Anti-PD-L1 antibodies and antibody-drug conjugates

Abstract

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

Description

Technical Field

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

Background Art

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

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

[0004] All references cited in this specification, including patent applications, patent publications, and scientific literature, are hereby incorporated by reference in their entirety as if each individual reference was specifically and individually indicated to be incorporated by reference. [Overview of the Initiative] [Means for solving the problem]

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

[0006] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to the human programmed death ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity to the human PD-L1 protein of 3 to 300 nM. In some embodiments, the antibody exhibits a binding affinity to the human PD-L1 protein of 3 to 15 nM.

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

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

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

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

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

[0012] In some embodiments, the antibody includes 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 includes 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 includes 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 includes the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 12.

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

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

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

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

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

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

[0019] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit includes a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker, resulting in the following structure: [ka] An antibody-drug conjugate is formed having the formula, where Ab represents the antibody and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0020] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit includes a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker, and the structure is: [ka] An antibody-drug conjugate is formed having the formula, where Ab represents the antibody and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0021] Also provided herein are antibodies or antigen-binding fragments that specifically bind to the human PD-L1 protein, wherein the antibody comprises a heavy chain CDR sequence of SEQ ID NOs. 3-5 and a light chain CDR sequence of SEQ ID NOs. 6-8, and the antibody contains one or more amino acid substitutions in one or more of the CDRs.

[0022] In some embodiments, the antibody exhibits a binding affinity to human PD-L1 protein at a concentration of 3 to 300 nM. In some embodiments, the antibody exhibits a binding affinity to human PD-L1 protein at a concentration of 3 to 15 nM.

[0023] In some embodiments, the antibody exhibits even higher total internalization than Ab1. In some embodiments, total internalization is a 9% to 155% increase in AUC compared to Ab1. In some embodiments, total internalization is determined by a FabFluor internalization assay.

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

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

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

[0027] In some embodiments, the antibody includes heavy chain CDR sequences of SEQ ID NOs. 13-15 and light chain CDR sequences of SEQ ID NOs. 16-18. In some embodiments, the antibody includes 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 includes 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 includes 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 includes the heavy chain variable region sequence of SEQ ID NO. 11 and the light chain variable region sequence of SEQ ID NO. 12.

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

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

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

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

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

[0033] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit includes a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker, resulting in the following structure: [ka] It forms an antibody-drug conjugate having the following characteristics: Ab represents the antibody and p is 2-10 It is a range. In some embodiments, p is 4. In some embodiments, p is 8.

[0034] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit includes a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker, and the structure is: [ka] An antibody-drug conjugate is formed having the formula, where Ab represents the antibody and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0035] 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 sequences of SEQ ID NOs. 13-15 and the light chain CDR sequences of SEQ ID NOs. 16-18, is provided herein.

[0036] In some embodiments, the antibody exhibits a binding affinity to human PD-L1 protein at a concentration of 3 to 300 nM. In some embodiments, the antibody exhibits a binding affinity to human PD-L1 protein at a concentration of 3 to 15 nM.

[0037] In some embodiments, the antibody exhibits even higher total internalization than Ab1. In some embodiments, total internalization is a 9% to 155% increase in AUC compared to Ab1. In some embodiments, total internalization is determined by a FabFluor internalization assay.

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

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

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

[0041] 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. The antibody includes a heavy chain variable region sequence. In some embodiments, the antibody includes 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 includes 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 includes the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 12.

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

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

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

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

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

[0047] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit includes a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker, resulting in the following structure: [ka] An antibody-drug conjugate is formed having the formula, where Ab represents the antibody and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0048] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzymatically cleavable linker unit. In some embodiments, the enzymatically cleavable linker unit includes a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker, and the structure is: [ka] An antibody-drug conjugate is formed having the formula, where Ab represents the antibody and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0049] An antibody or its antigen-binding fragment that specifically binds to the human PD-L1 protein, wherein the antibody is conjugated with camptothecin to form an antibody-drug conjugate, and the antibody-drug conjugate has the following structure: [ka] Also provided herein are antibodies or antigen-binding fragments having the formula, wherein Ab is an anti-PD-L1 antibody, y is 1, 2, 3 or 4, or 1 or 4, z is an integer from 2 to 12, or 2, 4, 8 or 12, and p is 1 to 16.

[0050] In some embodiments, the antibody-drug conjugate has the following structure: [ka] It holds.

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

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

[0053] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to human programmed death ligand 1 (PD-L1) protein, wherein the antibody exhibits a k dissoc , -1 , ,

[0055] , for human PD-L1 protein that is less than that of Ab1. In some embodiments, the antibody has a k 5 M -1 s -1 for human PD-L1 protein that is less than 5×10 assoc and is shown.

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

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

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

[0057] Pharmaceutical compositions comprising therapeutically effective amounts of the antibodies described herein and pharmaceutically acceptable excipients are also provided herein.

[0058] Methods for treating a target cancer, comprising administering one of the antibodies described herein to a target, are also provided herein. In some embodiments, the target 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.

[0059] Nucleic acids encoding any of the antibodies described herein are also provided herein.

[0060] Vectors containing any of the nucleic acids described herein are also provided herein.

[0061] Any of the host cells described herein, comprising any of the nucleic acids described herein, are also provided herein. In some embodiments, the host cells are Chinese hamster ovary (CHO) cells.

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

[0063] Methods for producing antibody-drug conjugates that specifically bind to the human PD-L1 protein are also provided herein, comprising culturing one of the host cells described herein under conditions suitable for antibody production, and conjugating the antibody with a cytotoxic agent. In some embodiments, the cytotoxic agent is MMAE or camptothecin.

[0064] Cancer (for example, PD-L1 + The use of any or any antibody-drug conjugate of the anti-PD-L1 antibodies described herein for use in the manufacture of pharmaceuticals for the treatment of cancers associated with the expression of PD-L1 is also provided herein.

[0065] Cancer (for example, PD-L1 + Anti-PD-L1 antibodies or antibody-drug conjugates described herein for use in the treatment of cancers related to expression are also provided herein.

[0066] Anti-PD-L1 antibodies or antibody-drug conjugates described herein for pharmaceutical use are also provided herein.

[0067] PD-L1 + In subjects where cell death is required, PD-L1 + A method for killing cells is also provided herein, which includes administering a therapeutically effective amount of any of the anti-PD-L1 antibodies described herein or any of the antibody-drug conjugates described herein to the target.

[0068] PD-L1 + In subjects where cell death is required, PD-L1 +The use of any of the anti-PD-L1 antibodies or any of the antibody-drug conjugates described herein for use in the manufacture of pharmaceuticals for killing cells is also provided herein.

[0069] The treatment involves administering a therapeutically effective dose of any of the anti-PD-L1 antibodies or antibody-drug conjugates described herein to a target solid tumor (e.g., PD-L1). + Methods for reducing the volume of solid tumors are also provided herein.

[0070] Target solid tumors (e.g., PD-L1 + Drugs for reducing the volume of solid tumors The use of any of the anti-PD-L1 antibodies or any of the antibody-drug conjugates described herein for use in manufacturing is also provided herein. [Brief explanation of the drawing]

[0071] [Figure 1] Figure 1 shows exemplary amino acid residues of Ab1 selected for the mutation.

[0072] [Figure 2-1] Figures 2A-2F show cytotoxicity of SG-559-xx ADCs in several cell lines. [Figure 2-2] Figures 2A-2F show cytotoxicity of SG-559-xx ADCs in several cell lines. [Figure 2-3] Figures 2A-2F show cytotoxicity of SG-559-xx ADCs in several cell lines.

[0073] [Figure 3] Figures 3A-3B show the internalization of SG-559-01 and SG-559-03 compared to the control antibody.

[0074] [Figure 4]Figures 4A-4B show the antitumor activity of SG-559-xx ADCs in the MDA-MB-231 mouse model.

[0075] [Figure 5] Figures 5A and 5B show the antitumor activity of SG-559-xx ADC in the BxPC3 mouse model.

[0076] [Figure 6-1] Figures 6A and 6B show the antitumor activity of SG-559-01 LALA ADC in the Karpas 299 mouse model. [Figure 6-2] Figures 6A and 6B show the antitumor activity of SG-559-01 LALA ADC in the Karpas 299 mouse model.

[0077] [Figure 7] Figure 7 shows the antitumor activity of SG-559-01 LALA ADC in the Calu-1 mouse model.

[0078] [Figure 8-1] Figures 8A and 8B show the antitumor activity of SG-559-01 LALA ADC in the EBC-1 mouse model. [Figure 8-2] Figures 8A and 8B show the antitumor activity of SG-559-01 LALA ADC in the EBC-1 mouse model.

[0079] [Figure 9] Figure 9 shows the in vitro PD-1 / PD-L1 blocking activity of the SG-559-01 LALA antibody and ADC.

[0080] [Figure 10-1] Figures 10A to 10D show the immunotoxicity of SG-559-01 and SG-559-01 LALA ADC in a human APC model. [Figure 10-2]Figures 10A to 10D show the immunotoxicity of SG-559-01 and SG-559-01 LALA ADC in a human APC model. [Figure 10-3] Figures 10A to 10D show the immunotoxicity of SG-559-01 and SG-559-01 LALA ADC in a human APC model. [Figure 10-4] Figures 10A to 10D show the immunotoxicity of SG-559-01 and SG-559-01 LALA ADC in a human APC model.

[0081] [Figure 11-1] Figures 11A to 11D show the immunotoxicity of SG-559-xx ADCs in a human APC model. [Figure 11-2] Figures 11A to 11D show the immunotoxicity of SG-559-xx ADCs in a human APC model. [Figure 11-3] Figures 11A to 11D show the immunotoxicity of SG-559-xx ADCs in a human APC model. [Figure 11-4] Figures 11A to 11D show the immunotoxicity of SG-559-xx ADCs in a human APC model.

[0082] [Figure 12-1] Figures 12A to 12D show the immune response to LPS stimulation in SG-559-01 ADC-treated human APCs in vitro. [Figure 12-2] Figures 12A to 12D show the immune response to LPS stimulation in SG-559-01 ADC-treated human APCs in vitro.

[0083] [Figure 13-1] Figures 13A to 13C show intratumoral immune cell infiltration in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Figure 13-2]Figures 13A to 13C show intratumoral immune cell infiltration in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC.

[0084] [Figure 14-1] Figures 14A to 14F show the intratumoral inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Figure 14-2] Figures 14A to 14F show the intratumoral inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Figure 14-3] Figures 14A to 14F show the intratumoral inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Figure 14-4] Figures 14A to 14F show the intratumoral inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Figure 14-5] Figures 14A to 14F show the intratumoral inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Figure 14-6] Figures 14A to 14F show the intratumoral inflammatory cytokine response in mice with Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. [Modes for carrying out the invention]

[0085] I. Definition To make this disclosure more easily understandable, certain terms are defined first. Where used in this application, unless otherwise expressly provided herein, each of the following terms shall have the meanings set forth below. Further definitions are provided throughout this application.

[0086] As used herein, the term "and / or" should be interpreted as the specific disclosure of each of two designated features or components, with or without the other. Accordingly, as used herein in terms such as "A and / or B," the term "and / or" is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Similarly, as used in terms such as "A, B, and / or C," the term "and / or" is intended to include the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and It is intended to include B; B and C; A (alone); B (alone); and C (alone).

[0087] The aspects and embodiments of the present invention described herein are understood to include aspects and embodiments "comprising," "consisting," and "consisting essentially of."

[0088] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to whom this disclosure relates. For example, see the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology,3rd ed.,1999,Academic The Oxford University Press and the Oxford Dictionary of Biochemistry and Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of many of the terms used in this disclosure for those skilled in the art.

[0089] Units, prefixes, and symbols are from the Systeme International de The data is presented in a format approved by Unites (SI). Numerical ranges include the number defining the range. The headings provided herein are not limitations on the various aspects of this disclosure that can be had by referring to this specification as a whole. Thus, the terms defined below are more fully defined by referring to this specification as a whole.

[0090] The terms “PD-L1,” “CD274,” “B7-H1,” and “Programmed Cell Death Ligand 1” are used interchangeably herein and, unless otherwise specified, include any variants, isoforms, and species homologs of human PD-L1 that are generally expressed by cells or on cells transfected with the PD-L1 gene.

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

[0092] The term "variable region" or "variable domain" refers to a domain in the heavy or light chain of an antibody that is involved in the binding of the antibody to an antigen. (V) H and V LThe antibody variable region (HVR) can be further subdivided into hypervariable regions, also called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs) (or hypervariable regions that can be hypervariable in the form of sequencely and / or structurally defined loops). The terms “complementarity-determining regions” and “CDR,” which are synonymous with “hypervariable region” or “HVR,” are known in the art to refer to discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). The terms “framework regions” and “FR” are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). H and V L Within, 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)).

[0093] In the context of this invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or any derivative thereof, having the ability to specifically bind to an antigen with a half-life of a significant period such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour (h), at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), about 24 hours or more, about 48 hours or more, about 3, 4, 5, 6, 7 days or more, or any other relevant functionally defined period (e.g., enough time to induce, promote, enhance and / or modulate the physiological response associated with antibody binding to the antigen and / or enough time for the antibody to mobilize effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain binding domains that interact with the antigen. The constant region (Ab) of an antibody can mediate the binding of immunoglobulins 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, the first component in the classical pathway of complement activation. Antibodies can also be bispecific antibodies, diabodies, multispecific antibodies, or similar molecules.

[0094] As used herein, the term “monoclonal antibody” refers to a recombinant antibody molecule preparation having a single primary amino acid sequence. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a specific epitope. Therefore, the term “human monoclonal antibody” refers to an antibody exhibiting a single binding specificity having variable and constant regions derived from a human germline immunoglobulin sequence. Human monoclonal antibodies can be produced by hybridomas containing B cells obtained from transgenic or transchromosomal non-human animals, such as transgenic mice having a genome containing human heavy chain and light chain transgenes fused to immortalized cells.

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

[0096] A “human antibody” (HuMAb) refers to an antibody in which both the FR and CDR have variable regions derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region also derives from a human germline immunoglobulin sequence. The human antibodies of this disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is grafted onto a human framework sequence. The terms “human antibody” and “fully human antibody” are used synonymously.

[0097] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody comprising a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology to the human variable domain. This can be achieved by grafting six non-human antibody complementarity-determining regions (CDRs) that together form an antigen-binding site onto a homologous human acceptor framework region (FR) (see International Publication 92 / 22653 and European Patent No. 0629240). To completely reconstruct the binding affinity and binding specificity of the parent antibody, substitution (reversion mutation) of framework residues from the parent antibody (i.e., the non-human antibody) to the human framework region may be required. Structural homology modeling can help identify amino acid residues within the framework region that are important for the antibody’s binding properties. Thus, a humanized antibody may comprise a non-human CDR sequence, a human framework region mainly containing one or more amino acid reverse mutations to the non-human amino acid sequence as needed, and a fully human constant domain. If necessary, further amino acid modifications, not necessarily revertant mutations, can be applied to obtain humanized antibodies with desirable characteristics such as affinity and biochemical properties.

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

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

[0100] The "antigen-binding site" or antigen-binding fragment of an antibody is specific to the antigen to which the entire antibody binds. This refers to one or more fragments of an antibody that retain the ability to bind to a target. Examples of antibody fragments (e.g., antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment whose name reflects its ability to readily crystallize. Pepsin treatment yields an F(ab')2 fragment having two antigen-binding sites and still being able to crosslink antigens.

[0101] "Percent (%) sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences to achieve maximum percent sequence identity, introducing gaps as necessary, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in various ways within the scope of the skills of the art, 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 the sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, the % sequence identity of a given amino acid sequence A to a given amino acid sequence B, or to a given amino acid sequence B (or, to put it another way, a given amino acid sequence A that has or contains a specific % sequence identity with a given amino acid sequence B, or to a given amino acid sequence B) is calculated as follows: 100 times the ratio X / Y In the formula, X is the number of amino acid residues scored as identical by their sequences in the alignment of programs A and B, and Y is the total number of amino acid residues in B. It will 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 of A to B is not equal to the % sequence identity of B to A.

[0102] As used herein, the terms “binding,” “conjugate,” or “specifically bind” in relation to the binding of an antibody to a given antigen typically mean approximately 10 in an Octet HTX instrument using an antibody as the ligand and an antigen as the analyte, as determined, for example, by biolayer interferometry (BLI) technology. -6 M or less, for example, 10 -7 M or less, for example, about 10 -8 M or less, for example, about 10 -9 M or less, about 10 -10 M or less or about 10 -11 M or even less than K D This is binding at an affinity corresponding to the antibody, and the antibody binds to, for example, a nonspecific antigen other than a given antigen or a closely related antigen (e.g., BSA, casein). D K is at most 1 / 10, for example at most 1 / 100, for example at most 1 / 1,000, for example at most 1 / 10,000, for example at most 1 / 100,000. D It binds to a predetermined antigen with the corresponding affinity. D Lower amounts of antibody K D Because it depends on the antibody K D If the K of antigen binding is very low, D K is the binding of nonspecific antigens D Amounts lower than this can be as low as 1 / 10,000 (i.e., antibodies are highly specific).

[0103] The term "K" used in this specification D The term (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity and K as used herein DThe opposite relationship exists, meaning that higher affinity corresponds to lower K D This is intended to refer to a lower affinity with a higher K D This is intended to refer to [something].

[0104] The term "ADC" refers to an antibody-drug conjugate, and in the context of this invention, This refers to an anti-PD-L1 antibody bound to the drug portion described in the application (e.g., MMAE or MMAF).

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

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

[0107] The abbreviation "MC" refers to maleimidocaproil in stretchers. [ka]

[0108] The abbreviation "MP" refers to maleimidopropionyl in stretchers. [ka]

[0109] As used herein, “PEG units” are organic moieties composed of repeating ethylene-oxy subunits (PEG or PEG subunits) and may be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEG is a heterogeneous mixture of size and molecular weight, while monodisperse PEG is typically purified from a heterogeneous mixture and therefore provides a single chain length and molecular weight. Preferred PEG units include discrete PEGs, which are compounds synthesized stepwise without a polymerization process. Discrete PEGs provide a single molecule with a defined specific chain length.

[0110] The PEG units provided herein comprise one or more polyethylene glycol chains, each consisting of one or more ethylene oxy subunits covalently bonded to one another. The polyethylene glycol chains can be linked to one another in linear, branched, or star-shaped configurations, for example. Typically, at least one of the polyethylene glycol chains before incorporation into a camptothecin conjugate is derivatized at one end with an electrophilic alkyl moiety for covalent bonding to the carbamate nitrogen of a methylene carbamate unit (i.e., representing an example of R). Typically, the terminal ethylene oxy subunits in each polyethylene glycol chain that do not participate in covalent bonding to the rest of the linker unit are modified with PEG cap-forming units, typically -CH3, CH2CH3, or CH2CH2CO2H, or an optionally substituted alkyl. A preferred PEG unit has a single polyethylene glycol chain in which 2 to 24 -CH2CH2O- subunits are covalently bonded in series, with one end terminated with a PEG cap-forming unit.

[0111] "Cancer" is a broad group of various diseases characterized by the uncontrolled proliferation of abnormal cells in the body. To refer to. "Cancer" or "cancer tissue" may include tumors. Uncontrolled cell division and proliferation result in the formation of malignant tumors, which can invade adjacent tissues and metastasize to distal parts of the body via the lymphatic system or bloodstream. After metastasis, a distal tumor can be said to "originate" what was the pre-metastatic tumor.

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

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

[0114] The term "complement-dependent cell injury" or CDC refers to a mechanism by which the Fc effector domain of a target-binding antibody activates a series of enzymatic reactions that ultimately lead to the formation of holes in the target cell membrane, thereby inducing cell death. Typically, an antigen-antibody complex, such as one on an antibody-coated target cell, binds to and activates complement component C1q, which in turn activates the complement cascade, resulting in target cell death. Complement activation can also lead to the deposition of complement components on the target cell surface, which promotes ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0115] "Cell proliferation inhibitory effect" refers to the inhibition of cell proliferation. "Cell proliferation inhibitors" refer to drugs that have a cell proliferation inhibitory effect on cells, thereby inhibiting the proliferation and / or expansion of a specific subset of cells. Cell proliferation inhibitors can be conjugated to antibodies or administered in combination with antibodies.

[0116] The “treatment” or “therapy” of the subject refers to any type of intervention or process performed on the subject, or administration of an activator to the subject, with the aim of reversing, alleviating, improving, inhibiting, slowing or preventing the onset, progression, occurrence, severity or recurrence of symptoms, complications, conditions or biochemical indicia associated with the disease. In some embodiments, the disease is cancer.

[0117] The term "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 human. The terms "subject," "patient," and "individual" are used interchangeably herein.

[0118] The “effective dose,” “therapeutic effective dose,” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of the drug, when used alone or in combination with another therapeutic agent, that protects a subject from the onset of the disease or promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of functional impairment or disability due to the distress of the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as by assaying the activity of the drug in human subjects during clinical trials, in predictive animal model systems of efficacy in humans, or in in vitro assays.

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

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

[0121] A therapeutically effective dose of a drug (e.g., an anti-PD-L1 antibody-drug conjugate) includes a “prophylactic effective dose,” which is any amount of the drug that, when administered alone or in combination with an anticancer agent, inhibits the development or recurrence of cancer in a subject at risk of developing cancer (e.g., a subject with a pre-malignant condition) or at risk of cancer recurrence. In some embodiments, the prophylactic effective dose completely prevents the development or recurrence of cancer. To “inhibit” the development or recurrence of cancer means either to reduce the likelihood of cancer development or recurrence, or to completely prevent cancer development or recurrence.

[0122] As used herein, “sub-therapeutic dose” means a dose of a therapeutic compound (e.g., an anti-PD-L1 antibody-drug conjugate) lower than the usual or typical dose of the therapeutic compound when administered alone for the treatment of a hyperproliferative disease (e.g., cancer).

[0123] The "immune-associated response pattern" refers to a clinical response pattern frequently observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or by modifying innate immune processes. This response pattern is characterized by a beneficial therapeutic effect followed by an initial increase in tumor burden or the appearance of new lesions, which is classified as disease progression in the evaluation of traditional chemotherapy agents and is synonymous with drug failure. Therefore, proper evaluation of immunotherapeutic agents may require long-term monitoring of their effects on the target disease.

[0124] For example, “anticancer drugs” promote the regression of cancer in a subject. In some embodiments, a therapeutically effective dose of the drug promotes cancer regression to the point of eliminating the cancer. “Promoting cancer regression” means that administration of an effective dose of the drug alone or in combination with an anticancer drug 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 disease-free periods, or prevention of functional impairment or disability due to the disease. Furthermore, the terms “effective” and “effectiveness” in relation to a treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or biological level resulting from the administration of the drug.

[0125] "Sustained response" refers to a sustained effect on the reduction of tumor growth after discontinuation of treatment. For example, tumor size may remain the same as or smaller than the size at the start of the administration phase. In some embodiments, the sustained response lasts for at least the same duration as the treatment period, or at least 1.5, 2.0, 2.5, or 3 times longer than the treatment period.

[0126] As used herein, “complete response” or “CR” means the disappearance of all target lesions. “Partial response” or “PR” means a reduction of at least 30% of the total longest diameter (SLD) of target lesions, with reference to baseline SLD. “Stable condition” or “SD” means neither a reduction in target lesions sufficient to qualify for PR nor an increase sufficient to qualify for PD, with reference to the smallest SLD since the start of treatment.

[0127] As used herein, “progression-free survival” or “PFS” refers to the length of time during and after treatment in which the treated disease (e.g., cancer) does not worsen. Progression-free survival may include the length of time in which the patient experiences a complete or partial response, as well as the length of time in which the patient experiences a stable condition.

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

[0129] As used herein, “overall survival” or “OS” refers to the percentage of individuals in a group that are likely to survive after a given period of time.

[0130] The phrase "pharmaceutically acceptable" indicates that a substance or composition must be chemically and / or toxicologically compatible with the other components of the formulation and / or the mammals treated with it.

[0131] The expression "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the present invention. Examples of salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acidic phosphates, isonicotinates, lactates, salicylates, acidic citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbicates, succinates, maleates, gentisinates, fumarates, glucons, glucurons, saccharates, formates, benzoates, glutamates, methanesulfonates "mesylate", ethanesulfons, benzenesulfons, p-toluenesulfons, pamoates (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoate)), alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt may include the inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic part that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. If multiple charged atoms are part of a pharmaceutically acceptable salt, it may have multiple counterions. Therefore, a pharmaceutically acceptable salt may have one or more charged atoms and / or one or more counterions.

[0132] "Administering" or "dosing" refers to the physical delivery of a therapeutic agent to a subject using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration of anti-PD-L1 antibody-drug conjugates include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes, for example, by injection or infusion (e.g., intravenous infusion). The term "parenteral administration," as used herein, means modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intrafocal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. The therapeutic agent may be administered via parenteral routes or orally. Other parenteral routes include topical, epidermal, or mucosal administration routes, e.g., intranasal, vaginal, rectal, tongue. Administration may be suppositories or topical administration. Administration may be, for example, one dose, multiple doses, and / or over a period of one or more times.

[0133] The terms “baseline” or “baseline value,” as used interchangeably herein, may refer to a measurement or characterization of symptoms before administration of treatment (e.g., an anti-PD-L1 antibody-drug conjugate as described herein) or at the start of treatment administration. The baseline value can be compared to a reference value to determine the reduction or improvement of symptoms of the PD-L1-related disease (e.g., cancer) as intended herein. The terms “reference” or “reference value,” as used interchangeably herein, may refer to a measurement or characterization of symptoms after administration of treatment (e.g., an anti-PD-L1 antibody-drug conjugate as described herein). The reference value may be measured once or more times during a dosing regimen or treatment cycle, or at the completion of a dosing regimen or treatment cycle. The “reference value” may be an absolute value, a relative value, a value with upper and / or lower limits, a range of values, an average value, a median, a mean value, or a value compared to the baseline value.

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

[0135] As used herein, the term “monotherapy” means that the anti-PD-L1 antibody-drug conjugate is the only anticancer agent administered to the subject during a treatment cycle. However, other therapeutic agents may be administered to the subject. For example, anti-inflammatory agents, or other drugs administered to a subject with cancer that treat cancer-related symptoms (e.g., inflammation, pain, weight loss, and general fatigue) but do not treat the underlying cancer itself, may be administered during the period of monotherapy.

[0136] As used herein, “adverse event” (AE) is any undesirable, generally unintended, or unwanted sign (including abnormal laboratory findings), symptom, or illness associated with the use of a medical procedure. A medical procedure may have one or more associated AEs, each AE may have the same or different levels of severity. “Modification of adverse events” refers to a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0137] As used herein, “Serious Adverse Event” or “SAE” refers to an adverse event that meets one of the following criteria: • Fatal or life-threatening (When used in the definition of a serious adverse event, “life-threatening” means an event in which there was a risk of death for the patient at the time of the event. More severely, it does not refer to an event that hypothetically could have caused death.) • Causes persistent or significant disability / incapacity • Constitutes a congenital anomaly / birth defect • An AE is defined as an event that is medically important, meaning it may require medical or surgical intervention to endanger the patient or prevent one of the outcomes listed above. Medical and scientific judgment must be exercised when determining whether an AE is "medically important." • Hospitalization of an inpatient or an existing inpatient, excluding: 1) routine management or monitoring of an underlying condition without worsening of the patient's condition; 2) selective or pre-planned treatment for an existing condition unrelated to the indication under study and which has not worsened since informed consent was signed; and 3) respite care when there is no social reason or worsening of the patient's overall condition. It requires an extension.

[0138] The use of alternative forms (e.g., "or") should be understood to mean either, both, or any combination thereof. Where used herein, the indefinite article "a" or "an" should be understood to refer to one or more enumerated or counted constituent elements.

[0139] The terms “about” or “comprising essentially of” refer to a value or composition that falls within an acceptable margin of error for a particular value or composition as determined by those skilled in the art, and this will depend in part on how the value or composition is measured or determined, i.e., on the limitations of the measuring system. For example, “about” or “comprising essentially of” may mean within or exceeding one standard deviation, according to convention in the art. Alternatively, “about” or “comprising essentially of” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, these terms may mean values ​​up to one order of magnitude or five times. Where a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable margin of error for that particular value or composition.

[0140] References to values ​​or parameters “about” in this specification include (and describe) embodiments relating to the value or parameter itself. For example, a statement referring to “about X” is a statement that encompasses “X”.

[0141] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values ​​within the listed range, and, where appropriate, a portion thereof (such as 1 / 10 and 1 / 100 of an integer), unless otherwise specified.

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

[0143] II.General The present invention provides antibodies and ADCs that specifically bind to PD-L1. The present invention is partly based on the discovery that antibody-drug conjugates, including MMAE antibody-drug conjugates and camptothecin antibody-drug conjugates targeted to PD-L1, are particularly effective in killing PD-L1+ expressing cells. 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.

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

[0145] IV. The Antibody of the Present Invention Previously, selective antibodies already used to treat cancer were conjugated with cytotoxic agents without sequence modification to create antibody-drug conjugates (ADCs). These ADCs have often proven to be more effective or more effective than unconjugated antibodies in killing tumor cells. Previously, when modifications to the antibody were attempted during the process of preparing the ADC, some possible modifications were to increase the binding affinity of the antibody or to enhance the antibody activity, such as ADCCs. However In at least some situations, it has been found that modifying or adjusting ADC antibodies, for example by reducing their binding affinity or their ADCC activity, improves the efficacy of ADCs compared to ADCs with unmodified antibodies. Some examples of this include ADCs with anti-PD-L1 antibodies (such as Ab1) that are remarkably optimized by modifying the antibodies, for example by reducing their binding affinity. For example, in some cases, anti-PD-L1 ADCs are more effective in killing tumor cells in vitro when the binding affinity of the antibody conjugated to a cytotoxic agent is reduced. In another example, in some cases, anti-PD-L1 ADCs are more effective in killing tumor cells in vitro and in vivo when the binding affinity of the antibody conjugated to a cytotoxic agent is reduced.

[0146] The present invention provides an antibody, such as a humanized antibody, that binds to PD-L1 with a binding affinity of 3 nM to 300 nM. In some embodiments, the antibodies described herein have a binding affinity of about 3 nM to 300 nM (e.g., about 3 nM to about 275 nM, about 3 nM to about 250 nM, about 3 nM to about 225 nM, about 3 nM to about 200 nM, about 3 nM to about 175 nM, about 3 nM to about 150 nM, about 3 nM to about 125 nM, about 3 nM to about 100 nM, about 3 nM to about 90 nM, about 3 nM to about 80 nM, about 3 nM to about 70 nM, about 3 nM to about 60 nM, about 3 nM to about 50 nM, about 3 nM to about 40 nM, about 3 nM to about 30 nM, about 3 nM to about 20 nM, about 3 nM to about 10 nM, about 10 nM to about 300 nM, about 10 nM to about 275 nM, about 10 nM to about 250 nM, about 10 nM to about 225 nM, about 10 nM to about 200 nM, about 10 nM to about 175 nM, about 10 nM to about 150 nM, about 10 nM to about 125 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 20 nM to about 300 nM, about 20 nM to about 275 nM, about 20 nM to about 250 nM, about 20 nM to about 225 nM, about 20 nM to about 200 nM, about 20 nM to about 175 nM, about 20 nM to about 150 nM, about 20 nM to about 125 nM, about 20 nM to about 100 nM, about 20 nM to about 90 nM, about 20 nM to about 80 nM, about 20 nM to about 70 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 20 nM to about 30 nM, about 30 nM to about 300 nM, about 30 nM to about 275 nM, about 30 nM to about 250 nM, about 30 nM to about 225 nM, about 30 nM to about 200 nM, about 30 nM to about 175 nM, about 30 nM to about 150 nM, about 30 nM to about 125 nM, about 30 nM to about 100 nM, about 30 nM to about 90 nM, about 30 nM to about 80 nM, about 30 nM to about 70 nM, about 30 nM to about 60 nM, about 30 nM to about 50 nM, about 30 nM to about 40 nM, about 40 nM to about 300 nM, about 40 nM to about 275 nM, about 40 nM to about 250 nM, about 40 nM to about 225 nM, about 40 nM to about 200 nM, about 40 nM to about 175 nM, about 40 nM to about 150 nM, about 40 nM to about 125 nM,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 M ~ approximately 175nM, approximately 50nM ~ approximately 150nM, approximately 50nM ~ approximately 125nM, approximately 50nM ~ approximately 100nM, approximately 50nM ~ approximately 90nM, approximately 50nM ~ approximately 80nM, approximately 50nM ~ approximately 70nM, approximately 50nM ~ approximately 60nM, approximately 60nM ~ approximately 300nM, approximately 60nM ~ approximately 275nM, approximately 60nM ~ approximately 250nM, approximately 60nM ~ approximately 225nM, approximately 60nM to approximately 200nM, approximately 60nM to approximately 175nM, approximately 60nM to approximately 150nM, approximately 60nM to approximately 125nM, approximately 60nM to approximately 100nM, approximately 60nM to approximately 90nM, approximately 60nM to approximately 80nM, approximately 60nM to approximately 70nM, approximately 70nM to approximately 300nM, approximately 70nM to approximately 275nM, approximately 70nM to approximately 25 0 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 approximately 150 nM, approximately 70 nM to approximately 125 nM, approximately 70 nM to approximately 100 nM, approximately 70 nM to approximately 90 nM, approximately 70 nM to approximately 80 nM, approximately 80 nM to approximately 300 nM, approximately 80 nM to approximately 275 nM, approximately 80 nM to approximately 250 nM, nM, about 80nM to about 225nM, about 80nM to about 200nM, about 80nM to about 175nM, about 80nM to about 150nM, about 80nM to about 125nM, about 80nM to about 100nM, about 80nM to about 90nM, about 9 0nM to about 300nM, about 90nM to about 275nM, about 90nM to about 250nM, about 90nM to about 225nM, about 90nM to about 200nM, about 90nM to about 175nM, about 90nM to about 150nM, about 90nM Approximately 125nM, approximately 90nM to approximately 100nM, approximately 100nM to approximately 300nM, approximately 100nM to approximately 275nM, approximately 100nM to approximately 250nM, approximately 100nM to approximately 225nM, approximately 100nM to approximately 200nM, approximately 100nM M ~ about 175nM, about 100nM to about 150nM, about 100nM to about 125nM, about 125nM to about 300nM, about 125nM to about 275nM, about 125nM to about 250nM, about 125nM to about 225nM, about 12 5nM to about 200nM, about 125nM to about 175nM, about 125nM to about 150nM, about 150nM to about 300nM, about 150nM to about 275nM, about 150nM to about 250nM, about 150nM to about 225nM, Approximately 150nM to approximately 200nM, approximately 150nM to approximately 175nM, approximately 175nM to approximately 300nM, approximately 175nM to approximately 275nM, approximately 175nM to approximately 250nM, approximately 175nM to approximately 225nM, approximately 175nM to approximately 200 K in nM, approximately 200nM to 300nM, approximately 200nM to 275nM, approximately 200nM to 250nM, approximately 200nM to 225nM, approximately 225nM to 300nM, approximately 225nM to 275nM, approximately 225nM to 250nM, approximately 250nM to 300nM, approximately 250nM to 275nM, or approximately 275nM to 30nM) (for example, when measured by biolayer interferometry (BLI) in phosphate-buffered saline) D It then binds to PD-L1.

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

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

[0149] In some embodiments, the anti-PD-L1 antibody provided herein may have a heavy chain CDR1 with one 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 of 3 nM to 300 nM. In some embodiments, the one amino acid substitution in SEQ ID NO: 3 is at amino acid position 2 of SEQ ID NO: 3. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 The amino acid substitution is a tyrosine to alanine substitution. In some embodiments, one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is a tyrosine to serine substitution. In some embodiments, one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is a tyrosine to glycine substitution. In some embodiments, one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is a tyrosine to threonine substitution. In some embodiments, one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is a tyrosine to valine substitution. In some embodiments, one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is a tyrosine to cysteine ​​substitution.

[0150] In some embodiments, the anti-PD-L1 antibodies provided herein are K3-300nM (or any of the sub-ranges of this range as described herein) D It binds to both glycosylated and non-glycosylated PD-L1.

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

[0152] In some embodiments, the anti-PD-L1 antibodies provided herein increased the rate of internalization by PD-L1+ cells compared to Ab1 (e.g., an increase of 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 an increase of 5% to 300% (or any of the sub-ranges of this range as described herein)).

[0153] In some embodiments, the anti-PD-L1 antibodies provided herein increased immune cell infiltration upon administration to mammals compared with Ab1 (e.g., an increase of 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 an increase of 5% to 300% (or any of the sub-ranges of this range as described herein)).

[0154] In some embodiments, the anti-PD-L1 antibodies provided herein, compared to Ab1, stimulate inflammatory cytokine production (e.g., the cytokines described herein) when administered to mammals. An increase of any one or more of the following (for example, an increase of at least 5%, an increase of at least 10%, an increase of at least 20%, an increase of at least 30%, an increase of at least 40%, an increase of at least 50%, an increase of at least 60%, an increase of at least 70%, an increase of at least 80%, an increase of at least 90%, an increase of at least 100%, an increase of at least 120%, an increase of at least 140%, an increase of at least 160%, an increase of at least 180%, an increase of at least 200%, an increase of at least 220%, an increase of at least 240%, an increase of at least 260%, an increase of at least 280%, an increase of at least 300%, or an increase of 5% to 300% (or any of the sub-ranges of this scope as described herein)).

[0155] In some embodiments, the anti-PD-L1 antibodies provided herein increased intracellular digestion by PD-L1+ cells compared to Ab1 (e.g., an increase of 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 an increase of 5% to 300% (or any of the sub-ranges of this range as described herein)).

[0156] In some embodiments, the anti-PD-L1 antibodies provided herein have less than 10% change in neutrophil and / or platelet counts (e.g., less than 8%, less than 6%, less than 4%, less than 2%, or less than 1%) when administered to mammals compared to Ab1.

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

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

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

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

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

[0162] Generally, the anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates of this disclosure bind to PD-L1, for example, human PD-L1, and exert cell proliferation inhibitory and cytotoxic effects against malignant cells such as cancer cells. Compared to untreated cells, this results in a 50% reduction in viability, or x50, or IC50. 50The concentration required to produce the desired result is one way to measure the cytotoxicity of anti-PD-L1 antibodies and / or anti-PD-ADCs. Preferred antibodies and / or ADCs of the present invention exhibit increased cytotoxicity and x50 compared to those of Ab1 antibodies and / or ADCs. In one embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 10 ng / mL to 30 ng / mL or 15 ng / mL to 25 ng / mL in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 15 ng / mL to 55 ng / mL or 20 ng / mL to 50 ng / mL in the MDA-MB-231 cell line. In yet another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits an x50 of 1 ng / mL to 7 ng / mL or 2 ng / mL to 5 ng / mL in the KARPAS 299 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention shows x50 concentrations of 15 ng / mL to 40 ng / mL or 20 ng / mL to 35 ng / mL in the L540CY cell line. In one embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention shows x50 concentrations of 12 ng / mL to 70 ng / mL or 15 ng / mL to 65 ng / mL in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention shows x50 concentrations of 3 ng / mL to 20 ng / mL or 5 ng / mL to 17 ng / mL in the MDA-MB-231 cell line. In yet another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention shows x50 concentrations of 1 ng / mL to 18 ng / mL or 3 ng / mL to 15 ng / mL in the KARPAS 299 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits x50 concentrations of 1 ng / mL to 20 ng / mL or 1 ng / mL to 15 ng / mL in the L540CY cell line.

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

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

[0165] In certain embodiments of this disclosure, the anti-PD-L1 antibody is an antigen-binding fragment as described herein (e.g., a human antigen-binding fragment), but is not limited to Fab, Fab' and F(ab')2, Fd, single-chain Fv(scFv), single-chain antibody, disulfide-bonded Fv(sdFv), and V L Domain or V H Examples include fragments containing any of the domains. Antigen-binding fragments containing a single-chain antibody may contain the variable region alone or in combination with all or part of the hinge region, CH1, CH2, CH3, and CL domains. Antigen-binding fragments containing any combination of the variable region having the hinge region, CH1, CH2, CH3, and CL domains are also included in this disclosure. In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment is from a human, mouse (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camelid, horse, or chicken.

[0166] The anti-PD-L1 antibodies of this disclosure may be monospecific, bispecific, tripspecific, or more highly multispecific. Multispecific antibodies may be specific to different epitopes of PD-L1, or to both PD-L1 and heterologous proteins. See, for example, PCT Publications WO93 / 17715, WO92 / 08802, WO91 / 00360, WO92 / 05793, Tutt et al., 1991, J.Immunol.147:60 69, U.S. Patents No. 4,474,893, No. 4,714,681, No. 4,925,648, No. 5,573,920, No. 5,601,819, and Kostelny et al., 1992, J.Immunol.148:1547 1553.

[0167] The anti-PD-L1 antibodies of this disclosure may be described or specified with respect to specific CDRs they contain. The exact amino acid sequence boundaries of a given CDR or FR are as follows: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (199 7) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J.Mol.Biol.262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J.Mol.Biol.262, 732-745. ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Dev Comp Immunol, 2003 Jan;27(1):55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, Yet another numbering The CDRs can be easily determined using any of the many well-known schemes, including those described by 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 may differ depending on the scheme used for identification. In some embodiments, the "CDR" or "complementarity-determining region" of a given antibody or region of it (e.g., its variable region) or individual specific CDRs (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass (or specific) CDRs as defined by any of the aforementioned schemes. For example, if a specific CDR (e.g., CDR-H3) is a given V H or V L When it is stated that a regional amino acid sequence contains the amino acid sequence of the corresponding CDR, such a CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the schemes described above. The scheme for identifying one or more specific CDRs may be specified, such as CDRs as defined by the Kabat, Chothia, AbM, or IMGT methods.

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

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

[0170] In one embodiment, an anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate is provided herein, 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 one embodiment, sequence number An anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate comprising a heavy chain containing the amino acid sequence of sequence number 9 and a light chain containing the amino acid sequence of sequence number 10 is provided herein.

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

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

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

[0174] In some embodiments, a light chain variable includes 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 respect to the amino acid sequence of SEQ ID NO: 2. Anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates containing a main agent are provided herein. In certain embodiments, 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 respect to the amino acid sequence of SEQ ID NO: 2 includes substitutions (e.g., conservative substitutions), insertions, or deletions with respect to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in SEQ ID NO: 2. In certain embodiments, the light chain contains one point mutation with respect to SEQ ID NO: 2. In further embodiments, the one point mutation is located in the CDR region.

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

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

[0177] Antibodies also include derivatives modified by covalent bonding of any type of molecule to the antibody, such that the covalent bond does not prevent the antibody from binding to PD-L1 or exerting a cell proliferation inhibitory or cytotoxic effect on the cell. For example, but not limited to, antibody derivatives include antibodies modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protective / blocking groups, proteolytic cleavage, or binding to cell ligands or other proteins. Any of many chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, or metabolic synthesis of tunicamycin. Furthermore, derivatives may contain one or more non-classical amino acids.

[0178] Humanized antibodies Humanized antibodies are genetically engineered antibodies in which a CDR derived from a non-human "donor" antibody is implanted into a human "acceptor" antibody sequence (see, for example, Queen, U.S. Patents 5,530,101 and 5,585,089; Winter, U.S. Patent 5,225,539; Carter, U.S. Patent 6,407,213; Adair, U.S. Patent 5,859,205; and Foote, U.S. Patent 6,881,557). The acceptor antibody sequence may be, for example, a mature human antibody sequence, a complex of such sequences, a consensus sequence of a human antibody sequence, or a germline region sequence. Preferred heavy chain acceptor sequences are germline V H Exxon V H 1-2 (also called HV1-2 in the literature) (Shin et al., 1991, EMBO J.10:3641~3645), and the hinge region (J H Regarding ), ExxonJ H -6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred acceptor sequence is exon VK2-30 (also called KV2-30 in the literature), and for the hinge region, it is exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Therefore, humanized antibodies are complete An antibody having some or all of its CDRs derived from a donor antibody, and, if present, a variable region framework sequence and a constant region that are entirely or substantially derived from a human antibody sequence. Similarly, a humanized heavy chain has at least one, two, usually all three CDRs derived entirely or substantially from a donor antibody heavy chain, and, if present, a heavy chain variable region framework sequence and a heavy chain constant region that are substantially derived from a human heavy chain variable region framework and constant region sequence. Similarly, a humanized light chain has at least one, two, usually all three CDRs derived entirely or substantially from a donor antibody light chain, and, if present, a light chain variable region framework sequence and a light chain constant region that are substantially derived from a human light chain variable region framework and constant region sequence. Except for nanobodies and dAbs, a humanized antibody includes a humanized heavy chain and a humanized light chain. A CDR in a humanized antibody is derived from the corresponding CDR in a non-human antibody if at least 60%, 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) are identical between each CDR. If at least 85%, 90%, 95%, or 100% of the corresponding residues defined by Kabat are identical, the variable region framework sequence or the constant region of the antibody chain is substantially derived from a human variable region framework sequence or a human constant region, respectively. In some embodiments, the PD-L1 antibody of the present invention is a humanized antibody.

[0179] Humanized antibodies often incorporate all six CDRs (preferably according to Kabat's definition) derived from mouse antibodies, but humanized antibodies can also be produced using fewer than all CDRs (e.g., at least three, four, or five) derived 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).

[0180] Selection of the steady-state region The heavy and light chain variable regions of humanized antibodies can be linked to at least a portion of the human constant region. The selection of the constant region depends, in part, on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotypes IgG1 and IgG3 have potent complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce potent cell-mediated effector function more strongly 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, as Fab, Fab', F(ab')2 and Fv, or as single-chain antibodies in which the heavy and light chain variable domains are linked via spacers.

[0181] The human constant region exhibits allotype and isoallotype diversity among different individuals; that is, the constant region can differ in different individuals at one or more polymorphic sites. Isoallotypes differ from allotypes in that the serum that recognizes the isoallotype binds to the non-polymorphic region of one or more other isotypes.

[0182] One or more amino acids at the amino or carboxyl termini of the light chain and / or heavy chain, such as the C-terminal lysine of the heavy chain, may be omitted or derivatized in part or all of the molecule. To reduce or increase effector functions 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 extend the half-life in humans (see, for example, Hinton et al., J. Biol. Chem. 279:6213, 2004), substitution can be introduced during the steady-state region.

[0183] Exemplary substitutions include amino acid substitutions of native amino acids to cysteine ​​residues, introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332 of the human IgG1 isotype, preferably the S239C mutation (U.S. Patent Application Publication No. 20100158909). The presence of additional cysteine ​​residues enables the formation of interchain disulfide bonds. The formation of such interchain disulfide bonds can cause steric hindrance, thereby reducing the affinity of the Fc domain-FcyR bond interaction. Cysteine ​​residues introduced in or near the Fc region of the IgG constant region can also serve as sites for conjugation to therapeutic agents (i.e., coupling cytotoxic drugs with thiol-specific reagents such as maleimide derivatives of the drug). The presence of the therapeutic agent causes steric hindrance, thereby further reducing the affinity of the Fc region-FcyR binding interaction. Other substitutions at any of positions 234, 235, 236 and / or 237 reduce the affinity to Fey receptors, particularly FcyRI receptors (see, e.g., U.S. Patent No. 6,624,821 and U.S. Patent No. 5,624,821).

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

[0185] Oligosaccharides covalently bound to the conserved Asn297 are involved in the ability of the IgG Fc domain to bind to FcyR (Lund et al., 1996, J.Immunol.157:4963-69; Wright and Morrison, 199', Trends Biotechnol.15:26-31). Manipulation of this glycoform on IgG can significantly improve IgG-mediated ADCC. Addition of bisecting N-acetylglucosamine modification to this glycoform (Umana et al., 1999, Nat.Biotechnol.17:176-180; Davies et al, 2001, Biotech.Bioeng.74:288-94) or fucose from this glycoform (Shields et al., 2002, J.Biol.Chem.277:26733- The removal of 40 (Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niva et al., 2004, Cancer Res. 64:2127-33) is one example of IgG Fc manipulation that improves the binding of IgG Fc to FcyR, thereby enhancing Ig-mediated ADCC activity.

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

[0187] The complement fixation activity of antibodies (both C1q binding and CDC activity) can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J.Immunol.166:2571-2575). The same substitutions on the human IgG2 skeleton can convert antibody isotypes that do not bind well to C1q and have a significant deficiency in complement activation activity into those that can bind to C1q and mediate CDC (Idusogie et al., 2001, J.Immunol.166:2571-75). Several other methods have also been applied to improve the complement fixation activity of antibodies. For example, grafting the 18-amino acid carboxyl-terminal tail fragment of IgM to the carboxyl terminus of IgG significantly enhances their CDC activity. This is also observed in IgG4, which normally does not have detectable CDC activity (Smith et al., 1995, J.Immunol.154:2226-36). Furthermore, substituting Ser444, located near the carboxyl terminus of the IgG1 heavy chain, with Cys induces tail-to-tail dimerization of IgG1, resulting in a 200-fold increase in CDC activity compared to monomeric IgG1 (Shopes et al., 1992, J.Immunol.148:2918-22). In addition, a bispecific diabody construct with specificity for C1q also confers CDC activity (Kontermann et al., 1997, Nat.Biotech.15:629-31).

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

[0189] Substitution of the 318(Glu) residue with a polar residue can alter, but not eliminate, C1q binding activity. Substitution of residue 297(Asn) with Ala leads to the removal of solubility, but only a slight decrease in affinity for C1q (about 3 times weaker). This change disrupts the glycosylation site and the presence of carbohydrates necessary for complement activation. Any other substitution at this site also disrupts the glycosylation site. The following mutations and any combination thereof also reduce C1q binding: D270A, K322A, P329A, and P31 IS (see WO06 / 036291). The L234A / L235A mutation (or LALA mutation) also reduces C1q binding and FcyR binding. In one embodiment, the anti-PD-L1 antibody of the present invention contains the L234A / L235A mutation.

[0190] References to human constant regions refer to constant regions with any natural allotype, or natural allotypes. This includes any rearrangement of residues occupying polymorphic positions in the ip. Furthermore, up to 1, 2, 5, or 10 mutations may exist in the native human constant region, e.g., shown above, to decrease Fc gamma receptor binding or increase binding to FcRN.

[0191] V. Expression of recombinant antibody Humanized antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically contain an expression regulatory sequence functionally linked to the coding sequence of the antibody chain, such as a naturally occurring or heterologous promoter region. Preferably, the expression regulatory sequence is a eukaryotic promoter system in a vector that can transform or translocate eukaryotic host cells. Once the vector is taken up into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and collection and purification of the cross-reacting antibody.

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

[0193] Once expressed, the antibody can be purified according to standard procedures in this field, such as HPLC purification, column chromatography, and gel electrophoresis (see Scopes, Protein Purification (Springer-Verlag, NY, 1982) in general).

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

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

[0196] The anti-PD-L1 antibodies described herein can be prepared by known recombinant techniques using well-known expression vector systems and host cells. In one embodiment, the antibody is prepared by De la Cruz Edmunds et al., 2006, Molecular Biotechnology. As disclosed in 34;179-190, European Patent No. 216846, U.S. Patent No. 5,981,216, WO87 / 04462, European Patent No. 323997, U.S. Patent No. 5,591,639, U.S. Patent No. 5,658,759, European Patent No. 338841, U.S. Patent No. 5,879,936 and U.S. Patent No. 5,891,693, the GS expression vector system is prepared in CHO cells.

[0197] The monoclonal anti-PD-L1 antibodies described herein may be produced by the hybridoma method, for example, first described by Kohler et al., Nature, 256, 495 (1975), or by the recombinant DNA method. Monoclonal antibodies may also be isolated from phage antibody libraries using the 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. For example, monoclonal antibodies can be obtained from hybridomas prepared from mouse spleen B cells obtained from mice immunized with the antigen of interest, for example, in the form of cells expressing the antigen on the surface, or from nucleic acids encoding the antigen of interest. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals such as rats, dogs, and primates.

[0198] Antibody-drug conjugates Antibody-drug conjugates (ADCs) can be formed by conjugating anti-PD-L1 antibodies to cytotoxic or cell proliferation inhibitory moieties (including pharmaceutically compatible salts thereof). Particularly suitable moieties for conjugation to 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 to cytotoxic agents such as chemotherapeutic agents or toxins (e.g., cell proliferation inhibitors or cell-destroying agents such as abrin, lysine A, pseudomonas exotoxin, or diphtheria toxin).

[0199] Anti-PD-L1 antibodies can be conjugated to prodrug-converting enzymes. Prodrug-converting enzymes can be recombinantly fused to antibodies or chemically conjugated to antibodies using known methods. Exemplary prodrug-converting enzymes include carboxypeptidase G2, β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

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

[0201] Therapeutic agents can be conjugated in such a way that the activity of the antibody is reduced unless the antibody is cleaved (for example, by hydrolysis, antibody degradation, or by a cleavage agent). Such therapeutic agents are conjugated to antibodies 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, so that the conjugate is cleaved from the antibody when taken up by PD-L1-expressing cancer cells (for example, in endosomes, or, for example, in the lysosomal environment, or in the caveolear environment, via pH sensitivity or protease sensitivity).

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

[0203] A cleavable linker may be pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers hydrolyzable in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide, orthoester, acetal, ketal, etc.) can be used. (See, for example, U.S. Patent Nos. 5,122,368, 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers can be hydrolyzed in neutral pH environments such as blood. It is relatively stable under 1H conditions, but unstable at pH 5.5 or below 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether conjugated to the therapeutic agent via an acylhydrazone linkage (see, for example, U.S. Patent No. 5,622,929)).

[0204] Other linkers can be cleaved under reducing conditions (e.g., disulfide linkers). Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyldithio)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 (CWVogel ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935).

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

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

[0207] Typically, linkers are substantially insensitive to the extracellular environment; that is, when ADCs are present in an extracellular environment (e.g., plasma), approximately 20% or less, typically approximately 15% or less, more typically approximately 10% or less, even more typically approximately 5% or less, approximately 3% or less, or approximately 1% or less of the linkers in a sample of ADCs are cleaved.

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

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

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

[0211] An exemplary antibody-drug conjugate is an auristatin-based antibody-drug conjugate (i.e., the drug component is an auristatin drug). Auristatin has been shown to bind to tubulin and interfere with microtubule dynamics as well as nuclear and cell division, and possesses anticancer activity. Typically, an auristatin-based antibody-drug conjugate contains a linker between the auristatin drug and the anti-PD-L1 antibody. The linker may be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or an incleavable linker (e.g., a linker released by antibody degradation). Examples of auristatins include auristatin T, MMAF, and MMAE. Exemplary auristatin synthesis and structure are described in U.S. Public Appeals Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in whole for all purposes.

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

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

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

Chemical formula

Chemical formula

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

[0216] In some embodiments, the camptothecin ADC is expressed by formula (IC): [ka] or a pharmaceutically acceptable salt thereof (In the formula, Ab is an anti-PD-L1 antibody, y is 1, 2, 3 or 4, or 1 or 4, and z is an integer between 2 and 12, or 2, 4, 8, or 12. (and p is between 1 and 16) It holds.

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

[0218] In some embodiments, camptothecin ADC is given by formula: [ka] or a pharmaceutically acceptable salt thereof The formula has the following characteristics (wherein p is 2, 4, or 8, preferably p is 8).

[0219] In some embodiments, camptothecin ADC is given by formula: [ka] or a pharmaceutically acceptable salt thereof The formula has the following characteristics (wherein p is 2, 4, or 8, preferably p is 8).

[0220] In some embodiments, the camptothecin drug-linker is of the formula: [ka] or a pharmaceutically acceptable salt thereof (In the formula, y is 1, 2, 3, or 4, or is 1 or 4, and z is an integer from 2 to 12, or is 2, 4, 8, or 12).

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

Chemical formula

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

Chemical formula

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

Chemical formula

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

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

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

[0227] The PEG (polyethylene glycol) portion of the drug linker may be in the range of 2 to 36. In all of 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.

[0228] Polydispersible PEGS, monodispersible PEGS, and discontinuous PEG can be used to prepare the PEGylated antibody-drug conjugates of the present invention. Polydispersible PEG has a size and molecular weight. Although a heterogeneous mixture, monodisperse PEG is typically purified from a heterogeneous mixture and therefore provides a single chain length and molecular weight. A preferred PEG unit is discontinuous PEG, which is a compound synthesized stepwise without a polymerization process. Discontinuous PEG provides a single molecule with a defined specific chain length. Similar to the subscript "p", when referring to a population of antibody-drug conjugates, the value of the subscript "n" may be an average number and may be an integer or a non-integer.

[0229] Examples of cytotoxic agents useful for conjugating anti-PD-L1 antibodies include antitubulins, DNA ligatures, DNA replication inhibitors, and chemotherapeutic sensitizers. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecin, duocalmycin, etoposides, mytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA ligators (e.g., enediyne and lexitropsin), duocalmycin, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulicin M, doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

[0230] Cytotoxic agents may include chemotherapeutic agents such as doxorubicin, paclitaxel, melphalan, vinca alkaloids, methotrexate, mitomycin C, or etoposide. The agents may also be CC-1065 analogs, calicheamicin, meitansine, drastatin 10, rhizoxin, or palytoxin analogs.

[0231] Cytotoxic agents can also be auristatins. Auristatins may be auristatin E derivatives, for example, esters formed between auristatin E and a keto acid. For example, auristatin E can be reacted with paraacetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatins include auristatin T, AFP, MMAF, and MMAE. The synthesis and structures of various auristatins are described, for example, in U.S. Patent Application Publication No. 2005-0238649 and U.S. Patent Application Publication No. 2006-0074008.

[0232] The cytotoxic agent may be a DNA sulcus binding agent. (See, for example, U.S. Patent No. 6,130,237.) For example, the sulcus binding agent may be a CBI compound or an enediyne (e.g., calicheamycin).

[0233] Cytotoxic agents or cell proliferation inhibitors may be antitubulins. Examples of antitubulins include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik), vinca alkiloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Exemplary auristatins are shown in formulas III-XIII below. Other suitable antitubulins include, for example, baccatin derivatives, taxane analogs (e.g., epothyron A and B), nocodazole, colchicine and corcimide, estramustine, cryptophycin, semadotin, mytansinoids, combretastatin, discodermoids, and eleuthrobin.

[0234] Cytotoxic agents may be mytansinoids or other groups of antitubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the mytansinoid may be meitansin or meitansin containing a drug linker such as DM-1 or DM-4 (ImmunoGen, Inc.; Chari et al., 1992, Cancer Res.).

[0235] VIII. Therapeutic applications The antibodies of the present invention can be used to treat cancer, either alone or as an anti-PD-L1 antibody-drug conjugate. Some such cancers exhibit detectable levels of PD-L1, measured either at the protein level (e.g., by an immunoassay using one of the exemplary antibodies) or at the mRNA level. Some such cancers preferably exhibit elevated levels of PD-L1 compared to non-cancerous tissue of the same type from the same patient. An exemplary level of PD-L1 on cancer cells suitable for treatment is 5,000 to 500,000 PD-L1 molecules per cell, but higher or lower levels can be treated. If necessary, the level of PD-L1 in the cancer is measured before treatment is performed.

[0236] Examples of cancers suitable for treatment due to PD-L1 expression 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 antibody or antibody-drug conjugate of the present invention is used in a method for treating melanoma. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating NSCLC. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating SCLC. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating head and neck cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating TNBC. Triple-negative breast cancer is a technical term for cancer lacking detectable estrogen and progesterone receptors and lacking HER2 / neu overexpression. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating ovarian cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating urothelial carcinoma. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating HCC. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating gastric cancer. In some embodiments, the antibody or antibody-drug conjugate of the present invention is used in a method for treating cervical cancer. The treatment can be applied to patients with primary or metastatic tumors of these types. The treatment can also be applied to patients who are refractory to conventional treatments or who have relapsed after responding to such treatments.

[0237] The antibodies of the present invention, such as humanized antibodies, either alone or as a conjugate thereof, are administered in an effective regimen meaning a dose, route of administration, and frequency of administration that delays the onset of cancer, reduces its severity, inhibits further progression, and / or improves at least one sign or symptom. If the patient already has cancer, the regimen may be called a therapeutically effective regimen. If the patient is at higher risk of cancer than the general population but has not yet experienced symptoms, the regimen may be called a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy can be observed in individual patients compared to historical controls or the past experience of the same patients. In other cases, therapeutic or prophylactic efficacy can be demonstrated in preclinical or clinical trials in a population of treated patients compared to a control population of untreated patients.

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

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

[0240] The frequency of administration depends, among other factors, particularly the half-life of the circulating antibody or conjugate, the patient's condition, and the route of administration. The frequency may be daily, weekly, monthly, four times a year, or at irregular intervals depending on the patient's condition or changes in the progression of the treated cancer. An exemplary frequency for intravenous administration is between two times a week and four times a year over the course of treatment, but higher or lower frequencies are also possible. Another exemplary frequency for intravenous administration is between three times a week or every four weeks over the course of treatment, but higher or lower frequencies are also possible. For subcutaneous administration, an exemplary frequency is daily to monthly, but higher or lower frequencies are also possible.

[0241] The number of doses administered depends on the nature of the cancer (e.g., whether it presents as acute or chronic) and the patient's response to treatment. For acute complications or acute exacerbations of chronic complications, 1 to 10 doses are often sufficient. In some cases, a single bolus dose, divided as needed, is sufficient for acute complications or acute exacerbations of chronic complications. Treatment can be repeated for recurrences or acute exacerbations of acute complications. For chronic complications, antibodies can be administered at regular intervals, for example, weekly, every two weeks, monthly, every three months, or every six months for at least 1, 5, or 10 years, or throughout the patient's lifetime.

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

[0243] Treatment with the antibody of the present invention is used in chemotherapy, radiation therapy, stem cell therapy, surgery, and treatment of the patient. It can be combined with other effective treatments for the disorder being treated, including standard treatments for the disorder. Useful classes of other drugs that can be administered with antibodies against PD-L1 and antibody-drug conjugates as described herein include, for example, antibodies against other receptors expressed on cancer cells, antitubulins (e.g., auristatin), DNA sulcus binding agents, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, e.g., cisplatin, mono(platinum), bis(platinum) and trinuclear platinum complexes and carboplatin), anthracyclines, antibiotics, folate antimetabolites, antimetabolites, chemotherapeutic sensitizers, duocalmycin, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosourea, platinol, preforming compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, and the like.

[0244] Treatment using an anti-PD-L1 antibody or antibody-drug conjugate, either alone or in combination with any other drug or regimen described as an antibody-drug conjugate, can increase the median progression-free survival or overall survival 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), particularly in cases of relapsed or refractory tumors, by at least 30% or 40%, preferably 50%, 60% to 70%, or even 100% or more compared to the same treatment (e.g., chemotherapy) without using an anti-PD-L1 antibody alone or as a conjugate. In addition, or alternatively, treatments including an anti-PD-L1 antibody alone or as a conjugate (e.g., standard chemotherapy) can increase the complete response rate, partial response rate, or objective response rate (complete + partial) in patients with tumors by at least 30% or 40%, preferably 50%, 60% to 70%, or even 100%, compared to the same treatment (e.g., chemotherapy) without using an anti-PD-L1 antibody alone or as a conjugate.

[0245] Typically, in clinical trials (e.g., Phase II, Phase II / III, or Phase III trials), the aforementioned increase in median progression-free survival and / or objective response rates in patients treated with anti-PD-L1 antibodies alone or as a conjugate in addition to standard treatment, compared to a control group of patients receiving standard treatment alone (or with placebo), is statistically significant, e.g., at the level of p=0.05, 0.01, or even 0.001. Complete and partial response rates are determined by objective criteria commonly used in cancer clinical trials, such as those enumerated or approved by the National Cancer Institute and / or the Food and Drug Administration.

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

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

[0248] The product or kit may further include a label or accompanying leaflet, which may be on or associated with the container and may indicate instructions for recombining and / or using the formulation. The label or accompanying leaflet may further indicate that the formulation is useful or intended for subcutaneous, intravenous (e.g., intravenous infusion), or other modes of administration for treating target cancers (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 multi-use vial that allows for repeated administration of the recombined formulation. The product or kit may further include a second container containing an appropriate diluent. The product or kit may further include other materials desirable from a commercial, therapeutic, and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying leaflets containing instructions for use.

[0249] The products or kits described herein may further include, as necessary, a container containing a second pharmaceutical product, where the first pharmaceutical product is an anti-PD-L1 antibody or an anti-PD-L1 antibody-drug conjugate, and the articles or kits further include label or package insert instructions for treating a subject with the second pharmaceutical product in an effective amount. In some embodiments, the second pharmaceutical product is intended to eliminate or reduce the severity of one or more adverse events.

[0250] In some embodiments, the anti-PD-L1 antibody or anti-PD-L1 antibody-drug conjugate is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is in a sealed container such as a vial, ampoule, or sachet indicating the amount of the activator. If the pharmaceutical is administered by injection, ampoules of sterile water for injection or saline can be provided as part of the kit, if necessary, so that the components can be mixed before administration. Such a kit may further include, if desired, one or more different conventional pharmaceutical components, such as containers containing one or more pharmaceutically acceptable carriers, additional containers, etc. Printed instructions, as inserts or labels, indicating the amount of the component to be administered, guidelines for administration, and / or guidelines for mixing the components may also be included in the kit.

[0251] X. Other applications Anti-PD-L1 antibodies described herein, such as humanized anti-PD-L1 antibodies, can be used to detect PD-L1 in the context of clinical diagnosis and treatment, or in research. PD-L1 expression in cancer provides an indicator that the cancer is suitable for treatment with the antibodies of the present invention. The antibodies may also be marketed as research reagents for experimental studies in detecting cells possessing PD-L1 and their responses to various stimuli. In such use, monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotypes and may be supplied in kit form with all the reagents necessary to carry out the PD-L1 assay. The antibodies described herein can be used to detect PD-L1 protein expression and determine whether cancer is suitable for treatment with PD-L1 ADCs.

[0252] All patent applications, websites, other publications, acceptance numbers, etc., cited above or below are incorporated by reference in whole for all purposes to the same extent that each individual item is specifically and individually indicated to be incorporated by reference. If different versions of the invention are associated with an acceptance number at different points in time, the version associated with the acceptance number on the effective filing date of this application is meant. The effective filing date means the earlier of the actual filing date or, where applicable, the filing date of the priority application referring to the acceptance number. Similarly, if different versions of a publication, website, etc., are published at different times, unless otherwise specified, the most recently published version on the effective filing date of this application is meant. Any feature, step, element, embodiment, or aspect of the invention may be used in combination with any other unless specifically stated otherwise. For clarity and understanding, the invention has been described in some detail by examples and illustrations, but it will be obvious that certain changes and modifications can be made within the scope of the appended claims. [Examples]

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

[0254] method Antibody production PD-L1-targeted SG-559-xx antibodies were produced by introducing point mutations into the CDR of fully human Ab1 to reduce affinity. Briefly, residues in the CDR adjacent to the PD-L1 binding epitope were mutated to different amino acids. Four selected exemplary residues are shown in Figure 1. For initial screening purposes, SG-559-xx antibodies were generated in ATUM bio using transient transfection in HEK293 cells.

[0255] For follow-up studies, antibodies were produced in-house according to the following protocol. The antibody variable domain sequence and constant domain sequence were synthesized using non-template PCR. In short, the hypothetical gene sequence was converted to an oligonucleotide sequence using Genewiz bioinformatics tools. Oligonucleotides were synthesized, pooled, and amplified using PCR. Full-length amplicons from the PCR reaction were cloned into a vector, and the product was then transformed into E. coli to isolate unique colonies. The colonies were grown overnight in liquid medium, plasmid DNA was isolated, purified, and sequenced using Sanger sequencing. The light and heavy chains were cloned into the pcDNA3.4 vector.

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

[0257] GE HiTrap mAb Select SuRe columns were used for the purification of 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 using 100 mM acetate pH 3 buffer. The samples were buffer-replaced with PBS using a 26 / 60 HiPrep desalting column. The samples underwent a final polishing step on a HiPrep Superdex 200 26 / 600 column performed in PBS. After filter-sterilization, the samples were taken for characterization. For characterization, A280 concentrated This includes aSEC HPLC, aHIC HPLC, and reduced PLRP-MS (QToF).

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

[0259] Production of antibody-drug conjugates (ADCs) SG-559-xx antibodies were conjugated to MDpr-PEG(12)-gluc-MMAE at a mean drug-to-antibody ratio (DAR) of 8, as described in U.S. Patent Application Publication No. 20180092984. SG-559-xx antibodies were conjugated to vc-MMAE at a mean DAR of 4, as described in U.S. Patent Application Publication No. 20050238649. SG-559-xx antibodies were conjugated to MP-PEG8-VKG-camptothecin at a mean DAR of 8, as described in PCT / US2019 / 025968 (filed April 5, 2019).

[0260] In vitro cell injury assay Cell lines were seeded 24 hours prior to antibody-drug conjugate (ADC) treatment to acclimate the cells. Where indicated, 500 IU / mL of interferon-γ was also added at this point to induce PD-L1 expression. Cells were then treated with the indicated doses of ADC and incubated at 37°C for 96 hours. Other PD-L1-targeting antibodies and isotype controls were included as ADCs for comparison. Cell viability of the cell lines was measured using CellTiter-Glo (Promega Corporation, Madison, WI) 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 are reported as x50 (the concentration of the compound required to produce a 50% decrease in viability compared to untreated cells).

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

[0262] In vivo activity study Nude mouse, 5.0 x 10 6 Individual BxPC3 pancreatic adenocarcinoma cells or 1.0 × 10⁶ 6EBC-1 NSCLC cells were subcutaneously inoculated into NSG mice. 5.0 × 10⁶ cells were then administered. 5 Individual MDA-MB-231 triple-negative breast cancer cells were subcutaneously inoculated into SCID mice. 1.0 × 10⁶ cells were then administered to SCID mice. 6 Individual Karpas 299 ALCL cells or 1.0 × 10⁶ 6 Nucleic Calu-1 NSCLC cells were subcutaneously inoculated. Tumor growth was monitored with calipers, and the average tumor volume was calculated using the formula (0.5 × [length × width]). 2 The calculation was performed using ]). The average tumor volume was approximately 100 mm². 3 When this was reached, the mice were either untreated or administered intraperitoneally as indicated by ADC. Non-conjugate antibodies and vc-MMAE ADC were administered once a week in a total of three doses. MP-PEG8-VKG-Camptothecin ADC was administered only once. Mice were treated when the tumor volume reached approximately 750 mm². 3 Euthanasia was performed when the condition reached a certain level. For immunocharacterization studies in animals with Karpas 299, the average tumor volume was 200 mm. 3 These mice were then treated with a single dose of non-conjugated antibody or ADC and euthanized after 6 days. Tumors were characterized exovivo by immunohistochemistry and cytokine analysis (Luminex). All animal handling was carried out in accordance with protocols approved by the Institutional Animal Care Committee, which is certified by the Association for Assessment and Accreditation of Laboratory Animal Care.

[0263] PD-L1 Blocked In vitro evaluation of PD-L1 blockade was performed using the PD-1 / PD-L1 blockade bioassay (Promega Corporation) according to the manufacturer's instructions. Briefly, PD-L1+ aAPC / CHO-K1 cells were seeded and acclimated for 16 hours. Then, the indicated concentrations of antibody or ADC were added to the seeded cells, followed by the addition of PD-1+ effector cells. In the absence of PD-1 / PD-L1 signaling, the interaction between aAPC / CHO-K1 cells and effector cells results in a bioluminescent signal. Therefore, more effective inhibition of the PD-1 / PD-L1 interaction results in a higher luminescence signal, quantified as fold induction compared to untreated cells. A PD-1-binding antibody (Promega Corporation) was included as a positive control, and an unbinding isotype antibody was included as a negative control.

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

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

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

[0267] The binding affinity of SG-559-xx antibodies or ADCs to glycosylated or deglycosylated PD-L1 was determined by biolayer interferometry using the Octet Red 384 system (ForteBio). Following a subsequent baselineization step, glycosylated and deglycosylated human PD-L1 at concentrations ranging from 500 nM to 0.69 nM (1×PBS pH 7.4 containing 1% BSA and 0.2% Tween-20) were incubated with loading probes for 150 seconds for the association step. Subsequently, a dissociation step was performed for 1000 seconds in the same buffer lacking human PD-L1. association and k dissociation This was then fitted to the bond curve obtained according to an established method.

[0268] result Example 1: Design and characterization of SG-559-xx antibodies As described in the method, 17 SG-559-xx antibodies were produced from the parental Ab1 antibody. The CDRs, including the mutations of these antibodies, are shown in Table 1. Sixteen of these antibodies were evaluated for their monovalent binding affinity to hPD-L1 by biolayer interferometry compared to Ab1 (Table 2). The measured affinities of the SG-559-xx antibodies ranged in approximately two-order-of-magnitude ranges, and K D The values ​​range from 4nM to 297nM. [Table 1] [Table 2]

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

[0270] Four SG-559-xx antibodies that consistently showed the highest efficacy in initial screening were selected. Their potency was further characterized as vc-MMAE and MP-PEG8-VKG-camptothecin ADCs (Table 3). In most cell lines tested, these ADCs were significantly more potent than Ab1. They also lacked activity in antigen-negative cell lines (SU-DHL-4), suggesting that this was not due to nonspecific binding. [Table 3]

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

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

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

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

[0275] Example 6: In vitro immunotoxicity of human APC SG-559-01 and SG-559-01 LALA were evaluated for immunotoxicity against APCs (e.g., macrophages and dendritic cells (DCs)). APCs were stimulated with IFNγ to upcontrol PD-L1 levels before treatment as described in the methods. SG-559-01 LALA ADCs showed immunotoxicity against human APCs similar in size to or within an order of magnitude of the isotype control in both macrophages and DCs (Figures 10A-10D).

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

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

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

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

[0280] Example 10: Binding affinity for glycosylated PD-L1 and deglycosylated PD-L1 The binding affinity of SG-559-01 to the glycosylated and deglycosylated forms of PD-L1 was evaluated. As described in the methods, the binding affinity was evaluated using biolayer interferometry on an Octet Red 384 system (ForteBio). The study evaluated the binding affinity of SG-559-01 to deglycosylated PD-L1 and control glycosylated PD-L1 (treated under the same conditions as described in the methods, but without PNGase F treatment). A difference of approximately twofold was observed in the binding affinity of SG-559-01 to deglycosylated PD-L1 compared to glycosylated PD-L1 (Table 5). The glycosylation status of PD-L1 was verified using mass spectrometry. [Table 5]

[0281] Unofficial sequence list SEQ ID NO: 1-Ab1 Heavy Chain Variable Region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS Sequence ID 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 Sequence ID 7-Ab1 Light Chain CDR2 Protein DASNRAT SEQ ID NO: 8-Ab1 Light Chain CDR3 Protein QQRSNWPT Sequence ID 9-SG-559-01 LALA hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 10-SG-559-01 Kappa light chain protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11-SG-559-01 Heavy Chain Variable Region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS Sequence ID 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 Sequence ID 16-SG-559-01 Light Chain CDR1 Protein RASQSVSSYLA Sequence ID 17-SG-559-01 Light Chain CDR2 Protein DASNRAT Sequence ID 18-SG-559-01 Light Chain CDR3 Protein QQRSNWPT Sequence ID 19-SG-559-02 LALA hIgG monoheavy-chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 20-SG-559-02 Kappa light chain protein EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 21-SG-559-02 Heavy Chain Variable Region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQA PGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS Sequence ID 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 Sequence ID 26-SG-559-02 Light Chain CDR1 Protein RASQSVSSALA Sequence ID 27-SG-559-02 Light Chain CDR2 Protein DASNRAT Sequence ID 28-SG-559-02 Light Chain CDR3 Protein QQRSNWPT Sequence ID 29-SG-559-03 LALA hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 30-SG-559-03 Kappa light chain protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNLPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence ID 31-SG-559-03 Heavy Chain Variable Region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS Sequence ID 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 Sequence ID 36-SG-559-03 Light Chain CDR1 Protein RASQSVSSYLA Sequence ID 37-SG-559-03 Light Chain CDR2 Protein DASNRAT Sequence ID 38-SG-559-03 Light Chain CDR3 Protein QQRSNLPT Sequence ID 39-SG-559-04 LALA hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 40-SG-559-04 Kappa light chain protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 41-SG-559-04 Heavy Chain Variable Region - Protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSS Sequence ID 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 Sequence ID 46-SG-559-04 Light Chain CDR1 Protein RASQSVSSYLA Sequence ID 47-SG-559-04 Light Chain CDR2 Protein DASNRAT Sequence ID 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 Sequence ID 56-SG-559-12 Light Chain CDR3 Protein QQASNWPT Sequence ID 57-SG-559-13 Light Chain CDR3 Protein QQKSNWPT Sequence ID 58-SG-559-14 Light Chain CDR3 Protein QQRSAWPT Sequence ID 59-SG-559-15 Light Chain CDR3 Protein QQRSQWPT Sequence ID 60-SG-559-16 Light Chain CDR3 Protein QQRSNAPT Sequence ID 61-SG-559-17 Light Chain CDR3 Protein QQRSNFPT Sequence ID 62-SG-559-01 hIgG1 heavy-chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 63-SG-559-02 hIgG monoheavy-chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 64-SG-559-03 hIgG monoheavy-chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 65-SG-559-04 hIgG monoheavy-chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 66-SG-559-01 Variable heavy chain region - nucleic acid caggtccagctggtgcagtctggggctgaggtgaagaagcctggggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcaccgccgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcac agaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca Sequence ID 67-SG-559-01 Variable light chain region - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 68 - SG - 559 - 02 Variable Heavy Chain Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO: 69 - SG - 559 - 02 Variable Light Chain Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcgccttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 70 - SG - 559 - 03 Variable Heavy Chain Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO: 71 - SG - 559 - 03 Variable Light Chain Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaacctgccgacgttcggccaagggaccaaggtggaaatcaaa SEQ ID NO: 72-SG-559-04 Variable Heavy Chain Region - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagcggcttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca SEQ ID NO: 73-SG-559-04 Variable Light Chain Region - Nucleic Acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa Sequence number: 74 - SG - 559 - 01 LALA hIgG1 heavy chain - nucleic acid SEQ ID NO: 75-SG-559-01 κ light chain - nucleic acid gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtc tgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt SEQ ID NO: 76-SG-559-01 hIgG1 heavy chain - nucleic acid Sequence ID 77-SG-559-02 LALA hIgG1 heavy-chain nucleic acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcc 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 caggtccagctggtgcagtctggggctgaggtgaagaagc Accession No. 80-SG-559-03 LALA hIgG1 Heavy Chain - Nucleic Acid caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggagccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagca acaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaagctgctgggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtttacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa 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 caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcacctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctcagctagcaccaagggcccatctgtcttccccctggcaccctcctccaagagcacctctgggggcacagctgccctgggctgcctggtcaaggactacttccctgaacctgtgacagtgtcctggaactcaggagccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactc cctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa Sequence number 83 - SG - 559 - 04 LALA hIgG1 heavy chain - nucleic acid ggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa 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 cgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtggggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccg acggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacacagaagagcctctccctgtctccgggcaaa SEQ ID NO: 86-Ab1 hIgG1 heavy chain protein QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence ID 87-Ab1 Kappa Light Chain Protein EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. A nucleic acid encoding an antibody or its antigen-binding fragment, wherein the antibody or its antigen-binding fragment specifically binds to the human PD-L1 protein, and the antibody comprises a heavy chain variable region and a light chain variable region, 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 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.

2. A vector comprising the nucleic acid described in claim 1.

3. A host cell comprising the vector according to claim 2.

4. The host cell according to claim 3, wherein the host cell is a Chinese hamster ovary (CHO) cell.

5. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to the human PD-L1 protein, comprising culturing the host cells described in claim 3 or 4 under conditions suitable for the production of the antibody.

6. A method for producing an antibody-drug conjugate that specifically binds to the human PD-L1 protein, comprising culturing the host cells described in claim 3 or 4 under conditions suitable for antibody production, and conjugating the antibody to a cytotoxic agent.

7. The method according to claim 6, wherein the cytotoxic agent is MMAE or camptothecin.

Images