CN111356702B

CN111356702B - anti-PD-L1 antibodies and antigen binding fragments thereof

Info

Publication number: CN111356702B
Application number: CN201880076255.2A
Authority: CN
Inventors: 张正平; 徐宏江; 杨玲; 张喜全; 应树松; 施伟; 赵凯迪; 宋伟; 张颖
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Nanjing Shunxin Pharmaceutical Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Nanjing Shunxin Pharmaceutical Co Ltd
Priority date: 2017-12-06
Filing date: 2018-12-06
Publication date: 2022-07-26
Anticipated expiration: 2038-12-06
Also published as: CN111356702A; WO2019109974A1

Abstract

A humanized anti-PD-L1 antibody and antigen binding fragment thereof are provided, as well as corresponding nucleotides, expression vectors comprising the polynucleotides, cells comprising the expression vectors, pharmaceutical compositions comprising the antibody or antigen binding fragment thereof, useful for reducing tumors or inhibiting tumor cell growth.

Description

anti-PD-L1 antibodies and antigen binding fragments thereof

Technical Field

The present invention relates to the field of isolated monoclonal antibodies, and in particular to antibodies that bind PD-Ll, antigen binding fragments thereof, murine antibodies comprising the CDR regions of said PD-Ll antibodies, chimeric antibodies, humanized antibodies, nucleic acids encoding the same, compositions comprising the same, and methods of using such antibodies and antigen binding fragments.

Background

Tumor immunotherapy is an innovative approach in the field of tumor therapy, and aims to improve the immune system response of patients to tumors to the greatest extent and relieve the inhibition of tumor cells to T cells. The killer T cells are cells which play a main role in killing tumor cells, however, a very complex immune regulation mechanism exists in a tumor microenvironment, the tumor killing activity of the killer T cells is inhibited along with the development of tumors, and the tumors grow and deteriorate continuously. Activation of T cells requires two signals: MHC-antigen peptide presentation signals and co-stimulatory signals. Meanwhile, a co-suppression signal also exists in the process of regulating T cell activation, so that over-activation of T cells is prevented. Among them, one of the most critical signaling pathways is the programmed death receptor 1 (PD-1)/programmed death receptor ligand 1(PD-L1) signaling pathway.

Programmed death receptor ligand 1(PD-L1) is a ligand for programmed death receptor 1 (PD-1). PD-L1 is also known as cluster of differentiation 274(CD274) or B7 homolog 1(B7-H1) and is a type 1 transmembrane protein encoded by the CD274 gene and having a molecular weight of 40 kDa. Both PD-L1 and PD-1 belong to the immunoglobulin superfamily and both consist of two extracellular Ig domains, i.e., an N-terminal V domain and a C-terminal constant domain. PD-L1 binds to its receptor PD-1 present on activated T cells, B cells and myeloid cells to modulate the activation or suppression of immune cells. In various tumors, such as melanoma, lung cancer, bladder cancer and other cancer cells, the expression of PD-L1 is obviously up-regulated. PD-L1 inhibits activation of T cells, production of cytokines and proliferation of T cells by binding to PD-1 on the surface of T cells (Fife et al (2011) Nature Immunology 10: 1185-1193); induction of depletion or anergy in cognate antigen-specific T cells (Hofmeyer et al (2011) Journal of Biomedicine and Biotechnology 2011: 1-9); and inducing apoptosis of effector T cells. Disruption of the PD-L1 gene resulted in an upregulated T cell response and the generation of autoreactive T cells (Latehman et al (2004) PNAS 101: 10691-10696). WO2016061142 discloses that antibody blockade of PD-L1 results in an increase in tumor-infiltrating lymphocytes, an increase in T cell receptor-mediated proliferation, and a decrease in immune evasion by cancer cells; WO2016022630 demonstrates that a class of anti-PD-L1 antibodies and fragments thereof restore the ability of effector T cells to proliferate and/or produce cytokines in the presence of regulatory T cells. Therefore, the blocking of the binding between PD-L1/PD-1 by the PD-L1 antibody is a very effective method in the field of tumor immunotherapy.

The invention provides an antibody which has a brand new sequence, is high in affinity, selectivity and bioactivity and is combined with PD-L1 and an antigen binding fragment thereof.

Disclosure of Invention

In one aspect, the invention provides an isolated antigen binding protein that binds PD-L1, or an antigen binding fragment thereof. In some embodiments, the present invention provides an isolated anti-PD-L1 antibody, or antigen-binding fragment thereof, that binds to human PD-L1 and exhibits superior properties. In some embodiments, the antibody or antigen binding fragment thereof binds to PD-L1 and blocks the binding of PD-1 to PD-L1. In another embodiment, the antibody or antigen-binding fragment thereof is capable of blocking the interaction between cells producing PD-1 and PD-L1, respectively. In another aspect, the invention provides a method of using such antibodies and antigen binding fragments, for example, to treat cancer and/or infectious disease, to increase T cell activation, or to reduce tumor immune escape, to reduce tumor, or to inhibit tumor cell growth in a subject using such antibodies and antigen binding fragments. In yet another aspect, the invention provides polynucleotides encoding such antigen binding proteins (e.g., antibodies) and antigen binding fragments thereof, expression vectors comprising the polynucleotides, recombinant cells comprising the expression vectors, and compositions comprising such antigen binding proteins (e.g., antibodies) and antigen binding fragments thereof, among others.

In one aspect, the invention provides an isolated anti-PD-L1 antibody or antigen-binding fragment thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12. 14-22 and/or SEQ ID NOs: 27. 29-38 or a mutant sequence thereof.

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region selected from the group consisting of SEQ ID NOs: 12. 17-22 and/or SEQ ID NOs: 29-34 or a mutated sequence thereof.

In some embodiments, an anti-PD-L1 antibody or antigen-binding fragment thereof according to the invention comprises an amino acid sequence selected from any one of SEQ ID NOs: 14-16 and/or SEQ ID NOs: 27. 35-38 or a mutated sequence thereof.

In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR1 sequence that is identical to a sequence selected from SEQ ID NOs: 17. 18, 29 or 30 has at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology or at least 99% homology.

In one embodiment, the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR2 sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NO: 12. 31 or 32 has at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In one embodiment, the anti-PD-L1 antibody or fragment thereof comprises a heavy chain HCDR3 sequence that hybridizes to a sequence selected from the group consisting of SEQ ID NOs: 19-22 or 33-34 has at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR1 sequence that is identical to a light chain LCDR1 sequence selected from SEQ ID NOs: 14. 35 or 36 has at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR2 sequence that is identical to a light chain LCDR2 sequence selected from SEQ ID NOs: 15 or 27 has at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In some embodiments, the anti-PD-L1 antibody or fragment thereof comprises a light chain LCDR3 sequence that is identical to a light chain LCDR3 sequence selected from SEQ ID NOs: 16. 37 or 38 has at least 80% homology, at least 81% homology, at least 82% homology, at least 83% homology, at least 84% homology, at least 85% homology, at least 86% homology, at least 87% homology, at least 88% homology, at least 89% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain HCDR1, HCDR2, and HCDR3, the heavy chain HCDR1 comprising an amino acid sequence identical to SEQ ID NO: 17. 18, 29 or 30, said heavy chain HCDR2 comprising a sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 12. 31 or 32 and said heavy chain HCDR3 comprises a sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 19-22 or 33-34, or a sequence having at least 80% homology in amino acid sequence; and a light chain LCDR1, LCDR2, and LCDR3, the light chain LCDR1 comprising a sequence identical to SEQ ID NO: 14. 35 or 36, and said light chain LCDR2 comprises a sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 15 or 27 and said light chain LCDR3 comprises a sequence having at least 80% homology to the amino acid sequence of SEQ ID NO: 16. 37 or 38, or a sequence having at least 80% homology thereto.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chains HCDR1, HCDR2 and HCDR3 and light chains LCDR1, LCDR2 and LCDR3, the heavy chains HCDR1, HCDR2 and HCDR3 and light chains LCDR1, LCDR2 and LCDR3 are selected from the following sequences or sequences having at least 80% homology thereto:

HCDR1 is selected from: SDYWX ₁ SEQ ID NO：11

Or X ₃ YWMN SEQ ID NO：23

HCDR2 is selected from: YISYTGSTYY NPSLKS SEQ ID NO: 12

Or MIHPSDSETX ₄ LNQKFKD SEQ ID NO：24

HCDR3 is selected from: GEAWDRRTLDX ₂ SEQ ID NO：13

Or X ₅ MVWX ₆ RX ₇ VMDY SEQ ID NO：25

LCDR1 is selected from: RASQDITNYLN SEQ ID NO:14

Or KSSQSLLDSDGX ₈ TYLN SEQ ID NO：26

LCDR2 is selected from: YTSRLHS SEQ ID NO: 15

Or LVSKLDS SEQ ID NO: 27

LCDR3 is selected from: QQVYTLPWT SEQ ID NO: 16

Or LQX ₉ THFPYT SEQ ID NO：28；

Wherein X ₁ Selected from N or S, X ₂ Selected from Y, C, A or V, X ₃ Selected from S or F, X ₄ Selected from R or W, X ₅ Selected from S or N, X ₆ Selected from T or S, X ₇ Selected from Q or H, X ₈ Selected from E or N, X ₉ Is selected from S or A.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain HCDR1, HCDR2, and HCDR 3; the heavy chain HCDR1 sequence selected from SEQ ID NO: 17. 18, 29 or 30; the heavy chain HCDR2 sequence selected from SEQ ID NO: 12. 31 or 32; the heavy chain HCDR3 sequence selected from SEQ ID NO: 19-22 or 33-34. In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a light chain LCDR1, LCDR2, and LCDR 3; the light chain LCDR1 sequence selected from SEQ ID NO: 14. 35 or 36; the light chain LCDR2 sequence selected from SEQ ID NO: 15 or 27; the light chain LCDR3 sequence selected from SEQ ID NO: 16. 37 or 38, or a pharmaceutically acceptable salt thereof.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain HCDR1, HCDR2, and HCDR3, the heavy chain HCDR1 selected from the group consisting of SEQ ID NOs: 17 or 18 or a sequence which is at least 80% homologous thereto, and said heavy chain HCDR2 is selected from the group consisting of SEQ ID NOs: 12 or a sequence thereof which is at least 80% homologous thereto, and the heavy chain HCDR3 is selected from the group consisting of SEQ ID NOs: 19-22 or a sequence at least 80% homologous thereto; and light chains LCDR1, LCDR2 and LCDR3, said light chains LCDR1, LCDR2 and LCDR3 being selected from SEQ ID NOs: 14. 15 and 16 or a sequence which is at least 80% homologous thereto.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain HCDR1, HCDR2, and HCDR3, the heavy chain HCDR1 selected from the group consisting of SEQ ID NOs: 29 or 30 or a sequence thereof which is at least 80% homologous thereto, and said heavy chain HCDR2 is selected from the group consisting of SEQ ID NOs: 31 or 32 or a sequence which is at least 80% homologous thereto, and said heavy chain HCDR3 is selected from the group consisting of SEQ ID NOs: 33 or 34 or a sequence at least 80% homologous thereto; and a light chain LCDR1, LCDR2, and LCDR3, the light chain LCDR1 selected from the group consisting of SEQ ID NO: 35 or 36 or a sequence thereof which is at least 80% homologous thereto, and the light chain LCDR2 is selected from the group consisting of SEQ ID NOs: 27 or a sequence at least 80% homologous thereto, and said light chain LCDR3 is selected from the group consisting of SEQ ID NOs: 37 or 38 or a sequence which is at least 80% homologous thereto.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2, and HCDR3, which heavy chain HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence that differs from the amino acid sequence according to SEQ ID NOs: 17. 12 and 19, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and light chains LCDR1, LCDR2 and LCDR3, said light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain variable region substantially identical to the light chain variable region according to SEQ ID NOs: 14. 15 and 16, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 17. 12 and 19, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 14. 15 and 16, LCDR1, LCDR2, and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2 and HCDR3, the heavy chain HCDR1, HCDR2 and HCDR3 comprising an amino acid sequence identical to a sequence according to SEQ ID NOs: 18. 12 and 20, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain LCDR1, LCDR2 and LCDR3 which respectively have a heavy chain identity to a light chain LCDR according to SEQ ID NOs: 14. 15 and 16, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 18. 12 and 20, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 14. 15 and 16, LCDR1, LCDR2, and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2, and HCDR3, the heavy chain HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence identical to a sequence according to SEQ ID NOs: 17. 12 and 20, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and light chains LCDR1, LCDR2 and LCDR3, said light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain variable region substantially identical to the light chain variable region according to SEQ ID NOs: 14. 15 and 16, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 17. 12 and 20, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 14. 15 and 16, LCDR1, LCDR2, and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2, and HCDR3, which heavy chain HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence that differs from the amino acid sequence according to SEQ ID NOs: 17. 12 and 21, an amino acid sequence having at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain LCDR1, LCDR2 and LCDR3 which respectively have a heavy chain identity to a light chain LCDR according to SEQ ID NOs: 14. 15 and 16, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 17. 12 and 21, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 14. 15 and 16, LCDR1, LCDR2, and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence identical to the sequence according to SEQ ID NOs: 17. 12 and 22, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and light chains LCDR1, LCDR2 and LCDR3, said light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain variable region substantially identical to the light chain variable region according to SEQ ID NOs: 14. 15 and 16, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 17. 12 and 22, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 14. 15 and 16, LCDR1, LCDR2 and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2, and HCDR3, the heavy chain HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence identical to a sequence according to SEQ ID NOs: 29. 31 and 33, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and light chains LCDR1, LCDR2 and LCDR3, said light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain variable region substantially identical to the light chain variable region according to SEQ ID NOs: 35. 27 and 37, an amino acid sequence having at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 29. 31 and 33, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 35. 27 and 37, LCDR1, LCDR2, and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises heavy chain HCDR1, HCDR2, and HCDR3, the heavy chain HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence identical to a sequence according to SEQ ID NOs: 30. 32 and 34, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; and light chains LCDR1, LCDR2 and LCDR3 comprising a heavy chain LCDR1, LCDR2 and LCDR3 which respectively have a heavy chain identity to a light chain LCDR according to SEQ ID NOs: 36. 27 and 38, an amino acid sequence having at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In another embodiment, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises an amino acid sequence according to SEQ ID NOs: 30. 32 and 34, and HCDR1, HCDR2 and HCDR3 according to SEQ ID NOs: 36. 27 and 38, LCDR1, LCDR2, and LCDR 3.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence identical to a sequence selected from the group consisting of seq ID NOs: 5. 7, 9, 45, 47, 48, 49 and 50, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and a light chain variable region comprising a heavy chain variable region substantially identical to a light chain variable region selected from the group consisting of SEQ ID NOs: 6. 8, 10, 46, 51 and 52, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 91% homologous, at least 92% homologous, at least 93% homologous, at least 94% homologous, at least 95% homologous, at least 96% homologous, at least 97% homologous, at least 98% homologous, or at least 99% homologous. In other embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof comprises, consists essentially of, or consists of a heavy chain variable region comprising, consisting of seq id no: selected from the group consisting of SEQ ID NOs: 5. 7, 9, 45, 47, 48, 49 and 50; and a light chain variable region comprising, consisting essentially of, or consisting of the sequence of seq id no: selected from the group consisting of SEQ ID NOs: 6. 8, 10, 46, 51 and 52.

In some embodiments, the invention provides an anti-PD-L1 antibody or antigen-binding fragment thereof comprising a heavy chain variable region comprising SEQ ID NO:5 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 6; comprises the amino acid sequence shown in SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8; comprises the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10, or a light chain variable region of the amino acid sequence of seq id No. 10.

In one embodiment, the invention provides a humanized anti-PD-L1 antibody comprising a variable heavy chain of an antibody selected from the group consisting of hu-Chi1-2.3, hu-Chi1-3.3, hu-Chi1-3.4, hu-Chi1-3.5 and hu-Chi1-3.6 and a variable light chain of an antibody selected from the group consisting of hu-Chi1-2.3, hu-Chi1-3.3, hu-Chi1-3.4, hu-Chi1-3.5 and hu-Chi 1-3.6. In some embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region having an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 45. 47, 48, 49 and 50, at least 80% homologous, at least 85% homologous, at least 90% homologous, at least 91% homologous, at least 92% homologous, at least 93% homologous, at least 94% homologous, at least 95% homologous, at least 96% homologous, at least 97% homologous, at least 98% homologous, or at least 99% homologous. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a light chain variable region having an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 46. 51 and 52, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous thereto.

In some embodiments, the present invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that is substantially identical to SEQ ID NO:45 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region that is substantially identical to SEQ ID NO:46 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In other embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that differs from the heavy chain variable region of SEQ ID NO:47 has at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region that is identical to SEQ ID NO:51 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In other embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that hybridizes to SEQ ID NO:48 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region that is identical to SEQ ID NO:52 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In other embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that hybridizes to SEQ ID NO:49 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region that is substantially identical to SEQ ID NO:51 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology. In some embodiments, the present invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises a heavy chain variable region that hybridizes to SEQ ID NO:50 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology; and a light chain variable region that is substantially identical to SEQ ID NO:51 have at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In some embodiments, the invention provides a humanized anti-PD-L1 antibody comprising a heavy chain variable region comprising SEQ ID NO:45 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 46; or comprises SEQ ID NO:47 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51; or comprises SEQ ID NO:48 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52; or comprises SEQ ID NO:49 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51; or a polypeptide comprising SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 51.

In some embodiments, the invention provides chimeric anti-PD-L1 antibodies, wherein the antibodies comprise a heavy chain having an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 39. 41 and 43, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous; and a light chain having an amino acid sequence substantially identical to a sequence selected from the group consisting of SEQ ID NOs: 40. 42 and 44, or a pharmaceutically acceptable salt thereof, having at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology.

In some embodiments, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an intact heavy chain having an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 53. 57, 61 and 62, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous thereto. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an intact light chain having an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 55 and 59, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homologous thereto.

In one embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an amino acid sequence according to SEQ ID NO:53 and a heavy chain according to SEQ ID NO:55, light chain. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an amino acid sequence according to SEQ ID NO:57 and a heavy chain according to SEQ ID NO:59, and a light chain. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an amino acid sequence according to SEQ ID NO:61 and a heavy chain according to SEQ ID NO:55, and a light chain. In another embodiment, the invention provides a humanized anti-PD-L1 antibody, wherein the antibody comprises an amino acid sequence according to SEQ ID NO:62 and a heavy chain according to SEQ ID NO:55, and a light chain.

In some embodiments, the invention provides a humanized anti-PD-L1 antibody or fragment thereof, preferably a humanized antibody hu-Chi1 having heavy chain FR region sequences from a human germline heavy chain, specifically from a combined sequence of human germline heavy chain IGHV4-30-4 '01 and IGHJ 1' 01. In some embodiments, the heavy chain variable region sequence of the humanized antibody hu-Chi1 has a back mutation of 0 to 10 amino acids, preferably one or more back mutations of amino acids selected from S35N, Q39R, K43N, G44K, W47Y, I48M, V71R; more preferably one or more amino acid back mutations selected from S35N, W47Y and V71R; most preferred are back mutations comprising S35N, W47Y, V71R. In some embodiments, the humanized antibody hu-Chi1 heavy chain variable region further comprises an amino acid mutation of C102Y. In some embodiments, the light chain FR region sequences on the humanized antibody hu-Chi1 light chain variable region are derived from a human germline light chain, specifically from a combined sequence of human germline light chains IGKV1-39 × 01 and IGKJ2 × 02. In some embodiments, the light chain variable region sequence of humanized antibody hu-Chi1 has a back mutation of 0-10 amino acids, preferably one or more back mutations of amino acids selected from the group consisting of I2F, P8T, G41D, P44V, Y87F, and Q100R; more preferably one or more amino acid back mutations selected from the group consisting of I2F, G41D and P44V; most preferred are amino acid back mutations comprising I2F, G41D, and P44V.

In one embodiment, the invention provides an anti-PD-L1 antibody or fragment thereof that binds to the same epitope on PD-L1 as any of the exemplary antibodies provided herein. In one embodiment, the antibody or fragment thereof competes with any of the exemplary antibodies provided herein for binding to PD-L1. Binding to PD-L1 can be measured by ELISA, flow cytometry, Surface Plasmon Resonance (SPR) assay, or any other method known in the art. In some embodiments of the invention, the PD-L1 antibody or fragment thereof binds to human PD-L1 protein and binds to cynomolgus monkey (cynomolgous) PD-L1 protein.

In some embodiments, the invention provides a binding affinity K that is between about 15nM and about 0.05nM _D anti-PD-L1 antibodies and fragments thereof that bind to PD-L1. In another embodiment, the present inventionThe anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 10nM to about 0.1nM _D Combined with PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 8nM to about 0.2nM _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 6nM to about 0.3nM _D Combined with PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 15nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein bind with a binding affinity, K, of about 10nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 6nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 4nM or less _D Combined with PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 1nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 0.70nM or less _D Combined with PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 0.50nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 0.30nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 0.10nM or less _D Binds to PD-L1. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein have a binding affinity, K, of about 0.05nM or less _D Binds to PD-L1. In some embodiments, the binding affinity, K, of the anti-PD-L1 antibodies and fragments provided herein is measured by a Biacore assay _D 。

In some embodiments, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein IC50 is about 1ng/mL to about 1500 ng/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein IC50 is about 10ng/mL to about 1200 ng/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein IC50 is about 20ng/mL to about 800 ng/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein IC50 is about 50ng/mL to about 500 ng/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the binding of PD-L1/PD-1, wherein IC50 is about 50ng/mL to about 200 ng/mL. In some embodiments, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit binding of PD-L1/PD-1, wherein IC50 is about 1200ng/mL or less, about 1000ng/mL or less, about 800ng/mL or less, about 400ng/mL or less, about 300ng/mL or less, about 250ng/mL or less, about 200ng/mL or less, about 150ng/mL or less, about 100ng/mL or less, about 75ng/mL or less, about 60ng/mL or less, about 50ng/mL or less, about 40ng/mL or less, about 30ng/mL or less, about 20ng/mL or less, or about 10ng/mL or less. In some embodiments, the IC50 of the anti-PD-L1 antibodies and fragments provided herein is measured by ELISA.

In other embodiments, provided herein are anti-PD-L1 antibodies and fragments thereof that inhibit interactions between cells expressing PD-L1 and PD-1 proteins, respectively, on their surfaces. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the interaction between cells expressing PD-L1 and PD-1 protein, respectively, on their surfaces, wherein IC50 is about 1ng/mL to about 500 ng/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the interaction between cells expressing PD-L1 and PD-1 proteins, respectively, on their surfaces, wherein IC50 is about 10ng/mL to about 400 g/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the interaction between cells that express PD-L1 and PD-1 proteins, respectively, on their surfaces, wherein IC50 is about 20ng/mL to about 300 ng/mL. In another embodiment, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the interaction between cells that express PD-L1 and PD-1 proteins, respectively, on their surfaces, wherein IC50 is about 50ng/mL to about 200 ng/mL. In some embodiments, the anti-PD-L1 antibodies and fragments thereof provided herein inhibit the interaction between cells that express PD-L1 and PD-1 proteins, respectively, on their surfaces, wherein IC50 is about 500ng/mL or less, about 450ng/mL or less, about 400ng/mL or less, about 350ng/mL or less, about 300ng/mL or less, about 250ng/mL or less, about 200ng/mL or less, about 150ng/mL or less, about 100ng/mL or less, or about 50ng/mL or less. In some embodiments, IC50 of the anti-PD-L1 antibodies and fragments thereof provided herein is measured by FACS.

In one embodiment, the anti-PD-L1 antibody provided herein is a chimeric antibody having an amino acid sequence according to SEQ ID NO:5 and the heavy chain variable region according to the amino acid sequence of SEQ ID NO: 6; or has an amino acid sequence according to SEQ ID NO: 7 and a heavy chain variable region according to the amino acid sequence of SEQ ID NO: 8; or has a sequence according to seq id NO: 9 and the heavy chain variable region according to the amino acid sequence of SEQ ID NO: 10, a light chain variable region of the amino acid sequence of seq id no; wherein the anti-PD-L1 antibody has the following PD-L1 binding EC50 as measured by ELISA or FACS: about 200ng/mL or less, or about 150ng/mL or less, or about 100ng/mL or less, or about 80ng/mL or less, or about 60ng/mL or less, or about 50ng/mL or less.

In some embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof of the invention is a chimeric antibody or antibody fragment. An anti-PD-L1 chimeric antibody or fragment thereof provided according to the invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, or a variant thereof. The humanized antibody light chain further comprises a constant region of a human kappa, lambda chain or variant thereof. Thus, in other embodiments, the anti-PD-L1 antibody or antigen-binding fragment thereof is a humanized antibody or antibody fragment.

In one embodiment, the anti-PD-L1 antibody or fragment thereof is a monoclonal antibody, scFv, Fab fragment, Fv fragment, F (ab ') ' fragment, F (ab ')2 fragment, bispecific antibody, immunoconjugate, or a combination thereof.

In one aspect, an isolated anti-PD-L1 antibody or fragment thereof is provided, wherein the antibody is produced by a hybridoma selected from the group consisting of the hybridomas designated herein as Chi1, Chi2-1, and Chi 2-2. Accordingly, the invention also includes hybridomas Chi1, Chi2-1, and Chi2-2, as well as any hybridomas that produce the antibodies disclosed herein. The invention also provides isolated polynucleotides encoding the antibodies and fragments thereof provided herein, e.g., the nucleotide sequence encoding the heavy chain of humanized antibody hu-Chi1-3.4 is set forth in SEQ ID NO: 54, and the nucleotide sequence of the coding light chain is shown as SEQ ID NO: 56; the nucleotide sequence of the heavy chain of the encoding humanized antibody hu-Chi1-2.3 is shown as SEQ ID NO: 58 and the nucleotide sequence of the coding light chain is shown as SEQ ID NO: shown at 60. In another aspect, the present invention provides expression vectors, such as the pMD-19T vector and pcDNA3.1 vector, among others, comprising the isolated polynucleotides. In a particular embodiment, the invention provides an expression vector comprising a nucleotide sequence encoding a humanized anti-PD-L1 antibody, for example, an expression vector encoding hu-Chi1-2.3 heavy and light chain nucleotide sequences. The present invention also provides host cells comprising the expression vector, which may be eukaryotic or prokaryotic cells, for example, the host cells include but are not limited to mammalian cells, insect cells, yeast cells, bacteria, and the like, and may preferably be selected from Chinese Hamster Ovary (CHO) cells, ExpiCHO cells, CHO-S cells, and CHO-DG44 cells, pichia pastoris, escherichia coli, and the like. In one embodiment, the present invention provides a method of producing the humanized anti-PD-L1 antibody or fragment thereof, the method comprising culturing a host cell comprising the expression vector.

In one embodiment, the invention provides anti-PD-L1 antibody immunoconjugates. Accordingly, the present invention provides antibodies or fragments thereof that bind to PD-L1 and are linked or conjugated to a therapeutic agent. Therapeutic agents that may be linked or conjugated to the anti-PD-L1 antibody may include, but are not limited to, cytotoxic drugs, radioisotopes, signaling pathway inhibitors, immunomodulators or antibodies. In one embodiment, the antibodies or fragments thereof provided herein are immunoconjugates comprising an anti-PD-L1 antibody or fragment thereof, and further comprising an antibody selected from the group consisting ofAgents from the group of therapeutic agents, cytotoxic agents, immunoadhesion molecules and imaging agents. In some embodiments, the imaging agent is selected from the group consisting of a radiolabel, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, and biotin. In some embodiments, the imaging agent is a radiolabel selected from the group consisting of: ³ H、 ¹⁴ C、 ³⁵ S、 ⁶² Cu、 ⁶⁴ Cu、 ⁸⁹ Zr、 ⁹⁰ Y、 ⁹⁹ Tc、 ¹¹¹ In、 ¹²⁵ I、 ¹³¹ I、 ¹⁷⁷ Lu、 ¹⁶⁶ ho and ¹⁵³ sm. In some embodiments, the therapeutic or cytotoxic agent is selected from the group comprising: chemotherapy, immunosuppressive agents, immunostimulating agents, antimetabolites, alkylating agents, antibiotics, growth factors, cytokines, anti-angiogenic agents, anti-mitotic agents, anthracyclines, toxins, and apoptotic agents. In some embodiments, the antigen binding protein is conjugated directly to the agent. In other embodiments, the antigen binding protein is conjugated to the agent through a linker. Suitable linkers include, but are not limited to, amino acid and polypeptide linkers reported in the literature. The linker may be cleavable or non-cleavable.

In one embodiment, the invention provides bispecific or multispecific antibodies specific for PD-L1 and at least one other antigen or epitope. The anti-PD-L1 antibodies and fragments thereof provided herein can be tested for binding to PD-L1 using the binding assays provided herein or any other binding assay known in the art.

In one aspect, the invention provides a composition comprising one or more anti-PD-L1 antibodies or fragments thereof provided herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable stabilizer, buffer, or excipient.

In one aspect, the invention provides a method for modulating an immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof provided herein. In one embodiment, the invention provides a method for increasing T cell activation, comprising contacting a T cell with an anti-PD-L1 antibody or fragment thereof provided herein. In one embodiment, the invention provides a method for treating or preventing a disease or disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof provided herein. In one embodiment, the invention provides a method for enhancing an anti-tumor response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention. In another embodiment, the invention provides a method for reducing tumor immune escape, reducing a tumor, or inhibiting the growth of tumor cells in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention. In another embodiment, the invention provides a method for treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the invention. In another embodiment, the cancer is selected from the group consisting of: lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, intestinal cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer (stomach cancer), testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head or neck cancer, stomach cancer (gastic cancer), pancreatic cancer, or a combination thereof.

In some embodiments, the present invention provides a method for treating an infectious disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-PD-L1 antibody or fragment thereof of the present invention. In another embodiment, the infectious disease is selected from the group consisting of: candidiasis, candidemia, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal conditions, gram-negative sepsis, tuberculosis, mononucleosis, influenza, respiratory diseases caused by respiratory syncytial virus, malaria, schistosomiasis and trypanosomiasis.

In some embodiments, the antibodies and fragments thereof provided herein are useful for treating diseases mediated by T helper type 2(Th2) T cells, such as, for example, asthma, allergy, or graft-versus-host disease.

In some embodiments, the antibodies and fragments thereof provided herein can be used to stimulate an immune response in a subject in need thereof. For example, in some embodiments, anti-PD-L1 antibodies and fragments thereof can be administered with an antigen of interest for use in eliciting an immune response to the antigen. The antigen of interest may be an antigen associated with a pathogen, such as a virus or a bacterium. Thus, in some embodiments, the invention provides a vaccine comprising an anti-PD-L1 antibody and an antigen, wherein the vaccine elicits an antigen-specific immune response.

In some embodiments, the antibodies and fragments thereof disclosed herein can be administered to a subject in need thereof in combination with one or more additional therapeutic agents. In some embodiments, the antibodies and fragments thereof can be administered to the subject before, during, and/or after the administration of the additional therapeutic agent to the subject. In one embodiment, the additional therapeutic agent is a chemotherapeutic agent, a radiotherapeutic agent, a cytokine, an antibody or fragment thereof, or any other additional therapeutic agent indicative of the disease to be treated. In some embodiments, the anti-PD-L1 antibody and the additional therapeutic agent exhibit therapeutic synergy when administered together, whether simultaneously or sequentially. In some embodiments, the anti-PD-L1 antibody and the additional therapeutic agent are administered in separate formulations. In another embodiment, the anti-PD-L1 antibody and the additional therapeutic agent are administered in the same formulation. In one embodiment, the anti-PD-L1 antibodies and fragments provided herein enhance the immunomodulatory effect of one or more additional therapeutic agents. In other embodiments, one or more additional therapeutic agents potentiate the effect of an anti-PD-L1 antibody or fragment thereof.

The invention provides isolated antibodies and antigen-binding fragments thereof, as well as nucleic acids encoding the antibodies and fragments, and compositions comprising the isolated antibodies, fragments, or nucleic acids. The term "isolated" refers to a compound of interest (e.g., an antibody or nucleic acid) that has been isolated from its natural environment. The invention also provides pharmaceutical compositions comprising an isolated antibody or fragment thereof, or a nucleic acid encoding the antibody or fragment, and further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.

As used herein, the term "antibody" refers to an antigen binding protein having at least one antigen binding domain. The antibodies and fragments thereof of the present invention may be whole antibodies or any fragment thereof. Thus, the antibodies and antigen-binding fragments of the invention include monoclonal antibodies or fragments thereof and antibody variants or fragments thereof, as well as immunoconjugates. Examples of antibody fragments include Fab fragments, Fab 'fragments, F (ab')2 fragments, Fv fragments, isolated CDR regions, single chain Fv molecules (scFv), and other antibody fragments known in the art. Antibodies and fragments thereof can also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The anti-PD-L1 antibodies and fragments thereof disclosed herein can be of the IgG1, IgG2, IgG3, or IgG4 isotype. The term "isotype" refers to the class of antibodies encoded by the heavy chain constant region gene. In one embodiment, the anti-PD-L1 antibodies and fragments thereof disclosed herein are of the IgG1 or IgG4 isotype. The PD-L1 antibodies and fragments thereof of the present invention may be derived from any species, including but not limited to mouse, rat, rabbit, primate, llama, and human. The anti-PD-L1 antibodies and fragments thereof can be chimeric, humanized, or fully human antibodies. In some embodiments, the anti-PD-L1 antibody is an antibody produced by a mouse-derived hybridoma cell line. Thus, in some embodiments, the anti-PD-L1 antibody is a murine antibody. In other embodiments, the anti-PD-L1 antibody is a chimeric antibody. In other embodiments, the chimeric antibody is a mouse-human chimeric antibody. In other embodiments, the antibody is a humanized antibody. In other embodiments, the antibody is derived from a murine antibody and is humanized.

A "Fab fragment" consists of the CH1 domains and the variable regions of one light and one heavy chain. The heavy chain of a Fab molecule is unable to form a disulfide bond with another heavy chain molecule.

A "Fab ' fragment" contains a portion of one light chain and one heavy chain comprising the VH domain and the CH1 domain and the region between the CHI domain and the CH2 domain, whereby an interchain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form a F (ab ')2 molecule.

An "F (ab')2 fragment" contains two light chains and two heavy chains comprising part of the constant region between the CH1 domain and the CH2 domain, whereby an interchain disulfide bond is formed between the two heavy chains. Thus, a F (ab ')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The "Fv region" comprises variable regions from both the heavy and light chains, but lacks a constant region.

"Single chain Fv antibody" (or "scFv antibody") refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, Fv polypeptides additionally comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For an overview OF scFv, see Pluckthun (1994) THE PHARMACOLOGY OF MONOCLONAL ANTIBODIES (MABIAL), Vol.113, eds. Rosenburg and Moore, Springer-Verlag, NewYork, p.269-315. See also international patent application publication No. WO88/01649 and U.S. patent nos. 4,946,778 and 5,260,203. The contents of the above documents are incorporated herein by reference.

"diabodies", also known as "bispecific antibodies", are small antibody fragments with two antigen-binding sites. The fragments comprise a heavy chain variable domain (VH) (VH-VL or VL-VH) linked to a light chain variable domain (VL) in the same polypeptide chain. By using linkers that are too short to pair between two domains of the same chain, the domains are forced to pair with complementary domains of another chain and form two antigen binding sites.

"chimeric antibody" is an antibody that: the antibody has at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species; and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody can comprise a murine variable region and a human constant region.

"humanized antibodies" are the following antibodies: the antibody comprises Complementarity Determining Regions (CDRs) derived from a non-human antibody; and framework regions and constant regions derived from human antibodies. For example, an anti-PD-L1 antibody provided herein can comprise CDRs derived from one or more murine antibodies as well as human framework and constant regions. Thus, in one embodiment, the humanized antibodies provided herein bind to the same epitope on PD-L1 as the murine antibody from which the CDRs of the antibody are derived. Exemplary humanized antibodies are provided herein. Additional anti-PD-L1 antibodies or variants thereof comprising the heavy and light chain CDRs provided herein can be produced using any human framework sequence and are also included in the invention. In one embodiment, suitable for use in the invention of the framework sequences include the structure and the provision of framework sequences similar to those of the framework sequences. Additional modifications may be made in the framework regions to improve the properties of the antibodies provided herein. Such additional framework modifications may include chemical modifications; point mutations to reduce immunogenicity or to remove T cell epitopes; or reverting the mutation to a residue in the original germline sequence. In some embodiments, such modifications include those corresponding to the mutations exemplified herein, including back mutations to germline sequences. For example, in one embodiment, one or more amino acids in the human framework regions of the VH and/or VL of the humanized antibodies provided herein are back mutated to the corresponding amino acids in the parent murine antibody. For example, for the VH and VL of humanized hu-Chi, several positions of the framework amino acids of the template human antibody described above were back mutated to the corresponding amino acid sequences in the mouse Chi antibody. In one embodiment, the amino acids at positions 35 and/or 39 and/or 43 and/or 44 and/or 48 and/or 71 of the heavy chain variable region of humanized antibody hu-Chi1 are back mutated to the corresponding amino acids found at those positions in mouse Chi1 heavy chain variable region. In another embodiment, the amino acid at position 2 and/or 8 and/or 41 and/or 44 and/or 87 and/or 100 of the light chain variable region is back mutated to the corresponding amino acid found at that position in the mouse Chi1 light chain variable region. And amino acids of S35N, Q39R, K43N, G44K, W47Y, I48M and V71R are subjected to back mutation to be corresponding amino acids in the corresponding mouse Chi antibody. In one embodiment, the humanized hu-Chi1-3.4 antibody comprises a heavy chain variable region wherein the amino acid at position 47 is mutated from trp (w) to try (y), the amino acid at position 35 is mutated from ser(s) to asn (n), and the amino acid at position 71 is mutated from val (v) to arg (r); and a light chain variable region wherein the amino acid at position 2 is mutated from ile (i) to phe (f), the amino acid at position 41 is mutated from gly (g) to asp (d), and the amino acid at position 44 is mutated from pro (p) to val (v). Additional or alternative back mutations can be made in the framework regions of the humanized antibodies provided herein to improve the properties of the antibody. The invention also includes humanized antibodies that bind PD-L1 and comprise framework modifications corresponding to the exemplary modifications described herein relative to any suitable framework sequence, as well as other framework modifications that otherwise improve the antibody properties, e.g., the amino acid at position 102 of the humanized antibody hu-Chi1 heavy chain variable region is mutated from cys (c) to try (y), ala (a), or val (v).

The terms "human PD-L1", "hPD-L1", and "hu-PD-L1", and the like, are used interchangeably herein and refer to variants or isoforms of human PD-L1 and human PD-L1. By "specific to" is meant that the antibody and fragments thereof bind PD-L1 with greater affinity than any other target. As used herein, the term "EC 50" refers to the effective concentration, 50% of the maximal response of an antibody. As used herein, the term "IC 50" refers to the inhibitory concentration, 50% of the maximal response of an antibody. Both EC50 and IC50 may be measured by ELISA or FACS analysis or any other method known in the art.

The anti-PD-L1 antibodies disclosed herein having one or more amino acid substitutions, insertions, deletions, or combinations thereof in the CDR or light chain variable region or heavy chain variable region retain the biological activity of the corresponding anti-PD-L1 antibody without amino acid substitutions, insertions, or deletions. Thus, the variant anti-PD-L1 antibodies provided herein retain binding to PD-L1. As used herein, percent homology refers to the number of identical amino acid sequences that are common to two reference sequences divided by the total number of amino acid positions, multiplied by 100. In some embodiments, the CDR or light chain variable region or heavy chain variable region amino acid sequence variant, wherein the variant comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more amino acid substitutions, insertions, or deletions, or combinations thereof. In another embodiment, the amino acid substitution is a conservative substitution.

In one aspect, the invention provides methods for treating a disease or condition in a subject that responds to an enhanced, stimulated or elicited immune response. As used herein, the term "treatment" refers to both therapeutic treatment as well as preventative or prophylactic measures. Subjects in need of treatment include those already with the disease or condition, as well as those who may have the disease or condition and whose purpose is to prevent, delay or attenuate the disease or condition. As used herein, the term "subject" means a mammal, such as a rodent, feline, canine, and primate. Preferably, the subject according to the invention is a human.

As used herein, the term "therapeutically effective amount" refers to the amount of a compound or composition necessary to provide a therapeutic and/or prophylactic benefit to a subject.

The terms "mutant sequence" and "mutant sequence" are used interchangeably herein to refer to a sequence that has been altered by substitution, deletion, or insertion, and the "mutant sequence" and "mutant sequence" may have more than 60% homology, for example more than 70% homology, further for example more than 80% homology to the sequence before mutation.

The use of the singular includes the plural unless specifically stated otherwise. The words "a" or "an" mean "at least one" unless specifically stated otherwise. The use of "or" means "and/or" unless stated otherwise. The meaning of the phrase "at least one" is equivalent to the meaning of the phrase "one or more". Furthermore, the use of the term "including/comprising/containing" as well as other forms such as "includes/comprising/containing" and "includes/containing" is not limiting. In addition, terms such as "element" or "component" include both elements or components that comprise one unit and elements and components that comprise more than one unit unless specifically stated otherwise.

The term "about" as used herein means that a numerical value is within an acceptable error range for the particular value determined by one of ordinary skill in the art, which numerical value depends in part on how it is measured or determined (i.e., the limits of the measurement system). For example, "about" in each practice in the art may mean within 1 or over 1 standard deviation. Alternatively, "about" or "substantially comprising" may mean a range of up to 20%. Furthermore, particularly for biological systems or processes, the term may mean at most an order of magnitude or at most 5 times the value. Unless otherwise indicated, when a particular value appears in the application and claims, the meaning of "about" or "consisting essentially of" should be assumed to be within an acceptable error range for that particular value.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily identify a variety of noncritical parameters that may be changed or modified to produce substantially similar results.

Drawings

FIG. 1: detecting binding of the chimeric anti-PD-L1 antibody to PD-L1 by ELISA over a range of concentrations;

FIG. 2: detecting the blocking effect of the chimeric anti-PD-L1 antibody on the binding of PD-1/PD-L1 in a certain antibody concentration range by ELISA;

FIG. 3: detecting the blocking effect of the chimeric anti-PD-L1 antibody on the binding of PD-1/PD-L1 in a certain antibody concentration range by FACS;

FIG. 4 is a schematic view of: the blocking effect of the chimeric anti-PD-L1 antibody on the binding of PD-1/PD-L1 in a certain antibody concentration range is detected through cell activity;

FIG. 5: blocking of binding of PD-1/PD-L1 by the humanized anti-PD-L1 antibody was examined by cellular activity over a range of antibody concentrations.

Detailed Description

Example 1PD-L1 antigen and detection protein preparation

A human PD-L1 full-length gene (HG 10084-M) of Unit Programmed Cell Death 1 Ligand 1(PD-L1) isotype 1(SEQ ID NO: 1) is used as a template of PD-L1 to obtain a gene sequence for encoding the antigen and the protein for detection, and the PD-L1 extracellular Domain (ECD) can be selected to be recombined and connected with an antibody heavy chain Fc fragment (such as mouse IgG2a) to form PD-L1-mFc or recombined and connected with His Tag to form PD-L1-His for immunization or later screening detection of mice. cDNAs encoding PD-L1 extracellular domain recombinant protein (hPD-L1(ECD) -mFc, SEQ ID NO: 2) containing a mouse antibody heavy chain Fc tag and PD-L1 extracellular domain recombinant protein (hPD-L1-His, SEQ ID NO: 3) containing a histidine tag, and PD-1 extracellular domain recombinant protein (hPD-1-mFc, SEQ ID NO: 4) containing a mouse antibody heavy chain Fc tag were obtained by PCR and subcloned into expression vector pcDNA3.1(Invitrogen, V-790), respectively. The vector constructed above was transfected into Exi-CHO cells (ThermoFisher A29133) for transient expression. Passage of cells to cell density 6 x 10 at transfection ⁶ Preparing a transfection compound according to the proportion of a transfection reagent to DNA (deoxyribonucleic acid) of 3: 1 per ml of cells, incubating for 5min at room temperature, slowly adding the transfection compound into the cells, and adding ExpicHO Enhancer and ExpicHO Feed according to the proportion after 20-24 h. After transient expression in ExpicHO cells for 7-14 days, hPD-L1-HisTag protein was purified using NTA column (GE healthcare), hPD-L1-mFc and hPD1-mFc recombinant proteins were purified using ProteinA column (GE healthcare). Or constructing the expression fragment in CHO-S (Thermo Fisher, A1155701) or CHO-DG44(Thermo Fisher, A1100001) cells for stable expression, and purifying.

EXAMPLE 2 preparation of anti-human PD-L1 monoclonal antibody hybridoma

The expression purified hPD-L1(ECD) -mFc recombinant protein (at 100. mu.g/mouse) was mixed well and emulsified with an equal volume of complete Freund's adjuvant (prime) or incomplete Freund's adjuvant (boost) and BALB/c mice were immunized subcutaneously every 2 weeks for 8 weeks. Sorting machineMice with high antibody titers in serum and titers tending towards plateau were selected for splenocyte fusion. 3 days prior to fusion, mice were immunized by spiking with hPD-L1(ECD) -mFc antigen without adjuvant (50. mu.g/mouse). Spleen lymphocytes were fused with myeloma Sp2/0 cells using an optimized PEG-mediated fusion procedure to obtain hybridoma cells. Splenocytes from immunized mice (1X 10) ⁸ Individual cells) and SP2/0 myeloma cells (2X 10) ⁷ Individual cells) were fused. After fusion, cells were resuspended in HAT complete medium, dispensed into 96-well plates at 0.1 ml/well, and 5% CO at 37 ℃ ₂ The incubator of (2) for cultivation. On day 5 after the fusion, the half-change was carried out with fresh HAT complete medium at 37 ℃ with 5% CO ₂ And (5) continuing culturing. On 7-8 days after fusion, the total liquid change is carried out according to the cell growth density, and the adopted culture medium is HT complete culture medium with 200 mul/hole, 37 ℃ and 5 percent CO ₂ And (5) continuing to culture. Around day 14 after fusion, detection was performed by ELISA using PD-L1 binding according to cell growth density (detection method 1.1). And performing ELISA detection (detection method 1.2) of blocking of PD-L1/PD-1 binding on the detected positive well cells, selecting wells capable of binding to PD-L1 and blocking binding to PD-1, and expanding the wells into a 24-well plate in time according to cell density. The cell lines transferred into the 24-well plate were retested for seed preservation and first subcloning. And (4) performing seed preservation on the positive subclone selected for the first time, and performing secondary subcloning. And performing seed preservation and protein expression when the secondary subcloning is positive.

Detection method 1.1 ELISA detection of binding PD-L1

Hybridoma antibodies were screened for PD-L1 binding and analyzed by ELISA using PD-L1-his protein. PD-L1 antigen (100. mu.L, 2. mu.g/ml) was coated in a high adsorption 96-well plate (Costar, 9018) at 4 ℃ overnight. After washing off the non-adsorbed antigen sufficiently, the non-specific binding sites were blocked using blocking buffer (PBS containing 2% bovine serum albumin). After washing the plates three times with washing buffer (PBS with 0.05% (v/v) Tween 20), 100. mu.L/well of the sample to be tested was added and incubated for 1 hour at room temperature. Plates were washed and incubated for an additional 60 minutes with a secondary antibody conjugated to horseradish peroxidase (HRP). Washing flatAfter plating, 100. mu.L/well of substrate TMB solution (eBioscience, 00-4201-56) was added and the plates incubated for 2min at room temperature. Add 100. mu.L/well of stop solution (2 NH) ₂ SO ₄ ) To stop the reaction. Colorimetric signals were generated and read at 450nm using a microplate reader (Thermo Fisher, MK3), with a 2-fold higher reading than the blank serum after background subtraction, as positive wells.

Detection method 1.2 ELISA detection of blockade of PD-L1/PD-1 binding

PD-L1 antigen (100. mu.L, 2. mu.g/ml) was coated in high adsorption 96-well plates at 4 ℃ overnight. After washing off the non-adsorbed antigen sufficiently, the non-specific binding sites were blocked using blocking buffer (PBS containing 2% bovine serum albumin). After washing the plate three times with washing buffer (PBS with 0.05% Tween 20), 100. mu.L/well of the sample to be tested was added, while 100. mu.l of biotin-labeled PD-1-mFc (0.1. mu.g/ml) was added to each well, and incubated at 37 ℃ for 2 h. After washing the plates 3 times, secondary antibody Avidin HRP (eBioscience E07418-1632) diluted 1: 500 was added at 100. mu.l/well and incubated at room temperature for 1 hour. After washing the plate, 100. mu.L/well of substrate solution TMB (eBioscience, 00-4201-56) was added and the plate was incubated at room temperature for 3 min. Colorimetric signals were generated and read at 450nm using a microplate reader (Thermo Fisher, MK3), wells with lower readings were selected.

Example 3 cDNA acquisition and chimeric antibody construction of anti-human PD-L1 antibody

Total RNA was isolated from the above hybridoma cells having binding and blocking functions using TRIzol as a template, and first strand cDNA was synthesized using superscript II reverse transcriptase (Life Technology, 18064-14) according to the instructions. The variable region sequences of the antibodies were then amplified by a PCR reaction using degenerate mouse IgG primers.

The PCR mixture was electrophoretically separated on a 1% agarose/Tris-borate gel containing 0.5. mu.g/ml ethidium bromide. A DNA fragment having the expected size (about 500bp for heavy and light chains) was excised from the gel and purified. The purified PCR product was cloned into pMD-19T vector (Takara, 6013) and transformed into DH 5. alpha. competent E.coli cells (Takara, 9057). 2 colonies from LB solid medium plate were picked for DNA sequencing. Obtaining the heavy chain variable region sequence and the light chain variable region sequence of the antibody (Chi1 variable region is shown as SEQ ID NOs: 5-6, Chi2 variable region is shown as SEQ ID NOs: 7-10). These antibodies show specific functions such as high affinity binding to PD-L1 and are capable of blocking the binding of PD-L1 to PD-1.

Construction and expression of chimeric antibody 1(Chi1) and chimeric antibody 2(Chi 2): the mouse VL region gene synthesis fragment is connected to a nucleotide sequence encoding a human kappa chain constant region through double enzyme digestion reaction to construct coding sequences of Chi1, Chi2-1 and Chi2-2 chimeric light chains (SEQ ID NOs: 40, 42 and 44 respectively). The gene synthesis fragment of the mouse VH region was ligated to the nucleotide sequence encoding human IgG1 constant region by double digestion to construct the coding sequence for Chi1, Chi2-1, Chi2-2 chimeric heavy chains (SEQ ID NOs: 39, 41 and 43).

Expi CHO cells (50mL system, 6X 10) were transfected with the DNA vector encoding the chimeric antibody ⁶ Individual cells/mL, DNA 1 μ g/mL) were transiently expressed and cultured for 7 days. The chimeric antibody in the supernatant was then purified using a ProteinA column (GEhealthcare).

Example 4 evaluation of Activity of chimeric antibody against human PD-L1

The binding of the chimeric antibody to PD-L1 was measured by the following ELISA, Biacore and flow cytometry methods and it was verified at the cellular level whether the chimeric antibody could block the binding of PD-1 to PD-L1.

4.1 affinity of anti-PD-L1 chimeric antibody

The affinity of the PD-L1 chimeric antibody to PD-L1 antigen was determined using Biacore, using an anti-human antibody capture kit (GE, BR-1008-39) to capture the PD-L1 antibody of the invention by covalently coupling the anti-human capture antibody to a CM5 biochip (GE, BR-1000-12) according to the methods described in the specification. Then, a series of concentrations of human PD-L1 antigen (hPD-L1-his, Nano biological 10084-H08H-200) or cynomolgus monkey PD-L1 antigen (Cyno-PD-L1-his, Nano biological 90251-C08H-200) are flowed on the surface of the chip, a Biacore instrument (GE, Biacore T200) is used for detecting reaction signals in real time to obtain a binding curve and a dissociation curve, and affinity numbers are obtained by software fittingThe value is obtained. After each cycle of dissociation in the experiment was completed, the biochip was regenerated using regeneration solution followed by the next capture, and the cycle was repeated to complete the determination of the affinity of the different antibodies to PD-L1. Finally, ka (kon), kd (koff) and K were determined by analyzing the data using GE BIAevaluation software in a 1: 1(Langmuir) binding model _D The value is obtained. By K _D Calculating the equilibrium dissociation constant K ═ kd/ka _D . The table below shows the affinity data of the chimeric antibody for human PD-L1 antigen, cynomolgus monkey PD-L1 antigen.

Table 1: affinity data for anti-PD-L1 antibody with PD-L1

ND: not tested

*: a humanized PD-L1 antibody disclosed in WO2016022630

4.2 Activity of anti-human PD-L1 chimeric antibodies to bind to PD-L1

The assay for PD-L1 binding of the chimeric antibody was performed by ELISA method, specifically referring to detection method 1.1 in example 2, using hPD-L1-mFc instead of PD-L1-his protein. Finally, the colorimetric signals read by the microplate reader at 450nm were analyzed using GraphPad Prism5 and the EC50 was calculated, see table 2, chimeric antibody 1(Chi1) and chimeric antibody 2-1(Chi2-1) having comparable PD-L1 binding activity compared to the control antibody, with chimeric antibody 1 being slightly superior.

Table 2: antigen binding data for chimeric antibodies Chi1 and Chi2

PD-L1 binding	Chi1	Chi2-1	Tecentriq
				EC50(ng/ml)	107.3	145.2	142.3

4.3 blocking of the binding Activity of PD-L1/PD-1 by anti-human PD-L1 chimeric antibodies

Based on the ELASA method, it was examined whether the chimeric antibody was able to block the binding of PD-1 to PD-L1. Referring to the detection method 1.2 in example 1, the colorimetric signal read by the microplate reader at 450nm was finally analyzed using GraphPad Prism5 and the IC50 was calculated, as shown in table 3, and chimeric antibody 1 and chimeric antibody 2-1 had comparable activity of blocking the binding of PD-1 to PD-L1, and chimeric antibody 1 and chimeric antibody 2-1 had slightly superior activity, compared to the control antibody.

Table 3: IC50 data of chimeric antibodies Chi1 and Chi2 for blocking PD-1 binding

PD-L1 blockade	Chi1	Chi2-1	Chi2-2	Tecentriq
					IC50(ng/ml)	514.5	462.1	1436	553.2

4.4 cell-based binding assays for anti-PD-L1 chimeric antibodies

The ability of anti-PD-L1 chimeric antibodies to bind to naturally expressed PD-L1 was analyzed by analyzing binding experiments with a U2OS cell line (PD-L1-U2OS) that stably expresses PD-L1. 2 x 10 to ⁵ Individual PD-L1-U2OS cells were added to each well of a 96-well plate, and a gradient of diluted anti-PD-L1 antibody was added to the cell suspension, along with biotin-labeled PD-1 protein to compete for binding to PD-L1. After incubation at 4 ℃ for 120min, cells were washed 3 times with PBS and incubated with avidin-FITC (eBioscience, 11-4317-87) for 60min at 4 ℃. The fluorescent signal was then detected by analysis using flow cytometry (BD, Accuri C6). Competitive binding of anti-PD-L1 antibody to PD-1 to PD-L1 was measured by mean fluorescence intensity of staining (MFI). Using GraphPad Prism5 analysis data, the calculated IC50 shown in table 4 that chimeric antibody 1, chimeric antibody 2-1 had comparable activity of blocking the binding of PD-1 to cell surface PD-L1 compared to the control antibody, and that chimeric antibody 1, chimeric antibody 2-1 were slightly superior in activity.

Table 4: IC50 data of chimeric antibodies Chi1 and Chi2 for blocking PD-1 binding

U2OS PD-L1 block	Chi1	Chi2-1	Chi2-2	Tecentriq
					IC50(ng/ml)	195.3	248.3	2167	342.1

4.5 analysis of anti-PD-L1 chimeric antibodies to block cell/cell interactions

In the detection system, modified Jurkat cells (DiscoverX, 93-1104C19) stably expressing PD-1 and U2OS cells (DiscoverX, 93-1066C3) stably expressing PD-L1 are respectively used. When PD-L1 on the surface of U2OS cell binds to PD-1 on the surface of Jurkat, the modified Jurkat cell induces downstream pathway and reacts with the detection substrate to generate chemiluminescence signal. Thus, when an anti-PD-L1 antibody is present in the system and is able to block the binding of PD-1 to PD-L1, no or a weak chemical signal will be generated. In this assay, both PD-L1 and PD-1 are expressed on the cell surface in native conformation, as distinguished from the use of recombinant proteins as screens in the previous examples. In the experiment, ligand cells (U2OS PD-L1 cells, 9.6X 10 cells) in the exponential growth phase were selected ⁵ Individual cells/ml), added to 384-well plates using an automatic dispenser, 12.5 μ L/well; adding a gradient of diluted antibody to each well; meanwhile, the receptor cells (Jurkat PD-1 cells) in the exponential growth phase were selected at 6.4X 10 ⁵ Each well was inoculated to 384 well plates at 12.5. mu.L/well using an automatic dispenser, mixed, incubated at room temperature for 120min, 2.5. mu.L of Bioassay Reagent 1(discover X, 93-0933) was added to each well, mixed, and incubated at room temperature for 15min in the absence of light. Then, 10. mu.L of Bioassay Reagent 2(DiscoverX, 93-0933) was added to each well, mixed well and incubated for 60min at room temperature in the dark. The microplate reader reads the chemiluminescent signal. Data were analyzed using GraphPad Prism5 and IC50 values were calculated. The results in Table 5 show that the chimeric antibodiesChi1 has comparable activity of blocking the binding of U2OS surface PD-L1 and Jurkat surface PD-1 compared with a control antibody, wherein Chi1 has better blocking activity.

Table 5: IC50 data for chimeric antibody Chi1 blocking cell/cell binding

Example 5 humanization of anti-human PD-L1 hybridoma monoclonal antibody

Based on the activity of the chimeric antibody, the optimal Chi1 was selected for humanization. The CDR regions of the murine antibody were grafted into the corresponding human template by CDR grafting method by searching and comparing IMGT human antibody heavy and light chain variable region Germline gene database (http:// www.imgt.org/3D structure-DB/cgi/DomainGapAlign. cgi), selecting heavy and light chain variable region Germline genes (Germine sequence) with high homology to Chi1 as template, forming variable region sequences in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (wherein the amino acid residues are determined and annotated by Kabat numbering system). The heavy chain humanized template of the murine antibody Chi1 is a combination of IGHV4-30-4 x 01 and IGHJ1 x 01; the light chain humanized template is a combination of IGKV1-39 × 01 and IGKJ2 × 02, and the sequence of the CDR after direct grafting is SEQ ID NOs: 45 and 46. The affinity of the antibody after direct humanization generally decreases to a great extent, and a back mutation needs to be further designed to restore the affinity and the function, namely, several sites of the framework amino acid of the humanized template antibody are back mutated into the amino acid sequence in the corresponding framework region in the mouse Chi1 antibody. For the heavy chain variable region of the humanized Chi1 antibody, ser(s) at position 35 (according to Kabat numbering, the same below) was mutated back to asn (n), trp (w) at position 47 was mutated back to tyr (y), and val (v) at position 71 was mutated back to arg (r); for the light chain variable region of humanized Chi1 antibody, I1e (I) at position 2 was mutated back to phe (f), gly (g) at position 41 was mutated to asp (d), and pro (p) at position 44 was mutated to val (v). The sequences encoding the VH and VL of the humanized hu-Chi1 antibody are SEQ ID NOs: 47 and 51, and to human antibody constant regions, to construct a complete antibody format, SEQ ID NOs: 53 and 55.

The DNA sequences described above (SEQ ID NOs: 54 and 56) encoding the light and heavy chains of the humanized hu-Chi1-3.4 antibody were used. Was cloned into the expression vector pcDNA3.1(Invitrogen, V-790). The above DNA vector was transfected into Expi CHO cells (50mL system, 5X 10) ⁶ mL cells, 1. mu.g/mL DNA), and cultured for 7 days. The humanized antibody in the supernatant was then purified using a ProteinA column (GE healthcare).

Table 6: humanized back mutation site of Chi1 antibody

And (4) Grafted: indicates that the murine CDRs were grafted directly into human FRs.

Antibody numbering convention according to Kabat nomenclature, e.g., W47Y indicates that W at position 47 is mutated to Y.

Humanized PD-L1 antibodies were numbered according to table 6 as shown in table 7.

Table 7: humanized PD-L1 antibody numbering

Example 6 evaluation of Activity of humanized anti-PD-L1 antibody

6.1 affinity of the anti-PD-L1 humanized antibody

Biacore measures the affinity of the anti-PD-L1 humanized antibody to the PD-L1 antigen, and the specific procedures refer to example 4.1, and the following Table 8 shows the affinity data of the humanized hu-Chi1, hu-Chi1-3.3, hu-Chi1-3.4 and chimeric antibody Chi1, which indicates that the humanized PD-L1 antibody better maintains the affinity to the PD-L1 in the reversion process.

Table 8: affinity data for humanized Chi1 antibody

anti-PD-L1 antibody	Heavy chain variable region	Light chain variable region	K _D (humanization)
				hu-Chi1	VH.1	VL.1	3.14E-09
hu-Chi1-3.3	Chi1_VL.1C	Chi1_VH.1C	8.15E-10
				hu-Chi1-3.4	Chi1_VL.1C	Chi1_VH.1D	7.99E-10
Chi1	VH	VL	6.74E-10

6.2 analysis of blocking of cell/cell interactions by anti-PD-L1 humanized antibodies

The blocking effect of the anti-PD-L1 humanized antibody was analyzed by cell/cell interaction, the specific procedure being as in example 4.5. The results in Table 9 show that the humanized antibody has a blocking activity comparable to that of the parent chimeric antibody.

Table 9: comparison of IC50 data for blocking cell interaction by humanized and chimeric antibodies

Sequence listing

<110> Zhengda Ningqing pharmaceutical industry group Nanjing Shunxin pharmaceutical Co., Ltd

CHIA TAI TIANQING PHARMACEUTICAL GROUP Co.,Ltd.

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Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Ala Lys

225 230 235 240

Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr

245 250 255

Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro

260 265 270

Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val

275 280 285

His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser

290 295 300

Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys

305 310 315 320

Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu

325 330 335

Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala

340 345 350

Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile

355 360 365

Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val

370 375 380

Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val

385 390 395 400

Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp

405 410 415

Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln

420 425 430

Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp

435 440 445

Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val

450 455 460

Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr

465 470 475 480

Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu

485 490 495

Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr

500 505 510

Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr

515 520 525

Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr

530 535 540

Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys

545 550 555 560

Ser Phe Ser Arg Thr Pro Gly Lys

565

<210> 3

<211> 244

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu

1 5 10 15

Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu

35 40 45

Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile

50 55 60

Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser

65 70 75 80

Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val

115 120 125

Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val

130 135 140

Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr

145 150 155 160

Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser

165 170 175

Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn

180 185 190

Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr

195 200 205

Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu

210 215 220

Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg His His

225 230 235 240

His His His His

<210> 4

<211> 500

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Ala Lys Thr Thr Ala Pro

165 170 175

Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser

180 185 190

Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr

195 200 205

Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro

210 215 220

Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val

225 230 235 240

Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His

245 250 255

Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro

260 265 270

Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu

275 280 285

Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu

290 295 300

Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser

305 310 315 320

Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu

325 330 335

Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr

340 345 350

Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser

355 360 365

Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro

370 375 380

Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln

385 390 395 400

Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val

405 410 415

Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val

420 425 430

Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu

435 440 445

Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg

450 455 460

Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val

465 470 475 480

Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg

485 490 495

Thr Pro Gly Lys

500

<210> 5

<211> 119

<212> PRT

<213> mouse Chi1 VH (Mus musculus)

<400> 5

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ala Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Arg Phe Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Lys Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Cys Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser

115

<210> 6

<211> 107

<212> PRT

<213> mouse Chi1 VL (Mus musculus)

<400> 6

Asp Phe Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Tyr Ala Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 7

<211> 120

<212> PRT

<213> mouse Chi2-1 VH (Mus musculus)

<400> 7

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Phe Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asn Met Val Trp Ser Arg Gln Val Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 8

<211> 112

<212> PRT

<213> mouse Chi2-1 VL (Mus musculus)

<400> 8

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser

35 40 45

Pro Lys Leu Leu Leu Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Gly Arg Val Glu Ala Ala Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 9

<211> 120

<212> PRT

<213> mouse Chi2-2 VH (Mus musculus)

<400> 9

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Asn Leu Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Trp Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val His

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys

85 90 95

Val Arg Ser Met Val Trp Thr Arg His Val Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 10

<211> 112

<212> PRT

<213> mouse Chi2-2 VL (Mus musculus)

<400> 10

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Glu Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Gly Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ala

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 11

<211> 5

<212> PRT

<213> mouse HCDR1(Mus musculus)

<400> 11

Ser Asp Tyr Trp Xaa

1 5

<210> 12

<211> 16

<212> PRT

<213> mouse HCDR2(Mus musculus)

<400> 12

Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 13

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 13

Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Xaa

1 5 10

<210> 14

<211> 11

<212> PRT

<213> mouse LCDR1(Mus musculus)

<400> 14

Arg Ala Ser Gln Asp Ile Thr Asn Tyr Leu Asn

1 5 10

<210> 15

<211> 7

<212> PRT

<213> mouse LCDR2(Mus musculus)

<400> 15

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 16

<211> 9

<212> PRT

<213> mouse LCDR3(Mus musculus)

<400> 16

Gln Gln Val Tyr Thr Leu Pro Trp Thr

1 5

<210> 17

<211> 5

<212> PRT

<213> mouse HCDR1(Mus musculus)

<400> 17

Ser Asp Tyr Trp Asn

1 5

<210> 18

<211> 5

<212> PRT

<213> mouse HCDR1(Mus musculus)

<400> 18

Ser Asp Tyr Trp Ser

1 5

<210> 19

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 19

Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Tyr

1 5 10

<210> 20

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 20

Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Cys

1 5 10

<210> 21

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 21

Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Ala

1 5 10

<210> 22

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 22

Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Val

1 5 10

<210> 23

<211> 5

<212> PRT

<213> mouse HCDR1(Mus musculus)

<400> 23

Xaa Tyr Trp Met Asn

1 5

<210> 24

<211> 17

<212> PRT

<213> mouse HCDR2(Mus musculus)

<400> 24

Met Ile His Pro Ser Asp Ser Glu Thr Xaa Leu Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 25

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 25

Xaa Met Val Trp Xaa Arg Xaa Val Met Asp Tyr

1 5 10

<210> 26

<211> 16

<212> PRT

<213> mouse LCDR1(Mus musculus)

<400> 26

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Xaa Thr Tyr Leu Asn

1 5 10 15

<210> 27

<211> 7

<212> PRT

<213> mouse LCDR2(Mus musculus)

<400> 27

Leu Val Ser Lys Leu Asp Ser

1 5

<210> 28

<211> 9

<212> PRT

<213> mouse LCDR3(Mus musculus)

<400> 28

Leu Gln Xaa Thr His Phe Pro Tyr Thr

1 5

<210> 29

<211> 5

<212> PRT

<213> mouse HCDR1(Mus musculus)

<400> 29

Phe Tyr Trp Met Asn

1 5

<210> 30

<211> 5

<212> PRT

<213> mouse HCDR1(Mus musculus)

<400> 30

Ser Tyr Trp Met Asn

1 5

<210> 31

<211> 17

<212> PRT

<213> mouse HCDR2(Mus musculus)

<400> 31

Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 32

<211> 17

<212> PRT

<213> mouse HCDR2(Mus musculus)

<400> 32

Met Ile His Pro Ser Asp Ser Glu Thr Trp Leu Asn Gln Lys Phe Lys

1 5 10 15

Asp

<210> 33

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 33

Asn Met Val Trp Ser Arg Gln Val Met Asp Tyr

1 5 10

<210> 34

<211> 11

<212> PRT

<213> mouse HCDR3(Mus musculus)

<400> 34

Ser Met Val Trp Thr Arg His Val Met Asp Tyr

1 5 10

<210> 35

<211> 16

<212> PRT

<213> mouse LCDR1(Mus musculus)

<400> 35

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 36

<211> 16

<212> PRT

<213> mouse LCDR1(Mus musculus)

<400> 36

Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Glu Thr Tyr Leu Asn

1 5 10 15

<210> 37

<211> 9

<212> PRT

<213> mouse LCDR3(Mus musculus)

<400> 37

Leu Gln Ser Thr His Phe Pro Tyr Thr

1 5

<210> 38

<211> 9

<212> PRT

<213> mouse LCDR3(Mus musculus)

<400> 38

Leu Gln Ala Thr His Phe Pro Tyr Thr

1 5

<210> 39

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> Chi1 chimeric antibody heavy chain sequence

<400> 39

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ala Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Arg Phe Pro Gly Asn Lys Leu Glu Tyr Met

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu

65 70 75 80

Gln Leu Lys Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Cys Trp Gly Gln Gly

100 105 110

Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 40

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> Chi1 chimeric antibody light chain sequence

<400> 40

Asp Phe Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Ser Asp Tyr Ala Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Arg Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 41

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> Chi2-1 chimeric antibody heavy chain sequence

<400> 41

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Phe Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Arg Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Asn Met Val Trp Ser Arg Gln Val Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 42

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> Chi2-1 chimeric antibody light chain sequence

<400> 42

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser

35 40 45

Pro Lys Leu Leu Leu Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Gly Arg Val Glu Ala Ala Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ser

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 43

<211> 450

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> Chi2-2 chimeric antibody heavy chain sequence

<400> 43

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Asn Leu Ser Cys Lys Thr Ser Gly Tyr Ser Phe Thr Ser Tyr

20 25 30

Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Met Ile His Pro Ser Asp Ser Glu Thr Trp Leu Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Val His

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys

85 90 95

Val Arg Ser Met Val Trp Thr Arg His Val Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 44

<211> 219

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> Chi2-2 chimeric antibody light chain sequence

<400> 44

Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Glu Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Glu Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Gly Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Leu Gln Ala

85 90 95

Thr His Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 45

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain variable region Chi1 hVH-CDR graft

<400> 45

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 46

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized light chain variable region Chi1 hVL-CDR graft

<400> 46

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 47

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain variable region-reverse mutation Chi1 hVH.1D

<400> 47

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 48

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain variable region-reverse mutation Chi1 hVH.1C

<400> 48

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 49

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain variable region-reversion Chi1 hVH.1E

<400> 49

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Ala Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 50

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain variable region-reverse mutation Chi1 hVH.1F

<400> 50

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 51

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized light chain variable region-back mutation Chi1 hVL.1C

<400> 51

Asp Phe Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 52

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized light chain variable region-back mutation Chi1 hVL.1B

<400> 52

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 53

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain sequence Chi1 hVH.1D-IgG1

<400> 53

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 54

<211> 1401

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> variation

<223> humanized heavy chain encoding gene sequence Chi1 hVH.1D-IgG1-DNA

<400> 54

atgggctgga gctgcatcat cctgttcctg gtggctaccg gagtgcactc ccaggtgcag 60

ctgcaggaga gcggaccagg actggtgaag ccttcccaga ccctgagcct gacctgtaca 120

gtgagcggct actctatcac atccgactat tggaattgga tcagacagca tccaggcaag 180

ggcctggagt acatcggcta catctcttat accggctcca catactataa ccccagcctg 240

aagtctcgcg tgaccatcag ccgggacaca tctaagaatc agttttctct gaagctgtcc 300

agcgtgaccg ccgctgatac agccgtgtac tattgcgcta ggggagaggc ttgggacagg 360

cggaccctgg attattgggg ccagggcacc ctggtgacag tgtcttccgc ttccacaaag 420

ggcccttccg tgttcccact ggctccctct tccaagtcta catccggagg aaccgccgct 480

ctgggatgcc tggtgaagga ttatttccca gagcccgtga ccgtgtcttg gaactccggc 540

gccctgacaa gcggagtgca tacctttcct gctgtgctgc agagctctgg cctgtattct 600

ctgtccagcg tggtgacagt gccatcttcc agcctgggca cccagacata catctgcaac 660

gtgaatcaca agcctagcaa taccaaggtg gacaagaagg tggagccaaa gtcttgtgat 720

aagacccata catgcccccc ttgtcctgct ccagagctgc tgggaggacc atccgtgttc 780

ctgtttccac ccaagcccaa ggacaccctg atgatctccc gcacaccaga ggtgacctgc 840

gtggtggtgg acgtgagcca cgaggatccc gaggtgaagt ttaactggta cgtggatggc 900

gtggaggtgc ataatgctaa gaccaagcca agagaggagc agtataacag cacataccgc 960

gtggtgtctg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 1020

aaggtgtcta ataaggccct gcccgctcct atcgagaaga caatctccaa ggccaagggc 1080

cagcctaggg agccacaggt gtataccctg cctccatctc gggacgagct gacaaagaac 1140

caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc tgtggagtgg 1200

gagtctaatg gccagcctga gaacaattat aagaccacac cccctgtgct ggacagcgat 1260

ggctctttct ttctgtactc taagctgaca gtggataagt ccaggtggca gcagggcaac 1320

gtgtttagct gctctgtgat gcatgaggct ctgcacaatc attacaccca gaagtccctg 1380

agcctgtctc ccggcaagtg a 1401

<210> 55

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized light chain sequence Chi1 hVL.1C-kappa

<400> 55

Asp Phe Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 56

<211> 696

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> variation

<223> humanized light chain-encoding gene sequence Chi1 hVL.1C-kappa-DNA

<400> 56

atgggctggt cttgcatcat cctgttcctg gtggccaccg gcgtgcactc cgacttccag 60

atgacacaga gcccttccag cctgagcgcc tctgtgggcg acagggtgac catcacatgt 120

cgggcttccc aggatatcac caactacctg aattggtatc agcagaagcc cgacaaggcc 180

gttaagctgc tgatctacta tacatccaga ctgcatagcg gagtgccatc tcgcttctcc 240

ggaagcggat ctggaaccga ctttaccctg acaatctctt ccctgcagcc agaggatttc 300

gctacatact attgccagca ggtgtacacc ctgccctgga catttggcca gggcaccaag 360

ctggagatca agcgtacggt ggccgctccc agcgtgttca tctttccccc ttctgacgag 420

cagctgaagt ctggcaccgc ttccgtggtg tgcctgctga acaatttcta ccccagagag 480

gccaaggtgc agtggaaggt ggataacgct ctgcagtccg gcaatagcca ggagtctgtg 540

accgagcagg actccaagga tagcacatat tctctgtctt ccaccctgac actgtctaag 600

gccgactacg agaagcacaa ggtgtatgct tgcgaggtga cccatcaggg cctgagctct 660

cctgtgacaa agtcctttaa tcgcggcgag tgttga 696

<210> 57

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain sequence Chi1 hVH.1C-IgG1

<400> 57

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Cys Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 58

<211> 1401

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> variation

<223> humanized heavy chain encoding gene sequence Chi1 hVH.1C-IgG1-DNA

<400> 58

atgggctgga gctgcatcat cctgttcctg gtggctaccg gagtgcactc ccaggtgcag 60

ctgcaggaga gcggaccagg actggtgaag ccttcccaga ccctgagcct gacctgtaca 120

gtgagcggct actctatcac atccgactat tggaattgga tcagacagca tccaggcaag 180

ggcctggagt acatcggcta catctcttat accggctcca catactataa ccccagcctg 240

aagtctcgcg tgaccatcag ccgggacaca tctaagaatc agttttctct gaagctgtcc 300

agcgtgaccg ccgctgatac agccgtgtac tattgcgcta ggggagaggc ttgggacagg 360

cggaccctgg attgttgggg ccagggcacc ctggtgacag tgtcttccgc ttccacaaag 420

ggcccttccg tgttcccact ggctccctct tccaagtcta catccggagg aaccgccgct 480

ctgggatgcc tggtgaagga ttatttccca gagcccgtga ccgtgtcttg gaactccggc 540

gccctgacaa gcggagtgca tacctttcct gctgtgctgc agagctctgg cctgtattct 600

ctgtccagcg tggtgacagt gccatcttcc agcctgggca cccagacata catctgcaac 660

gtgaatcaca agcctagcaa taccaaggtg gacaagaagg tggagccaaa gtcttgtgat 720

aagacccata catgcccccc ttgtcctgct ccagagctgc tgggaggacc atccgtgttc 780

ctgtttccac ccaagcccaa ggacaccctg atgatctccc gcacaccaga ggtgacctgc 840

gtggtggtgg acgtgagcca cgaggatccc gaggtgaagt ttaactggta cgtggatggc 900

gtggaggtgc ataatgctaa gaccaagcca agagaggagc agtataacag cacataccgc 960

gtggtgtctg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 1020

aaggtgtcta ataaggccct gcccgctcct atcgagaaga caatctccaa ggccaagggc 1080

cagcctaggg agccacaggt gtataccctg cctccatctc gggacgagct gacaaagaac 1140

caggtgtccc tgacctgtct ggtgaagggc ttctacccca gcgatatcgc tgtggagtgg 1200

gagtctaatg gccagcctga gaacaattat aagaccacac cccctgtgct ggacagcgat 1260

ggctctttct ttctgtactc taagctgaca gtggataagt ccaggtggca gcagggcaac 1320

gtgtttagct gctctgtgat gcatgaggct ctgcacaatc attacaccca gaagtccctg 1380

agcctgtctc ccggcaagtg a 1401

<210> 59

<211> 214

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized light chain sequence Chi1 hVL.1B-kappa

<400> 59

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Thr Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Lys Ala Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Val Tyr Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 60

<211> 696

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> variation

<223> humanized light chain-encoding Gene sequence Chi1 hVL.1B-kappa-DNA

<400> 60

atgggctggt cttgcatcat cctgttcctg gtggccaccg gcgtgcactc cgacatccag 60

atgacacaga gcccttccag cctgagcgcc tctgtgggcg acagggtgac catcacatgt 120

cgggcttccc aggatatcac caactacctg aattggtatc agcagaagcc cgacaaggcc 180

gttaagctgc tgatctacta tacatccaga ctgcatagcg gagtgccatc tcgcttctcc 240

ggaagcggat ctggaaccga ctttaccctg acaatctctt ccctgcagcc agaggatttc 300

gctacatact attgccagca ggtgtacacc ctgccctgga catttggcca gggcaccaag 360

ctggagatca agcgtacggt ggccgctccc agcgtgttca tctttccccc ttctgacgag 420

cagctgaagt ctggcaccgc ttccgtggtg tgcctgctga acaatttcta ccccagagag 480

gccaaggtgc agtggaaggt ggataacgct ctgcagtccg gcaatagcca ggagtctgtg 540

accgagcagg actccaagga tagcacatat tctctgtctt ccaccctgac actgtctaag 600

gccgactacg agaagcacaa ggtgtatgct tgcgaggtga cccatcaggg cctgagctct 660

cctgtgacaa agtcctttaa tcgcggcgag tgttga 696

<210> 61

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain sequence Chi1 hVH.1E-IgG 1

<400> 61

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Ala Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 62

<211> 449

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> VARIANT

<223> humanized heavy chain sequence Chi1 hVH.1F-IgG 1

<400> 62

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile

35 40 45

Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys

50 55 60

Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Glu Ala Trp Asp Arg Arg Thr Leu Asp Val Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds PD-L1, wherein the antibody or antigen-binding fragment thereof comprises the sequence:

(a) heavy chains HCDR1, HCDR2 and HCDR3, wherein the heavy chains HCDR1, HCDR2 and HCDR3 are respectively amino acid sequences shown as SEQ ID NO 17, 12 and 19; and light chain LCDR1, LCDR2 and LCDR3, wherein the light chain LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16; or

(b) Heavy chains HCDR1, HCDR2 and HCDR3, wherein the heavy chains HCDR1, HCDR2 and HCDR3 are respectively amino acid sequences shown in SEQ ID NO 18, 12 and 20; and light chain LCDR1, LCDR2 and LCDR3, wherein the light chain LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16; or

(c) Heavy chains HCDR1, HCDR2 and HCDR3, wherein the heavy chains HCDR1, HCDR2 and HCDR3 are respectively amino acid sequences shown as SEQ ID NO 17, 12 and 20; and light chain LCDR1, LCDR2 and LCDR3, wherein the light chain LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16; or

(d) Heavy chains HCDR1, HCDR2 and HCDR3, wherein the heavy chains HCDR1, HCDR2 and HCDR3 are respectively amino acid sequences shown in SEQ ID NO 17, 12 and 21; and light chain LCDR1, LCDR2 and LCDR3, wherein the light chain LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16; or

(e) Heavy chains HCDR1, HCDR2 and HCDR3, wherein the heavy chains HCDR1, HCDR2 and HCDR3 are respectively amino acid sequences shown in SEQ ID NO 17, 12 and 22; and light chains LCDR1, LCDR2 and LCDR3, wherein the light chains LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof that binds PD-L1 comprises heavy chains HCDR1, HCDR2, and HCDR3, which heavy chains HCDR1, HCDR2, and HCDR3 are the amino acid sequences set forth in SEQ ID NOs 17, 12, and 19, respectively; and light chains LCDR1, LCDR2 and LCDR3, wherein the light chains LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof that binds PD-L1 comprises heavy chains HCDR1, HCDR2, and HCDR3, which heavy chains HCDR1, HCDR2, and HCDR3 are the amino acid sequences set forth in SEQ ID NOs 18, 12, and 20, respectively; and light chains LCDR1, LCDR2 and LCDR3, wherein the light chains LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof that binds PD-L1 comprises heavy chains HCDR1, HCDR2, and HCDR3, which heavy chains HCDR1, HCDR2, and HCDR3 are the amino acid sequences set forth in SEQ ID NOs 17, 12, and 20, respectively; and light chains LCDR1, LCDR2 and LCDR3, wherein the light chains LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof that binds PD-L1 comprises heavy chains HCDR1, HCDR2, and HCDR3, which are the amino acid sequences set forth in SEQ ID NOs 17, 12, and 21, respectively, HCDR1, HCDR2, and HCDR 3; and light chains LCDR1, LCDR2 and LCDR3, wherein the light chains LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16.

6. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof that binds PD-L1 comprises heavy chains HCDR1, HCDR2, and HCDR3, which are the amino acid sequences set forth in SEQ ID NOs 17, 12, and 22, respectively, HCDR1, HCDR2, and HCDR 3; and light chains LCDR1, LCDR2 and LCDR3, wherein the light chains LCDR1, LCDR2 and LCDR3 are respectively amino acid sequences shown in SEQ ID NO. 14, 15 and 16.

7. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is a chimeric or humanized antibody or antigen-binding fragment thereof.

8. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 5; and a light chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 6; or

(b) A heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 45; and a light chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 46; or

(c) A heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 47; and a light chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 51; or

(d) A heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 48; and a light chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 52; or

(e) A heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 49; and a light chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 51; or

(f) A heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 50; and a light chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID NO: 51; or

(g) A heavy chain variable region comprising an amino acid sequence having at least 80% homology to SEQ ID No. 48; and a light chain variable region comprising an amino acid sequence having at least 80% homology with SEQ ID NO: 51.

9. The isolated antibody or antigen-binding fragment thereof of claim 8, wherein the antibody or antigen-binding fragment thereof comprises:

(a) the heavy chain variable region shown as SEQ ID NO. 5 and the light chain variable region shown as SEQ ID NO. 6; or

(b) The heavy chain variable region shown as SEQ ID NO. 45 and the light chain variable region shown as SEQ ID NO. 46; or

(c) The heavy chain variable region shown as SEQ ID NO. 47 and the light chain variable region shown as SEQ ID NO. 51; or

(d) The heavy chain variable region shown as SEQ ID NO. 48 and the light chain variable region shown as SEQ ID NO. 52; or

(e) The heavy chain variable region shown as SEQ ID NO. 49 and the light chain variable region shown as SEQ ID NO. 51; or

(f) The heavy chain variable region shown as SEQ ID NO. 50 and the light chain variable region shown as SEQ ID NO. 51; or

(g) The heavy chain variable region shown as SEQ ID NO. 48 and the light chain variable region shown as SEQ ID NO. 51.

10. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:45 and a light chain variable region as set forth in SEQ ID NO: 46.

11. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 47 and a light chain variable region as set forth in SEQ ID No. 51.

12. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID No. 48 and a light chain variable region as set forth in SEQ ID No. 52.

13. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO. 49 and a light chain variable region as set forth in SEQ ID NO. 51.

14. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises the heavy chain variable region as set forth in SEQ ID No. 50 and the light chain variable region as set forth in SEQ ID No. 51.

15. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region as set forth in SEQ ID NO:48 and a light chain variable region as set forth in SEQ ID NO: 51.

16. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 39; and a light chain comprising an amino acid sequence having at least 80% homology with SEQ ID No. 40; or

(b) A heavy chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 53; and a light chain comprising an amino acid sequence having at least 80% homology with SEQ ID No. 55; or

(c) A heavy chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 57; and a light chain comprising an amino acid sequence having at least 80% homology with SEQ ID No. 59; or

(d) A heavy chain comprising an amino acid sequence having at least 80% homology to SEQ ID NO 61; and a light chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 55; or

(e) A heavy chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 62; and a light chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 55; or

(f) A heavy chain comprising an amino acid sequence having at least 80% homology to SEQ ID No. 57; and a light chain comprising an amino acid sequence having at least 80% homology with SEQ ID No. 55.

17. The antibody or antigen-binding fragment thereof of claim 16, wherein the antibody or antigen-binding fragment thereof comprises:

(a) a heavy chain as shown in SEQ ID NO. 39 and a light chain as shown in SEQ ID NO. 40; or

(b) A heavy chain as shown in SEQ ID NO. 53 and a light chain as shown in SEQ ID NO. 55; or

(c) A heavy chain as shown in SEQ ID NO. 57 and a light chain as shown in SEQ ID NO. 59; or

(d) A heavy chain as set forth in SEQ ID NO 61 and a light chain as set forth in SEQ ID NO 55; or

(e) The heavy chain shown as SEQ ID NO. 62 and the light chain shown as SEQ ID NO. 55; or

(f) The heavy chain shown as SEQ ID NO. 57 and the light chain shown as SEQ ID NO. 55.

18. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO. 53 and a light chain as set forth in SEQ ID NO. 55.

19. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO. 57 and a light chain as set forth in SEQ ID NO. 59.

20. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO 61 and a light chain as set forth in SEQ ID NO 55.

21. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO:62 and a light chain as set forth in SEQ ID NO: 55.

22. An isolated antibody or antigen-binding fragment thereof that binds to PD-L1, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO. 57 and a light chain as set forth in SEQ ID NO. 55.

23. The antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, scFv, Fab fragment, Fv fragment, F (ab) 'fragment, F (ab')2 fragment, bispecific antibody, immunoconjugate, or a combination thereof.

24. The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof is linked or conjugated to a therapeutic agent.

25. The antibody or antigen-binding fragment thereof of claim 24, wherein the therapeutic agent is a cytotoxic drug, a radioisotope, an immunomodulator or an antibody.

26. The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof has an affinity of 10nM to 0.1nM for PD-L1.

27. The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof inhibits the binding of PD-L1 to PD-1 with an IC50 that inhibits the binding of PD-L1 to PD-1 of 20ng/mL to 800 ng/mL.

28. The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof inhibits the interaction between cells that express PD-L1 and PD-1 protein, respectively, on their surfaces.

29. The antibody or antigen-binding fragment thereof of any one of claims 1-22, wherein the antibody or antigen-binding fragment thereof has an IC50 of 10ng/mL to 400ng/mL that inhibits the interaction between cells expressing PD-L1 and PD-1 protein, respectively, on a surface.

30. A composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-29 and a pharmaceutically acceptable carrier.

31. An isolated polynucleotide encoding the antibody or antigen-binding fragment thereof of any one of claims 1-29.

32. An expression vector comprising the isolated polynucleotide of claim 31.

33. A host cell comprising the expression vector of claim 32.

34. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-29 for the manufacture of a medicament for promoting T cell activation.

35. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-29 for the manufacture of a medicament for reducing a tumor or inhibiting the growth of tumor cells in a subject.

36. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-29 for the manufacture of a medicament for treating cancer in a subject in need thereof.

37. The use of claim 36, wherein the cancer is selected from the group consisting of: lymphoma, leukemia, melanoma, glioma, breast cancer, lung cancer, intestinal cancer, bone cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, stomach cancer, testicular cancer, salivary gland cancer, thyroid cancer, thymus cancer, epithelial cancer, head or neck cancer, pancreatic cancer, or a combination thereof.

38. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1-29 for the preparation of a medicament for treating an infectious disease in a subject in need thereof, wherein the infectious disease is selected from the group consisting of: candidiasis, aspergillosis, streptococcal pneumonia, streptococcal skin and oropharyngeal conditions, gram-negative sepsis, tuberculosis, mononucleosis, influenza, malaria, schistosomiasis and trypanosomiasis.