Anti-PD-L1 antibody and antibody-drug conjugate

Abstract

A novel anti-PD-L1 antibody and antibody-drug conjugate and a method for using the anti-PD-L1 antibody and antibody-drug conjugate to treat cancer are provided.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. Patent Application No. 62 / 910,988, filed on October 4, 2019, the full text of which is incorporated herein by reference.

[0003] Technology field

[0004] The present invention relates to a novel anti-PD-L1 antibody and an antibody-drug conjugate, and a method of using the anti-PD-L1 antibody and the antibody-drug conjugate to treat cancer. Background Technology

[0005] PD-L1, also known as programmed death-ligand 1, B7-H1, or CD274, is a protein that has been shown to be expressed in various cancer cells. PD-L1 is a transmembrane protein that can interact with PD-1 and acts as an "off" switch to inactivate T cells. PD-L1 is typically overexpressed on tumor cells, and binding to PD-1 allows the tumor to evade the T cell immune response.

[0006] 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 melanoma, and 59,800 died. The 5-year survival rate for stage IV disease is less than 10%, and the median survival is only 6 to 12 months. Therefore, there is a need for improved treatment for melanoma as well as other cancers that express PD-L1. One type of treatment for cancers expressing PD-L1 involves administering anti-PD-L1 antibodies as immunotherapy. Immuno-oncology is a promising field for cancer treatment, but there is room for improvement in current therapies.

[0007] All references cited herein, including patent applications, patent publications, and scientific literature, are incorporated herein by reference in their entirety as indicated by each individual reference specifically and individually.

[0008] summation

[0009] Anti-PD-L1 antibodies and PD-L1-designated antibody-drug conjugates (ADCs) are provided herein. In particular, PD-L1-designated camptothecin ADCs and MMAE ADCs are provided herein. Additionally, methods for using anti-PD-L1-designated antibodies and ADCs to treat PD-L1 expression disorders are provided herein. Preferred anti-PD-L1 antibodies exhibit binding affinities for human PD-L1 protein ranging from 3 nM to 300 nM. Other preferred anti-PD-L1 antibodies comprise heavy chain CDR sequences of SEQ ID NO: 3 to 5 and light chain CDR sequences of SEQ ID NO: 6 to 8, wherein the antibody comprises one or more amino acid substitutions within one or more of the CDRs. Another preferred anti-PD-L1 antibody comprises heavy chain CDR sequences of sequence identification numbers: 13 to 15 and light chain CDR sequences of sequence identification numbers: 16 to 18.

[0010] Additionally, an antibody or its antigen-binding fragment that specifically binds to a human PD-L1 protein, which exhibits a binding affinity for the human programmed death-ligand 1 (PD-L1) protein at 3 to 300 nM, is provided herein. In some embodiments, the antibody exhibits a binding affinity for the human PD-L1 protein at 3 to 15 nM.

[0011] In some embodiments, the antibody further exhibits a total internalization higher than the total internalization of Ab1. In some embodiments, the total internalization is an increase of 9% to 155% of the AUC compared to the AUC of Ab1. In some embodiments, the total internalization is determined by the FabFluor internalization test.

[0012] In some embodiments, the antibody further exhibits a x50 lower than the x50 of Ab1. In some embodiments, the antibody is conjugated to monomethylauristatin E (MMAE), where x50 is 3 ng / mL to 20 ng / mL in the MDA-MB-231 cell line.

[0013] In some embodiments, the antibody is conjugated to camptothecin, where x50 is 15 ng / mL to 55 ng / mL in the MDA-MB-231 cell line.

[0014] In some embodiments, the antibody comprises heavy chain CDR sequences of sequence identification numbers: 13 to 15 and light chain CDR sequences of sequence identification numbers: 16 to 18.

[0015] In some embodiments, the antibody comprises heavy chain CDR sequences of sequence identification numbers: 3 to 5 and light chain CDR sequences of sequence identification numbers: 6 to 8, wherein the antibody comprises one or more amino acid substitutions within one or more of the CDRs.

[0016] In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 80% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 90% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 95% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises the heavy chain variable region sequence of sequence identification number: 11 and the light chain variable region sequence of sequence identification number: 12.

[0017] In some embodiments, the antibody comprises the light chain of sequence identification number: 9 and the heavy chain of sequence identification number: 10.

[0018] In some embodiments, the fragment is a Fab, Fab', F(ab')2, Fab'-SH, Fv, Diabadi, linear antibody, or single-strand antibody fragment.

[0019] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0020] In some embodiments, the heavy chain constant region is of the IgG1 isotype.

[0021] In some embodiments, the antibody is a humanized or chimeric antibody.

[0022] In some embodiments, the antibody is conjugated to the cytotoxic agent through a linker.

[0023] In some embodiments, the antibody is conjugated to monomethylauristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure:

[0024]

[0025] In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0026] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker to form an antibody-drug conjugate having the following structure:

[0027]

[0028] In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0029] Additionally, the present invention provides an antibody or its antigen-binding fragment that specifically binds to a human PD-L1 protein, wherein the antibody comprises a heavy chain CDR sequence of sequence identification numbers: 3 to 5 and a light chain CDR sequence of sequence identification numbers: 6 to 8, and the antibody comprises one or more amino acid substitutions within one or more of the CDRs.

[0030] In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein of 3 to 300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein of 3 to 15 nM.

[0031] In some embodiments, the antibody further exhibits a total internalization higher than the total internalization of Ab1. In some embodiments, the total internalization is an increase of 9% to 155% of the AUC compared to the AUC of Ab1. In some embodiments, the total internalization is determined by a popfluoride internalization test.

[0032] In some embodiments, the antibody additionally exhibits a x50 higher than the x50 of Ab1.

[0033] In some embodiments, the antibody is conjugated to monomethylauristatin E (MMAE), where x50 is 3 ng / mL to 20 ng / mL in the MDA-MB-231 cell line.

[0034] In some embodiments, the antibody is conjugated to camptothecin, where x50 is 15 ng / mL to 55 ng / mL in the MDA-MB-231 cell line.

[0035] In some embodiments, the antibody comprises heavy chain CDR sequences of sequence identification numbers: 13 to 15 and light chain CDR sequences of sequence identification numbers: 16 to 18. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 80% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 90% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 95% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises the heavy chain variable region sequence of sequence identification number: 11 and the light chain variable region sequence of sequence identification number: 12.

[0036] In some embodiments, the antibody comprises the light chain of sequence identification number: 9 and the heavy chain of sequence identification number: 10.

[0037] In some embodiments, the fragment is a Fab, Fab', F(ab')2, Fab'-SH, Fv, Diabadi, linear antibody, or single-strand antibody fragment.

[0038] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0039] In some embodiments, the heavy chain constant region is of the IgG1 isotype.

[0040] In some embodiments, the antibody is a humanized or chimeric antibody.

[0041] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethylauristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure:

[0042]

[0043] In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0044] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker to form an antibody-drug conjugate having the following structure:

[0045]

[0046] In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0047] Additionally, an antibody or its antigen-binding fragment that specifically binds to a human PD-L1 protein is provided herein, wherein the antibody comprises the heavy chain CDR sequence of sequence identification numbers: 13 to 15 and the light chain CDR sequence of sequence identification numbers: 16 to 18.

[0048] In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein of 3 to 300 nM. In some embodiments, the antibody exhibits a binding affinity for human PD-L1 protein of 3 to 15 nM.

[0049] In some embodiments, the antibody further exhibits a total internalization higher than the total internalization of Ab1. In some embodiments, the total internalization is an increase of 9% to 155% of the AUC compared to the AUC of Ab1. In some embodiments, the total internalization is determined by a popfluoride internalization test.

[0050] In some embodiments, the antibody additionally exhibits a x50 higher than the x50 of Ab1.

[0051] In some embodiments, the antibody is conjugated to monomethylauristatin E (MMAE), where x50 is 3 ng / mL to 20 ng / mL in the MDA-MB-231 cell line.

[0052] In some embodiments, the antibody is conjugated to camptothecin, where x50 is 15 ng / mL to 55 ng / mL in the MDA-MB-231 cell line.

[0053] In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 80% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 90% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity with sequence identification number: 11 and a light chain variable region sequence having at least 95% sequence identity with sequence identification number: 12. In some embodiments, the antibody comprises the heavy chain variable region sequence of sequence identification number: 11 and the light chain variable region sequence of sequence identification number: 12.

[0054] In some embodiments, the antibody comprises the light chain of sequence identification number: 9 and the heavy chain of sequence identification number: 10.

[0055] In some embodiments, the fragment is a Fab, Fab', F(ab')2, Fab'-SH, Fv, Diabadi, linear antibody, or single-strand antibody fragment.

[0056] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0057] In some embodiments, the heavy chain constant region is of the IgG1 isotype.

[0058] In some embodiments, the antibody is a humanized or chimeric antibody.

[0059] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethylauristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure:

[0060]

[0061] In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0062] In some embodiments, the antibody is conjugated to camptothecin. In some embodiments, the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Lys-Gly linker. In some embodiments, the antibody is conjugated to camptothecin via a linker to form an antibody-drug conjugate having the following structure:

[0063]

[0064] In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0065] Additionally, the present invention provides an antibody or its antigen-binding fragment that specifically binds to a human PD-L1 protein, wherein the antibody is conjugated to camptothecin to form an antibody-drug conjugate, and the antibody-drug conjugate has the following structure:

[0066]

[0067] In the above formula, 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.

[0068] In some embodiments, the antibody-drug conjugate has the following structure:

[0069]

[0070] In some embodiments, p is in the range of 2 to 10.

[0071] Additionally, an antibody or its antigen-binding fragment that specifically binds to the human PD-L1 protein is provided herein, wherein the antibody exhibits a binding affinity for the human programmed death-ligand 1 (PD-L1) protein greater than that of Ab1. In some embodiments, the antibody exhibits a binding affinity greater than 2.7 nM.

[0072] In addition, the antibody k against the human programmed death-ligand 1 (PD-L1) protein is less than that of Ab1 assoc An antibody or its antigen-binding fragment that specifically binds to the human PD-L1 protein, representing, is provided herein. In some embodiments, the antibody is 5 x 10 5 M -1 s -1 k for human PD-L1 protein less than assoc It represents.

[0073] In addition, the antibody k against the human programmed death-ligand 1 (PD-L1) protein, which is larger than that of Ab1 dissoc An antibody or its antigen-binding fragment that specifically binds to the human PD-L1 protein, representing, is provided herein. In some embodiments, the antibody is 2 x 10 3 s -1 k for excess human PD-L1 protein dissoc It represents.

[0074] Additionally, an antibody-drug conjugate comprising an antibody or its antigen-binding fragment that specifically binds to a human PD-L1 protein, wherein the antibody comprises a heavy chain CDR sequence of sequence identification numbers: 3 to 5 and a light chain CDR sequence of sequence identification numbers: 6 to 8, wherein the antibody comprises one or more amino acid substitutions within one or more of the CDRs, wherein the antibody exhibits a binding affinity for a human PD-L1 protein of 5 nM to 15 nM, and wherein the antibody is conjugated to MMAE.

[0075] Additionally, an antibody-drug conjugate comprising an antibody or its antigen-binding fragment that specifically binds to a human PD-L1 protein, wherein the antibody comprises a heavy chain CDR sequence of sequence identification numbers: 3 to 5 and a light chain CDR sequence of sequence identification numbers: 6 to 8, wherein the antibody comprises one or more amino acid substitutions within one or more of the CDRs, wherein the antibody exhibits a binding affinity for a human PD-L1 protein of 5 nM to 15 nM, and wherein the antibody is conjugated to camptothecin.

[0076] In addition, a pharmaceutical composition comprising a therapeutically effective amount of the antibody described herein and pharmaceutically acceptable excipients is provided herein.

[0077] Additionally, a method for treating cancer in a subject is provided herein, comprising administering any of the antibodies described herein to the subject. In some embodiments, the subject is a human subject. In some embodiments, the cancer is melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0078] In addition, nucleic acids encoding any antibody described herein are provided herein.

[0079] Additionally, a vector comprising any nucleic acid described herein is provided herein.

[0080] Additionally, any host cell described herein comprising any nucleic acid described herein is provided herein. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0081] Additionally, the present invention provides a method for producing an antibody that specifically binds to a human PD-L1 protein or its antigen-binding fragment, comprising culturing any host cell described herein under conditions suitable for the production of the antibody.

[0082] Additionally, the present invention provides a method for producing an antibody-drug conjugate that specifically binds to human PD-L1 protein, comprising culturing any host cell described herein under conditions suitable for the production of an antibody; and conjugating the antibody to a cytotoxic agent. In some embodiments, the cytotoxic agent is MMAE or camptothecin.

[0083] Also, cancer (e.g., PD-L1 + Uses of any anti-PD-L1 antibody or any antibody-drug conjugate described herein for use in the manufacture of medicines for the treatment of cancers associated with expression are provided herein.

[0084] Also, cancer (e.g., PD-L1 + The anti-PD-L1 antibody or the antibody-drug conjugate described herein for use in the treatment of cancer associated with expression) is provided herein.

[0085] Additionally, the anti-PD-L1 antibody or the antibody-drug conjugate described herein for medical use is provided herein.

[0086] In addition, PD-L1 comprising administering to a subject a therapeutically effective amount of any anti-PD-L1 antibody or any antibody-drug conjugate described herein. + PD-L1 in subjects requiring cell killing + A method for killing cells is provided at this institution.

[0087] Also, PD-L1 + PD-L1 in subjects requiring cell killing + Uses of any anti-PD-L1 antibody or any antibody-drug conjugate described herein for use in the manufacture of a drug for killing cells are provided herein.

[0088] In addition, a solid tumor (e.g., PD-L1) in a subject comprising administering to the subject a therapeutically effective amount of any anti-PD-L1 antibody or any antibody-drug conjugate described herein. + A method for reducing the volume of a solid tumor is provided at this institution.

[0089] In addition, solid tumors in subjects (e.g., PD-L1 + The use of any anti-PD-L1 antibody or any antibody-drug conjugate described herein for use in the manufacture of medicines for reducing the volume of solid tumors is provided herein. Brief explanation of the drawing

[0090] Figure 1 shows example amino acid residues of Ab1 selected for the mutation. Figures 2a to 2f show the cytotoxicity of SG-559-xx ADC in several cell lines. Figures 3a and 3b show the internalization of SG-559-01 and SG-559-03 compared to the control antibody. Figures 4a and 4b show the anti-tumor activity of SG-559-xx ADC in an MDA-MB-231 mouse model. Figures 5a and 5b show the anti-tumor activity of SG-559-xx ADC in a BxPC3 mouse model. Figures 6a and 6b show the anti-tumor activity of SG-559-01 LALA ADC in a Karpas 299 mouse model. Figure 7 shows the anti-tumor activity of SG-559-01 LALA ADC in a Calu-1 mouse model. Figures 8a and 8b show the anti-tumor activity of SG-559-01 LALA ADC in an EBC-1 mouse model. Figure 9 shows the in vitro PD-1 / PD-L1 blocking activity of the SG-559-01 LALA antibody and ADC. Figures 10a to 10d show the immunotoxicity of SG-559-01 and SG-559-01 LALA ADC in a human APC model. Figures 11a to 11d show the immunotoxicity of SG-559-xx ADC in a human APC model. Figures 12a to 12d show the immune response of human APCs treated with SG-559-01 ADC to LPS stimulation in vitro. Figures 13a to 13c show intratumoral immune cell infiltration in mice with Carpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. Figures 14a to 14f show the intratumoral inflammatory cytokine response in mice with Carpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADC. Specific details for implementing the invention

[0091] I. Definition

[0092] To facilitate a better understanding of the present disclosure, specific terms are defined first. As used in this application, each of the following terms will have the meaning set forth below, except as otherwise clearly provided herein. Additional definitions are provided throughout this application.

[0093] As used herein, the term “and / or” is taken as a specific disclosure of each of two specific features or components with or without the other. Accordingly, the term “and / or” as used herein in phrases such as “A and / or B” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0094] It is understood that the aspects and embodiments of the invention described herein include aspects and embodiments that are “comprising,” “constituted,” and “essentially constituted.”

[0095] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the relevant technical field to which this disclosure relates. For example, the literature [Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press]; [The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press]; and [Oxford Dictionary of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press] provides a general dictionary of many terms used in this disclosure to a person skilled in the art.

[0096] Units, prefixes, and symbols are indicated in their Systeme International de Unites (SI) accepted forms. Numerical values ​​include numbers that limit a range. The headings provided herein are not a limitation to the various aspects of the disclosure that may be taken by reference to the entire specification. Accordingly, terms defined immediately below are more fully defined by reference to the entire specification.

[0097] 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 homologues of human PD-L1 that are generally expressed by cells or expressed on cells transfected with the PD-L1 gene.

[0098] The term "immunoglobulin" refers to a class of structurally related glycoproteins composed of two pairs of polypeptide chains, consisting of one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. The structures of immunoglobulins have been widely characterized. For example, refer to the literature [Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989))]. Briefly, each heavy chain typically has a heavy chain variable region (V in this application). H or abbreviated as VH) and heavy chain invariant region (C H It consists of or CH). The heavy chain invariant region is typically composed of three domains, namely, C H 1, C H 2, and C H It consists of 3. The heavy chains are generally interconnected via disulfide bonds at the so-called "hinge region." Each light chain is typically in the light chain variable region (V in this application). L or abbreviated as VL) and light chain invariant region (C LIt consists of or CL). The light chain invariant region is typically one domain, i.e., C L It consists of. CL may be of the κ (kappa) or λ (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. Immunoglobulin may be derived from any commonly known isotype, including but not limited to IgA, secreted IgA, IgG, and IgM. IgG subclasses are also widely known to those skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3, and IgG4. "Isotype" refers to a class of antibodies or subclasses (e.g., IgM or IgG1) encoded by the heavy chain constant region gene.

[0099] The term "variable region" or "variable domain" refers to the domain of the antibody heavy or light chain involved in binding the antibody to an antigen. Variable regions of the heavy and light chains of natural antibodies (V, respectively) H and V LThe ) may be further subdivided into a region of supervariability, termed the complementarity-determining region (CDR), which is interposed with a more conserved region, termed the framework region (FR) (or a supervariability region that may be supervariable in the form of a sequence and / or structure of a structurally confined loop). The terms "complementarity-determining region" and "CDR," which are synonymous with "supervariability region" or "HVR," are known in the art to refer to non-contiguous sequences of amino acids within an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are 4 FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and 4 FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region. Each V H and V L Within, 3 CDRs and 4 FRs are typically arranged from amino-terminal to carboxy-terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (also in the literature [Chothia and Lesk J. Mot. Biol [See ., 195, 901-917 (1987)]).

[0100] In the context of the present invention, the term “antibody” (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof having the ability to specifically bind to an antigen under typical physiological conditions having a significant period, e.g., a half-life of at least about 30 minutes, at least about 45 minutes, at least about 1 hour (h), at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours (h), at least about 24 hours, at least about 48 hours, at least about 3, 4, 5, 6, 7 days, etc., or any other related functionally defined period (e.g., sufficient time to induce, promote, enhance, or modulate the physiological response associated with antibody binding to the antigen and / or sufficient time for the antibody to mobilize effector activity). Variable regions of the heavy chain and light chain of the immunoglobulin molecule contain binding domains that interact with the antigen. The constant region of an antibody (Ab) can mediate the binding of immunoglobulin to various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation, to host tissues or factors. The antibody may also be a bispecific antibody, a diabodi, a multispecific antibody, or a similar molecule.

[0101] As used herein, the term "monoclonal antibody" refers to a preparation of an antibody molecule produced recombinantly with a single primary amino acid sequence. Monoclonal antibody compositions exhibit single-binding specificity and affinity for specific epitopes. Accordingly, the term "human monoclonal antibody" refers to an antibody exhibiting single-binding specificity having variable and constant regions derived from human germline immunoglobulin sequences. Human monoclonal antibodies may be produced by a hybridoma comprising B cells obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome containing human heavy chain transgenes and light chain transgenes fused to immortalized cells.

[0102] "Isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind to antigens other than PD-L1). However, an isolated antibody that specifically binds to PD-L1 may be cross-reactive to other antigens, such as PD-L1 molecules from different species. Furthermore, the isolated antibody may be substantially free of other cellular substances and / or chemicals. In one embodiment, the isolated antibody comprises an antibody conjugate attached to another agent (e.g., a small molecule drug). In some embodiments, the isolated anti-PD-L1 antibody comprises a conjugate of the anti-PD-L1 antibody to a small molecule drug (e.g., MMAE or MMAF).

[0103] "Human antibody" (HuMAb) refers to an antibody having a variable region in which both the FR and CDR are derived from the human wiring immunoglobulin sequence. Furthermore, if the antibody contains a constant region, the constant region is also derived from the human wiring immunoglobulin sequence. The human antibodies of this disclosure may contain amino acid residues not encoded by the human wiring immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which a CDR sequence derived from the wiring of another mammalian species, e.g., mouse, is grafted onto the human framework sequence. The terms "human antibody" and "fully human antibody" are used synonymously.

[0104] As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to contain a high degree of sequence homology to the human variable domain. This can be achieved by grafting six non-human antibody complementation-determining regions (CDRs) that together form an antigen binding site onto a homologous human recipient framework region (FR) (see WO92 / 22653 and EP0629240). To fully reconstruct the binding affinity and specificity of the parent antibody, substitution (reverse-mutation) of framework residues from the parent antibody (i.e., non-human antibody) into the human framework region may be required. Structural homology modeling can help identify amino acid residues in the framework region that are critical to the binding properties of the antibody. Thus, the humanized antibody may contain a non-human CDR sequence, one or more amino acid reverse-mutations, primarily of randomly non-human amino acid sequences, and a human framework region containing a completely human constant domain. Optionally, additional amino acid modifications that are not necessarily reverse mutations may be applied to obtain humanized antibodies having desirable characteristics, such as affinity and biochemical properties.

[0105] As used herein, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species (e.g., rodents) and the constant region is derived from a different species, e.g., humans. Chimeric antibodies may be produced by antibody manipulation. "Antibody manipulation" is a term used comprehensively for different types of antibody modification and is a process widely known to those skilled in the art. In particular, chimeric antibodies [Sambrook et al.It can be generated by standard DNA techniques as described in [Molecular Cloning: A Laboratory Manual, 1989, New York: Cold Spring Harbor Laboratory Press, Ch. 15]. Thus, the chimeric antibody may be a genetically or enzymatically engineered recombinant antibody. Generating a chimeric antibody is within the knowledge of a person skilled in the art, and therefore, the generation of a chimeric antibody according to the present invention may be carried out by methods other than those described herein. Chimeric monoclonal antibodies for therapeutic applications that reduce antibody immunogenicity have been developed. These may typically contain a non-human (e.g., murine) variable region specific to the antigen of interest, and human constant antibody heavy and light chain domains. The terms "variable region" or "variable domain" as used in the context of chimeric antibodies refer to a region comprising the CDR and framework regions of both the heavy and light chains of the immunoglobulin.

[0106] "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.

[0107] The “antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen bound by the whole antibody. Examples of antibody fragments (e.g., antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabadies; linear antibodies; single-stranded antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of the antibody produces two identical antigen-binding fragments, each referred to as the “Fab” fragment having a single antigen-binding site and the remaining “Fc” fragment, the name of which reflects the ability to easily crystallize. Pepsin treatment produces an F(ab')2 fragment having two antigen-binding sites and still capable of cross-linking the antigen.

[0108] With respect to a reference polypeptide sequence, “percent (%) sequence identity” is defined as the percentage of amino acid residues in candidate sequences identical to amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve maximum percentage sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percentage amino acid sequence identity may be achieved in various ways within the art of the relevant art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A person 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 with respect to, with respect to, or with respect to a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A having or containing a specific % sequence identity with respect to, with respect to, or with respect to a given amino acid sequence B) is calculated as follows:

[0109] 100 multiplied by fraction X / Y

[0110] In the above formula, X is the number of amino acid residues scored as identical matches based on the sequences in the alignment of the programs of A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equivalent to the length of amino acid sequence B, it will be acknowledged that the % sequence identity of A with respect to B will not be equivalent to the % sequence identity of B with respect to A.

[0111] In the context of the binding of an antibody to a predetermined antigen, the terms “bind,” “bind,” or “specifically bind” as used herein typically refer to about 10 when determined, for example, by biolayer interferometric (BLI) techniques on an Octet HTX instrument using an antibody as the ligand and an antigen as the analyte. -6 M or less, e.g., 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 even about 10 -11 K less than or equal to M D It is a binding having an affinity corresponding to, where the antibody is the K of binding to a non-specific antigen (e.g., BSA, casein) other than a predetermined antigen or a closely related antigen. D K that is at least 10 times lower than, for example, at least 100 times lower, for example, at least 1,000 times lower, for example, at least 10,000 times lower, for example, at least 100,000 times lower. D It binds to a predetermined antigen with an affinity corresponding to . Binding K D The lower amount is the antibody's K D Since it depends on, the antibody's K D When the value is very low, the K of binding to the antigen D K of binding to non-specific antigens D A lower amount can be at least 10,000 times (i.e., antibodies are highly specific).

[0112] The term "K" used in this document D " (M) refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. The affinities, and K used herein D They have an inverse relationship; that is, a higher affinity corresponds to a lower K D It is intended to refer to, and lower affinity is higher K D It is intended to refer to.

[0113] The term "ADC" refers to an antibody-drug conjugate, which refers to an anti-PD-L1 antibody coupled to a drug moiety (e.g., MMAE or MMAF) as described in this application in the context of the present invention.

[0114] The abbreviations "vc" and "val-cit" refer to the dipeptide linker valine-citrulline.

[0115] The abbreviation VKG refers to the tripeptide linker valine-lysine-glycine.

[0116] The abbreviation "MC" refers to stretcher maleimidocaproil:

[0117]

[0118] The abbreviation "MP" refers to the stretcher maleimidopropionil:

[0119]

[0120] As used herein, the "PEG unit" is an organic moiety composed of repeating ethylene-oxy subunits (PEG or PEG subunits) and may be polydisperse, monodisperse, or separable (i.e., having distinct numbers of ethylene-oxy subunits). Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, whereas monodisperse PEG is typically purified from a heterogeneous mixture and thus provides a single chain length and molecular weight. Preferred PEG units include separable PEG, which is a compound synthesized in a stepwise manner and not synthesized through a polymerization process. Separable PEG provides a single molecule having a limited and specified chain length.

[0121] The PEG units provided herein comprise one or more polyethylene glycol chains, each consisting of one or more ethyleneoxy subunits covalently attached to one another. The polyethylene glycol chains may be linked together, for example, in a linear, branched, or star-shaped arrangement. Typically, at least one of the polyethylene glycol chains prior to incorporation into the camphothecin conjugate is derivatized at one end to an alkyl moiety (i.e., an example of R) substituted with an electrophilic group for covalent attachment to the carbamate nitrogen of the methylene carbamate unit. Typically, the terminal ethyleneoxy subunit in each polyethylene glycol chain that does not participate in covalent attachment to the remainder of the linker unit is modified to a PEG capping unit, typically an optionally substituted alkyl, such as -CH3, CH2CH3, or CH2CH2CO2H. The preferred PEG unit has a single polyethylene glycol chain having 2 to 24 -CH2CH2O- subunits that are covalently attached in series and terminated at one end as PEG capping units.

[0122] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancer tissue" may include tumors. Uncontrolled cell division and growth result in the formation of malignant tumors that can invade neighboring tissues and also metastasize to distant parts of the body via the lymphatic system or bloodstream. After metastasis, the distal tumor may be described as originating from the "pre-metastatic tumor."

[0123] The term "antibody-dependent cytotoxicity," or ADCC, is a mechanism that induces apoptosis relying on the interaction of antibody-coated target cells with lytically active immune cells (also referred to as effector cells). These effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to the Fc effector domain(s) of Ig bound to target cells via their antigen-coating sites. The death of antibody-coated target cells occurs as a result of effector cell activity.

[0124] The term "antibody-dependent cellular phagocytosis," or ADCP, refers to the process in which antibody-coated cells are wholly or partially internalized by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to the Fc effector domain(s) of Ig.

[0125] The term "complement-dependent cytotoxicity," or CDC, refers to a mechanism in which the Fc effector domain(s) of a target-bound antibody activate a series of enzymatic reactions that ultimately induce apoptosis, leading to the formation of holes in the target cell membrane. Typically, antigen-antibody complexes, such as those on antibody-coated target cells, bind to and activate the complement component C1q, which in turn activates the complement cascade, resulting in target cell death. Complement activation can also cause the deposition of complement components on the surface of target cells, which facilitates ADCC, by binding to complement receptors on leukocytes (e.g., CR3).

[0126] "Cell arrest effect" refers to the inhibition of cell proliferation. "Cell arrest agent" refers to an agent that inhibits the growth and / or expansion of a specific subset of cells by having a cell arrest effect on the cells. Cell arrest agents may be conjugated to antibodies or administered in combination with antibodies.

[0127] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject, or the administration of an activator to a subject, for the purpose of reversing, alleviating, improving, suppressing, slowing down, or preventing the onset, progression, development, severity, or recurrence of symptoms, complications, conditions, or biochemical signs associated with the disease. In some embodiments, the disease is cancer.

[0128] “Subject” includes any human or non-human animal. The term “non-human animal” includes, but is not limited to, vertebrates, e.g., non-human primates, sheep, dogs, and rodents, e.g., mice, rats, and guinea pigs. In some embodiments, the subject is a human. The terms “subject,” “patient,” and “individual” are used interchangeably herein.

[0129] The “effective dose,” “therapeutic effective dose,” or “therapeutic effective dosage” of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, promotes disease regression, as evidenced by protecting the subject from the onset of the disease, reducing the severity of disease symptoms, increasing the frequency and duration of disease symptom-free periods, or preventing damage or disability caused by disease suffering. The ability of a therapeutic agent to promote disease regression may be evaluated using various methods known to ordinary practitioners, for example, in human subjects during clinical trials, in animal model systems that serve as predictors of efficacy in humans, or by testing the activity of the agent in in vitro assays.

[0130] As an example of the treatment of a tumor, a therapeutically effective dose of an anticancer agent inhibits cell growth or tumor growth in treated subjects(s) (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% compared to untreated subjects(s) (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective dose of an anticancer agent inhibits cell growth or tumor growth in treated subjects(s) (e.g., one or more treated subjects) by 100% compared to untreated subjects(s).

[0131] In another embodiment of the present disclosure, tumor regression may be observed and continued for a period of 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.

[0132] A therapeutically effective dose of a drug (e.g., an anti-PD-L1 antibody-drug conjugate) includes a “preventively effective dose,” which is any amount of the drug that inhibits the development or recurrence of cancer when administered alone or in combination with an anticancer agent to a subject at risk of developing cancer (e.g., a subject with a premature state) or experiencing a recurrence of cancer. In some embodiments, the preventively effective dose completely prevents the development or recurrence of cancer. “Inhibiting” the development or recurrence of cancer means reducing the likelihood of the development or recurrence of cancer, or completely preventing the development or recurrence of cancer.

[0133] As used herein, "sub-therapeutic dose" refers to a dose of a therapeutic compound (e.g., anti-PD-L1 antibody-drug conjugate) lower than the usual or typical dose of the therapeutic compound when administered alone for the treatment of proliferative diseases (e.g., cancer).

[0134] "Immune-related response patterns" refer to clinical response patterns often observed in cancer patients treated with immunotherapies that generate antitumor effects by inducing cancer-specific immune responses or by modifying natural immune processes. These response patterns are characterized by beneficial therapeutic effects followed by an early increase in tumor burden or the appearance of new lesions, which would be classified as disease progression and synonymous with drug failure in the evaluation of traditional chemotherapy. Therefore, the proper evaluation of immunotherapies may require long-term monitoring of the effects of these agents on the target disease.

[0135] For example, an "anticancer agent" promotes cancer regression 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 administering an effective dose of the drug, either alone or in combination with an anticancer agent, results in a reduction in tumor growth or size, tumor necrosis, a reduction in the severity of at least one disease symptom, an increase in the frequency and duration of symptom-free periods, or the prevention of damage or disability caused by disease suffering. Additionally, regarding treatment, the terms "effective" and "effectiveness" include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of the drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other harmful physiological effects (adverse effects) at the cellular, organ, and / or organism level resulting from the administration of the drug.

[0136] "Sustained response" refers to a sustained effect on reducing tumor growth after discontinuation of treatment. For example, the tumor size may remain the same or smaller compared to the size at the start of administration. In some embodiments, the sustained response has a duration at least equal to the duration of treatment, or at least 1.5, 2.0, 2.5, or 3 times longer than the duration of treatment.

[0137] As used herein, “complete response” or “CR” refers to the disappearance of all target lesions; “partial response” or “PR” refers to a reduction of at least 30% of the sum of the longest diameters (SLD) of the target lesions, with reference to the baseline SLD; and “stable disease” or “SD” refers to no sufficient shrinkage of the target lesions qualifying for PR and no sufficient increase qualifying for PD, with reference to the smallest SLD since the start of treatment.

[0138] As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not worsen. Progression-free survival may include the amount of time during which the patient experienced a complete or partial response, as well as the amount of time during which the patient experienced stable disease.

[0139] As used herein, "Total Reaction Rate" or "ORR" refers to the sum of the complete reaction (CR) rate and the partial reaction (PR) rate.

[0140] As used herein, "total survival" or "OS" refers to the percentage of individuals in a population likely to be alive after a specific duration of time.

[0141] The phrase “pharmaceutical acceptable” indicates that the substance or composition must be chemically and / or toxicologically miscible with other components including the formulation and / or mammals treated with it.

[0142] As used herein, the phrase "pharmaceutical acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of the compound of the present invention. Exemplary salts include sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucoronate, saccharate, formate, benzoate, glutamate, methanesulfonate "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate, pamoate (i.e., 4,4'-methylene-bis-(2-hydroxy-3-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts are included but not limited thereto. Pharmaceutically acceptable salts may involve the inclusion of another molecule, e.g., acetate ions, succinate ions, or other counterions. Counterions may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, pharmaceutically acceptable salts may have more than one charged atom in their structure. If multiple charged atoms are part of the pharmaceutically acceptable salt, they may have multiple counterions. Thus, pharmaceutically acceptable salts may have one or more charged atoms and / or one or more counterions.

[0143] “Administering” or “administration” refers to the physical introduction of a therapeutic agent into a subject using any various methods and delivery systems known to a person skilled in the art. Exemplary routes of administration for anti-PD-L1 antibody-drug conjugates include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral administration routes by, for example, injection or infusion (e.g., intravenous infusion). As used herein, the term “parenteral administration” means a mode of administration other than intestinal and local administration, typically by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intradural, intralymphatic, intralesional, intra-articular, intra-orbital, intra-cardiac, intradermal, intraperitoneal, trans-tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intravertebral, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. The therapeutic agent may be administered via non-parenteral routes or orally. Other parenteral routes include routes of topical, epidermal, or mucosal administration, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration may also be performed, e.g., once, multiple times, and / or over one or more extended periods.

[0144] The terms “baseline” or “baseline value” as used interchangeably herein may refer to the measurement or characterization of symptoms prior to administration of a therapy (e.g., an anti-PD-L1 antibody-drug conjugate as described herein) or at the start of administration of the therapy. The baseline value may be compared to a reference value to determine a reduction or improvement in symptoms of a PD-L1-associated disease (e.g., cancer) considered herein. The terms “reference” or “reference value” as used interchangeably herein may refer to the measurement or characterization of symptoms after administration of a therapy (e.g., an anti-PD-L1 antibody-drug conjugate as described herein). The reference value may be measured one or more times during a dose regimen or treatment cycle or at the completion of a dose 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:average value; or a value compared to the baseline value.

[0145] Similarly, "baseline 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: average value; or a value compared to a reference value. The reference value and / or baseline value may be obtained from a single object, from two different objects, or from a group of objects (e.g., a group of 2, 3, 4, 5 or more objects).

[0146] 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 the treatment cycle. However, other therapeutic agents may be administered to the subject. For example, anti-inflammatory agents or other agents administered to a subject with cancer to treat symptoms associated with the cancer but not the underlying cancer itself, such as inflammation, pain, weight loss, and general problems, may be administered during the period of monotherapy.

[0147] As used herein, “Adverse Event” (AE) is any undesirable and generally unintended or undesirable sign (including abnormal laboratory results), symptom, or condition associated with the use of a medical treatment. A medical treatment may have one or more associated AEs, and each AE may have the same or different levels of severity. Reference to a method that may “change an adverse event” means a treatment regimen that reduces the occurrence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0148] As used herein, "Serious adverse event" or "SAE" is an adverse event that meets one of the following criteria:

[0149] · As used in the definition of a serious adverse event, "fatal or life-threatening" refers to an event in which the patient was at risk of death at the time of the event; this does not refer to an event that could have hypothetically caused death in more severe cases.

[0150] · Causes persistent or significant impairment / disability

[0151] · Constitutes a congenital abnormality / birth defect

[0152] · Medically significant, that is, defined as an event that may require medical or surgical intervention to endanger the patient or prevent one of the outcomes listed above. Medical and scientific judgment must be trained in determining whether an AE is "medically significant."

[0153] · Requiring admission of an inpatient or extension of existing hospitalization, excluding the following: 1) routine treatment or monitoring of an underlying condition not associated with any deterioration of the condition; 2) selection or pre-planned treatment for a pre-existing condition unrelated to the indications under study or not deteriorated since the informed consent was signed; and 3) social reasons and temporary treatment in the absence of any deterioration of the patient's general condition.

[0154] The use of alternatives (e.g., "or") should be understood to mean one, both, or any combination thereof. The indefinite article used herein should be understood to refer to "one or more" of any listed or enumerated components.

[0155] The terms “about” or “essentially containing” refer to a value or composition that is within an acceptable margin of error for a specific value or composition as determined by a person skilled in the art who relies in part on the manner in which the value or composition is measured or determined, namely, the limitations of the measurement system. For example, “about” or “essentially containing” may mean within one or more than one standard deviation according to the practice of the art. Alternatively, “about” or “essentially containing” may mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the terms may mean up to one digit of the value or up to five times. Where a specific value or composition is provided in this application and claims, unless otherwise stated, the meaning of “about” or “essentially containing” should be assumed to be within an acceptable margin of error for that specific value or composition.

[0156] References to "about" a value or parameter herein include (and describe) an embodiment relating to the value or parameter itself. For example, a description referring to "about X" includes and describes "X".

[0157] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the listed range, and, where appropriate, its fraction (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise indicated.

[0158] Various aspects of the present disclosure are further described in detail in the following subsections.

[0159] II. General Information

[0160] The present invention provides antibodies and ADCs that specifically bind to PD-L1. The present invention is based on the finding that antibody-drug conjugates, including MMAE antibody-drug conjugates and camptothecin antibody-drug conjugates partially targeted at PD-L1, are particularly effective in killing PD-L1+ expressing cells. PD-L1 has been shown to be expressed in various cancers, including melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer.

[0161] III. Target Molecule

[0162] Unless otherwise indicated, PD-L1 refers to human PD-L1. An exemplary human protein sequence is assigned UniProt identification number Q9NZQ7.

[0163] IV. Antibodies of the present invention

[0164] Previously, selected antibodies already used to treat cancer were conjugated to cytotoxic agents without sequence modification to produce antibody-drug conjugates (ADCs). These ADCs have often proven effective in killing tumor cells or even more effective than unconjugated antibodies. Previously, when modifications to antibodies were considered during the ADC manufacturing process, some possible modifications involved increasing the antibody's binding affinity or enhancing antibody activity, such as ADCC. However, in at least some situations, modifying or adjusting the ADC antibody—for example, by decreasing its binding affinity or reducing its ADCC activity—results in an improvement in the efficacy of the ADC compared to an ADC with an unmodified antibody. Some examples of this include ADCs with surprisingly optimized anti-PD-L1 antibodies (e.g., Ab1) by modifying the antibodies, for instance, by reducing their binding affinity. For instance, 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 the cytotoxic agent is reduced. In another example, in some cases, anti-PD-L1 ADCs are more effective at killing tumor cells in vitro and in vivo when the binding affinity of the antibody conjugated to the cytotoxic agent is reduced.

[0165] The present invention provides an antibody that binds to PD-L1 with a binding affinity of 3 nM to 300 nM, such as a humanized antibody. In some embodiments, the antibody described herein is 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, about 40 nM to about 100 nM, about 40 nM to about 90 nM, about 40 nM to about 80 nM, about 40 nM to about 70 nM, about 40 nM to about 60 nM, about 40 nM to about 50 nM, about 50 nM to about 300 nM, about 50 nM to about 275 nM, about 50 nM to about 250 nM, about 50 nM to about 225 nM, about 50 nM to about 200 nM, about 50 nM to about 175 nM, about 50 nM to about 150 nM, about 50 nM to about 125 nM, about 50 nM to about 100 nM, about 50 nM to about 90 nM, about 50 nM to about 80 nM, about 50 nM to about 70 nM, about 50 nM to about 60 nM,About 60 nM to about 300 nM, about 60 nM to about 275 nM, about 60 nM to about 250 nM, about 60 nM to about 225 nM, about 60 nM to about 200 nM, about 60 nM to about 175 nM, about 60 nM to about 150 nM, about 60 nM to about 125 nM, about 60 nM to about 100 nM, about 60 nM to about 90 nM, about 60 nM to about 80 nM, about 60 nM to about 70 nM, about 70 nM to about 300 nM, about 70 nM to about 275 nM, about 70 nM to about 250 nM, about 70 nM to about 225 nM, about 70 nM to about 200 nM, about 70 nM to about 175 nM, about 70 nM to about 150 nM, about 70 nM to about 125 nM, about 70 nM to about 100 nM, about 70 nM to about 90 nM, about 70 nM to about 80 nM, about 80 nM to about 300 nM, about 80 nM to about 275 nM, about 80 nM to about 250 nM, about 80 nM to about 225 nM, about 80 nM to about 200 nM, about 80 nM to about 175 nM, about 80 nM to about 150 nM, about 80 nM to about 125 nM, about 80 nM to about 100 nM, about 80 nM to about 90 nM, about 90 nM to about 300 nM, about 90 nM to about 275 nM, about 90 nM to about 250 nM, about 90 nM to about 225 nM, about 90 nM to about 200 nM, about 90 nM to about 175 nM, about 90 nM to about 150 nM, about 90 nM to about 125 nM, about 90 nM to about 100 nM, about 100 nM to about 300 nM, about 100 nM to about 275 nM, about 100 nM to about 250 nM, about 100 nM to about 225 nM, about 100 nM to about 200 nM,About 100 nM to about 175 nM, about 100 nM to about 150 nM, about 100 nM to about 125 nM, about 125 nM to about 300 nM, about 125 nM to about 275 nM, about 125 nM to about 250 nM, about 125 nM to about 225 nM, about 125 nM to about 200 nM, about 125 nM to about 175 nM, about 125 nM to about 150 nM, about 150 nM to about 300 nM, about 150 nM to about 275 nM, about 150 nM to about 250 nM, about 150 nM to about 225 nM, about 150 nM to about 200 nM, about 150 nM to about 175 nM, about 175 nM to about 300 nM, about 175 nM to about 275 nM, about 175 nM to about 250 nM, about 175 nM to about 225 nM, about 175 nM to about 200 nM, about 200 nM to about 300 nM, about 200 nM to about 275 nM, about 200 nM to about 250 nM, about 200 nM to about 225 nM, about 225 nM to about 300 nM, about 225 nM to about 275 nM, about 225 nM to about 250 nM, about 250 nM to about 300 nM, about 275 nM to about 275 nM, about 225 nM to about 250 nM, about 250 nM to about 300 nM, about 250 nM to about 275 nM, or about 275 nM to about K of 30 nM) (e.g., as measured by biolayer interferometry (BLI) in phosphate-buffered saline), D It can bind to PD-L1.

[0166] In some embodiments, binding affinity 1 is the binding affinity. In some embodiments, these antibodies are entirely point mutants of the human anti-PD-L1 antibody Ab1. Ab1 is defined by the CDR region of SEQ ID NOs: 3 to 5 and SEQ ID NOs: 6 to 8, the variable region 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 mutant is found in the CDR region. In some embodiments, the point mutant exhibits reduced binding affinity and / or increased cytotoxicity and / or internalization rate compared to Ab1. In some embodiments, the point mutant exhibits reduced binding affinity and increased cytotoxicity in vitro. In some embodiments, the point mutant exhibits reduced binding affinity and increased cytotoxicity in vivo. In some embodiments, the point mutant exhibits reduced binding affinity and increased cytotoxicity both in vitro and in vivo. In some embodiments, the point mutant exhibits reduced binding affinity and increased internalization rate in vitro. In some embodiments, the point mutant exhibits reduced binding affinity and increased internalization rate in vivo. In some embodiments, the point mutant exhibits reduced binding affinity and increased internalization rate both in vitro and in vivo.

[0167] In some embodiments, the anti-PD-L1 antibody provided herein may have one or two total amino acid substitutions in a set of six CDRs of heavy chain CDRs of sequence identification no. 3 to 5 and light chain CDRs of sequence identification no. 6 to 8, and binds to PD-L1 with 3 nM to 300 nM of KD. In some embodiments, the anti-PD-L1 antibody provided herein may have one amino acid substitution in a set of six CDRs of heavy chain CDRs of sequence identification no. 3 to 5 and light chain CDRs of sequence identification no. 6 to 8, and binds to PD-L1 with 3 nM to 300 nM of KD.

[0168] In some embodiments, the anti-PD-L1 antibody provided herein may have a heavy chain CDR1 having one amino acid substitution at sequence number: 3, a heavy chain CDR2 at sequence number: 4, a heavy chain CDR3 at sequence number: 5, a light chain CDR1 at sequence number: 6, a light chain CDR2 at sequence number: 7, and a light chain CDR3 at sequence number: 8, and binds to PD-L1 at 3 nM to 300 nM of KD. In some embodiments, one amino acid substitution at sequence number: 3 is at amino acid position 2 of sequence number: 3. In some embodiments, one amino acid substitution at amino acid position 2 of sequence number: 3 is an amino acid substitution of tyrosine to alanine. In some embodiments, one amino acid substitution at amino acid position 2 of sequence number: 3 is an amino acid substitution of tyrosine to serine. In some embodiments, one amino acid substitution at amino acid position 2 of sequence identification number: 3 is an amino acid substitution of tyrosine to glycine. In some embodiments, one amino acid substitution at amino acid position 2 of sequence identification number: 3 is an amino acid substitution of tyrosine to threonine. In some embodiments, one amino acid substitution at amino acid position 2 of sequence identification number: 3 is an amino acid substitution of tyrosine to valine. In some embodiments, one amino acid substitution at amino acid position 2 of sequence identification number: 3 is an amino acid substitution of tyrosine to cysteine.

[0169] In some embodiments, the anti-PD-L1 antibody provided herein is K at a concentration of 3 nM to 300 nM (or any sub-range of this range described herein). D It binds to both glycosylated and non-glycosylated PD-L1.

[0170] In some embodiments, the anti-PD-L1 antibody provided herein increases 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% to 300% increase, 5% to 280% increase, 5% to 260% increase, 5% to 240% increase, 5% 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, 60% increase to 260% increase, 60% increase to 240% increase, 60% increase to 220% increase, 60% increase to 200% increase, 60% increase to 180% increase, 60% increase to 160% increase, 60% increase to 140% increase, 60% increase to 120% increase, 60% increase to 100% increase, 60% increase to 80% increase, 80% increase to 300% increase, 80% increase to 280% increase, 80% increase to 260% increase, 80% increase to 240% increase, Increase by 80% to 220%, increase by 80% to 200%, increase by 80% to 180%, increase by 80% to 160%, increase by 80% to 140%, increase by 80% to 120%, increase by 80% to 100%, increase by 100% to 300%, increase by 100% to 280%, increase by 100% to 260%, increase by 100% to 240%, increase by 100% to 220%, increase by 100% to 200%, increase by 100% to 180%,100% increase to 160% increase, 100% increase to 140% increase, 100% increase to 120% increase, 120% increase to 300% increase, 120% increase to 280% increase, 120% increase to 260% increase, 120% increase to 240% increase, 120% increase to 220% increase, 120% increase to 200% increase, 120% increase to 180% increase, 120% increase to 160% increase, 120% increase to 140% increase, 140% increase to 300% increase, 140% increase to 280% increase, 140% increase to 260% increase, 140% increase to 240% increase, 140% increase to 220% increase, 140% increase to 200% increase, 140% increase to 180% increase, 140% increase to 160% increase, 160% increase to 300% increase, 160% increase to 280% increase, 160% increase to 260% increase, 160% increase to 240% increase, 160% increase to 220% increase, 160% increase to 200% increase, 160% increase to 180% increase, 180% increase to 300% increase, 180% increase to 280% increase, 180% increase to 260% increase, 180% increase to 240% increase, 180% increase to 220% increase, 180% increase to 200% increase, 200% increase to 300% increase, 200% increase to 280% increase, 200% 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) has in vitro and / or in vivo cytotoxicity.

[0171] In some embodiments, the anti-PD-L1 antibody provided herein has an increased (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 (or any sub-range of this range described herein)) cell internalization rate by PD-L1+ cells compared to Ab1.

[0172] In some embodiments, the anti-PD-L1 antibody provided herein has increased immune cell infiltration upon administration to mammals 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 (or any sub-range of this range described herein)).

[0173] In some embodiments, the anti-PD-L1 antibody provided herein has increased (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% to 300% increase (or any sub-range of this range described herein)) inflammatory cytokine production (e.g., one or more of any cytokines described herein).

[0174] In some embodiments, the anti-PD-L1 antibody provided herein has increased (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 (or any sub-range of this range described herein)) intracellular digestion by PD-L1+ cells compared to Ab1.

[0175] In some embodiments, the anti-PD-L1 antibody provided herein has a change of less than 10% in neutrophil and / or platelet counts (e.g., a change of less than 8%, a change of less than 6%, a change of less than 4%, a change of less than 2%, or a change of less than 1%) when administered to mammals compared to Ab1.

[0176] Binding affinity of the PD-L1 antibody of the present invention (i.e., dissociation constant, K D ) is preferably larger than that of Ab1. The preferred PD-L1 antibody binds to and / or competes with the same epitope as 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 additional embodiments, k assoc is 5.5 x 10 5 M -1 s -1 It is less than. In another embodiment, k dissoc (or off-rate) is greater than that of Ab1. In further embodiments, k dissoc is 1.50 x 10 3 s -1 It is an excess.

[0177] The anti-PD-L1 antibodies of the present invention may also be described or specified in terms of their binding affinity to PD-L1 (e.g., human PD-L1). In some embodiments, preferred binding affinities are 2.7 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 200 nM, 250 nM, 300 nM, 400 nM, or dissociation constants or K greater than 500 nM. D Includes those having. In some embodiments, the preferred PD-L1 antibody has a binding affinity of 3 nM to 300 nM, 3 nM to 200 nM, 3 nM to 100 nM, 3 nM to 50 nM, 3 nM to 40 nM, 3 nM to 20 nM, 3 nM to 15 nM, 5 nM to 300 nM, and 5 nM to 15 nM. In some embodiments, the preferred PD-L1 antibody has a binding affinity that is at least 2, 3, 3.7, 4, or 5 times greater than the binding affinity of Ab1. In some of the embodiments, the binding affinity is a monovalent binding affinity.

[0178] In some embodiments, the binding of the anti-PD-L1 antibody of the present invention is pH-dependent, so that the antibody exhibits differential binding over a pH gradient. In some embodiments, the anti-PD-L1 antibody exhibits maximum binding at a pH of about 4 to about 10. In some embodiments, maximum binding is at a pH of about 6 to about 9. In some embodiments, maximum binding is at a pH of about 6.5 to about 8.

[0179] The preferred antibodies of the present invention inhibit cancer (e.g., cell growth, metastasis, and / or lethality to the organism) as indicated for cancerous cells proliferating in culture in animal models or clinical trials. Animal models may be formed by inserting PD-L1-expressing human tumor cell lines into suitable immunodeficient rodent strains, e.g., athymic nude mice or SCID mice. These tumor cell lines may be established in immunodeficient rodent hosts as solid tumors by subcutaneous injection or as disseminated tumors by intravenous injection.

[0180] Once established within a host, these tumor models can be applied to evaluate the therapeutic efficacy of anti-PD-L1 antibodies or their conjugated forms as described in the examples.

[0181] Generally, the anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate of the present disclosure binds to PD-L1, e.g., human PD-L1, and exerts cell arrest and cytotoxic effects on malignant cells, e.g., cancer cells. The concentration required to produce a 50% reduction in viability compared to untreated cells, or x50, or IC 50This is one method of measuring the cytotoxicity of anti-PD-L1 antibodies and / or anti-PD-ADCs. The 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 a x50 of 10 ng / mL to 30 ng / mL, or 15 ng / mL to 25 ng / mL, in BXPC3 cell lines. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits a x50 of 15 ng / mL to 55 ng / mL, or 20 ng / mL to 50 ng / mL, in MDA-MB-231 cell lines. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits a x50 of 1 ng / mL to 7 ng / mL or 2 ng / mL to 5 ng / mL in the Carpas 299 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to vcMMAE of the present invention exhibits a x50 of 15 ng / mL to 40 ng / mL or 20 ng / mL to 35 ng / mL in the L540CY cell line. In one embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits a x50 of 12 ng / mL to 70 ng / mL or 15 ng / mL to 65 ng / mL in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits an x50 of 3 ng / mL to 20 ng / mL, or 5 ng / mL to 17 ng / mL, in the MDA-MB-231 cell line. In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits an x50 of 1 ng / mL to 18 ng / mL, or 3 ng / mL to 15 ng / mL, in the Carpas 299 cell line.In another embodiment, the anti-PD-L1 antibody conjugated to camptothecin of the present invention exhibits x50 of 1 ng / mL to 20 ng / mL, or 1 ng / mL to 15 ng / mL in L540CY cell lines.

[0182] Generally, the anti-PD-L1 antibody and / or anti-PD-L1 antibody-drug conjugate of the present disclosure is internalized into cells, such as cancer cells. One method of measuring internalization is to use 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 of the fluorescent signal over time (or AUC). The popfluor (IncuCyte®) internalization assay can be used for this quantification. The preferred antibody and / or ADC of the present invention exhibits increased total internalization compared to that of Ab1 and / or the ADC. In one embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase of 9% to 155% in AUC compared to the AUC of Ab1. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 40% to 130%, or 40% to 50%, compared to the AUC of Ab1, as tested in the 786-O cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 90% to 100%, or 90% to 95%, compared to the AUC of Ab1, as tested in the A375 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 85% to 155%, or 85% to 90%, compared to the AUC of Ab1, as tested in the BXPC3 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 9% to 40%, or 9% to 13%, compared to the AUC of Ab1, as tested in the ES-2 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present invention exhibits an increase in AUC of 75% to 145%, or 75% to 80%, compared to the AUC of Ab1, as tested in the MDA-MB-231 cell line.

[0183] The anti-PD-L1 antibody of the present disclosure is preferably monoclonal and may be a multispecific, human, humanized or chimeric antibody, a single-strand antibody, a Fab fragment, an F(ab') fragment, a fragment produced by a Fab expression library, and a PD-L1 binding fragment of any of the above. In some embodiments, the anti-PD-L1 antibody of the present disclosure specifically binds to PD-L1. The immunoglobulin molecule of the present disclosure may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of an immunoglobulin molecule. In one embodiment, the anti-PD-L1 antibody of the present disclosure is of the IgG1 type.

[0184] In specific embodiments of the present disclosure, the anti-PD-L1 antibody is an antigen-binding fragment (e.g., human antigen-binding fragment) as described herein, and Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibody, disulfide-linked Fv (sdFv) and V L or V H The antigen-binding fragment comprising any one of the domains is included, but is not limited thereto. An antigen-binding fragment comprising a single-strand antibody may comprise variable region(s) alone or in combination with all or part of the following: a hinge region, CH1, CH2, CH3, and CL domains. Additionally, an antigen-binding fragment comprising any combination of the hinge region, CH1, CH2, CH3, and CL domains and the variable region(s) is included in the present disclosure. In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment is human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken.

[0185] The anti-PD-L1 antibodies of the present disclosure may be monospecific, bispecific, trispecific, or of greater multiple specificity. Multiple specific antibodies may be specific to different epitopes of PD-L1, or may be specific to both PD-L1 and heterologous proteins. For example, PCT publications WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; literature [Tutt, et al. , 1991, J. Immunol. 147:60 69]; U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Literature [Kostelny et al. See , 1992, J. Immunol. 148:1547 1553.

[0186] The anti-PD-L1 antibodies of this disclosure may be described or specified in terms of the specific CDRs they comprise. The exact amino acid sequence boundaries of a given CDR or FR are referenced in the literature [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. , (1997) JMB 273,927-948] ("Chothia" numbering scheme); [MacCallum et al. , J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding 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 scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70], ("Aho" numbering scheme); and [Martin et al. It can be easily determined using any number of widely known schemes, including those described in ["Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23):9268-9272], ("AbM" numbering scheme). The boundaries of a given CDR may vary depending on the scheme used for identification. In some embodiments, the "CDR" or "complementarity determining region" of a given antibody or its region (e.g., its variable region), or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass one (or specific) CDR as defined by any of the aforementioned schemes. For example, a specific CDR (e.g., CDR-H3) given V H or V LWhen a region is stated to contain the amino acid sequence of a corresponding CDR, it is understood that such a CDR has the sequence of a corresponding CDR (e.g., CDR-H3) within a variable region, as defined by any of the aforementioned schemes. A scheme for identifying a specific CDR or CDRs, such as those defined by the Kavat, Cotia, AbM, or IMGT methods, can be specified.

[0187] The CDR sequences of the anti-PD-L1 antibody and the anti-PD-L1 antibody-drug conjugate described herein are from the literature [Kabat et al. It follows the kavat numbering scheme as described in [(1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD].

[0188] In one aspect, an anti-PD-L1 antibody and / or an 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 sequence identification number:13, (ii) CDR-H2 comprising the amino acid sequence of sequence identification number:14, and (iii) CDR-H3 comprising the amino acid sequence of sequence identification number:15; and the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of sequence identification number:16, (ii) CDR-L2 comprising the amino acid sequence of sequence identification number:17, and (iii) CDR-L3 comprising the amino acid sequence of sequence identification number:18, and the CDR of the anti-PD-L1 antibody is defined by the kavat numbering scheme.

[0189] In one aspect, an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate is provided herein, comprising a heavy chain variable domain having the amino acid sequence of sequence identification number: 11 and a light chain variable domain having the amino acid sequence of sequence identification number: 12. In one aspect, an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate is provided herein, comprising a heavy chain having the amino acid sequence of sequence identification number: 9 and a light chain having the amino acid sequence of sequence identification number: 10.

[0190] In some embodiments, an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate is provided herein, comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO:11. In certain embodiments, 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 the amino acid sequence of SEQ ID NO:11 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted from SEQ ID NO:11. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the CDR (i.e., in the FR). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain sequence of sequence identification number:11, which includes a post-translational modification of the sequence.

[0191] In some embodiments, an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate is provided herein, comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 12. In certain embodiments, 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 the amino acid sequence of SEQ ID NO:12 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted from SEQ ID NO:12. In certain embodiments, the substitutions, insertions, or deletions (e.g., 1, 2, 3, 4, or 5 amino acids) occur in a region outside the CDR (i.e., in the FR). In some embodiments, the anti-PD-L1 antibody comprises the light chain variable domain sequence of sequence identification number:12, which includes a post-translational modification of the sequence.

[0192] In some embodiments, an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate is provided herein, comprising a heavy chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of sequence identification number:1. In certain embodiments, 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 the amino acid sequence of SEQ ID NO:1 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted from SEQ ID NO:1. In certain embodiments, the heavy chain contains one point mutation relative to SEQ ID NO:1. In additional embodiments, the one point mutation is located in the CDR region.

[0193] In some embodiments, an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate is provided herein, comprising a light chain variable domain comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence of sequence identification number:2. In certain embodiments, 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 the amino acid sequence of SEQ ID NO:2 contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence and retains the ability to bind to PD-L1 (e.g., human PD-L1). In certain embodiments, a total of 1 to 10 amino acids are substituted, inserted, or deleted from SEQ ID NO:2. In certain embodiments, the light chain contains one point mutation relative to SEQ ID NO:2. In additional embodiments, the one point mutation is located in the CDR region.

[0194] In some embodiments, the anti-PD-L1 antibody or the anti-PD-L1 antibody of the anti-PD-L1 antibody-drug conjugate is a monoclonal antibody.

[0195] There are five classes of immunoglobulins having heavy chains designated as α, δ, ε, γ, and μ, respectively: IgA, IgD, IgE, IgG, and IgM. The γ and α classes are further divided into subclasses, and, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. IgG1 antibodies may exist as multiple polymorphic variants termed as homozygotes (see reference [Jefferis and Lefranc 2009. mAbs(Reviewed in Vol 1 Issue 4 1-7), any of these are suitable for use in parts of the embodiments of the present invention. Common homozygous variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any embodiment of the present invention, the antibody may comprise a heavy chain Fc region comprising a human IgG Fc region. In further embodiments, the human IgG Fc region comprises human IgG1.

[0196] The antibody also includes derivatives modified, that is, derivatives formed by the covalent attachment of any type of molecule to the antibody such that the covalent attachment does not prevent the antibody from binding to PD-L1 or from exerting a cell arrest or cytotoxic effect on the cell. For example (but not limited to), antibody derivatives include antibodies modified by derivatization, for example, glycosylation, acetylation, PEGylation, phosphorylation, amidation, known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any number of chemical modifications may be performed by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

[0197] Humanized antibodies

[0198] Humanized antibodies are genetically engineered antibodies in which a CDR from a non-human "donor" antibody is grafted into a human "recipient" antibody sequence (see, e.g., Queen, US 5,530,101 and 5,585,089; Winter, US 5,225,539; Carter, US 6,407,213; Adair, US 5,859,205; and Foote, US 6,881,557). The recipient antibody sequence may be, for example, a mature human antibody sequence, a composite of such sequences, a consensus sequence of human antibody sequences, or a wiring region sequence. A preferred recipient sequence for the heavy chain is wiring V H Exxon V H 1-2 (also referred to as HV1-2 in the literature) and (Shin et al., 1991, EMBO J. 10:3641-3645), hinge region (J H Regarding ), Exxon J H-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred recipient sequence is exon VK2-30 (also referred to as 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). Thus, the humanized antibody is an antibody having all or substantially all of the CDRs from the donor antibody, and, where present, the variable region framework sequence and the constant region from all or substantially the human antibody sequence. Similarly, the humanized heavy chain has at least one, two, and typically all three CDRs from all or substantially the donor antibody heavy chain, and, where present, the heavy chain variable region framework sequence and the heavy chain constant region from substantially the human heavy chain variable region framework and constant region sequence. Similarly, the humanized light chain has at least one, two, and typically all three CDRs from the donor antibody light chain, either wholly or substantially, and, if present, a light chain variable region framework sequence and a light chain constant region from a substantially human light chain variable region framework and constant region sequence. In addition to nanobodies and dAbs, the humanized antibody comprises a humanized heavy chain and a humanized light chain. The CDRs in the humanized antibody are substantially from the corresponding CDRs in the non-human antibody if at least 60%, 85%, 90%, 95%, or 100% of the corresponding residues (as defined by the kavat) are identical between the respective CDRs. The variable region framework sequence of the antibody chain or the constant region of the antibody chain is substantially from the human variable region framework sequence or the human constant region, respectively, if at least 85%, 90%, 95%, or 100% of the corresponding residues defined by the kavat are identical. In some embodiments, the PD-L1 antibody of the present invention is a humanized antibody.

[0199] Humanized antibodies often incorporate all six CDRs from mouse antibodies (preferably as defined by kavat), but they can also be produced to have fewer than all CDRs from mouse antibodies (e.g., at least three, four, or five) CDRs (e.g., literature [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]).

[0200] Selection of invariant regions

[0201] The heavy and light chain variable regions of the humanized antibody may 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 cellular phagocytosis, and / or complement-dependent cytotoxicity are desired. For example, human isotype IgG1 and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity, and human IgG4 lacks complement-dependent cytotoxicity. Human IgG1 and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region may be lambda or kappa. The antibody may be expressed as distinct heavy and light chains, as Fab, Fab', F(ab')2, and Fv, or as a tetramer containing two light chains and two heavy chains as a single-chain antibody in which the heavy and light chain variable domains are linked via a spacer.

[0202] Human invariant regions exhibit homozygous and isozygous variations among different individuals; that is, invariant regions may differ in different individuals at one or more polymorphic sites. Isozygous differs from invariant in that serum recognizing isozygous binds to one or more non-polymorphic regions of different isotypes.

[0203] One or several amino acids at the amino or carboxyl terminus of the light and / or heavy chains, such as C-terminal lysine of the heavy chain, may be lost or derivatized in part or all of the molecule. Substitutions may be made in the invariant region to decrease or increase effector function, such as complement-mediated cytotoxicity or ADCC (e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and see Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend the half-life in humans (e.g., see Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0204] Exemplary substitutions include amino acid substitution of a natural amino acid with a cysteine ​​residue introduced at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332, preferably the S239C mutation in human IgG1 isotype (US 20100158909). The presence of additional cysteine ​​residues allows for the formation of interchain disulfide bonds. Such interchain disulfide bond formation can reduce the affinity of Fc domain-FcyR binding interactions by causing steric hindrance. Cysteine ​​residue(s) introduced into or adjacent to the Fc region of the IgG constant region may also function as a site for conjugation to therapeutic agents (i.e., coupling cytotoxic drugs using thiol-specific reagents, e.g., 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 positions 234, 235, 236, and / or 237 reduce the affinity for Fey receptors, particularly FcyRI receptors {see, e.g., US 6,624,821, US 5,624,821).

[0205] 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 regulates the catabolism of IgG and their passage and exocytosis 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 enables IgG to be regenerated into circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The regions on human IgG1 involved in FcRn binding have been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitution at human IgG1 sites Pro238, Thr256, Thr307, Gln311, Asp312, Glu380, Glu382, or Asn434 promotes FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgG1 molecules having these substitutions have a longer serum half-life. Consequently, these modified IgG1 molecules can perform their effector functions over a longer period compared to unmodified IgG1 and thus exert their therapeutic efficacy. Other exemplary substitutions to increase binding to FcRn include Gin at position 250 and / or Leu at position 428. EU numbering is used for all positions in the invariant region.

[0206] Oligosaccharides covalently attached to conserved Asn297 are involved in the ability of the Fc domain of IgG 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 glycotype on IgG can significantly improve IgG-mediated ADCC. The addition of a bisected 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 the removal of fucose from this glycoform (Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc manipulation that enhances Ig-mediated ADCC activity by improving the binding between IgG Fc and FcyR.

[0207] Systemic substitution of solvent-exposed amino acids in the human IgG1 Fc region produced IgG variants with altered FcyR binding affinities (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Compared to parental IgG1, subsets of these variants involving substitutions for Ala at Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 Lys334 demonstrate increased binding affinities for both FcγR and ADCC activity (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

[0208] The complement fixation activity of antibodies (both C1q binding and CDC activity) can be improved by substitutions at Lys326 and Glu333 (Idusogie et al., 2001, J. Immunol. 166:2571-2575). The same substitutions on the human IgG2 backbone can convert antibody isotypes that bind poorly to C1q and are severely deficient in complement activation activity into those capable of both binding to C1q and mediating 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 onto the carboxyl-terminus of IgG significantly enhances their CDC activity. This is observed even in IgG4, which typically lacks detectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Furthermore, substituting Ser444, located near the carboxy-terminus of the IgG1 heavy chain, with Cys induced 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). Additionally, bispecific diabody constructs with specificity for C1q also confer CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0209] Complement activity can be reduced by mutating at least one of the heavy chain amino acid residues 318, 320, and 322 to a residue having a different side chain, e.g., Ala. Instead of any one of the three residues, other alkyl-substituted non-ionic residues, e.g., Gly, Leu, or Val, or aromatic non-polar residues such as Phe, Tyr, Trp, and Pro can also reduce or eliminate C1q bonds. Ser, Thr, Cys, and Met can be used at residues 320 and 322 (but not 318) to reduce or eliminate C1q bond activity.

[0210] Substitution of residue 318 (Glu) with a polar residue modifies C1q binding activity but may not eliminate it. Replacing residue 297 (Asn) with Ala results in the elimination of lytic activity but only slightly reduces the affinity for C1q (about 3 times weaker). This alteration destroys the glycosylation site and the presence of carbohydrates required for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combinations thereof also reduce C1q binding: D270A, K322A, P329A, and P31 IS (see WO 06 / 036291). The L234A / L235A mutation (or LALA mutation) also reduces C1q binding, as well as FcyR binding. In one embodiment, the anti-PD-L1 antibody of the present invention contains the L234A / L235A mutation.

[0211] References to human invariant regions include invariant regions having any natural homozygos or any permutation of residues occupying polymorphic positions in natural homozygos. Additionally, up to 1, 2, 5, or 10 mutations, such as those indicated above that decrease Fc gamma receptor binding or increase binding to FcRN, may exist relative to natural human invariant regions.

[0212] V. Expression of Recombinant Antibodies

[0213] Humanized antibodies are typically produced by recombinant expression. A recombinant polynucleotide construct typically comprises an expression control sequence operably linked to the coding sequence of an antibody chain, which includes a naturally-associated or heterologous promoter region. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting eukaryotic host cells. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high-level expression of the nucleotide sequence and for the collection and purification of cross-reactive antibodies.

[0214] Mammalian cells are preferred hosts for expressing nucleotide fragments encoding immunoglobulin or fragments thereof. See the literature [Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987)]. A number of suitable host cell lines capable of secreting intact xenoproteins have been developed in the relevant art and include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myeloma including Sp2 / 0 and NS0. Preferably, the cells are non-human. Expression vectors for these cells may include expression control sequences, such as a replication origin, promoter, enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and essential processing information sites, such as a ribosome binding site, RNA splice site, polyadenylation site, and transcription terminator sequence. Desirable expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papillomavirus, etc. See literature [Co et al., J. Immunol. 148:1149 (1992)].

[0215] Once expressed, the antibody can be purified according to standard procedures in the relevant technical field, including HPLC purification, column chromatography, and gel electrophoresis (generally, see [Scopes, Protein Purification (Springer-Verlag, NY, 1982)]).

[0216] VI. Nucleic Acids

[0217] The present invention further provides a nucleic acid encoding any of the humanized heavy and light chains described above. Typically, the nucleic acid also encodes a signal peptide fused to the mature heavy and light chains. A coding sequence on the nucleic acid may be operablely linked to a regulatory sequence, such as a promoter, enhancer, ribosome binding site, transcription termination signal, etc., to ensure the expression of the coding sequence. The nucleic acid encoding the heavy and light chains may occur in an isolated form or may be cloned into one or more vectors. The nucleic acid may be synthesized, for example, by solid-state synthesis or by PCR of overlapping oligonucleotides. The nucleic acid encoding the heavy and light chains may be bound, for example, as an adjacent nucleic acid within an expression vector, or may be separate, for example, each cloned into its own expression vector.

[0218] In some aspects, a nucleic acid encoding an anti-PD-L1 antibody or its antigen-binding fragment as described herein is also provided herein. Additionally, a vector comprising a nucleic acid encoding an anti-PD-L1 antibody or its antigen-binding fragment as described herein is provided herein. Additionally, a host cell expressing a nucleic acid encoding an anti-PD-L1 antibody or its antigen-binding fragment as described herein is provided herein. Additionally, a host cell comprising a vector comprising a nucleic acid encoding an anti-PD-L1 antibody or its antigen-binding fragment as described herein is provided herein.

[0219] The anti-PD-L1 antibody described herein can be produced by widely known expression vector systems and widely known recombinant techniques using host cells. In one embodiment, the antibody is from the literature [De la Cruz Edmunds et al. , 2006, Molecular Biotechnology It is prepared in CHO cells using a GS expression vector system as disclosed in EP216846, U.S. Patent No. 5,981,216, WO 87 / 04462, EP323997, U.S. Patent No. 5,591,639, U.S. Patent No. 5,658,759, EP338841, U.S. Patent No. 5,879,936, and U.S. Patent No. 5,891,693.

[0220] The monoclonal anti-PD-L1 antibody described herein is, for example, from the literature [Kohler et al. , Nature Monoclonal antibodies can be produced by the hybridoma method first described by [ , 256, 495 (1975)] or by the recombinant DNA method. Monoclonal antibodies are also, for example, [Clackson et al. , Nature , 352, 624-628 (1991)] and [Marks et al., JMol, Biol Monoclonal antibodies can be isolated from a phage antibody library using the technique described in [., 222(3):581-597 (1991)]. Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from a hybridoma prepared from murine spleen B cells obtained from a mouse immunized with the antigen of interest, for example, in the form of a cell expressing an antigen on its surface, or in the form of a nucleic acid encoding the antigen of interest. Monoclonal antibodies can also be obtained from a hybridoma derived from antibody-expressing cells of immunized human or non-human mammals, such as rats, dogs, primates, etc.

[0221] Antibody-drug conjugate

[0222] Anti-PD-L1 antibodies can be conjugated to cytotoxic or cell-suspending moietyes (including their pharmaceutically miscible salts) to form antibody-drug conjugates (ADCs). Particularly suitable moietyes for conjugation to antibodies are cytotoxic agents (e.g., chemotherapy agents), prodrug-converting enzymes, radioisotopes or compounds, or toxins (these moietyes are collectively referred to as therapeutic agents). For example, anti-PD-L1 antibodies can be conjugated to cytotoxic agents, such as chemotherapy agents, or toxins (e.g., cell-suspending or cytotoxic agents, such as abrine, lysine A, Pseudomonas exotoxin, or diphtheria toxin).

[0223] Anti-PD-L1 antibodies can be conjugated to pro-drug converting enzymes. Pro-drug converting enzymes can be recombinantly fused to antibodies or chemically conjugated thereto using known methods. Exemplary pro-drug converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, β-lactamase, β-glucosidase, nitroreductase, and carboxypeptidase A.

[0224] The technology of conjugating therapeutic agents to proteins, and particularly to antibodies, is widely known. (See, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,” in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., Alan R. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,” in Controlled Drug Delivery (Robinson et al. eds., Marcel Dekker, Inc., 2nd ed., 1987)]; [Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological And Clinical Applications (Pinchera et al. eds., 1985)]; Cancer Detection And Therapy (Baldwin et al. [eds., Academic Press, 1985]; and see [Thorpe et al., 1982, Immunol. Rev. 62:119-58]. Also see, for example, PCT Publication WO 89 / 12624.)

[0225] The therapeutic agent may be conjugated in a manner that reduces the activity of the antibody unless it is cleaved from the antibody (e.g., by hydrolysis, by antibody degradation, or by a cleavage agent). Such a therapeutic agent is attached to an antibody with a cleavable linker that is sensitive to cleavage in the intracellular environment of PD-L1-expressing cancer cells but substantially not sensitive in the extracellular environment, such that the conjugate is cleaved from the antibody when the antibody is internalized by PD-L1-expressing cancer cells (e.g., in endosomes, or in a lysosome or vesicular environment, for example, by pH sensitivity or protease sensitivity).

[0226] Typically, an ADC includes a linker region between the therapeutic agent and the anti-PD-L1 antibody. As mentioned above, typically, the linker is cleavable under intracellular conditions so that the cleavage of the linker releases the therapeutic agent from the antibody in an intracellular environment (e.g., within a lysosome, endosome, or vesicle). The linker may be a peptidyl linker that is cleavable by intracellular peptidases or protease enzymes, including, for example, lysosome or endosome proteases. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. The cleavage agents may include cathepsin B and D and plasmin (see, for example, the literature [Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123]). Most typical examples are peptidyl linkers that are cleavable by enzymes present in PD-L1-expressing cells. For example, a peptidyl linker cleavable by cathepsin-B, a thiol-dependent protease highly expressed in cancerous tissues (e.g., a linker comprising a Phe-Leu or Gly-Phe-Leu-Gly peptide) may be used. Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In specific embodiments, the peptidyl linker cleavable by intracellular protease comprises a Val-Cit linker or a Phe-Lys dipeptide (e.g., see U.S. Patent 6,214,345 describing the synthesis of doxorubicin into a Val-Cit linker). One advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

[0227] Cleatable linkers can be pH-sensitive, that is, sensitive to hydrolysis at specific pH values. Typically, pH-sensitive linkers are hydrolyzable under acidic conditions. For example, acid-unstable linkers that are hydrolyzable in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic acid amide, orthoester, acetal, ketal, etc.) may be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; and literature [Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123]; and [Neville et al., 1989, Biol. Chem. 264: 14653-14661].) These linkers are relatively stable under neutral pH conditions, e.g., those in blood, but are unstable at pH 5.5 or below 5.0, which is approximately the pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to a therapeutic agent via an acylhydrazone linkage (e.g., see U.S. Patent No. 5,622,929)).

[0228] Other linkers are cleavable under reducing conditions (e.g., disulfide linkers). Disulfide linkers include SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), and those that can be formed using SPDB and SMPT. {e.g., reference [Thorpe et al., 1987, Cancer Res. 47:5924-5931]; [See Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (CW Vogel ed., Oxford U. Press, 1987). Also see U.S. Patent No. 4,880,935.]

[0229] The linker may also be a malonate linker (Johnson 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 malonate linker (Johnson 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).

[0230] The linker may also be a non-cleavable linker directly attached to the therapeutic agent (e.g., drug), such as a maleimido-alkylene- or maleimide-aryl linker. The active drug-linker is released by the degradation of the antibody.

[0231] Typically, the linker is not substantially sensitive to the extracellular environment, which means that when the ADC is present in the extracellular environment (e.g., plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, and more typically about 5% or less, about 3% or less, or about 1% or less of the linker in the sample of the ADC is cleaved.

[0232] Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by independently incubating both (a) an ADC ("ADC sample") and (b) equimolar amounts of unconjugated antibody or therapeutic agent ("control sample") with plasma for a predetermined period (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with that present in the control sample, as measured, for example, by high-performance liquid chromatography.

[0233] The linker can also promote cell internalization. The linker can promote cell internalization when conjugated to a therapeutic agent (i.e., in the context of a linker-therapeutic moiety of an ADC or ADC derivative as described herein). Alternatively, the linker can promote cell internalization when conjugated to both a therapeutic agent and an anti-PD-L1 antibody (i.e., in the context of an ADC as described herein).

[0234] Anti-PD-L1 antibodies can be conjugated to a linker via heteroatoms of the antibody. These heteroatoms may exist on the antibody in their natural state or may be introduced into the antibody. In some aspects, the anti-PD-L1 antibody will be conjugated to the linker via nitrogen atoms of lysine residues. In other aspects, the anti-PD-L1 antibody will be conjugated to the linker via sulfur atoms of cysteine ​​residues. Cysteine ​​residues may be naturally occurring or engineered into the antibody. Methods of conjugating linkers and drug-linkers to antibodies via lysine and cysteine ​​residues are known in the relevant art.

[0235] 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, interfere with microtubule dynamics and nuclear and cell division, and possesses anticancer activity. Typically, an auristatin-based antibody-drug conjugate comprises a linker between the auristatin drug and the anti-PD-L1 antibody. The linker may be, for example, a cleavable linker (e.g., peptidyl linker, carbohydrate linker) or a non-cleavable linker (e.g., a linker released by the degradation of the antibody). Auristatins include auristatin T, MMAF, and MMAE. Exemplary synthesis and structure of auristatin are described in U.S. Publications Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated herein by reference in its entirety and for all purposes.

[0236] Exemplary antibody-drug conjugates also include camptothecin-based antibody-drug conjugates (i.e., the drug component is the camptothecin drug). Camptothecin is a topoisomerase inhibitor that has been shown to possess anticancer activity. Typically, camptothecin-based antibody-drug conjugates include a linker between the camptothecin 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 a non-cleavable linker (e.g., a linker released by the degradation of the antibody). The synthesis and structure of an exemplary camptothecin drug-linker are described in PCT / US19 / 025968 (filed April 5, 2019), the full text of which is incorporated herein by reference for all purposes.

[0237] Other exemplary antibody-drug conjugates include metansinoid antibody-drug conjugates (i.e., the drug component is a metansinoid 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)).

[0238] An exemplary antibody-drug conjugate comprises the following vcMMAE and mcMMAF antibody-drug conjugates or their pharmaceutically acceptable salts, where p represents the drug load and Ab represents the anti-PD-L1 antibody:

[0239]

[0240]

[0241] An exemplary anti-PD-L1 antibody-drug conjugate comprises a camptothecin antibody-drug conjugate as follows, where p represents the drug load and Ab represents the anti-PD-L1 antibody:

[0242] In some embodiments, the camptothecin ADC has the chemical formula (IC) or a pharmaceutically acceptable salt thereof:

[0243]

[0244] In the above formula

[0245] Ab is an anti-PD-L1 antibody;

[0246] y is 1, 2, 3, or 4, or 1 or 4;

[0247] z is an integer from 2 to 12, or 2, 4, 8, or 12;

[0248] p is 1 to 16.

[0249] 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.

[0250] In some embodiments, the camptothecin ADC has the following chemical formula or a salt that is pharmaceutically acceptable thereto:

[0251]

[0252] In the above formula, p is 2, 4, or 8, and preferably p is 8.

[0253] In some embodiments, the camptothecin ADC has the following chemical formula or a salt that is pharmaceutically acceptable thereto:

[0254]

[0255] In the above formula, p is 2, 4, or 8, and preferably p is 8.

[0256] In some embodiments, the camptothecin drug-linker has the following chemical formula or a pharmaceutically acceptable salt thereof:

[0257]

[0258] In the above formula

[0259] y is 1, 2, 3, or 4, or 1 or 4;

[0260] z is an integer from 2 to 12, or 2, 4, 8, or 12.

[0261] In some embodiments, the camptothecin drug-linker has the following chemical formula:

[0262]

[0263] MP-PEG8-VKG-Camptothecine

[0264] In some embodiments, the camptothecin drug-linker has the following chemical formula:

[0265]

[0266] MP-PEG4-VKG-Camptothecine

[0267] In some embodiments, the camptothecin drug-linker has the following chemical formula:

[0268]

[0269] MP-PEG12-VKG-Camptothecine

[0270] With respect to PD-L1 targeted antibody-drug conjugates, the subscript p indicates the drug load and, depending on the context, may indicate the number of drug-linker molecules attached to individual antibody molecules and thus be an integer value, or may indicate the average drug load and thus be an integer or non-integer value, but typically a non-integer value. The average drug load indicates the average number of drug-linker molecules per antibody in the population. Often, but not always, when the inventors refer to antibodies, e.g., monoclonal antibodies, they are referring to a population of antibody molecules. In a composition comprising a population of antibody-drug conjugate molecules, the average drug load is an important quality attribute because it determines the amount of drug that can be delivered to target cells. The percentage of unconjugated antibody molecules in the composition is included in the average drug load value.

[0271] In a preferred aspect of the present invention, the average drug load is 1 to about 16, preferably about 2 to about 14, more preferably about 2 to about 10 when referring to a composition comprising a group of antibody-drug conjugate compounds.

[0272] For MMAE and camptothecin ADCs, e.g. those exemplified herein, the preferred average drug load is about 2, 4, or 8, and the particularly preferred average drug load is about 8. In one embodiment, the preferred average drug load for the MMAE ADC is 2 or 4. In one embodiment, the preferred average drug load for the camptothecin ADC is 4 or 8. In an exemplary embodiment, the drug-linker is conjugated to a cysteine ​​residue of reduced interchain disulfide. In some aspects, the actual drug load for individual antibody molecules in a group of antibody-drug conjugate compounds is 1 to 10 (or 6 to 10 or 6 to 8), and the dominant drug loading is 8. Higher drug loads may be achieved, for example, when the drug-linker is conjugated to an introduced cysteine ​​residue (e.g., a cysteine ​​residue introduced at position 239 according to the EU index) in addition to the interchain disulfide.

[0273] The PEG (polyethylene glycol) portion of the drug linker may be in the range of 2 to 36. In all the above embodiments, the subscript z is preferably 2 to 12, 4 to 12, 8 to 14, 8 to 12, 10 to 12, or 10 to 14, more preferably 2, 4, 8, or 12, and most preferably 8.

[0274] Polydisperse PEG, monodisperse PEG, and isolating PEG can be used to prepare the PEGylated antibody-drug conjugates of the present invention. Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, whereas monodisperse PEG is typically purified from a heterogeneous mixture and thus provides a single chain length and molecular weight. A preferred PEG unit is isolating PEG, which is a compound synthesized in a stepwise manner and not synthesized through a polymerization process. Isolating PEG provides a single molecule having a limited and specified chain length. Regarding the subscript "p," when referring to a group of antibody-drug conjugates, the value for the subscript "n" may be an average number, an integer, or a non-integer number.

[0275] Useful classes of cytotoxic agents for conjugates to anti-PD-L1 antibodies include, for example, antitubulin agonists, DNA minor groove binders, DNA replication inhibitors, chemotherapy sensitizers, etc. Other exemplary classes of cytotoxic agents include anthracyclines, auristatin, camptothecin, duocarmycin, etoposide, metansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binders (e.g., endiine and lexitropsin), duocarmycin, taxanes (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulisin M, doxorubicin, morpholino-doxorubicin, and cyanomorpholino-doxorubicin.

[0276] The cytotoxic agent may be a chemotherapy agent, e.g., doxorubicin, paclitaxel, melphalan, vinca alkaloid, methotrexate, mitomycin C, or etoposide. The agent may also be a CC-1065 analog, caliceamicin, meitansin, an analog of dolastatin 10, lyzoxin, or palitoxin.

[0277] The cytotoxic agent may also be auristatin. Auristatin may be an auristatin E derivative, for example, an ester formed between auristatin E and a keto acid. For example, auristatin E can react 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 structure of various auristatins are described, for example, in US 2005-0238649 and US 2006-0074008.

[0278] The cytotoxic agent may be a DNA minor groove binder. (See, for example, U.S. Patent No. 6,130,237.) For example, the minor groove binder may be a CBI compound or an endiine (e.g., caliceamicin).

[0279] Cytotoxic agents or cell arrest agents may be anti-tubulin agonists. Examples of anti-tubulin agonists include taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik)), vinca alkaloids (e.g., vincristine, vinblastine, vindesin, and vinorelbine), and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Exemplary auristatins are shown in Formulas III through XIII below. Other suitable antitubulin agonists include, for example, bacatine derivatives, taxane analogs (e.g., epotillon A and B), nocodazole, colchicine and colsimide, estramustine, cryptopicin, semadotine, meitansinoids, combretastatin, discordermoid, and eleutrobin.

[0280] The cytotoxic agent may be a metansinoid, which is another group of anti-tubulin agonists (e.g., DM1, DM2, DM3, DM4). For example, the metansinoid may be a metansin or a metansin-containing drug linker, such as DM-1 or DM-4 (ImmunoGen, Inc.; see also [Chari et al., 1992, Cancer Res.]).

[0281] VIII. Application of Treatment

[0282] The antibody of the present invention may be used to treat cancer, either alone or as its anti-PD-L1 antibody-drug conjugate. Some of these cancers exhibit detectable levels of PD-L1 measured at the protein level (e.g., by immunoassay using one of the exemplary antibodies) or mRNA level. Some of these cancers preferably exhibit elevated levels of PD-L1 compared to the same type of non-cancerous tissue from the same patient. Exemplary levels of PD-L1 on cancer cells that may be treated are 5,000 to 500,000 PD-L1 molecules per cell, but higher or lower levels may be treated. Optionally, the level of PD-L1 in the cancer is measured before performing treatment.

[0283] Examples of cancers associated with PD-L1 expression and treatable 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 term in the relevant art for cancer lacking detectable estrogen and progesterone receptors and lacking overexpression of HER2 / neu. 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. Treatment may be applied to patients with primary or metastatic tumors of these types. Treatment may also be applied to patients who are refractory to conventional treatment or who have relapsed after responding to such treatment.

[0284] The antibody of the present invention, e.g., a humanized antibody, is administered as an effective regimen, either alone or as a conjugate thereof, comprising a dosage, route of administration, and frequency of administration that delays the onset of cancer and / or reduces its severity, prevents further deterioration, or improves at least one of its signs or symptoms. If the patient already has cancer, the regimen may be referred to as a therapeutically effective regimen. If the patient has an elevated risk of cancer compared to the general population but has not yet experienced symptoms, the regimen may be referred to as a prophylactically effective regimen. In some cases, therapeutic or prophylactic efficacy may be observed in the individual patient compared to a historical control group or past experience in the same patient. In other cases, therapeutic or prophylactic efficacy may be demonstrated in preclinical or clinical trials in a group of treated patients compared to a control group of untreated patients.

[0285] An exemplary dose for a monoclonal antibody is a patient's body weight of 0.1 mg / kg to 50 mg / kg, more typically 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, or 1 mg / kg to 10 mg / kg, or 2 mg / kg to 30 mg / kg, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, or 2 mg / kg to 10 mg / kg, or 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 12 mg / kg, or 3 mg / kg to 10 mg / kg. Exemplary dosages for a monoclonal antibody or its antibody-drug conjugate are fixed doses of 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg, or 3 mg / kg to 7.5 mg / kg of the subject's body weight, or 0.1 to 20, or 0.5 to 5 mg / kg of body weight (e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg), or 10 to 1500, or 200 to 1500 mg. In some methods, the patient is administered a dose of at least 1.5 mg / kg, at least 2 mg / kg, or at least 3 mg / kg administered at least once every 3 weeks. The dosage depends, among other factors, on the frequency of administration, the patient's condition and response to prior treatment if present, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic.

[0286] Administration may be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intradural, intraperitoneal, local, intranasal, or intramuscular. Administration may also be localized directly into the tumor. Administration into the systemic circulation by intravenous or subcutaneous administration is preferred. Intravenous administration may be, for example, by an infusion over a period such as 30 to 90 minutes or by a single bolus injection.

[0287] The frequency of administration depends, among other factors, on the half-life of the antibody or conjugate in circulation, the patient's condition, and the route of administration. The frequency may be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient's condition or the progression of the cancer being treated. An exemplary frequency for intravenous administration is twice a week to quarterly over a continuous course of treatment, but more or less frequent administration is also possible. Another exemplary frequency for intravenous administration is three times a week or every four weeks over a continuous course of treatment, but more or less frequent administration is also possible. For subcutaneous administration, an exemplary frequency is daily to monthly, but more or less frequent administration is also possible.

[0288] The number of doses administered depends on the nature of the cancer (e.g., whether it presents with acute or chronic symptoms) and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, 1 to 10 doses are often sufficient. Sometimes, a single bolus dose in a randomly divided form is sufficient for acute disorders or acute exacerbations of chronic disorders. Treatment may be repeated for recurrences or acute exacerbations of acute disorders. For chronic disorders, the antibody may be administered at regular intervals, for example, for at least 1, 5, or 10 years, or for the patient's lifetime, weekly, bi-weekly, monthly, quarterly, or every 6 months.

[0289] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and are manufactured under GMP conditions. Pharmaceutical compositions may be provided in unit dosage forms (i.e., doses for a single administration). Pharmaceutical compositions may be formulated using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants. The formulation depends on the selected route of administration. For injection, antibodies may be formulated in an aqueous solution, preferably in a physiologically miscible buffer, such as Hank's 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 reconstitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. The concentration of the antibody in the liquid formulation may be, for example, 1 to 100 mg / ml, e.g. 10 mg / ml.

[0290] Treatment with the antibody of the present invention may be combined with chemotherapy, radiation, stem cell therapy, surgery, and other treatments effective for the disorder being treated. A useful class of other agents that may be administered with antibodies against PD-L1 and antibody-drug conjugates as described herein includes, for example, antibodies against other receptors expressed on cancer cells, antitubulin agonists (e.g., auristatin), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes, such as cisplatin, mono(platinum), bis(platinum), and tri-nucleus platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycin, etoposide, fluorinated pyrimidines, ionophores, lexitropsin, nitrosourea, platinol, pre-forming compounds, purine antimetabolites, puromycin, radiosensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, etc.

[0291] Treatment with an anti-PD-L1 antibody or antibody-drug conjugate, either alone or in combination with any other agent or therapy described above as an antibody-drug conjugate, can increase the median progression-free survival or overall survival time of a patient with a tumor (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer), particularly in relapsed or refractory cases, by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100% or more, compared to the same treatment without the anti-PD-L1 antibody (e.g., chemotherapy), either alone or as a conjugate. Additionally or alternatively, a treatment containing an anti-PD-L1 antibody alone or as a conjugate (e.g., standard chemotherapy) can increase the complete response rate, partial response rate, or objective response rate (complete + partial) of a patient with a tumor by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100% compared to the same treatment without the anti-PD-L1 antibody alone or as a conjugate (e.g., chemotherapy).

[0292] 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 response rate in patients treated with standard therapy plus an anti-PD-L1 antibody, either alone or as a conjugate, compared to a control group of patients receiving standard therapy alone (or plus placebo), is statistically significant, for example, at the level of p = 0.05, 0.01, or even 0.001. Complete and partial response rates are determined by objective criteria typically used in clinical trials for cancer, as listed or permitted by, for example, the National Cancer Institute and / or the U.S. Food and Drug Administration.

[0293] IX. Manufactured Products and Kits

[0294] In another aspect, a manufactured product or kit comprising the anti-PD-L1 antibody or the anti-PD-L1 antibody-drug conjugate described herein is provided. The manufactured product or kit may further comprise instructions for the use of the anti-PD-L1 antibody or the anti-PD-L1 antibody-drug conjugate described herein in the method of the present invention. Accordingly, in certain embodiments, the manufactured product or kit comprises instructions for the use of the anti-PD-L1 antibody or the anti-PD-L1 antibody-drug conjugate described herein in a method for treating 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) described herein in a method comprising administering an effective amount of the anti-PD-L1 antibody or the anti-PD-L1 antibody-drug conjugate described herein to a subject. In some embodiments, the subject is a human.

[0295] The manufactured product or kit may additionally include a container. Suitable containers include, for example, bottles, vials (e.g., double-chamber vials), syringes (e.g., 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.

[0296] The product or kit may further comprise a label or package insert on or associated with the container that may indicate instructions for the reconstitution and / or use of the formulation. The label or package insert may further indicate that the formulation is useful or intended for other modes of administration to treat cancer in a subject (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial 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 allowing for repeated administration of the reconstituted formulation. The product or kit may further comprise a second container containing a suitable diluent. The manufactured product or kit may additionally include other materials desirable from a commercial, therapeutic, and user perspective, including other buffers, diluents, filters, needles, syringes, and package inserts having instructions for use.

[0297] The manufactured product or kit of the present invention optionally further comprises a container containing a second medicine, wherein an anti-PD-L1 antibody or an anti-PD-L1 antibody-drug conjugate is the first medicine, and the article or kit further comprises instructions on a label or package insert for treating a subject with the second medicine in an effective amount. In some embodiments, the second medicine is intended to eliminate or reduce the severity of one or more adverse events.

[0298] In some embodiments, the anti-PD-L1 antibody or the anti-PD-L1 antibody-drug conjugate is present in a container as a lyophilized powder. In some embodiments, the lyophilized powder is in a hermetically sealed container, such as a vial, ampoule, or sachet, indicating the amount of active agent. Where the pharmaceutical is administered by injection, an ampoule of sterile water for injection or saline solution may be provided, for example, optionally as part of a kit so that the components can be mixed before administration. If desired, such a kit may further include one or more of various conventional pharmaceutical components, for example, a container having one or more pharmaceutically acceptable carriers, additional containers, etc., as would be readily apparent to a person skilled in the art. Instructions printed as inserts or labels indicating the amount of the component to be administered, instructions for administration, and / or instructions for mixing the components may also be included in the kit.

[0299] X. Other applications

[0300] The anti-PD-L1 antibodies described herein, such as humanized anti-PD-L1 antibodies, may be used to detect PD-L1 in the context of clinical diagnosis or treatment, or in research. Expression of PD-L1 on cancer provides an indication that the cancer can be treated with the antibodies of the present invention. The antibodies may also be sold as research reagents for laboratory studies to detect cells containing PD-L1 and their response to various stimuli. For such uses, the monoclonal antibodies may be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes, and may be provided in the form of a kit containing all the essential reagents for performing a assay for PD-L1. The antibodies described herein may be used to detect PD-L1 protein expression and to determine whether cancer can be treated with PD-L1 ADCs.

[0301] All patent applications, websites, other publications, accession numbers, etc. cited above or below are incorporated by reference in their entirety for all purposes to the same extent as each individual item is indicated to be incorporated by reference specifically and individually. Where different versions of a sequence are associated with an accession number at different times, the version associated with the accession number at 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 accession number. Likewise, where different versions of publications, websites, etc. are disclosed at different times, unless otherwise indicated, the version most recently disclosed at 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 indicated otherwise. Although the invention has been described in some detail by way of example and illustration for the sake of clarity and understanding, it will be apparent that certain variations and modifications may be practiced within the scope of the appended claims.

[0302] Examples

[0303] The cell lines described in the following examples were maintained in culture under conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DMSZ) in Braunschweig, Germany, or otherwise known.

[0304] method

[0305] Antibody production

[0306] SG-559-xx antibodies designated for PD-L1 were generated by introducing point mutations entirely into the CDR of human Ab1 to reduce affinity. Briefly, residues in the CDR located close to the PD-L1 binding epitope were mutated to different amino acids. Four selected example residues are Fig. 1 It is illustrated in [figure]. For initial screening purposes, SG-559-xx antibodies were produced at ATUM Bio using transient transfection in HEK293 cells.

[0307] For follow-up studies, antibodies were produced in-house according to the following protocol. The antibody variable and constant domain sequences were synthesized using non-template PCR. Briefly, virtual gene sequences were converted into oligonucleotide sequences using bioinformatics tools from Genewiz. Oligonucleotides were synthesized, pooled, and amplified using PCR. The full-length amplicon from the PCR reaction was cloned into a vector, the product was subsequently transformed into E. coli, and the unique colonies were isolated. Colonies were grown overnight in liquid medium, the plasmid DNA was isolated, purified, and its sequence verified using Sanger sequencing. The light and heavy chains were cloned into the pcDNA3.4 vector.

[0308] A 1:1 ratio of antibody heavy and light chain vectors was diluted in ThermoFisher OptiPRO SFM medium containing ExpiFectamine CHO transfection reagent. Subsequently, the DNA / transfection reagent was added to the ExpiCHO culture in ThermoFisher ExpiCHO expression medium and cultured for 9 days with the ExpiCHO enhancer added on day 1 and the ExpiCHO feed added on days 1 and 2. The culture was collected by centrifugation and 0.2 µm filtration or by depth filtration using Millipore X0HC and D0HC pods followed by 0.2 µm filtration.

[0309] GE HiTrap mAb Select SuRe columns were used for the purification of each IgG. Prior to elution, the resin was washed with 5CV PBS + 0.1% Triton, 5CV PBS + 0.5M NaCl, and 7.5CV PBS. IgG was eluted using 100 mM acetic acid pH 3 buffer. The samples were buffer-exchanged with PBS using a 26 / 60 HiPrep desalting column. Samples that had undergone the final polishing step on a HiPrep Superdex 200 26 / 600 column were run in PBS. Subsequently, the samples were filter-sterilized and collected for characterization. Characterization included A280 concentration, aSEC HPLC, aHIC HPLC, and reduced PLRP-MS (QToF).

[0310] Biolayer interferometry

[0311] The binding affinity of the SG-559-xx antibody was determined by performing biolayer interferometric analysis using an Octet Red 384 system (ForteBio). The anti-human Fab-CH1 (FAB2G) biosensor (ForteBio) was loaded with 4 μg / mL of SG-559-xx antibody for 100 seconds. Following a subsequent baseline step, human PD-L1 (Acro Biosciences) was incubated with the probe loaded for the association step for 150 seconds at concentrations ranging from 500 nM to 0.69 nM (1x PBS pH 7.4 containing 1% casein and 0.2% Tween-20). Subsequently, a dissociation step was performed for 1000 seconds in the same buffer lacking human PD-L1. 회합 and k 해리 It was fitted to the combination curve generated according to the established method.

[0312] Production of Antibody-Drug Conjugates (ADCs)

[0313] The SG-559-xx antibody was conjugated to MDpr-PEG(12)-gluc-MMAE with an average drug-to-antibody ratio (DAR) of 8 as described in US20180092984. The SG-559-xx antibody was conjugated to vc-MMAE with an average DAR of 4 as described in US20050238649. The SG-559-xx antibody was conjugated to MP-PEG8-VKG-camptothecin with an average DAR of 8 as described in PCT / US2019 / 025968 (filed April 5, 2019).

[0314] In vitro cytotoxicity test

[0315] Cell lines were plated for 24 hours prior to antibody-drug conjugate (ADC) treatment to allow the cells to acclimatize. Where indicated, 500 IU / mL of interferon-γ was also added at this time to induce PD-L1 expression. Subsequently, cells were treated with the indicated dose of ADC and incubated at 37°C for 96 hours. Other PD-L1-specific antibodies as well as isotype controls were included as ADCs for comparison. Cell viability for the cell lines was measured using CellTiter-Glo (Promega Corporation, Madison, Wisconsin, USA) 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, Massachusetts, USA). The results are reported as x50, which is the concentration of the compound required to produce a 50% reduction in viability compared to untreated cells.

[0316] Internalization test

[0317] Internalization of PD-L1-designated antibodies was performed using a popfluorescence pH-sensitive conjugate on IncuSite (Sartorius). The antibodies were conjugated to a pH-sensitive dye having a fluorescence signal that increases as pH decreases from the cell surface and into the endosome / lysosome compartment. Adherent cells were plated for 24 hours (with 500 IU / mL interferon-γ to induce PD-L1 expression) and then incubated with these conjugates. Suspension cells were plated for 3 hours and then incubated with these conjugates. Subsequently, 0.5 μg / mL of the designated dye-antibody conjugate was administered to the cells, and they were incubated for 48 hours. Using IncuSite S3 software (Sartorius), the total integrated intensity of the fluorescence signal was normalized to the percentage of total growth rate per well per time point. The results are reported as the area under the curve of normalized integrated intensity versus time.

[0318] In vivo activity research

[0319] Nude mouse 5.0 x 10 6 1 BxPC3 pancreatic adenocarcinoma cell or 1.0 x 10⁶ 6 Canine EBC-1 NSCLC cells were inoculated subcutaneously. NSG mice were 5.0 x 10⁶ 5 100 MDA-MB-231 triple-negative breast cancer cells were inoculated subcutaneously. SCID mice were 1.0 x 10 6 299 ALCL cells or 1.0 x 10⁶ Carpas 6 Canine Calu-1 NSCLC cells were inoculated subcutaneously. Tumor growth was monitored with calipers, and the average tumor volume was calculated using the formula (0.5 x [length x width 2 It was calculated using ]). The average tumor volume was approximately 100 mm². 3When [the threshold] was reached, mice were left untreated or administered intraperitoneally as directed with the ADC. Unconjugated antibodies and vc-MMAE ADCs were administered weekly for a total of three doses. MP-PEG8-VKG-camptothecin ADCs were administered only once. Tumor volume was approximately 750 mm³ 3 Mice were euthanized when [the threshold] was reached. For the study to characterize immune features in animals containing Carpas 299, the average tumor volume was 200 mm² 3 These mice were brought to a certain level. Subsequently, these mice were treated with a single dose of unconjugated antibody or ADC and euthanized after 6 days. Tumors were characterized in vitro by immunohistochemistry and cytokine analysis (Luminex). All animal procedures were performed under protocols approved by the Institutional Animal Care and Use Committee at facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care.

[0320] PD-L1 blocking

[0321] In vitro PD-L1 blockade Evaluations were performed using the PD-1 / PD-L1 blocking bioassay (Promega Corporation) according to the manufacturer's instructions. Briefly, PD-L1+ aAPC / CHO-K1 cells were plated and acclimatized for 16 hours. Subsequently, the indicated concentrations of antibodies or ADCs were added to the plated cells, followed by PD-1+ effector cells. In the absence of PD-1 / PD-L1 signaling, interactions between aAPC / CHO-K1 cells and effector cells generate bioluminescent signals. Therefore, more effective inhibition of PD-1 / PD-L1 interactions generates higher luminescence signals, which are quantified as a fold change compared to untreated cells. A PD-1 binding antibody (Promega Corporation) was included as a positive control, and a non-binding isotype antibody was included as a negative control.

[0322] Immunotoxicity in a human APC model stimulated by IFNγ upregulating PD-L1

[0323] The immunotoxicity of antibodies or ADCs against human antigen-presenting cells in vitro was measured using human antigen-presenting cells (APCs) stimulated with interferon-γ (IFNγ) and subsequently treated with any of the antibodies or ADCs described herein. Human APCs were stimulated with IFNγ (R&D Systems) at 500 IU / mL for 24 hours in vitro to upregulate PD-L1, and then treated with SG-559-xx ADCs. Immunotoxicity was calculated as a percentage of the survival rate of untreated APCs at different antibody or ADC concentrations.

[0324] Suppression of immune response in human APC model

[0325] Immunosuppression was measured using human antigen-presenting cells (APCs) stimulated with lipopolysaccharide (LPS) and subsequently treated with any of the antibodies or ADCs described herein. Human APCs were stimulated in vitro with IFNγ (R&D Systems) at 500 IU / mL for 24 hours to upregulate PD-L1. Subsequently, human APCs were treated with SG-559-xx ADCs as indicated for 24 hours. Then, human APCs were stimulated in vitro with LPS (Sigma Aldrich) at 100 ng / mL for 48 hours. The response to LPS was measured by flow cytometry staining of MHC class II and CD86 (Biolegend). The intensity of immune function was calculated as a fold change in MHC class II or CD86 in response to LPS stimulation in APCs at different antibody or ADC concentrations.

[0326] Deglycosylation of human PD-L1 using PNGase F

[0327] To generate deglycosylated hPD-L1, human PD-L1 was treated with PNGase F enzyme (New England Biolabs) combined with a denaturation protocol. PNGase F catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex oligosaccharides from N-linked glycoproteins. Human PD-L1 was treated with the denaturation protocol in the absence of PNGase F to provide a reaction control. 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. Glycosylation status was confirmed by mass spectrometry.

[0328] Biolayer interferometrics was performed using an Octet Red 384 system (ForteBio) to determine the binding affinity of SG-559-xx antibodies or ADCs to glycosylated or deglycosylated PD-L1. Following a subsequent baseline step, glycosylated and deglycosylated human PD-L1 at concentrations ranging from 500 nM to 0.69 nM (1x PBS pH 7.4 containing 1% BSA and 0.2% Tween-20) were incubated for 150 seconds with probes loaded for the association step. Subsequently, a dissociation step was performed for 1000 seconds in the same buffer lacking human PD-L1. 회합 and k 해리 It was fitted to the combination curve generated according to the established method.

[0329] result

[0330] Example 1: Design and Characterization of SG-559-xx Antibody

[0331] Seventeen SG-559-xx antibodies were generated from the parental Ab1 antibody as described in the method. The CDRs containing mutations of these antibodies are Table 1 It is indicated in ). Sixteen of these antibodies were evaluated for their monovalent binding affinity to hPD-L1 by biolayer interferometry compared to Ab1 ( Table 2 The measured affinity of the SG-559-xx antibody spanned nearly double digits, and K D The values ​​ranged from 4 nM to 297 nM.

[0332] Table 1. SG-559-XX variant sequences

[0333]

[0334] Table 2. SG-559-XX Binding Affinity

[0335]

[0336] Example 2: In vitro cytotoxicity

[0337] The cytotoxicity of SG-559-xx antibodies as ADCs was evaluated on PD-L1-expressing cancer cell lines, including 786-O, BxPC3, ES-2, MDA-MB-231, Carpas 299, and L540cy, as described in the methods. In some experiments, SU-DHL-4 (PD-L1 negative cancer cell line) was included as a control. An initial screening of 15 SG-559-xx ADCs with the MDpr-PEG(12)-gluc-MMAE load (DAR 8) (excluding the two with the lowest affinity) demonstrated that some SG-559-xx antibodies exhibited significantly improved cytotoxicity compared to the parental Ab1 ( FIGS. 2a to 2f ).

[0338] Four SG-559-xx antibodies that consistently showed the highest efficacy in the initial screen were further characterized as vc-MMAE and MP-PEG8-VKG-camptothecin ADCs for their efficacy ( Table 3 In most cell lines tested, these ADCs were significantly more potent than Ab1. They also exhibited no activity in antigen-negative cell lines (SU-DHL-4), suggesting that this was not due to non-specific binding.

[0339] Table 3. SG-559-XX x50 values ​​(ng / mL)

[0340]

[0341]

[0342] Example 3: Internalization

[0343] To confirm the cytotoxicity results, the internalization of SG-559-01 and SG-559-03 was further investigated using the IncuSite imaging system and pH-sensitive dye conjugates as described in the methods. The internalization of SG-559-01 and SG-559-03 was consistently higher across most cell lines tested. This was measured by the percentage increase in the area under the curve (AUC) of normalized integrated intensity over time ( Table 4 ). The example curve is MDA-MB-231 ( Fig. 3a ) and Carpas 299 ( Fig. 3b It is represented for ).

[0344] Table 4. SG-559-XX Internalization

[0345]

[0346] Example 4: In vivo anti-tumor activity

[0347] Four SG-559-xx antibodies characterized by in vitro screening were also tested for anti-tumor efficacy in two mouse xenograft models. In the MDA-MB-231 model, SG-559-xx antibodies as ADCs exhibited significant anti-tumor activity with two drug-linkers ( FIGS. 4a to 4b ). In the BxPC3 model, the SG-559-xx antibody as an ADC exhibited moderate anti-tumor activity ( FIGS. 5a to 5b In almost all cases, SG-559-xx ADCs were more effective than Ab1 ADCs, suggesting that the observed in vitro phenotype translates to the in vivo environment.

[0348] Anti-tumor efficacy was further observed in additional models using one of the inventors' most promising antibodies as an Fc effector-reduced variant (SG-559-01 LALA). As an ADC, the SG-559-01 LALA antibody is Carpas 299 ( FIGS. 6a to 6b ), Calu-1 ( Fig. 7 ), and EBC-1 ( FIGS. 8a to 8bSignificant anti-tumor activity was demonstrated with one or two drug linkers in the ) model. Note that this activity is distinct from that of the unconjugated SG-559-01 LALA antibody.

[0349] Example 5: PD-L1 blocking

[0350] The ability of SG-559-01 LALA to block PD-1 / PD-L1 checkpoints in vitro was further characterized. Compared to the PD-1 antibody control, SG-559-01 can inhibit PD-1 / PD-L1 signaling more effectively. Furthermore, unconjugated SG-559-01 LALA was equivalent to SG-559-01 LALA conjugated to two types of drug linkers, demonstrating that conjugation does not affect PD-1 / PD-L1 blockade ( Fig. 9 ).

[0351] Example 6: Immunotoxicity to human APCs in vitro

[0352] SG-559-01 and SG-559-01 LALA were evaluated for immunotoxicity against APCs (e.g., macrophages and dendritic cells (DCs)). PD-L1 was upregulated in APCs by stimulating them with IFNγ prior to treatment as described in the methods. SG-559-01 LALA ADCs exhibited immunotoxicity against human APCs in both macrophages and DCs that was similar to or within a single order of magnitude of the isotype control ( FIGS. 10a to 10d ).

[0353] The four SG-559-xx antibodies characterized by in vitro screening were also tested for immunotoxicity against APCs (i.e., dendritic cells and macrophages). The immunotoxicity against human APCs was similar to or within a single digit of that of each SG-559-xx ADC ( FIGS. 11a to 11d ).

[0354] Example 7: Suppression of immune response

[0355] The SG-559-01 ADC was further characterized by measuring the suppression of the immune response in LPS-treated human APCs. As described in the methods, human APCs in vitro were stimulated with LPS after ADC treatment, and the subsequent upregulation of MHC Class II and CD86 was quantified as a measure of the immune response. Treatment with the SG-559-01 ADC [induced] MHC Class II ( FIGS. 12a to 12b ) and CD86 ( FIGS. 12c to d As measured by ), it caused suppression of the immune response in both DCs and macrophages in a manner similar to the isotype control, or within a single digit of it.

[0356] Example 8: Increase in immune infiltration

[0357] The SG-559-01 LALA vc-MMAE ADC was further characterized by evaluating immune infiltration in mice with Carpas 299 tumors. Mice containing tumors were treated as directed, and the tumors were characterized after 6 days. Compared to the untreated control group and both the SG-559-01 antibody, the SG-559-01 vc-MMAE ADC induced immune infiltration in mice with Carpas 299 tumors ( FIGS. 13a to c ). Fig. 13a It indicates an increase in mCD45+ cells (pan-leukocyte marker). Fig. 13b It indicates an increase in mCD11c+ cells (a marker for a subset of dendritic cells and macrophages). Fig. 13c indicates an increase in mF4 / 80+ cells (macrophage marker).

[0358] Example 9: Inflammatory cytokine response

[0359] The ability of the SG-559-01 LALA vc-MMAE ADC to induce the production of inflammatory cytokines in the tumor microenvironment (TME) was further characterized. Compared to both the untreated control and the SG-559-01 LALA antibody, the SG-559-01 LALA vc-MMAE ADC induced eotaxin (a chemokine against eosinophils; Fig. 14a ), MIP1a (pro-inflammatory macrophage cytokine; Fig. 14b ), MIP1b (pro-inflammatory macrophage cytokine; Fig. 14c ), MIG / CXCL9 (induced by IFNγ, affects the migration and differentiation of immune cells; Fig. 14d ), MCP1 (chemokine against monocytes / macrophages; Fig. 14e ), and Rantes (chemokines against monocytes, T cells, and eosinophils; Fig. 14f It induces inflammatory cytokines in the TME as measured by the intratumoral concentration of ).

[0360] Example 10: Binding affinity for glycosylated PD-L1 and deglycosylated PD-L1

[0361] The binding affinity of SG-559-01 to glycosylated and deglycosylated forms of PD-L1 was evaluated. Binding affinity was assessed using biolayer interferometry on an Octet Red 384 system (ForteBio) as described in the methods. PD-L1 was deglycosylated as described in the methods using the PNGase F enzyme and denaturation protocol. The binding affinity of SG-559-01 to deglycosylated PD-L1 and control glycosylated PD-L1 (identical treatment conditions as described in the methods, but without PNGase F treatment). A ~2-fold difference in the binding affinity of SG-559-01 to deglycosylated PD-L1 was observed compared to glycosylated PD-L1 ( Table 5 The glycosylation status of PD-L1 was confirmed using mass spectrometry.

[0362] Table 5. Binding of SG-559-01 to glycosylated versus deglycosylated hPD-L1

[0363]

[0364] Abbreviated sequence list

[0365] Sequence ID: 1 - Ab1 heavy chain variable region - Protein

[0366]

[0367] Sequence Identification Number: 2 - Ab1 light chain variable region - Protein

[0368]

[0369] Sequence Identification Number: 3 - Ab1 heavy chain CDR1 - protein

[0370]

[0371] Sequence Identification Number: 4 - Ab1 heavy chain CDR2 - protein

[0372]

[0373] Sequence Identification Number: 5 - Ab1 heavy chain CDR3 - protein

[0374]

[0375] Sequence Identification Number: 6 - Ab1 light chain CDR1 - protein

[0376]

[0377] Sequence Identification Number: 7 - Ab1 light chain CDR2 - protein

[0378]

[0379] Sequence Identification Number: 8 - Ab1 light chain CDR3 - protein

[0380]

[0381] Sequence Identification Number: 9 - SG-559-01 LALA hIgG1 heavy chain - protein

[0382]

[0383] Sequence Identification Number: 10 - SG-559-01 Kappa light chain - protein

[0384]

[0385] Sequence Identification Number: 11 - SG-559-01 Heavy chain variable region - Protein

[0386]

[0387] Sequence Identification Number: 12 - SG-559-01 Light chain variable region - Protein

[0388]

[0389] Sequence Identification Number: 13 - SG-559-01 Heavy Chain CDR1 - Protein

[0390]

[0391] Sequence Identification Number: 14 - SG-559-01 Heavy Chain CDR2 - Protein

[0392]

[0393] Sequence Identification Number: 15 - SG-559-01 Heavy Chain CDR3 - Protein

[0394]

[0395] Sequence Identification Number: 16 - SG-559-01 Light Chain CDR1 - Protein

[0396]

[0397] Sequence Identification Number: 17 - SG-559-01 Light Chain CDR2 - Protein

[0398]

[0399] Sequence Identification Number: 18 - SG-559-01 Light Chain CDR3 - Protein

[0400]

[0401] Sequence Identification Number: 19 - SG-559-02 LALA hIgG1 heavy chain - protein

[0402]

[0403] Sequence Identification Number: 20 - SG-559-02 Kappa light chain - protein

[0404]

[0405] Sequence Identification Number: 21 - SG-559-02 Heavy chain variable region - Protein

[0406]

[0407] Sequence Identification Number: 22 - SG-559-02 Light chain variable region - Protein

[0408]

[0409] Sequence Identification Number: 23 - SG-559-02 Heavy Chain CDR1 - Protein

[0410]

[0411] Sequence Identification Number: 24 - SG-559-02 Heavy Chain CDR2 - Protein

[0412]

[0413] Sequence Identification Number: 25 - SG-559-02 Heavy Chain CDR3 - Protein

[0414]

[0415] Sequence Identification Number: 26 - SG-559-02 Light Chain CDR1 - Protein

[0416]

[0417] Sequence Identification Number: 27 - SG-559-02 Light chain CDR2 - Protein

[0418]

[0419] Sequence Identification Number: 28 - SG-559-02 Light chain CDR3 - Protein

[0420]

[0421] Sequence Identification Number: 29 - SG-559-03 LALA hIgG1 heavy chain - protein

[0422]

[0423] Sequence Identification Number: 30 - SG-559-03 Kappa light chain - protein

[0424]

[0425] Sequence Identification Number: 31 - SG-559-03 Heavy chain variable region - Protein

[0426]

[0427] Sequence ID: 32 - SG-559-03 Light chain variable region - Protein

[0428]

[0429] Sequence Identification Number: 33 - SG-559-03 Heavy Chain CDR1 - Protein

[0430]

[0431] Sequence Identification Number: 34 - SG-559-03 Heavy Chain CDR2 - Protein

[0432]

[0433] Sequence Identification Number: 35 - SG-559-03 Heavy Chain CDR3 - Protein

[0434]

[0435] Sequence Identification Number: 36 - SG-559-03 Light Chain CDR1 - Protein

[0436]

[0437] Sequence Identification Number: 37 - SG-559-03 Light Chain CDR2 - Protein

[0438]

[0439] Sequence Identification Number: 38 - SG-559-03 Light chain CDR3 - Protein

[0440]

[0441] Sequence Identification Number: 39 - SG-559-04 LALA hIgG1 heavy chain - protein

[0442]

[0443] Sequence Identification Number: 40 - SG-559-04 Kappa light chain - protein

[0444]

[0445] Sequence Identification Number: 41 - SG-559-04 Heavy chain variable region - Protein

[0446]

[0447] Sequence Identification Number: 42 - SG-559-04 Light chain variable region - Protein

[0448]

[0449] Sequence Identification Number: 43 - SG-559-04 Heavy Chain CDR1 - Protein

[0450]

[0451] Sequence Identification Number: 44 - SG-559-04 Heavy Chain CDR2 - Protein

[0452]

[0453] Sequence Identification Number: 45 - SG-559-04 Heavy Chain CDR3 - Protein

[0454]

[0455] Sequence Identification Number: 46 - SG-559-04 Light chain CDR1 - Protein

[0456]

[0457] Sequence Identification Number: 47 - SG-559-04 Light chain CDR2 - Protein

[0458]

[0459] Sequence Identification Number: 48 - SG-559-04 Light chain CDR3 - Protein

[0460]

[0461] Sequence Identification Number: 49 - SG-559-05 Heavy Chain CDR2 - Protein

[0462]

[0463] Sequence Identification Number: 50 - SG-559-06 Heavy Chain CDR2 - Protein

[0464]

[0465] Sequence Identification Number: 51 - SG-559-07 Heavy Chain CDR2 - Protein

[0466]

[0467] Sequence Identification Number: 52 - SG-559-08 Heavy Chain CDR2 - Protein

[0468]

[0469] Sequence Identification Number: 53 - SG-559-09 Heavy Chain CDR2 - Protein

[0470]

[0471] Sequence Identification Number: 54 - SG-559-10 Heavy Chain CDR3 - Protein

[0472]

[0473] Sequence Identification Number: 55 - SG-559-11 Heavy Chain CDR3 - Protein

[0474]

[0475] Sequence Identification Number: 56 - SG-559-12 Light Chain CDR3 - Protein

[0476]

[0477] Sequence Identification Number: 57 - SG-559-13 Light Chain CDR3 - Protein

[0478]

[0479] Sequence Identification Number: 58 - SG-559-14 Light Chain CDR3 - Protein

[0480]

[0481] Sequence Identification Number: 59 - SG-559-15 Light Chain CDR3 - Protein

[0482]

[0483] Sequence Identification Number: 60 - SG-559-16 Light Chain CDR3 - Protein

[0484]

[0485] Sequence Identification Number: 61 - SG-559-17 Light Chain CDR3 - Protein

[0486]

[0487] Sequence Identification Number: 62 - SG-559-01 hIgG1 heavy chain - protein

[0488]

[0489] Sequence Identification Number: 63 - SG-559-02 hIgG1 heavy chain - protein

[0490]

[0491] Sequence Identification Number: 64 - SG-559-03 hIgG1 heavy chain - protein

[0492]

[0493] Sequence Identification Number: 65 - SG-559-04 hIgG1 heavy chain - protein

[0494]

[0495] Sequence Identification Number: 66 - SG-559-01 Variable heavy chain region - Nucleic acid

[0496]

[0497] Sequence Identification Number: 67 - SG-559-01 Variable light chain region - Nucleic acid

[0498]

[0499] Sequence Identification Number: 68 - SG-559-02 Variable heavy chain region - Nucleic acid

[0500]

[0501] Sequence Identification Number: 69 - SG-559-02 Variable light chain region - Nucleic acid

[0502]

[0503] Sequence Identification Number: 70 - SG-559-03 Variable heavy chain region - Nucleic acid

[0504]

[0505] Sequence Identification Number: 71 - SG-559-03 Variable light chain region - Nucleic acid

[0506]

[0507] Sequence Identification Number: 72 - SG-559-04 Variable heavy chain region - Nucleic acid

[0508]

[0509] Sequence Identification Number: 73 - SG-559-04 Variable light chain region - Nucleic acid

[0510]

[0511] Sequence Identification Number: 74 - SG-559-01 LALA hIgG1 heavy chain - nucleic acid

[0512]

[0513] Sequence Identification Number: 75 - SG-559-01 Kappa light chain - Nucleic acid

[0514]

[0515] Sequence Identification Number: 76 - SG-559-01 hIgG1 heavy chain - nucleic acid

[0516]

[0517] Sequence Identification Number: 77 - SG-559-02 LALA hIgG1 heavy chain - nucleic acid

[0518]

[0519] Sequence Identification Number: 78 - SG-559-02 Kappa light chain - Nucleic acid

[0520]

[0521] Sequence Identification Number: 79 - SG-559-02 hIgG1 heavy chain - nucleic acid

[0522]

[0523] Sequence Identification Number: 80 - SG-559-03 LALA hIgG1 heavy chain - nucleic acid

[0524]

[0525] Sequence Identification Number: 81 - SG-559-03 Kappa light chain - nucleic acid

[0526]

[0527] Sequence Identification Number: 82 - SG-559-03 hIgG1 heavy chain - nucleic acid

[0528]

[0529] Sequence Identification Number: 83 - SG-559-04 LALA hIgG1 heavy chain - nucleic acid

[0530]

[0531] Sequence Identification Number: 84 - SG-559-04 Kappa light chain - nucleic acid

[0532]

[0533] Sequence Identification Number: 85 - SG-559-04 hIgG1 heavy chain - nucleic acid

[0534]

[0535] Sequence Identification Number: 86 - Ab1 hIgG1 heavy chain - protein

[0536]

[0537] Sequence Identification Number: 87 - Ab1 kappa light chain - protein

[0538]

Claims

Claim 1 An antibody or its antigen-binding fragment that specifically binds to human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequence of SEQ ID NOs: 13 to 15 and the light chain CDR sequence of SEQ ID NOs: 16 to 18. Claim 2 An antibody or its antigen-binding fragment that exhibits a binding affinity for human PD-L1 protein of 3 to 300 nM in claim 1. Claim 3 An antibody or its antigen-binding fragment according to claim 1 or 2, wherein the antibody exhibits a binding affinity for human PD-L1 protein of 3 to 15 nM. Claim 4 An antibody or its antigen-binding fragment according to claim 1, wherein the antibody further exhibits a total internalization higher than the total internalization of an antibody comprising the heavy chain variable region sequence of sequence identification number: 1 and the light chain variable region sequence of sequence identification number:

2. Claim 5 In paragraph 4, an antibody or its antigen-binding fragment in which total internalization is an increase of 9% to 155% of the AUC compared to the AUC of an antibody comprising the heavy chain variable region sequence of sequence identification number: 1 and the light chain variable region sequence of sequence identification number:

2. Claim 6 In paragraph 5, an antibody or its antigen-binding fragment in which total internalization is determined by a popfluoride internalization assay. Claim 7 An antibody or its antigen-binding fragment according to claim 1, wherein the antibody further exhibits an IC50 higher than the IC50 of an antibody comprising the heavy chain variable region sequence of sequence identification number: 1 and the light chain variable region sequence of sequence identification number:

2. Claim 8 In claim 7, the antibody or its antigen-binding fragment is conjugated to monomethylauristatin E (MMAE) and has an IC50 of 3 ng / mL to 20 ng / mL in the MDA-MB-231 cell line. Claim 9 In claim 7, the antibody or its antigen-binding fragment is conjugated to camptothecin and has an IC50 of 15 ng / mL to 55 ng / mL in the MDA-MB-231 cell line. Claim 10 An antibody or its antigen-binding fragment according to claim 1, wherein the antibody comprises the heavy chain variable region sequence of sequence identification number: 11 and the light chain variable region sequence of sequence identification number:

12. Claim 11 An antibody or its antigen-binding fragment according to claim 1, wherein the antibody comprises a light chain of sequence identification number: 9 and a heavy chain of sequence identification number:

10. Claim 12 In claim 1, the fragment is an antibody or its antigen-binding fragment that is Fab, Fab', F(ab')2, Fab'-SH, Fv, Diabadi, a linear antibody, or a single-strand antibody fragment. Claim 13 In paragraph 1, the antibody or its antigen-binding fragment is one in which the antibody contains L234A and L235A mutations in the heavy chain of the antibody. Claim 14 In claim 1, an antibody or its antigen-binding fragment in which the heavy chain constant region is of an IgG1 isotype. Claim 15 In paragraph 1, the antibody is a humanized or chimeric antibody or its antigen-binding fragment. Claim 16 In paragraph 1, an antibody or its antigen-binding fragment in which the antibody is conjugated to a cytotoxic agent through a linker. Claim 17 In paragraph 16, an antibody or its antigen-binding fragment in which the antibody is conjugated to monomethylauristatin E (MMAE). Claim 18 In paragraph 17, an antibody or its antigen-binding fragment in which the antibody is conjugated to MMAE through an enzyme-cleavable linker unit. Claim 19 In paragraph 18, an antibody or its antigen-binding fragment wherein the enzyme-cleavable linker unit comprises a Val-Cit linker. Claim 20 In paragraph 16, an antibody or its antigen-binding fragment wherein the antibody is conjugated to MMAE through a linker to form an antibody-drug conjugate having the following structure: In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. Claim 21 In paragraph 20, an antibody in which p is 4 or its antigen-binding fragment. Claim 22 In paragraph 20, an antibody in which p is 8 or its antigen-binding fragment. Claim 23 In paragraph 16, an antibody or its antigen-binding fragment in which the antibody is conjugated to camptothecin. Claim 24 In paragraph 23, an antibody or its antigen-binding fragment in which the antibody is conjugated to camptothecin through an enzyme-cleavable linker unit. Claim 25 In paragraph 24, an antibody or its antigen-binding fragment wherein the enzyme-cleavable linker unit comprises a Val-Lys-Gly linker. Claim 26 In paragraph 23, an antibody or its antigen-binding fragment wherein the antibody is conjugated to camptothecin through a linker to form an antibody-drug conjugate having the following structure: In the above formula, Ab represents an antibody, and p is in the range of 2 to 10. Claim 27 In paragraph 26, an antibody in which p is 4 or its antigen-binding fragment. Claim 28 In paragraph 26, an antibody in which p is 8 or its antigen-binding fragment. Claim 29 A pharmaceutical composition comprising the antibody of claim 1 or its antigen-binding fragment for treating cancer in a subject. Claim 30 In paragraph 29, a pharmaceutical composition in which the subject is a human subject. Claim 31 A pharmaceutical composition according to claim 29 or 30, wherein the cancer is melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple-negative breast cancer (TNBC), ovarian cancer, urothelial carcinoma, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer. Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete Claim 63 delete Claim 64 delete Claim 65 delete Claim 66 delete Claim 67 delete Claim 68 delete Claim 69 delete Claim 70 delete Claim 71 delete Claim 72 delete Claim 73 delete Claim 74 delete Claim 75 delete Claim 76 delete Claim 77 delete Claim 78 delete Claim 79 delete Claim 80 delete Claim 81 delete Claim 82 delete Claim 83 delete Claim 84 delete Claim 85 delete Claim 86 delete Claim 87 delete Claim 88 delete Claim 89 delete Claim 90 delete Claim 91 delete Claim 92 delete Claim 93 delete Claim 94 delete Claim 95 delete Claim 96 delete Claim 97 delete Claim 98 delete Claim 99 delete Claim 100 delete Claim 101 delete Claim 102 delete Claim 103 delete Claim 104 delete Claim 105 delete Claim 106 delete Claim 107 delete Claim 108 delete Claim 109 delete Claim 110 delete Claim 111 delete Claim 112 delete Claim 113 delete Claim 114 delete Claim 115 delete Claim 116 delete Claim 117 delete Claim 118 delete Claim 119 delete Claim 120 delete Claim 121 delete Claim 122 delete Claim 123 delete Claim 124 delete Claim 125 delete Claim 126 delete Claim 127 delete Claim 128 delete Claim 129 delete Claim 130 delete Claim 131 delete Claim 132 delete Claim 133 delete Claim 134 delete Claim 135 delete Claim 136 delete Claim 137 delete Claim 138 delete Claim 139 delete

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