Anti-pd-l1 antibodies and antibody-drug conjugates

Abstract

Provided are novel anti-PD-L1 antibodies and antibody-drug conjugates and methods of using such anti-PD-L1 antibodies and antibody-drug conjugates to treat cancer.

Description

[0001] Cross-references to related applications

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

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

[0004] PD-L1 (also known as programmed death-ligand 1, B7-H1, or CD274) is a protein that has been shown to be expressed in various cancer cells. PD-L1 is a transmembrane protein that can interact with PD-1 and acts as an "off" switch to inactivate T cells. PD-L1 is typically overexpressed on tumor cells, and its binding to PD-1 allows tumors to evade T cell immune responses.

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

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

[0007] Provided herein are anti-PD-Ll antibodies and PD-Ll directed antibody-drug conjugates (ADCs). In particular, provided herein are PD-Ll directed camptothecin ADCs and MMAE ADCs. Also provided herein are methods of using anti-PD-Ll directed antibodies and ADCs to treat disorders expressing PD-Ll. Preferred anti-PD-Ll antibodies exhibit a binding affinity to human PD-Ll protein of between 3 nM and 300 nM. Other preferred anti-PD-Ll antibodies comprise the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs. Other preferred anti-PD-Ll antibodies comprise the heavy chain CDR sequences of SEQ ID NOs: 13-15 and the light chain CDR sequences of SEQ ID NOs: 16-18.

[0008] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human Programmed Death Ligand 1 (PD-Ll) protein, wherein the antibody exhibits a binding affinity to human PD-Ll protein of between 3 nM and 300 nM. In some embodiments, the antibody exhibits a binding affinity to human PD-Ll protein of between 3 nM and 15 nM.

[0009] In some embodiments, the antibody further exhibits a total internalization that is higher than the total internalization of Ab 1. In some embodiments, the total internalization has an AUC increase of between 9% and 155% relative to the AUC of Ab 1. In some embodiments, the total internalization is determined by a FabFluor internalization assay.

[0010] In some embodiments, the antibody further exhibits an x50 that is lower than the x50 of Ab 1. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE), and wherein the x50 is between 3 ng / mL and 20 ng / mL in a MDA-MB-231 cell line.

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

[0012] In some embodiments, the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 13-15 and the light chain CDR sequences of SEQ ID NOs: 16-18.

[0013] In some embodiments, the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs.

[0014] In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 11 and a light chain variable region sequence of SEQ ID NO: 12.

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

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

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

[0018] In some embodiments, the heavy chain constant region is of IgGl isotype.

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

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

[0021] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker, forming an antibody-drug conjugate having the following structure:

[0022]

[0023] wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0024] 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 structure:

[0025] wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0026] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs.

[0027] In some embodiments, the antibody exhibits a binding affinity to a human PD-L1 protein of between 3 and 300 nM. In some embodiments, the antibody exhibits a binding affinity to a human PD-L1 protein of between 3 and 15 nM.

[0028] In some embodiments, the antibody further exhibits a total internalization higher than that of Ab1. In some embodiments, the total internalization has an AUC increase of between 9% and 155% relative to the AUC of Ab1. In some embodiments, the total internalization is determined by a FabFluor internalization assay.

[0029] In some embodiments, the antibody further exhibits an x50 higher than that of Ab1.

[0030] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE), and wherein the x50 is between 3 ng / mL and 20 ng / mL in a MDA-MB-231 cell line.

[0031] In some embodiments, the antibody is conjugated to camptothecin, and wherein the x50 is between 15 ng / mL and 55 ng / mL in a MDA-MB-231 cell line.

[0032] In some embodiments, the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 13-15 and the light chain CDR sequences of SEQ ID NOs: 16-18. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises the heavy chain variable region sequence of SEQ ID NO: 11 and the light chain variable region sequence of SEQ ID NO: 12.

[0033] In some embodiments, the antibody comprises a light chain of SEQ ID NO: 9 and a heavy chain of SEQ ID NO: 10.

[0034] In some embodiments, the fragment is a Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, linear antibody, or single-chain antibody fragment.

[0035] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0036] In some embodiments, the heavy chain constant region is of IgGl isotype.

[0037] In some embodiments, the antibody is a humanized or chimeric antibody.

[0038] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker, forming an antibody-drug conjugate having the structure:

[0039]

[0040] wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0041] 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 structure:

[0042] wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0043] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 13-15 and the light chain CDR sequences of SEQ ID NOs: 16-18.

[0044] In some embodiments, the antibody exhibits a binding affinity to a human PD-L1 protein of between 3 and 300 nM. In some embodiments, the antibody exhibits a binding affinity to a human PD-L1 protein of between 3 and 15 nM.

[0045] In some embodiments, the antibody further exhibits a total internalization higher than that of Ab1. In some embodiments, the total internalization has an AUC increase of between 9% and 155% relative to the AUC of Ab1. In some embodiments, the total internalization is determined by a FabFluor internalization assay.

[0046] In some embodiments, the antibody further exhibits an x50 higher than that of Ab1.

[0047] In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE), and wherein the x50 is between 3 ng / mL and 20 ng / mL in a MDA-MB-231 cell line.

[0048] In some embodiments, the antibody is conjugated to camptothecin, and wherein the x50 is between 15 ng / mL and 55 ng / mL in a MDA-MB-231 cell line.

[0049] In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 80% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 80% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 90% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 11 and a light chain variable region sequence having at least 95% sequence identity to SEQ ID NO: 12. In some embodiments, the antibody comprises a heavy chain variable region sequence of SEQ ID NO: 11 and a light chain variable region sequence of SEQ ID NO: 12.

[0050] In some embodiments, the antibody comprises a light chain of SEQ ID NO: 9 and a heavy chain of SEQ ID NO: 10.

[0051] In some embodiments, the fragment is a Fab, Fab', F(ab')2, Fab'-SH, Fv, diabody, linear antibody, or single-chain antibody fragment.

[0052] In some embodiments, the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

[0053] In some embodiments, the heavy chain constant region is of IgGl isotype.

[0054] In some embodiments, the antibody is a humanized or chimeric antibody.

[0055] In some embodiments, the antibody is conjugated to a cytotoxic agent via a linker. In some embodiments, the antibody is conjugated to monomethyl auristatin E (MMAE). In some embodiments, the antibody is conjugated to MMAE via an enzyme-cleavable linker unit. In some embodiments, the enzyme-cleavable linker unit comprises a Val-Cit linker. In some embodiments, the antibody is conjugated to MMAE via a linker, forming an antibody-drug conjugate having the structure:

[0056]

[0057] wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0058] 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:

[0059] wherein Ab represents the antibody and p ranges from 2 to 10. In some embodiments, p is 4. In some embodiments, p is 8.

[0060] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human PD-L1 protein, wherein the antibody is conjugated to camptothecin to form an antibody-drug conjugate, wherein the antibody-drug conjugate has the following structure:

[0061] wherein Ab is an anti-PD-L1 antibody; y is 1, 2, 3, or 4, or is 1 through 4; and z is an integer from 2 to 12, or is 2, 4, 8, or 12; and p is 1-16.

[0062] In some embodiments, the antibody-drug conjugate has the following structure:

[0063]

[0064] In some embodiments, p ranges from 2 to 10.

[0065] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human Programmed Death Ligand 1 (PD-L1) protein, wherein the antibody exhibits a binding affinity to the human PD-L1 protein that is greater than that of Ab1. In some embodiments, the antibody exhibits a binding affinity that is greater than 2.7 nM.

[0066] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human Programmed Death Ligand 1 (PD-L1) protein, wherein the antibody exhibits a k assoc of the human PD-L1 protein that is less than that of Ab1. In some embodiments, the antibody exhibits a k assoc of the human PD-L1 protein that is less than 5 x 10 5 M -1 s -1 of the human PD-L1 protein that is less than 5 x 10 assoc .

[0067] Also provided herein are antibodies or antigen-binding fragments thereof that specifically bind to a human Programmed Death Ligand 1 (PD-L1) protein, wherein the antibody exhibits a kdissoc k dissoc In some embodiments, the antibody exhibits a k 3 s -1 of 2 x 10 dissoc .

[0068] Also provided herein are antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof that specifically binds to a human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs, and wherein the antibody exhibits a binding affinity for the human PD-L1 protein of between 5 nM and 15 nM, and wherein the antibody is conjugated to MMAE.

[0069] Also provided herein are antibody-drug conjugates comprising an antibody or antigen-binding fragment thereof that specifically binds to a human PD-L1 protein, wherein the antibody comprises the heavy chain CDR sequences of SEQ ID NOs: 3-5 and the light chain CDR sequences of SEQ ID NOs: 6-8, wherein the antibody comprises one or more amino acid substitutions within one or more CDRs, and wherein the antibody exhibits a binding affinity for the human PD-L1 protein of between 5 nM and 15 nM, and wherein the antibody is conjugated to camptothecin.

[0070] Also provided herein are pharmaceutical compositions comprising a therapeutically effective amount of any of the antibodies described herein and a pharmaceutically acceptable excipient.

[0071] Also provided herein are methods of treating cancer in a subject, the method comprising administering to the subject any of the antibodies described herein. In some embodiments, the subject is a human subject. In some embodiments, the cancer is melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, or cervical cancer.

[0072] Also provided herein are nucleic acids encoding any of the antibodies described herein.

[0073] Also provided herein are vectors comprising any of the nucleic acids described herein.

[0074] Also provided herein are any of the host cells described herein comprising any of the nucleic acids described herein. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell.

[0075] This document also provides a method for generating an antibody or antigen-binding fragment thereof that specifically binds to the human PD-L1 protein, the method comprising culturing any host cell described herein under conditions suitable for generating the antibody.

[0076] This document also provides a method for generating an antibody-drug conjugate that specifically binds to the human PD-L1 protein, the method comprising culturing the host cells described herein under conditions suitable for generating the antibody; and conjugating the antibody to a cytotoxic agent. In some embodiments, the cytotoxic agent is MMAE or camptothecin.

[0077] This article also provides information on any anti-PD-L1 antibody or any antibody-drug conjugate described herein in the manufacture of drugs for the treatment of cancer (e.g., with PD-L1). + Use in drugs that express related cancer.

[0078] This article also provides the anti-PD-L1 antibody or antibody-drug conjugate described herein for the treatment of cancer (e.g., with PD-L1). + (Expression related to cancer).

[0079] This article also provides information on the anti-PD-L1 antibody or antibody-drug conjugate described herein for medical use.

[0080] This article also provides PD-L1 inhibitors for killing subjects who require it. + A cellular method comprising administering to the subject a therapeutically effective amount of any of the anti-PD-L1 antibodies or any of the antibody-drug conjugates described herein.

[0081] This article also provides information on any anti-PD-L1 antibody or any antibody-drug conjugate described herein for the manufacture of PD-L1 for killing subjects in need. + Uses in cell-based pharmaceuticals.

[0082] This article also provides information on reducing solid tumors in subjects (e.g., PD-L1). + A method for measuring the volume of a solid tumor, the method comprising administering to the subject a therapeutically effective amount of any antiPD-L1 antibody or any antibody-drug conjugate described herein.

[0083] This article also provides information on any anti-PD-L1 antibody or any antibody-drug conjugate described herein in the manufacture of drugs intended to reduce the size of solid tumors (e.g., PD-L1) in subjects. + The use of drugs to measure the volume of solid tumors. Attached Figure Description

[0084] Figure 1Exemplary amino acid residues selected for mutation for Ab1 are shown.

[0085] Figures 2A-2F Cytotoxicity of SG-559-xx ADCs in several cell lines is shown.

[0086] Figures 3A-3B Internalization of SG-559-01 and SG-559-03 compared to control antibodies is shown.

[0087] Figures 4A-4B Anti-tumor activity of SG-559-xx ADCs in MDA-MB-231 mouse models is shown.

[0088] Figures 5A-5B Anti-tumor activity of SG-559-xx ADCs in BxPC3 mouse models is shown.

[0089] Figures 6A-6B Anti-tumor activity of SG-559-01LALA ADC in Karpas 299 mouse models is shown.

[0090] Figure 7 Anti-tumor activity of SG-559-01LALA ADC in Calu-1 mouse models is shown.

[0091] Figures 8A-8B Anti-tumor activity of SG-559-01LALA ADC in EBC-1 mouse models is shown.

[0092] Figure 9 In vitro PD-1 / PD-L1 blocking activity of SG-559-01LALA antibody and ADC is shown.

[0093] Figures 10A-10D Immunotoxicity of SG-559-01 and SG-559-01LALA ADC in human APC models is shown.

[0094] Figures 11A-11D Immunotoxicity of SG-559-xx ADCs in human APC models is shown.

[0095] Figures 12A-12D LPS-stimulated immune response in vitro of human APC treated with SG-559-01 ADC is shown.

[0096] Figures 13A-13C Intratumoral immune cell infiltration in mice with Karpas 299 tumors treated with SG-559-01LALA vc-MMAE ADC is shown.

[0097] Figures 14A-14FIntratumoral inflammatory cytokine responses in mice bearing Karpas 299 tumors treated with SG-559-01 LALA vc-MMAE ADCs are shown. DETAILED DESCRIPTION

[0098] I. DEFINITIONS

[0099] To facilitate a better understanding of the present disclosure, certain terms are defined first. As used in the present application, and unless specifically defined in the present specification, each of the following terms shall have the meaning set forth therein. Additional definitions are set forth throughout the present application.

[0100] The term "and / or," where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, for example, "A and / or B" is a specific disclosure of "A" and "B," of "A" and "not B," of "not A" and "B," and of "not A" and "not B." Likewise, the term "and / or" as used herein, such as in a phrase such as "A, B, and / or C," is to be taken as specific disclosure of each of the following: A, B, and C; A, B, and not C; A, and not B, and C; not A, B, and C; not A, B, and not C; not A, and not B, and C; A, and not B, and not C; not A, B, and C; not A, B, and not C; not A, and not B, and C; A, B, and C; A, B, and not C; A, and not B, and C; not A, B, and C; not A, B, and not C; not A, and not B, and C; A, and B; A and C; B and C; not A and B; not A and C; not B and C; not A, not B, and not C; A; B; C; not A; not B; and not C.

[0101] It is to be understood that the aspects and embodiments of the present application described herein encompass "comprising multiple aspects and embodiments," "consisting of multiple aspects and embodiments," and "consisting essentially of multiple aspects and embodiments."

[0102] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is directed. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0103] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0104] The terms "PD-L1," "CD274," "B7-H1," and "Programmed cell death ligand 1" are used interchangeably herein and include any variant, isoform, and species homolog of human PD-L1 generally expressed by a cell or on a cell transfected with a PD-L1 gene, unless otherwise indicated.

[0105] The term "immunoglobulin" refers to a class of structurally related glycoproteins that are composed of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four connected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain typically is comprised of a heavy chain variable region (abbreviated herein as V H or VH) and a heavy chain constant region (C H or CH). The heavy chain constant region typically is comprised of three domains, C H 1, C H 2, and C H 3. The heavy chains are typically interconnected by disulfide bonds in the so-called "hinge" region. Each light chain typically is comprised of a light chain variable region (abbreviated herein as V L or VL) and a light chain constant region (C L or CL). The light chain constant region typically is comprised of one domain, C L . The CL can be of kappa (kappa) or lambda (lambda) isotype. The terms "constant domain" and "constant region" are used interchangeably herein. The immunoglobulin can be derived from any of the known isotypes, including but not limited to IgA, secretory IgA, IgG, and IgM. IgG subclasses are also well known to those skilled in the art and include but are not limited to human IgGl, IgG2, IgG3, and IgG4. "Isotype" refers to the antibody class or subclass (e.g., IgM or IgGl) that is encoded by heavy chain constant region genes.

[0106] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable regions of the heavy chain and light chain (V H and V L) can be further subdivided into regions of hypervariability (or hypervariable regions, which when in the form of sequence and / or structure defining loops can be hypervariable), also called complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to non-contiguous sequences of amino acids within the variable region of an antibody that impart antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). "Framework regions" and "FRs" are known in the art to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). In each V H and V L In each V

[0107] In the context of the present application, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either, having the ability to specifically bind to an antigen under typical physiological conditions, with a half-life that is a significant period of time, such as at least about 30 min, at least about 45 min, at least about one hour (h), at least about two hours, at least about four hours, at least about eight hours, at least about 12 hours (h), about 24 hours or more, about 48 hours or more, about three, four, five, six, seven days or more, etc., or any other relevant functionally defined period of time (such as a period of time sufficient to induce, promote, enhance, and / or modulate a physiological response associated with the bound antigen by the antibody and / or sufficient to allow the antibody to recruit effector activity). The variable regions of the heavy and light chains of an immunoglobulin molecule contain the binding domain that interacts with an antigen. The constant regions of an antibody (Ab) can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (such as effector cells) and components of the complement system (such as Clq, the first component in the classical pathway of complement activation). An antibody can also be a bispecific antibody, diabody, multispecific antibody, or similar molecule.

[0108] The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules produced by a single primary amino acid sequence recombinantly. A monoclonal antibody composition displays single binding specificity and affinity to a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody displaying single binding specificity that has variable and constant regions of immunoglobulin sequences derived from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by a hybridoma including a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.

[0109] An "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-L1 is substantially free of antibodies that specifically bind antigens other than PD-L1). An isolated antibody that specifically binds to PD-L1 may, however, have cross-reactivity to other antigens, such as PD-L1 molecules from different species. Moreover, an isolated antibody can be substantially free of other cellular material and / or chemicals. In one embodiment, an isolated antibody includes an antibody conjugate attached to another agent (e.g., a small molecule drug). In some embodiments, an isolated anti-PD-L1 antibody includes a conjugate of an anti-PD-L1 antibody with a small molecule drug (e.g., MMAE or MMAF).

[0110] A "human antibody" (HuMAb) refers to an antibody having variable regions in which both the FR and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which

[0111] The term "humanized antibody" as used herein refers to a genetically engineered non-human antibody that contains human antibody constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting the six non-human antibody complementarity determining regions (CDRs) that collectively form the antigen binding site onto cognate human acceptor framework regions (FRs) (see WO 92 / 22653 and EP 0629240). In order to fully reconstitute the binding affinity and specificity of the parent antibody, it can be necessary to substitute framework residues from the parent antibody (i.e. the non-human antibody) into the human framework regions (back mutations). Structural homology modeling can aid in the identification of amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized antibody can comprise non-human CDR sequences, a primarily human framework region optionally containing one or more amino acid back mutations of non-human amino acid sequences, and a fully human constant region. Optionally, additional amino acid modifications (which are not necessarily back mutations) can be applied to obtain a humanized antibody with preferred characteristics such as affinity and biochemical properties.

[0112] The term "chimeric antibody" as used herein refers to an antibody in which the variable region is derived from a non-human species (e.g. from a rodent) and the constant region is derived from a different species (such as human). Chimeric antibodies can be generated by antibody engineering. "Antibody engineering" is a generic term for modifications of antibodies of different species and is a process well known to the skilled person. In particular, chimeric antibodies can be generated by using standard DNA techniques as described in Sambrook et al., 1989, Molecular Cloning: A laboratory Manual, New York: Cold Spring Harbor Laboratory Press, Chapter 15. Thus, the chimeric antibody can be a genetically or enzymatically engineered recombinant antibody. The generation of chimeric antibodies is within the knowledge of the skilled person and thus, the generation of a chimeric antibody according to the present application can be performed by other methods than described herein. Chimeric monoclonal antibodies have been developed for therapeutic applications to reduce the immunogenicity of the antibody. They can typically contain non-human (e.g. murine) variable regions that are specific for the antigen of interest and human constant antibody heavy and light chain domains. The term "variable region" or "variable domain" used in the context of a chimeric antibody refers to the region comprising the CDRs and framework regions of the heavy and light chains of an immunoglobulin.

[0113] "Anti-antigen antibody" refers to an antibody that binds to an antigen. For example, an anti-PD-Ll antibody is an antibody that binds to the antigen PD-L1.

[0114] An "antigen-binding portion" or "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen to which the whole antibody binds. Examples of antibody fragments (e.g., antigen-binding fragments) include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each of which has one antigen-binding site, and a residual "Fc" fragment, which has no antigen-binding site. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0115] "Percentage (%) sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, the percent sequence identity of a given amino acid sequence A to / with / relative to a given amino acid sequence B (or this can be expressed in the phrase as a given amino acid sequence A that has or comprises a certain % sequence identity to / with / relative to a given amino acid sequence B) is calculated as follows:

[0116] Score X / Y times 100

[0117] where X is the number of amino acid residues scored as identical matches by the sequence alignment program across the alignment length of the two sequences being compared, and where Y is the total number of amino acid residues in sequence B. It will be appreciated that where amino acid sequences A and B do not have the same length, the % sequence identity of A relative to B will not equal the % sequence identity of B relative to A.

[0118] As used herein, in the context of an antibody binding to a predetermined antigen, the terms "binding" or "bind" or "specifically binding" typically are with an affinity corresponding to a KDof about 10 -6 M or less (e.g., 10-7 M or less, such as about 10 -8 M or less, such as about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 M or even less) of K D , and wherein the antibody binds to a predetermined antigen with an affinity corresponding to a K D at least ten-fold lower (such as at least 100-fold, for example at least 1,000-fold, such as at least 10,000-fold, for example at least 100,000-fold) than its K D , wherein the K D lowered to a degree depending on the K D , thus when the K D is very low, then the K D lower than the K D The degree to which the K

[0119] As used herein, the term "K D " (M) refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Affinity and K D are inversely related, i.e. a higher affinity is intended to refer to a lower K D , and a lower affinity is intended to refer to a higher K D .

[0120] The term "ADC" refers to an antibody-drug conjugate, which in the context of the present application refers to an anti-PD-Ll antibody coupled to a drug moiety (e.g. MMAE or MMAF) as described in the present application.

[0121] The abbreviations "vc" and "val-cit" refer to the dipeptide linker valine-citrulline.

[0122] The abbreviation VKG refers to the tripeptide linker valine-lysine-glycine.

[0123] The abbreviation "MC" refers to the maleimidocaproyl extender:

[0124]

[0125] The abbreviation "MP" refers to the maleimidopropionyl extender:

[0126]

[0127] A "PEG unit" as used herein is an organic moiety composed of repeating ethylene- oxy subunits (PEG or PEG subunits) and can be polydisperse, monodisperse, or discrete (i.e., having a discrete number of ethylene-oxy subunits). Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from the heterogeneous mixture, thus providing a single chain length and molecular weight. Preferred PEG units comprise discrete PEG, i.e., compounds synthesized in a stepwise fashion rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length.

[0128] The PEG units provided herein comprise one or more polyethylene glycol chains, each polyethylene glycol chain composed of one or more ethyleneoxy subunits covalently attached to one another. The polyethylene glycol chains can be linked together, for example, in a linear, branched, or star configuration. Typically, at least one polyethylene glycol chain prior to incorporation into the camptothecin conjugate is derivatized at one end with an alkyl moiety substituted with an electrophilic group for covalent attachment to the carbamate nitrogen of the methylenecarbamate unit (i.e., an example of R). Typically, the terminal ethyleneoxy subunit in each polyethylene glycol chain that will not participate in covalent attachment to the remaining portion of the linker unit is modified with a PEG capping unit (typically an optionally substituted alkyl group such as -CH3, CH2CH3, or CH2CH2CO2H). Preferred PEG units have a single polyethylene glycol chain having from 2 to 24 -CH2CH2- subunits covalently attached in series and terminated at one end with a PEG capping unit.

[0129] "Cancer" refers to a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. "Cancer" or "cancerous tissue" can include a tumor. Unregulated cell division and growth results in the formation of malignant tumors, which invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream. After metastasis, the distant tumor can be said to be "derived" from the pre-metastatic tumor.

[0130] The term "antibody-dependent cellular cytotoxicity" or ADCC is a mechanism of inducing cell death that relies on the interaction of antibody-coated target cells with immune cells (also called effector cells) that have lytic activity. Such effector cells include natural killer cells, monocytes / macrophages, and neutrophils. Effector cells attach to one or more Fc effector domains of Ig, which is bound to the target cell via its antigen-binding site. Death of the antibody-coated target cell occurs as a result of effector cell activity.

[0131] The term“antibody-dependent cellular phagocytosis” or ADCP refers to the process by which antibody-coated cells are internalized, in whole or in part, by phagocytic immune cells (e.g., macrophages, neutrophils, and dendritic cells) that bind to one or more Fc effector domains of Ig.

[0132] The term“complement-dependent cytotoxicity” or CDC refers to a mechanism of inducing cell death in which one or more Fc effector domains of a target-bound antibody activates a series of enzymatic reactions that ultimately results in the formation of a pore in the target cell membrane. Typically, antigen-antibody complexes, such as those on antibody-coated target cells, bind and activate complement component Clq, which in turn activates the complement cascade, leading to target cell death. Activation of complement can also result in the deposition of complement components on the surface of target cells that promote ADCC by binding to complement receptors (e.g., CR3) on leukocytes.

[0133] “Cytostatic effect” refers to the inhibition of cell proliferation. A“cytostatic agent” refers to an agent that has a cytostatic effect on cells, thereby inhibiting the growth and / or expansion of a particular cell subpopulation. A cytostatic agent can be conjugated to an antibody or administered in combination with an antibody.

[0134] “Treatment” or“therapy” of a subject refers to any type of intervention or process performed on a subject, or administration of an active agent to a subject, with the objective to reverse, alleviate, improve, inhibit, slow down or prevent the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. In some embodiments, the disease is cancer.

[0135] A“subject” includes any human or non-human animal. The term“non-human animal” includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents (e.g., mice, rats, and hamsters). In some embodiments, the subject is a human. The terms“subject” and“patient” and“individual” are used interchangeably herein.

[0136] An“effective amount” or“therapeutically effective amount” or“therapeutically effective dose” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes disease regression as evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or prevention of impairment or disability due to the disease. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as evaluation in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0137] For example, for the treatment of a tumor, a therapeutically effective amount of an anti-cancer agent inhibits cell growth or tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, or at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% in treated subjects (e.g., one or more treated subjects) relative to untreated subjects (e.g., one or more untreated subjects). In some embodiments, a therapeutically effective amount of an anti-cancer agent inhibits cell growth or tumor growth 100% in treated subjects (e.g., one or more treated subjects) relative to untreated subjects (e.g., one or more untreated subjects).

[0138] In other embodiments of the disclosure, tumor regression can be observed and sustained 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.

[0139] A therapeutically effective amount of a drug includes a "prophylactically effective amount," which is any amount of a drug (e.g., an anti-PD-Ll antibody-drug conjugate) that, when administered alone or in combination with an anti-cancer agent to a subject at risk of developing a cancer (e.g., a subject having a precancerous condition) or suffering a recurrence of a cancer, inhibits the development or recurrence of the cancer. In some embodiments, a prophylactically effective amount completely prevents the occurrence or recurrence of a cancer. To "inhibit" the occurrence or recurrence of a cancer means to reduce the likelihood of the occurrence or recurrence of a cancer, or to completely prevent the occurrence or recurrence of a cancer.

[0140] As used herein, "subtherapeutic dose" means a dose of a therapeutic compound (e.g., an anti-PD-Ll antibody-drug conjugate) that is lower than the usual or typical dose of the therapeutic compound when administered alone to treat a hyperproliferative disease (e.g., a cancer).

[0141] "Immune-related response pattern" refers to a pattern of clinical response often observed in cancer patients treated with immunotherapeutic agents that exert an anti-tumor effect by inducing a cancer-specific immune response or by modifying natural immune processes. This response pattern is characterized by a beneficial therapeutic effect after an initial increase in tumor burden or appearance of new lesions, which would be classified as progressive disease in the evaluation of traditional chemotherapeutic agents and would be synonymous with drug failure. Thus, proper evaluation of immunotherapeutic agents can require long-term monitoring of the impact of these agents on the disease of interest.

[0142] For example, an "anti-cancer agent" promotes regression of cancer in a subject. In some embodiments, a therapeutically effective amount of a drug promotes regression of cancer to the extent that the cancer is eliminated. To "promote regression of cancer" means that administration of an effective amount of a drug, alone or in combination with an anti-cancer agent, results in a decrease in tumor growth or size, tumor necrosis, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. In addition, the terms "effective" and "effectiveness" with respect to a treatment include both pharmacological effectiveness and physiological safety. Pharmacological effectiveness is the ability of a drug to promote regression of cancer in a patient. Physiological safety is the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ, and / or organism level resulting from the administration of a drug.

[0143] A "durable response" refers to a sustained effect on reducing tumor growth after cessation of treatment. For example, the size of a tumor can remain the same or be smaller compared to the size at the beginning of the administration period. In some embodiments, the duration of a durable response is at least as long as the duration of treatment, or is at least 1.5, 2.0, 2.5, or 3 times longer than the duration of treatment.

[0144] As used herein, a "complete response" or "CR" refers to disappearance of all target lesions; a "partial response" or "PR" refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD; and "stable disease" or "SD" refers to neither sufficient shrinkage of target lesions to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started.

[0145] As used herein, "progression-free survival" or "PFS" refers to the length of time during and after treatment that the disease being treated (e.g., cancer) does not get worse. Progression-free survival can include the amount of time patients experience complete response or partial response as well as the amount of time patients experience stable disease.

[0146] As used herein, "overall response rate" or "ORR" refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0147] As used herein, "overall survival" or "OS" refers to the percentage of individuals in a group that can survive for a specified duration.

[0148] The phrase "pharmaceutically acceptable" indicates that the substance or composition must be compatible chemically and / or toxicologically with the other ingredients of a formulation and / or the mammal being treated with it.

[0149] As used herein, the phrase "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound of the present application. Exemplary salts include, but are not limited to, sulfates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, bisulfates, phosphates, acid phosphates, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, succinates, maleates, gentisates, fumarates, gluconates, glucuronates, saccharates, formates, benzoates, glutamates, methanesulfonates "mesylates", ethanesulfonates, benzenesulfonates, p-toluenesulfonates, pamoates (i.e., 4,4'-methylene-bis-(3-hydroxy-2-naphthoate)) salts, alkali metal (e.g., sodium and potassium) salts, alkaline earth metal (e.g., magnesium) salts, and ammonium salts. A pharmaceutically acceptable salt can involve inclusion of another molecule, such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt can have more than one charged atom and therefore can have more than one counterion. Examples of a pharmaceutically acceptable salt of a compound of the present application having a plurality of charged atoms can have a plurality of counterions. Thus, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterions.

[0150] "Administering" or "administration" refer to the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration of the anti-PD-Ll antibody-drug conjugate include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, e.g., by injection or infusion (e.g., intravenous infusion). The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection as well as intradermal, subcutaneous, intramuscular, and mucosal, such as nasal, vaginal, rectal, sublingual, or topical, administration. A therapeutic agent can be administered via a non-parenteral route or orally. Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasally, vaginally, rectally, sublingually, or topically. Administration can be, for example, one, multiple, and / or over one or more extended periods of time.

[0151] The terms "baseline" or "baseline value," as used interchangeably herein, can refer to a measurement or characterization of a symptom prior to or at the start of administration of a therapy (e.g., an anti-PD-Ll antibody-drug conjugate as described herein). A baseline value can be compared to a reference value to determine a reduction or improvement in a symptom of a PD-Ll -associated disease (e.g., a cancer) contemplated herein. The terms "reference" or "reference value," as used interchangeably herein, can refer to a measurement or characterization of a symptom after administration of the therapy (e.g., an anti-PD-Ll antibody-drug conjugate as described). A reference value can be measured one or more times during a dosage regimen or treatment cycle or at the completion of a dosage regimen or treatment cycle. A "reference value" can be an absolute value; a relative value; a value with an upper and / or lower limit; a range of values; a mean value; a median value; a mean; or a value compared to a baseline value.

[0152] Similarly, a "baseline value" can be an absolute value; a relative value; a value with an upper and / or lower limit; a range of values; a mean value; a median value; a mean; or a value compared to a reference value. A reference value and / or a baseline value can be obtained from one individual, two different individuals, or a group of individuals (e.g., a group of two, three, four, five, or more individuals).

[0153] The term "monotherapy" as used herein means that the anti-PD-Ll antibody-drug conjugate is the only anti-cancer agent administered to a subject during a treatment cycle. However, other therapeutic agents can be administered to the subject. For example, an anti-inflammatory agent or other agent that is administered to a subject with a cancer to treat symptoms associated with the cancer, including, for example, inflammation, pain, weight loss, and general malaise, but not the underlying cancer itself, can be administered during a monotherapy period.

[0154] An "adverse event" (AE) as used herein is any unfavorable and generally unintended sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medicinal product. A medicinal product can have one or more associated AEs, and each AE can have the same or different levels of severity. Reference to a method that is capable of "altering an adverse event" means a treatment regimen that reduces the incidence and / or severity of one or more AEs associated with the use of a different treatment regimen.

[0155] A "serious adverse event" or "SAE" as used herein is an adverse event that meets one of the following criteria:

[0156] • Results in death or life-threatening (As used in the definition of serious adverse event, "life-threatening" is an event in which the patient was at risk of death at the time of the event; it is not an event that, had it been any more severe, might have caused death.

[0157] • Results in persistent or significant disability / incapacity

[0158] • Constitutes a congenital anomaly / birth defect

[0159] • Has medical significance, defined as an event that jeopardizes the patient or that can require medical or surgical intervention to prevent one of the outcomes listed above. Medical and scientific judgment must be exercised in deciding whether an AE is “of medical significance”

[0160] • Requires inpatient hospitalization or prolongation of existing hospitalization, excluding the following: 1) routine therapeutic or monitoring of an underlying disease, independent of any worsening of the condition; 2) elective or pre-planned treatment of a pre-existing condition unrelated to the indication under study and not worsened since signing of informed consent; and 3) social reasons and temporary custodial care in the absence of any worsening of the patient’s overall condition.

[0161] Use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the indefinite articles “a” or “an” should be understood to refer to “one or more” of any stated or included component.

[0162] The term “about” or “consisting essentially of’ refers to a value or composition that is within an acceptable error range for the particular value or composition determined by one of ordinary skill in the art to be within the scope of the embodiments as defined by the variable and / or parameters appropriate for the measurement or determination of the value or composition, i.e., the limits of standard measurement error. For example, “about” or “consisting essentially of’ can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” or “consisting essentially of’ can mean up to 20% of a given value. Furthermore, particularly with regard to biological systems or processes, the term can mean up to an order of magnitude or up to 5-fold of a value. When providing a particular value or composition in the application and claims, unless otherwise indicated, the meaning of “about” or “consisting essentially of’ should be assumed to be within an acceptable error range of the particular value or composition.

[0163] Reference to “about” a value or parameter herein includes (and describes) embodiments with that value or parameter per se. For example, description in terms of “about X” includes and describes “X”.

[0164] As described herein, any concentration range, percentage range, ratio range, or integer range is intended to include the values expressly stated in that particular range, and, where appropriate, to include fractions of the statement values (e.g., one-tenth of an integer part, and one-hundredth of a percentage) to the extent that such fractions are potentially useful, unless otherwise indicated.

[0165] Various aspects of the disclosure are described in further detail in the following subsections.

[0166] II. SUMMARY

[0167] The present application provides antibodies and ADCs that specifically bind PD-L1. The present application is based in part on the discovery that antibody-drug conjugates targeting PD-L1, including MMAE antibody-drug conjugates and camptothecin antibody-drug conjugates, are particularly effective at killing cells expressing PD-L1+. PD-L1 has been shown to be expressed in a variety of cancers, including melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer.

[0168] III. Target Molecule

[0169] Unless otherwise indicated, PD-L1 refers to human PD-L1. An exemplary human protein sequence is assigned UniProt ID NO. Q9NZQ7.

[0170] IV. Antibodies of the Present Invention

[0171] Previously, selected antibodies that have been used to treat cancer have been conjugated to cytotoxic agents without sequence modification to produce antibody-drug conjugates (ADCs). These ADCs have generally proven to be as effective or more effective at killing tumor cells than the unconjugated antibodies. Previously, if modification of the antibody was considered in the process of making the ADC, some possible modifications would increase the binding affinity of the antibody or increase the antibody's action such as ADCC. However, it has been discovered that at least in some cases, modifying or adjusting the ADC antibody, for example, by decreasing its binding affinity or decreasing its ADCC activity, results in improved effectiveness of the ADC compared to the ADC with the unmodified antibody. Some examples of this include ADCs with anti-PD-Ll antibodies, such as Ab1, that are surprisingly optimized by modifying the antibody, for example, by decreasing its binding affinity. For example, in some cases, anti-PD-Ll ADCs are more effective at killing tumor cells in vitro when the binding affinity of the antibody conjugated to the cytotoxic agent is decreased. In another example, in some cases, anti-PD-Ll 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 decreased.

[0172] The present application provides antibodies, such as humanized antibodies, that bind PD-L1 with a binding affinity of between 3 nM and 300 nM. In some embodiments, the antibodies described herein can bind to PD-L1 with a K Dbetween 3 nM and 300 nM (e.g., about 3 nM to about 275 nM, about 3 nM to about 250 nM, about 3 nM to about 225 nM, about 3 nM to about 200 nM, about 3 nM to about 175 nM, about 3 nM to about 150 nM, about 3 nM to about 125 nM, about 3 nM to about 100 nM, about 3 nM to about 90 nM, about 3 nM to about 80 nM, about 3 nM to about 70 nM, about 3 nM to about 60 nM, about 3 nM to about 50 nM, about 3 nM to about 40 nM, about 3 nM to about 30 nM, about 3 nM to about 20 nM, about 3 nM to about 10 nM, about 10 nM to about 300 nM, about 10 nM to about 275 nM, about 10 nM to about 250 nM, about 10 nM to about 225 nM, about 10 nM to about 200 nM, about 10 nM to about 175 nM, about 10 nM to about 150 nM, about 10 nM to about 125 nM, about 10 nM to about 100 nM, about 10 nM to about 90 nM, about 10 nM to about 80 nM, about 10 nM to about 70 nM, about 10 nM to about 60 nM, about 10 nM to about 50 nM, about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 20 nM to about 300 nM, about 20 nM to about 275 nM, about 20 nM to about 250 nM, about 20 nM to about 225 nM, about 20 nM to about 200 nM, about 20 nM to about 175 nM, about 20 nM to about 150 nM, about 20 nM to about 125 nM, about 20 nM to about 100 nM, about 20 nM to about 90 nM, about 20 nM to about 80 nM, about 20 nM to about 70 nM, about 20 nM to about 60 nM, about 20 nM to about 50 nM, about 20 nM to about 40 nM, about 20 nM to about 30 nM, about 30 nM to about 300 nM, about 30 nM to about 275 nM, about 30 nM to about 250 nM, about 30 nM to about 225 nM, about 30 nM to about 200 nM, about 30 nM to about 175 nM, about 30 nM to about 150 nM, about 30 nM to about 125 nM, about 30 nM to about 100 nM, about 30 nM to about 90 nM, about 30 nM to about 80 nM, about 30 nM to about 70 nM, about 30 nM to about 60 nM, about 30 nM to about 50 nM, 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,Approximately 50 nm to approximately 300 nm, approximately 50 nm to approximately 275 nm, approximately 50 nm to approximately 250 nm, approximately 50 nm to approximately 225 nm, approximately 50 nm to approximately 200 nm, approximately 50 nm to approximately 175 nm, approximately 50 nm to approximately 150 nm, approximately 50 nm to approximately 125 nm, approximately 50 nm to approximately 100 nm, approximately 50 nm to approximately 90 nm, approximately 50 nm to approximately 80 nm, approximately 50 nm to approximately 70 nm, approximately 50 nm to approximately 60 nm, approximately 60 nm to approximately 300 nm, approximately 60 nm to approximately 275 nm, approximately 60 nm to approximately 250 nm, approximately 60 nm to approximately 225 nm, approximately 60 nm to approximately 200 nm, approximately 60 nm to approximately 175 nm, approximately 60 nm to Approximately 150 nm, approximately 60 nm to approximately 125 nm, approximately 60 nm to approximately 100 nm, approximately 60 nm to approximately 90 nm, approximately 60 nm to approximately 80 nm, approximately 60 nm to approximately 70 nm, approximately 70 nm to approximately 300 nm, approximately 70 nm to approximately 275 nm, approximately 70 nm to approximately 250 nm, approximately 70 nm to approximately 225 nm, approximately 70 nm to approximately 200 nm, approximately 70 nm to approximately 175 nm, approximately 70 nm to approximately 150 nm, approximately 70 nm to approximately 125 nm, approximately 70 nm to approximately 100 nm, approximately 70 nm to approximately 90 nm, approximately 70 nm to approximately 80 nm, approximately 80 nm to approximately 300 nm, approximately 80 nm to approximately 275 nm, approximately 80 nm to approximately 250 nm, approximately 80nM to about 225nM, about 80nM to about 200nM, about 80nM to about 175nM, about 80nM to about 150nM, about 80nM to about 125nM, about 80nM to about 100nM, about 80nM to about 90nM, about 90nM to about 300nM, about 90nM to about 275nM, about 90nM to about 250nM, about 90nM to about 225nM, about 90nM to about 200nM, about 90nM to about 175nM, about 90nM to about 150nM, about 90nM to about 125nM, about 90nM to about 100nM, about 100nM to about 300nM, about 100nM to about 275nM, about 100nM to about 250nM, about 1 00 nm to about 225 nm, about 100 nm to about 200 nm, about 100 nm to about 175 nm, about 100 nm to about 150 nm, about 100 nm to about 125 nm, about 125 nm to about 300 nm, about 125 nm to about 275 nm, about 125 nm to about 250 nm, about 125 nm to about 225 nm, about 125 nm to about 200 nm, about 125 nm to about 175 nm, about 125 nm to about 150 nm, about 150 nm to about 300 nm, about 150 nm to about 275 nm, about 150 nm to about 250 nm, about 150 nm to about 225 nm, about 150 nm to about 200 nm, about 150 nm to about 175 nm.about 175 nM to about 300 nM, about 175 nM to about 275 nM, about 175 nM to about 250 nM, about 175 nM to about 225 nM, about 175 nM to about 200 nM, about 200 nM to about 300 nM, about 200 nM to about 275 nM, about 200 nM to about 250 nM, about 200 nM to about 225 nM, about 225 nM to about 300 nM, about 225 nM to about 275 nM, about 225 nM to about 250 nM, about 250 nM to about 300 nM, about 250 nM to about 275 nM, or about 275 nM to about 30 nM) (e.g., as measured by Bio-Layer Interferometry (BLI) in phosphate buffered saline).

[0173] In some embodiments, the binding affinity is monovalent binding affinity. In some embodiments, the antibodies are point mutants of fully human anti-PD-Ll antibody Ab1. Ab1 is defined by the CDR regions of SEQ ID NOs: 3-5 and 6-8, the variable regions of SEQ ID NOs: 1 and 2, and the heavy and light chains of SEQ ID NOs: 86 and 87. In further embodiments, the point mutations are present in the CDR regions. In some embodiments, the point mutants exhibit reduced binding affinity and / or increased cytotoxicity and / or internalization rate compared to Ab1. In some embodiments, the point mutants exhibit reduced binding affinity and increased cytotoxicity in vitro. In some embodiments, the point mutants exhibit reduced binding affinity and increased cytotoxicity in vivo. In some embodiments, the point mutants exhibit reduced binding affinity and increased cytotoxicity both in vitro and in vivo. In some embodiments, the point mutants exhibit reduced binding affinity and increased internalization rate in vitro. In some embodiments, the point mutants exhibit reduced binding affinity and increased internalization rate in vivo. In some embodiments, the point mutants exhibit reduced binding affinity and increased internalization rate both in vitro and in vivo.

[0174] In some embodiments, the anti-PD-Ll antibodies provided herein can have one or two total amino acid substitutions in the six CDRs of the set of heavy chain CDRs of SEQ ID NOs. 3-5 and light chain CDRs of SEQ ID NOs. 6-8, and bind to PD-L1 with a KD between 3 nM and 300 nM. In some embodiments, the anti-PD-Ll antibodies provided herein can have one amino acid substitution in the six CDRs of the set of heavy chain CDRs of SEQ ID NOs. 3-5 and light chain CDRs of SEQ ID NOs. 6-8, and bind to PD-L1 with a KD between 3 nM and 300 nM.

[0175] In some embodiments, an anti-PD-L1 antibody provided herein can have a heavy chain CDR1 having one amino substitution in SEQ ID NO: 3, a heavy chain CDR2 of SEQ ID NO: 4, a heavy chain CDR3 of SEQ ID NO: 5, a light chain CDR1 of SEQ ID NO: 6, a light chain CDR2 of SEQ ID NO: 7, and a light chain CDR3 of SEQ ID NO: 8 and binds to PD-L1 with a KD between 3 nM and 300 nM. In some embodiments, the one amino acid substitution in SEQ ID NO: 3 is at amino acid position 2 of SEQ ID NO: 3. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution of tyrosine to alanine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution of tyrosine to serine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution of tyrosine to glycine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution of tyrosine to threonine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution of tyrosine to valine. In some embodiments, the one amino acid substitution at amino acid position 2 of SEQ ID NO: 3 is an amino acid substitution of tyrosine to cysteine.

[0176] In some embodiments, an anti-PD-L1 antibody provided herein binds to both glycosylated and non-glycosylated PD-L1 with a KD between 3 nM and 300 nM (or any subranges of this range described herein). D

[0177] ​In some embodiments, an anti-PD-L1 antibody provided herein has an increase (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 between a 5% increase and a 300% increase, between a 5% increase and a 280% increase, between a 5% increase and a 260% increase, between a 5% increase and a 240% increase, between a 5% increase and a 220% increase, between a 5% increase and a 200% increase, between a 5% increase and a 180% increase, between a 5% increase and a 160% increase, between a 5% increase and a 140% increase, between a 5% increase and a 120% increase, between a 5% increase and a 100% increase, between a 5% increase and a 80% increase, between a 5% increase and a 60% increase, between a 5% increase and a 40% increase, between a 5% increase and a 20% increase, between a 5% increase and a 10% increase, between a 10% increase and a 300% increase, between a 10% increase and a 280% increase, between a 10% increase and a 260% increase, between a 10% increase and a 240% increase, between a 10% increase and a 220% increase, between a 10% increase and a 200% increase, between a 10% increase and a 180% increase, between a 10% increase and a 160% increase, between a 10% increase and a 140% increase, between a 10% increase and a 120% increase, between a 10% increase and a 100% increase, between a 10% increase and a 80% increase, between a 10% increase and a 60% increase, between a 10% increase and a 40% increase, between a 10% increase and a 20% increase, between a 20% increase and a 300% increase, between a 20% increase and a 280% increase, between a 20% increase and a 260% increase, between a 20% increase and a 240% increase, between a 20% increase and a 220% increase, between a 20% increase and a 200% increase, between a 20% increase and a 180% increase, between a 20% increase and a 160% increase, between a 20% increase and a 140% increase, between a 20% increase and a 120% increase, between a 20% increase and a 100% increase, between a 20% increase and a 80% increase, between a 20% increase and a 60% increase, between a 20% increase and a 40% increase, between a 40% increase and a 300% increase, between a 40% increase and a 280% increase, between a 40% increase and a 260% increase, between a 40% increase and a 240% increase, between a 40% increase and a 220% increase, between a 40% increase and a 200% increase, between a 40% increase and a 180% increase, between a 40% increase and a 160% increase, between a 40% increase and a 140% increase, between a 40% increase and a 120% increase, between a 40% increase and a 100% increase, between a 40% increase and a 80% increase, between a 40% increase and a 60% increase, in vitro and / or in vivo, compared to Ab1.between 40% increase and 80% increase, between 40% increase and 60% increase, between 60% increase and 300% increase, between 60% increase and 280% increase, between 60% increase and 260% increase, between 60% increase and 240% increase, between 60% increase and 220% increase, between 60% increase and 200% increase, between 60% increase and 180% increase, between 60% increase and 160% increase, between 60% increase and 140% increase, between 60% increase and 120% increase, between 60% increase and 100% increase, between 60% increase and 80% increase, between 80% increase and 300% increase, between 80% increase and 280% increase, between 80% increase and 260% increase, between 80% increase and 240% increase, between 80% increase and 220% increase, between 80% increase and 200% increase, between 80% increase and 180% increase, between 80% increase and 160% increase, between 80% increase and 140% increase, between 80% increase and 120% increase, between 80% increase and 100% increase, between 100% increase and 300% increase, between 100% increase and 280% increase, between 100% increase and 260% increase, between 100% increase and 240% increase, between 100% increase and 220% increase, between 100% increase and 200% increase, between 100% increase and 180% increase, between 100% increase and 160% increase, between 100% increase and 140% increase, between 100% increase and 120% increase, between 120% increase and 300% increase, between 120% increase and 280% increase, between 120% increase and 260% increase, between 120% increase and 240% increase, between 120% increase and 220% increase, between 120% increase and 200% increase, between 120% increase and 180% increase, between 120% increase and 160% increase, between 120% increase and 140% increase, between 140% increase and 300% increase, between 140% increase and 280% increase, between 140% increase and 260% increase, between 140% increase and 240% increase, between 140% increase and 220% increase, between 140% increase and 200% increase, between 140% increase and 180% increase, between 140% increase and 160% increase, between 160% increase and 300% increase, between 160% increase and 280% increase,cytotoxicity of PD-L1+ cells between 160% increase and 260% increase, between 160% increase and 240% increase, between 160% increase and 220% increase, between 160% increase and 200% increase, between 160% increase and 180% increase, between 180% increase and 300% increase, between 180% increase and 280% increase, between 180% increase and 260% increase, between 180% increase and 240% increase, between 180% increase and 220% increase, between 180% increase and 200% increase, between 200% increase and 300% increase, between 200% increase and 280% increase, between 200% increase and 260% increase, between 200% increase and 240% increase, between 200% increase and 220% increase, between 220% increase and 300% increase, between 220% increase and 280% increase, between 220% increase and 260% increase, between 220% increase and 240% increase, between 240% increase and 300% increase, between 240% increase and 280% increase, between 240% increase and 260% increase, between 260% increase and 300% increase, between 260% increase and 280% increase, or between 280% increase and 300% increase.

[0178] In some embodiments, an 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 between 5% increase and 300% increase (or any subranges of this range described herein)) intracellularization rate by PD-L1+ cells compared to Ab1.

[0179] In some embodiments, the anti-PD-Ll antibodies provided herein have an increase (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or between a 5% increase and a 300% increase (or any sub-range of this range described herein)) in immune cell infiltration upon administration to a mammal compared to Ab1.

[0180] In some embodiments, the anti-PD-Ll antibodies provided herein have an increase (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or between a 5% increase and a 300% increase (or any sub-range of this range described herein)) in inflammatory cytokine production (e.g., one or more of any of the cytokines described herein) upon administration to a mammal compared to Ab1.

[0181] In some embodiments, the anti-PD-Ll antibodies provided herein have an increase (e.g., at least a 5% increase, at least a 10% increase, at least a 20% increase, at least a 30% increase, at least a 40% increase, at least a 50% increase, at least a 60% increase, at least a 70% increase, at least a 80% increase, at least a 90% increase, at least a 100% increase, at least a 120% increase, at least a 140% increase, at least a 160% increase, at least a 180% increase, at least a 200% increase, at least a 220% increase, at least a 240% increase, at least a 260% increase, at least a 280% increase, at least a 300% increase, or between a 5% increase and a 300% increase (or any sub-range of this range described herein)) in intracellular digestion by PD-L1+ cells compared to Ab1.

[0182] In some embodiments, the anti-PD-Ll antibodies provided herein have less than 10% change in neutrophil and / or platelet count (e.g., less than 8% change, less than 6% change, less than 4% change, less than 2% change, or less than 1% change) after administration to a mammal compared to AbI.

[0183] The binding affinity (i.e., dissociation constant, K D ) of the PD-L1 antibodies of the present application is preferably greater than the binding affinity of AbI. Preferred PD-L1 antibodies bind to the same epitope as AbI and / or compete for binding to human PD-L1. In one embodiment, the binding affinity of the PD-L1 antibodies of the present application is greater than 2.7 nM. In a further embodiment, the monovalent binding affinity of the PD-L1 antibodies of the present application is greater than 2.7 nM. In another embodiment, the k assoc (on rate) is less than the k assoc of AbI. In a further embodiment, the k assoc is less than 5.5 x 10 5 M -1 s -1 In another embodiment, the k dissoc (off rate) is greater than the k dissoc of AbI. In a further embodiment, the k dissoc is greater than 1.50 x 10 3 s -1 .

[0184] The anti-PD-Ll antibodies of the present application can also be described or specified according to their binding affinity for PD-L1 (e.g., human PD-L1). In some embodiments, preferred binding affinities include those having a dissociation constant or K Dthose. In some embodiments, preferred PD-L1 antibodies have a binding affinity of between 3 nM and 300 nM, between 3 nM and 200 nM, between 3 nM and 100 nM, between 3 nM and 50 nM, between 3 nM and 40 nM, between 3 nM and 20 nM, between 3 nM and 15 nM, between 5 nM and 300 nM, and between 5 nM and 15 nM. In some embodiments, preferred PD-L1 antibodies have a binding affinity that is at least 2-fold, 3-fold, 3.7-fold, 4-fold, or 5-fold greater than the binding affinity of Ab1. In some of the above embodiments, the binding affinity is monovalent binding affinity.

[0185] In some embodiments, the binding of the anti-PD-L1 antibodies of the application is pH dependent, such that the antibodies exhibit different binding across a pH gradient. In some embodiments, the anti-PD-L1 antibodies exhibit maximal binding between a pH of about 4 and a pH of about 10. In some embodiments, the maximal binding is between a pH of about 6 and a pH of about 9. In some embodiments, the maximal binding is between a pH of about 6.5 and a pH of about 8.

[0186] In animal models or clinical trials, preferred antibodies of the application inhibit cancer (e.g., cell growth, metastasis, and / or lethality to an organism), as shown on cancer cells propagated in culture. Animal models can be formed by implanting human tumor cell lines expressing PD-L1 into an appropriate immunodeficient rodent strain (e.g., athymic nude mice or SCID mice). These tumor cell lines can be established as solid tumors by subcutaneous injection or as disseminated tumors by intravenous injection in the immunodeficient rodent host.

[0187] Once established in a host, these tumor models can be used to evaluate the therapeutic efficacy of anti-PD-L1 antibodies or conjugated forms thereof as described in the Examples.

[0188] In general, the anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates of the present disclosure bind to PD-L1, e.g., human PD-L1, and exert cytostatic and cytotoxic effects on malignant cells, such as cancer cells. The concentration required for a 50% reduction in viability compared to untreated cells, or x50 or IC 50is one way to measure cytotoxicity of anti-PD-Ll antibodies and / or anti-PD-ADCs. Preferred antibodies and / or ADCs of the present invention exhibit increased cytotoxicity and x50 compared to the cytotoxicity and x50 of the Ab1 antibody and / or ADC. In one embodiment, anti-PD-Ll antibodies of the present invention conjugated to vcMMAE exhibit an x50 in the BXPC3 cell line of between 10 ng / mL and 30 ng / mL or between 15 ng / mL and 25 ng / mL. In another embodiment, anti-PD-Ll antibodies of the present invention conjugated to vcMMAE exhibit an x50 in the MDA-MB-231 cell line of between 15 ng / mL and 55 ng / mL or between 20 ng / mL and 50 ng / mL. In another embodiment, anti-PD-Ll antibodies of the present invention conjugated to vcMMAE exhibit an x50 in the KARPAS 299 cell line of between 1 ng / mL and 7 ng / mL or between 2 ng / mL and 5 ng / mL. In another embodiment, anti-PD-Ll antibodies of the present invention conjugated to vcMMAE exhibit an x50 in the L540CY cell line of between 15 ng / mL and 40 ng / mL or between 20 ng / mL and 35 ng / mL. In one embodiment, anti-PD-Ll antibodies of the present invention conjugated to camptothecin exhibit an x50 in the BXPC3 cell line of between 12 ng / ml and 70 ng / ml or between 15 ng / ml and 65 ng / ml. In another embodiment, anti-PD-Ll antibodies of the present invention conjugated to camptothecin exhibit an x50 in the MDA-MB-231 cell line of between 3 ng / mL and 20 ng / mL or between 5 ng / mL and 17 ng / mL. In another embodiment, anti-PD-Ll antibodies of the present invention conjugated to camptothecin exhibit an x50 in the KARPAS 299 cell line of between 1 ng / mL and 18 ng / mL or between 3 ng / mL and 15 ng / mL. In another embodiment, anti-PD-Ll antibodies of the present invention conjugated to camptothecin exhibit an x50 in the L540CY cell line of between 1 ng / mL and 20 ng / mL or between 1 ng / mL and 15 ng / mL.

[0189] Generally, anti-PD-Ll antibodies and / or anti-PD-Ll antibody-drug conjugates of the present disclosure are internalized into cells, such as cancer cells. One way to measure internalization is to utilize a pH-sensitive antibody conjugate that emits a fluorescent signal when internalized in a cell-based assay. The total internalization of such an antibody can be quantified by the area under the curve (or AUC) of the fluorescent signal over time. FabFluor Internalization assays can be used for this quantification. Preferred antibodies and / or ADCs of the present disclosure show increased total internalization compared to total internalization of Ab1 and / or ADC. In one embodiment, the anti-PD-L1 antibody or ADC of the present disclosure exhibits an increase in AUC between 9% and 155% relative to the AUC of Ab1. In another embodiment, the anti-PD-L1 antibody or ADC of the present disclosure exhibits an increase in AUC between 40% and 130% or between 40% and 50% relative to the AUC of Ab1, as tested in the 786-0 cell line. In another embodiment, the anti-PD-L1 antibody or ADC of the present disclosure exhibits an increase in AUC between 90% and 100% or between 90% and 95% relative 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 disclosure exhibits an increase in AUC between 85% and 155% or between 85% and 90% relative 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 disclosure exhibits an increase in AUC between 9% and 40% or between 9% and 13% relative 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 disclosure exhibits an increase in AUC between 75% and 145% or between 75% and 80% relative to the AUC of Ab1, as tested in the MDA-MB-231 cell line.

[0190] The anti-PD-L1 antibodies of the present disclosure are preferably monoclonal, and can be multispecific, human, humanized or chimeric, single-chain, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, and PD-L1 binding fragments of any of the above. In some embodiments, the anti-PD-L1 antibodies of the present disclosure specifically bind PD-L1. The immunoglobulin molecules of the present disclosure can 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. In one embodiment, the anti-PD-L1 antibodies of the present disclosure are of the IgG1 type.

[0191] In certain embodiments of the present disclosure, the anti-PD-L1 antibodies are antigen binding fragments (e.g., human antigen binding fragments) as described herein, and include, but are not limited to, Fabs, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), and V L or V HFragments of Domains. Antigen-binding fragments that comprise single-chain antibodies can comprise one or more variable regions, alone or in combination with all or a portion of a hinge region, CH1, CH2, CH3, and CL domain. The present disclosure also includes antigen-binding fragments that comprise one or more variable regions in any combination with a hinge region, CH1, CH2, CH3, and CL domain. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is a human, murine (e.g., mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken antibody.

[0192] The anti-PD-L1 antibodies of the present disclosure can be mono-specific, bi-specific, tri-specific, or of greater multi-specificity. Multi-specific antibodies can have specificity for different epitopes of PD-L1, or can have specificity for both PD-L1 as well as a heterologous protein. See, e.g., PCT publications WO 93 / 17715; WO 92 / 08802; WO 91 / 00360; WO 92 / 05793; 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; Kostelny et al., 1992, J. Immunol. 148:1547 1553.

[0193] The anti-PD-L1 antibodies of the present disclosure can be described or specified in terms of the particular CDRs they comprise. The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5thEd. 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(l):55-77 (“IMGT” numbering scheme); Honegger A and Pluckthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme). The boundaries of a given CDR can vary depending on the scheme used for identification.In some embodiments, a "CDR" or "complementarity determining region" or individually designated CDR (e.g., CDR-H1, CDR-H2, CDR-H3) of a given antibody or region thereof (e.g., a variable region thereof) shall be understood to encompass the amino acid sequence of one (or a particular) CDR as defined by any of the above schemes. For example, in the case of a statement that a particular CDR (e.g., CDR-H3) contains the amino acid sequence of a corresponding CDR in the amino acid sequence of a V. H or V L region, it shall be understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region as defined by any of the above schemes. Schemes for identifying a particular CDR or CDRs (e.g., CDRs as defined by the Kabat, Chothia, AbM, or IMGT methods) can be specified.

[0194] The CDR sequences of the anti-PD-L1 antibodies and anti-PD-L1 antibody-drug conjugates described herein are in accordance with the Kabat numbering scheme as described below: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD.

[0195] In one aspect, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (iii) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15; and / or wherein the light chain variable region comprises (i) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 16, (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 17, and (iii) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 18, wherein the CDRs of the anti-PD-L1 antibody are defined by the Kabat numbering scheme.

[0196] In one aspect, provided herein is an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate, comprising a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 11 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 12. In one aspect, provided herein is an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 9 and a light chain comprising the amino acid sequence of SEQ ID NO: 10.

[0197] In some embodiments, provided herein is an anti-PD-L1 antibody and / or an anti-PD-L1 antibody-drug conjugate, comprising a heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 11 retains the ability to bind to PD-L1 (e.g., human PD-L1) and contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence. In certain embodiments, a total of 1 to 10 amino acids have been substituted, inserted and / or deleted in SEQ ID NO: 11. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4 or 5 amino acids) occur in regions outside of the CDRs (i.e., in the FRs). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 11, including post-translational modifications of that sequence.

[0198] In some embodiments, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 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 in SEQ ID NO: 1 are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4 or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 1, including post-translational modifications of that sequence.

[0199] In some embodiments, provided herein are anti-PD-L1 antibodies and / or anti-PD-L1 antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 1 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 in SEQ ID NO: 1 are substituted, inserted and / or deleted. In certain embodiments, the substitutions, insertions or deletions (e.g., 1, 2, 3, 4 or 5 amino acids) occur in regions outside the CDRs (i.e., in the FRs). In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable domain sequence of SEQ ID NO: 1, including post-translational modifications of that sequence.

[0200] In some embodiments, provided herein are anti-PD-Ll antibodies and / or anti-PD-Ll antibody-drug conjugates comprising a heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2. In certain embodiments, a heavy chain variable domain comprising an amino acid sequence that has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 2 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 have been substituted, inserted and / or deleted in SEQ ID NO: 2. In certain embodiments, the light chain contains one point mutation relative to SEQ ID NO: 2. In further embodiments, the single point mutation is in a CDR region.

[0201] In some embodiments, the anti-PD-Ll antibody or anti-PD-Ll antibody of the anti-PD-Ll antibody-drug conjugate is a monoclonal antibody.

[0202] There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes are further divided based on their subclasses, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. IgGl antibodies can exist in a variety of polymorphic variants, known as allotypes (reviewed in Jefferis and Lefranc 2009. mAbs vol. 1 no. 4 1-7), any of which are suitable for use in some embodiments herein. Common allotype variants in the human population are those designated by the letters a, f, n, z, or combinations thereof. In any of the embodiments herein, the antibody can comprise a heavy chain Fc region comprising a human IgG Fc region. In other embodiments, the human IgG Fc region comprises human IgGl.

[0203] Antibodies also include modified derivatives, the modifications being 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 inhibitory or cytotoxic effect on cells. For example, and without limitation, antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cell ligand or other protein, etc. Any of a wide variety of chemical modifications can be made to the antibody by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of a neoglycoprotein, etc. Furthermore, the derivatives can contain one or more non-classical amino acids.

[0204] Humanized antibodies

[0205] Humanized antibodies are genetically engineered antibodies in which CDRs from a non-human "donor" antibody are grafted into a human "acceptor" 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 acceptor antibody sequence can be, e.g., a mature human antibody sequence, a composite of such sequences, a consensus sequence or a germline region sequence of human antibodies. A preferred acceptor sequence for the heavy chain is the germline V H Exon V H l-2 (also referred to in the literature as HV1-2) (Shin et al., 1991, EMBO J. 10:3641-3645) and for the hinge region (J H ) is Exon J H-6 (Mattila et al., 1995, Eur. J. Immunol. 25:2578-2582). For the light chain, the preferred acceptor sequence is exon VK2-30 (also referred to in the literature as KV2-30) and for the hinge region, the preferred acceptor sequence is exon JK-4 (Hieter et al., 1982, J. Biol. Chem. 257:1516-1522). Thus, a humanized antibody is an antibody having some or all of the CDRs from a donor antibody and all or substantially the variable region framework sequences and constant regions (if present) from a human antibody sequence. Similarly, a humanized heavy chain has at least one, two, and usually all three CDRs from a donor antibody heavy chain and substantially the heavy chain variable region framework sequences and heavy chain constant regions (if present) from a human heavy chain variable region framework and constant region sequences. Similarly, a humanized light chain has at least one, two, and usually all three CDRs from a donor antibody light chain and substantially the light chain variable region framework sequences and light chain constant regions (if present) from a human light chain variable region framework and constant region sequences. Except for nanobodies and dAbs, a humanized antibody comprises a humanized heavy chain and a humanized light chain. CDRs in a humanized antibody are substantially from corresponding CDRs in a non-human antibody when at least 60%, 85%, 90%, 95%, or 100% of the corresponding residues (as defined by Kabat) between the respective CDRs are the same. A variable region framework sequence of an antibody chain or a constant region of an antibody chain is substantially from a human variable region framework sequence or a human constant region, respectively, when at least 85%, 90%, 95%, or 100% of the corresponding residues are the same as defined by Kabat. In some embodiments, the PD-L1 antibodies of the application are humanized antibodies.

[0206] Although humanized antibodies typically comprise all six CDRs from a mouse antibody (preferably as defined by Kabat), they can also be made with fewer than all (e.g., at least 3, 4, or 5) CDRs from a mouse antibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos et al., Journal of Molecular Biology, 320:415-428, 2002; Iwahashi et al., Mol. Immunol. 36:1079-1091, 1999; Tamura et al., Journal of Immunology, 164:1432-1441, 2000).

[0207] Selection of constant regions

[0208] The heavy and light chain variable regions of the humanized antibody can be linked to at least a portion of a human constant region. The choice of constant region depends in part on whether antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, and / or complement-dependent cytotoxicity is desired. For example, human isotypes IgGl and IgG3 have strong complement-dependent cytotoxicity, human isotype IgG2 has weak complement-dependent cytotoxicity and human IgG4 lacks complement-dependent cytotoxicity. Human IgGl and IgG3 also induce stronger cell-mediated effector functions than human IgG2 and IgG4. The light chain constant region can be lambda or kappa. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy chains, light chains, such as Fab, Fab', F(ab')2, and Fv, or as a single chain antibody in which the heavy and light chain variable domains are linked by a spacer.

[0209] Human constant regions show allotypic and isotypic variation between different individuals, that is, the constant region can differ at one or more polymorphic positions in different individuals. Isotypes differ from allotypes in that the serum that recognizes the isotype binds to non-polymorphic regions of one or more other isotypes.

[0210] One or several amino acids at the amino or carboxy terminus of the light and / or heavy chain, such as the C-terminal lysine of the heavy chain, can be missing or derivatized in whole or in part. Substitutions can be made in the constant region to decrease or increase effector function, such as complement-mediated cytotoxicity or ADCC (see, e.g., Winter et al., U.S. Patent No. 5,624,821; Tso et al., U.S. Patent No. 5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006), or to extend half-life in humans (see, e.g., Hinton et al., J. Biol. Chem. 279:6213, 2004).

[0211] Exemplary substitutions include amino acid substitutions at amino acid positions 234, 235, 237, 239, 267, 298, 299, 326, 330, or 332 to a cysteine residue, preferably the S239C mutation in the human IgGl isotype (US20100158909). The presence of additional cysteine residues allows for interchain disulfide bond formation. This interchain disulfide bond formation can cause steric hindrance, thereby decreasing the affinity of the Fc region-FcyR binding interaction. One or more cysteine residues introduced in or near the Fc region of the IgG constant region can also serve as a site for conjugation to a therapeutic agent (i.e., a cytotoxic drug using a thiol-specific reagent such as a maleimide derivative of a drug). The presence of the therapeutic agent causes steric hindrance, thereby further decreasing the affinity of the Fc region-FcyR binding interaction. Other substitutions at any of positions 234, 235, 236, and / or 237 decrease the affinity for Fey receptors, in particular the FcyRI receptor (see, e.g., US 6,624,821, US 5,624,821).

[0212] The in vivo half-life of an antibody can also affect its effector functions. The half-life of an antibody can be increased or decreased to alter its therapeutic activity. FcRn is a receptor that is structurally similar to MHC class I antigens, which is noncovalently associated with β2-microglubulin. FcRn regulates the catabolism of IgG and its transcytosis across tissues (Ghetie and Ward, 2000, Annu. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). IgG-FcRn interactions occur at pH 6.0 (the pH of intracellular vesicles) but not at pH 7.4 (the pH of blood); this interaction enables IgG to be recycled back into the circulation (Ghetie and Ward, 2000, Ann. Rev. Immunol. 18:739-766; Ghetie and Ward, 2002, Immunol. Res. 25:97-113). The region on human IgGl involved in FcRn binding has been mapped (Shields et al., 2001, J. Biol. Chem. 276:6591-604). Alanine substitutions at positions Pro238, Thr256, Thr307, Gln311, Aspl312, Glu380, Glu382, or Asn434 of human IgGl enhance FcRn binding (Shields et al., 2001, J. Biol. Chem. 276:6591-604). IgGl molecules with these substitutions have a longer serum half-life. Thus, these modified IgGl molecules can be able to perform their effector functions for a longer period of time compared to unmodified IgGl, and thus exert their therapeutic efficacy. Other exemplary substitutions for increasing binding to FcRn include Gin at position 250 and / or Leu at position 428. EU numbering is used for all positions in the constant region.

[0213] Oligosaccharides covalently attached to conserved Asn297 are involved in the ability of the Fc region of IgG to bind FcyRs (Lund et al., 1996, J. Immunol. 157:4963-69; Wright and Morrison, 199, Trends Biotechnol. 15:26-31). Engineering of this glycoform on IgG can significantly improve IgG-mediated ADCC. Addition of bisected N-acetylglucosamine modifications (Umana et al., 1999, Nat. Biotechnol. 17:176-180; Davies et al., 2001, Biotech. Bioeng. 74:288-94) to or removal of fucose from this glycoform (Shields et al., 2002, J. Biol. Chem. 277:26733-40; Shinkawa et al., 2003, J. Biol. Chem. 278:6591-604; Niwa et al., 2004, Cancer Res. 64:2127-33) are two examples of IgG Fc engineering that improve binding between IgG Fc and FcyRs, thereby increasing Ig-mediated ADCC activity.

[0214] Systematic substitution of solvent-exposed amino acids of the human IgGl Fc region has resulted in IgG variants with altered FcyR binding affinities (Shields et al., 2001, J. Biol. Chem. 276:6591-604). A subset of these variants including substitutions of Thr256 / Ser298, Ser298 / Glu333, Ser298 / Lys334, or Ser298 / Glu333 Lys334 to Ala showed both increased binding affinities to FcyRs and ADCC activity when compared to the parent IgGl (Shields et al., 2001, J. Biol. Chem. 276:6591-604; Okazaki et al., 2004, J. Mol. Biol. 336:1239-49).

[0215] The complement-fixing activity of antibodies (both Clq 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 a human IgG2 backbone can convert an antibody isotype that binds Clq poorly and lacks complement-activating activity severely to one that both can bind Clq and mediate CDC (Idusogie et al., 2001, J. Immunol. 166:2571-75). Several other approaches have also been used to improve the complement-fixing activity of antibodies. For example, grafting the 18-amino acid carboxy-terminal tailpiece fragment of IgM to the carboxy terminus of IgG greatly enhances its CDC activity. This enhancement is observed even for IgG4, which normally has no detectable CDC activity (Smith et al., 1995, J. Immunol. 154:2226-36). Furthermore, substitution of Cys for Ser444 near the carboxy terminus of IgG 1 induces tail-to-tail dimerization of IgG 1, with a 200-fold increase in CDC activity over monomeric IgGl (Shopes et al., 1992, J. Immunol. 148:2918-22). In addition, bispecific diabody constructs specific for Clq also confer CDC activity (Kontermann et al., 1997, Nat. Biotech. 15:629-31).

[0216] Complement activity can be reduced by mutating at least one of amino acid residues 318, 320, and 322 of the heavy chain to a residue with a different side chain, such as Ala. Other alkyl-substituted nonpolar residues (such as Gly, Leu, or Val) or aromatic nonpolar residues (such as Phe, Tyr, Trp, and Pro) in place of any of the three residues also reduce or eliminate Clq binding. Ser, Thr, Cys, and Met can be used at residues 320 and 322 but not at 318 to reduce or eliminate Clq binding activity.

[0217] Substitution of the 318 (Glu) residue with a polar residue can alter, but not abrogate, the C1q binding activity. Substitution of residue 297 (Asn) with Ala results in the ablation of the cleavage activity, but only a slight decrease (about three-fold weaker) in the affinity for C1q. This change destroys the glycosylation site and the need for the presence of carbohydrate for complement activation. Any other substitution at this site also destroys the glycosylation site. The following mutations and any combination thereof also reduce C1q binding: D270A, K322A, P329A, and P31 IS (see WO06 / 036291). The L234A / L235A mutations (or LALA mutations) also reduce C1q binding as well as FcyR binding. In one embodiment, the anti-PD-L1 antibodies of the application include the L234A / L235A mutations.

[0218] Reference to a human constant region includes any constant region with any substitution of residues that have any natural allotype or polymorphic position that occupies a natural allotype. In addition, there can be up to 1, 2, 5, or 10 mutations relative to the native human constant region, such as those indicated above, to reduce Fcy receptor binding or increase binding to the FcRN.

[0219] V. Expression of Recombinant Antibodies

[0220] Humanized antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically comprise expression control sequences, including naturally associated or heterologous promoter regions, operably linked to coding sequences for antibody chains. Preferably, the expression control sequences are eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the cross-reacting antibodies.

[0221] Mammalian cells are the preferred host for expressing nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, From Genes to Clones, (VCH Publishers, New York, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art and include CHO cell lines (e.g., DG44), various COS cell lines, HeLa cells, HEK293 cells, L cells, and myeloma cells that do not produce antibodies (including Sp2 / 0 and NSO). Preferably, the cells are non-human. Expression vectors for these cells can include expression control sequences such as origins of replication, promoters, enhancers (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like. See Co et al., J. Immunol. 148:1149 (1992).

[0222] Once expressed, the antibodies can be purified according to standard procedures in the art including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally Scopes, Protein Purification (Springer-Verlag, NY, 1982)).

[0223] VI. Nucleic Acids

[0224] The present application also provides nucleic acids encoding any of the above humanized heavy and light chains. Typically, the nucleic acids also encode a signal peptide fused to the mature heavy and light chains. The coding sequences on the nucleic acids can be operably linked to regulatory sequences to ensure expression of the coding sequences, such as promoters, enhancers, ribosome binding sites, transcriptional termination signals, and the like. The nucleic acids encoding the heavy and light chains can be in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized, for example, by solid state synthesis of overlapping oligonucleotides or by PCR. The nucleic acids encoding the heavy and light chains can be linked as a contiguous nucleic acid, for example, within an expression vector, or can be separate, for example, each cloned into its own expression vector.

[0225] In some aspects, provided herein are nucleic acids encoding an anti-PD-L1 antibody or antigen binding fragment thereof as described herein. Also provided herein are vectors comprising a nucleic acid encoding an anti-PD-L1 antibody or antigen binding fragment thereof as described herein. Further provided herein are host cells expressing a nucleic acid encoding an anti-PD-L1 antibody or antigen binding fragment thereof as described herein. Also provided herein are host cells comprising a vector containing a nucleic acid encoding an anti-PD-L1 antibody or antigen binding fragment thereof as described herein.

[0226] The anti-PD-L1 antibodies described herein can be produced by well-known recombinant techniques using well-known expression vector systems and host cells. In one embodiment, the antibodies are produced in CHO cells using the GS expression vector system as disclosed in De la Cruz Edmunds et al., 2006, Molecular Biotechnology 34; 179-190, EP216846, U.S. Patent No. 5,981,216, WO 87 / 04462, EP323997, U.S. Patent No. 5,591,639, U.S. Patent No. 5,658,759, EP338841, U.S. Patent No. 5,879,936, and U.S. Patent No. 5,891,693.

[0227] The monoclonal anti-PD-L1 antibodies described herein can be produced, for example, by the hybridoma method first described by Kohler et al., Nature, 256, 495 (1975), or can be produced by recombinant DNA methods. Monoclonal antibodies can also be isolated from a phage antibody library using, for example, the techniques described in Clackson et al., Nature, 352, 624-628 (1991) and Marks et al., J Mol. Biol., 222(3):581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, a monoclonal antibody can be obtained from a hybridoma prepared from murine splenic B cells obtained from a mouse immunized with an antigen of interest, e.g., in the form of a cell expressing the antigen on the surface, or a nucleic acid encoding the antigen of interest. A monoclonal antibody can also be obtained from a hybridoma of antibody-expressing cells derived from an immunized human or non-human mammal, such as a rat, dog, primate, etc.

[0228] Antibody-drug conjugates

[0229] The anti-PD-Ll antibodies can be conjugated to cytotoxic or cytostatic moieties, including pharmaceutically compatible salts thereof, to form antibody drug conjugates (ADCs). Particularly suitable moieties for conjugation to the antibodies are cytotoxic agents (e.g., chemotherapeutic agents), prodrug converting enzymes, radioisotopes, or compounds or toxins (these moieties are collectively referred to as therapeutic agents). For example, the anti-PD-Ll antibodies can be conjugated to cytotoxic agents such as chemotherapeutic agents or toxins (e.g., cytostatic or cytocidal, such as, e.g., abrin, ricin A, Pseudomonas exotoxin, or diphtheria toxin).

[0230] The anti-PD-Ll antibodies can be conjugated to prodrug converting enzymes. The prodrug converting enzymes can be recombinantly fused to the antibodies or chemically conjugated thereto using known methods. Exemplary prodrug converting enzymes are carboxypeptidase G2, beta-glucuronidase, penicillin-V-amidase, penicillin-G-amidase, beta-lactamase, beta-glucosidase, nitroreductase, and carboxypeptidase A.

[0231] Techniques for conjugating therapeutic agents to proteins, particularly antibodies, are well 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 Edition 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); "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody In Cancer Therapy," in Monoclonal Antibodies For Cancer Detection And Therapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al., 1982, Immunol. Rev. 62:119-58. See also, e.g., PCT Publication WO 89 / 12624.

[0232] The therapeutic agent can be conjugated in a manner that reduces its activity unless it is cleaved from the antibody (e.g., by hydrolysis, by antibody degradation, or by a cleaving agent). Such a therapeutic agent is attached to the antibody with a cleavable linker that is susceptible to cleavage in the intracellular environment of a PD-L1 -expressing cancer cell, but is substantially insensitive to the extracellular environment, such that when the conjugate is internalized by a PD-L1 -expressing cancer cell it is cleaved from the antibody (e.g., in an endosome or cleaved in a lysosomal environment or in a caveolae environment, e.g., due to pH sensitivity or protease sensitivity).

[0233] Typically, the ADC comprises a linker region between the therapeutic agent and the anti-PD-Ll antibody. As described above, typically, the linker is cleavable under intracellular conditions, such that cleavage of the linker releases the therapeutic agent from the antibody in the intracellular environment (e.g., within a lysosome or endosome or caveolae). The linker can be, for example, a peptidyl linker cleavable by intracellular peptidases or proteases, including lysosomal or endosomal proteases. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleavage agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Most typically, the peptidyl linker is cleavable by enzymes present in cells expressing PD-L1. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue (e.g., a linker comprising a Phe-Leu or Gly-Phe-Leu-Gly peptide) can be used. Other such linkers are described, e.g., in U.S. Patent No. 6,214,345. In particular embodiments, a peptidyl linker cleavable by intracellular proteases includes a Val-Cit linker or a Phe-Lys dipeptide (see, e.g., U.S. Patent 6,214,345, which describes synthesis of doxorubicin with a Val-Cit linker). One advantage of using intracellular proteolysis to release the therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is generally high.

[0234] The cleavable linker can be pH sensitive, i.e., susceptible to hydrolysis at certain pH values. Typically, the pH sensitive linker is hydrolyzable under acidic conditions. For example, an acid-labile linker that is hydrolyzable in lysosomes (e.g., hydrazone, hemiketal, aminobenzothiazole, cis-aconitate, orthoester, acetal, ketal, etc.) can be used. (See, e.g., U.S. Patent Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0 (the approximate pH of lysosomes). In certain embodiments, the hydrolyzable linker is a thioether linker (e.g., a thioether attached to the therapeutic agent via an acyl hydrazone bond) (see, e.g., U.S. Patent No. 5,622,929).

[0235] Other linkers (e.g., disulfide linkers) are cleavable under reducing conditions. Disulfide linkers include those that can be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB, and SMPT. {See, e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel, ed., Oxford U. Press, 1987. See also U.S. Patent No. 4,880,935.)

[0236] The linker can 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 can 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).

[0237] The linker can also be a non-cleavable linker, such as a maleimidyl-alkylene- or maleimid-aryl linker attached directly to the therapeutic agent (e.g., drug). The active drug-linker is released by degradation of the antibody.

[0238] Typically, the linker is substantially insensitive to the extracellular environment, meaning that no more than about 20%, typically no more than about 15%, more typically no more than about 10%, even more typically no more than about 5%, no more than about 3%, or no more than about 1% of the linkers are cleaved in a sample of the ADC when the ADC is present in the extracellular environment (e.g., in the plasma).

[0239] Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) the ADC ("ADC sample") and (b) an equimolar amount of unconjugated antibody or therapeutic agent ("control sample") independently in plasma for a predetermined period of time (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 to the amount present in the control sample, as determined, for example, by high performance liquid chromatography.

[0240] The linker can also facilitate cellular internalization. The linker can facilitate cellular internalization when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic moiety of an ADC or ADC derivative as described herein). Alternatively, the linker can facilitate cellular internalization when conjugated to both a therapeutic agent and an anti-PD-Ll antibody (i.e., in the context of an ADC as described herein).

[0241] The anti-PD-Ll antibody can be conjugated to the linker via a heteroatom of the antibody. These heteroatoms can be present on the antibody in its native state or can be introduced into the antibody. In some aspects, the anti-PD-Ll antibody will be conjugated to the linker via a nitrogen atom of a lysine residue. In other aspects, the anti-PD-Ll antibody will be conjugated to the linker via a sulfur atom of a cysteine residue. The cysteine residue can be a naturally occurring or a residue engineered into the antibody. Methods of conjugating linkers and drug-linkers to antibodies via lysine and cysteine residues are known in the art.

[0242] Exemplary antibody-drug conjugates include auristatin-based antibody-drug conjugates (i.e., the drug component is an auristatin drug). Auristatins bind tubulin, have been shown to interfere with microtubule dynamics as well as nuclear and cellular division, and have anti-cancer activity. Typically, auristatin-based antibody-drug conjugates comprise a linker between the auristatin drug and the anti-PD-Ll antibody. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker that is released by degradation of the antibody). Auristatins include auristatin T, MMAF, and MMAE. Exemplary synthesis and structures of auristatins are described in U.S. Publication Nos. 7,659,241, 7,498,298, 2009-0111756, 2009-0018086, and 7,968,687, each of which is incorporated by reference herein in its entirety and for all purposes.

[0243] Exemplary antibody-drug conjugates also include camptothecin-based antibody-drug conjugates (i.e., the drug component is a camptothecin drug). Camptothecins are topoisomerase inhibitors that have been shown to have anti-cancer activity. Typically, camptothecin-based antibody-drug conjugates comprise a linker between the camptothecin drug and the anti-PD-Ll antibody. The linker can be, for example, a cleavable linker (e.g., a peptidyl linker, a carbohydrate linker) or a non-cleavable linker (e.g., a linker that is released by degradation of the antibody). The synthesis and structure of exemplary camptothecin drug linkers are described in PCT / US19 / 025968 (filed April 5, 2019), which is incorporated by reference herein in its entirety and for all purposes.

[0244] Other exemplary antibody-drug conjugates include maytansinoid antibody-drug conjugates (i.e., the drug component is a maytansinoid drug) and benzodiazepine antibody-drug conjugates (i.e., the drug component is a benzodiazepine (e.g., pyrrolo[l,4]benzodiazepine dimers (PBD dimers), indolinobenzodiazepine dimers, and oxazolidine benzodiazepine dimers)).

[0245] Exemplary antibody-drug conjugates include vcMMAE and mcMMAF antibody-drug conjugates as follows, where p represents the drug payload and Ab represents an anti-PD-Ll antibody:

[0246]

[0247] vcMMAE

[0248]

[0249] mcMMAF

[0250] or a pharmaceutically acceptable salt thereof.

[0251] Exemplary anti-PD-Ll antibody-drug conjugates include camptothecin antibody-drug conjugates as follows, where p represents the drug payload and Ab represents an anti-PD-Ll antibody:

[0252] In some embodiments, the camptothecin ADC has the formula (IC):

[0253]

[0254] or a pharmaceutically acceptable salt thereof;

[0255] wherein

[0256] Ab is an anti-PD-Ll antibody;

[0257] y is 1, 2, 3, or 4, or is 1 or 4; and

[0258] z is an integer from 2 to 12, or is 2, 4, 8, or 12;

[0259] and p is 1-16.

[0260] 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.

[0261] In some embodiments, the camptothecin ADC has the formula:

[0262]

[0263] or a pharmaceutically acceptable salt thereof;

[0264] wherein p is 2, 4, or 8, preferably p is 8.

[0265] In some embodiments, the camptothecin ADC has the formula:

[0266] or a pharmaceutically acceptable salt thereof;

[0267] wherein p is 2, 4, or 8, preferably p is 8.

[0268] In some embodiments, the camptothecin drug linker has the following formula:

[0269] or a pharmaceutically acceptable salt thereof;

[0270] wherein

[0271] y is 1, 2, 3, or 4, or is 1 or 4; and

[0272] z is an integer from 2 to 12, or is 2, 4, 8, or 12.

[0273] In some embodiments, the camptothecin drug linker has the following formula:

[0274]

[0275] MP-PEG8-VKG-Camptothecin

[0276] In some embodiments, the camptothecin drug linker has the following formula:

[0277]

[0278] MP-PEG4-VKG-Camptothecin

[0279] In some embodiments, the camptothecin drug linker has the following formula:

[0280]

[0281] MP-PEG12-VKG-camptothecin

[0282] With reference to antibody-drug conjugates targeting PD-L1, subscript p represents drug loading, and depending on the context, can represent the number of molecules of drug-linker molecule attached to an individual antibody molecule, and thus is an integer value; or can represent the average drug loading, and thus can be an integer or non-integer value, but typically is a non-integer value. The average drug loading represents the average number of drug-linker molecules per antibody in a population. Typically, but not always, when we refer to an antibody, e.g., a monoclonal antibody, we are referring to a population of antibody molecules. In a composition comprising a population of antibody-drug conjugate molecules, the average drug loading is an important quality attribute because it determines the amount of drug that can be delivered to a target cell. The percentage of un-conjugated antibody molecules in a composition is included in the average drug loading value.

[0283] In preferred aspects of the application, when referring to a composition comprising a population of antibody-drug conjugate compounds, the average drug loading is from 1 to about 16, preferably about 2 to about 14, more preferably about 2 to about 10.

[0284] For MMAE and camptothecin ADCs, such as those exemplified herein, a preferred average drug loading is about 2, 4, or 8, and a particularly preferred average drug loading is about 8. In one embodiment, a preferred average drug loading for MMAE ADCs is 2 or 4. In one embodiment, a preferred average drug loading for camptothecin ADCs is 4 or 8. In exemplary embodiments, the drug-linker is conjugated to a cysteine residue of a reduced interchain disulfide. In some aspects, the actual drug loading of individual antibody molecules in a population of antibody-drug conjugate compounds is from 1 to 10 (or from 6 to 10 or from 6 to 8), with a predominant drug loading of 8. Higher drug loadings can be achieved, for example, if the drug-linker is conjugated to an introduced cysteine residue (such as a cysteine residue introduced at position 239 according to the EU index) in addition to the interchain disulfide.

[0285] The PEG (polyethylene glycol) portion of the drug linker can range from 2 to 36. Subscript z in all of the above embodiments 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.

[0286] Polydisperse PEG, monodisperse PEG, and discrete PEG can be used to make the pegylated antibody drug conjugates of the application. Polydisperse PEG is a heterogeneous mixture of sizes and molecular weights, while monodisperse PEG is typically purified from the heterogeneous mixture, thus providing a single chain length and molecular weight. The preferred PEG unit is discrete PEG, i.e., a compound synthesized in a stepwise fashion rather than via a polymerization process. Discrete PEG provides a single molecule with a defined and specified chain length. As with the subscript "p", the value of the subscript "n" can be an average number and can be an integer or non-integer when referring to a population of antibody-drug conjugates.

[0287] Useful classes of cytotoxic agents for conjugation to anti-PD-Ll antibodies include, for example, anti-tubulin agents, DNA minor groove binders, DNA replication inhibitors, chemotherapy sensitizers, and the like. Other exemplary classes of cytotoxic agents include anthracyclines, auristatins, camptothecins, duocarmycins, etoposides, maytansinoids, and vinca alkaloids. Some exemplary cytotoxic agents include auristatins (e.g., auristatin T, auristatin E, AFP, monomethyl auristatin F (MMAF), lipophilic monomethyl auristatin F, monomethyl auristatin E (MMAE)), DNA minor groove binders (e.g., enediyne and lexitropsins), duocarmycins, taxoids (e.g., paclitaxel and docetaxel), vinca alkaloids, nicotinamide phosphoribosyltransferase inhibitors (NAMPTi), tubulysins M, doxorubicin, morpholino-doxorubicin, and cyclic morpholino-doxorubicin.

[0288] The cytotoxic agent can be a chemotherapeutic agent, such as, for example, doxorubicin, paclitaxel, melphalan, vinca alkaloid, methotrexate, mitomycin C, or etoposide. The agent can also be a CC-1065 analog, a calicheamicin, a maytansinoid, an analog of dolastatin 10, a rhizoxin, or a pumitrep.

[0289] The cytotoxic agent can also be an auristatin. The auristatin can be an auristatin E derivative, such as an ester formed between auristatin E and a keto acid. For example, auristatin E can be reacted with p-acetylbenzoic acid or benzoyl valeric acid to produce AEB and AEVB, respectively. Other exemplary auristatins include auristatin T, AFP, MMAF, and MMAE. The synthesis and structure of various auristatins are described in, for example, US 2005-0238649 and US 2006-0074008.

[0290] The cytotoxic agent can be a DNA minor groove binder. (See, e.g., U.S. Patent No. 6,130,237.) For example, the minor groove binder can be a CBI compound or an enediyne (e.g., a calicheamicin).

[0291] The cytotoxic or cytostatic agent can be an anti-tubulin agent. Examples of anti-tubulin agents include taxanes (e.g., paclitaxel (Taxol®), (pegylated liposomal doxorubicin), (docetaxel), T67 (Tularik), vinca alkaloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine), and auristatins (e.g., auristatin E, AFP, MMAF, MMAE, AEB, AEVB). Exemplary auristatins are shown in the following Formulas III-XIII. Other suitable anti-tubulin agents include, for example, baccatin derivatives, taxane analogs (e.g., epothilones A and B), nocodazole, colchicine and colchicinide, estramustine, cryptophysin, symadexin, maytansinoid, combretastin, discodermoide, and eleuthrobin.

[0292] The cytotoxic agent can be a maytansinoid, i.e., another group of anti-tubulin agents (e.g., DM1, DM2, DM3, DM4). For example, the maytansinoid can be maytansine or a drug linker containing maytansine (e.g., DM-1 or DM-4) (ImmunoGen, Inc.; see also Chari et al., 1992, Cancer Res.).

[0293] VIII. Therapeutic Applications

[0294] The antibodies of the application, alone or as anti-PD-Ll antibody-drug conjugates thereof, can be used to treat cancer. Some such cancers show detectable levels of PD-L1 measured at the protein (e.g., by immunoassay using one of the exemplified antibodies) or mRNA level. Some such cancers show elevated levels of PD-L1 relative to non-cancerous tissue of the same type, preferably from the same patient. Exemplary levels of PD-L1 on treatable cancer cells are 5,000-500,000 PD-L1 molecules per cell, but higher or lower levels can be treated. Optionally, the level of PD-L1 in the cancer is measured prior to treatment.

[0295] Examples of cancers that are associated with PD-L1 expression and amenable to treatment include melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating melanoma. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating NSCLC. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating SCLC. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating head and neck cancer. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating TNBC. Triple negative breast cancer is a specialized term for a cancer that lacks detectable estrogen and progesterone receptors and lacks HER2 / neu overexpression. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating ovarian cancer. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating urothelial cancer. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating HCC. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating gastric cancer. In some embodiments, the antibodies or antibody-drug conjugates of the application are used in methods of treating cervical cancer. The treatment can be applied to patients with primary or metastatic tumors of these kinds. The treatment can also be applied to patients refractory to conventional therapy or who have relapsed after responding to such therapy.

[0296] The antibodies of the application, such as humanized antibodies, are administered alone or as conjugates thereof in an effective regimen, which means a dosage, route of administration, and frequency of administration that can delay onset, reduce severity, inhibit further progression, and / or improve at least one sign or symptom of cancer. The regimen can be referred to as therapeutically effective if the patient already has cancer. The regimen can be referred to as prophylactically effective if the patient is at increased risk for developing cancer relative to the general population, but has not yet experienced symptoms. In some cases, therapeutic or prophylactic efficacy can be observed in an individual patient, relative to historical controls in the same patient or past experience. In other cases, therapeutic or prophylactic efficacy can be demonstrated in a pre-clinical or clinical trial, relative to a control population of untreated patients.

[0297] Exemplary doses of a monoclonal antibody are 0.1 mg / kg to 50 mg / kg of patient body weight, more typically 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg to 15 mg / kg, 1 mg / kg to 12 mg / kg, or 1 mg / kg to 10 mg / kg 1, or 2 mg / kg to 30 mg / kg, 2 mg / kg to 20 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 12 mg / kg, or 2 mg / kg to 10 mg / kg, or 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 12 mg / kg, or 3 mg / kg to 10 mg / kg. Exemplary doses of a monoclonal antibody or antibody drug conjugate thereof are 1 mg / kg to 7.5 mg / kg, or 2 mg / kg to 7.5 mg / kg or 3 mg / kg to 7.5 mg / kg of subject body weight, or 0.1-20 mg / kg body weight or 0.5-5 mg / kg body weight (e.g., 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg or 10 mg / kg) or as a flat dose of 10-1500 mg or 200-1500 mg. In some methods, a dose of at least 1.5 mg / kg, at least 2 mg / kg, or at least 3 mg / kg is administered to the patient once every three weeks or more often. The dose depends on the frequency of administration, the condition of the patient, and the response to prior treatment, if any, whether the treatment is prophylactic or therapeutic, and whether the disorder is acute or chronic, among other factors.

[0298] Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Administration can also be directly into a tumor. Preferably, administration is into the systemic circulation, by intravenous or subcutaneous administration. Intravenous administration can be, for example, by infusion over a period of time (such as 30-90 min) or by single bolus injection.

[0299] The frequency of administration depends on factors such as the half-life of the antibody or conjugate in circulation, the condition of the patient, and the route of administration. The frequency can be once a day, once a week, once a month, once a quarter, or at irregular intervals in response to changes in the condition of the patient or the progression of the cancer being treated. An exemplary frequency for intravenous administration in a continuous course of treatment is between twice a week and once a quarter, but administration can also be at a higher or lower frequency. Another exemplary frequency for intravenous administration in a continuous course of treatment is between once a week or three times a four weeks, but administration can also be at a higher or lower frequency. For subcutaneous administration, an exemplary frequency of administration is once a day to once a month, but administration can also be at a higher or lower frequency.

[0300] The number of doses administered depends on the nature of the cancer (e.g., whether acute or chronic symptoms are present) and the response of the disorder to treatment. For acute disorders or acute exacerbations of chronic disorders, between 1 and 10 doses are usually sufficient. Sometimes, a single bolus dose (optionally in divided form) is sufficient for acute disorders or acute exacerbations of chronic disorders. For relapses or acute exacerbations of acute disorders, repeated treatments are possible. For chronic disorders, the antibody can be administered at regular intervals, e.g., once a week, once every two weeks, once a month, once a quarter, once every six months, for at least 1, 5, or 10 years or the lifetime of the patient.

[0301] Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic, and are manufactured under GMP conditions. The pharmaceutical composition can be provided in unit dosage form, i.e., a dose for a single administration. The pharmaceutical composition can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, the antibody can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the antibody can be lyophilized for use with an appropriate medium (e.g., sterile, pyrogen-free water) for construction prior to use. The concentration of the antibody in liquid formulations can be, e.g., 1-100 mg / ml, such as 10 mg / ml.

[0302] Treatment with the antibodies of the application can be combined with chemotherapy, radiation, stem cell therapy, surgery, other treatments effective for the disorder being treated. Useful classes of other agents that can be administered with antibodies and antibody-drug conjugates against PD-L1 as described herein include, for example, antibodies against other receptors expressed on cancer cells, anti-tubulin agents (e.g., auristatins), DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono-, di-, and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluoropyrimidines, ionophores, lexitropsins, nitrosoureas, cisplatin, preformed compounds, purine antimetabolites, puromycins, radiosensitizers, steroids, taxoids, topoisomerase inhibitors, vinca alkaloids, and the like.

[0303] Treatment with an anti-PD-L1 antibody or antibody-drug conjugate, optionally in combination with any of the other agents or regimens described above, either alone or as a conjugate, can increase the median progression-free survival or overall survival time of a patient having a tumor (e.g., melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, triple negative breast cancer (TNBC), ovarian cancer, urothelial cancer, hepatocellular carcinoma (HCC), gastric cancer, and cervical cancer), especially in the case of a relapsed or refractory tumor, by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100% or more, compared to the same treatment (e.g., chemotherapy) without the anti-PD-L1 antibody alone or as a conjugate. Additionally or alternatively, treatment (e.g., standard chemotherapy) including an anti-PD-L1 antibody alone or as a conjugate can increase the complete response rate, partial response rate, or objective response rate (complete + partial) of a patient having a tumor by at least 30% or 40%, but preferably 50%, 60% to 70%, or even 100%, compared to the same treatment (e.g., chemotherapy) without the anti-PD-L1 antibody alone or as a conjugate.

[0304] Typically, in a clinical trial (e.g., a Phase II, Phase II / III, or Phase III trial), the foregoing increases in median progression-free survival and / or response rate of patients treated with a standard therapy plus an anti-PD-L1 antibody alone or as a conjugate, relative to a control group of patients receiving the standard therapy alone (or plus placebo), are statistically significant, e.g., at the p = 0.05 or 0.01 or even 0.001 level. Complete and partial response rates are determined by objective criteria commonly used in cancer clinical trials (e.g., as set forth or accepted by the National Cancer Institute and / or the Food and Drug Administration).

[0305] IX. Articles of manufacture and kits

[0306] In another aspect, an article of manufacture or kit comprising an anti-PD-Ll antibody or an anti-PD-Ll antibody-drug conjugate described herein is provided. The article of manufacture or kit can further comprise instructions for use of an anti-PD-Ll antibody or an anti-PD-Ll antibody-drug conjugate described herein in a method of the present application. Thus, in certain embodiments, the article of manufacture or kit comprises instructions for use of an anti-PD-Ll antibody or an anti-PD-Ll antibody-drug conjugate described herein in a method of 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 cancer, hepatocellular cancer (HCC), gastric cancer, and cervical cancer) in a subject, the method comprising administering to the subject an effective amount of an anti-PD-Ll antibody or an anti-PD-Ll antibody-drug conjugate described herein. In some embodiments, the subject is a human.

[0307] The article of manufacture or kit can further comprise a container. Suitable containers include, for example, bottles, vials (e.g., dual-chamber vials), syringes (e.g., single or dual-chamber syringes), and test tubes. In some embodiments, the container is a vial. The containers can be formed from a variety of materials such as glass or plastic. The container holds a formulation.

[0308] The article of manufacture or kit can further comprise a label or package insert on or associated with the container, indicating that the formulation can be used or intended to be used in subcutaneous, intravenous (e.g., intravenous infusion), or other modes of administration 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 cancer, hepatocellular cancer (HCC), gastric cancer, and cervical cancer) in a subject. The container(s) can be a single unit-dose or multi-dose vial that allows for repeated administration of the reconstituted formulation. The article of manufacture or kit can further comprise a second container comprising a suitable diluent. The article of manufacture or kit can also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

[0309] The article of manufacture or kit herein also optionally comprises a container comprising a second agent, wherein the anti-PD-Ll antibody or anti-PD-Ll antibody-drug conjugate is a first agent, and the article of manufacture or kit further comprises instructions on a label or package insert for treating a subject with an effective amount of the second agent. In some embodiments, the second agent is used to eliminate or reduce the severity of one or more adverse events.

[0310] In some embodiments, the anti-PD-Ll antibody or anti-PD-Ll antibody-drug conjugate is present in the container as a lyophilized powder. In some embodiments, the lyophilized powder is in a gas- sealed container (such as a vial, ampule, or pouch) that indicates the amount of active agent. In cases where the drug is to be administered by injection, an ampule of sterile water for injection or normal saline can optionally be provided as part of the kit, so that the components can be mixed prior to administration. Such kits can also include, if desired, one or more of various conventional pharmaceutical components, such as, for example, containers of one or more of the pharmaceutically-acceptable carriers, additional containers and the like, as will be apparent to those skilled in the art. Also included in such kits can be a printed instruction sheet which

[0311] X. Other Uses

[0312] The anti-PD-Ll antibodies described herein, such as humanized anti-PD-Ll antibodies, can be used to detect PD-L1 in a clinical diagnostic or therapeutic setting or in research. Expression of PD-L1 on a cancer provides an indication that the cancer can be treated with the antibodies of the present application. The antibodies can also be sold as reagents for laboratory studies that detect cells bearing PD-L1 and their responses to various stimuli. In such uses, the monoclonal antibodies can be labeled with fluorescent molecules, spin-labeled molecules, enzymes, or radioisotopes and can be provided in kit form with all reagents necessary to carry out assays for PD-L1. The antibodies described herein can be used to detect PD-L1 protein expression and determine whether a cancer can be treated with a PD-L1 ADC.

[0313] All patent applications, websites, other publications, accession numbers, and the like cited above or below are incorporated by reference in their entireties for all purposes as if each individual item was specifically and individually indicated to be incorporated by reference. If different versions of a sequence are associated with different dates of accession numbers, the version is referred to that is associated with the date of the effective filing date of this application. The effective filing date means the earlier of the actual filing date with respect to the accession number (as applicable) or the filing date of the priority application. Likewise, if different versions of a publication, website, and the like are published at different times, the version that is published closest to the effective filing date of the application is referred to, unless otherwise specified. Unless specifically indicated otherwise, any feature, step, element, embodiment, or aspect of the application can be used in combination with any other. Although the application has been described in some detail to facilitate understanding, it will be apparent that certain changes and modifications can be practiced within the scope of the appended claims.

[0314] Examples

[0315] The cell lines described in the following examples were maintained in culture medium according to conditions specified by the American Type Culture Collection (ATCC) or Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany (DMSZ) or as otherwise known.

[0316] Methods

[0317] Antibody production

[0318] SG-559-xx antibodies against PD-L1 were generated by introducing point mutations into the CDRs of the fully human Ab1 in order to reduce affinity. Briefly, residues in the CDRs immediately adjacent to the PD-L1 binding epitope were mutated to dissimilar amino acids. Figure 1 Four exemplary selected residues are illustrated in the middle panel. For preliminary screening purposes, SG-559-xx antibodies were produced by ATUM bio using transient transfection in HEK293 cells.

[0319] For follow-up studies, antibodies were generated in-house according to the following protocol. Antibody variable and constant domain sequences were synthesized using template-free PCR. Briefly, virtual gene sequences were converted to oligonucleotide sequences using Genewiz’s bioinformatics tools. Oligonucleotides were synthesized, pooled, and amplified using PCR. Full-length amplicons from the PCR reaction were cloned into a vector, and then the product was transformed into E. coli and unique colonies were isolated. Colonies were grown overnight in liquid media, and plasmid DNA was isolated, purified, and sequence verified using Sanger sequencing. Light and heavy chain clones were cloned into pcDNA3.4 vector.

[0320] Antibody heavy and light chain vectors at a 1:1 ratio were diluted into ThermoFisher OptiPRO SFM media with ExpiFectamine CHO transfection reagent. DNA / transfection reagent was then added to ExpiCHO cultures in ThermoFisher ExpiCHO Expression media and incubated for nine days with ExpiCHO enhancer added on day one and ExpiCHO feed added on days one and two. Cultures were harvested by centrifugation and 0.2um filtration or by depth filtration using Millipore X0HC and D0HC pods followed by 0.2um filtration.

[0321] GE HiTrap mAb Select SuRe columns were used to purify each IgG. Prior to elution, resins were washed with 5CV PBS + 0.1% Triton, 5CV PBS + 0.5M NaCl, and 7.5CV of PBS. IgG was eluted using 100mM acetic acid pH3 buffer. Sample buffer was exchanged to PBS using a 26 / 60 HiPrep Desalt column. Samples were subjected to a fine purification step on a HiPrep Superdex 200 26 / 600 column run in PBS. Samples were then filter sterilized and sampled for characterization. Characterization included A280 concentration, aSEC HPLC, aHIC HPLC, and reduced PLRP-MS (QToF).

[0322] Biolayer interferometry

[0323] Bio-layer interferometry was performed using an Octet Red 384 system (ForteBio) to determine the binding affinity of SG-559-xx antibodies. Anti-human Fab-CH1 (FAB2G) biosensors (ForteBio) were loaded with 4 pg / mL SG-559-xx antibody for 100 s. Following a subsequent baseline step, human PD-L1 (Acro Biosciences) at a concentration range of 500 nM to 0.69 nM (1 x PBS pH 7.4 with 1% casein, 0.2% Tween-20) was incubated with the loaded probes for 150 s for an association step. This was followed by a dissociation step for 1000 s in the absence of human PD-L1 in the same buffer. The k 缔合 and k 解离 were fitted to the resulting binding curves.

[0324] Production of antibody-drug conjugates (ADCs)

[0325] SG-559-xx antibodies were conjugated to MDpr-PEG(12)-gluc-MMAE at an average drug-to-antibody ratio (DAR) of 8, as described in US20180092984. SG-559-xx antibodies were conjugated to vc-MMAE at an average DAR of 4, as described in US20050238649. SG-559-xx antibodies were conjugated to MP-PEG8-VKG-camptothecin at an average DAR of 8, as described in PCT / US2019 / 025968 (filed April 5, 2019).

[0326] In vitro cytotoxicity assays

[0327] Cell lines were plated 24 hours prior to antibody-drug conjugate (ADC) treatment to allow cells to acclimate. Interferon-gamma at 500 IU / mL was also added at this time to induce PD-L1 expression, where indicated. Cells were then treated with the indicated dose of ADC and incubated at 37°C for 96 hours. Additional PD-L1 -directed antibodies as well as isotype controls were included as ADCs for comparison. CellTiter-Glo (Promega Corporation, Madison, WI) was used to measure cell viability of cell lines according to the manufacturer’s instructions. Briefly, cells were incubated with CellTiter-Glo reagent for 30 minutes at room temperature and luminescence was measured using an Envision plate reader (Perkin Elmer, Waltham, MA). Results are reported as x50, the concentration of compound required to produce a 50% reduction in viability compared to untreated cells.

[0328] Internalization assay

[0329] Internalization of PD-L1 directed antibodies was performed on Incucyte (Sartorius) using FabFluor pH sensitive conjugates. Antibodies were conjugated to a pH sensitive dye that has an incremental fluorescent signal from the cell surface and into the endosomal / lysosomal compartment as the pH decreases. Adherent cells were plated for 24h (with 500 IU / mL interferon-g to induce PD-L1 expression) and then incubated with these conjugates. Suspended cells were plated for 3h and then incubated with these conjugates. Cells were then dosed with 0.5 pg / mL of the indicated dye-antibody conjugates and allowed to incubate for 48h. The total integrated intensity of the fluorescent signal was normalized to % confluence per well per time point using Incucyte S3 software (Sartorius). Results are reported as the area under the curve of normalized integrated intensity versus time.

[0330] In vivo activity studies

[0331] Nude mice were inoculated subcutaneously with 5.0 x 10 6 BxPC3 pancreatic adenocarcinoma cells or 1.0 x 10 6 EBC-1 NSCLC cells. NSG mice were inoculated subcutaneously with 5.0 x 10 5 MDA-MB-231 triple negative breast cancer cells. SCID mice were inoculated subcutaneously with 1.0 x 10 6 Karpas 299 ALCL cells or 1.0 x 10 6 Calu-1 NSCLC cells. Tumor growth was monitored with calipers and the average tumor volume was calculated using the formula (0.5 x [length x width 2 ]). When the average tumor volume reached approximately 100 mm 3 , mice were either left untreated or dosed intraperitoneally with ADCs as indicated. Unconjugated antibody and vc-MMAE ADCs were dosed weekly for a total of three doses. MP-PEG8-VKG-camptothecin ADCs were dosed only once. Mice were euthanized when the tumor volume reached approximately 750 mm 3 . For immunophenotyping studies of Karpas 299 tumor bearing animals, the average tumor volume was allowed to reach 200 mm 3These mice were then treated with a single dose of unconjugated antibody or ADC, and euthanized six days later. Tumors were characterized ex vivo by immunohistochemistry and cytokine analysis (Luminex). All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities.

[0332] PD-L1 blockade

[0333] In vitro assessment of PD-L1 blockade was performed using the PD-1 / PD-L1 Blockade Bioassay (Promega Corporation) according to the manufacturer’s instructions. Briefly, PD-L1+aAPC / CHO-K1 cells were plated and allowed to adapt for 16 hours. Antibodies or ADCs at the indicated concentrations were then added to the plated cells, followed by the addition of PD-1+effector cells. In the absence of PD-1 / PD-L1 signaling, the interaction between aAPC / CHO-K1 cells and effector cells produces a bioluminescent signal. Thus, more effective inhibition of the PD-1 / PD-L1 interaction results in a higher luminescent signal, quantified as fold induction relative to untreated cells. A PD-1 binding antibody (Promega Corporation) was included as a positive control, and a non-conjugating isotype antibody was included as a negative control.

[0334] Immunotoxicity in human APC models stimulated with IFNy to upregulate PD-L1

[0335] Immunotoxicity of antibodies or ADCs on human antigen presenting cells in vitro was measured using human antigen presenting cells (APCs) stimulated with interferon-gamma (IFNy), then treated with any of the antibodies or ADCs described herein. Human APCs were stimulated with 500 IU / mL of IFNy (R&D Systems) for 24 hours in vitro to upregulate PD-L1 prior to treatment with SG-559-xx ADC. Immunotoxicity was calculated as a percentage of the viability of untreated APCs at different antibody or ADC concentrations.

[0336] Immunoregulatory inhibition in human APC models

[0337] Immune response suppression was measured using human antigen presenting cells (APCs) stimulated with lipopolysaccharide (LPS) and then treated with any of the antibodies or ADCs described herein. Human APCs were stimulated with 500 IU / mL of IFNy (R&D Systems) for 24 hours to upregulate PD-L1 in vitro. Human APCs were then treated with SG-559-xx ADC for 24 hours as indicated. Human APCs were then stimulated with 100 ng / mL of LPS (Sigma Aldrich) for 48 hours in vitro. The response to LPS was measured by flow cytometry staining for MHC class II and CD86 (Biolegend). Immune function strength was calculated as the fold change in MHC class II or CD86 in APCs in response to LPS stimulation at different antibody or ADC concentrations.

[0338] Deglycosylation of human PD-L1 using PNGase F

[0339] To generate deglycosylated hPD-L1, human PD-L1 was treated with PNGase F enzyme (New England Biolabs) in conjunction with a denaturation protocol. PNGase F catalyzes the cleavage of the N-linked oligosaccharide between the most internal GlcNAc and the asparagine residue from high mannose, hybrid, and complex oligosaccharides of N-linked glycoproteins. Human PD-L1 was subjected to the denaturation protocol in the absence of PNGase F to provide a reaction control. The deglycosylation protocol included combining human PD-L1 (Acro Biosciences) with Rapid PNGase F Buffer, heating the human PD-L1 at 75 °C for 5 minutes, cooling the denatured human PD-L1 on ice, adding PNGase F, and overnight at 37 °C. Glycosylation status was confirmed with mass spectrometry.

[0340] Biolayer interferometry 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. After a baseline step, glycosylated human PD-L1 and deglycosylated human PD-L1 at a concentration range of 500 nM to 0.69 nM (1x PBS pH 7.4 with 1% BSA, 0.2% Tween-20) were incubated with the loaded probes for 150 s for the association step. This was followed by a dissociation step of 1000 s in the same buffer lacking human PD-L1. The k 缔合 and k 解离 were fitted to the resulting binding curves.

[0341] Results

[0342] Example 1: Design and characterization of SG-559-xx antibodies

[0343] Seventeen SG-559-xx antibodies were generated from the parental Ab1 antibody as described in the Methods. The CDRs containing mutations for these antibodies are listed in Table 1. Sixteen of these antibodies were evaluated for monovalent binding affinity to hPD-L1 by biolayer interferometry compared to Ab1 (Table 2). The measured affinities of the SG-559-xx antibodies spanned almost two orders of magnitude, and the Kdvalues ranged from 4 nM to 297 nM. D Values are from 4 nM to 297 nM.

[0344] Table 1. SG-559-XX variant sequences

[0345] SG-559-XX variants Mutated CDRs SEQ ID NO. SG-559-01 HC CDR1 13 SG-559-02 LC CDR1 26 SG-559-03 LC CDR3 38 SG-559-04 HC CDR3 45 SG-559-05 HC CDR2 49 SG-559-06 HC CDR2 50 SG-559-07 HC CDR2 51 SG-559-08 HC CDR2 52 SG-559-09 HC CDR2 53 SG-559-10 HC CDR3 54 SG-559-11 HC CDR3 55 SG-559-12 LC CDR3 56 SG-559-13 LC CDR3 57 SG-559-14 LC CDR3 58 SG-559-15 LC CDR3 59 SG-559-16 LC CDR3 60 SG-559-17 LC CDR3 61

[0346] Table 2. SG-559-XX binding affinities

[0347]

[0348]

[0349] Example 2: In vitro cytotoxicity

[0350] The cytotoxicity of SG-559-xx antibodies as ADCs was evaluated against cancer cell lines expressing PD-L1, including 786-O, BxPC3, ES-2, MDA-MB-231, Karpas 299, and L540cy, as described in the Methods. In some experiments, SU-DHL-4, a PD-L1 negative cancer cell line, was included as a control. An initial screen of fifteen SG-559-xx ADCs with MDpr-PEG(12)-gluc-MMAE payloads (DAR 8), excluding the two with the lowest affinities, indicated that several SG-559-xx antibodies exhibited significantly improved cytotoxicity relative to the parental Ab1 (Figure 1). Figures 2A-2F

[0351] The four SG-559-xx antibodies that consistently exhibited the highest potency in the initial screen were further characterized for their potency as vc-MMAE and MP-PEG8-VKG-camptothecin ADCs (Table 3). These ADCs were significantly more potent than Ab1 in most of the cell lines tested. They also had no activity in the antigen negative cell line (SU-DHL-4), indicating that this was not due to non-specific binding.

[0352] Table 3. SG-559-XX x50 values (ng / mL)

[0353]

[0354] ​

[0355] Example 3: Internalization

[0356] To confirm the cytotoxicity results, the internalization of SG-559-01 and SG-559-03 was further examined using the Incucyte imaging system and pH sensitive dye conjugates as described in the methods. The internalization of SG-559-01 and SG-559-03 was consistently high in most of the cell lines tested. This was measured by the percent increase in area under the curve (AUC) of normalized integrated intensity versus time (Table 4). Exemplary curves are shown for MDA-MB-231 Figure 3A ) and Karpas 299 Figure 3B ).

[0357] Table 4. SG-559-XX internalization

[0358]

[0359] Example 4: Anti-tumor activity in vivo

[0360] The four SG-559-xx antibodies characterized for in vitro screening were also tested for anti-tumor efficacy in two mouse xenograft models. In the MDA-MB-231 model, the SG-559-xx antibodies as ADCs exhibited significant anti-tumor activity Figures 4A-4B ) with two drug linkers. In the BxPC3 model, the SG-559-xx antibodies as ADCs exhibited moderate anti-tumor activity Figures 5A-5B ). In almost all cases, the SG-559-xx ADCs were more efficacious than the Ab1 ADC, indicating that the observed in vitro phenotype translated to the in vivo setting.

[0361] The anti-tumor efficacy was further observed in additional models using one of our most promising antibodies as a Fc effector function reduced variant (SG-559-01LALA). The SG-559-01LALA antibody as ADCs exhibited significant anti-tumor activity in the Karpas 299 Figures 6A-6B ), Calu-1 Figure 7 ), and EBC-1 Figures 8A-8B ) models with one or two drug linkers. Note that this activity was distinct from that of the unconjugated SG-559-01LALA antibody.

[0362] Example 5: PD-L1 blockade

[0363] SG-559-01LALA was further characterized for its ability to block the PD-1 / PD-L1 checkpoint in vitro. SG-559-01 was able to more effectively inhibit PD-1 / PD-L1 signaling relative to the PD-1 antibody control. In addition, unconjugated SG-559-01LALA was comparable to SG-559-01LALA conjugated to two drug linkers, demonstrating that conjugation did not impact PD-1 / PD-L1 blockade Figure 9

[0364] Example 6: Immunotoxicity on human APCs in vitro

[0365] SG-559-01 and SG-559-01LALA were evaluated for immunotoxicity on APCs (e.g., macrophages and dendritic cells (DCs)). APCs were stimulated with IFNy prior to treatment to upregulate PD-L1 as described in the Methods. SG-559-01LALA ADCs exhibited immunotoxicity on human APCs that was similar or within an order of magnitude of the isotype control in both macrophages and DCs Figures 10A-10D

[0366] The four SG-559-xx antibodies characterized for in vitro screening were also tested for immunotoxicity against APCs (i.e., dendritic cells and macrophages). For each SG-559-xx ADC, the immunotoxicity on human APCs was similar or within an order of magnitude Figures 11A-11D

[0367] Example 7: Immune response suppression

[0368] SG-559-01 ADCs were further characterized by measuring immune response suppression in human APCs treated with LPS. Human APCs were stimulated with LPS in vitro after ADC treatment as described in the Methods, and the amount of upregulation of MHC class II and CD86 was quantified as a measure of immune response. Treatment with SG-559-01 ADCs resulted in immune response suppression in DCs and macrophages in a similar manner or within an order of magnitude of the isotype control as measured by MHC class II Figures 12A-12B ) and CD86 Figures 12C-12D

[0369] Example 8: Increase in immune infiltration

[0370] ​​​​SG-559-01vc-MMAE ADC was further characterized by assessing immune infiltration in mice with Karpas 299 tumors. Tumor-bearing mice were treated as indicated and tumors were characterized six days later. SG-559-01vc-MMAE ADC induced immune infiltration in mice with Karpas 299 tumors relative to both untreated controls and SG-559-01 antibody Figures 13A-13C Figure 13A An increase in mCD45+ cells (pan leukocyte marker) is shown. Figure 13B An increase in mCD11c+ cells (marker for dendritic cell and macrophage subsets) is shown. Figure 13C An increase in mF4 / 80+ cells (macrophage marker) is shown.

[0371] Example 9: Inflammatory cytokine response

[0372] SG-559-01LALA vc-MMAE ADC was further characterized for its ability to induce inflammatory cytokine production in the tumor microenvironment (TME). SG-559-01LALA vc-MMAE ADC induced inflammatory cytokines in the TME relative to both untreated controls and SG-559-01LALA antibody as measured by intratumoral concentrations of: eotaxin (chemotactic factor for eosinophils; Figure 14A ), MIP1a (pro-inflammatory macrophage cytokine; Figure 14B ), MIP1b (pro-inflammatory macrophage cytokine; Figure 14C ), MIG / CXCL9 (induced by IFNy, influences migration and differentiation of immune cells; Figure 14D ), MCP1 (monocyte / macrophage chemotactic factor; Figure 14E ), and regulated on activation, normal T cell expressed and secreted factor (rantes) (monocyte, T cell, and eosinophil chemotactic factor; Figure 14F ).

[0373] Example 10: Binding affinity to glycosylated and deglycosylated PD-L1

[0374] ​Evaluate the binding affinity of SG-559-01 to glycosylated and deglycosylated forms of PD-L1. Evaluate the binding affinity on an Octet Red 384 system (ForteBio) using biolayer interferometry as described in the Methods. Deglycosylate PD-L1 using PNGase F enzyme and denaturing protocol as described in the Methods. Evaluate the binding affinity of SG-559-01 to deglycosylated PD-L1 and to control glycosylated PD-L1 (same treatment conditions but without PNGase F treatment as described in the Methods). Observe a ~2-fold difference in the binding affinity of SG-559-01 to deglycosylated PD-L1 compared to glycosylated PD-L1 (Table 5). Verify the glycosylation status of PD-L1 using mass spectrometry.

[0375] Table 5. Binding of SG-559-01 to glycosylated and deglycosylated hPD-L1

[0376]

[0377] Informal Sequence Listing

[0378] SEQ ID NO: 1 - Ab1 heavy chain variable region - protein

[0379] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS

[0380] SEQ ID NO: 2 - Ab1 light chain variable region - protein

[0381] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK

[0382] SEQ ID NO: 3 - Ab1 heavy chain CDR1 - protein

[0383] TYAIS

[0384] SEQ ID NO: 4 - Ab1 heavy chain CDR2 - protein

[0385] GIIPIFGKAHYAQKFQG

[0386] SEQ ID NO: 5 - Ab1 heavy chain CDR3 - protein

[0387] KFHFVSGSPFGMDV

[0388] SEQ ID NO:6 - Ab1 light chain CDR1 - protein

[0389] RASQSVSSYLA

[0390] SEQ ID NO:7 - Ab1 light chain CDR2 - protein

[0391] DASNRAT

[0392] SEQ ID NO:8 - Ab1 light chain CDR3 - protein

[0393] QQRSNWPT

[0394] SEQ ID NO:9 - SG-559-01 LALA hlgGl heavy chain - protein

[0395] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0396] SEQ ID NO: 10 - SG-559-01 kappa light chain - protein

[0397] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0398] SEQ ID NO: 11 - SG-559-01 Heavy Chain Variable Region - Protein

[0399] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS

[0400] SEQ ID NO: 12 - SG-559-01 Light Chain Variable Region - Protein

[0401] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK

[0402] SEQ ID NO: 13 - SG-559-01 Heavy Chain CDR1 - Protein

[0403] TAAIS

[0404] SEQ ID NO: 14 - SG-559-01 Heavy Chain CDR2 - Protein

[0405] GIIPIFGKAHYAQKFQG

[0406] SEQ ID NO: 15 - SG-559-01 Heavy Chain CDR3 - Protein

[0407] KFHFVSGSPFGMDV

[0408] SEQ ID NO: 16 - SG-559-01 Light Chain CDR1 - Protein

[0409] RASQSVSSYLA

[0410] SEQ ID NO: 17 - SG-559-01 Light Chain CDR2 - Protein

[0411] DASNRAT

[0412] SEQ ID NO: 18 - SG-559-01 Light Chain CDR3 - Protein

[0413] QQRSNWPT

[0414] SEQ ID NO: 19 - SG-559-02 LALA hlgGl Heavy Chain - Protein

[0415] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0416] SEQ ID NO: 20 - SG-559-02 Kappa Light Chain - Protein

[0417] EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0418] SEQ ID NO: 21 - SG-559-02 Heavy chain variable region - Protein

[0419] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS

[0420] SEQ ID NO: 22 - SG-559-02 Light chain variable region - Protein

[0421] EIVLTQSPATLSLSPGERATLSCRASQSVSSALAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK

[0422] SEQ ID NO: 23 - SG-559-02 Heavy chain CDR1 - Protein

[0423] TYAIS

[0424] SEQ ID NO: 24 - SG-559-02 Heavy chain CDR2 - Protein

[0425] GIIPIFGKAHYAQKFQG

[0426] SEQ ID NO: 25 - SG-559-02 Heavy chain CDR3 - Protein

[0427] KFHFVSGSPFGMDV

[0428] SEQ ID NO: 26 - SG-559-02 Light chain CDR1 - Protein

[0429] RASQSVSSALA

[0430] SEQ ID NO: 27 - SG-559-02 light chain CDR2 - protein

[0431] DASNRAT

[0432] SEQ ID NO: 28 - SG-559-02 light chain CDR3 - protein

[0433] QQRSNWPT

[0434] SEQ ID NO: 29 - SG-559-03 LALA hlgGl heavy chain - protein

[0435] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0436] SEQ ID NO: 30 - SG-559-03 kappa light chain - protein

[0437] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNLPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0438] SEQ ID NO: 31 - SG-559-03 Heavy chain variable region - Protein

[0439] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSS

[0440] SEQ ID NO: 32 - SG-559-03 Light chain variable region - Protein

[0441] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNLPTFGQGTKVEIK

[0442] SEQ ID NO: 33 - SG-559-03 Heavy chain CDR1 - Protein

[0443] TYAIS

[0444] SEQ ID NO: 34 - SG-559-03 Heavy chain CDR2 - Protein

[0445] GIIPIFGKAHYAQKFQG

[0446] SEQ ID NO: 35 - SG-559-03 Heavy chain CDR3 - Protein

[0447] KFHFVSGSPFGMDV

[0448] SEQ ID NO: 36 - SG-559-03 Light chain CDR1 - Protein

[0449] RASQSVSSYLA

[0450] SEQ ID NO: 37 - SG-559-03 light chain CDR2 - Protein

[0451] DASNRAT

[0452] SEQ ID NO: 38 - SG-559-03 light chain CDR3 - Protein

[0453] QQRSNLPT

[0454] SEQ ID NO: 39 - SG-559-04 LALA hlgGl heavy chain - Protein

[0455] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0456] SEQ ID NO: 40 - SG-559-04 kappa light chain - Protein

[0457] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0458] SEQ ID NO: 41 - SG-559-04 Heavy chain variable region - Protein

[0459] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSS

[0460] SEQ ID NO: 42 - SG-559-04 Light chain variable region - Protein

[0461] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIK

[0462] SEQ ID NO: 43 - SG-559-04 Heavy chain CDR1 - Protein

[0463] TYAIS

[0464] SEQ ID NO: 44 - SG-559-04 Heavy chain CDR2 - Protein

[0465] GIIPIFGKAHYAQKFQG

[0466] SEQ ID NO: 45 - SG-559-04 Heavy chain CDR3 - Protein

[0467] KFHFVSGSGFGMDV

[0468] SEQ ID NO: 46 - SG-559-04 Light chain CDR1 - Protein

[0469] RASQSVSSYLA

[0470] SEQ ID NO:47 - SG-559-04 Light Chain CDR2 - Protein

[0471] DASNRAT

[0472] SEQ ID NO:48 - SG-559-04 Light Chain CDR3 - Protein

[0473] QQRSNWPT

[0474] SEQ ID NO:49 - SG-559-05 Heavy Chain CDR2 - Protein

[0475] GIIPIAGKAHYAQKFQG

[0476] SEQ ID NO:50 - SG-559-06 Heavy Chain CDR2 - Protein

[0477] GIIPIFGAAHYAQKFQG

[0478] SEQ ID NO:51 - SG-559-07 Heavy Chain CDR2 - Protein

[0479] GIIPIFGRAHYAQKFQG

[0480] SEQ ID NO:52 - SG-559-08 Heavy Chain CDR2 - Protein

[0481] GIIPIFGKAAYAQKFQG

[0482] SEQ ID NO:53 - SG-559-09 Heavy Chain CDR2 - Protein

[0483] GIIPIFGKAFYAQKFQG

[0484] SEQ ID NO:54 - SG-559-10 Heavy Chain CDR3 - Protein

[0485] KFHFVSGAPFGMDV

[0486] SEQ ID NO:55 - SG-559-11 Heavy Chain CDR3 - Protein

[0487] KFHFVSGSPAGMDV

[0488] SEQ ID NO:56 - SG-559-12 Light Chain CDR3 - Protein

[0489] QQASNWPT

[0490] SEQ ID NO:57 - SG-559-13 light chain CDR3 - protein

[0491] QQKSNWPT

[0492] SEQ ID NO:58 - SG-559-14 light chain CDR3 - protein

[0493] QQRSAWPT

[0494] SEQ ID NO:59 - SG-559-15 light chain CDR3 - protein

[0495] QQRSQWPT

[0496] SEQ ID NO:60 - SG-559-16 light chain CDR3 - protein

[0497] QQRSNAPT

[0498] SEQ ID NO:61 - SG-559-17 light chain CDR3 - protein

[0499] QQRSNFPT

[0500] SEQ ID NO:62 - SG-559-01 hlgGl heavy chain - protein

[0501] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTAAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0502] SEQ ID NO:63 - SG-559-02 hlgGl heavy chain - protein

[0503] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0504] SEQ ID NO:64 - SG-559-03 hlgGl heavy chain - protein

[0505] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0506] SEQ ID NO:65 - SG-559-04 hlgGl heavy chain - protein

[0507] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSGFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0508] SEQ ID NO: 66 - SG-559-01 Heavy chain variable region - nucleic acid

[0509] caggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaagacttctggagacaccttcagcaccgccgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatatttggtaaagcacactacgcacagaagttccagggcagagtcacgattaccgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca

[0510] SEQ ID NO: 67 - SG-559-01 Light chain variable region - nucleic acid

[0511] gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaa

[0512] SEQ ID NO: 68 - SG-559-02 Heavy chain variable region - nucleic acid

[0513] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGACTTCTGGAGACACCTTCAGCACCTATGCTATCAGCTGGGTGC GACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTAAAGCACACTACGCACAGAAGTTCCAGGGCAGAGTCACGAT TACCgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca

[0514] SEQ ID NO: 69 - SG-559-02 Light Chain Variable Region - Nucleic Acids

[0515] GAAATTGTGTTGACACAGTCTCCAGCCACCCGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCGCCTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCAC TCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTATTACTGTCAGCAGCGTAGCACTGGCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0516] SEQ ID NO: 70 - SG-559-03 Heavy Chain Variable Region - Nucleic Acids

[0517] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGACTTCTGGAGACACCTTCAGCACCTATGCTATCAGCTGGGTGC GACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTAAAGCACACTACGCACAGAAGTTCCAGGGCAGAGTCACGAT TACCgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagccccttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca

[0518] SEQ ID NO: 71 - SG-559-03 light chain variable region - nucleic acid

[0519] GAAATTGTGTTGACACAGTCTCCAGCCACCCGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCA GTTATTACTGTCAGCAGCGTAGCAACCTGCCGACGTT CGGCCAAGGGACCAAGGTGGA AATCAA A

[0520] SEQ ID NO: 72 - SG-559-04 heavy chain variable region - nucleic acid

[0521] CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGACTTCTGGAGACACCTTCAGCACCTATGCTATCAGCTGGGTGC GACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTAAAGCACACTACGCACAGAAGTTCCAGGGCAGAGTCACGAT TACCgcggacgaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtatttttgtgcgagaaagtttcactttgtttcggggagcggcttcggtatggacgtctggggccaagggaccacggtcaccgtctcctca

[0522] SEQ ID NO: 73 - SG-559-04 Light Chain Variable Region - Nucleic Acids

[0523] GAAATTGTGTTGACACAGTCTCCAGCCACCCGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCTACTTAGCCTG GTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTC ACTCTCACCATCAGCAGCCTAGAGCCTGAAGATT TTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCGACG TTCGGCCAAGGGACCAAGGTGGA AATCAA A

[0524] SEQ ID NO: 74 - SG-559-01 LALA hlgGl Heavy Chain - Nucleic Acids

[0525]

[0526] SEQ ID NO:75 - SG-559-01 kappa light chain - nucleic acid

[0527] gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

[0528] SEQ ID NO:76 - SG-559-01 hlgGl heavy chain - nucleic acid

[0529]

[0530] SEQ ID NO: 77 - SG-559-02 LALA hlgG1 heavy chain - nucleic acid

[0531]

[0532] SEQ ID NO: 78 - SG-559-02 kappa light chain - nucleic acid

[0533] gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcgccttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

[0534] SEQ ID NO: 79 - SG-559-02 hlgGl heavy chain - nucleic acid

[0535]

[0536] SEQ ID NO: 80 - SG-559-03 LALA hlgGl heavy chain - nucleic acid

[0537]

[0538] SEQ ID NO: 81 - SG-559-03 kappa light chain - nucleic acid

[0539] gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaacctgccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

[0540] SEQ ID NO: 82 - SG-559-03 hlgGl heavy chain - nucleic acid

[0541]

[0542] SEQ ID NO: 83 - SG-559-04 LALA hlgG1 heavy chain - nucleic acid

[0543]

[0544] SEQ ID NO: 84 - SG-559-04 kappa light chain - nucleic acid

[0545] gaaattgtgttgacacagtctccagccaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacctggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagccaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcctgaagattttgcagtttattactgtcagcagcgtagcaactggccgacgttcggccaagggaccaaggtggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

[0546] SEQ ID NO: 85 - SG-559-04 hlgGl heavy chain - nucleic acid

[0547]

[0548] SEQ ID NO: 86 - Ab1 hlgG1 heavy chain - protein

[0549] QVQLVQSGAEVKKPGSSVKVSCKTSGDTFSTYAISWVRQAPGQGLEWMGGIIPIFGKAHYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYFCARKFHFVSGSPFGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0550] SEQ ID NO: 87 - Ab1 kappa light chain - protein

[0551] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQUENCE LISTING <110> PatSnap Co. Ltd. <120> Anti-PD-L1 antibodies and antibody-drug conjugates <130> 49223-0051WO1 <150> 62 / 910,988 <151> 04-OCT-2019 <160> 87 <170> PatentIn Version 3.5 <210> 1 <211> 123 <212> PRT <213> Artificial <220> <223> Variable region of Ab1 heavy chain <400> 1 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 2 <211> 106 <212> PRT <213> Artificial <220> <223> Ab1 light chain variable region <400> 2 Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 5 <212> PRT <213> Artificial <220> <223> Ab1 heavy chain CDR1 <400> 3 Thr Tyr Ala Ile Ser 1 5 <210> 4 <211> 17 <212> PRT <213> Artificial <220> <223> Ab1 heavy chain CDR2 <400> 4 Gly lie lie Pro lie Phe Gly Lys Ala His Tyr Ala Gin Lys Phe Gin 1 5 10 15 Gly <210> 5 <211> 14 <212> PRT <213> Artificial <220> <223> Ab1 heavy chain CDR3 <400> 5 Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 6 <211> 11 <212> PRT <213> Artificial <220> <223> Ab1 light chain CDR1 <400> 6 Arg Ala Ser Gin Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 7 <211> 7 <212> PRT <213> Artificial <220> <223> Ab1 light chain CDR2 <400> 7 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 8 <211> 8 <212> PRT <213> Artificial <220> <223> Ab1 light chain CDR3 <400> 8 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 9 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-01 LALA hlgG1 heavy chain <400> 9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Ala 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 10 <211> 213 <212> PRT <213> Artificial <220> <223> SG-559-01 kappa light chain <400> 10 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160 Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 11 <211> 123 <212> PRT <213> Artificial <220> <223> SG-559-01 Heavy Chain Variable Region <400> 11 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Ala 20 25 30 Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gin Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 12 <211> 106 <212> PRT <213> Artificial <220> <223> SG-559-01 light chain variable region <400> 12 Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu lie Lys 100 105 <210> 13 <211> 5 <212> PRT <213> Artificial <220> <223> CDR1 of SG-559-01 heavy chain <400> 13 Thr Ala Ala lie Ser 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 of SG-559-01 heavy chain <400> 14 Gly lie lie Pro lie Phe Gly Lys Ala His Tyr Ala Gin Lys Phe Gin 1 5 10 15 Gly <210> 15 <211> 14 <212> PRT <213> artificial <220> <223> CDR3 of SG-559-01 heavy chain <400> 15 Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 16 <211> 11 <212> PRT <213> artificial <220> <223> CDR1 of SG-559-01 light chain <400> 16 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 17 <211> 7 <212> PRT <213> artificial <220> <223> CDR2 of SG-559-01 light chain <400> 17 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 18 <211> 8 <212> PRT <213> artificial <220> <223> CDR3 of SG-559-01 light chain <400> 18 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 19 <211> 453 <212> PRT <213> artificial <220> <223> SG-559-02 LALA hlgGl heavy chain <400> 19 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 20 <211> 213 <212> PRT <213> Artificial <220> <223> SG-559-02 kappa light chain <400> 20 Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160 Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 21 <211> 123 <212> PRT <213> Artificial <220> <223> SG-559-02 Heavy Chain Variable Region <400> 21 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gin Lys Phe 50 55 60 Gln Gly Arg Val Thr lie Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 22 <211> 106 <212> PRT <213> Artificial <220> <223> SG-559-02 light chain variable region <400> 22 Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 23 <211> 5 <212> PRT <213> Artificial <220> <223> CDR1 of heavy chain of SG-559-02 <400> 23 Thr Tyr Ala Ile Ser 1 5 <210> 24 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 of heavy chain of SG-559-02 <400> 24 Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 25 <211> 14 <212> PRT <213> Artificial <220> <223> CDR3 of heavy chain of SG-559-02 <400> 25 Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 26 <211> 11 <212> PRT <213> Artificial <220> <223> CDR1 of SG-559-02 light chain <400> 26 Arg Ala Ser Gln Ser Val Ser Ser Ala Leu Ala 1 5 10 <210> 27 <211> 7 <212> PRT <213> Artificial <220> <223> CDR2 of SG-559-02 light chain <400> 27 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 28 <211> 8 <212> PRT <213> Artificial <220> <223> CDR3 of SG-559-02 light chain <400> 28 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 29 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-03 LALA hlgGl heavy chain <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala lie Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly lie lie Pro lie Phe Gly Lys Ala His Tyr Ala Gin Lys Phe 50 55 60 Gln Gly Arg Val Thr lie Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gin Thr Tyr He Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 30 <211> 213 <212> PRT <213> Artificial <220> <223> SG-559-03 kappa light chain <400> 30 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Leu Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 31 <211> 123 <212> PRT <213> Artificial <220> <223> SG-559-03 Heavy Chain Variable Region <400> 31 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gin Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 32 <211> 106 <212> PRT <213> Artificial <220> <223> SG-559-03 light chain variable region <400> 32 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Leu Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 33 <211> 5 <212> PRT <213> Artificial <220> <223> CDR1 of SG-559-03 heavy chain <400> 33 Thr Tyr Ala Ile Ser 1 5 <210> 34 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 of SG-559-03 heavy chain <400> 34 Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 35 <211> 14 <212> PRT <213> Artificial <220> <223> CDR3 of SG-559-03 heavy chain <400> 35 Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 1 5 10 <210> 36 <211> 11 <212> PRT <213> Artificial <220> <223> CDR1 of SG-559-03 light chain <400> 36 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 37 <211> 7 <212> PRT <213> Artificial <220> <223> CDR2 of SG-559-03 light chain <400> 37 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial <220> <223> SG-559-03 light chain CDR3 <400> 38 Gln Gln Arg Ser Asn Leu Pro Thr 1 5 <210> 39 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-04 LALA hIgG1 heavy chain <400> 39 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Gly Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala 225 230 235 240 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 40 <211> 213 <212> PRT <213> Artificial <220> <223> SG-559-04 kappa light chain <400> 40 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 41 <211> 123 <212> PRT <213> Artificial <220> <223> SG-559-04 Heavy Chain Variable Region <400> 41 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Gly Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 42 <211> 106 <212> PRT <213> Artificial <220> <223> SG-559-04 light chain variable region <400> 42 Glu Ile Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gin Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu Ile Lys 100 105 <210> 43 <211> 5 <212> PRT <213> Artificial <220> <223> CDR1 of SG-559-04 heavy chain <400> 43 Thr Tyr Ala Ile Ser 1 5 <210> 44 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 of SG-559-04 heavy chain <223> CDR3 of SG-559-04 heavy chain<400> 44 Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 45 <211> 14 <212> PRT <213> Artificial <220> <223> CDR3 of heavy chain of SG-559-04 <400> 45 Lys Phe His Phe Val Ser Gly Ser Gly Phe Gly Met Asp Val 1 5 10 <210> 46 <211> 11 <212> PRT <213> Artificial <220> <223> CDR1 of light chain of SG-559-04 <400> 46 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial <220> <223> CDR2 of light chain of SG-559-04 <400> 47 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 48 <211> 8 <212> PRT <213> Artificial <220> <223> CDR3 of light chain of SG-559-04 <400> 48 Gln Gln Arg Ser Asn Trp Pro Thr 1 5 <210> 49 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 Hinge SG-559-05 Heavy Chain <400> 49 Gly Ile Ile Pro Ile Ala Gly Lys Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 50 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 Hinge SG-559-06 Heavy Chain <400> 50 Gly Ile Ile Pro Ile Phe Gly Ala Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 51 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 Hinge SG-559-07 Heavy Chain <400> 51 Gly Ile Ile Pro Ile Phe Gly Arg Ala His Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 52 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 Hinge SG-559-08 Heavy Chain <400> 52 Gly lie lie Pro lie Phe Gly Lys Ala Ala Tyr Ala Gin Lys Phe Gin 1 5 10 15 Gly <210> 53 <211> 17 <212> PRT <213> Artificial <220> <223> CDR2 of heavy chain of SG-559-09 <400> 53 Gly lie lie Pro lie Phe Gly Lys Ala Phe Tyr Ala Gin Lys Phe Gin 1 5 10 15 Gly <210> 54 <211> 14 <212> PRT <213> Artificial <220> <223> CDR3 of heavy chain of SG-559-10 <400> 54 Lys Phe His Phe Val Ser Gly Ala Pro Phe Gly Met Asp Val 1 5 10 <210> 55 <211> 14 <212> PRT <213> Artificial <220> <223> CDR3 of heavy chain of SG-559-11 <400> 55 Lys Phe His Phe Val Ser Gly Ser Pro Ala Gly Met Asp Val 1 5 10 <210> 56 <211> 8 <212> PRT <213> Artificial <220> <223> SG-559-12 light chain CDR3 <400> 56 Gln Gln Ala Ser Asn Trp Pro Thr 1 5 <210> 57 <211> 8 <212> PRT <213> Artificial <220> <223> SG-559-13 light chain CDR3 <400> 57 Gln Gln Lys Ser Asn Trp Pro Thr 1 5 <210> 58 <211> 8 <212> PRT <213> Artificial <220> <223> SG-559-14 light chain CDR3 <400> 58 Gln Gln Arg Ser Ala Trp Pro Thr 1 5 <210> 59 <211> 8 <212> PRT <213> Artificial <220> <223> SG-559-15 light chain CDR3 <400> 59 Gln Gln Arg Ser Gln Trp Pro Thr 1 5 <210> 60 <211> 8 <212> PRT <213> Artificial <220> <223> SG-559-16 light chain CDR3 <400> 60 Gln Gln Arg Ser Asn Ala Pro Thr 1 5 <210> 61 <211> 8 <212> PRT <213> Artificial <220> <223> CDRH3 of SG-559-17 <400> 61 Gln Gln Arg Ser Asn Phe Pro Thr 1 5 <210> 62 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-01 hlgG1 heavy chain <400> 62 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Ala 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 63 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-02 hlgG1 Heavy Chain <400> 63 Gln Val Gin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gin Ala Pro Gly Gin Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gin Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 64 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-03 hlgG1 heavy chain <400> 64 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 65 <211> 453 <212> PRT <213> Artificial <220> <223> SG-559-04 hlgG1 heavy chain <400> 65 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Gly Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 66 <211> 369 <212> DNA <213> Artificial <220> <223> SG-559-01 Heavy Chain Variable Region <400> 66 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc accgccgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt atttttgtgc gagaaagttt 300 cactttgttt cggggagccc cttcggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctca 369 <210> 67 <211> 318 <212> DNA <213> Artificial <220> <223> SG-559-01 light chain variable region <400> 67 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggccgacgtt cggccaaggg 300 accaaggtgg aaatcaaa 318 <210> 68 <211> 369 <212> DNA <213> Artificial <220> <223> SG-559-02 heavy chain variable region <400> 68 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 TCTGGAGACACCTTCAGC ACC ATGCTATC AGCTGGGT GCGACAGGCC 120 CCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTAA 180 GCACAGAAGTTCCAGGGCAGAGTCACGATTA CC GCGGAAT ATCCACGAGC AC AGCCTAC 240 ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTTTTGTGC 300 C ACTTTGTTTCGGGGAGCCCC T TCGGTATGGACGTCTGGGGCCAAGGGACC AC GGTCA CC 360 GTCTCCTCA 369 <210> 69 <211> 318 <212> PRT <213> Artificial <220> <223> SG-559-02 light chain variable region <400> 69 Gly Ala Ala Ala Thr Thr Gly Thr Gly Thr Thr Gly Ala Cys Ala Cys 1 5 10 15 Ala Gly Thr Cys Thr Cys Cys Ala Gly Cys Cys Ala Cys Cys Cys Thr 20 25 30 Gly Thr Cys Thr Thr Thr Gly Thr Cys Thr Cys Cys Ala Gly Gly Gly 35 40 45 Gly Ala Ala Ala Gly Ala Gly Cys Cys Ala Cys Cys Cys Thr Cys Thr 50 55 60 Gly Ala Ala Ala Gly Ala Gly Cys Cys Ala Cys Cys Cys Thr Cys ThrCys Cys Thr Gly Cys Ala Gly Gly Gly Cys Cys Ala Gly Thr Cys Ala 65 70 75 80 Gly Ala Gly Thr Gly Thr Thr Ala Gly Cys Ala Gly Cys Gly Cys Cys 85 90 95 Thr Thr Ala Gly Cys Cys Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys 100 105 110 Ala Gly Ala Ala Ala Cys Cys Thr Gly Gly Cys Cys Ala Gly Gly Cys 115 120 125 Thr Cys Cys Cys Ala Gly Gly Cys Thr Cys Cys Thr Cys Ala Thr Cys 130 135 140 Thr Ala Thr Gly Ala Thr Gly Cys Ala Thr Cys Cys Ala Ala Cys Ala 145 150 155 160 Gly Gly Gly Cys Cys Ala Cys Thr Gly Gly Cys Ala Thr Cys Cys Cys 165 170 175 Ala Gly Cys Cys Ala Gly Gly Thr Thr Cys Ala Gly Thr Gly Gly Cys 180 185 190 Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Cys Ala Gly 195 200 205 Ala Cys Thr Thr Cys Ala Cys Thr Cys Thr Cys Ala Cys Cys Ala Thr 210 215 220 Cys Ala Gly Cys Ala Gly Cys Cys Thr Ala Gly Ala Gly Cys Cys Thr 225 230 235 240 Gly Ala Ala Gly Ala Thr Thr Thr Thr Gly Cys Ala Gly Thr Thr Thr 245 250 255 Ala Thr Thr Ala Cys Thr Gly Thr Cys Ala Gly Cys Ala Gly Cys Gly 260 265 270 Thr Ala Gly Cys Ala Ala Cys Thr Gly Gly Cys Cys Gly Ala Cys Gly 275 280 285 Thr Thr Cys Gly Gly Cys Cys Ala Ala Gly Gly Gly Ala Cys Cys Ala 290 295 300 Ala Gly Gly Thr Gly Gly Ala Ala Ala Thr Cys Ala Ala Ala 305 310 315 <210> 70 <211> 369 <212> DNA <213> Artificial <220> <223> SG-559-03 Heavy Chain Variable Region <400> 70 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc acctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 GACAGAAGTT CCAGGGCAGC GTCACGATTA CCAGCAGAAG ATCCACGAGC ACAGCCTAC 240 ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACAGCCGTGT ATTTTTGTGC GAGAAAGTTT 300 CCTTCTTGTG TCGGGGAGCC CCTTCGCTAT GGACGTCTGG GGCCAAGGGA CCACGGTCAC 360 GTCTCCTCA 369 <210> 71 <211> 318 <212> DNA <213> Artificial <220> <223> SG-559-03 light chain variable region <400> 71 GAAATTGTGT TGACACAGTC TCCAGCCACC CTGTCTTTGT CTCCAGGGGA AAGAGCCACC 60 CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTG GTACCA ACAGAAACCT 120 GGCCAGGCTC CCAGGCTCCT CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC 180 AGGTTCAAGT GGCAGTGGGT CTTGGACAGA CTTCAC TCTCACCATC AGCAGCCTAG AGCCT 240 GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACC TGCCGACGTT CGGCCAAGGG 300 ACCAAGGTGG AAATCAA 318 <210> 72 <211> 369 <212> DNA <213> Artificial <220> <223> SG-559-04 heavy chain variable region <400> 72 CAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTC 60 TCCTGCAAGACTTCTGGAGACACCTTCAGCACCTATGCTATCAGCTGGGTGCGACAGGCC 120 CCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATATTTGGTAAAGCACACTAC 180 GCACAGAAGTTCCAGGGCAGAGTCACGAT TACC GC GGA TGA ATCC AC GAG C AC AGC CT AC 240 ATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTTTTGTGCGGAGAAAGTTT 300 C ACTTTGT TTC GGGGAGCGGCTTCG GT ATGGACGTCTGGGGCCAAGGGACCACGGTCACC 360 GTCTCCTCA 369 <210> 73 <211> 318 <212> DNA <213> Artificial <220> <223> SG-559-04 light chain variable region <400> 73 GAAATTGTGTTGACACAGTC TCCAGCCAC CCTGTCTTTGTCTCCAGGGGAAAGAGCCACC 60 CTCTCCTGCA GGGCCAGTCA GAGTGTAGC AGCTACTTAG CCTGGTACCA ACAGAAACCT 120 GGCCAGGCTCCCA GGCTCCTCATCTATGATGCATCCAACAGGGCCACTGGCATCCCAGCC 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggccgacgtt cggccaaggg 300 accaaggtgg aaatcaaa 318 <210> 74 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-01 LALA hIgG1 heavy chain <400> 74 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc accgccgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt atttttgtgc gagaaagttt 300 cactttgttt cggggagccc cttcggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag ctagcaccaa gggcccatct gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagctgc cctgggctgc ctggtcaagg actacttccc tgaacctgtg 480 acagtgtcct ggaactcagg agccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaagct 720 gctgggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tttacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 AGCGACATCG CCgtggagtgg agagcaatgg gcagccggag aacaactaca agaccacg 1200 CCTCCCgtgtggactccgacggctccttcttcctctacagcaagctcaccgtggacaag 1260 AGCAGgtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaac 1320 Cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 75 <211> 639 <212> DNA <213> Artificial <220> <223> SG-559-01 kappa light chain <400> 75 GAAATTGTGTT GACACAGTCT CCAGCCACCC TGTCTTTGTC TCCAGGGGAA AGAGCCACC 60 CTCTCCTGCA GGGCCAGTCA GAGTGTTAGC AGCTACTTAG CCTGgtacca acagaaacct 120 GGCCAGGCTC CCAGGCTCCT CATCTATGAT Gcatccaaca gggccactgg catcccagcc 180 AGGTTcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 GAAGATTTTG CAGTTTATTA CTGTCAGCAG CGTAGCAACT GGCCGACGTT CGGccaaggg 300 ACCAAGGTGG AAATCAAACG TACGgtggct gcaccatctg tcttcatctt CCCGCCATCT 360 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600 agctcgcccg tcacaaagag cttcaacagg ggagagtgt 639 <210> 76 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-01 hlgG1 heavy chain <400> 76 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc accgccgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt atttttgtgc gagaaagttt 300 cactttgttt cggggagccc cttcggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag ctagcaccaa gggcccatct gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagctgc cctgggctgc ctggtcaagg actacttccc tgaacctgtg 480 acagtgtcct ggaactcagg agccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 77 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-02 LALA hlgG1 heavy chain <400> 77 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc acctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180atggagctga gcagcctgag atctgaggac acggccgtgt atttttgtgc gagaaagttt 300 cactttgttt cggggagccc cttcggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag ctagcaccaa gggcccatct gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagctgc cctgggctgc ctggtcaagg actacttccc tgaacctgtg 480 acagtgtcct ggaactcagg agccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaagct 720 gctgggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tttacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 78 <211> 639 <212> DNA <213> Artificial <220> <223> SG-559-02 kappa light chain <400> 78 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agcgccttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggccgacgtt cggccaaggg 300 accaaggtgg aaatcaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 360 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600 agctcgcccg tcacaaagag cttcaacagg ggagagtgt 639 <210> 79 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-02 hlgG1 heavy chain <400> 79 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc acctatgcta tcagctgggt gcgacaggcc 120 CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TATTTGGTAA AGCACACTAC 180 GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACG AATCCACGAG CACAGCCTAC 240 ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTTTTGTGC GAGAAAGTTT 300 CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TATTTGGTAA AGCACACTAC 180 GTCTCCTCAG CTCAGCACCA AGGGCCCATC TGTCTTCCCC CTGGCACCCT CTCCTAAGAG 420 ACCTCTGGGG GCACAGCTGC CCTGGGCTGC CTGgtCAAGG ACTACTTCC CTGAACCTGT G 480 ACAGTGTCTT GGAACCTGAG GCCCTGACCA GCggCgtGC ACACCTTCCC GGCTGTCCTA 540 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTGgtGAcc GTGCCCTCCA GCAGCTTGGG C 600 ACCCAGACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGT GGACAAGAAA 660 GTTGAGCCCA AATCTTGTGA CAAACTCAC ACA TGC CCAC CGTGCCCAG CACCTGAAC T C 720 CTGGGGGGAC CGTCAGTCTT CCTCTTCCCC CCAAACCCA AGGACACCC T CATGATCTC C 780 CggACCCCTG AGGTCAcATG CgtGgtGgtG GACGTGAGCC ACGAAGACCC TGAGGTCAAG 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 80 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-03 LALA hlgG1 heavy chain <400> 80 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 TCCTGCAAGA CTTCTGGAGA CCCTTCAGC A CCCTATGCTA TCAGCTGGGT GCGACAGGCC 120 CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TTATTGGTAA AGCACACTAC 180 GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGCGGACG AATCCACGAG CACAGCCTAC 240 ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTTTTGTGC GAGAAAGTTT 300 C ACTTTGTTT CGGGGAGCCC CTT CGGTATG GACGTCTGGG GCCAAGGGAC CACG GTCACC 360 GTCTCCTCAG C TAGCACCAAG GGCCCATCT GTCTTCCCCC TGGCACCCTC CTCCAAGAGC 420 ACCTCTGGGG GCACAGCTGC CCTGGGCTGC CTG GTCAAGG ACTACTTCC CTGAACCTGTG 480 ACAGTGT CCT GGAAC T C AGGAGCCCT GACC AGCGGC GTGC ACACCTTCCC GGCTGTCCTA 540 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTG GT AC C GTGCCCTCC AGC AGCTTGGGC 600 ACCCAGACCT ACATCTGCAA CGTGAATCAC AAGCCCAGCA ACACCAAGGT GGACAAGAAA 660 GTTGAGCCCA AATCTTGTGA CAA AACTCAC ACATGCCCAC C GTGCCCAGC ACCTGAAGCT 720 GCTGGGGGAC C GT C AGTCTT CCTCTTCCCC CCAAACCCA AGGACACCC T CATGATCTCC 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tttacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 81 <211> 639 <212> DNA <213> Artificial <220> <223> SG-559-03 kappa light chain <400> 81 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaacc tgccgacgtt cggccaaggg 300 accaaggtgg aaatcaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 360 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600 agctcgcccg tcacaaagag cttcaacagg ggagagtgt 639 <210> 82 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-03 hlgG1 heavy chain <400> 82 CAGGTCCAGC TG GTGCAGTCT GGGGCTGAGG TGAAGAAGCC TGGGTCCTCG GTGAAGGTC 60 TCCTGCAAGA CTTCTGGAGA CCCTTCAGC ACCTATGCTA TCAGCTGGGT GCGACAGGCC 120 CCTGGACAAG GGCTTGAGTG GATGGGAGGG ATCATCCCTA TTATTGGTAA AGCACACTAC 180 GCACAGAAGT TCCAGGGCAG AGTCACGATT ACCGC GGACGAATCCACGAGCACAGCCTAC 240 ATGGAGCTGA GCAGCCTGAG ATCTGAGGAC ACGGCCGTGT ATTTTTGTGC GAGAAAGTTT 300 C ACTTTGTTT CGGGGAGCCC CTT CGGTATGGACGTCTGGGGCCAAGGGACCACGGTCACC 360 GTCTCCTCAG C TAGCACCAAG GGCCCATCT GTCTTCCCCC TGGCACCCTC CTCCAAGAGC 420 ACCTCTGGGG GCACAGCTGC CCTGGGCTGC CTG GTCAAGGACTACTTCCCT GAACCTGTG 480 ACAGTGT CCT GGAAC T C AGGAGCCCT GACC AGCGGC GTGC ACACCTTCCCGGCTGTCTA 540 CAGTCCTCAG GACTCTACTC CCTCAGCAGC GTG GT AC C GTGCCCTCCAG C AGCTTGGGC 600 ACCCAGACCT ACATCTGCAACGTGAA TCACAAGCCCAGCAACACCAAGGTGGACAAGAAA 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 83 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-04 LALA hlgG1 heavy chain <400> 83 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc acctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt atttttgtgc gagaaagttt 300 cactttgttt cggggagcgg cttcggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag ctagcaccaa gggcccatct gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagctgc cctgggctgc ctggtcaagg actacttccc tgaacctgtg 480 acagtgtcct ggaactcagg agccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 GAGAATAGCA ATGACCAGAC TACATCTGCA ACgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaagct 720 gctgggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tttacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 84 <211> 639 <212> DNA <213> Artificial <220> <223> SG-559-04 kappa light chain <400> 84 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggccgacgtt cggccaaggg 300 accaaggtgg aaatcaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 360 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 420 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 480 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 540 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 600 agctcgcccg tcacaaagag cttcaacagg ggagagtgt 639 <210> 85 <211> 1359 <212> DNA <213> Artificial <220> <223> SG-559-04 hlgG1 heavy chain <400> 85 caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaaga cttctggaga caccttcagc acctatgcta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa agcacactac 180 gcacagaagt tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt atttttgtgc gagaaagttt 300 cactttgttt cggggagcgg cttcggtatg gacgtctggg gccaagggac cacggtcacc 360 gtctcctcag ctagcaccaa gggcccatct gtcttccccc tggcaccctc ctccaagagc 420 acctctgggg gcacagctgc cctgggctgc ctggtcaagg actacttccc tgaacctgtg 480 acagtgtcct ggaactcagg agccctgacc agcggcgtgc acaccttccc ggctgtccta 540 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320 cactacacac agaagagcct ctccctgtct ccgggcaaa 1359 <210> 86 <211> 453 <212> PRT <213> Artificial <220> <223> Ab1 hIgG1 heavy chain <400> 86 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Asp Thr Phe Ser Thr Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gin Gin Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser 435 440 445 Leu Ser Pro Gly Lys 450 <210> 87 <211> 213 <212> PRT <213> Artificial <220> <223> Ab1 kappa light chain <400> 87 Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Leu Leu lie 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly lie Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Thr 85 90 95 Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160 Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human PD-L1 protein, wherein the antibody comprises the heavy chain variable region sequence of SEQ ID NO:11 and the light chain variable region sequence of SEQ ID NO:

12.

2. The antibody according to claim 1, wherein the antibody exhibits a binding affinity for human PD-L1 protein between 3 and 300 nM.

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

4. The antibody according to claim 1, wherein the antibody further exhibits a higher total internalization than the antibody comprising the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:

87.

5. The antibody according to claim 4, wherein the total internalization has an AUC increase between 9% and 155% relative to the AUC of the antibody comprising the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:

87.

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

7. The antibody of claim 1, wherein the antibody further exhibits a higher x50 than that of an antibody comprising the heavy chain of SEQ ID NO:86 and the light chain of SEQ ID NO:

87.

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

9. The antibody of claim 7, wherein the antibody is conjugated with camptothecin, and wherein the x50 in the MDA-MB-231 cell line is between 15 ng / mL and 55 ng / mL.

10. The antibody according to any one of claims 1-9, wherein the antibody comprises the heavy chain of SEQ ID NO:9 and the light chain of SEQ ID NO:

10.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1-9, wherein the fragment is Fab, Fab', F(ab')2, Fab'-SH, Fv, a bispecific antibody, a linear antibody, or a single-chain antibody fragment.

12. The antibody according to any one of claims 1-9, wherein the antibody contains L234A and L235A mutations in the heavy chain of the antibody.

13. The antibody according to any one of claims 1-9, wherein the heavy chain constant region is of the IgG1 isotype.

14. The antibody according to any one of claims 1-9, wherein the antibody is a humanized or chimeric antibody.

15. The antibody according to any one of claims 1-9, wherein the antibody is conjugated to a cytotoxic agent via a linker.

16. The antibody of claim 15, wherein the antibody is conjugated with monomethylaurestatin E (MMAE).

17. The antibody of claim 16, wherein the antibody is conjugated to MMAE via an enzyme-cleavable linker unit.

18. The antibody of claim 17, wherein the enzyme-cleavable adapter unit comprises a Val-Cit adapter.

19. The antibody of claim 15, wherein the antibody is conjugated to MMAE via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10.

20. The antibody according to claim 19, wherein p is 4.

21. The antibody according to claim 19, wherein p is 8.

22. The antibody according to claim 15, wherein the antibody is conjugated with camptothecin.

23. The antibody of claim 22, wherein the antibody is conjugated to camptothecin via an enzyme-cleavable linker unit.

24. The antibody of claim 23, wherein the enzyme-cleavable adapter unit comprises a Val-Lys-Gly adapter.

25. The antibody according to any one of claims 22-24, wherein the antibody is conjugated with camptothecin via a linker to form an antibody-drug conjugate having the following structure: Where Ab represents the antibody and p ranges from 2 to 10.

26. The antibody according to claim 25, wherein p is 4.

27. The antibody according to claim 25, wherein p is 8.

28. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1-27 and a pharmaceutically acceptable excipient.

29. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-27 in the preparation of a medicament for treating a subject with a PD-L1-expressing cancer, wherein said 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.

30. The use according to claim 29, wherein the subject is a human subject.

31. A nucleic acid encoding an antibody according to any one of claims 1-27.

32. A vector comprising the nucleic acid according to claim 31.

33. A host cell comprising the nucleic acid according to claim 32.

34. The host cell according to claim 33, wherein the host cell is a Chinese hamster ovary (CHO) cell.

35. A method for generating an antibody or an antigen-binding fragment thereof that specifically binds to human PD-L1 protein, the method comprising culturing a host cell according to claim 33 or 34 under conditions suitable for generating the antibody.

36. A method for generating an antibody-drug conjugate that specifically binds to human PD-L1 protein, the method comprising culturing a host cell according to claim 33 or 34 under conditions suitable for generating the antibody; and conjugating the antibody to a cytotoxic agent.

37. The method of claim 36, wherein the cytotoxic agent is MMAE or camptothecin.

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