Anti-PD-l1 antibodies and antibody-drug conjugates

Abstract

To provide novel anti-PD-L1 antibodies and antibody-drug conjugates and methods of using such anti-PD-L1 antibodies and antibody-drug conjugates to treat cancer.SOLUTION: Anti-PD-L1 antibodies and PD-L1-directed antibody-drug conjugates (ADCs) are provided herein. In particular, PD-L1-directed camptothecin ADCs and MMAE ADCs are provided herein. Also provided herein are methods of using anti-PD-L1-directed antibodies and ADCs to treat PD-L1-expressing disorders. Preferred anti-PD-L1 antibodies exhibit a binding affinity to the human PD-L1 protein that is between 3 nM and 300 nM.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Patent Application No. 62 / 910,988, filed Oct. 4, 2019, which is hereby incorporated by reference in its entirety. The present invention relates to novel anti - PD - L1 antibodies and antibody - drug conjugates, and methods of using such anti - PD - L1 antibodies and antibody - drug conjugates for treating cancer.

Background Art

[0002] PD - L1, also known as programmed death ligand 1, B7 - H1 or CD274, is a protein that has been shown to be expressed in various cancer cells. PD - L1 is a transmembrane protein that can interact with PD - 1 and act as an “off” switch to inactivate T cells. PD - L1 is generally overexpressed on tumor cells, and by binding to PD - 1, tumors can evade the T - cell immune response.

[0003] There are several cancers that express PD - L1, including melanoma. Melanoma is the most dangerous type of skin cancer. In 2015, there were 3.1 million people with active disease, and melanoma caused 59,800 deaths. The 5 - year survival rate for stage IV disease is less than 10%, and the median survival is only 6 - 12 months. Thus, improved treatments are needed for melanoma and other cancers that express PD - L1. One type of treatment for cancers that express PD - L1 involves administering anti - PD - L1 antibodies as immunotherapy. Immuno - oncology is a promising area of cancer treatment, but there is room for improvement in current therapies.

[0004] All references cited herein, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference.

SUMMARY OF THE INVENTION

MEANS FOR SOLVING THE PROBLEM

[0005] Anti-PD-L1 antibodies and PD-L1-directed antibody-drug conjugates (ADCs) are provided herein. In particular, PD-L1-directed camptothecin ADCs and MMAE ADCs are provided herein. Methods of using anti-PD-L1-directed antibodies and ADCs for treating PD-L1 expression disorders are also provided herein. Preferred anti-PD-L1 antibodies exhibit a binding affinity for human PD-L1 protein that is from 3 nM to 300 nM. Other preferred anti-PD-L1 antibodies comprise heavy chain CDR sequences of SEQ ID NOs: 3-5 and light chain CDR sequences of SEQ ID NOs: 6-8, wherein the antibody comprises 1 or more amino acid substitutions in 1 or more of the CDRs. Other preferred anti-PD-L1 antibodies comprise heavy chain CDR sequences of SEQ ID NOs: 13-15 and light chain CDR sequences of SEQ ID NOs: 16-18.

[0006] Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to human programmed death ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity for human PD-L1 protein that is from 3 to 300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein that is from 3 to 15 nM.

[0007] In some embodiments, the antibody further exhibits a total internalization that is higher than that of Ab1. In some embodiments, the total internalization is an increase in AUC of 9% to 155% relative to the AUC of Ab1. In some embodiments, the total internalization is determined by the FabFluor internalization assay.

[0008] In some embodiments, the antibody further exhibits an x50 lower than x50 of Ab1. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE), and in the MDA-MB-231 cell line, the x50 is 3 ng / mL to 20 ng / mL.

[0009] In some embodiments, the antibody is conjugated to camptothecin, and in the MDA-MB-231 cell line, the x50 is 15 ng / mL to 55 ng / mL.

[0010] In some embodiments, the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 13-15 and the light chain CDR sequences of SEQ ID NOs: 16-18.

[0011] In some embodiments, the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, and the antibody comprises one or more amino acid substitutions in one or more of the CDRs.

[0012] In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity with SEQ ID NO: 11 and a light chain variable region sequence having at least 80% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity with SEQ ID NO: 11 and a light chain variable region sequence having at least 90% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with SEQ ID NO: 11 and a light chain variable region sequence having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 12.

[0013] In some embodiments, the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0014] In some embodiments, the fragment is a Fab, Fab’, F(ab’)2, Fab’-SH, Fv, diabody, linear antibody, or single-chain antibody fragment.

[0015] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0016] In some embodiments, the heavy chain constant region is of the IgG1 isotype.

[0017] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0018] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker.

[0019] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit includes a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker and has the following structure:

Chemical formula

[0020] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker and has the structure: [Chemical formula] to form an antibody-drug conjugate, wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0021] An antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody comprises heavy chain CDR sequences of SEQ ID NOs: 3-5 and light chain CDR sequences of SEQ ID NOs: 6-8, and the antibody comprises one or more amino acid substitutions in one or more of the CDRs, is also provided herein.

[0022] In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein that is 3-300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein that is 3-15 nM.

[0023] In some embodiments, the antibody further exhibits a total internalization that is higher than the total internalization of Ab1. In some embodiments, the total internalization is an increase of 9%-155% of the AUC relative to the AUC of Ab1. In some embodiments, the total internalization is determined by the FabFluor internalization assay.

[0024] In some embodiments, the antibody further exhibits an x50 that is higher than the x50 of Ab1.

[0025] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE), and in the MDA-MB-231 cell line, x50 is from 3 ng / mL to 20 ng / mL.

[0026] In some embodiments, the antibody is conjugated to camptothecin, and in the MDA-MB-231 cell line, x50 is from 15 ng / mL to 55 ng / mL.

[0027] In some embodiments, the antibody comprises heavy chain CDR sequences of SEQ ID NOs: 13-15 and light chain CDR sequences of SEQ ID NOs: 16-18. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity with SEQ ID NO: 11 and a light chain variable region sequence having at least 80% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity with SEQ ID NO: 11 and a light chain variable region sequence having at least 90% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with SEQ ID NO: 11 and a light chain variable region sequence having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 12.

[0028] In some embodiments, the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0029] In some embodiments, the fragment is a Fab, Fab’, F(ab’)2, Fab’-SH, Fv, diabody, linear antibody, or single-chain antibody fragment.

[0030] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0031] In some embodiments, the heavy chain constant region is of the IgG1 isotype.

[0032] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0033] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker and has the following structure: [Chemical formula] to form an antibody-drug conjugate, wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0034] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit comprises a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker and has the structure: [Chemical formula] to form an antibody-drug conjugate, wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0035] An antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody comprises a heavy chain CDR sequence of SEQ ID NOs: 13-15 and a light chain CDR sequence of SEQ ID NOs: 16-18, and the antibody or antigen-binding fragment thereof is provided herein.

[0036] In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein that is 3-300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein that is 3-15 nM.

[0037] In some embodiments, the antibody further exhibits a total internalization that is higher than that of Ab1. In some embodiments, the total internalization is an increase of 9%-155% of the AUC relative to the AUC of Ab1. In some embodiments, the total internalization is determined by the FabFluor internalization assay.

[0038] In some embodiments, the antibody further exhibits an x50 that is higher than that of Ab1.

[0039] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE), and in the MDA-MB-231 cell line, the x50 is 3 ng / mL - 20 ng / mL.

[0040] In some embodiments, the antibody is conjugated to camptothecin, and in the MDA-MB-231 cell line, the x50 is 15 ng / mL - 55 ng / mL.

[0041] In some embodiments, the antibody has a heavy chain variable region sequence having at least 80% sequence identity with SEQ ID NO: 11 and a light chain It includes a variable region array of the lock. In some embodiments, the antibody includes a heavy chain variable region array having at least 90% sequence identity with SEQ ID NO: 11 and a light chain variable region array having at least 90% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody includes a heavy chain variable region array having at least 95% sequence identity with SEQ ID NO: 11 and a light chain variable region array having at least 95% sequence identity with SEQ ID NO: 12. In some embodiments, the antibody includes the heavy chain variable region array of SEQ ID NO: 11 and the light chain variable region array of SEQ ID NO: 12.

[0042] In some embodiments, the antibody includes the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0043] In some embodiments, the fragment is a Fab, Fab’, F(ab’)2, Fab’-SH, Fv, diabody, linear antibody, or single-chain antibody fragment.

[0044] In some embodiments, the antibody contains L234A mutation and L235A mutation in the heavy chain of the antibody.

[0045] In some embodiments, the heavy chain constant region is of the IgG1 isotype.

[0046] In some embodiments, the antibody is a humanized antibody or a chimeric antibody.

[0047] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit includes a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker and has the following structure:

Chemical formula

[0048] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit comprises a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker and has the structure:

Chemical formula

[0049] An antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody is conjugated to camptothecin to form an antibody-drug conjugate, and the antibody-drug conjugate has the structure:

Chemical formula

[0050] In some embodiments, the antibody-drug conjugate has the structure:

Chemical formula

[0051] In some embodiments, p ranges from 2 to 10.

[0052] Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to human programmed death ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity for human PD-L1 protein that is greater than Ab1. In some embodiments, the antibody exhibits a binding affinity greater than 2.7 nM.

[0053] Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to human programmed death ligand 1 (PD-L1) protein, wherein the antibody has a k assoc less than that of Ab1 for human PD-L1 protein. In some embodiments, the antibody has a k 5 M -1 s -1 less than 5×10 assoc for human PD-L1 protein.

[0054] Also provided herein is an antibody or antigen-binding fragment thereof that specifically binds to human programmed death ligand 1 (PD-L1) protein, wherein the antibody has a k dissoc greater than that of Ab1 for human PD-L1 protein. In some embodiments, the antibody has a k 3 s -1 greater than 2×10 dissoc for human PD-L1 protein.

[0055] Also provided herein is an antibody-drug conjugate that is an antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, the antibody comprises one or more amino acid substitutions in one or more of the CDRs, the antibody exhibits a binding affinity for human PD-L1 protein that is 5 nM to 15 nM, and the antibody is conjugated to MMAE.

[0056] An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody comprises the heavy-chain CDR sequences of SEQ ID NOs: 3-5 and the light-chain CDR sequences of SEQ ID NOs: 6-8, the antibody comprises one or more amino acid substitutions in one or more of the CDRs, the antibody exhibits a binding affinity for human PD-L1 protein that is 5 nM to 15 nM, the antibody is conjugated to camptothecin, and the antibody-drug conjugate is provided herein.

[0057] Also provided herein is a pharmaceutical composition comprising a therapeutically effective amount of the antibody described herein and a pharmaceutically acceptable excipient.

[0058] Also provided herein is a method of treating cancer in a subject, comprising administering to the subject any of the antibodies described herein. In some embodiments, the subject is a human subject. In some embodiments, the cancer is melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0059] Also provided herein is a nucleic acid encoding any of the antibodies described herein.

[0060] Also provided herein is a vector comprising any of the nucleic acids described herein.

[0061] Also provided herein is any of the host cells described herein comprising any of the nucleic acids described herein. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0062] Also provided herein is a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, comprising culturing any of the host cells described herein under conditions suitable for antibody production.

[0063] Also provided herein is a method for producing an antibody-drug conjugate that specifically binds to human PD-L1 protein, comprising culturing any of the host cells described herein under conditions suitable for antibody production and conjugating the antibody to a cytotoxic agent. In some embodiments, the cytotoxic agent is MMAE or camptothecin.

[0064] cancer (e.g., PD-L1 + Also provided herein is the use of any of the anti-PD-L1 antibodies described herein or any antibody-drug conjugate for the manufacture of a medicament for the treatment of cancer (e.g., cancer associated with PD-L1 expression).

[0065] cancer (e.g., PD-L1 + Also provided herein is the use of an anti-PD-L1 antibody described herein or an antibody-drug conjugate described herein for the treatment of cancer (e.g., cancer associated with PD-L1 expression).

[0066] Also provided herein is the use of an anti-PD-L1 antibody described herein or an antibody-drug conjugate described herein for use in a medicament.

[0067] PD-L1 + A method of killing PD-L1 + cells in a subject in need of cell death, the method comprising administering to the subject a therapeutically effective amount of any of the anti-PD-L1 antibodies described herein or any of the antibody-drug conjugates described herein is also provided herein.

[0068] PD-L1 + A method of killing PD-L1 +Also provided herein is the use of any of the anti-PD-L1 antibodies described herein or any of the antibody-drug conjugates described herein for use in the manufacture of a medicament for killing cells.

[0069] A method of reducing the volume of a solid tumor in a subject (e.g., a PD-L1 + solid tumor) comprising administering to the subject a therapeutically effective amount of any of the anti-PD-L1 antibodies described herein or any of the antibody-drug conjugates described herein is also provided herein.

[0070] A solid tumor in a subject (e.g., a PD-L1 + solid tumor) for reducing the volume of the Also provided herein is the use of any of the anti-PD-L1 antibodies described herein or any of the antibody-drug conjugates described herein for use in the manufacture of a medicament. BRIEF DESCRIPTION OF THE DRAWINGS

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Modes for Carrying Out the Invention

[0085] I. Definitions To enable the present disclosure to be more readily understood, certain terms are first defined. As used in this application, unless otherwise explicitly provided herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth throughout this application.

[0086] As used herein, the term "and / or" shall be construed as a specific disclosure of each of the two specified features or components, whether or not the other is present. Thus, the term "and / or" as used in terms such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in terms such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0087] It is understood that the aspects and embodiments of the invention described herein include those "comprising," "consisting of," and "consisting essentially of" aspects and embodiments.

[0088] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provides those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0089] Units, prefixes, and symbols are shown in the form approved by the Systeme International de Unites (SI). Numerical ranges include the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure that can be had by reference to the specification as a whole. Accordingly, the terms defined below are more fully defined by reference to the specification as a whole.

[0090] The terms "PD-L1", "CD274", "B7-H1", and "programmed cell death ligand 1" are used interchangeably herein and, unless otherwise specified, generally include any variant, isoform, and species homolog of human PD-L1 that is expressed by a cell or expressed on a cell transfected with the PD-L1 gene.

[0091] The term "immunoglobulin" refers to a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structure of immunoglobulins is well characterized. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as V H or VH) and a heavy chain constant region (C H or CH). The heavy chain constant region typically consists of three domains, C H 1, C H 2, and C HIt consists of 3. The heavy chains are generally linked to each other via disulfide bonds in the so-called "hinge region". Each light chain typically consists of a light chain variable region (abbreviated as V L or VL herein) and a light chain constant region (C L or CL). The light chain constant region typically consists of one domain C L from. CL can be of the κ (kappa) or λ (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. The immunoglobulin can be derived from any of the generally known isotypes including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0092] The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. The variable regions of the heavy and light chains of a natural antibody (V H and V LIt can be further subdivided into hypervariable regions (or hypervariable regions that can be hypervariable in the form of arrays and / or structurally defined loops), also called complementarity-determining regions (CDRs), in which more conserved regions, called framework regions (FRs), are scattered. The terms "complementarity-determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known in the art to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy-chain variable region (CDR-H1, CDR-H2, CDR-H3), and three CDRs in each light-chain variable region (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs in each full-length heavy-chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light-chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). Within each V H and V L , the three CDRs and four FRs are typically arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol., 195, 901-917 (1987)).

[0093] As used herein, the term "antibody" (Ab) in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, that has the ability to specifically bind to an antigen with a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour (h), at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, etc., for a significant period of time, or any other relevant functionally defined period (e.g., a time sufficient to induce, facilitate, enhance and / or modulate a physiological response related to antibody binding to an antigen and / or a time sufficient for the antibody to mobilize effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain binding domains that interact with the antigen. The constant region of the antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (such as effector cells) and components of the complement system such as C1q, which is the first component in the classical pathway of complement activation. Antibodies can also be bispecific antibodies, diabodies, multispecific antibodies or similar molecules.

[0094] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules made recombinantly that have a single primary amino acid sequence. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice having a genome containing a human heavy chain transgene and a light chain transgene fused to immortalized cells.

[0095] "Isolated antibody" refers to an antibody that substantially does not contain other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-L1 substantially does not contain antibodies that specifically bind to antigens other than PD-L1). However, an isolated antibody that specifically binds to PD-L1 can have cross-reactivity with other antigens such as PD-L1 molecules from different species. Further, the isolated antibody may not substantially contain other cellular materials and / or chemical substances. In one embodiment, the isolated antibody comprises an antibody conjugate bound to another agent (e.g., for example, a small molecule drug). In some embodiments, the isolated anti-PD-L1 antibody comprises a conjugate of an anti-PD-L1 antibody and a small molecule drug (e.g., MMAE or MMAF).

[0096] "Human antibody" (HuMAb) refers to an antibody having a variable region in which both the FR and CDR are derived from human germline immunoglobulin sequences. Further, when the antibody comprises a constant region, that constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure can contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species such as a mouse are grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used synonymously.

[0097] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody that includes a human antibody constant domain and a non-human variable domain that has been modified to contain a high level of sequence homology to a human variable domain. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that together form the antigen binding site, into a homologous human acceptor framework region (FR) (see International Publication No. WO 92 / 22653 and European Patent No. 0629240). Framework residue substitutions (back mutations) from the parental antibody (i.e., non-human antibody) to the human framework region may be required to fully reconstruct the binding affinity and binding specificity of the parental antibody. Structural homology modeling can be useful in identifying amino acid residues within the framework region that are important for the binding properties of the antibody. Thus, a humanized antibody can include non-human CDR sequences, a human framework region that includes, optionally, one or more amino acid back mutations to non-human amino acid sequences, and a fully human constant domain. Optionally, additional amino acid modifications that are not necessarily back mutations can be applied to obtain a humanized antibody having desired characteristics such as affinity and biochemical properties.

[0098] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., derived from a rodent), and the constant region is derived from a different species such as a human. Chimeric antibodies can be generated by antibody engineering. "Antibody engineering" is a term commonly used for the modification of different types of antibodies and is a process well-known to those skilled in the art. In particular, chimeric antibodies can be generated using standard DNA techniques such as those described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Ch. 15. Thus, chimeric antibodies can be genetically or enzymatically engineered recombinant antibodies. Generating chimeric antibodies is within the knowledge of those skilled in the art, and thus, the generation of chimeric antibodies according to the present invention can be performed by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic applications have been developed to reduce antibody immunogenicity. They can typically include a non-human (e.g., mouse) variable region specific for the antigen of interest, as well as the heavy chain domain and light chain domain of a human constant antibody. The term "variable region" or "variable domain" as used in the context of chimeric antibodies refers to a region that includes both the CDR and the framework regions of both the heavy and light chains of an immunoglobulin.

[0099] "Antigen-binding antibody" refers to an antibody that binds to an antigen. For example, an anti-PD-L1 antibody is an antibody that binds to the antigen PD-L1.

[0100] The "antigen-binding portion" or antigen-binding fragment of an antibody is specific for the antigen bound by the whole antibody refers to one or more fragments of an antibody that retain the ability to bind specifically. Examples of antibody fragments (e.g., antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called “Fab” fragments, each having a single antigen-binding site, and a residual “Fc” fragment whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-binding sites and can still cross-link antigens.

[0101] “Percent (%) sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence after the sequences are aligned to achieve maximum percent sequence identity and gaps are introduced, if necessary, with no consideration of any conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (or, equivalently, a given amino acid sequence A having or including a particular percent sequence identity to, with, or against a given amino acid sequence B) can be calculated as follows. 100 times the ratio X / Y In the formula, X is the number of amino acid residues scored as identical matches by the alignment of the sequences in the programs of A and B, and Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % sequence identity of A to B is not equal to the % sequence identity of B to A.

[0102] As used herein, the terms "binding," "binds," or "specifically binds" in the context of the binding of an antibody to a given antigen typically refer to a K of about 10 -6 M or less, e.g., 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less or about 10 -11 M or even less of K D corresponding to binding with an affinity such that the antibody binds to a given antigen with an affinity that is, for example, at most 1 / 10, e.g., at most 1 / 100, e.g., at most 1 / 1,000, e.g., at most 1 / 10,000, e.g., at most 1 / 100,000 of the K D for binding to a non-specific antigen (e.g., BSA, casein) other than the given antigen or an antigen closely related thereto. Since the lower the K D of the binding depends on the K D of the antibody, when the K D of the antibody is very low, the K D of the binding to the antigen may be at most 1 / 10,000 of the K D of the binding to a non-specific antigen (i.e., the antibody is highly specific). D The term "K

[0103] D " (M) as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. The affinity as used herein and K D Dis in an inverse relationship, i.e., a higher affinity means a lower K D is intended to refer to, and a lower affinity means a higher K D is intended to refer to.

[0104] The term "ADC" refers to an antibody-drug conjugate and, in the context of the present invention, refers to an anti-PD-L1 antibody conjugated to a drug moiety (e.g., MMAE or MMAF) as described in the present application.

[0105] The abbreviations "vc" and "val-cit" refer to valine-citrulline of a dipeptide linker.

[0106] The abbreviation VKG refers to valine-lysine-glycine of a tripeptide linker.

[0107] The abbreviation "MC" refers to maleimidocaproyl of a spacer:

Chemical formula

[0108] The abbreviation "MP" refers to maleimidopropionyl of a spacer:

Chemical formula

[0109] As used herein, a "PEG unit" is an organic moiety composed of repeating ethyleneoxy subunits (PEG or PEG subunits) and can be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture and thus provides a single chain length and molecular weight. Preferred PEG units include discrete PEGs, which are compounds synthesized stepwise without a polymerization process. Discrete PEG provides a single molecule with a defined specific chain length.

[0110] The PEG units provided herein include one or more polyethylene glycol chains, each composed of one or more ethyleneoxy subunits covalently bonded to each other. The polyethylene glycol chains can be linked to each other, for example, in a linear, branched or star configuration. Typically, at least one of the polyethylene glycol chains before incorporation into the camptothecin conjugate is derivatized at one end with an alkyl moiety substituted with an electrophilic group for covalent attachment to the carbamate nitrogen of a methylene carbamate unit (i.e., representing an example of R). Typically, the terminal ethyleneoxy subunit in each polyethylene glycol chain that is not involved in the covalent attachment to the remainder of the linker unit is modified with a PEG capping unit, typically an optionally substituted alkyl such as -CH3, CH2CH3 or CH2CH2CO2H. Preferred PEG units have a single polyethylene glycol chain in which 2 to 24 -CH2CH2O- subunits are covalently bonded in series and terminated at one end with a PEG capping unit.

[0111] "Cancer" refers to a wide group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancer tissue" may include tumors. Uncontrolled cell division and growth lead to the formation of malignant tumors that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, the distant tumor can be said to "derive" from the pre - metastatic tumor.

[0112] The term "antibody - dependent cell - mediated cytotoxicity" or ADCC is a mechanism that induces cell death depending on the interaction between antibody - coated target cells and immune cells with lytic activity (also called effector cells). Such effector cells include natural killer cells, monocytes / macrophages and neutrophils. Effector cells bind to the Fc effector domain of Ig bound to the target cell via their antigen - binding sites. The death of antibody - coated target cells occurs as a result of effector cell activity.

[0113] The term "antibody-dependent cell phagocytosis" or ADCP refers to the process by which antibody-coated cells are internalized, either wholly or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc effector domain of the Ig.

[0114] The term "complement-dependent cytotoxicity" or CDC refers to a mechanism that induces cell death in which the Fc effector domain of a target-binding antibody activates a series of enzymatic reactions that ultimately lead to the formation of pores in the target cell membrane. Typically, antigen-antibody complexes, such as those on an antibody-coated target cell, bind to and activate complement component C1q, which then activates the complement cascade to bring about target cell death. Activation of the complement can also result in the deposition on the surface of the target cell of complement components that promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0115] "Cell growth inhibitory effect" refers to the inhibition of cell growth. A "cell growth inhibitor" refers to an agent that has a cell growth inhibitory effect on cells and thereby inhibits the growth and / or expansion of a particular subset of cells. A cell growth inhibitor can be conjugated to an antibody or administered in combination with an antibody.

[0116] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on, or administration of an active agent to, a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical indicia associated with a disease. In some embodiments, the disease is cancer.

[0117] "Subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents such as mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms "subject", "patient", and "individual" are used interchangeably herein.

[0118] An "effective amount" or "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent, when used alone or in combination with another therapeutic agent, protects the subject against the onset of a disease, or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or prevention of functional or disability impairment due to the pain of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those of skill in the art, such as in human subjects during clinical trials, in predictive animal model systems of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0119] As an example for the treatment of tumors, a therapeutically effective amount of an anti-cancer agent inhibits cell growth or tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% in a treated subject (e.g., one or more treated subjects) compared to an untreated subject (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective amount of an anti-cancer agent inhibits cell growth or tumor growth by 100% in a treated subject (e.g., one or more treated subjects) compared to an untreated subject (e.g., one or more untreated subjects).

[0120] In other embodiments of the disclosure, tumor regression can be observed and can continue for at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days.

[0121] A therapeutically effective amount of a drug (e.g., an anti-PD-L1 antibody-drug conjugate) includes any amount of the drug that inhibits the development or recurrence of cancer when administered alone or in combination with an anti-cancer agent to a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or at risk of suffering from cancer recurrence, which is a "preventive effective amount". In some embodiments, the preventive effective amount completely prevents the development or recurrence of cancer. "Inhibiting" the development or recurrence of cancer means either reducing the likelihood of the development or recurrence of cancer or completely preventing the development or recurrence of cancer.

[0122] As used herein, "a dose below the therapeutic amount" means a dose of a therapeutic compound (e.g., an anti-PD-L1 antibody-drug conjugate) that is lower than the normal or typical dose of the therapeutic compound when administered alone for the treatment of a hyperproliferative disorder (e.g., cancer).

[0123] "Immune-related response pattern" refers to a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce an anti-tumor effect by inducing a cancer-specific immune response or by modifying the natural immune process. This response pattern is characterized by an initial increase in tumor burden or the appearance of new lesions followed by a beneficial therapeutic effect, which is classified as disease progression in the evaluation of traditional chemotherapeutic agents and is synonymous with drug failure. Thus, an appropriate evaluation of immunotherapeutic agents may require long-term monitoring of the effects of these agents on the target disease.

[0124] As an example, an "anticancer agent" promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of a drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means that administration of an effective amount of the drug alone or in combination with an anticancer agent results in a decrease in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of periods without disease symptoms, or prevention of dysfunction or disability due to disease. Further, the terms "effective" and "efficacy" with respect to a treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or organism level resulting from administration of the drug.

[0125] "Sustained response" refers to a persistent effect on the reduction of tumor growth after cessation of treatment. For example, the tumor size may remain the same or smaller compared to the size at the start of the dosing phase. In some embodiments, the sustained response has a duration that is at least as long as the treatment period or at least 1.5-fold, 2.0-fold, 2.5-fold, or 3-fold longer than the treatment period.

[0126] As used herein, "complete response" or "CR" refers to the disappearance of all target lesions. "Partial response" or "PR" refers to a decrease of at least 30% in the sum of the longest diameters (SLD) of target lesions, with reference to the baseline SLD. "Stable disease" or "SD" refers to neither a reduction in target lesions sufficient to qualify for PR nor an increase sufficient to qualify for PD, with reference to the minimum SLD after the start of treatment.

[0127] As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not progress. Progression-free survival can include the length of time a patient experiences a complete response or partial response, as well as the length of time a patient experiences stable disease.

[0128] As used herein, "overall response rate" or "ORR" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0129] As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group who are likely to survive after a specified period.

[0130] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients that make up the formulation and / or the mammal being treated therewith.

[0131] The expression "pharmaceutically acceptable salts" as used herein refers to pharmaceutically acceptable organic or inorganic salts of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharinate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts can include the inclusion of another molecule such as an acetate ion, succinate ion or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Further, pharmaceutically acceptable salts can have more than one charged atom in their structure. When multiple charged atoms are part of a pharmaceutically acceptable salt, multiple counterions can be present. Thus, pharmaceutically acceptable salts can have one or more charged atoms and / or one or more counterions.

[0132] "Administering" or "administration" refers to the physical introduction of a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration of an anti-PD-L1 antibody-drug conjugate include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration by injection or infusion (e.g., intravenous infusion). As used herein, the phrase "parenteral administration" generally means a mode of administration other than enteral and topical administration by injection, and includes, but is not limited to, intravenous, intramuscular, arterial, intrathecal, lymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural and intrasternal injections and infusions, as well as in vivo electroporation. A therapeutic agent can be administered via a parenteral route or orally. Other parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasal, vaginal, rectal, sublingual or topical administration. Administration can be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0133] As used interchangeably herein, the terms "baseline" or "baseline value" can refer to the measurement or characterization of symptoms before the administration of a treatment (e.g., an anti-PD-L1 antibody-drug conjugate as described herein) or at the start of administration of the treatment. By comparing the baseline value to a reference value, it is possible to determine the reduction or improvement of symptoms of a PD-L1 related disease (e.g., cancer) contemplated herein. As used interchangeably herein, the terms "reference" or "reference value" can refer to the measurement or characterization of symptoms after the administration of a treatment (e.g., the anti-PD-L1 antibody-drug conjugate described). The reference value can be measured once or more during the dosing regimen or treatment cycle or at the completion of the dosing regimen or treatment cycle. A "reference value" can be an absolute value, a relative value, a value having an upper and / or lower limit, a range of values, an average value, a median value, a mean value, or a value compared to the baseline value.

[0134] Similarly, a "baseline value" may be an absolute value, a relative value, a value having an upper and / or lower limit, a range of values, an average value, a median value, an average value, or a value compared to a reference value. The reference value and / or the baseline value can be obtained from one individual, two different individuals, or a population of individuals (e.g., a group of 2, 3, 4, 5 or more individuals).

[0135] As used herein, the term "monotherapy" means that the anti-PD-L1 antibody-drug conjugate is the only anti-cancer agent administered to a subject during a treatment cycle. However, other therapeutic agents can be administered to the subject. For example, an anti-inflammatory agent, or another agent that is administered to a subject having cancer to treat symptoms associated with cancer (e.g., inflammation, pain, weight loss, and general malaise), but does not treat the underlying cancer itself, can be administered during the period of monotherapy.

[0136] As used herein, an "adverse event" (AE) is any undesirable, generally unintended, or unwanted sign (including abnormal laboratory findings), symptom, or disease associated with the use of a medical treatment. A medical treatment can have one or more related AEs, and each AE can have the same or different levels of severity. References to "changes in adverse events" refer to treatment regimens that reduce the incidence and / or severity of one or more AEs associated with the use of different treatment regimens.

[0137] As used herein, a "serious adverse event" or "SAE" is an adverse event that meets one of the following criteria. · Fatal or life-threatening (when used in the definition of a serious adverse event, "life-threatening" refers to an event where there was a risk that the patient could die at the time of the event. It does not refer to an event that hypothetically could have caused death if it were more severe). · Results in persistent or significant disability / incapacity · Constitutes a congenital anomaly / birth defect · It is defined as an event that is medically important, i.e., an event that may endanger the patient or require medical or surgical intervention to prevent one of the outcomes listed above. When determining whether an AE is "medically important", medical and scientific judgment must be exercised. · Excluding (1) routine treatment or monitoring of underlying diseases without deterioration of the condition, (2) elective or pre-planned treatment for an existing condition that is unrelated to the indication under study and has not deteriorated since signing the informed consent, and (3) respite care in the absence of social reasons and deterioration of the patient's general condition, hospitalization of inpatients or extension of an existing hospitalization is required.

[0138] The use of alternative forms (e.g., "or") should be understood to mean any, both, or any combination of the alternative forms. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more than one" of the recited or enumerated components.

[0139] The terms "about" or "comprising essentially of" refer to a value or composition that is within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, "about" or "comprising essentially of" can mean within or exceeding one standard deviation, according to the conventions of the art. Alternatively, "about" or "comprising essentially of" can mean a range of up to 20%. Further, especially with respect to biological systems or processes, these terms can mean a value within one order of magnitude or up to five-fold. When a particular value or composition is provided in the present application and claims, unless otherwise specified, the meaning of "about" or "comprising essentially of" should be assumed to be within the acceptable error range for that particular value or composition.

[0140] References to a value or parameter “about” in this specification include (and describe) embodiments that target the value or parameter itself. For example, a description that refers to “about X” describes “X” inclusively.

[0141] As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

[0142] Various aspects of the present disclosure are described in further detail in the following subsections.

[0143] II. General The present invention provides antibodies and ADCs that specifically bind to PD-L1. The present invention is based in part on the discovery that antibody-drug conjugates, including an MMAE antibody-drug conjugate and a camptothecin antibody-drug conjugate targeted to PD-L1, are particularly effective at killing PD-L1+ expressing cells. PD-L1 has been shown to be expressed in various cancers, including melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer.

[0144] III. Target Molecule Unless otherwise indicated, PD-L1 refers to human PD-L1. The exemplary human protein sequence is assigned UniProt ID NO. Q9NZQ7.

[0145] IV. Antibodies of the Invention Previously, the select antibodies that were already being used to treat cancer were conjugated to cytotoxic agents without sequence modification to make antibody-drug conjugates (ADCs). These ADCs have often been shown to be more effective or more effective than unconjugated antibodies in killing tumor cells. Previously, if modifications to the antibody were contemplated during the process of preparing the ADC, some of the possible modifications were to increase the binding affinity of the antibody or to increase antibody functions such as ADCC. However , it has been found that in at least some situations, for example, by modifying or tuning the ADC antibody by decreasing its binding affinity or decreasing its ADCC activity, the efficacy of the ADC is improved compared to an ADC with an unmodified antibody. Some of these examples include ADCs having anti-PD-L1 antibodies (such as Ab1) that are surprisingly optimized by modifying the antibodies, for example, by decreasing their binding affinity. For example, in some cases, an anti-PD-L1 ADC is more effective at killing tumor cells in vitro when the binding affinity of the antibody conjugated to the cytotoxic agent is decreased. In another example, in some cases, an anti-PD-L1 ADC is more effective at killing tumor cells in vitro and in vivo when the binding affinity of the antibody conjugated to the cytotoxic agent is decreased.

[0146] The present invention provides an antibody, such as a humanized antibody, that binds to PD-L1 with a binding affinity of 3 nM to 300 nM. In some embodiments, the antibodies described herein have a binding affinity of about 3 nM to 300 nM (e.g., about 3 nM to about 275 nM, about 3 nM to about 250 nM, about 3 nM to about 225 nM, about 3 nM to about 200 nM, about 3 nM to about 175 nM, about 3 nM to about 150 nM, about 3 nM to about 125 nM, about 3 nM to about 100 nM, about 3 nM to about 90 nM, about 3 nM to about 80 nM, about 3 nM to about 70 nM, about 3 nM to about 60 nM, about 3 nM to about 50 nM, about 3 nM to about 40 nM, about 3 nM to about 30 nM, about 3 nM to about 20 nM, about 3 nM to about 10 nM, about 10 nM to about 300 nM, about 10 nM to about 275 nM, about 10 nM to about 250 nM, about 10 nM to about 225 nM, about 10 nM to about 200 nM, about 10 nM to about 175 nM, about 10 nM to about 150 nM, about 10 nM to about 125 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 20 nM to about 300 nM, about 20 nM to about 275 nM, about 20 nM to about 250 nM, about 20 nM to about 225 nM, about 20 nM to about 200 nM, about 20 nM to about 175 nM, about 20 nM to about 150 nM, about 20 nM to about 125 nM, about 20 nM to about 100 nM, about 20 nM to about 90 nM, about 20 nM to about 80 nM, about 20 nM to about 70 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 20 nM to about 30 nM, about 30 nM to about 300 nM, about 30 nM to about 275 nM, about 30 nM to about 250 nM, about 30 nM to about 225 nM, about 30 nM to about 200 nM, about 30 nM to about 175 nM, about 30 nM to about 150 nM, about 30 nM to about 125 nM, about 30 nM to about 100 nM, about 30 nM to about 90 nM, about 30 nM to about 80 nM, about 30 nM to about 70 nM, about 30 nM to about 60 nM, about 30 nM to about 50 nM, about 30 nM to about 40 nM, about 40 nM to about 300 nM, about 40 nM to about 275 nM, about 40 nM to about 250 nM, about 40 nM to about 225 nM, about 40 nM to about 200 nM, about 40 nM to about 175 nM, about 40 nM to about 150 nM, about 40 nM to about 125 nM,From about 40 nM to about 100 nM, from about 40 nM to about 90 nM, from about 40 nM to about 80 nM, from about 40 nM to about 70 nM, from about 40 nM to about 60 nM, from about 40 nM to about 50 nM, from about 50 nM to about 300 nM, from about 50 nM to about 275 nM, from about 50 nM to about 250 nM, from about 50 nM to about 225 nM, from about 50 nM to about 200 nM, from about 50 nM to about 175 nM, from about 50 nM to about 150 nM, from about 50 nM to about 125 nM, from about 50 nM to about 100 nM, from about 50 nM to about 90 nM, from about 50 nM to about 80 nM, from about 50 nM to about 70 nM, from about 50 nM to about 60 nM, from about 60 nM to about 300 nM, from about 60 nM to about 275 nM, from about 60 nM to about 250 nM, from about 60 nM to about 225 nM, from about 60 nM to about 200 nM, from about 60 nM to about 175 nM, from about 60 nM to about 150 nM, from about 60 nM to about 125 nM, from about 60 nM to about 100 nM, from about 60 nM to about 90 nM, from about 60 nM to about 80 nM, from about 60 nM to about 70 nM, from about 70 nM to about 300 nM, from about 70 nM to about 275 nM, from about 70 nM to about 250 nM, from about 70 nM to about 225 nM, from about 70 nM to about 200 nM, from about 70 nM to about 175 nM, from about 70 nM to about 150 nM, from about 70 nM to about 125 nM, from about 70 nM to about 100 nM, from about 70 nM to about 90 nM, from about 70 nM to about 80 nM, from about 80 nM to about 300 nM, from about 80 nM to about 275 nM, from about 80 nM to about 250, nM, about 80 nM to about 225 nM, about 80 nM to about 200 nM, about 80 nM to about 175 nM, about 80 nM to about 150 nM, about 80 nM to about 125 nM, about 80 nM to about 100 nM, about 80 nM to about 90 nM, about 90 nM to about 300 nM, about 90 nM to about 275 nM, about 90 nM to about 250 nM, about 90 nM to about 225 nM, about 90 nM to about 200 nM, about 90 nM to about 175 nM, about 90 nM to about 150 nM, about 90 nM to about 125 nM, about 90 nM to about 100 nM, about 100 nM to about 300 nM, about 100 nM to about 275 nM, about 100 nM to about 250 nM, about 100 nM to about 225 nM, about 100 nM to about 200 nM, about 100 nM to about 175 nM, about 100 nM to about 150 nM, about 100 nM to about 125 nM, about 125 nM to about 300 nM, about 125 nM to about 275 nM, about 125 nM to about 250 nM, about 125 nM to about 225 nM, about 125 nM to about 200 nM, about 125 nM to about 175 nM, about 125 nM to about 150 nM, about 150 nM to about 300 nM, about 150 nM to about 275 nM, about 150 nM to about 250 nM, about 150 nM to about 225 nM, about 150 nM to about 200 nM, about 150 nM to about 175 nM, about 175 nM to about 300 nM, about 175 nM to about 275 nM, about 175 nM to about 250 nM, about 175 nM to about 225 nM, about 175 nM to about 200 nM, about 200 nM to about 300 nM, about 200 nM to about 275 nM, about 200 nM to about 250 nM, about 200 nM to about 225 nM, about 225 nM to about 300 nM, about 225 nM to about 275 nM, about 225 nM to about 250 nM, about 250 nM to about 300 nM, about 250 nM to about 275 nM, or about 275 nM to about 30 nM) (for example, when measured by biolayer interferometry (BLI) in phosphate buffered saline) of K D binds to PD-L1 at

[0147] In some embodiments, the binding affinity is a monovalent binding affinity. In some embodiments, these antibodies are point mutants of the full-length human anti-PD-L1 antibody Ab1. Ab1 is defined by the CDR regions of SEQ ID NOs: 3-5 and SEQ ID NOs: 6-8, the variable regions of SEQ ID NOs: 1 and 2, and the heavy and light chains of SEQ ID NOs: 86 and 87. In further embodiments, the point mutations are found in the CDR regions. In some embodiments, the point mutants exhibit a decreased binding affinity and / or an increased cytotoxicity and / or internalization rate as compared to Ab1. In some embodiments, the point mutants exhibit a decreased binding affinity and an increased cytotoxicity in vitro. In some embodiments, the point mutants exhibit a decreased binding affinity and an increased cytotoxicity in vivo. In some embodiments, the point mutants exhibit a decreased binding affinity and an increased cytotoxicity both in vitro and in vivo. In some embodiments, the point mutants exhibit a decreased binding affinity and an increased internalization rate in vitro. In some embodiments, the point mutants exhibit a decreased binding affinity and an increased internalization rate in vivo. In some embodiments, the point mutants exhibit a decreased binding affinity and an increased internalization rate both in vitro and in vivo.

[0148] In some embodiments, the anti-PD-L1 antibodies provided herein can have one or two total amino acid substitutions in the set of six CDRs of the heavy chain CDRs of SEQ ID NOs: 3-5 and the light chain CDRs of SEQ ID NOs: 6-8, and bind to PD-L1 with a KD of 3 nM to 300 nM. In some embodiments, the anti-PD-L1 antibodies provided herein can have one amino acid substitution in the set of six CDRs of the heavy chain CDRs of SEQ ID NOs: 3-5 and the light chain CDRs of SEQ ID NOs: 6-8, and bind to PD-L1 with a KD of 3 nM to 300 nM.

[0149] In some embodiments, the anti-PD-L1 antibody provided herein can have a heavy chain CDR1 having one amino acid substitution at SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 4, a heavy chain CDR3 of SEQ ID NO: 5, a light chain CDR1 of SEQ ID NO: 6, a light chain CDR2 of SEQ ID NO: 7, and a light chain CDR3 of SEQ ID NO: 8, and binds to PD-L1 with a KD of 3 nM to 300 nM. In some embodiments, the one amino acid substitution in SEQ ID NO: 3 is at amino acid position 2 of SEQ ID NO: 3. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution from tyrosine to alanine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution from tyrosine to serine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution from tyrosine to glycine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution from tyrosine to threonine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution from tyrosine to valine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution from tyrosine to cysteine.

[0150] In some embodiments, the anti-PD-L1 antibody provided herein binds to both glycosylated PD-L1 and non-glycosylated PD-L1 with a K D D of 3 nM to 300 nM (or any of the sub-ranges of this range described herein).

[0151] In some embodiments, the anti-PD-L1 antibodies provided herein exhibit increased in vitro and / or in vivo cytotoxicity of PD-L1+ cells compared to Ab1 (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or a 5% to 300% increase, a 5% to 280% increase, a 5% to 260% increase, a 5% to 240% increase, a 5% to 220% increase, a 5% to 200% increase, a 5% to 180% increase, a 5% to 160% increase, a 5% to 140% increase, a 5% to 120% increase, a 5% to 100% increase, a 5% to 80% increase, a 5% to 60% increase, a 5% to 40% increase, a 5% to 20% increase, a 5% to 10% increase, a 10% to 300% increase, a 10% to 280% increase, a 10% to 260% increase, a 10% to 240% increase, a 10% to 220% increase, a 10% to 200% increase, a 10% to 180% increase, a 10% to 160% increase, a 10% to 140% increase, a 10% to 120% increase, a 10% to 100% increase, a 10% to 80% increase, a 10% to 60% increase, a 10% to 40% increase, a 10% to 20% increase, a 20% to 300% increase, a 20% to 280% increase, a 20% to 260% increase, a 20% to 240% increase, a 20% to 220% increase, a 20% to 200% increase, a 20% to 180% increase, a 20% to 160% increase, a 20% to 140% increase, a 20% to 120% increase, a 20% to 100% increase, a 20% to 80% increase, a 20% to 60% increase, a 20% to 40% increase,An increase of 40% to 300%, an increase of 40% to 280%, an increase of 40% to 260%, an increase of 40% to 240%, an increase of 40% to 220%, an increase of 40% to 200%, an increase of 40% to 180%, an increase of 40% to 160%, an increase of 40% to 140%, an increase of 40% to 120%, an increase of 40% to 100%, an increase of 40% to 80%, an increase of 40% to 60%, an increase of 60% to 300%, an increase of 60% to 280%, an increase of 60% to 260%, an increase of 60% to 240%, an increase of 60% to 220%, an increase of 60% to 200%, an increase of 60% to 180%, an increase of 60% to 160%, an increase of 60% to 140%, an increase of 60% to 120%, an increase of 60% to 100%, an increase of 60% to 80%, an increase of 80% to 300%, an increase of 80% to 280%, an increase of 80% to 260%, an increase of 80% to 240%, an increase of 80% to 22, 0% increase, 80% increase to 200% increase, 80% increase to 180% increase, 80% increase to 160% increase, 80% increase to 140% increase, 80% increase to 120% increase, 80% increase to 100% increase, 100% increase to 300% increase, 100% increase to 280% increase, 100% increase to 260% increase, 100% increase to 240% increase, 100% increase to 220% increase, 100% increase to 200% increase, 100% increase to 180% increase, 100% increase to 160% increase, 100% increase to 140% increase, 100% increase to 120% increase, 120% increase to 300% increase, 120% increase to 280% increase, 120% increase to 260% increase, 120% increase to 240% increase, 120% increase to 220% increase, 120% increase to 200% increase, 120% increase to 180% increase, 120% increase to 160% increase, 120% increase to 140% increase, 140% increase to 300% increase, 140% increase to 280% increase, 140% increase to 260% increase, 140% increase to 240% increase, 140% increase to 220% increase, 140% increase to 200% increase, 140% increase to 180% increase, 140% increase to 160% increase, 160% increase to 300% increase, 160% increase to 280% increase, 160% increase to 260% increase, 160% increase to 240% increase, 160% increase to 220% increase, 160% increase to 200% increase, 160% increase to 180% increase, 180% increase to 300% increase, 180% increase to 280% increase, 180% increase to 260% increase, 180% increase to 240% increase, 180% increase to 220% increase, 180% increase to 200% increase, 200% increase to 300% increase, 200% increase to 280% increase, 200% increase to 260% increase, 200% increase to 240% increase, 200% increase to 220% increase, 220% increase to 300% increase, 220% increase to 280% increase, 220% increase to 260% increase, 220% increase to 240% increase, 240% increase to 300% increase, 240% increase to 280% increase, 240% increase to 260% increase, 260% increase to 300% increase, 260% increase to 280% increase, or 280% increase to 300% increase).

[0152] In some embodiments, the anti-PD-L1 antibodies provided herein have an increased rate of internalization by PD-L1+ cells compared to Ab1 (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least an 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least an 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or a 5% to 300% increase (or any sub-range of this range described herein)).

[0153] In some embodiments, the anti-PD-L1 antibodies provided herein have an increased immune cell infiltration upon administration to a mammal compared to Ab1 (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least an 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least an 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or a 5% to 300% increase (or any sub-range of this range described herein)).

[0154] In some embodiments, the anti-PD-L1 antibodies provided herein have an increased inflammatory cytokine production (e.g., the cytokines described herein) upon administration to a mammal compared to Ab1. increased (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least an 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least an 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or a 5% increase to 300% increase (or any sub-range of this range described herein)) in any one or more of

[0155] In some embodiments, the anti-PD-L1 antibody provided herein increased intracellular digestion by PD-L1+ cells (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least an 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least an 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or a 5% increase to 300% increase (or any sub-range of this range described herein)) compared to Ab1.

[0156] In some embodiments, the anti-PD-L1 antibody provided herein has a change of less than 10% (e.g., less than 8%, less than 6%, less than 4%, less than 2%, or less than 1%) in neutrophil and / or platelet counts upon administration to a mammal compared to Ab1.

[0157] The binding affinity of the PD-L1 antibody of the invention (i.e., dissociation constant K D) preferably exceeds the binding affinity of Ab1. Preferred PD-L1 antibodies bind to the same epitope and / or compete with Ab1 for binding to human PD-L1. In one embodiment, the binding affinity of the PD-L1 antibody of the present invention is greater than 2.7 nM. In a further embodiment, the monovalent binding affinity of the PD-L1 antibody of the present invention is greater than 2.7 nM. In another embodiment, k assoc (or on rate) is less than that of Ab1. In a further embodiment, k assoc is 5.5×10 5 M -1 s -1 less than. In another embodiment, k dissoc (or off rate) exceeds the k dissoc (or off rate) of Ab1. In a further embodiment, k dissoc is greater than 1.50×10 3 s -1 greater than.

[0158] The anti-PD-L1 antibodies of the present invention can also be described or specified with respect to their binding affinity for PD-L1 (e.g., human PD-L1). In some embodiments, preferred binding affinities include a dissociation constant or K greater than 2.7 nM, greater than 5 nM, greater than 6 nM, greater than 7 nM, greater than 8 nM, greater than 9 nM, greater than 10 nM, greater than 15 nM, greater than 20 nM, greater than 25 nM, greater than 30 nM, greater than 40 nM, greater than 50 nM, greater than 60 nM, greater than 70 nM, greater than 80 nM, greater than 90 nM, greater than 100 nM, greater than 110 nM, greater than 120 nM, greater than 130 nM, greater than 140 nM, greater than 150 nM, greater than 200 nM, greater than 250 nM, greater than 300 nM, greater than 400 nM or greater than 500 nM DThose having [the relevant property] are included. In some embodiments, preferred PD-L1 antibodies have a binding affinity of 3 nM to 300 nM, 3 nM to 200 nM, 3 nM to 100 nM, 3 nM to 50 nM, 3 nM to 40 nM, 3 nM to 20 nM, 3 nM to 15 nM, 5 nM to 300 nM, and 5 nM to 15 nM. In some embodiments, preferred PD-L1 antibodies have a binding affinity that is at least 2-fold, 3-fold, 3.7-fold, 4-fold or 5-fold greater than the binding affinity of Ab1. In some of the above embodiments, the binding affinity is a monovalent binding affinity.

[0159] In some embodiments, the binding of the anti-PD-L1 antibody of the present invention is pH-dependent, and thus the antibody exhibits different bindings across a pH gradient. In some embodiments, the anti-PD-L1 antibody exhibits maximum binding at a pH of about 4 to about 10. In some embodiments, the maximum binding is at a pH of about 6 to about 9. In some embodiments, the maximum binding is at a pH of about 6.5 to about 8.

[0160] Preferred antibodies of the present invention inhibit cancer (e.g., cell growth, metastasis and / or lethality to an organism) as shown on cancerous cells growing in culture in an animal model or clinical trial. Animal models can be formed by transplanting PD-L1-expressing human tumor cell lines into a suitable immunodeficient rodent strain, such as athymic nude mice or SCID mice. These tumor cell lines can be established as solid tumors by subcutaneous injection or as disseminated tumors by intravenous injection in an immunodeficient rodent host.

[0161] Once established in the host, these tumor models can be applied to evaluate the therapeutic efficacy of an anti-PD-L1 antibody or its conjugate form as described in the examples.

[0162] Generally, the anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate of the present disclosure binds to PD-L1, for example, human PD-L1, and exerts an inhibitory effect on cell proliferation and a cytotoxic effect on malignant cells such as cancer cells. A 50% decrease in viability compared to untreated cells, or x50, or IC 50The concentration required to effect is one method of measuring the cytotoxicity of the anti-PD-L1 antibody and / or anti-PD-ADC. The preferred antibodies and / or ADCs of the present invention exhibit increased cytotoxicity and x50 as compared to those of the Ab1 antibody and / or ADC. In one embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 10 ng / mL to 30 ng / mL or 15 ng / mL to 25 ng / mL in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 15 ng / mL to 55 ng / mL or 20 ng / mL to 50 ng / mL in the MDA-MB-231 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 1 ng / mL to 7 ng / mL or 2 ng / mL to 5 ng / mL in the KARPAS 299 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 15 ng / mL to 40 ng / mL, or 20 ng / mL to 35 ng / mL in the L540CY cell line. In one embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits an x50 of 12 ng / ml to 70 ng / ml, or 15 ng / ml to 65 ng / ml in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits an x50 of 3 ng / mL to 20 ng / mL or 5 ng / mL to 17 ng / mL in the MDA-MB-231 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits an x50 of 1 ng / mL to 18 ng / mL or 3 ng / mL to 15 ng / mL in the KARPAS 299 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits an x50 of 1 ng / mL to 20 ng / mL or 1 ng / mL to 15 ng / mL in the L540CY cell line.

[0163] Generally, the anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates of the present disclosure are internalized into cells such as cancer cells. One way to measure internalization is to utilize a pH-sensitive antibody conjugate that emits a fluorescent signal upon internalization in a cell-based assay. This total internalization of the antibody can be quantified by the area under the curve (or AUC) of the fluorescent signal over time. The FabFluor (IncuCyte®) internalization assay can be used for this quantification. Preferred antibodies and / or ADCs of the present invention exhibit increased total internalization as compared to the total internalization of Ab1 and / or ADC. In one embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 9% to 155% relative to the AUC of Ab1. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 40% to 130%, or 40% to 50%, relative to the AUC of Ab1 when tested in the 786-O cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 90% to 100%, or 90% to 95%, relative to the AUC of Ab1 when tested in the A375 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 85% to 155%, or 85% to 90%, relative to the AUC of Ab1 when tested in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 9% to 40%, or 9% to 13%, relative to the AUC of Ab1 when tested in the ES-2 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 75% to 145%, or 75% to 80%, relative to the AUC of Ab1 when tested in the MDA-MB-231 cell line.

[0164] The anti-PD-L1 antibodies of the present disclosure are preferably monoclonal and can be multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, Fab fragments, F(ab’) fragments, fragments produced by a Fab expression library, and any PD-L1 binding fragment of the above. In some embodiments, the anti-PD-L1 antibodies of the present disclosure specifically bind to PD-L1. The immunoglobulin molecules of the present disclosure can be of any type of immunoglobulin molecule (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass. In one embodiment, the anti-PD-L1 antibody of the present disclosure is of the IgG1 type.

[0165] In certain embodiments of the present disclosure, the anti-PD-L1 antibody is an antigen-binding fragment (e.g., a human antigen-binding fragment) as described herein, including but not limited to Fab, Fab’, and F(ab’)2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-bonded Fv (sdFv), and fragments containing either the L V domain or the H V domain. Antigen-binding fragments containing single-chain antibodies can include the variable region alone or in combination with all or part of the hinge region, CH1, CH2, CH3, and CL domains. Antigen-binding fragments containing any combination of variable regions with hinge region, CH1, CH2, CH3, and CL domains are also included in the present disclosure. In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment is human, murine (e.g., mouse and rat), equine, ovine, rabbit, caprine, guinea pig, camelid, equine or avian.

[0166] The anti-PD-L1 antibodies of the present disclosure can be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies can be specific for different epitopes of PD-L1 or can be specific for both PD-L1 and a heterologous protein. See, for example, PCT Publications WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., 1991, J. Immunol. 147:6069, U.S. Patent Nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920, 5,601,819, Kostelny et al., 1992, J. Immunol. 148:1547 - 1553.

[0167] The anti-PD-L1 antibodies of the present disclosure can be described or specified with respect to the particular CDRs they contain. The exact amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), ’’Sequences of Proteins of Immunological Interest,’’ 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering scheme); Al-Lazikani et al., (199 7) JMB 273,927-948 (the “Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), ’’Antibody-antigen interactions: Contact analysis and binding site topography,’’ J. Mol. Biol. 262, 732-745.’’ (the “Contact” numbering scheme); Lefranc MP et al., ’’IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,’’Dev Comp Immunol,2003 Jan;27(1):55-77(「IMGT」 numbering scheme);Honegger A and Plueckthun A,’’Yet another numbering scheme for immunoglobulin variable domains:an automatic modeling and analysis tool,’’J Mol Biol,2001 Jun 8;309(3):657-70,(「Aho」 numbering scheme); and Martin et al.,’’Modeling antibody hypervariable loops:a combined algorithm,’’PNAS,1989,86(23):9268-9272,(「AbM」 numbering scheme), can be readily determined using any of a number of well-known schemes, including those described by. The boundaries of a given CDR can vary depending on the scheme used for identification. In some embodiments, the “CDR” or “complementary determining region” or individual specific CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., its variable region) are to be understood to encompass CDRs as defined by any of the foregoing schemes (or specific). For example, if a particular CDR (e.g., CDR-H3) is stated to include the amino acid sequence of the corresponding CDR in a given V H or V L region amino acid sequence, such a CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the foregoing schemes. The scheme for identifying one or more particular CDRs may be specified, such as CDRs as defined by the Kabat, Chothia, AbM, or IMGT methods.

[0168] The CDR sequences of the anti-PD-L1 antibodies and anti-PD-L1 antibody-drug conjugates described herein follow the Kabat numbering scheme as described in Kabat et al., (1991), ’’Sequences of Proteins of Immunological Interest,’’ 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.

[0169] In one aspect, an anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and / or the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, and the CDRs of the anti-PD-L1 antibody are defined by the Kabat numbering scheme, an anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate are provided herein.

[0170] In one aspect, an anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12 are provided herein. In one aspect, the sequence number An anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10 are provided herein.

[0171] In some embodiments, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 comprises substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., FRs). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 11 including post-translational modifications of the sequence.

[0172] In some embodiments, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 12 includes substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 12 are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDRs (i.e., FRs). In some embodiments, the anti-PD-L1 antibody comprises the light chain variable domain sequence of SEQ ID NO: 12 including post-translational modifications of the sequence.

[0173] In some embodiments, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 includes substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 1. In certain embodiments, the heavy chain comprises one point mutation relative to SEQ ID NO: 1. In a further embodiment, the one point mutation is located in the CDR region.

[0174] In some embodiments, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a light chain variable do main comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 includes substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted and / or deleted in SEQ ID NO: 2. In certain embodiments, the light chain comprises one point mutation relative to SEQ ID NO: 2. In a further embodiment, the one point mutation is located in the CDR region.

[0175] In some embodiments, the anti-PD-L1 antibody of the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate is a monoclonal antibody.

[0176] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each having a heavy chain called α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in multiple polymorphic variants called allotypes (reviewed in Jefferis and Lefranc 2009. mAbs Vol 1 Issue 4 1-7), any of which are suitable for use in part of the embodiments herein. Common allotype variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody may include a heavy chain Fc region that includes a human IgG Fc region. In further embodiments, the human IgG Fc region includes human IgG1.

[0177] The antibody also includes derivatives modified by covalent attachment of any type of molecule to the antibody such that the covalent bond does not prevent the antibody from binding to PD-L1 or exerting an antiproliferative or cytotoxic effect on cells. For example, without limitation, antibody derivatives include antibodies modified by, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, binding to cell ligands or other proteins, and the like. Any of a number of chemical modifications can be performed by known techniques including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Further, the derivative may contain one or more non-classical amino acids.

[0178] Humanized antibody A humanized antibody is a genetically engineered antibody in which the CDRs derived from a non-human "donor" antibody are transplanted into a human "acceptor" antibody sequence (see, e.g., Queen, U.S. Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539; Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. No. 5,859,205; and Foote, U.S. Pat. No. 6,881,557). The acceptor antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of human antibody sequences, or a germline region sequence. Preferred acceptor sequences for the heavy chain are germline V H exon V H 1-2 (also referred to as HV1-2 in the literature) (Shin et al., 1991, EMBO J. 10:3641-3645), and for the hinge region (J H ), exon J H -6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred acceptor sequence is exon VK2-30 (also referred to as KV2-30 in the literature), and for the hinge region, exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Thus, a humanized antibody is fully An antibody having some or all of the CDRs derived entirely or substantially from a donor antibody, and, if present, a variable region framework sequence and a constant region derived entirely or substantially from a human antibody sequence. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor heavy chain antibody, and, if present, a heavy chain variable region framework sequence and a heavy chain constant region substantially derived from a human heavy chain variable region framework and constant region sequence. Similarly, a humanized light chain has at least one, two, and usually all three CDRs derived entirely or substantially from a donor antibody light chain, and, if present, a light chain variable region framework sequence and a light chain constant region substantially derived from a human light chain variable region framework and constant region sequence. Except for nanobodies and dAbs, humanized antibodies include a humanized heavy chain and a humanized light chain. The CDRs in a humanized antibody are derived from the corresponding CDRs in a non-human antibody when at least 60%, 85%, 90%, 95% or 100% of the corresponding residues (as defined by Kabat) are identical between each CDR. The variable region framework sequence of an antibody chain or the constant region of an antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively, when at least 85%, 90%, 95% or 100% of the corresponding residues as defined by Kabat are identical. In some embodiments, the PD-L1 antibody of the invention is a humanized antibody.

[0179] Humanized antibodies often incorporate all six CDRs (preferably as defined by Kabat) from a mouse antibody, but it is also possible to make humanized antibodies using fewer than all of the CDRs (e.g., at least 3, 4 or 5) from a mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0180] Selection of Constant Region The heavy and light chain variable regions of a humanized antibody can be linked to at least a portion of a human constant region. The selection of the constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce more potent cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be λ or κ. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the heavy and light chain variable domains are linked through a spacer.

[0181] Human constant regions exhibit allotypic and isotypic diversity among different individuals, i.e., the constant region can vary among different individuals at one or more polymorphic positions. An isotype differs from an allotype in that sera that recognize the isotype bind to non-polymorphic regions of one or more other isotypes.

[0182] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be deleted or derivatized in part or all of the molecule. To decrease or increase effector functions such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or To extend the half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004), substitutions can be made in the constant region.

[0183] Exemplary substitutions include amino acid substitutions to the cysteine residue of a natural amino acid, which are introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330 or 332 of the human IgG1 isotype, preferably the S239C mutation (U.S. Patent Application Publication No. 20100158909). The presence of additional cysteine residues allows for the formation of interchain disulfide bonds. The formation of such interchain disulfide bonds can cause steric hindrance, thereby reducing the affinity of the Fc region-FcγR binding interaction. The cysteine residue(s) introduced in or near the Fc region of the IgG constant region can also serve as a site for conjugation to a therapeutic agent (i.e., coupling a cytotoxic drug using a thiol-specific reagent such as a maleimide derivative of the drug). The presence of the therapeutic agent causes steric hindrance, thereby further reducing the affinity of the Fc region-FcγR binding interaction. Other substitutions at any of positions 234, 235, 236 and / or 237 reduce the affinity for Fcγ receptors, particularly the FcγRI receptor (see, e.g., U.S. Patent No. 6,624,821, U.S. Patent No. 5,624,821).

[0184] The in vivo half-life of an antibody can also affect its effector function. The half-life of an antibody can be increased or decreased to modify its therapeutic activity. FcRn is a receptor that is structurally similar to MHC class I antigens that non-covalently associate with β2-microglobulin. FcRn regulates the catabolism of IgG across tissues and their transcytosis (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The IgG-FcRn interaction occurs at pH 6.0 (the pH of intracellular vesicles), but not at pH 7.4 (the pH of blood). This interaction enables IgG to be recycled back into circulation (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The regions on human IgG1 involved in FcRn binding have been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 of human IgG1 enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules with these substitutions have a longer serum half-life. As a result, these modified IgG1 molecules can perform their effector functions over a longer period compared to unmodified IgG1, and thus can exert their therapeutic efficacy. Other exemplary substitutions for increasing binding to FcRn include Gln at position 250 and / or Leu at position 428. EU numbering is used for all positions within the constant region.

[0185] The oligosaccharide covalently attached to conserved Asn297 is involved in the ability of the Fc region of IgG to bind to FcγR (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 1997, Trends Biotechnol. 15:26-31). Manipulation of this glycoform on IgG can significantly improve IgG-mediated ADCC. Addition of bisecting N-acetylglucosamine modification to this glycoform (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) or removal of fucose from this glycoform (Shields et al., 2002, J. Biol. Chem. 277:26733- 40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niva et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc engineering that improve the binding of IgG Fc to FcγR and thereby enhance IgG-mediated ADCC activity.

[0186] Systematic substitution of solvent-exposed amino acids in the Fc region of human IgG1 generated IgG variants with altered FcγR binding affinity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). A subset of these variants containing substitutions at Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 Lys334 for Ala showed increased binding affinity for FcγR and ADCC activity when compared to parental IgG1 (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

[0187] The complement fixation activity of antibodies (both C1q binding and CDC activity) can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575). The same substitutions on the human IgG2 backbone can convert an antibody isotype that binds poorly to C1q and has significantly impaired complement activation activity into one that can bind to C1q and mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methods have also been applied to improve the complement fixation activity of antibodies. For example, grafting of the 18-amino acid carboxyl-terminal tail piece of IgM to the carboxyl terminus of IgG greatly enhances their CDC activity. This has also been observed for IgG4, which normally does not have detectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Substituting Ser444, which is located near the carboxyl terminus of the IgG1 heavy chain, with Cys induces tail-to-tail dimerization of IgG1 and increases the CDC activity 200-fold compared to monomeric IgGl (Shopes et al., 1992, J. Immunol. 148:2918-22). Furthermore, bispecific diabody constructs with specificity for C1q also confer CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0188] Complement activity can be reduced by mutating at least one of amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted nonionic residues such as Gly, Leu, or Val, or aromatic nonpolar residues such as Phe, Tyr, Trp, and Pro in place of any one of the three residues also reduce or abolish C1q binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322 to reduce or abolish C1q binding activity, but not at 318.

[0189] Substitution of residue 318 (Glu) with a polar residue may alter, but not abolish, C1q binding activity. Substitution of residue 297 (Asn) with Ala leads to the elimination of lytic activity, but only slightly reduces affinity for C1q (approximately 3-fold weaker). This change destroys the glycosylation site and the presence of carbohydrate required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination thereof also reduce C1q binding: D270A, K322A, P329A and P31 IS (see WO06 / 036291). The L234A / L235A mutation (or LALA mutation) also reduces C1q binding as well as FcyR binding. In one embodiment, the anti-PD-L1 antibody of the invention comprises a L234A / L235A mutation.

[0190] Reference to a human constant region includes constant regions with any naturally occurring allotypes or naturally occurring allotypes. Any permutation of residues occupying polymorphic positions in the constant region may be used, including any permutation of residues occupying polymorphic positions in the constant region. Also, there may be up to 1, 2, 5 or 10 mutations relative to a native human constant region, such as those shown above, to decrease Fc gamma receptor binding or increase binding to Fc RNA.

[0191] V. Expression of Recombinant Antibodies Humanized antibodies are usually produced by recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences, such as naturally associated or heterologous promoter regions, operably linked to the coding sequences of the antibody chains. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and for the collection and purification of cross-reacting antibodies.

[0192] Mammalian cells are preferred hosts for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting the heterologous protein in its complete state have been developed in the art and include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2 / 0 and NS0. Preferably, the cells are non-human. Expression vectors for these cells can include an origin of replication, expression control sequences such as promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See Co et al., J. Immunol. 148:1149 (1992).

[0193] Once expressed, the antibody can be purified according to standard techniques in the art such as HPLC purification, column chromatography, gel electrophoresis, etc. (generally, see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0194] VI. Nucleic Acids The present invention further provides a nucleic acid encoding either the humanized heavy chain or the light chain described herein. Typically, the nucleic acid also encodes a signal peptide fused to the mature heavy chain and light chain variable regions. The coding sequences on the nucleic acid can be operably linked to control sequences, such as promoters, enhancers, ribosome binding sites, transcription termination signals, etc., to ensure the expression of the coding sequences. The nucleic acids encoding the heavy chain and the light chain can exist in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized, for example, by solid-phase synthesis or by PCR of overlapping oligonucleotides. The nucleic acids encoding the heavy chain and the light chain can be ligated, for example, as one continuous nucleic acid within an expression vector or can be separate, for example, each can be cloned into its own expression vector.

[0195] In some embodiments, nucleic acids encoding an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein are also provided herein. Vectors comprising a nucleic acid encoding an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein are further provided herein. Host cells expressing a nucleic acid encoding an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein are further provided herein. Host cells comprising a vector comprising a nucleic acid encoding an anti-PD-L1 antibody or antigen-binding fragment thereof as described herein are further provided herein.

[0196] The anti-PD-L1 antibodies described herein can be prepared by well-known recombinant techniques using well-known expression vector systems and host cells. In one embodiment, the antibody is described in De la Cruz Edmunds et al., 2006, Molecular Biotechnology 34; As disclosed in European Patent No. 216846, US Patent No. 5,981,216, WO87 / 04462, European Patent No. 323997, US Patent No. 5,591,639, US Patent No. 5,658,759, European Patent No. 338841, US Patent No. 5,879,936, and US Patent No. 5,891,693, it is prepared in CHO cells using a GS expression vector system.

[0197] The monoclonal anti-PD-L1 antibodies described herein can be prepared by, for example, the hybridoma method first described by Kohler et al., Nature, 256, 495 (1975), or by recombinant DNA methods. Monoclonal antibodies can also be isolated from phage antibody libraries using, for example, the techniques described by Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol Biol., 222(3):581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained from mice immunized with the antigen of interest, for example, in the form of cells expressing the antigen on the surface or from nucleic acids encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized human or non-human mammals such as rats, dogs, and primates.

[0198] Antibody-drug conjugate An anti-PD-L1 antibody can be conjugated to a cytotoxic moiety or a cell growth inhibitory moiety (including its pharmaceutically acceptable salts) to form an antibody-drug conjugate (ADC). Moieties particularly suitable for conjugation to the antibody are cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radioisotopes or compounds or toxins (collectively referred to as therapeutic agents). For example, the anti-PD-L1 antibody can be conjugated to a cytotoxic agent such as a chemotherapeutic agent or a toxin (e.g., a cell growth inhibitor or cytoclasis such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin).

[0199] The anti-PD-L1 antibody can be conjugated to a prodrug converting enzyme. The prodrug converting enzyme can be recombinantly fused to the antibody or chemically conjugated to the antibody using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.

[0200] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well known. (e.g., Arnon et al., ’’Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,’’ in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., ’’Antibodies For Drug Delivery,’’ in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed. 1987); Thorpe, ’’Antibody Carrier "Use of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies ’84: Biological And Clinical Applications (Pinchera et al. eds., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and see Thorpe et al., 1982, Immunol. Rev. 62:119-58. For example, see also PCT Publication WO89 / 12624.)

[0201] The therapeutic agent can be conjugated to the antibody such that (e.g., by hydrolysis, by antibody degradation, or by a cleaving agent) its activity is reduced if the antibody is not excised. The conjugate is cleaved from the antibody when taken up internally by cancer cells expressing PD-L1 (e.g., in endosomes or, for example, by pH sensitivity or protease sensitivity, in the lysosomal environment or in the caveolear environment), using a cleavable linker that is sensitive to cleavage in the intracellular environment of cancer cells expressing PD-L1 but substantially insensitive to the extracellular environment, to bind such a therapeutic agent to the antibody.

[0202] Typically, the ADC comprises a linker region between the therapeutic agent and the anti-PD-L1 antibody. As noted above, typically the linker is cleavable under intracellular conditions such that cleavage of the linker releases the therapeutic agent from the antibody within the intracellular environment (e.g., within a lysosome or endosome or caveola). The linker can be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme including, for example, a lysosomal or endosomal protease. Typically, the peptidyl linker is at least 2 amino acids in length or at least 3 amino acids in length. Cleaving agents can include cathepsin B and D and plasmin (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typically, it is a peptidyl linker that is cleavable by an enzyme present in PD-L1-expressing cells. For example, a peptidyl linker cleavable by the thiol-dependent protease cathepsin B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising the Phe-Leu or Gly-Phe-Leu-Gly peptide). Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In certain embodiments, the peptidyl linker cleavable by an intracellular protease comprises a Val-Cit linker or a Phe-Lys dipeptide (see, e.g., U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin using a Val-Cit linker). One advantage of using intracellular proteolytic release of the therapeutic agent is that the agent is typically attenuated when conjugated and the serum stability of the conjugate is usually high.

[0203] Cleavable linkers can be pH-sensitive, i.e., sensitive to hydrolysis at specific pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-labile linkers that are hydrolyzable in lysosomes (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic acid amides, orthoesters, acetals, ketals, etc.) can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are relatively stable under neutral p H conditions such as those in blood, but are unstable at pH 5.5 or less than 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether linked to a therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Patent No. 5,622,929)).

[0204] Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT (see, for example, Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935).

[0205] The linker can also be a malonic acid linker (Johnsonra et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12). The linker can also be a malonic acid linker (Johnsonra et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0206] The linker can also be a non-cleavable linker such as a maleimide-alkylene- or maleimide-aryl linker that is directly attached to a therapeutic agent (e.g., a drug). The active drug-linker is released by degradation of the antibody.

[0207] Typically, the linker is substantially insensitive to the extracellular environment, i.e., when the ADC is present in the extracellular environment (e.g., in plasma), less than about 20%, typically less than about 15%, more typically less than about 10%, even more typically less than about 5%, about 3%, or about 1% of the linker in the sample of the ADC is cleaved.

[0208] Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) the ADC “ADC sample” and (b) an equimolar amount of the unconjugated antibody or therapeutic agent “control sample” independently of plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample, e.g., as measured by high performance liquid chromatography, to that present in the control sample.

[0209] The linker can also promote endocytosis. The linker can promote endocytosis when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent moiety of an ADC or ADC derivative as described herein). Alternatively, the linker can promote endocytosis when conjugated to both a therapeutic agent and an anti-PD-L1 antibody (i.e., in the context of an ADC as described herein).

[0210] The anti-PD-L1 antibody can be conjugated to a linker via a heteroatom of the antibody. These heteroatoms can be present on the antibody in its native state or can be introduced into the antibody. In some embodiments, the anti-PD-L1 antibody will be conjugated to the linker via the nitrogen atom of a lysine residue. In other embodiments, the anti-PD-L1 antibody will be conjugated to the linker via the sulfur atom of a cysteine residue. The cysteine residue can be either a naturally occurring one or one engineered into the antibody. Methods of conjugating linkers and drug-linkers to antibodies via lysine and cysteine residues are known in the art.

[0211] Exemplary antibody-drug conjugates include auristatin-based antibody-drug conjugates (i.e., the drug component is an auristatin drug). Auristatins have been shown to bind to tubulin and interfere with microtubule dynamics and nuclear and cell division and have anti-cancer activity. Typically, auristatin-based antibody-drug conjugates include a linker between the auristatin drug and the anti-PD-L1 antibody. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). Auristatins include auristatin T, MMAF, and MMAE. Exemplary auristatin syntheses and structures are described in U.S. Patent Publications Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in its entirety for all purposes.

[0212] Exemplary antibody-drug conjugates also include camptothecin-based antibody-drug conjugates (i.e., the drug component is a camptothecin drug). Camptothecin is a topoisomerase inhibitor that has been shown to have anti-cancer activity. Typically, camptothecin-based antibody-drug conjugates include a linker between the camptothecin drug and the anti-PD-L1 antibody. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). Exemplary syntheses and structures of camptothecin drug-linkers are described in PCT / US19 / 025968 (filed April 5, 2019), which are hereby incorporated by reference in their entirety for all purposes.

[0213] Other exemplary antibody-drug conjugates include maytansinoid antibody-drug conjugates (i.e., the drug component is a maytansinoid drug) and benzodiazepine antibody-drug conjugates (i.e., the drug component is a benzodiazepine (e.g., a pyrrolo[1,4]benzodiazepine dimer (PBD dimer), an indolinobenzodiazepine dimer, and an oxazolidinobenzodiazepine dimer)).

[0214] Exemplary antibody-drug conjugates include the following vcMMAE and mcMMAF antibody-drug conjugates (wherein p represents the drug load and Ab represents the anti-PD-L1 antibody),

Chem.

Chem.

[0215] Exemplary anti-PD-L1 antibody-drug conjugates include the following camptothecin antibody-drug conjugates (wherein p represents the drug load and Ab represents the anti-PD-L1 antibody).

[0216] In some embodiments, the camptothecin ADC has the formula (IC):

Chemical formula

[0217] In some aspects of these embodiments, p is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some aspects, p is 2, 4 or 8.

[0218] In some embodiments, the camptothecin ADC has the formula:

Chemical formula

[0219] In some embodiments, the camptothecin ADC has the formula:

Chemical formula

[0220] In some embodiments, the camptothecin drug-linker has the formula:

Chemical formula

[0221] In some embodiments, the camptothecin drug-linker has the formula:

Chemical formula

[0222] In some embodiments, the camptothecin drug-linker has the formula:

Chemical formula

[0223] In some embodiments, the camptothecin drug-linker has the formula:

Chemical formula

[0224] Referring to the PD-L1-targeted antibody-drug conjugate, the subscript p represents the drug load and, depending on the context, can represent the number of drug-linker molecules bound to an individual antibody molecule and is thus an integer value, or can represent the average drug load and is thus an integer or non-integer value, but is typically a non-integer value. The average drug load represents the average number of drug-linker molecules per antibody in a population. Often, but not always, when referring to an antibody, e.g., a monoclonal antibody, it refers to a population of antibody molecules. In a composition comprising a population of antibody-drug conjugate molecules, the average drug load is an important quality attribute as it determines the amount of drug that can be delivered to target cells. The percentage of unconjugated antibody molecules in the composition is included in the average drug load value.

[0225] In a preferred embodiment of the present invention, the average drug load when referring to a composition comprising a population of antibody-drug conjugate compounds is from 1 to about 16, preferably from about 2 to about 14, more preferably from about 2 to about 10.

[0226] In the case of MMAE ADC and camptothecin ADC (such as the ADCs exemplified herein), the preferred average drug load is about 2, 4 or 8, and the particularly preferred average drug load is about 8. In one embodiment, the preferred average drug load of MMAE ADC is 2 or 4. In one embodiment, the preferred average drug load of camptothecin ADC is 4 or 8. In an exemplary embodiment, the drug-linker is conjugated to a cysteine residue of a reduced interchain disulfide. In some embodiments, the actual drug load of an individual antibody molecule in the population of antibody-drug conjugate compounds is from 1 to 10 (or 6 to 10 or 6 to 8), and the predominant drug load is 8. For example, in addition to the interchain disulfide, a higher drug load can be achieved when the drug-linker is conjugated to an introduced cysteine residue (such as a cysteine residue introduced at position 239 according to the EU index).

[0227] The PEG (polyethylene glycol) moiety of the drug linker can range from 2 to 36. The subscript z in all of the above embodiments is preferably from 2 to 12, 4 to 12, 8 to 14, 8 to 12, 10 to 12 or 10 to 14, more preferably 2, 4, 8 or 12, and most preferably 8.

[0228] Polydisperse PEGs, monodisperse PEGs and discontinuous PEGs can be used to prepare the PEGylated antibody drug conjugates of the present invention. Polydisperse PEGs have a size and molecular weight Although a heterogeneous mixture, monodisperse PEG is typically purified from a heterogeneous mixture and thus provides a single chain length and molecular weight. Preferred PEG units are discontinuous PEGs, which are compounds synthesized stepwise without a polymerization process. Discontinuous PEGs provide single molecules with defined specific chain lengths. Similar to the subscript "p", when referring to a population of antibody-drug conjugates, the value of the subscript "n" may be an average number and can be an integer or non-integer.

[0229] Examples of classes of cytotoxic agents useful for conjugating to anti-PD-L1 antibodies include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, chemotherapy sensitizers, and the like. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecin, duocarmycin, etoposide, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binders (e.g., enediynes and lexitropsins), duocarmycin, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulysin M, doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

[0230] The cytotoxic agent can be a chemotherapeutic agent such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent can also be a CC-1065 analog, calicheamicin, maytansine, dolastatin 10, lysocine, or a paritoxin analog.

[0231] The cytotoxin can also be an auristatin. The auristatin can be, for example, an auristatin E derivative which is an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with para-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include auristatin T, AFP, MMAF and MMAE. The synthesis and structure of various auristatins are described, for example, in US Patent Application Publication No. 2005-0238649 and US Patent Application Publication No. 2006-0074008.

[0232] The cytotoxin can be a DNA minor groove binder. (See, for example, US Patent No. 6,130,237.) For example, the minor groove binder can be a CBI compound or an enediyne (e.g., calicheamicin).

[0233] The cytotoxin or cytostatic agent can be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T 67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Exemplary auristatins are shown in the following Formulas III-XIII. Other suitable anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colcemid, estramustine, cryptophycin, sermadotin, maytansinoid, combretastatin, discodermoide and eleuthrobin.

[0234] The cytotoxic agent can be a maytansinoid, another group of anti-tubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can be maytansine or maytansine containing a drug linker such as DM-1 or DM-4 (ImmunoGen, Inc.; Chari et al., 1992, Cancer Res.).

[0235] VIII. Therapeutic Applications The antibodies of the invention can be used to treat cancer, either alone or as their anti-PD-L1 antibody-drug conjugates. Some such cancers exhibit a detectable level of PD-L1 measured either at the protein level (e.g., by an immunoassay using one of the exemplified antibodies) or at the mRNA level. Some such cancers preferably exhibit elevated levels of PD-L1 compared to the same type of non-cancerous tissue from the same patient. Exemplary levels of PD-L1 on cancer cells suitable for treatment are 5,000 to 500,000 PD-L1 molecules per cell, although higher or lower levels can be treated. Optionally, the level of PD-L1 in the cancer is measured prior to treatment.

[0236] Examples of cancers associated with PD-L1 expression and suitable for treatment include melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating melanoma. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating NSCLC. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating SCLC. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating head and neck cancer. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating TNBC. Triple-negative breast cancer is a technical term for cancer that lacks detectable estrogen and progesterone receptors and lacks overexpression of HER2 / neu. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating ovarian cancer. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating urothelial cancer. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating HCC. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating gastric cancer. In some embodiments, the antibodies or antibody-drug conjugates of the invention are used in methods of treating cervical cancer. Treatment can be applied to patients having these types of primary or metastatic tumors. Treatment can also be applied to patients refractory to conventional treatments, or patients who have relapsed after response to such treatments.

[0237] The antibodies of the present invention, such as humanized antibodies alone or as conjugates thereof, are administered in an effective regimen that means a dosage, route of administration, and frequency of administration that delays the onset of cancer, reduces the severity, inhibits further progression, and / or improves at least one sign or symptom. When a patient already has cancer, this regimen can be said to be a therapeutically effective regimen. When a patient has a high risk of cancer compared to the general population but has not yet experienced symptoms, the regimen can be said to be a prophylactically effective regimen. In some examples, the therapeutic or prophylactic efficacy can be observed in individual patients compared to historical controls or the patient's past experience. In other examples, the therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials in a population of treated patients compared to a control population of untreated patients.

[0238] Exemplary dosages of monoclonal antibodies are from 0.1 mg to 50 mg per kg of patient body weight, more typically 1 mg to 30 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, or 1 mg to 10 mg 1, or 2 mg to 30 mg, 2 mg to 20 mg, 2 mg to 15 mg, 2 mg to 12 mg, or 2 mg to 10 mg, or 3 mg to 30 mg, 3 mg to 20 mg, 3 mg to 15 mg, 3 mg to 12 mg, or 3 mg to 10 mg. Exemplary dosages of monoclonal antibodies or antibody-drug conjugates thereof are from 1 mg to 7.5 mg, or 2 mg to 7.5 mg, or 3 mg to 7.5 mg per kg of subject body weight, or 0.1 to 20, or 0.5 to 5 mg body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mg), or a fixed dosage of 10 to 1500 or 200 to 1500 mg. In some methods, a patient is administered a dose of at least 1.5 mg / kg, at least 2 mg / kg or at least 3 mg / kg, once every 3 weeks or more. The dosage depends on, among other factors, the frequency of administration, the patient's condition and response to previous treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0239] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Administration can also be directly localized within the tumor. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration can be, for example, by infusion or single bolus injection over a period such as 30 to 90 minutes.

[0240] The frequency of administration depends, among other factors, on the half-life of the antibody or conjugate in circulation, the patient's condition and the route of administration. The frequency can be daily, weekly, monthly, four times a year or at irregular intervals depending on changes in the patient's condition or the progression of the cancer being treated. Exemplary frequencies for intravenous administration are between twice a week and four times a year over the course of the treatment, although higher or lower frequencies of administration are also possible. Other exemplary frequencies for intravenous administration are between once a week and three times every four weeks over the course of the treatment, although higher or lower frequencies of administration are also possible. For subcutaneous administration, exemplary administration frequencies are daily to monthly, although higher or lower frequencies of administration are also possible.

[0241] The number of doses administered depends on the nature of the cancer (e.g., whether presenting acute or chronic symptoms) and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often sufficient. In some cases, a single bolus dose, in divided form if necessary, is sufficient for acute disorders or acute exacerbations of chronic disorders. Treatment can be repeated for recurrences or acute exacerbations of acute disorders. In the case of chronic disorders, the antibody can be administered at regular intervals, for example, at least once, five or ten years, or over the patient's lifetime, weekly, every two weeks, monthly, every three months, every six months.

[0242] Pharmaceutical compositions for parenteral administration are preferably sterile, substantially isotonic, and manufactured under GMP conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., dosages for single administration). Pharmaceutical compositions can be formulated with one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the route of administration selected. For injection, the antibody can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution (to reduce discomfort at the site of injection), or physiological saline or acetate buffer. The solution can contain formulating agents such as suspending, stabilizing, and / or dispersing agents. Alternatively, the antibody can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. The concentration of the antibody in the liquid formulation can be, for example, 1 to 100 mg / mL, such as 10 mg / mL.

[0243] Treatment with the antibodies of the invention may be achieved by chemotherapy, radiation, stem cell treatment, surgery, or other treatments. It may be combined with other treatments effective for the disorder being treated, including standard of care therapy for the disorder. Useful classes of other agents that may be administered with antibodies to PD-L1 and antibody-drug conjugates as described herein include, for example, antibodies to other receptors expressed on cancerous cells, antitubulin agents (e.g., auristatins), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposide, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinol, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0244] Treating a patient having a tumor (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer), particularly in the case of recurrence or refractoriness, with an anti-PD-L1 antibody or an antibody-drug conjugate, optionally alone or in combination with any of other agents or regimens described as an antibody-drug conjugate, can increase the median progression-free survival or overall survival of the patient by at least 30% or 40%, preferably 50%, 60% - 70% or even 100% or longer, compared to the same treatment (e.g., chemotherapy) without using the anti-PD-L1 antibody alone or as a conjugate. Additionally, or alternatively, a treatment comprising an anti-PD-L1 antibody alone or as a conjugate (e.g., standard chemotherapy) can increase the complete response rate, partial response rate or objective response rate (complete + partial) of a patient having a tumor by at least 30% or 40%, preferably 50%, 60% - 70% or even 100%, compared to the same treatment (e.g., chemotherapy) without using the anti-PD-L1 antibody alone or as a conjugate.

[0245] Typically, in a clinical trial (e.g., a Phase II, Phase II / III or Phase III trial), the aforementioned increases in the median progression-free survival and / or response rate of patients treated with an anti-PD-L1 antibody alone or as a conjugate in addition to standard treatment, compared to a control group of patients receiving standard treatment alone (or plus placebo), are statistically significant, e.g., at a level of p = 0.05 or 0.01 or even 0.001. The complete response rate and partial response rate are determined by objective criteria commonly used in cancer clinical trials, as enumerated or approved, for example, by the National Cancer Institute of the United States and / or the U.S. Food and Drug Administration.

[0246] IX. Manufactured Articles and Kits In another aspect, there is provided a manufactured article or kit comprising an anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate described herein. The manufactured article or kit may further comprise instructions for using the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate described herein in the methods of the invention. Thus, in certain embodiments, the manufactured article or kit comprises instructions for use of the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate described herein in a method for treating a cancer of a subject (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer) comprising administering an effective amount of the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate described herein to the subject. In some embodiments, the subject is human.

[0247] The manufactured article or kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (such as single or dual chamber syringes) and test tubes. In some embodiments, the container is a vial. The container may be formed from a variety of materials such as glass or plastic. The container holds the formulation.

[0248] The manufactured product or kit may further include a label or package insert, which may be on the container or associated with the container and may indicate instructions for reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous (e.g., intravenous infusion), or other modes of administration for treating a target cancer (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer). The container holding the formulation may be a single-use vial or a multi-use vial that allows for repeated administration of the reconstituted formulation. The manufactured product or kit may further include a second container containing a suitable diluent. The manufactured product or kit may further include other materials desirable from a commercial, therapeutic, and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use.

[0249] The manufactured product or kit herein may further include, optionally, a container containing a second pharmaceutical, the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate is the first pharmaceutical, and the article or kit further includes instructions on a label or package insert for treating a subject with the second pharmaceutical in an effective amount. In some embodiments, the second pharmaceutical is for eliminating or reducing the severity of one or more adverse events.

[0250] In some embodiments, the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate is present in a container as a lyophilized powder. In some embodiments, the lyophilized powder is within a sealed container, such as a vial, ampule, or sachet, and indicates the amount of the active agent. When the medicament is administered by injection, an ampule of, for example, sterile water for injection or physiological saline can be provided as part of the kit, if necessary, so that the components can be mixed prior to administration. Such a kit can further include, if desired, various conventional pharmaceutical ingredients, such as, for example, a container containing 1 or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. Printed instructions in the form of an insert or label indicating the amount of the components to be administered, instructions for administration, and / or instructions for mixing the components can also be included in the kit.

[0251] X. Other Applications Anti-PD-L1 antibodies described herein, such as humanized anti-PD-L1 antibodies, can be used to detect PD-L1 in the context of clinical diagnosis or treatment, or in research. The expression of PD-L1 in cancer provides an indication that the cancer is suitable for treatment with the antibodies of the present invention. The antibodies can also be sold as research reagents for experimental studies in detecting cells having PD-L1 and their responses to various stimuli. In such uses, the monoclonal antibodies can be labeled with a fluorescent molecule, a spin-labeled molecule, an enzyme, or a radioactive isotope and provided in kit form together with all of the reagents necessary to perform an assay for PD-L1. The antibodies described herein can be used to detect PD-L1 protein expression and to determine whether a cancer is suitable for treatment with a PD-L1 ADC.

[0252] All patent applications, websites, other publications, accession numbers, etc., cited above or below are incorporated by reference in their entirety for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference. Sequences When different versions are associated with the acceptance number at different times, it means the version associated with the acceptance number on the effective filing date of the present application. The effective filing date means the earlier of the actual filing date or, if applicable, the filing date of the priority application referred to in the acceptance number. Similarly, when different versions of publications, websites, etc. are published at different times, unless otherwise specified, it means the version most recently published on the effective filing date of the application. Any feature, step, element, embodiment or aspect of the present invention can be used in combination with any other, unless specifically stated otherwise. For clarity and understanding, the present invention has been described in some detail by way of illustration and example, but it is obvious that certain changes and modifications can be made within the scope of the appended claims.

Example

[0253] The cell lines described in the following examples were maintained in culture according to the conditions specified by the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig, Germany (DMSZ) or as otherwise known.

[0254] Method Antibody production The SG-559-xx antibody directed against PD-L1 was produced by introducing point mutations into the CDRs of the fully human Ab1 to reduce affinity. Briefly, the residues in the CDRs proximal to the PD-L1 binding epitope were mutated to different amino acids. Four selected exemplary residues are shown in Figure 1. For initial screening purposes, the SG-559-xx antibody was made at ATUM bio using transient transfection in HEK293 cells.

[0255] For the follow-up study, antibodies were prepared in-house according to the following protocol. Antibody variable domain sequences and constant domain sequences were synthesized using non-template PCR. Briefly, virtual gene sequences were converted to oligonucleotide sequences using Genewiz's bioinformatics tools. Oligonucleotides were synthesized, pooled, and amplified using PCR. Full-length amplicons from the PCR reaction were cloned into vectors, and then the products were transformed into E. coli, and unique colonies were isolated. Colonies were grown overnight in liquid medium, plasmid DNA was isolated, purified, and the sequence was verified using Sanger sequencing. The light and heavy chains were cloned into the pcDNA3.4 vector.

[0256] Antibody heavy and light chain vectors in a 1:1 ratio were diluted into ThermoFisher OptiPRO SFM medium containing ExpiFectamine CHO transfection reagent. Then, the DNA / transfection reagent was added to ExpiCHO cultures in ThermoFisher ExpiCHO expression medium, ExpiCHO enhancer was added on day 1, and ExpiCHO feeds were added on days 1 and 2 and cultured for 9 days. The cultures were harvested by centrifugation and 0.2um filtration, or by depth filtration using Millipore X0HC and D0HC pods followed by 0.2um filtration.

[0257] The GE HiTrap mAb Select SuRe column was used for the purification of each IgG. Before elution, the resin was washed with 5CV PBS+0.1% Triton, 5CV PBS+0.5M NaCl, and 7.5CV of PBS. IgG was eluted using 100 mM acetic acid pH3 buffer. The sample was buffer-exchanged into PBS using a 26 / 60 HiPrep desalting column. The sample underwent a final polishing step on a HiPrep Superdex 200 26 / 600 column run in PBS. Then, the sample was filter-sterilized and samples were taken for characterization. For characterization, the A280 concentration Degree, aSEC HPLC, aHIC HPLC, and reduced PLRP-MS (QToF) are included.

[0258] Biolayer interferometry Biolayer interferometry was performed using an Octet Red 384 system (ForteBio) to determine the binding affinity of the SG-559-xx antibody. An anti-human Fab-CH1 (FAB2G) biosensor (ForteBio) was loaded with 4 μg / mL of the SG-559-xx antibody for 100 seconds. After the subsequent baselining step, human PD-L1 (Acro Biosciences) at concentrations ranging from 500 nM to 0.69 nM (1× PBS pH 7.4 containing 1% casein, 0.2% Tween-20) was incubated with the probe loaded for the association step for 150 seconds. Subsequently, a dissociation step was performed for 1000 seconds in the same buffer lacking human PD-L1. k association and k dissociation were fitted to the binding curves obtained according to the established method.

[0259] Production of antibody-drug conjugate (ADC) The SG-559-xx antibody was conjugated to MDpr-PEG(12)-gluc-MMAE at an average drug:antibody ratio (DAR) of 8 as described in U.S. Patent Application Publication No. 20180092984. The SG-559-xx antibody was conjugated to vc-MMAE at an average DAR of 4 as described in U.S. Patent Application Publication No. 20050238649. The SG-559-xx antibody was conjugated to MP-PEG8-VKG-camptothecin at an average DAR of 8 as described in PCT / US2019 / 025968 (filed on April 5, 2019).

[0260] In vitro cell injury assay Cell lines were seeded 24 hours prior to antibody-drug conjugate (ADC) treatment to acclimatize the cells. Where indicated, 500 IU / mL of interferon-γ was also added at this time to induce PD-L1 expression. The cells were then treated with the indicated doses of ADC and incubated at 37 °C for 96 hours. Other PD-L1-directed antibodies and isotype controls were included as ADCs for comparison. The cell viability of the cell lines was measured using CellTiter-Glo (Promega Corporation, Madison, WI) according to the manufacturer's instructions. Briefly, the cells were incubated with the CellTiter-Glo reagent for 30 minutes at room temperature, and luminescence was measured using an Envision plate reader (Perkin Elmer, Waltham, MA). Results are reported as x50 (the concentration of the compound required to cause a 50% decrease in viability compared to untreated cells).

[0261] Internalization assay Internalization of the PD-L1-directed antibody was performed using a FabFluor pH-sensitive conjugate on an Incucyte (Sartorius). The antibody was conjugated to a pH-sensitive dye that increases in fluorescence signal as the pH decreases from the cell surface to the endosome / lysosome compartment. Adherent cells were plated 24 hours prior to incubation with these conjugates (along with 500 IU / mL of interferon-γ to induce PD-L1 expression). Suspension cells were seeded 3 hours prior to incubation with these conjugates. The cells were then given 0.5 μg / mL of the indicated dye-antibody conjugate and incubated for 48 hours. Using Incucyte S3 software (Sartorius), the total integrated intensity of the fluorescence signal was normalized to the % confluence per well per time point. Results are reported as the area under the curve of the normalized integrated intensity versus time.

[0262] In vivo activity study Nude mice were injected with 5.0×10 6 individual BxPC3 pancreatic adenocarcinoma cells or 1.0×10 6Individual EBC-1 NSCLC cells were subcutaneously inoculated. NSG mice were subcutaneously inoculated with 5.0×10 5 individual MDA-MB-231 triple-negative breast cancer cells. SCID mice were subcutaneously inoculated with 1.0×10 6 individual Karpas 299 ALCL cells or 1.0×10 6 individual Calu-1 NSCLC cells. Tumor growth was monitored with calipers, and the mean tumor volume was calculated using the formula (0.5 × [length × width 2 ). When the mean tumor volume reached approximately 100 mm 3 , the mice were either untreated or intraperitoneally administered as indicated by the ADC. The unconjugated antibody and vc-MMAE ADC were administered once weekly for a total of 3 doses. The MP-PEG8-VKG-camptothecin ADC was administered only once. The mice were euthanized when the tumor volume reached approximately 750 mm 3 . For the immunophenotyping study in animals bearing Karpas 299, the mean tumor volume was allowed to reach 200 mm 3 . These mice were then treated with a single dose of the unconjugated antibody or ADC and euthanized 6 days later. Tumors were characterized ex vivo by immunohistochemistry and cytokine analysis (Luminex). The handling of all animals was conducted based on a protocol approved by the Institutional Animal Care and Use Committee of a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

[0263] PD-L1 blockade In vitro evaluation of PD-L1 blockade was performed using a PD-1 / PD-L1 blockade bioassay (Promega Corporation) according to the manufacturer's instructions. Briefly, PD-L1 + aAPC / CHO-K1 cells were seeded and allowed to acclimate for 16 hours. Antibodies or ADCs at the indicated concentrations were then added to the seeded cells, followed by the addition of PD-1 + effector cells. In the absence of PD-1 / PD-L1 signaling, the interaction between aAPC / CHO-K1 cells and effector cells results in a bioluminescence signal. Thus, more effective inhibition of PD-1 / PD-L1 interaction results in a higher luminescence signal, which is quantified as fold induction relative to untreated cells. A PD-1 binding antibody (Promega Corporation) was included as a positive control, and an unbound isotype antibody was included as a negative control.

[0264] Immunotoxicity in a human APC model that upregulates PD-L1 upon stimulation with IFNγ Antibody or ADC immunotoxicity to human antigen-presenting cells in vitro was measured using human antigen-presenting cells (APCs) treated with any of the antibodies or ADCs described herein after stimulation with interferon-γ (IFNγ). Human APCs were stimulated in vitro with 500 IU / mL of IFNγ (R&D Systems) for 24 hours to upregulate PD-L1 prior to treatment with SG-559-xx ADC. Immunotoxicity was calculated as the percentage of viability of untreated APCs at different antibody or ADC concentrations.

[0265] Inhibition of immune response in a human APC model Inhibition of the immune response was measured using human antigen-presenting cells (APCs) treated with either the antibodies or ADCs described herein after stimulation with lipopolysaccharide (LPS). Human APCs were stimulated in vitro with 500 IU / mL of IFNγ (R&D Systems) for 24 hours to upregulate PD-L1. The human APCs were then treated with the SG-559-xx ADC as indicated for 24 hours. The human APCs were then stimulated in vitro with 100 ng / mL of LPS (Sigma Aldrich) for 48 hours. The response to LPS was measured by flow cytometry staining for MHC class II and CD86 (Biolegend). The strength of the immune function was calculated as the fold change in MHC class II or CD86 in response to LPS stimulation in APCs at different antibody or ADC concentrations.

[0266] Deglycosylation of human PD-L1 using PNGase F To produce deglycosylated hPD-L1, human PD-L1 was treated with PNGase F enzyme (New England Biolabs) in combination with a denaturation protocol. PNGase F catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc residue and the asparagine residue of high mannose-type oligosaccharides, hybrid-type oligosaccharides, and complex-type oligosaccharides derived from N-linked glycoproteins. Human PD-L1 was subjected to the denaturation protocol in the absence of PNGase F to obtain a reaction control. The deglycosylation protocol included combining human PD-L1 (Acro Biosciences) with Rapid PNGase F buffer, heating the human PD-L1 at 75 °C for 5 minutes, cooling the denatured human PD-L1 on ice, adding PNGase F, and incubating overnight at 37 °C. The glycosylation status was confirmed by mass spectrometry.

[0267] The bio-layer interferometry was performed using the Octet Red 384 system (ForteBio) to determine the binding affinity of the SG-559-xx antibody or ADC to glycosylated or deglycosylated PD-L1. After subsequent baseline steps, glycosylated human PD-L1 and deglycosylated human PD-L1 at concentrations ranging from 500 nM to 0.69 nM (1×PBS pH 7.4 containing 1% BSA, 0.2% Tween-20) were incubated with the loading probe for 150 seconds for the association step. Subsequently, a dissociation step was performed for 1000 seconds in the same buffer lacking human PD-L1. k association and k dissociation were fitted to the binding curves obtained according to established methods.

[0268] Results Example 1: Design and Characterization of SG-559-xx Antibodies As described in the method, 17 SG-559-xx antibodies were produced from the parental Ab1 antibody. The CDRs containing the mutations of these antibodies are shown in Table 1. Sixteen of these antibodies were evaluated for their monovalent binding affinity to hPD-L1 by bio-layer interferometry compared to Ab1 (Table 2). The measured affinities of the SG-559-xx antibodies were in the approximately two-digit range and the K D values were 4 nM to 297 nM.

Table 1

Table 2

[0269] Example 2: In Vitro Cytotoxicity As described in the method for cancer cell lines expressing PD-L1, including 786-O, BxPC3, ES-2, MDA-MB-231, Karpas 299 and L540CY, the cytotoxicity of SG-559-xx antibody as an ADC was evaluated. In some experiments, SU-DHL-4 (PD-L1 negative cancer cell line) was included as a control. Initial screening of 15 SG-559-xx ADCs (excluding the two with the lowest affinity) using the MDpr-PEG(12)-gluc-MMAE payload (DAR8) demonstrated that some SG-559-xx antibodies showed significantly improved cytotoxicity compared to the parental Ab1 (Figures 2A - 2F).

[0270] Four SG-559-xx antibodies that consistently showed the highest potency in the initial screening were further characterized for their potency as vc-MMAE and MP-PEG8-VKG-camptothecin ADCs (Table 3). In most of the cell lines tested, these ADCs were significantly more potent than Ab1. They also showed no activity in the antigen-negative cell line (SU-DHL-4), suggesting that this was not due to non-specific binding.

Table 3

[0271] Example 3: Internalization To confirm the cytotoxicity results, the internalization of SG-559-01 and SG-559-03 was further investigated using the Incucyte imaging system and a pH-sensitive dye conjugate as described in the method. The internalization of SG-559-01 and SG-559-03 was consistently high across most of the cell lines tested. This was measured by the percent increase in the area under the curve (AUC) of the normalized integrated intensity over time (Table 4). Exemplary curves are shown for MDA-MB-231 (Figure 3A) and Karpas 299 (Figure 3B).

Table 4

[0272] Example 4: In Vivo Antitumor Activity Four SG-559-xx antibodies characterized for in vitro screening were also tested for antitumor efficacy in two mouse xenograft models. In the MDA-MB-231 model, the SG-559-xx antibodies as ADCs showed significant antitumor activity with two drug linkers (Figures 4A - 4B). In the BxPC3 model, the SG-559-xx antibodies as ADCs showed moderate antitumor activity (Figures 5A - 5B). In almost all cases, the SG-559-xx ADCs were more effective than the Ab1 ADCs, suggesting that the observed in vitro phenotype was translated into an in vivo setting.

[0273] Antitumor efficacy was further observed in an additional model using one of the inventors' most promising antibodies as an Fc effector function-reduced variant (SG-559-01 LALA). The SG-559-01 LALA antibody as an ADC showed significant antitumor activity with one or two drug linkers in the Karpas 299 (Figures 6A - 6B), Calu-1 (Figure 7), and EBC-1 (Figures 8A - 8B) models. It should be noted that this activity was different from that of the unconjugated SG-559-01 LALA antibody.

[0274] Example 5: PD-L1 Blockade SG-559-01 LALA was further characterized for its ability to block the PD-1 / PD-L1 checkpoint in vitro. Compared to the PD-1 antibody control, SG-559-01 was able to more effectively inhibit PD-1 / PD-L1 signaling. Furthermore, the unconjugated SG-559-01 LALA was equivalent to SG-559-01 LALA conjugated to two drug linkers, demonstrating that conjugation did not affect PD-1 / PD-L1 blocking (Figure 9).

[0275] Example 6: Immunotoxicity to Human APCs In Vitro SG-559-01 and SG-559-01 LALA were evaluated for immunotoxicity against APCs (e.g., macrophages and dendritic cells (DCs)). APCs were stimulated with IFNγ to upregulate PD-L1 prior to treatment as described in the method. The SG-559-01 LALA ADC showed immunotoxicity against human APCs that was similar to or within one order of magnitude of the isotype control in both macrophages and DCs (Figures 10A - 10D).

[0276] Four SG-559-xx antibodies characterized for in vitro screening were also tested for immunotoxicity against APCs (i.e., dendritic cells and macrophages). Immunotoxicity against human APCs was similar to or within one order of magnitude of each of the SG-559-xx ADCs (Figures 11A - 11D).

[0277] Example 7: Inhibition of immune response The SG-559-01 ADC was further characterized by measuring the inhibition of the immune response in human APCs treated with LPS. As described in the method, in vitro human APCs were stimulated with LPS after ADC treatment, and the subsequent upregulation of MHC class II and CD86 was quantified as a measure of the immune response. Treatment with the SG-559-01 ADC resulted in immune response inhibition in both DCs and macrophages in a similar manner or within one order of magnitude of the isotype control as measured by MHC class II (Figures 12A - 12B) and CD86 (Figures 12C - D).

[0278] Example 8: Increase in immune infiltration The SG-559-01 LALA vc-MMAE ADC was further characterized by evaluating immune infiltration in mice bearing Karpas 299 tumors. Mice bearing tumors were treated as shown and the tumors were characterized 6 days later. Compared to both untreated controls and the SG-559-01 antibody, the SG-559-01 vc-MMAE The ADC induced immune infiltration in mice bearing Karpas 299 tumors (Figures 13A - C). Figure 13A shows an increase in mCD45+ cells (a pan - leukocyte marker). Figure 13B shows an increase in mCD11c+ cells (a marker for a subset of dendritic cells and macrophages). Figure 13C shows an increase in mF4 / 80+ cells (a macrophage marker).

[0279] Example 9: Inflammatory Cytokine Response The SG - 559 - 01 LALA vc - MMAE ADC was further characterized for its ability to induce the production of inflammatory cytokines in the tumor microenvironment (TME). Compared to both untreated controls and the SG - 559 - 01 LALA antibody, the SG - 559 - 01 LALA vc - MMAE ADC induces inflammatory cytokines in the TME as measured by the intratumoral concentrations of eotaxin (a chemokine for eosinophils, Figure 14A), MIP1a (a pro - inflammatory macrophage cytokine, Figure 14B), MIP1b (a pro - inflammatory macrophage cytokine, Figure 14C), MIG / CXCL9 (induced by IFNγ and affecting the migration and differentiation of immune cells, Figure 14D), MCP1 (a chemokine for monocytes / macrophages, Figure 14E) and Rantes (a chemokine for monocytes, T cells and eosinophils, Figure 14F).

[0280] Example 10: Binding Affinity for Glycosylated PD - L1 and Deglycosylated PD - L1 SG - 559 - 01 was evaluated for its binding affinity to glycosylated and deglycosylated forms of PD - L1. As described in the method, the binding affinity was evaluated using biolayer interferometry with the Octet Red 384 system (ForteBio). It was valued. Using PNGase F enzyme and the denaturation protocol, PD-L1 was deglycosylated as described in the method. SG-559-01 was evaluated for its binding affinity to deglycosylated PD-L1 and control glycosylated PD-L1 (under the same treatment conditions as described in the method but without PNGase F treatment). A difference of approximately 2-fold was observed in the SG-559-01 binding affinity to deglycosylated PD-L1 compared to glycosylated PD-L1 (Table 5). Mass spectrometry was used to verify the glycosylation status of PD-L1.

Table 5

[0281] Unofficial Sequence Listing SEQ ID NO: 1 - Ab1 Heavy Chain Variable Region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS SEQ ID NO: 2 - Ab1 Light Chain Variable Region - Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK SEQ ID NO: 3 - Ab1 Heavy Chain CDR1 - Protein TYAIS SEQ ID NO: 4 - Ab1 Heavy Chain CDR2 - Protein GIIPIFGKAHYAQKFQG SEQ ID NO: 5 - Ab1 Heavy Chain CDR3 - Protein KFHFVSGSPFGMDV SEQ ID NO: 6 - Ab1 Light Chain CDR1 - Protein RASQSVSSYLA SEQ ID NO: 7 - Ab1 Light Chain CDR2 - Protein DASNRAT Array No. 8 - Ab1 light chain CDR3 - protein QQRSNWPT Array No. 9 - SG - 559 - 01 LALA hIgG1 heavy chain - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Array No. 10 - SG - 559 - 01 kappa light chain - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Array No. 11 - SG - 559 - 01 heavy chain variable region - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS Sequence number 12-SG-559-01 light chain variable region - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK Sequence number 13-SG-559-01 heavy chain CDR1 - protein TAAIS Sequence number 14-SG-559-01 heavy chain CDR2 - protein GIIPIFGKAHYAQKFQG Sequence number 15-SG-559-01 heavy chain CDR3 - protein KFHFVSGSPFGMDV Sequence number 16-SG-559-01 light chain CDR1 - protein RASQSVSSYLA Sequence number 17-SG-559-01 light chain CDR2 - protein DASNRAT Sequence number 18-SG-559-01 light chain CDR3 - protein QQRSNWPT Sequence number 19-SG-559-02 LALA hIgG1 heavy chain - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20-SG-559-02 kappa light chain - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 21-SG-559-02 heavy chain variable region - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQA PGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS SEQ ID NO: 22-SG-559-02 light chain variable region - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK Sequence number 23-SG-559-02 heavy chain CDR1-protein TYAIS Sequence number 24-SG-559-02 heavy chain CDR2-protein GIIPIFGKAHYAQKFQG Sequence number 25-SG-559-02 heavy chain CDR3-protein KFHFVSGSPFGMDV Sequence number 26-SG-559-02 light chain CDR1-protein RASQSVSSALA Sequence number 27-SG-559-02 light chain CDR2-protein DASNRAT Sequence number 28-SG-559-02 light chain CDR3-protein QQRSNWPT Sequence number 29-SG-559-03 LALA hIgG1 heavy chain-protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 30 - SG - 559 - 03 kappa light chain - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNLPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 31 - SG - 559 - 03 heavy chain variable region - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS SEQ ID NO: 32 - SG - 559 - 03 light chain variable region - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNLPTFGQGTKVEIK SEQ ID NO: 33-SG-559-03 Heavy Chain CDR1-Protein TYAIS SEQ ID NO: 34-SG-559-03 Heavy Chain CDR2-Protein GIIPIFGKAHYAQKFQG SEQ ID NO: 35-SG-559-03 Heavy Chain CDR3-Protein KFHFVSGSPFGMDV SEQ ID NO: 36-SG-559-03 Light Chain CDR1-Protein RASQSVSSYLA SEQ ID NO: 37-SG-559-03 Light Chain CDR2-Protein DASNRAT SEQ ID NO: 38-SG-559-03 Light Chain CDR3-Protein QQRSNLPT SEQ ID NO: 39-SG-559-04 LALA hIgG1 Heavy Chain-Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 40 - SG - 559 - 04 kappa light chain - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 41 - SG - 559 - 04 heavy chain variable region - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSS SEQ ID NO: 42 - SG - 559 - 04 light chain variable region - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK Sequence number 43-SG-559-04 heavy chain CDR1-protein TYAIS Sequence number 44-SG-559-04 heavy chain CDR2-protein GIIPIFGKAHYAQKFQG Sequence number 45-SG-559-04 heavy chain CDR3-protein KFHFVSGSGFGMDV Sequence number 46-SG-559-04 light chain CDR1-protein RASQSVSSYLA Sequence number 47-SG-559-04 light chain CDR2-protein DASNRAT Sequence number 48-SG-559-04 light chain CDR3-protein QQRSNWPT Sequence number 49-SG-559-05 heavy chain CDR2-protein GIIPIAGKAHYAQKFQG Sequence number 50-SG-559-06 heavy chain CDR2-protein GIIPIFGAAHYAQKFQG Sequence number 51-SG-559-07 heavy chain CDR2-protein GIIPIFGRAHYAQKFQG Sequence number 52-SG-559-08 heavy chain CDR2 -protein GIIPIFGKAAYAQKFQG Sequence number 53-SG-559-09 heavy chain CDR2-protein GIIPIFGKAFYAQKFQG Sequence number 54-SG-559-10 heavy chain CDR3-protein KFHFVSGAPFGMDV Sequence number 55-SG-559-11 heavy chain CDR3-protein KFHFVSGSPAGMDV Light chain CDR3 - protein of Accession No. 56 - SG - 559 - 12 QQASNWPT Light chain CDR3 - protein of Accession No. 57 - SG - 559 - 13 QQKSNWPT Light chain CDR3 - protein of Accession No. 58 - SG - 559 - 14 QQRSAWPT Light chain CDR3 - protein of Accession No. 59 - SG - 559 - 15 QQRSQWPT Light chain CDR3 - protein of Accession No. 60 - SG - 559 - 16 QQRSNAPT Light chain CDR3 - protein of Accession No. 61 - SG - 559 - 17 QQRSNFPT hIgG1 heavy chain - protein of Accession No. 62 - SG - 559 - 01 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK hIgG1 heavy chain - protein of Accession No. 63 - SG - 559 - 02 QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 64-SG-559-03 hIgG1 heavy chain-protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Array No. 65-SG-559-04 hIgG1 heavy chain - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Array No. 66-SG-559-01 variable heavy chain region - nucleic acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcaccgccgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca Array No. 67-SG-559-01 variable light chain region - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 68 - SG - 559 - 02 Variable Heavy Chain Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO: 69 - SG - 559 - 02 Variable Light Chain Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcgccttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 70 - SG - 559 - 03 Variable Heavy Chain Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO: 71 - SG - 559 - 03 Variable Light Chain Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaacctgccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 72 - SG - 559 - 04 Variable Heavy Chain Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagcggcttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO: 73 - SG - 559 - 04 Variable Light Chain Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 74-SG-559-01 LALA hIgG1 heavy chain-nucleic acid SEQ ID NO: 75-SG-559-01 kappa light chain-nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtc tgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 76-SG-559-01 hIgG1 heavy chain-nucleic acid Accession No. 77-SG-559-02 LALA hIgG1 heavy chain-nucleic acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcc SEQ ID NO: 78-SG-559-02 kappa light chain - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcgccttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 79-SG-559-02 hIgG1 heavy chain - nucleic acid caggtccagctggtgcagtctggggctgaggtgaagaagc Accession No. 80-SG-559-03 LALA hIgG1 Heavy Chain - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggagccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagca acaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagctgctgggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtttacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa SEQ ID NO: 81-SG-559-03 kappa light chain - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaacctgccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt Sequence number 82 - SG - 559 - 03 hIgG1 heavy chain - nucleic acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggagccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactc cctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa SEQ ID NO: 83 - SG - 559 - 04 LALA hIgG1 heavy chain - nucleic acid ggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa SEQ ID NO: 84 - SG - 559 - 04 kappa light chain - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 85 - SG - 559 - 04 hIgG1 heavy chain - nucleic acid cgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa Sequence No. 86 - Ab1 hIgG1 heavy chain - protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence No. 87 - Ab1 kappa light chain - protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

**Claim 1** A nucleic acid encoding an antibody or an antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof specifically binds to human PD-L1 protein, and the antibody comprises heavy chain CDR sequences of SEQ ID NOs: 13 to 15 and light chain CDR sequences of SEQ ID NOs: 16 to 18, a nucleic acid encoding an antibody or an antigen-binding fragment thereof. **Claim 2** A vector comprising the nucleic acid according to claim 1. **Claim 3** A host cell comprising the nucleic acid according to claim 2. **Claim 4** The host cell according to claim 3, wherein the host cell is a Chinese hamster ovary (CHO) cell. **Claim 5** A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, comprising culturing the host cell according to claim 3 or 4 under conditions suitable for the production of the antibody. **Claim 6** A method for producing an antibody-drug conjugate that specifically binds to human PD-L1 protein, comprising culturing the host cell according to claim 3 or 4 under conditions suitable for the production of the antibody, and conjugating the antibody to a cytotoxic agent. **Claim 7** The method according to claim 6, wherein the cytotoxic agent is MMAE or camptothecin.

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