10-Fc (92 kDa), 4.9 mg / kg anti-PD-L1 antibody (avelumab) (150 kDa), 36 mg / kg anti-CSF1R / IL10 fusion (185.5 kDa), 5.8 mg / kg anti-PD-L1 / TGFpR fusion (M7824) (177 kDa), or 6 mg / kg anti-PD-L1 / IL10 fusion protein (avelumab / IL10) (185.5 kDa) twice per week. Tumor volume was measured by caliper measurements twice per week until the end of the study.

[0421] Results

[0422] As shown by the assays plotted in Figure 8A and Figure 8BExemplary anti-PD-L1 / IL10 fusion proteins of the present disclosure exhibited very strong anti-tumor activity during the course of the study, showing the smallest tumor volumes of all treatments in both the CT26 and EMT6 tumor models, as shown by the tumor volume data plotted.

[0423] While the appended claims set forth the broadest disclosure of the present disclosure, the disclosure described herein can also be defined by the claims below, which can be amended before and after grant by the Patent Office. The disclosure described herein, including embodiments, descriptions and implementations, is meant as illustrative only and is not to be construed as limiting the present disclosure. One skilled in the art could start with the disclosure described herein and, using routine experimentation, adapt the disclosure to various alternative embodiments, implementations and applications without departing from the spirit and scope of the present disclosure. Any and all such similar modifications and adaptations of the disclosure described herein are intended to fall within the scope of the present disclosure and appended claims.

[0424] While the foregoing disclosure of the present application has been described in some detail by way of example and illustration, it is not to be construed as limited thereto. The disclosure includes embodiments, descriptions and implementations described herein, which are intended as illustrative only and are not to be construed as limiting the present disclosure. One skilled in the art could start with the disclosure described herein and, using routine experimentation, adapt the disclosure to various alternative embodiments, implementations and applications without departing from the spirit and scope of the present disclosure. Any and all such similar modifications and adaptations of the disclosure described herein are intended to fall within the scope of the present disclosure and appended claims.

[0425] Other embodiments of the application are set forth in the following claims.

[0426] The disclosures of all publications, patent applications, patents or other documents mentioned in this text are expressly incorporated herein by reference for the purpose of providing illustrative support for some embodiments of the present disclosure. In the case of inconsistencies between the disclosure of this document and the disclosure of the documents incorporated by reference, the disclosure of this document will prevail.

Claims

1. An anti-PDLl antibody, characterized in that, complementarity determining region (CDR-L1), a second light chain complementarity determining region (CDR-L2), and a third light chain complementarity determining region (CDR-L3), and (ii) a first heavy chain complementarity determining region (CDR-H1), a second heavy chain complementarity determining region (CDR-H2), and a third heavy chain complementarity determining region (CDR-H3), wherein: (a) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 49, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 50, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 51, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 55; (b) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 87, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 88, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 89, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 91; (c) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 93, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 94, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 97; (d) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 99, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 100, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 101, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 103; (e) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 105, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 106, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 108; (f) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 93, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 110, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 111, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 113; (g) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 93, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 115, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 117; (h) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 119, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 120, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 122; or (i) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 124, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 125, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO:

55.

2. The antibody of claim 1, wherein the antibody comprises a heavy chain variable domain (V H ) amino acid sequence and a light chain variable domain (V L ) amino acid sequence, wherein the antibody comprises: (a) V that is at least 90% identical to SEQ ID NO: 52 H an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 56 L an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 56 (b) V having an amino acid sequence of SEQ ID NO: 90 H (b) V having an amino acid sequence of SEQ ID NO: 90 L (b) V having an amino acid sequence of SEQ ID NO: 90 (c) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 96 H (c) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 96 L (c) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 96 (d) V having an amino acid sequence of SEQ ID NO: 102 H (d) V having an amino acid sequence of SEQ ID NO: 102 L (d) V having an amino acid sequence of SEQ ID NO: 102 (e) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 107 H (e) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 107 L (e) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 107 (f) V that is at least 90% identical to SEQ ID NO: 112 H an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 114 L an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 114 (g) V having an amino acid sequence of SEQ ID NO: 116 H (g) V having an amino acid sequence of SEQ ID NO: 116 L (g) V having an amino acid sequence of SEQ ID NO: 116 (h) V that is at least 90% identical to SEQ ID NO: 121 H an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 123 L an amino acid sequence; or (i) V has an amino acid sequence that is at least 90% identical to SEQ ID NO: 126 H (ii) V has an amino acid sequence that is at least 90% identical to SEQ ID NO: 56 L (ii) V has an amino acid sequence that is at least 90% identical to SEQ ID NO: 56 3. The antibody of claim 1, wherein the antibody comprises a heavy chain (HC) amino acid sequence and a light chain (LC) amino acid sequence, wherein the antibody comprises: (a) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 149, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 142; (b) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 154, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 128; (c) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 155, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 130; (d) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 156, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 132; (e) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 157, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 134; (f) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 158, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 136; (g) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 159, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 138; (h) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 160, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 140; or (i) a HC amino acid sequence that is at least 90% identical to SEQ ID NO: 161, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO:

142.

4. A recombinant anti-PD-L1 / IL10 fusion protein, characterized in that, The antibody according to any one of claims 1-3, wherein the IL10 is fused to the C-terminus of the antibody heavy chain (HC) via a linker, wherein the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 74, 75, 76, 77, 78, and 79.

5. The fusion protein of claim 4, wherein: The amino acid sequence of the IL10 is set forth in SEQ ID NO:

73.

6. The fusion protein of claim 5, wherein the antibody comprises: (a) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 83, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 142; (b) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 127, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 128; (c) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 129, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 130; (d) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 131, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 132; (e) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 133, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 134; (f) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 135, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 136; (g) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 137, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 138; (h) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 139, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 140; or (i) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 141, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO:

142.

7. An anti-PD-Ll antibody fusion protein, characterized in that, the HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 135, and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 136; (b) the CDR-H1 amino acid sequence of SEQ ID NO: 87, the CDR-H2 amino acid sequence of SEQ ID NO: 88, the CDR-H3 amino acid sequence of SEQ ID NO: 89, the CDR-L1 amino acid sequence of SEQ ID NO: 53, the CDR-L2 amino acid sequence of SEQ ID NO: 54, and the CDR-L3 amino acid sequence of SEQ ID NO: 91; and (b) the CDR-H1 amino acid sequence of SEQ ID NO: 87, the CDR-H2 amino acid sequence of SEQ ID NO: 88, the CDR-H3 amino acid sequence of SEQ ID NO: 89, the CDR-L1 amino acid sequence of SEQ ID NO: 53, the CDR-L2 amino acid sequence of SEQ ID NO: 54, and the CDR-L3 amino acid sequence of SEQ ID NO: 91; and (c) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 93, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 94, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 97; (d) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 99, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 100, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 101, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 103; (e) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 105, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 106, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 108; (f) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 93, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 110, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 111, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 113; (g) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 93, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 115, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 117; (h) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 119, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 120, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO: 122; or (i) the amino acid sequence of CDR-H1 is set forth in SEQ ID NO: 124, the amino acid sequence of CDR-H2 is set forth in SEQ ID NO: 125, the amino acid sequence of CDR-H3 is set forth in SEQ ID NO: 95, the amino acid sequence of CDR-L1 is set forth in SEQ ID NO: 53, the amino acid sequence of CDR-L2 is set forth in SEQ ID NO: 54, and the amino acid sequence of CDR-L3 is set forth in SEQ ID NO:

55.

8. The anti-PD-Ll antibody fusion protein of claim 7, wherein the antibody comprises a heavy chain variable domain (V H ) amino acid sequence and a light chain variable domain (V L ) amino acid sequence, wherein the antibody comprises: (a) V that is at least 90% identical to SEQ ID NO: 52 H an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 56 L an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 56 (b) V having an amino acid sequence of SEQ ID NO: 90 H (b) V having an amino acid sequence of SEQ ID NO: 90 L (b) V having an amino acid sequence of SEQ ID NO: 90 (c) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 96 H (c) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 96 L (c) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 96 (d) V having an amino acid sequence of SEQ ID NO: 102 H (d) V having an amino acid sequence of SEQ ID NO: 102 L (d) V having an amino acid sequence of SEQ ID NO: 102 (e) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 107 H (e) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 107 L (e) V having an amino acid sequence that is at least 90% identical to SEQ ID NO: 107 (f) V that is at least 90% identical to SEQ ID NO: 112 H an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 114 L an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 114 (g) V having an amino acid sequence of SEQ ID NO: 116 H (g) V having an amino acid sequence of SEQ ID NO: 116 L (g) V having an amino acid sequence of SEQ ID NO: 116 (h) V that is at least 90% identical to SEQ ID NO: 121 H an amino acid sequence; and V that is at least 90% identical to SEQ ID NO: 123 L an amino acid sequence; or (i) V has an amino acid sequence that is at least 90% identical to SEQ ID NO: 126 H (ii) V has an amino acid sequence that is at least 90% identical to SEQ ID NO: 56 L (ii) V has an amino acid sequence that is at least 90% identical to SEQ ID NO: 56 9. The anti-PD-Ll antibody fusion protein of claim 7, wherein the antibody fusion protein comprises a HC-IL10 fusion amino acid sequence and a light chain (LC) amino acid sequence, wherein the antibody fusion protein comprises (a) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 83 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 142; (b) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 127 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 128; (c) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 129 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 130; (d) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 131 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 132; (e) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 133 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 134; (f) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 135 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 136; (g) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 137 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 138; (h) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 139 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO: 140; or (i) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 141 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO:

142. (i) a HC-IL10 fusion amino acid sequence that is at least 90% identical to SEQ ID NO: 141 and a LC amino acid sequence that is at least 90% identical to SEQ ID NO:

142.

10. An isolated polynucleotide, comprising: an antibody of any one of claims 1-3; or a fusion protein of any one of claims 4-9.

11. An isolated host cell, wherein, comprising the polynucleotide of claim 10.

12. A method of producing an antibody, characterized by, comprising culturing the isolated host cell of claim 11 to produce the antibody.

13. A pharmaceutical composition, characterized by, comprising the antibody of any one of claims 1-3 or the fusion protein of any one of claims 4-9, and a pharmaceutically acceptable carrier.

14. Use of the antibody of any one of claims 1-3, the fusion protein of any one of claims 4-9, or the pharmaceutical composition of claim 13 in the manufacture of a medicament for treating a disease; wherein the disease is colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, stomach cancer, head and neck cancer, or oral cancer.

15. Use of a PD-L1 antagonist and an IL10 agonist in the manufacture of a medicament for treating a cancer; wherein the PD-L1 antagonist comprises the antibody of any one of claims 1-3; wherein the IL10 agonist is an IL-10 having an amino acid sequence set forth in SEQ ID NO: 73; and the cancer is colon cancer, pancreatic cancer, ovarian cancer, liver cancer, kidney cancer, breast cancer, lung cancer, stomach cancer, head and neck cancer, or oral cancer.

16. The use of any one of claims 14-15, wherein the medicament further comprises T cells.

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