Anti-pd-l1 antibodies and antibody-drug conjugates

Abstract

The present invention provides 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.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202080082113.4, filed on October 2, 2020, entitled "Anti-PD-L1 Antibody and Antibody-Drug Conjugate".

[0002] Cross-reference to related applications This application claims priority to U.S. Patent Application No. 62 / 910,988, filed October 4, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention relates to novel anti-PD-L1 antibodies and antibody-drug conjugates, as well as methods of treating cancer using such anti-PD-L1 antibodies and antibody-drug conjugates. Background Technology

[0004] 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 acts as an "off" switch to inactivate T cells. PD-L1 is typically overexpressed on tumor cells, and its binding to PD-1 allows tumors to evade T cell immune responses.

[0005] Several cancers express PD-L1, including melanoma. Melanoma is the most dangerous type of skin cancer. In 2015, 59,800 people died from melanoma out of 3.1 million people with active disease. The five-year survival rate for stage IV disease is less than 10%, with a median survival of only 6–12 months. Therefore, improved treatment is needed for melanoma and other cancers that express PD-L1. One type of treatment for PD-L1-expressing cancers involves administering anti-PD-L1 antibodies as immunotherapy. Immuno-oncology is a promising field for cancer treatment, but there is still room for improvement in current therapies.

[0006] All references cited in this article (including patent applications, patent publications and scientific literature) are incorporated into this article in their entirety by reference, as if each individual reference were explicitly and individually indicated to be incorporated by reference. Summary of the Invention

[0007] This document provides anti-PD-L1 antibodies and PD-L1-targeted antibody-drug conjugates (ADCs). Specifically, this document provides PD-L1-targeted camptothecin ADCs and MMAE ADCs. This document also provides methods for treating PD-L1 expression disorders using anti-PD-L1-targeted antibodies and ADCs. Preferred anti-PD-L1 antibodies exhibit binding affinity to human PD-L1 protein in the range of 3 nM to 300 nM. Other preferred anti-PD-L1 antibodies comprise the heavy chain CDR sequences of SEQ ID NO: 3-5 and the light chain CDR sequences of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs. Other preferred anti-PD-L1 antibodies comprise the heavy chain CDR sequences of SEQ ID NO: 13-15 and the light chain CDR sequences of SEQ ID NO: 16-18.

[0008] This document also provides antibodies or antigen-binding fragments thereof that specifically bind to human programmed death-ligand 1 (PD-L1) protein, wherein the antibodies exhibit binding affinity to human PD-L1 protein in the range of 3 to 300 nM. In some embodiments, the antibodies exhibit binding affinity to human PD-L1 protein in the range of 3 to 15 nM.

[0009] In some embodiments, the antibody further exhibits a higher total internalization than Ab1. In some embodiments, the total internalization has an AUC increase between 9% and 155% relative to Ab1. In some embodiments, the total internalization is determined by a FabFluor internalization assay.

[0010] In some embodiments, the antibody further exhibits an x50 lower than that of Ab1. In some embodiments, the antibody is conjugated to monomethylaurestatin E (MMAE), and wherein the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

[0011] In some embodiments, the antibody is conjugated with camptothecin, and the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

[0012] In some embodiments, the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 13-15 and the light chain CDR sequence of SEQ ID NO: 16-18.

[0013] In some embodiments, the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 3-5 and the light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs.

[0014] 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.

[0015] In some embodiments, the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0016] In some implementations, the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a biantibody, a linear antibody, or a single-chain antibody fragment.

[0017] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0018] In some implementations, the heavy chain constant region is an IgG1 isotype.

[0019] In some implementations, the antibody is a humanized or chimeric antibody.

[0020] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker.

[0021] In some embodiments, the antibody is conjugated to monomethylaurestatin 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 comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0022] In some embodiments, the antibody is conjugated with camptothecin. In some embodiments, the antibody is conjugated with 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 with camptothecin via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0023] This document also provides antibodies or antigen-binding fragments thereof that specifically bind to human PD-L1 protein, wherein the antibodies comprise heavy chain CDR sequences of SEQ ID NO: 3-5 and light chain CDR sequences of SEQ ID NO: 6-8, wherein the antibodies comprise one or more amino acid substitutions within one or more CDRs.

[0024] In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 15 nM.

[0025] In some embodiments, the antibody further exhibits a higher total internalization than Ab1. In some embodiments, the total internalization has an AUC increase between 9% and 155% relative to Ab1. In some embodiments, the total internalization is determined by a FabFluor internalization assay.

[0026] In some implementations, the antibody further exhibits a higher x50 than Ab1.

[0027] In some embodiments, the antibody is conjugated to monomethylaurestatin E (MMAE), and the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

[0028] In some embodiments, the antibody is conjugated with camptothecin, and the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

[0029] In some embodiments, the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 13-15 and the light chain CDR sequence of SEQ ID NO: 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.

[0030] In some embodiments, the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0031] In some implementations, the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a biantibody, a linear antibody, or a single-chain antibody fragment.

[0032] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0033] In some implementations, the heavy chain constant region is an IgG1 isotype.

[0034] In some implementations, the antibody is a humanized or chimeric antibody.

[0035] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethylaurestatin 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 comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0036] In some embodiments, the antibody is conjugated with camptothecin. In some embodiments, the antibody is conjugated with 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 with camptothecin via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0037] This article also provides antibodies or antigen-binding fragments thereof that specifically bind to human PD-L1 protein, wherein the antibodies comprise the heavy chain CDR sequences of SEQ ID NO: 13-15 and the light chain CDR sequences of SEQ ID NO: 16-18.

[0038] In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 15 nM.

[0039] In some embodiments, the antibody further exhibits a higher total internalization than Ab1. In some embodiments, the total internalization has an AUC increase between 9% and 155% relative to Ab1. In some embodiments, the total internalization is determined by a FabFluor internalization assay.

[0040] In some implementations, the antibody further exhibits a higher x50 than Ab1.

[0041] In some embodiments, the antibody is conjugated to monomethylaurestatin E (MMAE), and the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

[0042] In some embodiments, the antibody is conjugated with camptothecin, and the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

[0043] 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.

[0044] In some embodiments, the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0045] In some implementations, the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a biantibody, a linear antibody, or a single-chain antibody fragment.

[0046] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0047] In some implementations, the heavy chain constant region is an IgG1 isotype.

[0048] In some implementations, the antibody is a humanized or chimeric antibody.

[0049] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethylaurestatin 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 comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0050] In some embodiments, the antibody is conjugated with camptothecin. In some embodiments, the antibody is conjugated with 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 with camptothecin via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0051] This article also provides antibodies or antigen-binding fragments thereof that specifically bind to human PD-L1 protein, wherein the antibody is conjugated with camptothecin to form an antibody-drug conjugate, wherein the antibody-drug conjugate has the following structure: , where Ab is an anti-PD-L1 antibody; y is 1, 2, 3 or 4, or 1 to 4; and z is an integer from 2 to 12, or 2, 4, 8 or 12; and p is 1 to 16.

[0052] In some embodiments, the antibody-drug conjugate has the following structure:

[0053] In some implementations, p ranges from 2 to 10.

[0054] This document also provides antibodies or antigen-binding fragments thereof that specifically bind to human programmed death-ligand 1 (PD-L1) protein, wherein the antibodies exhibit a binding affinity greater than Ab1 for human PD-L1 protein. In some embodiments, the antibodies exhibit a binding affinity greater than 2.7 nM.

[0055] This article also provides antibodies or antigen-binding fragments thereof that specifically bind to human programmed death-ligand 1 (PD-L1) protein, wherein the antibodies exhibit a k-value smaller than Ab1 for binding to human PD-L1 protein. assoc k assoc In some implementations, the antibody exhibits a size of less than 5 x 10⁻⁶. 5 M -1 s -1 The k-like properties of human PD-L1 protein assoc .

[0056] This article also provides antibodies or antigen-binding fragments thereof that specifically bind to human programmed death-ligand 1 (PD-L1) protein, wherein the antibodies exhibit a k-value greater than Ab1 for binding to human PD-L1 protein. dissoc k dissocIn some implementations, the antibody exhibits a size greater than 2 x 10⁻⁶. 3 s -1 The k-like properties of human PD-L1 protein dissoc .

[0057] This document also provides antibody-drug conjugates comprising an antibody or an antigen-binding fragment thereof that specifically binds to the human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 3-5 and the light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs, and wherein the antibody exhibits binding affinity to the human PD-L1 protein between 5 nM and 15 nM, and wherein the antibody is conjugated to MMAE.

[0058] This document also provides antibody-drug conjugates 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 sequence of SEQ ID NO: 3-5 and the light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs, and wherein the antibody exhibits a binding affinity to human PD-L1 protein between 5 nM and 15 nM, and wherein the antibody is conjugated with camptothecin.

[0059] This article also provides pharmaceutical compositions comprising a therapeutically effective amount of the antibody described herein and a pharmaceutically acceptable excipient.

[0060] This document also provides methods for treating a subject's cancer, the methods comprising administering any of the antibodies described herein to the subject. 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0061] This article also provides the nucleic acids encoding any of the antibodies described herein.

[0062] This article also provides vectors containing any of the nucleic acids described herein.

[0063] This document also provides any host cell described herein that contains any of the nucleic acids described herein. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0064] This document also provides a method for generating an antibody or antigen-binding fragment thereof that specifically binds to the human PD-L1 protein, the method comprising culturing any host cell described herein under conditions suitable for generating the antibody.

[0065] This document also provides a method for generating an antibody-drug conjugate that specifically binds to the human PD-L1 protein, the method comprising culturing the host cells described herein under conditions suitable for generating the antibody; and conjugating the antibody to a cytotoxic agent. In some embodiments, the cytotoxic agent is MMAE or camptothecin.

[0066] This article also provides information on any anti-PD-L1 antibody or any antibody-drug conjugate described herein in the manufacture of drugs for the treatment of cancer (e.g., with PD-L1). + Use in drugs that express related cancer.

[0067] This article also provides the anti-PD-L1 antibody or antibody-drug conjugate described herein for the treatment of cancer (e.g., with PD-L1). + (Expression related to cancer).

[0068] This article also provides information on the anti-PD-L1 antibody or antibody-drug conjugate described herein for medical use.

[0069] This article also provides PD-L1 inhibitors for killing subjects who require it. + A cellular method comprising administering to the subject a therapeutically effective amount of any of the anti-PD-L1 antibodies or any of the antibody-drug conjugates described herein.

[0070] This article also provides information on any anti-PD-L1 antibody or any antibody-drug conjugate described herein for the manufacture of PD-L1 for killing subjects in need. + Uses in cell-based pharmaceuticals.

[0071] This article also provides information on reducing solid tumors in subjects (e.g., PD-L1). + A method for measuring the volume of a solid tumor, the method comprising administering to the subject a therapeutically effective amount of any antiPD-L1 antibody or any antibody-drug conjugate described herein.

[0072] This article also provides information on any anti-PD-L1 antibody or any antibody-drug conjugate described herein in the manufacture of drugs intended to reduce the size of solid tumors (e.g., PD-L1) in subjects. + The use of pharmaceutical agents to measure the volume of solid tumors. Attached Figure Description

[0073] Figure 1Exemplary amino acid residues selected for mutation of Ab1 are shown.

[0074] Figures 2A-2F The cytotoxicity of SG-559-xx ADC in several cell lines is shown.

[0075] Figures 3A-3B The internalization of SG-559-01 and SG-559-03 compared to the control antibody is shown.

[0076] Figures 4A-4B The antitumor activity of SG-559-xx ADC in the MDA-MB-231 mouse model was demonstrated.

[0077] Figures 5A-5B The antitumor activity of SG-559-xx ADC in the BxPC3 mouse model was demonstrated.

[0078] Figures 6A-6B The antitumor activity of SG-559-01 LALA ADC in the Karpas 299 mouse model was demonstrated.

[0079] Figure 7 The antitumor activity of SG-559-01 LALA ADC in the Calu-1 mouse model was demonstrated.

[0080] Figures 8A-8B The antitumor activity of SG-559-01 LALA ADC in the EBC-1 mouse model was demonstrated.

[0081] Figure 9 The in vitro PD-1 / PD-L1 blocking activity of the SG-559-01 LALA antibody and ADC was demonstrated.

[0082] Figures 10A-10D The immunotoxicity of SG-559-01 and SG-559-01 LALA ADC in a human APC model was demonstrated.

[0083] Figures 11A-11D The immunotoxicity of SG-559-xx ADC in a human APC model is shown.

[0084] Figures 12A-12D The in vitro immune response of human APCs treated with SG-559-01 ADC to LPS stimulation is shown.

[0085] Figures 13A-13C Tumor-mediated immune cell infiltration is shown in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC.

[0086] Figures 14A-14FThe tumor inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC is shown. Detailed Implementation

[0087] I. Definition

[0088] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, each of the following terms shall have the meaning set forth below unless expressly provided otherwise. Further definitions are set forth throughout this application.

[0089] The term “and / or”, as used herein, is considered to refer to a specific disclosure of each of two specified features or components with or without the other. Thus, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Similarly, the term “and / or” as used herein in phrases such as “A, B, and / or C” is intended to cover each of the following: 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).

[0090] It should be understood that the aspects and embodiments of the present invention described herein include "comprising multiple aspects and embodiments", "consisting of multiple aspects and embodiments" and "substantially consisting of multiple aspects and embodiments".

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary of Biochemistry and Molecular Biology, revised edition, 2000, Oxford University Press provide a general dictionary for those skilled in the art of the use of many terms in this disclosure.

[0092] Units, prefixes, and symbols are represented in their International System of Units (SI) recognized form. Numerical ranges include the numbers defining the ranges. The headings provided herein are not intended to limit the various aspects of this disclosure, which can be obtained by referring to the specification as a whole. Thus, the terms defined below can be defined more fully by referring to the specification as a whole.

[0093] The terms “PD-L1”, “CD274”, “B7-H1” and “programmed cell death ligand 1” are used interchangeably herein and, unless otherwise stated, include any variant, isotype and material homolog of human PD-L1 generally expressed by cells or expressed on cells transfected with the PD-L1 gene.

[0094] The term "immunoglobulin" refers to a class of structure-associated glycoproteins consisting of two pairs of polypeptide chains: a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four chains being interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed., Raven Press, New York (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated as V in this paper). H or VH) and heavy chain constant region (C H Or CH) constitutes it. The heavy chain constant region typically consists of three structural domains, namely C H 1. C H 2 and C H 3. Composition. Heavy chains are typically interconnected via disulfide bonds in so-called "hinge regions." Each light chain typically consists of a light chain variable region (abbreviated as V in this paper). L or VL) and light chain constant region (C L Or CL) constitutes it. The light chain constant region typically consists of a structural domain C L Composition. CL can be a κ (kappa) or λ (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. Immunoglobulins can be derived from any well-known isotype, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to an antibody class or subclass (e.g., IgM or IgG1) encoded by a heavy chain constant region gene.

[0095] The term "variable region" or "variable domain" refers to the structural domain of the antibody heavy or light chain involved in antibody-antigen binding. The variable regions of the heavy and light chains of natural antibodies (V1 and V2, respectively) are... H and V LThe variable region can be further subdivided into highly variable regions (or hypervariable regions, which may be highly variable when in the form of sequence and / or structurally defined loops), also known as complementarity-determining regions (CDRs). These CDRs are interspersed with more conserved regions called frame regions (FRs). The terms “complementarity-determining region” and “CDR” are synonymous with “hypervariable region” or “HVR” and are known in the art to refer to a discontinuous sequence of amino acids within the antibody variable region that confers antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. “Frame 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. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. In each V... H and V L In this case, the three CDRs and four FRs are typically arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mot. Biol., 195, 901-917 (1987)).

[0096] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof that has the ability to specifically bind to an antigen under typical physiological conditions, wherein the half-life is a significant time period, such as at least about 30 min, at least about 45 min, at least about one hour (h), at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours (h), about 24 hours or longer, about 48 hours or longer, about three, four, five, six, seven days or longer, or any other relevant functionally defined time period (such as the time sufficient to induce, promote, enhance, and / or regulate the physiological response associated with the antibody binding to the antigen and / or the time sufficient to recruit 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 regions 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, i.e., the first component in the classical pathway of complement activation). Antibodies can also be bispecific antibodies, biantibodies, multispecific antibodies, or similar molecules.

[0097] As used herein, the term "monoclonal antibody" refers to a formulation of an antibody molecule recombined from a single primary amino acid sequence. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope. Therefore, the term "human monoclonal antibody" refers to an antibody exhibiting single binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas, including B cells fused with immortalized cells from transgenic or transchromosomal nonhuman animals (such as transgenic mice) that have a genome containing human heavy chain and light chain transgenes.

[0098] "Isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-L1). However, an isolated antibody that specifically binds to PD-L1 may be cross-reactive with other antigens, such as PD-L1 molecules from different species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In one embodiment, the isolated antibody comprises an antibody conjugate attached to another pharmaceutical agent (e.g., a small molecule drug). In some embodiments, the isolated anti-PD-L1 antibody comprises a conjugate of an anti-PD-L1 antibody with a small molecule drug (e.g., MMAE or MMAF).

[0099] "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. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. Human antibodies in this disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which a CDR sequence derived from another mammalian species (such as a mouse) has been grafted onto a human frame sequence. The terms "human antibody" and "fully human antibody" are used synonymously.

[0100] As used herein, “humanized antibody” refers to a genetically engineered nonhuman antibody containing a human antibody constant domain and a nonhuman variable domain modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six nonhuman antibody complementarity-determining regions (CDRs) that co-form the antigen-binding site into the homologous human receptor frame region (FR) (see WO 92 / 22653 and EP 0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace the frame residues from the parent antibody (i.e., the nonhuman antibody) with human frame regions (reversion mutations). Structural homology modeling may help identify amino acid residues in the frame regions that are important for the antibody's binding properties. Thus, a humanized antibody may contain a nonhuman CDR sequence, a major human frame region optionally containing one or more amino acid reversion mutations of a nonhuman amino acid sequence, and a fully human constant region. Optionally, additional amino acid modifications (not necessarily reversion mutations) may be applied to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0101] 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., rodents) and the constant region is derived from a different species (e.g., humans). Chimeric antibodies can be produced through antibody engineering. "Antibody engineering" is a general term used for the modification of various types of antibodies, and it is a process well known to those skilled in the art. In particular, chimeric antibodies can be produced using standard DNA techniques as described in Chapter 15 of Sambrook et al., 1989, *Molecular Cloning: Alaboratory Manual*, New York: Cold Spring Harbor Laboratory Press. Therefore, the chimeric antibody can be a genetically or enzymatically engineered recombinant antibody. The production of chimeric antibodies is within the knowledge of those skilled in the art, and therefore, the production of chimeric antibodies according to the invention can be carried out by methods other than those described herein. Chimeric monoclonal antibodies have been developed for therapeutic applications to reduce the immunogenicity of antibodies. They can typically contain a non-human (e.g., mouse) variable region specific to a target antigen, as well as human constant antibody heavy and light chain domains. In the context of chimeric antibodies, the term "variable region" or "variable domain" refers to the region containing the CDR and framework regions of the heavy and light chains of immunoglobulins.

[0102] "Antigen antibody" refers to an antibody that binds to an antigen. For example, anti-PD-L1 antibody is an antibody that binds to the antigen PD-L1.

[0103] An antibody's "antigen-binding portion" or "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen bound to the whole antibody. Examples of antibody fragments (e.g., antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; biantibodies; 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 with one antigen-binding site; and a residual "Fc" fragment, a name reflecting its tendency to crystallize. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and remains capable of cross-linking antigens.

[0104] The "sequence identity percentage (%)" of a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after alignment and cleavage (if necessary) to achieve the maximum sequence identity percentage, and without considering any conserved substitutions as part of the sequence identity. Alignment used to determine the amino acid sequence identity percentage can be performed in a variety of ways well known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared. For example, the sequence identity percentage of a given amino acid sequence A to / with / relative to a given amino acid sequence B (or this can be expressed in the phrase "a given amino acid sequence A having or containing a specific percentage of sequence identity to / with / relative to a given amino acid sequence B") is calculated as follows: Fraction X / Y multiplied by 100 Where X is the number of amino acid residues in the sequence that score as identical matches in the A and B alignments of this procedure, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the sequence identity percentage of A relative to B will not be equal to the sequence identity percentage of B relative to A.

[0105] As used herein, in the context of antibody binding to a predetermined antigen, the terms "binding" or "specific binding" typically refer to binding with an affinity corresponding to approximately 10 when the antibody is used as a ligand and the antigen as an analyte in an Octet HTX instrument, for example, by a biofilm layer interferometry (BLI) technique. -6 M or smaller (e.g., 10) -7 M or smaller, such as about 10-8 M or smaller, such as about 10 -9 M or smaller, approximately 10 -10 M or smaller or about 10 -11 K (M or even smaller) D And wherein the antibody binds to the predetermined antigen with an affinity corresponding to a K0 higher affinity than it binds to nonspecific antigens (e.g., BSA, casein) other than the predetermined antigen or closely related antigens. D K is at least ten times lower (e.g., at least 100 times, for example, at least 1,000 times, for example, at least 10,000 times, for example, at least 100,000 times). D Among them, the K that is combined D The lower level depends on the antibody's K D Therefore, when the antibody's K D When it is very low, then the K that binds to the antigen... D Below the K level that binds to non-specific antigens D The degree of specificity can be at least 10,000 times (i.e., the antibody is highly specific).

[0106] As used in this article, the term "K" D (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. For example, the affinity and K used in this paper... D It is an inverse correlation, meaning that higher affinity refers to lower K. D And lower affinity is intended to refer to higher K D .

[0107] The term "ADC" refers to an antibody-drug conjugate, which in the context of this invention refers to an anti-PD-L1 antibody conjugated to a drug moiety (e.g., MMAE or MMAF) as described in this application.

[0108] The abbreviations "vc" and "val-cit" refer to the dipeptide linker valine-citrulline.

[0109] The abbreviation VKG refers to the tripeptide linker valine-lysine-glycine.

[0110] The abbreviation "MC" refers to the maleimide hexanoyl extension:

[0111] The abbreviation "MP" refers to the maleimide propionyl extensor:

[0112] As used herein, a “PEG unit” is an organic moiety (PEG or PEG subunit) consisting of repeating ethylene-oxy groups and can be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy groups). Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from heterogeneous mixtures, thus providing a single chain length and molecular weight. Preferred PEG units comprise discrete PEG, i.e., compounds synthesized stepwise rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

[0113] The PEG unit provided herein comprises one or more polyethylene glycol chains, each consisting of one or more ethylene oxide subunits covalently attached to each other. The polyethylene glycol chains may be linked together, for example, in a linear, branched, or star configuration. Typically, at least one polyethylene glycol chain prior to incorporation with the camptothecin conjugate is derivatized at one end with an alkyl portion, said alkyl portion being substituted with an electrophilic group for covalent attachment to the urethane nitrogen of the methylene carbamate unit (i.e., an example representing R). Typically, the terminal ethylene oxide subunits in each polyethylene glycol chain not involved in covalent attachment to the remaining portion of the connector unit are modified with a PEG end-capping unit (typically an optionally substituted alkyl group, such as -CH3, CH2CH3, or CH2CH2CO2H). Preferred PEG units have a single polyethylene glycol chain having 2 to 24 -CH2CH2- subunits covalently attached in series and terminated at one end with a PEG end-capping unit.

[0114] "Cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancer tissue" can include tumors. Unregulated cell division and growth lead to the formation of malignant tumors that invade adjacent tissues and can also metastasize to distant sites of the body via the lymphatic system or bloodstream. After metastasis, a distant tumor can be said to "originate" from the pre-metastatic tumor.

[0115] The term "antibody-dependent cytotoxicity" or ADCC is a mechanism that induces cell death through the interaction of antibody-coated target cells with lytically active immune cells (also known as effector cells). These effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to one or more Fc effector domains of an Ig, which binds to the target cell via its antigen-binding site. The death of the antibody-coated target cell occurs as a result of effector cell activity.

[0116] The term “antibody-dependent phagocytosis” or ADCP refers to the process by which antibody-coated cells are internalized, either whole or partially, by phagocytic immune cells (such as macrophages, neutrophils, and dendritic cells) that bind to one or more Fc effector domains of Ig.

[0117] The term "complement-dependent cytotoxicity" or CDC refers to a mechanism that induces cell death in which one or more Fc effector domains of a target-binding antibody activate a series of enzymatic reactions that ultimately result in the formation of pores on the target cell membrane. Typically, antigen-antibody complexes (such as those on antibody-coated target cells) bind to and activate complement component C1q, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the target cell surface, which promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0118] "Cytostatic effect" refers to the inhibition of cell proliferation. "Cytostatic agent" refers to a drug that has a cytostatic effect on cells, thereby inhibiting the growth and / or expansion of specific cell subpopulations. Cytostatic agents can be conjugated with antibodies or administered in combination with antibodies.

[0119] "Treatment" or "therapy" for a subject refers to any type of intervention or treatment performed on a subject, or the administration of an active agent to a subject, with the aim of reversing, reducing, improving, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, signs, or biochemical indicators associated with a disease. In some implementations, the disease is cancer.

[0120] "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.

[0121] An "effective amount," "therapeutic effective amount," or "therapeutic dose" of a drug or therapeutic agent is any amount of the drug, used alone or in combination with another therapeutic agent, that protects a subject from the onset of disease or promotes disease remission, demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of injury or disability caused by disease distress. The ability of a therapeutic agent to promote disease remission can be evaluated using a variety of methods known to skilled practitioners, such as in human subjects during clinical trials, in animal model systems predicting efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0122] For example, in the treatment of tumors, a therapeutically effective amount of an anticancer agent, relative to untreated subjects (e.g., one or more untreated subjects), 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 some embodiments, a therapeutically effective amount of an anticancer agent, relative to untreated subjects (e.g., one or more untreated subjects), inhibits cell growth or tumor growth by 100% in treated subjects (e.g., one or more treated subjects).

[0123] In other embodiments of this disclosure, tumor regression can be observed and last 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.

[0124] Therapeutic effective amounts of a drug include “preventative effective amounts,” which are any amounts of a drug (e.g., an anti-PD-L1 antibody-drug conjugate) administered alone or in combination with an anticancer agent to subjects at risk of developing cancer (e.g., having a precancerous condition) or suffering cancer recurrence that inhibit the development or recurrence of cancer. In some implementations, preventative effective amounts completely prevent the occurrence or recurrence of cancer. “Inhibiting” the occurrence or recurrence of cancer means reducing the likelihood of cancer occurring or recurring, or completely preventing the occurrence or recurrence of cancer.

[0125] As used herein, “subtherapeutic dose” means a dose of a therapeutic compound (e.g., an anti-PD-L1 antibody-drug conjugate) that is lower than the usual or typical dose of the therapeutic compound when administered alone to treat a hyperproliferative disease (e.g., cancer).

[0126] "Immune-associated response patterns" refer to clinical response patterns frequently observed in cancer patients treated with immunotherapies that exert their antitumor effects by inducing cancer-specific immune responses or by modifying innate immune processes. This response pattern is characterized by beneficial treatment effects following an initial increase in tumor burden or the appearance of new lesions, which would be classified as disease progression and synonymous with drug failure in the evaluation of conventional chemotherapy. Therefore, appropriate evaluation of immunotherapies may require long-term monitoring of the effects of these agents on the target disease.

[0127] For example, an "anticancer agent" promotes cancer regression in a subject. In some implementations, a therapeutically effective amount of the drug promotes cancer regression to the point of elimination. "Promoting cancer regression" means that administration of an effective amount of the drug, alone or in combination with an anticancer agent, results in a reduction in tumor growth or size, tumor necrosis, a decrease in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free periods, or prevention of damage or disability caused by disease distress. Furthermore, the terms "effective" and "efficacy" in relation to 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 adverse physiological effects (adverse effects) at the cellular, organ, and / or organismal level caused by the administration of the drug.

[0128] "Sustained response" refers to a sustained effect on reducing tumor growth after treatment has been discontinued. For example, the tumor size may remain the same or smaller compared to its size at the start of the treatment period. In some implementations, the duration of sustained response is at least the same as, or at least 1.5, 2.0, 2.5, or 3 times longer than, the duration of treatment.

[0129] As used in this article, “complete response” or “CR” means the disappearance of all target lesions; “partial response” or “PR” means a reduction of at least 30% in the sum of the longest diameters (SLD) of the target lesions, with reference to the baseline SLD; and “stable disease” or “SD” means that the target lesions have neither shrunk sufficiently to meet PR nor increased sufficiently to meet PD, with reference to the minimum SLD since the start of treatment.

[0130] As used in this article, “progression-free survival” or “PFS” refers to the length of time during and after treatment when the treated disease (e.g., cancer) does not worsen. Progression-free survival can include the amount of time a patient experiences a complete or partial response as well as the amount of time a patient experiences disease stabilization.

[0131] As used in this article, “total response rate” or “ORR” refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0132] As used in this article, “overall survival” or “OS” refers to the percentage of individuals in a group who are likely to survive after a specific duration.

[0133] The phrase “pharmaceutically acceptable” indicates that a substance or composition must be chemically and / or toxicologically compatible with other components constituting the preparation and / or the mammals to which it is treated.

[0134] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the compounds of the present invention. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannins, pantothenates, bitartrates, ascorbic acid salts, succinates, maleates, gentianates, fumarates, gluconates, glucurons, glycosides, formates, benzoates, glutamates, methanesulfonates ("methanesulfonates"), ethanesulfonates, benzenesulfonates, p-toluenesulfonates, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthylcarboxylic acid)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. Pharmaceutically acceptable salts may involve the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterions. The counterion can be any organic or inorganic component that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. An example of a pharmaceutically acceptable salt having multiple charged atoms as part of its structure can be the presence of multiple counterions. Therefore, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0135] "Administering" or "administration" means the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the anti-PD-L1 antibody-drug conjugate include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, such as by injection or infusion (e.g., intravenous infusion). As used herein, the phrase "parenteral administration" means administration by injection, in addition to enteral and local administration, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intracardiac, intra-orbital, intracardiac, intratracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Therapeutic agents may be administered via non-parenteral routes or orally. Other non-parenteral routes include local, epidermal, or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual, or local administration. Application may also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0136] The terms “baseline” or “baseline value” used interchangeably herein may refer to a measurement or characterization of symptoms prior to or at the start of treatment (e.g., an anti-PD-L1 antibody-drug conjugate as described herein). Baseline values ​​may be compared to reference values ​​to determine the reduction or improvement of symptoms for PD-L1-related diseases (e.g., cancer) considered herein. The terms “reference” or “reference value” used interchangeably herein may refer to a measurement or characterization of symptoms after treatment (e.g., an anti-PD-L1 antibody-drug conjugate as described herein). Reference values ​​may be measured once or multiple times during a dosing regimen or treatment cycle or at the completion of a dosing regimen or treatment cycle. A “reference value” may be an absolute value; a relative value; a value with an upper and / or lower limit; a range of values; an average; a median; a mean; or a value compared to a baseline value.

[0137] Similarly, a “baseline value” can be an absolute value; a relative value; a value with an upper and / or lower limit; a range of values; an average; a median; a mean; or a value compared to a reference value. Reference and / or baseline values ​​can be obtained from one individual, two different individuals, or a group of individuals (e.g., a group of two, three, four, five, or more individuals).

[0138] As used herein, the term "monotherapy" means that the anti-PD-L1 antibody-drug conjugate is the only anticancer agent administered to the subject during a treatment cycle. However, other therapeutic agents may be administered to the subject. For example, an anti-inflammatory agent or other agent administered to a subject with cancer during a monotherapy period to treat cancer-related symptoms, but not the underlying cancer itself, such as inflammation, pain, weight loss, and general malaise, may be given.

[0139] As used herein, “adverse events” (AEs) are any unfavorable and often unintentional or unwanted signs (including abnormal laboratory findings), symptoms, or illnesses associated with the use of medication. Medication treatment may have one or more associated AEs, and each AE may have the same or different levels of severity. The reference to methods that can “modify adverse events” means treatment regimens that reduce the incidence and / or severity of one or more AEs associated with the use of different treatment regimens.

[0140] As used in this article, a “serious adverse event” or “SAE” is an adverse event that meets one of the following criteria: • Fatal or life-threatening (as used in the definition of serious adverse events, “life-threatening” means an event in which the patient is at risk of death at the time of the event; it does not mean an event that, if assumed to be more serious, could lead to death).

[0141] • Causes persistent or significant disability / incapacity • Constitutes congenital abnormalities / birth defects • Medically significant, defined as an event that harms a patient or may require medical or surgical intervention to prevent one of the outcomes listed above. Medical and scientific judgment must be made in determining whether an adverse event (AE) is "medically significant." • Hospitalization or extension of existing hospitalization is required, excluding the following: 1) routine treatment or monitoring of underlying diseases that are not related to any worsening of the condition; 2) selective or pre-planned treatment of a pre-existing condition that is not related to the indication in the study and has not worsened since the informed consent was signed; and 3) social reasons and temporary care in the absence of any deterioration in the patient’s overall condition.

[0142] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination of the alternatives. As used herein, the indefinite article "a" or "an" should be understood to mean "a or more" of any of the stated or enumerated components.

[0143] The terms “about” or “substantially constitute” refer to a value or composition within an acceptable error range for a particular value or composition as determined by a person skilled 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, according to practice in the art, “about” or “substantially constitute” may mean within one or more standard deviations. Alternatively, “about” or “substantially constitute” may mean a range up to 20%. Furthermore, particularly with respect to biological systems or processes, the term may mean a value up to an order of magnitude or up to five times. When a particular value or composition is provided in this application and claims, unless otherwise stated, it should be assumed that the meaning of “about” or “substantially constitute” is within an acceptable error range for that particular value or composition.

[0144] References to “about” a value or parameter in this document include (and describe) implementations for said value or parameter itself. For example, a reference to “about X” includes and describes “X”.

[0145] As stated herein, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the range, and, where appropriate, to include fractions thereof (such as tenths and hundredths of an integer), unless otherwise indicated.

[0146] The various aspects of this disclosure are described in further detail in the following sections.

[0147] II. Overview

[0148] This invention provides antibodies and ADCs that specifically bind to PD-L1. This invention is based in part on the finding that antibody-drug conjugates targeting PD-L1 (including MMAE antibody-drug conjugates and camptothecin antibody-drug conjugates) are particularly effective in killing cells expressing PD-L1+. PD-L1 has been shown to be expressed in a variety of 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer.

[0149] III. Target Molecules

[0150] Unless otherwise stated, PD-L1 refers to human PD-L1. UniProtID NO. Q9NZQ7 is assigned to the exemplary human protein sequence.

[0151] IV. The antibody of the present invention

[0152] Previously, selected antibodies used to treat cancer were conjugated with cytotoxic agents without sequence modifications to produce antibody-drug conjugates (ADCs). These ADCs have generally proven to be as effective or more effective as unconjugated antibodies in killing tumor cells. Previously, if antibody modification was considered during ADC preparation, some possible modifications would increase the antibody's binding affinity or enhance its activity, such as ADCC. However, it has been found that, at least in some cases, modifying or tuning ADC antibodies by, for example, reducing their binding affinity or ADCC activity, leads to improved ADC efficacy compared to ADCs with unmodified antibodies. Some examples include ADCs containing anti-PD-L1 antibodies (such as Ab1), which have been surprisingly optimized by modifying the antibodies (e.g., reducing their binding affinity). For example, in some cases, anti-PD-L1 ADCs have been more effective at killing tumor cells in vitro when the binding affinity of the antibody conjugated to the cytotoxic agent is reduced. In another example, in some cases, anti-PD-L1 ADCs are more effective at killing tumor cells in vitro and in vivo when the binding affinity of antibodies conjugated to cytotoxic agents is reduced.

[0153] This invention provides antibodies, such as humanized antibodies, that bind to PD-L1 with a binding affinity between 3 nM and 300 nM. In some embodiments, the antibodies described herein can bind to PD-L1, whose K DBetween 3 nM and 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 nM, approximately 30 nM to approximately 40 nM, approximately 40 nM to approximately 300 nM, approximately 40 nM to approximately 275 nM, approximately 40 nM to approximately 250 nM, approximately 40 nM to approximately 225nM, approximately 40 nM to approximately 200 nM, approximately 40 nM to approximately 175 nM, approximately 40 nM to approximately 150 nM, approximately 40 nM to approximately 125 nM, approximately 40 nM to approximately 100 nM, approximately 40 nM to approximately 90 nM, approximately 40 nM to approximately 80 nM, approximately 40 nM to approximately 70 nM, approximately 40 nM to approximately 60 nM, approximately 40 nM to approximately 50 nM, approximately 50 nM to approximately 300 nM, approximately 50 nM to approximately 275 nM, approximately 50 nM to approximately 250 nM, approximately 50 nM to approximately 225 nM, approximately 50 nM to approximately 200 nM, approximately 50 nM to approximately 175 nM, approximately 50 nM to approximately 150 nM, approximately 50 nM to approximately 125 nM, approximately 50 nM to approximately 100 nM, approximately 50 nM to approximately 90 nM, approximately 50 nM to approximately 80 nM, approximately 50 nM to approximately 70 nM, approximately 50 nM to approximately 60 nM, approximately 60 nM to approximately 300 nM, approximately 60 nM to approximately 275 nM, approximately 60 nM to approximately 250 nM, approximately 60 nM to approximately 225 nM, approximately 60 nM to approximately 200 nM, approximately 60 nM to approximately 175 nM, approximately 60 nM to approximately 150 nM, approximately 60 nM to approximately 125 nM, approximately 60 nM to approximately 100 nM, approximately 60 nM to approximately 90 nM, approximately 60 nM to approximately 80 nM, approximately 60 nM to approximately 70 nM, approximately 70 nM to approximately 300 nM, approximately 70 nM to approximately 275 nM, approximately 70 nM to approximately 250 nM, approximately 70 nM to approximately 225 nM, approximately 70 nM to approximately 200 nM, approximately 70 nM to approximately 175 nM, approximately 70 nM to about 150 nM, about 70 nM to about 125 nM, about 70 nM to about 100 nM, about 70 nM to about 90 nM, about 70 nM to about 80 nM, about 80 nM to about 300 nM, about 80 nM to about 275 nM, 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 nM, approximately 90 nM to approximately 150 nM, approximately 90 nM to approximately 125 nM, approximately 90 nM to approximately 100 nM, approximately 100 nM to approximately 300 nM, approximately 100 nM to approximately 275 nM, approximately 100nM 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 (e.g., as measured by biolayer interference measurement (BLI) in phosphate buffered saline).

[0154] In some embodiments, the binding affinity is a monovalent binding affinity. In some embodiments, these antibodies are point mutants of the fully human anti-PD-L1 antibody Ab1. Ab1 is defined by the CDR regions of SEQ ID NO: 3-5 and SEQ ID NO: 6-8, the variable regions of SEQ ID NO: 1 and 2, and the heavy and light chains of SEQ ID NO: 86 and 87. In a further embodiment, the point mutation is present in the CDR region. In some embodiments, the point mutant exhibits decreased binding affinity and / or increased cytotoxicity and / or internalization rate compared to Ab1. In some embodiments, the point mutant exhibits decreased binding affinity and increased cytotoxicity in vitro. In some embodiments, the point mutant exhibits decreased binding affinity and increased cytotoxicity in vivo. In some embodiments, the point mutant exhibits decreased binding affinity and increased cytotoxicity both in vitro and in vivo. In some embodiments, the point mutant exhibits decreased binding affinity and increased internalization rate in vitro. In some embodiments, the point mutant exhibits reduced binding affinity and increased internalization rate in vivo. In some embodiments, the point mutant exhibits reduced binding affinity and increased internalization rate both in vitro and in vivo.

[0155] In some embodiments, the anti-PD-L1 antibody provided herein may have one or two total amino acid substitutions in six CDRs of the heavy chain CDRs of SEQ ID NO. 3-5 and the light chain CDRs of SEQ ID NO. 6-8, and bind to PD-L1 with a KD between 3 nM and 300 nM. In some embodiments, the anti-PD-L1 antibody provided herein may have one amino acid substitution in six CDRs of the heavy chain CDRs of SEQ ID NO. 3-5 and the light chain CDRs of SEQ ID NO. 6-8, and bind to PD-L1 with a KD between 3 nM and 300 nM.

[0156] In some embodiments, the anti-PD-L1 antibody provided herein may have a heavy chain CDR1 having an amino substitution in SEQ ID NO: 3, a heavy chain CDR2 in SEQ ID NO: 4, a heavy chain CDR3 in SEQ ID NO: 5, a light chain CDR1 in SEQ ID NO: 6, a light chain CDR2 in SEQ ID NO: 7, and a light chain CDR3 in SEQ ID NO: 8, and bind to PD-L1 with a KD between 3 nM and 300 nM. In some embodiments, an amino acid substitution in SEQ ID NO: 3 is located at amino acid position 2 of SEQ ID NO: 3. In some embodiments, an amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution where tyrosine is replaced by alanine. In some embodiments, an amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution where tyrosine is replaced by serine. In some embodiments, an amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution where tyrosine is replaced by glycine. In some embodiments, the amino acid substitution at position 2 of SEQ ID NO: 3 is a substitution where tyrosine is replaced by threonine. In some embodiments, the amino acid substitution at position 2 of SEQ ID NO: 3 is a substitution where tyrosine is replaced by valine. In some embodiments, the amino acid substitution at position 2 of SEQ ID NO: 3 is a substitution where tyrosine is replaced by cysteine.

[0157] In some embodiments, the anti-PD-L1 antibody provided herein is used at a Kc concentration between 3 nM and 300 nM (or any subrange of this range as described herein). D It binds to both glycosylated and non-glycosylated PD-L1.

[0158] In some embodiments, the anti-PD-L1 antibody provided herein exhibits an increase (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, or between 5% and 300%, between 5% and 280%) compared to Ab1. Between increases, between 5% and 260% increases, between 5% and 240% increases, between 5% and 220% increases, between 5% and 200% increases, between 5% and 180% increases, between 5% and 160% increases, between 5% and 140% increases, between 5% and 120% increases, between 5% and 100% increases, between 5% and 80% increases, between 5% and 60% increases, between 5% and 40% increases, between 5% and 20% increases, between 5% and 10% increases, between 10% and 300% increases, between 10% and 280% increases, between 10% and... Between 260% increase, between 10% and 240% increase, between 10% and 220% increase, between 10% and 200% increase, between 10% and 180% increase, between 10% and 160% increase, between 10% and 140% increase, between 10% and 120% increase, between 10% and 100% increase, between 10% and 80% increase, between 10% and 60% increase, between 10% and 40% increase, between 10% and 20% increase, between 20% and 300% increase, between 20% and 280% increase, between 20% and 260% increase, and between 20%... Between 20% and 240% increase, between 20% and 220% increase, between 20% and 200% increase, between 20% and 180% increase, between 20% and 160% increase, between 20% and 140% increase, between 20% and 120% increase, between 20% and 100% increase, between 20% and 80% increase, between 20% and 60% increase, between 20% and 40% increase, between 40% and 300% increase, between 40% and 280% increase, between 40% and 260% increase, between 40% and 240% increase, between 40% and 220% increase.Between 40% and 200% increase, between 40% and 180% increase, between 40% and 160% increase, between 40% and 140% increase, between 40% and 120% increase, between 40% and 100% increase, between 40% and 80% increase, between 40% and 60% increase, between 60% and 300% increase, between 60% and 280% increase, between 60% and 260% increase, between 60% and 240% increase, between 60% and 220% increase, between 60% and 200% increase, between 60% and 180% increase, between 60% and 1... Between 60% and 140% increases, between 60% and 120% increases, between 60% and 100% increases, between 60% and 80% increases, between 80% and 300% increases, between 80% and 280% increases, between 80% and 260% increases, between 80% and 240% increases, between 80% and 220% increases, between 80% and 200% increases, between 80% and 180% increases, between 80% and 160% increases, between 80% and 140% increases, between 80% and 120% increases, between 80% and 100% increases. Between 100% and 300% increase, between 100% and 280% increase, between 100% and 260% increase, between 100% and 240% increase, between 100% and 220% increase, between 100% and 200% increase, between 100% and 180% increase, between 100% and 160% increase, between 100% and 140% increase, between 100% and 120% increase, between 120% and 300% increase, between 120% and 280% increase, between 120% and 260% increase, between 120% and 240% increase, between 120% and... Between 220% increase, between 120% and 200% increase, between 120% and 180% increase, between 120% and 160% increase, between 120% and 140% increase, between 140% and 300% increase, between 140% and 280% increase, between 140% and 260% increase, between 140% and 240% increase, between 140% and 220% increase, between 140% and 200% increase, between 140% and 180% increase, between 140% and 160% increase, between 160% and 300% increase, between 160% and 280% increase.Between 160% and 260% increase, between 160% and 240% increase, between 160% and 220% increase, between 160% and 200% increase, between 160% and 180% increase, between 180% and 300% increase, between 180% and 280% increase, between 180% and 260% increase, between 180% and 240% increase, between 180% and 220% increase, between 180% and 200% increase, between 200% and 300% increase, between 200% and 280% increase, between 200% increase... Cytotoxicity of PD-L1+ cells between 260% and 200% increases, between 240% and 220% increases, between 300% and 220% increases, between 280% and 220% increases, between 260% and 240% increases, between 300% and 240% increases, between 280% and 240% increases, between 260% and 300% increases, between 280% and 260% increases, between 260% and 280% increases, or between 280% and 300% increases.

[0159] In some implementations, the anti-PD-L1 antibody provided herein has an increased (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, or between 5% and 300% (or any subrange of this range described herein)) internalization rate in PD-L1+ cells compared to Ab1.

[0160] In some embodiments, compared to Ab1, the anti-PD-L1 antibody provided herein, upon administration to mammals, exhibits increased (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, or between 5% and 300% (or any subrange of this range described herein)) immune cell infiltration.

[0161] In some embodiments, compared to Ab1, upon administration to mammals, the anti-PD-L1 antibody provided herein exhibits increased (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, or between 5% and 300% (or any subrange of this range described herein)) production of inflammatory cytokines (e.g., one or more of any cytokines described herein).

[0162] In some implementations, the anti-PD-L1 antibody provided herein has an increased (e.g., at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 120%, at least 140%, at least 160%, at least 180%, at least 200%, at least 220%, at least 240%, at least 260%, at least 280%, at least 300%, or between 5% and 300% (or any subrange of this range described herein)) intracellular digestion by PD-L1+ cells compared to Ab1.

[0163] In some implementations, the anti-PD-L1 antibody provided herein, when administered to mammals, exhibits a change of less than 10% in neutrophil and / or platelet counts (e.g., less than 8%, less than 6%, less than 4%, less than 2%, or less than 1%) compared to Ab1.

[0164] The binding affinity (i.e., dissociation constant, K) of the PD-L1 antibody of the present invention D Preferably, the binding affinity is greater than that of Ab1. Preferred PD-L1 antibodies bind to the same epitopes as Ab1 and / or competitively bind 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, the k... assoc (or opening rate) less than Ab1 k assoc In a further implementation, the k assoc Less than 5.5 x 10 5 M -1 s -1 In another implementation, the k dissoc (or closure rate) greater than Ab1's k dissoc In a further implementation, the k dissoc Greater than 1.50 x 10 3 s -1 .

[0165] The anti-PD-L1 antibody of the present invention can also be described or specified according to its binding affinity to PD-L1 (e.g., human PD-L1). In some embodiments, preferred binding affinity includes a dissociation constant or KL greater than 2.7 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM, or 500 nM. DThose. In some embodiments, preferred PD-L1 antibodies have binding affinity between 3 nM and 300 nM, between 3 nM and 200 nM, between 3 nM and 100 nM, between 3 nM and 50 nM, between 3 nM and 40 nM, between 3 nM and 20 nM, between 3 nM and 15 nM, between 5 nM and 300 nM, and between 5 nM and 15 nM. In some embodiments, preferred PD-L1 antibodies have binding affinity at least 2, 3, 3.7, 4, or 5 times that of Ab1. In some of the above embodiments, the binding affinity is a monovalent binding affinity.

[0166] In some embodiments, the binding of the anti-PD-L1 antibody of the present invention is pH-dependent, such that the antibody exhibits different bindings across a pH gradient. In some embodiments, the anti-PD-L1 antibody exhibits maximum binding between a pH of about 4 and a pH of about 10. In some embodiments, the maximum binding is between a pH of about 6 and a pH of about 9. In some embodiments, the maximum binding is between a pH of about 6.5 and a pH of about 8.

[0167] In animal models or clinical trials, the preferred antibodies of the present invention inhibit cancer (e.g., cell growth, metastasis, and / or lethality to the organism), as demonstrated by cancer cells proliferating in cultures. Animal models can be established by implanting human tumor cell lines expressing PD-L1 into appropriate immunodeficient rodent strains (e.g., athymic nude mice or SCID mice). These tumor cell lines can be established as solid tumors in immunodeficient rodent hosts via subcutaneous injection or as disseminated tumors via intravenous injection.

[0168] Once established within the host, these tumor models can be used to evaluate the therapeutic efficacy of anti-PD-L1 antibodies or their conjugate forms as described in the examples.

[0169] Typically, the anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates disclosed herein bind to PD-L1, such as human PD-L1, and exert cytotoxic and inhibitory effects on malignant cells, such as cancer cells. The concentration required to achieve a 50% reduction in viability compared to untreated cells is either x50 or IC50. 50This is one method for measuring the cytotoxicity of anti-PD-L1 antibodies and / or anti-PD-ADCs. Compared to the cytotoxicity and x50 of Ab1 antibodies and / or ADCs, the preferred antibodies and / or ADCs of the present invention exhibit increased cytotoxicity and x50. In one embodiment, the vcMMAE-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 between 10 ng / mL and 30 ng / mL or between 15 ng / mL and 25 ng / mL in the BXPC3 cell line. In another embodiment, the vcMMAE-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 between 15 ng / mL and 55 ng / mL or between 20 ng / mL and 50 ng / mL in the MDA-MB-231 cell line. In yet another embodiment, the vcMMAE-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 between 1 ng / mL and 7 ng / mL or between 2 ng / mL and 5 ng / mL in the KARPAS 299 cell line. In another embodiment, the vcMMAE-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 in the L540CY cell line between 15 ng / mL and 40 ng / mL, or between 20 ng / mL and 35 ng / mL. In one embodiment, the camptothecin-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 in the BXPC3 cell line between 12 ng / mL and 70 ng / mL, or between 15 ng / mL and 65 ng / mL. In another embodiment, the camptothecin-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 in the MDA-MB-231 cell line between 3 ng / mL and 20 ng / mL, or between 5 ng / mL and 17 ng / mL. In another embodiment, the camptothecin-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 in the KARPAS 299 cell line between 1 ng / mL and 18 ng / mL, or between 3 ng / mL and 15 ng / mL. In another embodiment, the camptothecin-conjugated anti-PD-L1 antibody of the present invention exhibits an x50 in the L540CY cell line between 1 ng / mL and 20 ng / mL, or between 1 ng / mL and 15 ng / mL.

[0170] Typically, the anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates of this disclosure are internalized into cells such as cancer cells. One way to measure internalization is by using a pH-sensitive antibody conjugate that emits a fluorescent signal upon internalization in a cell-based assay. The total internalization of this antibody can be quantified by the area under the curve (AUC) of the fluorescence signal over time. The FabFluor (IncuCyte®) internalization assay can be used for this quantification. The preferred antibodies and / or ADCs of the present invention exhibit increased total internalization 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 between 9% and 155% relative to the AUC of Ab1. In another embodiment, as tested in the 786-O cell line, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC between 40% and 130% or between 40% and 50% relative to the AUC of Ab1. In another embodiment, as tested in the A375 cell line, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC between 90% and 100% or between 90% and 95% relative to the AUC of Ab1. In another embodiment, as tested in the BXPC3 cell line, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC between 85% and 155% or between 85% and 90% relative to the AUC of Ab1. In another embodiment, as tested in the ES-2 cell line, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC between 9% and 40% or between 9% and 13% relative to the AUC of Ab1. In another embodiment, as tested in the MDA-MB-231 cell line, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC between 75% and 145% or between 75% and 80% relative to the AUC of Ab1.

[0171] The anti-PD-L1 antibody of this disclosure is preferably monoclonal and may be a multispecific antibody, a human antibody, a humanized antibody or a chimeric antibody, a single-chain antibody, a Fab fragment, an F(ab') fragment, a fragment generated from a Fab expression library, or a PD-L1 binding fragment of any of the above. In some embodiments, the anti-PD-L1 antibody of this disclosure specifically binds to PD-L1. The immunoglobulin molecule of this disclosure may be any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. In one embodiment, the anti-PD-L1 antibody of this disclosure is of type IgG1.

[0172] In some embodiments of this disclosure, the anti-PD-L1 antibody is an antigen-binding fragment as described herein (e.g., a human antigen-binding fragment), and includes, but is not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv), and V-containing... L or V H Fragments of structural domains. An antigen-binding fragment comprising a single-chain antibody may contain one or more variable regions, alone or in combination with all or some of the following: a hinge region, CH1, CH2, CH3, and CL domains. This disclosure also includes antigen-binding fragments comprising one or more variable regions in any combination of a hinge region, CH1, CH2, CH3, and CL domains. In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment is an antibody of human, mouse (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0173] The anti-PD-L1 antibody disclosed herein may be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies may be specific to different epitopes of PD-L1, or specific to both PD-L1 and the heterologous protein. See, for example, PCT publications WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; Tutt, et al., 1991, J. Immunol. 147:60 69; US patents 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., 1992, J. Immunol. 148:1547 1553.

[0174] The anti-PD-L1 antibodies disclosed herein can be described or detailed according to the specific CDRs they contain. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described below: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding sitetopography,” J. Mol. Biol. 262, 732-745 (“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, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, June 8, 2001; 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). The boundaries of a given CDR can vary depending on the scheme used for identification.In some implementations, a “CDR” or “complementarity-determining region” or a separately designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or its region (e.g., its variable region) should be understood to encompass one (or a specific) CDR as defined by any of the above-described schemes. For example, a statement that a specific CDR (e.g., CDR-H3) contains a given V. H or V L In the case of the amino acid sequence of the corresponding CDR in the region amino acid sequence, it should be understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) in the variable region, as defined by any of the above-described schemes. Schemes for identifying a particular one or more CDRs (such as CDRs defined by the Kabat, Chothia, AbM, or IMGT methods) can be specified.

[0175] The CDR sequences of the anti-PD-L1 antibody and anti-PD-L1 antibody-drug conjugate described herein are based on the Kabat numbering scheme as follows: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD.

[0176] In one aspect, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising heavy chain variable regions and light chain variable regions, wherein the heavy chain variable region comprises (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 13, (ii) CDR-H2 containing the amino acid sequence of SEQ ID NO: 14, and (iii) CDR-H3 containing the amino acid sequence of SEQ ID NO: 15; and / or wherein the light chain variable region comprises (i) CDR-L1 containing the amino acid sequence of SEQ ID NO: 16, (ii) CDR-L2 containing the amino acid sequence of SEQ ID NO: 17, and (iii) CDR-L3 containing the amino acid sequence of SEQ ID NO: 18, wherein the CDR of the anti-PD-L1 antibody is defined by the Kabat numbering scheme.

[0177] In one aspect, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain containing the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain containing the amino acid sequence of SEQ ID NO: 12. In another aspect, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 9 and a light chain containing the amino acid sequence of SEQ ID NO: 10.

[0178] In some embodiments, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 11. In some embodiments, the heavy chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 11 contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 11 are substituted, inserted, and / or deleted. In some embodiments, substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDR (i.e., in the FR). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 11, which includes post-translational modifications of that sequence.

[0179] In some embodiments, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a light chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 12. In some embodiments, a heavy chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 12 contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 12 are substituted, inserted, and / or deleted. In some embodiments, substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in regions outside the CDR (i.e., in the FR). In some embodiments, the anti-PD-L1 antibody comprises the light chain variable domain sequence of SEQ ID NO: 12, which includes post-translational modifications of that sequence.

[0180] In some embodiments, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 1. In some embodiments, the heavy chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 1 contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 1 are substituted, inserted, and / or deleted. In some embodiments, the heavy chain contains a point mutation relative to SEQ ID NO: 1. In a further embodiment, the point mutation is located in the CDR region.

[0181] In some embodiments, this document provides anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a light chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 2. In some embodiments, a heavy chain variable domain containing 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 with the amino acid sequence of SEQ ID NO: 2 contains substitutions (e.g., conserved substitutions), insertions, or deletions relative to a reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In some embodiments, a total of 1 to 10 amino acids in SEQ ID NO: 2 are substituted, inserted, and / or deleted. In some embodiments, the light chain contains a point mutation relative to SEQ ID NO: 2. In a further embodiment, the point mutation is located in the CDR region.

[0182] In some embodiments, the anti-PD-L1 antibody or anti-PD-L1 antibody of the anti-PD-L1 antibody-drug conjugate is a monoclonal antibody.

[0183] Five classes of immunoglobulins exist: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain referred to as α, δ, ε, γ, and μ, respectively. The γ and α classes are further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies can exist in a variety of polymorphic variants, referred to as allotypes (see Jefferis and Lefranc 2009. mAbs Vol. 1, No. 4, pp. 1-7 for a review), any of which is suitable for use in some embodiments herein. Common allotype variants in the human population are those named by the letters a, f, n, z, or combinations thereof. In any embodiment herein, the antibody may comprise a heavy chain Fc region containing the human IgG Fc region. In other embodiments, the human IgG Fc region comprises human IgG1.

[0184] Antibodies also include modified derivatives, wherein the modification is achieved by covalently attaching any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to PD-L1 or exerting cytotoxic or inhibitory effects on cells. For example, but not limited to, antibody derivatives include antibodies that have been modified, for example, by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage to cellular ligands or other proteins. Any of a variety of chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, the derivatives may contain one or more non-classical amino acids.

[0185] Humanized antibodies

[0186] Humanized antibodies are genetically engineered antibodies in which a CDR derived from a non-human "donor" antibody is grafted into a human "recipient" antibody sequence (see, for example, Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The recipient antibody sequence can be, for example, a mature human antibody sequence, a complex of such sequences, a common sequence of human antibody sequences, or a germline region sequence. For heavy chains, the preferred recipient sequence is germline V. H Exon V H l-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 The preferred receptor sequence is exon J. H-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred receptor sequence is exon VK2-30 (also referred to as KV2-30 in the literature), and for the hinge region, the preferred receptor sequence is exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Therefore, a humanized antibody is an antibody having some or all of the CDRs, wholly or substantially derived from the donor antibody, and a variable region framework sequence and a constant region (if present), wholly or substantially derived from the human antibody sequence. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs, wholly or substantially derived from the donor antibody heavy chain, and a heavy chain variable region framework sequence and a heavy chain constant region (if present), wholly or substantially derived from the human heavy chain variable region framework and constant region sequences. Similarly, the humanized light chain has at least one, two, and usually all three CDRs that are entirely or substantially derived from the donor antibody light chain, as well as a light chain variable region framework sequence and a light chain constant region (if present) that are substantially derived from the human light chain variable region framework and constant region sequences. Except for nanobodies and dAbs, humanized antibodies comprise humanized heavy chains and humanized light chains. The CDRs in a humanized antibody are substantially 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 among their respective CDRs. The variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially derived from the human variable region framework sequence or the human constant region, respectively, when at least 85%, 90%, 95%, or 100% of the corresponding residues are identical as defined by Kabat. In some embodiments, the PD-L1 antibody of the present invention is a humanized antibody.

[0187] Although humanized antibodies typically contain all six CDRs from mouse antibodies (preferably as defined by Kabat), they can also be manufactured with fewer than all (e.g., at least 3, 4, or 5) CDRs from mouse antibodies (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).

[0188] Selection of constant region

[0189] The variable regions of the heavy and light chains of humanized antibodies can be linked to at least a portion of the human constant region. The choice of the constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent phagocytosis, and / or complement-dependent cytotoxicity are required. For example, human allotypes IgG1 and IgG3 have strong complement-dependent cytotoxicity, human allotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be λ or κ. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as separate heavy and light chains such as Fab, Fab', F(ab')2, and Fv, or as single-chain antibodies in which the variable domains of the heavy and light chains are linked by spacers.

[0190] Human constant regions exhibit both allotropic and heterotropic variations among individuals; that is, the constant region may differ at one or more polymorphic locations in different individuals. The difference between heterotropic and allotropic regions lies in the fact that serum identifying heterotropic regions binds to one or more non-polymorphic regions of other isotypes.

[0191] One or more amino acids at the amino or carboxyl terminus of the light and / or heavy chains (such as the C-terminal lysine of the heavy chain) may be deleted or derivatized in part or all of the molecule. Substitutions may be made in the constant region to reduce or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, for example, 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 prolong the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0192] Exemplary substitutions include the introduction of a native amino acid at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332 into a cysteine ​​residue, preferably the S239C mutation in the human IgG1 isotype (US20100158909). The presence of additional cysteine ​​residues allows for interchain disulfide bond formation. This interchain disulfide bond formation can cause steric hindrance, thereby reducing the affinity of the Fc region-FcyR binding interaction. One or more cysteine ​​residues introduced in or near the Fc region of the IgG constant region can also serve as sites for conjugation with therapeutic agents (i.e., cytotoxic drugs coupled with thiol-specific agents such as maleimide derivatives of drugs). The presence of therapeutic agents causes steric hindrance, thereby further reducing the affinity of the Fc region-FcyR binding interaction. Other substitutions at any of the positions 234, 235, 236 and / or 237 reduce affinity for Fey receptors, particularly FcyRI receptors (see, for example, US 6,624,821, US 5,624,821).

[0193] The in vivo half-life of an antibody can also affect its effector function. Increasing or decreasing the half-life of an antibody can alter its therapeutic activity. FcRn is a receptor structurally similar to an MHC class I antigen that non-covalently associates with β2-microglobulin. FcRn regulates the catabolic metabolism of IgG and its transtissue transcytosis (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). 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 allows IgG to recirculate back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). Regions on human IgG1 involved in FcRn binding have been localized (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 in 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. Therefore, these modified IgG1 molecules may be able to perform their effector functions over a longer period of time compared to unmodified IgG1, and thus exert their therapeutic efficacy. Other exemplary substitutions for increasing FcRn binding include Gin at position 250 and / or Leu at position 428. EU numbers are used for all positions in the constant region.

[0194] Oligosaccharides covalently attached to conserved Asn297 participate in the ability of IgG to bind FcyR in the Fc region (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 199, Trends Biotechnol. 15:26-31). Engineering this glycoform on IgG can significantly improve IgG-mediated ADCC. Adding bipartite N-acetylglucosamine modification (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotech.Bioeng. 74:288-94) to this glycoform or removing fucose from this glycoform (Shields et al., 2002, J.Biol. Chem. 277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc engineering that improves the binding between IgG Fc and FcyR, thereby enhancing Ig-mediated ADCC activity.

[0195] Systematic substitution of solvent-exposed amino acids in the Fc region of human IgG1 has produced IgG variants with altered FcyR binding affinity (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Subsets of these variants, including those substituted with Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 Lys334 to 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).

[0196] The complement-fixing 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). Identical substitutions on the human IgG2 backbone can convert antibody isoforms that are poorly bound to C1q and severely lack complement activation activity into isoforms that can both bind C1q and mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other methods have also been used to improve antibody complement-fixing activity. For example, transplanting an 18-amino acid C-terminal tail fragment from IgM to the C-terminus of IgG significantly enhances its CDC activity. This enhancement has been observed even for IgG4, which typically lacks detectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Furthermore, substitution of Ser444 near the C-terminus of the IgG1 heavy chain with Cys induced tail-to-tail dimerization of IgG1, with CDC activity increasing 200-fold compared to monomeric IgG1 (Shopes et al., 1992, J. Immunol. 148:2918-22). Additionally, bispecific biantibody constructs with C1q specificity also conferred CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0197] Complement activity can be reduced by mutating at least one of the amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain (such as Ala). Replacing any one of the three residues with other alkyl-substituted nonionic residues (such as Gly, Leu, or Val) or aromatic nonpolar residues (such as Phe, Tyr, Trp, and Pro) also reduces or eliminates C1q binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322 but not at 318 to reduce or eliminate C1q binding activity.

[0198] Substituting a polar residue for the 318 (Glu) residue alters, but does not eliminate, C1q binding activity. Substituting the 297 (Asn) residue for Ala results in the removal of cleavage activity, but only slightly reduces (approximately three-fold weaker) the affinity for C1q. This alteration disrupts the presence of the glycosylation site and the carbohydrate required for complement activation. Any other substitution at this site also disrupts the glycosylation site. The following mutations and any combinations thereof also reduce C1q binding: D270A, K322A, P329A, and P31IS (see WO06 / 036291). The L234A / L235A mutation (or the LALA mutation) also reduces C1q binding as well as FcyR binding. In one embodiment, the anti-PD-L1 antibody of the present invention comprises the L234A / L235A mutation.

[0199] References to human constant regions include constant regions with any substitutions of residues that are in any natural allotype or occupy polymorphic positions in natural allotypes. Furthermore, up to 1, 2, 5, or 10 mutations relative to the natural human constant region, as indicated above, may exist to reduce Fcγ receptor binding or increase binding to FcRN.

[0200] V. Expression of recombinant antibodies

[0201] Humanized antibodies are typically produced through recombinant expression. Recombinant polynucleotide constructs typically contain expression control sequences operatively linked to the coding sequence of the antibody chain, including natively associated or heterologous promoter regions. Preferably, the expression control sequence is a eukaryotic promoter system in a vector 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 sequence and for the collection and purification of cross-reactive antibodies.

[0202] Mammalian cells are the preferred host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, New York, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and antibody-non-antibody-producing myeloma cells (including Sp2 / 0 and NSO). Preferably, the cells are non-human. Expression vectors for these cells may include expression control sequences such as origin of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)) and necessary processing information sites such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator 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).

[0203] Once expressed, the antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, etc. (see Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0204] VI. Nucleic Acids

[0205] The present invention also provides nucleic acids encoding either of the aforementioned humanized heavy and light chains. Typically, the nucleic acid also encodes a signal peptide fused to the mature heavy and light chains. The coding sequence on the nucleic acid can be operatively linked to regulatory sequences such as promoters, enhancers, ribosome binding sites, transcription termination signals, etc., to ensure expression of the coding sequence. The nucleic acids encoding the heavy and light chains can exist in a separate form or can be cloned into one or more vectors. The nucleic acids can be synthesized, for example, by solid-state synthesis of overlapping oligonucleotides or by PCR. The nucleic acids encoding the heavy and light chains can, for example, be linked as a single continuous nucleic acid within an expression vector, or they can be separate, for example, each cloned into its own expression vector.

[0206] In some respects, this document also provides nucleic acids encoding anti-PD-L1 antibodies or antigen-binding fragments thereof as described herein. This document also provides vectors comprising nucleic acids encoding anti-PD-L1 antibodies or antigen-binding fragments thereof as described herein. This document also provides host cells expressing nucleic acids encoding anti-PD-L1 antibodies or antigen-binding fragments thereof as described herein. This document also provides host cells comprising vectors containing nucleic acids encoding anti-PD-L1 antibodies or antigen-binding fragments thereof as described herein.

[0207] The anti-PD-L1 antibody described herein can be prepared using well-known recombinant techniques, well-known expression vector systems, and host cells. In one embodiment, the antibody is prepared in CHO cells using a GS expression vector system as disclosed below: De la Cruz Edmunds et al., 2006, Molecular Biotechnology 34; 179-190, EP216846, U.S. Patent No. 5,981,216, WO 87 / 04462, EP323997, U.S. Patent No. 5,591,639, U.S. Patent No. 5,658,759, EP338841, U.S. Patent No. 5,879,936, and U.S. Patent No. 5,891,693.

[0208] The monoclonal anti-PD-L1 antibody described herein can be produced, for example, by the hybridoma method first described by Kohler et al., Nature, 256, 495 (1975), or by a recombinant DNA method. Monoclonal antibodies can also be isolated from phage antibody libraries using techniques described, for example, Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., JMol, 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 a target antigen (e.g., in the form of cells expressing the antigen on their surface, or nucleic acids encoding the target antigen). Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells from immunized human or non-human mammals (e.g., rats, dogs, primates, etc.).

[0209] Antibody-drug conjugates

[0210] Anti-PD-L1 antibodies can be conjugated with cytotoxic or cell-inhibiting moieties (including their pharmaceutically compatible salts) to form antibody-drug conjugates (ADCs). Particularly suitable moieties for conjugation with antibodies are cytotoxic agents (e.g., chemotherapeutic agents), prodrug-converting enzymes, radioisotopes, or compounds or toxins (these moieties are collectively referred to as therapeutic agents). For example, anti-PD-L1 antibodies can be conjugated with cytotoxic agents such as chemotherapeutic agents or toxins (e.g., cell inhibitors or cytokillants, such as abrinogen, ricin A, Pseudomonas exotoxin, or diphtheria toxin).

[0211] Anti-PD-L1 antibodies can be conjugated to prodrug-converting enzymes. The prodrug-converting enzyme can be recombinantly fused to 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.

[0212] The technique of conjugating therapeutic agents with proteins, especially antibodies, is well known. (See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy (edited by Reisfeld et al., Alan R. Liss, Inc., 1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (edited by Robinson et al., Marcel Dekker, Inc., 2nd edition, 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (edited by Pinchera et al., 1985); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (edited by Baldwin et al., Academic Press, (1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, for example, PCT Publication WO 89 / 12624.)

[0213] The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, by antibody degradation, or by a cleaving agent). This therapeutic agent is attached to the antibody using a cleavable linker that is sensitive to cleavage in the intracellular environment of PD-L1-expressing cancer cells but substantially insensitive to the extracellular environment, such that the conjugate is cleaved from the antibody when internalized by PD-L1-expressing cancer cells (e.g., in endosomes or, for example, in a lysosomal environment or in a pit environment due to pH sensitivity or protease sensitivity).

[0214] Typically, an ADC includes a linker region between a therapeutic agent and an anti-PD-L1 antibody. As described above, the linker is typically cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in an intracellular environment (e.g., within lysosomes, endosomes, or pits). The linker can be a peptide linker, for example, cleaved by intracellular peptidases or proteases (including lysosomal or endosomal proteases). Typically, the peptide linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D, as well as plasmin (see, for example, Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typically, the peptide linker is cleavable by enzymes present in cells expressing PD-L1. For example, a peptide linker cleavable by cathepsin-B, a thiol-dependent protease highly expressed in cancerous tissue (e.g., a linker containing Phe-Leu or Gly-Phe-Leu-Gly peptides) can be used. Other such connectors are described, for example, in U.S. Patent No. 6,214,345. In specific embodiments, peptide connectors cleavable by intracellular proteases include the Val-Cit connector or the Phe-Lys dipeptide (see, for example, U.S. Patent 6,214,345, which describes the synthesis of doxorubicin using the Val-Cit connector). One advantage of using intracellular proteolytically hydrolyzed therapeutic agents is that the agent typically decays upon conjugation, and the serum stability of the conjugate is generally high.

[0215] The cleavable adapter can be pH-sensitive, meaning it is sensitive to hydrolysis at certain pH values. Typically, pH-sensitive adapters are hydrolyzable under acidic conditions. For example, acid-labile adapters that are hydrolyzable in lysosomes (e.g., hydrazones, hemicarbazones, thiophene, cis-aconitamide, orthoesters, acetals, ketals, etc.) can be used. (See, for example, U.S. Patents 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 adapters are relatively stable under neutral pH conditions (such as those in blood) but unstable below pH 5.5 or 5.0 (the approximate pH of lysosomes). In some embodiments, the hydrolyzable connector is a thioether connector (such as a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, for example, U.S. Patent No. 5,622,929).

[0216] Other connectors (such as disulfide connectors) are cleavable under reducing conditions. Disulfide connectors 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-α-methyl-α-(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 (edited by CW Vogel, Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935.)}

[0217] The connector may also be a malonic acid ester connector (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl connector (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 connector may also be a malonic acid ester connector (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl connector (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).

[0218] The connector can also be an uncuttable connector, such as a maleimide-alkylene or maleimide-aryl connector that attaches directly to a therapeutic agent (e.g., a drug). The active drug-connector is released through antibody degradation.

[0219] Typically, the adapter is substantially insensitive to the extracellular environment, meaning that when the ADC is present in the extracellular environment (e.g., in plasma), no more than about 20%, typically no more than about 15%, more typically no more than about 10%, or even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the adapter in the ADC sample is cleaved.

[0220] Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by independently culturing (a) an ADC (“ADC sample”) and (b) an equimolar amount of unconjugated antibody or therapeutic agent (“control sample”) in plasma for a predetermined time 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, for example, by high-performance liquid chromatography, with the amount present in the control sample.

[0221] The linker can also promote cell internalization. When conjugated with a therapeutic agent (i.e., in the context of the linker-therapeutic agent portion of an ADC or ADC derivative as described herein), the linker can promote cell internalization. Alternatively, when conjugated with both a therapeutic agent and an anti-PD-L1 antibody (i.e., in the context of an ADC as described herein), the linker can promote cell internalization.

[0222] The anti-PD-L1 antibody can be conjugated to a linker via heteroatoms of the antibody. These heteroatoms can be present on the antibody in their native state or can be introduced into the antibody. In some aspects, the anti-PD-L1 antibody is conjugated to the linker via the nitrogen atom of a lysine residue. In other aspects, the anti-PD-L1 antibody is conjugated to the linker via the sulfur atom of a cysteine ​​residue. The cysteine ​​residue can be naturally occurring or engineered into the antibody. Methods of conjugating linkers and drug-linkers to antibodies via lysine and cysteine ​​residues are known in the art.

[0223] Exemplary antibody-drug conjugates include aurestatin-based antibody-drug conjugates (i.e., the drug component is the aurestatin drug). Aurestatin binds to tubulin and has been shown to interfere with microtubule dynamics as well as cell nucleus and cell division, and has anticancer activity. Typically, aurestatin-based antibody-drug conjugates contain a linker between the aurestatin drug and an antiPD-L1 antibody. The linker may be, for example, a cleavable linker (e.g., a peptide linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). Aurestatin includes aurestatin T, MMAF, and MMAE. Exemplary synthesis and structure of aurestatin are described in U.S. Publications 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 and for all purposes.

[0224] 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 anticancer activity. Typically, camptothecin-based antibody-drug conjugates contain a linker between the camptothecin drug and an anti-PD-L1 antibody. The linker may be, for example, a cleavable linker (e.g., a peptide linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker released by antibody degradation). The synthesis and structure of exemplary camptothecin drug linkers are described in PCT / US19 / 025968 (filed April 5, 2019), which is incorporated herein by reference in its entirety and for all purposes.

[0225] Other exemplary antibody-drug conjugates include maytansine alkaloid antibody-drug conjugates (i.e., the drug component is maytansine alkaloid drug) and benzodiazepine antibody-drug conjugates (i.e., the drug component is benzodiazepine (e.g., pyrrolo[l,4]benzodiazepine dimer (PBD dimer), indolebenzodiazepine dimer and oxazolidinylbenzodiazepine dimer)).

[0226] Exemplary antibody-drug conjugates include the following vcMMAE and mcMMAF antibody-drug conjugates, where p represents the drug load and Ab represents an anti-PD-L1 antibody:

[0227] vcMMAE

[0228] mcMMAF Or its pharmaceutically acceptable salt.

[0229] Exemplary anti-PD-L1 antibody drug conjugates include camptothecin antibody drug conjugates as follows, where p represents the drug load and Ab represents the anti-PD-L1 antibody:

[0230] In some embodiments, the camptothecin ADC has the formula (IC): (IC) Or its pharmaceutically acceptable salt; in Ab is an anti-PD-L1 antibody; y is 1, 2, 3, or 4, or 1 or 4; and z is an integer from 2 to 12, or 2, 4, 8 or 12; And p is 1-16.

[0231] In some aspects of these implementation schemes, p is 2, 3, 4, 5, 6, 7, 8, 9, or 10. In other aspects, p is 2, 4, or 8.

[0232] In some embodiments, the camptothecin ADC has the formula:

[0233] Or its pharmaceutically acceptable salt; Where p is 2, 4 or 8, preferably p is 8.

[0234] In some embodiments, the camptothecin ADC has the formula:

[0235] Or its pharmaceutically acceptable salt; Where p is 2, 4 or 8, preferably p is 8.

[0236] In some embodiments, the camptothecin drug connector has the following formula:

[0237] Or its pharmaceutically acceptable salt; in y is 1, 2, 3, or 4, or 1 or 4; and z is an integer from 2 to 12, or 2, 4, 8 or 12.

[0238] In some embodiments, the camptothecin drug connector has the following formula:

[0239] MP-PEG8-VKG-camptothecin

[0240] In some embodiments, the camptothecin drug connector has the following formula:

[0241] MP-PEG4-VKG-camptothecin

[0242] In some embodiments, the camptothecin drug connector has the following formula:

[0243] MP-PEG12-VKG-camptothecin

[0244] When referring to antibody-drug conjugates targeting PD-L1, the subscript 'p' indicates drug load, and depending on the context, it can represent the number of drug-adaptor molecules attached to an individual antibody molecule, and thus be an integer value; or it can represent the average drug load, and thus can be an integer or non-integer value, but typically a non-integer value. Average drug load represents the average number of drug-adaptor molecules per antibody in the population. Typically, but not always, when we refer to antibodies, such as monoclonal antibodies, we are referring to the population of antibody molecules. In compositions containing a population of antibody-drug conjugate molecules, average drug load is an important quality property because 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.

[0245] In a preferred aspect of the invention, when referring to a composition comprising a group of antibody-drug conjugate compounds, the average drug load is from about 1 to about 16, preferably about 2 to about 14, more preferably about 2 to about 10.

[0246] For MMAE and camptothecin ADCs, as exemplified herein, the preferred average drug load is about 2, 4, or 8, and particularly preferred is about 8. In one embodiment, the preferred average drug load for the MMAE ADC is 2 or 4. In one embodiment, the preferred average drug load for the camptothecin ADC is 4 or 8. In an exemplary embodiment, the drug-connector is conjugated to a cysteine ​​residue of the reduced interchain disulfide. In some aspects, the actual drug load of an individual antibody molecule in the antibody-drug conjugate compound group ranges from 1 to 10 (or from 6 to 10 or from 6 to 8), with the predominant drug load being 8. For example, a higher drug load can be achieved if, in addition to the interchain disulfide, the drug-connector is also conjugated to an introduced cysteine ​​residue (such as the cysteine ​​residue introduced at position 239 according to the EU index).

[0247] The PEG (polyethylene glycol) portion of the drug connector can range from 2 to 36. In all the above embodiments, the subscript z is preferably 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.

[0248] Polydisperse PEG, monodisperse PEG, and discrete PEG can be used to prepare the polyethylene glycol-modified antibody-drug conjugates of the present invention. Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from a heterogeneous mixture, thus providing a single chain length and molecular weight. The preferred PEG unit is discrete PEG, i.e., a compound synthesized stepwise rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length. As indicated by the subscript "p", when referring to a population of antibody-drug conjugates, the value of the subscript "n" can be an average and can be an integer or a non-integer.

[0249] Useful classes of cytotoxic agents conjugated to anti-PD-L1 antibodies include, for example, anti-tubulin agents, DNA minor groove binding agents, DNA replication inhibitors, and chemosensitizers. Other exemplary classes of cytotoxic agents include anthracyclines, auristatin, camptothecin, buprofen, etoposide, maytansine alkaloids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatin (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binding agents (e.g., enediyne and lexitropsin), buprofen, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulolysin M, doxorubicin, morpholino-doxorubicin, and cyclomorpholino-doxorubicin.

[0250] The cytotoxic agent may be a chemotherapeutic agent, such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agent may also be a CC-1065 analog, chachomycosis, maytansine, a serotonin 10 analog, rhizobium, or anemone toxin.

[0251] The cytotoxic agent may also be auristatin. Auristatin can be an auristatin E derivative, such as an ester formed between auristatin E and a keto acid. For example, auristatin E can react with p-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 2005-0238649 and US 2006-0074008.

[0252] The cytotoxic agent may be a DNA minor groove binder. (See, for example, U.S. Patent No. 6,130,237.) For example, the minor groove binder may be a CBI compound or an enediyne (e.g., cazithromycin).

[0253] The cytotoxic agent or cell inhibitor may be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., Taxol®), Taxotere®, T67, vinca alkaloids (e.g., vincristine, vinblastine, vinorelbine, and vinorelbine), and auristatin (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEB). Exemplary auristatins are shown in formulas III-XIII below. Other suitable anti-tubulin agents include, for example, berry gibberellin derivatives, taxane analogs (e.g., epothilone A and B), nocodazole, colchicine and colchicineamine, estradiol, cryptophysin, simmadol, maytansine alkaloids, compressormoide, and eleuthrobin.

[0254] The cytotoxic agent may be maytansine alkaloids, which are another group of anti-microtubule agents (e.g., DM1, DM2, DM3, DM4). For example, maytansine alkaloids may be maytansine or drug linkers containing maytansine (e.g., DM-1 or DM-4) (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.).

[0255] VIII. Therapeutic Use

[0256] The antibodies of the present invention, alone or as their anti-PD-L1 antibody-drug conjugates, can be used to treat cancer. Some such cancers exhibit detectable PD-L1 levels measured at the protein level (e.g., by immunoassay using one of the illustrated antibodies) or mRNA level. Some such cancers exhibit elevated PD-L1 levels relative to the same type of non-cancerous tissue (preferably from the same patient). Exemplary PD-L1 levels on treatable cancer cells are 5,000-500,000 PD-L1 molecules per cell, but higher or lower levels can be treated. Optionally, the level of PD-L1 in the cancer is measured prior to treatment.

[0257] 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating melanoma. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating NSCLC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating SCLC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating head and neck cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating TNBC. Triple-negative breast cancer is a specific term for cancers lacking detectable estrogen and progesterone receptors and lacking HER2 / neu overexpression. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating ovarian cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods of treating urothelial carcinoma. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating HCC. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating gastric cancer. In some embodiments, the antibodies or antibody-drug conjugates of the present invention are used in methods for treating cervical cancer. The treatment can be applied to patients with these types of primary or metastatic tumors. The treatment can also be applied to patients who are refractory to conventional treatment or who have relapsed after responding to such treatment.

[0258] The antibodies of the present invention (such as humanized antibodies) are administered alone or as conjugates thereof in an effective regimen, wherein an effective regimen means a dose, route of administration, and frequency of administration that can delay onset, reduce severity, inhibit further deterioration, and / or improve at least one sign or symptom of cancer. If the patient already has cancer, the regimen may be called a therapeutically effective regimen. If the patient has an increased risk of developing cancer relative to the general population but has not yet experienced symptoms, the regimen may be called a preventatively effective regimen. In some cases, therapeutic or preventative efficacy can be observed in a single patient relative to a historical control or past experience in the same patient. In other cases, therapeutic or preventative efficacy can be demonstrated in preclinical or clinical trials relative to a control group of untreated patients.

[0259] Exemplary doses of monoclonal antibodies are 0.1 mg / kg to 50 mg / kg of patient body weight, more typically 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, or 1 mg / kg to 10 mg / kg, or 2 mg / kg to 30 mg / kg, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, or 2 mg / kg to 10 mg / kg, or 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 12 mg / kg, or 3 mg / kg to 10 mg / kg. Exemplary doses of monoclonal antibodies or antibody-drug conjugates thereof are 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg, or 3 mg / kg to 7.5 mg / kg of the subject's body weight, or 0.1-20 mg / kg of body weight, or 0.5-5 mg / kg of body weight (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg), or as a fixed dose of 10-1500 mg or 200-1500 mg. In some methods, the patient is administered a dose of at least 1.5 mg / kg, at least 2 mg / kg, or at least 3 mg / kg every three weeks or longer. The dose depends on factors such as the frequency of administration, the patient's condition and response to prior treatment (if any), whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0260] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, local, intranasal, or intramuscular. Administration can also be directed directly to the tumor. It is preferably administered into the systemic circulation via intravenous or subcutaneous administration. Intravenous administration can be performed, for example, by infusion over a period of time (e.g., 30-90 minutes) or by a single bolus injection.

[0261] The frequency of administration depends on factors such as the half-life of the antibody or conjugate in circulation, the patient's condition, and the route of administration. The frequency can be once daily, once weekly, once monthly, once quarterly, or at irregular intervals in response to changes in the patient's condition or the progression of the cancer being treated. Exemplary frequencies for intravenous administration during continuous treatment are between twice weekly and once quarterly, but higher or lower frequencies are also possible. Other exemplary frequencies for intravenous administration during continuous treatment are between once weekly and three times every four weeks, but higher or lower frequencies are also possible. For subcutaneous administration, exemplary frequencies range from once daily to once monthly, but higher or lower frequencies are also possible.

[0262] The number of doses administered depends on the nature of the cancer (e.g., whether it presents with acute or chronic symptoms) and the disorder's response to treatment. For acute disorders or acute exacerbations of chronic disorders, one to ten doses are often sufficient. Sometimes, a single bolus dose (optionally in split doses) is sufficient for acute disorders or acute exacerbations of chronic disorders. For relapses or acute exacerbations of acute disorders, repeated treatment may be necessary. For chronic disorders, antibodies may be administered at regular intervals, such as weekly, bi-weekly, monthly, quarterly, or every six months, for at least one, five, or ten years, or the patient's lifespan.

[0263] Pharmaceutical compositions intended for parenteral administration are preferably sterile and substantially isotonic, and manufactured under GMP conditions. The pharmaceutical composition may be provided in unit dosage forms (i.e., doses for a single administration). The pharmaceutical composition may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the chosen route of administration. For injection, the antibody may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce injection site discomfort). The solution may contain formulation agents such as suspending agents, stabilizers, and / or dispersants. Alternatively, the antibody may be in lyophilized form prior to use for construction with a suitable medium (e.g., sterile, pyrogen-free water). The concentration of the antibody in the liquid formulation may be, for example, 1-100 mg / ml, such as 10 mg / ml.

[0264] Treatment with the antibodies of this invention can be combined with chemotherapy, radiation, stem cell therapy, surgery, and other treatments effective against ongoing disorders. Other useful classes of agents that can be administered with antibodies and antibody-drug conjugates targeting PD-L1 as described herein include, for example, antibodies against other receptors expressed on cancer cells, anti-microtubule agents (e.g., auratestatin), DNA minor groove binding agents, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum), and trinuclear platinum complexes, and carboplatin), anthracyclines, antibiotics, antifolate, antimetabolites, chemotherapy sensitizers, pyromalacin, etoposide, fluorinated pyrimidines, ionophores, lexiconazole, nitrosourea, cisplatin, preformed compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

[0265] Compared to the same treatment (e.g., chemotherapy) but without an anti-PD-L1 antibody alone or as a conjugate, treatment with an anti-PD-L1 antibody or antibody-drug conjugate, optionally combined with any other of the above-mentioned agents or regimens alone or as an antibody-drug conjugate, can increase median progression-free survival or overall survival by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100% or longer, in patients with tumors (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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer), especially in the case of recurrent or refractory tumors. Alternatively or alternatively, treatment including anti-PD-L1 antibodies, either alone or as conjugates, (e.g., standard chemotherapy) can increase the complete response rate, partial response rate, or objective response rate (complete + partial) in patients with tumors by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100%, compared to the same treatment (e.g., chemotherapy) but without anti-PD-L1 antibodies alone or as conjugates.

[0266] Typically, in clinical trials (e.g., phase II, II / III, or III trials), the aforementioned increase in median progression-free survival and / or response rate in patients treated with standard therapy plus an anti-PD-L1 antibody, either alone or as a conjugate, is statistically significant, for example, at the p = 0.05, 0.01, or even 0.001 level, compared to a control group receiving standard therapy alone (or plus placebo). Complete and partial response rates are determined using objective criteria commonly used in cancer clinical trials (e.g., those listed or accepted by the National Cancer Institute and / or the Food and Drug Administration).

[0267] IX. Products and Reagent Kits

[0268] In another aspect, an article or kit is provided comprising the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate described herein. The article or kit may also comprise instructions for use of the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate described herein in the methods of the present invention. Therefore, in some embodiments, the 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 of treating a subject with 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer), the method 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 a human.

[0269] The product or reagent kit may further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single-chamber or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The container can be made of various materials, such as glass or plastic. The container contains the preparation.

[0270] The product or kit may also include a label or packaging insert on or associated with the container indicating instructions regarding the reconstitution and / or use of the formulation. The label or packaging insert may further indicate that the formulation is intended for use subcutaneously, intravenously (e.g., intravenous infusion), or by other routes of administration to treat cancers in subjects (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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer). The container holding the formulation may be a single-use vial or a reusable vial, allowing for repeated administration of the reconstituted formulation. The product or kit may also include a second container containing a suitable diluent. The product or kit may also include other materials desired from a commercial, therapeutic, and user perspective, including additional buffers, diluents, filters, needles, syringes, and packaging inserts printed with instructions for use.

[0271] The article or kit may optionally also include a container containing a second agent, wherein the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate is the first agent, and the article or kit may also include instructions for use in treating a subject with an effective amount of the second agent, as indicated on a label or packaging insert. In some embodiments, the second agent is used to eliminate or reduce the severity of one or more adverse events.

[0272] In some embodiments, the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate is present as a lyophilized powder in the container. In some embodiments, the lyophilized powder is in an air-sealed container (such as a vial, ampoule, or pouch) indicating the amount of active agent. In cases where the drug is administered by injection, ampoules of sterile water or saline for injection may be optionally provided as part of the kit, allowing the components to be mixed prior to administration. If desired, such kits may also include one or more of various conventional pharmaceutical components, such as containers having one or more pharmaceutically acceptable carriers, additional containers, etc., as will be apparent to those skilled in the art. The kit may also include printed instructions as inserts or labels indicating the amount of components to be administered, administration guidelines, and / or instructions for mixing components.

[0273] X. Other Applications

[0274] The anti-PD-L1 antibodies described herein, such as humanized anti-PD-L1 antibodies, can be used to detect PD-L1 in clinical diagnostic or therapeutic settings or in research. PD-L1 expression in cancer provides an indication that the cancer can be treated with the antibodies of this invention. These antibodies are also commercially available as reagents for laboratory studies involving the detection of PD-L1-carrying cells and their responses to various stimuli. In such uses, monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and can be provided in kit form along with all reagents required for PD-L1 assays. The antibodies described herein can be used to detect PD-L1 protein expression and determine whether cancer can be treated with PD-L1 ADCs.

[0275] All patent applications, websites, other publications, registration numbers, etc., cited above or below are incorporated herein by reference in their entirety for all purposes, to the extent that each individual item is specifically and individually indicated as such. If different versions of a sequence are associated with registration numbers at different times, this refers to the version associated with the registration number at the effective filing date of this application. Effective filing date means the earlier of the actual filing date of the registration number (if applicable) or the filing date of the priority application. Similarly, if different versions of publications, websites, etc., are published at different times, this refers to the version published closest to the effective filing date of the application, unless otherwise stated. Unless otherwise specifically stated, any feature, step, element, embodiment, or aspect of the invention may be used in combination with any other. Although the invention has been described in somewhat detail by way of illustration and example for purposes of clarity and understanding, it is clear that certain changes and modifications may be made within the scope of the appended claims.

[0276] Example

[0277] The cell lines described in the following examples were maintained in culture medium according to the conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ), or as other known conditions.

[0278] method Antibody production

[0279] An SG-559-xx antibody against PD-L1 was generated by introducing a point mutation into the CDR of fully human Ab1 to reduce affinity. In short, residues in the CDR immediately adjacent to the PD-L1 binding epitope were mutated to dissimilar amino acids. Figure 1 Four selected exemplary residues are illustrated. For preliminary screening purposes, the SG-559-xx antibody was generated in HEK293 cells using transient transfection with ATUM bio.

[0280] For subsequent research, antibodies were generated internally according to the following protocol. The variable and constant domain sequences of the antibodies were synthesized using non-template PCR. In short, the virtual gene sequence was converted into an oligonucleotide sequence using Genewiz's bioinformatics tools. The oligonucleotides were synthesized, collected, and amplified using PCR. The full-length amplicons from the PCR reaction were cloned into a vector, and the product was then transformed into *E. coli*, and unique colonies were isolated. The colonies were allowed to grow overnight in liquid medium, and the plasmid DNA was isolated, purified, and sequenced using Sanger sequencing for validation. The light and heavy chains were cloned into the pcDNA3.4 vector.

[0281] The antibody heavy and light chain vectors were diluted 1:1 in ThermoFisher OptiPRO SFM medium containing ExpiFectamine CHO transfection reagent. DNA / transfection reagent was then added to the ExpiCHO culture in ThermoFisher ExpiCHO Expression medium and cultured for nine days, with ExpiCHO enhancer added on day 1 and ExpiCHO feed added on days 1 and 2. Cultures were harvested by centrifugation and 0.2 μm filtration, or by deep filtration using a Millipore X0HC and D0HC pod followed by 0.2 μm filtration.

[0282] A GE HiTrap mAb Select SuRe column was used to purify each IgG. Prior to elution, the resin was washed with 5 CV PBS + 0.1% Triton, 5 CV PBS + 0.5 M NaCl, and 7.5 CV PBS. IgG was eluted with 100 mM acetate pH3 buffer. The sample buffer was replaced with PBS using a 26 / 60 HiPrep Desalt column. The samples underwent a fine purification step using a HiPrep Superdex 200 26 / 600 column running in PBS. The samples were then filtered sterilized and then sampled for characterization. Characterization included A280 concentration, aSEC HPLC, aHIC HPLC, and reduction PLRP-MS (QToF).

[0283] Biological layer interferometry

[0284] The binding affinity of the SG-559-xx antibody was determined using a biolayer interferometry method with the Octet Red 384 system (ForteBio). The anti-human Fab-CH1 (FAB2G) biosensor (ForteBio) was loaded with 4 µg / mL SG-559-xx antibody for 100 s. Following the subsequent baseline step, human PD-L1 (Acro Biosciences) at concentrations ranging from 500 nM to 0.69 nM (1x PBS pH 7.4, containing 1% casein, 0.2% Tween-20) was incubated with the loaded probe for 150 s for the association step. This was followed by a dissociation step in the same buffer lacking human PD-L1 for 1000 s. Following established methods, k... 缔合 and k 解离 Fit it to the resulting binding curve.

[0285] Production of antibody-drug conjugates (ADCs)

[0286] As described in US20180092984, SG-559-xx antibodies are conjugated to MDpr-PEG(12)-gluc-MMAE with an average drug-to-antibody ratio (DAR) of 8. As described in US20050238649, SG-559-xx antibodies are conjugated to vc-MMAE with an average DAR of 4. As described in PCT / US2019 / 025968 (filed April 5, 2019), SG-559-xx antibodies are conjugated to MP-PEG8-VKG-camptothecin with an average DAR of 8.

[0287] In vitro cytotoxicity assay

[0288] Twenty-four hours before antibody-drug conjugate (ADC) treatment, cell lines were plated to allow for cell adaptation. Interferon-γ at this time was also added, as indicated, to induce PD-L1 expression. Cells were then treated with the indicated dose of ADC and incubated at 37ºC for 96 hours. Other PD-L1-targeting antibodies and isotype controls were included as ADCs for comparison. Cell viability was measured using CellTiter-Glo (Promega Corporation, Madison, Wisconsin) according to the manufacturer's instructions. Briefly, cells were incubated with CellTiter-Glo reagent at room temperature for 30 minutes, and luminescence was measured using an Envision plate reader (Perkin Elmer, Waltham, MA). Results were reported as x50, representing the compound concentration required to produce a 50% reduction in viability compared to untreated cells.

[0289] Internalization assay

[0290] Internalization of PD-L1-directed antibodies was performed using FabFluor pH-sensitive conjugates on Incucyte (Sartorius). The antibodies were conjugated to a pH-sensitive dye that exhibited an increasing fluorescence signal from the cell surface and into the endosome / lysosomal compartments as pH decreased. Adherent cells were plated for 24 h (with 500 IU / mL interferon-γ to induce PD-L1 expression) and then incubated with these conjugates. Suspension cells were plated for 3 h and then incubated with these conjugates. Cells were then given 0.5 µg / mL of the indicated dye-antibody conjugate and allowed to incubate for 48 h. The total integrated intensity of the fluorescence signal was normalized to confluence % / well / time point using Incucyte S3 software (Sartorius). Results are reported as the area under the curve of the normalized integrated intensity versus time.

[0291] In vivo activity study

[0292] Nude mice were used with 5.0 x 10 6 One BxPC3 pancreatic adenocarcinoma cell or 1.0 x 10 6 5.0 x 10T EBC-1 NSCLC cells were subcutaneously seeded into NSG mice. 5 1 MDA-MB-231 triple-negative breast cancer cells were subcutaneously inoculated into SCID mice. 6 299 Karpas ALCL cells or 1.0 x 10299 6 Calu-1 NSCLC cells were subcutaneously seeded. Tumor growth was monitored using calipers and calculated using the formula (0.5 x [length x width]). 2 ]) Calculate the average tumor volume. When the average tumor volume reaches approximately 100 mm 3 Mice were not treated or administered intraperitoneally with the ADC as directed. Unconjugated antibody and VC-MMAEADC were administered weekly for a total of three doses. MP-PEG8-VKG-camptothecin ADC was administered only once. When the tumor volume reached approximately 750 mm... 3 The mice were euthanized. For immunophenotyping studies of Karpas 299 tumor-bearing animals, an average tumor volume of 200 mm was allowed. 3These mice were then treated with a single dose of unconjugated antibody or ADC and euthanized six days later. Tumors were characterized in vitro by immunohistochemistry and cytokine analysis (Luminex). All animal procedures were performed in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, according to protocols approved by the Institutional Animal Care and Use Committee.

[0293] PD-L1 blockade

[0294] In vitro assessment of PD-L1 blockade was performed using the PD-1 / PD-L1 blocking bioassay (Promega Corporation), following the manufacturer's instructions. In short, PD-L1+ aAPC / CHO-K1 cells were plated and allowed to acclimatize for 16 hours. An antibody or ADC at an indicated concentration was then added to the plated cells, followed by PD-1+ effector cells. In the absence of PD-1 / PD-L1 signaling, the interaction between aAPC / CHO-K1 cells and effector cells produces a bioluminescent signal. Therefore, more effective inhibition of the PD-1 / PD-L1 interaction resulted in a higher bioluminescent signal, quantified as a fold increase in induction relative to untreated cells. A PD-1 binding antibody (Promega Corporation) was included as a positive control, and a non-binding allotype antibody was included as a negative control.

[0295] Immunotoxicity in a human APC model with upregulated PD-L1 stimulated by IFNγ stimulation

[0296] Immunotoxicity of human antigen-presenting cells (APCs) to the antibodies or ADCs described herein was measured in vitro using human antigen-presenting cells stimulated with interferon-γ (IFNγ) and then treated with any of the antibodies or ADCs described herein. Prior to treatment with the SG-559-xx ADC, human APCs were stimulated in vitro with 500 IU / mL IFNγ (R&D Systems) for at least 24 hours to modulate PD-L1. Immunotoxicity was calculated as the percentage of the activity of untreated APCs at different antibody or ADC concentrations.

[0297] Suppression of immune response in human APC model

[0298] Immunorepression was measured using human antigen-presenting cells (APCs) stimulated with lipopolysaccharide (LPS) and then treated with any of the antibodies or ADCs described herein. PD-L1 was regulated in vitro by stimulation with 500 IU / mL IFNγ (R&D Systems) for at least 24 hours. Then, human APCs were treated with SG-559-xx ADC for 24 hours as instructed. Subsequently, human APCs were stimulated in vitro with 100 ng / mL LPS (Sigma Aldrich) for 48 hours. Response to LPS was measured by flow cytometry staining for MHC class II and CD86 (Biolegend). Immunological strength was calculated as the fold change in MHC class II or CD86 in APCs in response to LPS stimulation at different antibody or ADC concentrations.

[0299] Human PD-L1 was deglycosylated using PNG enzyme F.

[0300] To generate deglycosylated hPD-L1, human PD-L1 was treated with a denaturation protocol using PNG enzyme F (New England Biolabs). PNG enzyme F catalyzes the cleavage of the innermost GlcNAc between the innermost GlcNAc and the asparagine residues of the high-mannose, heterozygous, and complex oligosaccharides from N-linked glycoproteins. A denaturation protocol was also provided in the absence of PNG enzyme F to serve as a reaction control. The deglycosylation protocol involved combining human PD-L1 (Acro Biosciences) with Rapid PNG enzyme F buffer, heating the human PD-L1 at 75ºC for 5 minutes, cooling the denatured human PD-L1 on ice, adding PNG enzyme F, and incubating overnight at 37ºC. The glycosylation status was confirmed by mass spectrometry.

[0301] Biolayer interferometry was performed using the Octet Red 384 system (ForteBio) to determine the binding affinity of SG-559-xx antibody or ADC to glycosylated or deglycosylated PD-L1. Following a baseline step, glycosylated and deglycosylated human PD-L1 at concentrations ranging from 500 nM to 0.69 nM (1x PBS pH 7.4, containing 1% BSA and 0.2% Tween-20) were incubated with the loaded probe for 150 s for an association step. This was followed by a dissociation step for 1000 s in the same buffer lacking human PD-L1. Following established methods, k... 缔合 and k 解离 Fit it to the resulting binding curve.

[0302] result Example 1: Design and characterization of SG-559-xx antibody

[0303] As described in the method, seventeen SG-559-xx antibodies were generated from the parental Ab1 antibody. The mutated CDRs of these antibodies are listed in Table 1. The monovalent binding affinity of sixteen of these antibodies to hPD-L1 was evaluated using the biolayer interference metric compared to Ab1 (Table 2). The measured affinity of the SG-559-xx antibodies spanned almost two orders of magnitude, and K... D The values ​​range from 4 nM to 297 nM.

[0304] Table 1. SG-559-XX variant sequences

[0305] Table 2. Binding Affinity of SG-559-XX

[0306] Example 2: In vitro cytotoxicity

[0307] As described in the methods, the cytotoxicity of SG-559-xx antibodies as ADCs was evaluated against PD-L1-expressing cancer cell lines (including 786-O, BxPC3, ES-2, MDA-MB-231, Karpas 299, and L540cy). In some experiments, SU-DHL-4 (a PD-L1-negative cancer cell line) was included as a control. Initial screening of fifteen SG-559-xx ADCs (excluding the two with the lowest affinity) with the MDpr-PEG(12)-gluc-MMAE payload (DAR 8) showed that several SG-559-xx antibodies exhibited significantly improved cytotoxicity relative to the parental Ab1. Figures 2A-2F ).

[0308] The four SG-559-xx antibodies that consistently demonstrated the highest potency in the initial screening were further characterized as vc-MMAE and MP-PEG8-VKG-camptothecin ADCs (Table 3). These ADCs were significantly more potent than Ab1 in most of the cell lines tested. They also showed no activity in the antigen-negative cell line (SU-DHL-4), indicating that this was not due to nonspecific binding.

[0309] Table 3. SG-559-XX x50 values ​​(ng / mL)

[0310] Example 3: Internalization

[0311] To confirm the cytotoxicity results, as described in the methods section, the internalization of SG-559-01 and SG-559-03 was further examined using the Incucyte imaging system and pH-sensitive dye conjugates. In most of the cell lines tested, the internalization of SG-559-01 and SG-559-03 was consistently high. This was measured by the percentage increase in the area under the curve (AUC) of the normalized integrated intensity relative to time (Table 4). MDA-MB-231 (…) is shown in… Figure 3A ) and Karpas 299 ( Figure 3B An example curve.

[0312] Table 4. Internalization of SG-559-XX

[0313] Example 4: In vivo antitumor activity

[0314] The antitumor efficacy of four SG-559-xx antibodies used for in vitro screening was also tested in two mouse xenograft models. In the MDA-MB-231 model, the SG-559-xx antibodies used as ADCs exhibited significant antitumor activity with two drug linkers. Figures 4A-4B In the BxPC3 model, the SG-559-xx antibody, used as an ADC, exhibited moderate antitumor activity. Figures 5A-5B In almost all cases, the SG-559-xx ADC was more effective than the Ab1 ADC, indicating that the observed exotype was translated into the in vivo environment.

[0315] Further antitumor efficacy was observed in another model using one of our most promising antibodies as an Fc effector-reduced variant (SG-559-01 LALA). The SG-559-01 LALA antibody, as an ADC, was used with one or two drug linkers in Karpas 299 (… Figures 6A-6B Calu-1 Figure 7 ) and EBC-1 ( Figures 8A-8B The model exhibited significant antitumor activity. Note that this activity differs from that of the unconjugated SG-559-01 LALA antibody.

[0316] Example 5: PD-L1 Blockade

[0317] Further characterization was conducted on the ability of SG-559-01 LALA to block PD-1 / PD-L1 checkpoints in vitro. Compared to the PD-1 antibody control, SG-559-01 was able to inhibit PD-1 / PD-L1 signaling more effectively. Furthermore, unconjugated SG-559-01 LALA was comparable to SG-559-01 LALA conjugated with both drug linkers, demonstrating that conjugation does not affect PD-1 / PD-L1 blockade. Figure 9 ).

[0318] Example 6: In vitro immunotoxicity to human APC

[0319] The immunotoxicity of SG-559-01 and SG-559-01 LALA to APCs (e.g., macrophages and dendritic cells (DCs)) was evaluated. APCs were stimulated with IFNγ to upregulate PD-L1 prior to treatment, as described in the methods. The SG-559-01 LALA ADC exhibited immunotoxicity to human APCs, with immunotoxicity in both macrophages and DCs similar to or within one order of magnitude of that in the isotype control. Figures 10A-10D ).

[0320] The immunotoxicity of four SG-559-xx antibodies used for in vitro screening against APCs (i.e., dendritic cells and macrophages) was also tested. For each SG-559-xx ADC, the immunotoxicity against human APCs was similar or within one order of magnitude. Figures 11A-11D ).

[0321] Example 7: Suppression of Immune Response

[0322] The SG-559-01 ADC was further characterized by measuring immunosuppression in human APCs treated with LPS. As described in the method, human APCs were stimulated in vitro with LPS after ADC treatment, and the upregulation of MHC class II and CD86 was subsequently quantified as a measure of immune response. For example, by measuring the upregulation of MHC class II (… Figures 12A-12B ) and CD86 ( Figures 12C-12D )Measured in DCs and macrophages, treatment with SG-559-01 ADC resulted in a similar or order-of-magnitude suppression of the immune response, or in the same type of control.

[0323] Example 8: Increasing Immune Infiltration

[0324] The SG-559-01LALA vc-MMAE ADC was further characterized by evaluating immune infiltration in mice with Karpas 299 tumors. Tumor-bearing mice were treated as instructed, and tumors were characterized after six days. Compared to both the untreated control and the SG-559-01 antibody, the SG-559-01 vc-MMAE ADC induced immune infiltration in mice with Karpas 299 tumors. Figures 13A-13C ). Figure 13A The increase was observed in mCD45+ cells (a marker of panleukocytes). Figure 13B An increase was observed in mCD11c+ cells (a marker of dendritic and macrophage subsets). Figure 13C An increase was observed in mF4 / 80+ cells (macrophage markers).

[0325] Example 9: Inflammatory Cytokine Response

[0326] Further characterization was conducted on the ability of SG-559-01 LALA vc-MMAE ADC to induce the production of inflammatory cytokines in the tumor microenvironment (TME). Compared to both the untreated control and SG-559-01 LALA antibody, SG-559-01 LALA vc-MMAE ADC induced inflammatory cytokines in the TME, as measured by the following intratumoral concentrations: eotaxin (eosinophil chemokine); Figure 14A MIP1a (pro-inflammatory macrophage cytokine); Figure 14B MIP1b (pro-inflammatory macrophage cytokine); Figure 14C MIG / CXCL9 (induced by IFNγ, affecting the migration and differentiation of immune cells); Figure 14D MCP1 (a chemokine for monocytes / macrophages); Figure 14E ) and rantes (chemokines of monocytes, T cells and eosinophils) that are regulated by activation and expressed and secreted by normal T cells; Figure 14F ).

[0327] Example 10: Binding affinity with glycosylated PD-L1 and deglycosylated PD-L1

[0328] The binding affinity of SG-559-01 for glycosylated and deglycosylated forms of PD-L1 was evaluated. Binding affinity was assessed using the biolayer interferometry method on an Octet Red 384 system (ForteBio) as described in the Methods section. PD-L1 was deglycosylated using PNG enzyme F and a denaturation protocol as described in the Methods section. The binding affinity of SG-559-01 for deglycosylated PD-L1 and control glycosylated PD-L1 (under the same treatment conditions but without PNG enzyme F treatment, as described in the Methods section) was evaluated. An approximately 2-fold difference was observed in the binding affinity of SG-559-01 for deglycosylated PD-L1 compared to glycosylated PD-L1 (Table 5). The glycosylation status of PD-L1 was verified using mass spectrometry.

[0329] Table 5. Binding of SG-559-01 to glycosylated and deglycosylated hPD-L1

[0330] This disclosure relates to the following implementation plan.

[0331] 1. An antibody or an antigen-binding fragment thereof that specifically binds to human programmed death-ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity to human PD-L1 protein between 3 and 300 nM.

[0332] 2. The antibody according to embodiment 1, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 15 nM.

[0333] 3. The antibody according to embodiment 1 or embodiment 2, wherein the antibody further exhibits a total internalization higher than that of Ab1.

[0334] 4. The antibody according to embodiment 3, wherein the total internalization has an AUC increase between 9% and 155% relative to the AUC of Ab1.

[0335] 5. The antibody according to embodiment 3, wherein the total internalization is determined by FabFluor internalization assay.

[0336] 6. The antibody according to any one of embodiments 1-5, wherein the antibody further exhibits a x50 lower than that of Ab1.

[0337] 7. The antibody according to embodiment 6, wherein the antibody is conjugated to monomethylaurestatin E (MMAE), and wherein the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

[0338] 8. The antibody according to embodiment 6, wherein the antibody is conjugated with camptothecin, and wherein the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

[0339] 9. The antibody according to any one of embodiments 1-8, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 13-15 and the light chain CDR sequence of SEQ ID NO: 16-18.

[0340] 10. The antibody according to any one of embodiments 1-8, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 3-5 and the light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs.

[0341] 11. The antibody according to any one of embodiments 1-10, wherein 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.

[0342] 12. The antibody according to any one of embodiments 1-11, wherein 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.

[0343] 13. The antibody according to any one of embodiments 1-12, wherein 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.

[0344] 14. The antibody according to any one of embodiments 1-13, wherein 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.

[0345] 15. The antibody according to any one of embodiments 1-14, wherein the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0346] 16. The antibody according to any one of embodiments 1-15, wherein the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a biantibody, a linear antibody, or a single-chain antibody fragment.

[0347] 17. The antibody according to any one of embodiments 1-16, wherein the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0348] 18. The antibody according to any one of embodiments 1-17, wherein the heavy chain constant region is of the IgG1 isotype.

[0349] 19. The antibody according to any one of embodiments 1-18, wherein the antibody is a humanized or chimeric antibody.

[0350] 20. The antibody according to any one of embodiments 1-19, wherein the antibody is conjugated to a cytotoxic agent via a linker.

[0351] 21. The antibody according to embodiment 20, wherein the antibody is conjugated with monomethylaurestatin E (MMAE).

[0352] 22. The antibody according to embodiment 21, wherein the antibody is conjugated to MMAE via an enzyme-cleavable linker unit.

[0353] 23. The antibody according to embodiment 22, wherein the enzyme-cleavable adapter unit comprises a Val-Cit adapter.

[0354] 24. The antibody according to any one of embodiments 20-23, wherein the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure:

[0355] Where Ab represents the antibody and p ranges from 2 to 10.

[0356] 25. The antibody according to implementation scheme 24, wherein p is 4.

[0357] 26. The antibody according to implementation scheme 24, wherein p is 8.

[0358] 27. The antibody according to embodiment 20, wherein the antibody is conjugated with camptothecin.

[0359] 28. The antibody according to embodiment 27, wherein the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit.

[0360] 29. The antibody according to embodiment 28, wherein the enzyme-cleavable adapter unit comprises a Val-Lys-Gly adapter.

[0361] 30. The antibody according to any one of embodiments 27-29, wherein the antibody is conjugated with camptothecin via a linker to form an antibody-drug conjugate having the following structure:

[0362] Where Ab represents the antibody and p ranges from 2 to 10.

[0363] 31. The antibody according to implementation scheme 30, wherein p is 4.

[0364] 32. The antibody according to embodiment 30, wherein p is 8.

[0365] 33. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of embodiments 1-32 and a pharmaceutically acceptable excipient.

[0366] 34. A method of treating a subject with cancer, the method comprising administering to the subject an antibody according to any one of embodiments 1-33.

[0367] 35. The method according to embodiment 34, wherein the subject is a human subject.

[0368] 36. The method according to embodiment 34 or embodiment 35, wherein 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0369] 37. A nucleic acid that encodes an antibody according to any one of embodiments 1-33.

[0370] 38. A vector comprising the nucleic acid according to embodiment 37.

[0371] 39. A host cell comprising the nucleic acid according to embodiment 38.

[0372] 40. The host cell according to embodiment 39, wherein the host cell is a Chinese hamster ovary (CHO) cell.

[0373] 41. A method for generating an antibody or an antigen-binding fragment thereof that specifically binds to a human PD-L1 protein, the method comprising culturing a host cell according to embodiment 39 or 40 under conditions suitable for generating the antibody.

[0374] 42. A method for generating an antibody-drug conjugate that specifically binds to human PD-L1 protein, the method comprising culturing host cells according to embodiment 39 or 40 under conditions suitable for generating the antibody; and conjugating the antibody to a cytotoxic agent.

[0375] 43. The method according to embodiment 42, wherein the cytotoxic agent is MMAE or camptothecin.

[0376] 44. 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 NO: 3-5 and a light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs.

[0377] 45. The antibody according to embodiment 44, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 300 nM.

[0378] 46. ​​The antibody according to embodiment 44 or 45, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 15 nM.

[0379] 47. The antibody according to any one of embodiments 44-46, wherein the antibody further exhibits a total internalization higher than that of Ab1.

[0380] 48. The antibody according to embodiment 47, wherein the total internalization has an AUC increase between 9% and 155% relative to the AUC of Ab1.

[0381] 49. The antibody according to embodiment 48, wherein the total internalization is determined by a FabFluor internalization assay.

[0382] 50. The antibody according to any one of embodiments 44-49, wherein the antibody further exhibits a x50 greater than that of Ab1.

[0383] 51. The antibody according to embodiment 50, wherein the antibody is conjugated to monomethylaurestatin E (MMAE), and wherein the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

[0384] 52. The antibody according to embodiment 50, wherein the antibody is conjugated with camptothecin, and wherein the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

[0385] 53. The antibody according to any one of embodiments 44-52, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 13-15 and the light chain CDR sequence of SEQ ID NO: 16-18.

[0386] 54. The antibody according to any one of embodiments 44-53, wherein 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.

[0387] 55. The antibody according to any one of embodiments 44-54, wherein 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.

[0388] 56. The antibody according to any one of embodiments 44-55, wherein 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.

[0389] 57. The antibody according to any one of embodiments 44-56, wherein 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.

[0390] 58. The antibody according to any one of embodiments 44-57, wherein the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0391] 59. The antibody according to any one of embodiments 44-58, wherein the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a biantibody, a linear antibody, or a single-chain antibody fragment.

[0392] 60. The antibody according to any one of embodiments 44-59, wherein the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0393] 61. The antibody according to any one of embodiments 44-60, wherein the heavy chain constant region is of the IgG1 isotype.

[0394] 62. The antibody according to any one of embodiments 44-61, wherein the antibody is a humanized or chimeric antibody.

[0395] 63. The antibody according to any one of embodiments 44-62, wherein the antibody is conjugated to a cytotoxic agent via a linker.

[0396] 64. The antibody according to embodiment 63, wherein the antibody is conjugated to monomethylaurestatin E (MMAE).

[0397] 65. The antibody according to embodiment 64, wherein the antibody is conjugated to MMAE via an enzyme-cleavable linker unit.

[0398] 66. The antibody according to embodiment 65, wherein the enzyme-cleavable adapter unit comprises a Val-Cit adapter.

[0399] 67. The antibody according to any one of embodiments 63-66, wherein the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure:

[0400] Where Ab represents the antibody and p ranges from 2 to 10.

[0401] 68. The antibody according to implementation scheme 67, wherein p is 4.

[0402] 69. The antibody according to implementation scheme 67, wherein p is 8.

[0403] 70. The antibody according to embodiment 63, wherein the antibody is conjugated with camptothecin.

[0404] 71. The antibody according to embodiment 70, wherein the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit.

[0405] 72. The antibody according to embodiment 71, wherein the enzyme-cleavable adapter unit comprises a Val-Lys-Gly adapter.

[0406] 73. The antibody according to any one of embodiments 70-72, wherein the antibody is conjugated with camptothecin via a linker to form an antibody-drug conjugate having the following structure:

[0407] Where Ab represents the antibody and p ranges from 2 to 10.

[0408] 74. The antibody according to implementation scheme 73, wherein p is 4.

[0409] 75. The antibody according to implementation scheme 73, wherein p is 8.

[0410] 76. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of embodiments 44-75 and a pharmaceutically acceptable excipient.

[0411] 77. A method of treating a subject with cancer, the method comprising administering to the subject an antibody according to any one of embodiments 44-76.

[0412] 78. The method according to embodiment 77, wherein the subject is a human subject.

[0413] 79. The method according to embodiment 77 or embodiment 78, wherein 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0414] 80. A nucleic acid that encodes an antibody according to any one of embodiments 44-76.

[0415] 81. A vector comprising the nucleic acid according to embodiment 80.

[0416] 82. A host cell comprising the nucleic acid according to embodiment 81.

[0417] 83. The host cell according to embodiment 82, wherein the host cell is a Chinese hamster ovary (CHO) cell.

[0418] 84. A method for generating an antibody or an antigen-binding fragment thereof that specifically binds to a human PD-L1 protein, the method comprising culturing a host cell according to embodiment 82 or 83 under conditions suitable for generating the antibody.

[0419] 85. A method for generating an antibody-drug conjugate that specifically binds to human PD-L1 protein, the method comprising culturing host cells according to embodiment 82 or 83 under conditions suitable for generating the antibody; and conjugating the antibody to a cytotoxic agent.

[0420] 86. The method according to embodiment 85, wherein the cytotoxic agent is MMAE or camptothecin.

[0421] 87. An antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 13-15 and the light chain CDR sequence of SEQ ID NO: 16-18.

[0422] 88. The antibody according to embodiment 87, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 300 nM.

[0423] 89. The antibody according to embodiment 87 or 88, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 15 nM.

[0424] 90. The antibody according to any one of embodiments 87-89, wherein the antibody further exhibits a total internalization higher than that of Ab1.

[0425] 91. The antibody according to embodiment 90, wherein the total internalization has an AUC increase between 9% and 155% relative to the AUC of Ab1.

[0426] 92. The antibody according to embodiment 91, wherein the total internalization is determined by FabFluor internalization assay.

[0427] 93. The antibody according to any one of embodiments 87-92, wherein the antibody further exhibits a x50 greater than that of Ab1.

[0428] 94. The antibody according to embodiment 93, wherein the antibody is conjugated to monomethylaurestatin E (MMAE), and wherein the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

[0429] 95. The antibody according to embodiment 93, wherein the antibody is conjugated with camptothecin, and wherein the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

[0430] 96. The antibody according to any one of embodiments 87-95, wherein 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.

[0431] 97. The antibody according to any one of embodiments 87-96, wherein 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.

[0432] 98. The antibody according to any one of embodiments 87-97, wherein 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.

[0433] 99. The antibody according to any one of embodiments 87-98, wherein 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.

[0434] 100. The antibody according to any one of embodiments 87-99, wherein the antibody comprises the light chain of SEQ ID NO: 9 and the heavy chain of SEQ ID NO: 10.

[0435] 101. The antibody according to any one of embodiments 87-100, wherein the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a biantibody, a linear antibody, or a single-chain antibody fragment.

[0436] 102. The antibody according to any one of embodiments 87-101, wherein the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0437] 103. The antibody according to any one of embodiments 87-102, wherein the heavy chain constant region is of the IgG1 isotype.

[0438] 104. The antibody according to any one of embodiments 87-103, wherein the antibody is a humanized or chimeric antibody.

[0439] 105. The antibody according to any one of embodiments 87-104, wherein the antibody is conjugated to a cytotoxic agent via a linker.

[0440] 106. The antibody according to embodiment 105, wherein the antibody is conjugated with monomethylaurestatin E (MMAE).

[0441] 107. The antibody according to embodiment 106, wherein the antibody is conjugated to MMAE via an enzyme-cleavable linker unit.

[0442] 108. The antibody according to embodiment 107, wherein the enzyme-cleavable adapter unit comprises a Val-Cit adapter.

[0443] 109. The antibody according to any one of embodiments 105-108, wherein the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure:

[0444] Where Ab represents the antibody and p ranges from 2 to 10.

[0445] 110. The antibody according to embodiment 109, wherein p is 4.

[0446] 111. The antibody according to implementation scheme 109, wherein p is 8.

[0447] 112. The antibody according to embodiment 105, wherein the antibody is conjugated with camptothecin.

[0448] 113. The antibody according to embodiment 112, wherein the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit.

[0449] 114. The antibody according to embodiment 113, wherein the enzyme-cleavable adapter unit comprises a Val-Lys-Gly adapter.

[0450] 115. The antibody according to any one of embodiments 112-114, wherein the antibody is conjugated with camptothecin via a linker to form an antibody-drug conjugate having the following structure:

[0451] Where Ab represents the antibody and p ranges from 2 to 10.

[0452] 116. The antibody according to embodiment 115, wherein p is 4.

[0453] 117. The antibody according to embodiment 115, wherein p is 8.

[0454] 118. A pharmaceutical composition comprising a therapeutically effective amount of an antibody according to any one of embodiments 87-117 and a pharmaceutically acceptable excipient.

[0455] 119. A method of treating a subject with cancer, the method comprising administering to the subject an antibody according to any one of embodiments 87-118.

[0456] 120. The method according to embodiment 119, wherein the subject is a human subject.

[0457] 121. The method according to embodiment 119 or 120, wherein 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 carcinoma, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0458] 122. A nucleic acid that encodes an antibody according to any one of embodiments 87-118.

[0459] 123. A vector comprising the nucleic acid according to embodiment 122.

[0460] 124. A host cell comprising the nucleic acid according to embodiment 123.

[0461] 125. The host cell according to embodiment 124, wherein the host cell is a Chinese hamster ovary (CHO) cell.

[0462] 126. A method for generating an antibody or an antigen-binding fragment thereof that specifically binds to a human PD-L1 protein, the method comprising culturing a host cell according to embodiment 124 or 125 under conditions suitable for generating the antibody.

[0463] 127. A method for generating an antibody-drug conjugate that specifically binds to a human PD-L1 protein, the method comprising culturing a host cell according to embodiment 124 or 125 under conditions suitable for generating the antibody; and conjugating the antibody to a cytotoxic agent.

[0464] 128. The method according to embodiment 127, wherein the cytotoxic agent is MMAE or camptothecin.

[0465] 129. An antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody is conjugated with camptothecin to form an antibody-drug conjugate, wherein the antibody-drug conjugate has the following structure:

[0466] in Ab is an anti-PD-L1 antibody; y is 1, 2, 3, or 4, or 1 or 4; and z is an integer from 2 to 12, or 2, 4, 8 or 12; And p is 1-16.

[0467] 130. The antibody according to embodiment 129, wherein the antibody-drug conjugate has the following structure:

[0468] 131. The antibody according to embodiment 129 or 130, wherein p ranges from 2 to 10.

[0469] 132. An antibody or an antigen-binding fragment thereof that specifically binds to human programmed death-ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity greater than Ab1 for human PD-L1 protein.

[0470] 133. The antibody according to embodiment 132, wherein the antibody exhibits a binding affinity greater than 2.7 nM.

[0471] 134. An antibody or an antigen-binding fragment thereof that specifically binds to human programmed death-ligand 1 (PD-L1) protein, wherein the antibody exhibits a k-value smaller than Ab1 for binding to human PD-L1 protein. assoc k assoc .

[0472] 135. The antibody according to embodiment 134, wherein the antibody exhibits a size of less than 5 x 10⁻⁶. 5 M -1 s -1 The k-like properties of human PD-L1 protein assoc .

[0473] 136. An antibody or an antigen-binding fragment thereof that specifically binds to human programmed death-ligand 1 (PD-L1) protein, wherein the antibody exhibits a k-value greater than Ab1 for binding to human PD-L1 protein. dissoc k dissoc .

[0474] 137. The antibody according to embodiment 136, wherein the antibody exhibits a size greater than 2 x 10⁻⁶. 3 s -1 The k-like properties of human PD-L1 protein dissoc .

[0475] 138. 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 a heavy chain CDR sequence of SEQ ID NO: 3-5 and a light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs, and wherein the antibody exhibits binding affinity to human PD-L1 protein between 5 nM and 15 nM, and wherein the antibody is conjugated to MMAE.

[0476] 139. 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 a heavy chain CDR sequence of SEQ ID NO: 3-5 and a light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs, and wherein the antibody exhibits a binding affinity to human PD-L1 protein between 5 nM and 15 nM, and wherein the antibody is conjugated with camptothecin.

[0477] Informal sequence list 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 SEQ ID NO:8 - Ab1 light chain CDR3 - protein QQRSNWPT SEQ ID NO:9 - SG-559-01 LALA hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:10 - SG-559-01 κ light chain - Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:11 - SG-559-01 heavy chain variable region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS SEQ ID NO:12 - SG-559-01 light chain variable region - Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK SEQ ID NO:13 - SG-559-01 Heavy chain CDR1 - Protein TAAIS SEQ ID NO:14 - SG-559-01 Heavy chain CDR2 - Protein GIIPIFGKAHYAQKFQG SEQ ID NO:15 - SG-559-01 Heavy chain CDR3 - Protein KFHFVSGSPFGMDV SEQ ID NO:16 - SG-559-01 Light chain CDR1 - Protein RASQSVSSYLA SEQ ID NO:17 - SG-559-01 Light chain CDR2 - Protein DASNRAT SEQ ID NO:18 - SG-559-01 Light chain CDR3 - Protein QQRSNWPT SEQ ID NO:19 - SG-559-02 LALA hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:20 - SG-559-02 κ light chain - Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:21 - SG-559-02 heavy chain variable region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS SEQ ID NO:22 - SG-559-02 light chain variable region - Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK SEQ ID NO:23 - SG-559-02 Heavy chain CDR1 - Protein TYAIS SEQ ID NO:24 - SG-559-02 Heavy chain CDR2 - Protein GIIPIFGKAHYAQKFQG SEQ ID NO:25 - SG-559-02 Heavy chain CDR3 - Protein KFHFVSGSPFGMDV SEQ ID NO:26 - SG-559-02 Light chain CDR1 - Protein RASQSVSSALA SEQ ID NO:27 - SG-559-02 Light chain CDR2 - Protein DASNRAT SEQ ID NO:28 - SG-559-02 Light chain CDR3 - Protein QQRSNWPT SEQ ID NO:29 - SG-559-03 LALA hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:30 - SG-559-03 κ 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 κ 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 SEQ ID NO:43 - SG-559-04 Heavy chain CDR1 - Protein TYAIS SEQ ID NO:44 - SG-559-04 Heavy chain CDR2 - Protein GIIPIFGKAHYAQKFQG SEQ ID NO:45 - SG-559-04 Heavy chain CDR3 - Protein KFHFVSGSGFGMDV SEQ ID NO:46 - SG-559-04 Light chain CDR1 - Protein RASQSVSSYLA SEQ ID NO:47 - SG-559-04 Light chain CDR2 - Protein DASNRAT SEQ ID NO:48 - SG-559-04 Light chain CDR3 - Protein QQRSNWPT SEQ ID NO:49 - SG-559-05 Heavy chain CDR2 - Protein GIIPIAGKAHYAQKFQG SEQ ID NO:50 - SG-559-06 Heavy chain CDR2 - Protein GIIPIFGAAHYAQKFQG SEQ ID NO:51 - SG-559-07 Heavy chain CDR2 - Protein GIIPIFGRAHYAQKFQG SEQ ID NO:52 - SG-559-08 Heavy chain CDR2 - Protein GIIPIFGKAAYAQKFQG SEQ ID NO:53 - SG-559-09 Heavy chain CDR2 - Protein GIIPIFGKAFYAQKFQG SEQ ID NO:54 - SG-559-10 Heavy chain CDR3 - Protein KFHFVSGAPFGMDV SEQ ID NO:55 - SG-559-11 Heavy chain CDR3 - Protein KFHFVSGSPAGMDV SEQ ID NO:56 - SG-559-12 Light chain CDR3 - Protein QQASNWPT SEQ ID NO:57 - SG-559-13 Light chain CDR3 - Protein QQKSNWPT SEQ ID NO:58 - SG-559-14 Light chain CDR3 - Protein QQRSAWPT SEQ ID NO:59 - SG-559-15 Light chain CDR3 - Protein QQRSQWPT SEQ ID NO:60 - SG-559-16 Light chain CDR3 - Protein QQRSNAPT SEQ ID NO:61 - SG-559-17 Light chain CDR3 - Protein QQRSNFPT SEQ ID NO:62 - SG-559-01 hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:63 - SG-559-02 hIgG1 Heavy Chain - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:64 - SG-559-03 hIgG1 Heavy Chain - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:65 - SG-559-04 hIgG1 Heavy Chain - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:66 - SG-559-01 Heavy Chain Variable Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcaccgccgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO:67 - SG-559-01 Light Chain Variable Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO:68 - SG-559-02 Heavy Chain Variable Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO:69 - SG-559-02 Light Chain Variable Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcgccttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO:70 - Nucleic acid of variable region of heavy chain of SG-559-03 caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO:71 - Nucleic acid of variable region of light chain of SG-559-03 gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaacctgccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO:72 - Nucleic acid of the variable region of the heavy chain of SG-559-04 caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagcggcttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO:73 - Nucleic acid of the variable region of the light chain of SG-559-04 gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO:74 - SG-559-01 LALA hIgG1 Heavy Chain - Nucleic Acid SEQ ID NO:75 - SG-559-01 κ light chain - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO:76 - SG-559-01 hIgG1 heavy chain - nucleic acid SEQ ID NO:77 - SG-559-02 LALA hIgG1 heavy chain - nucleic acid SEQ ID NO:78 - SG-559-02 κ light chain - Nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcgccttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO:79 - SG-559-02 hIgG1 heavy chain - Nucleic acid SEQ ID NO:80 - SG-559-03 LALA hIgG1 heavy chain - nucleic acid SEQ ID NO:81 - SG-559-03 κ light chain - Nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaacctgccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO:82 - SG-559-03 hIgG1 heavy chain - Nucleic acid SEQ ID NO:83 - SG-559-04 LALA hIgG1 heavy chain - nucleic acid SEQ ID NO:84 - SG-559-04 κ light chain - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO:85 - SG-559-04 hIgG1 heavy chain - nucleic acid SEQ ID NO:86 - Ab1 hIgG1 Heavy Chain - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:87 - Ab1 κ Light Chain - Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to human programmed death-ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity to human PD-L1 protein between 3 and 300 nM.

2. The antibody of claim 1, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 15 nM.

3. The antibody according to claim 1 or claim 2, wherein the antibody further exhibits a higher total internalization than Ab1.

4. The antibody according to claim 3, wherein the total internalization has an AUC increase between 9% and 155% relative to the AUC of Ab1.

5. The antibody according to claim 3, wherein the total internalization is determined by FabFluor internalization assay.

6. The antibody according to any one of claims 1-5, wherein the antibody further exhibits a x50 lower than that of Ab1.

7. The antibody of claim 6, wherein the antibody is conjugated with monomethylaurestatin E (MMAE), and wherein the x50 in the MDA-MB-231 cell line is between 3 ng / mL and 20 ng / mL.

8. The antibody according to claim 6, wherein the antibody is conjugated with camptothecin, and wherein the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

9. The antibody according to any one of claims 1-8, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 13-15 and the light chain CDR sequence of SEQ ID NO: 16-18.

10. The antibody according to any one of claims 1-8, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NO: 3-5 and the light chain CDR sequence of SEQ ID NO: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs.

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