RU2778085C2 - Antibody to pd-l1, its antigen-binding fragment, and their pharmaceutical use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: monoclonal antibody to PD-L1, its antigen-binding fragment, its production method, and its pharmaceutical use, as well as a nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment, a recombinant vector, a host cell, a pharmaceutical composition for the treatment of diseases associated with human PD-L1, and a method for the treatment of diseases associated with human PD-L1 are presented.

EFFECT: invention allows for obtaining an antibody to PD-L1 and its antigen-binding fragment, used for the effective treatment of diseases or disorders associated with PD-L1.

14 cl, 5 dwg, 16 tbl, 10 ex

Description

This patent application claims priority under 201810023267.0 filed January 10, 2018, the contents of which are incorporated herein in their entirety.

The technical field to which the invention belongs

The present invention relates to an antibody to PD-L1 and its antigen-binding fragment. In addition, the present invention also relates to a chimeric antibody and a humanized antibody containing the CDR of an anti-PD-L1 antibody, and the present invention also relates to a pharmaceutical composition containing an anti-PD-L1 antibody and an antigen-binding fragment thereof, and its use as a diagnostic agent. and a drug for the treatment of diseases associated with PD-L1.

State of the art

The statements in this document only provide background information relating to the present invention and do not necessarily constitute prior art.

Tumor immunotherapy is a trend in the field of tumor therapy that has been actively developing for a long time, in which T-cell immunotherapy of tumors is the main one. Tumor immunotherapy is the full use and mobilization of killer T cells in patients with tumors to destroy tumors. This may be the most effective and safe way to treat tumors. At the same time, tumor escape from immune surveillance is a huge obstacle to tumor immunotherapy. Tumor cells contribute to the rapid growth of tumors through their own suppressive effect on the immune system.

The relationship between the mechanism of tumor escape from immune surveillance and the body's immune response to the tumor is very complex. In the early stage of tumor immunotherapy, tumor-specific killer T cells were biologically active, but they lost their killer function at a later stage of tumor growth. Therefore, tumor immunotherapy consists in maximizing the response of the patient's own immune system to the tumor. The basic principle of tumor immunotherapy is not only to activate the initial response of the body's immune system, but also to maintain the duration and intensity of the immune response.

There are two systems of signaling pathways for T-cell activation in the human body. In addition to providing a first signal by presenting complexes of MHC molecules with antigenic peptides to T cells by antigen presenting cells, a number of co-stimulatory molecules are also required to provide a second signal in order for T cells to mount a normal immune response. This dual signaling pathway system plays a vital role in balancing the body's immune system and precisely controls the body's initiation of various immune responses to autologous antigen and exogenous antigens. The absence of a second signal provided by the co-stimulatory molecule leads to a lack of response or a slowdown in the specific immune response involving T cells, thus resulting in tolerance. Therefore, the second signaling pathway plays a key regulatory role in the entire immune response process.

In 1992, programmed cell death molecule 1 (PD-1) was discovered to be a protein receptor expressed on the surface of T cells that is involved in cell apoptosis. PD-1 belongs to the CD28 family and shares 23% amino acid homology with cytotoxic T-lymphocyte antigen 4 (CTLA-4), but its expression, unlike CTLA, occurs mainly on activated T cells, B cells, and myeloid cells. . There are two PD-1 ligands, PD-L1 and PD-L2, respectively. PD-L1 is expressed mainly on T cells, B cells, macrophages and dendritic cells (DC), and its expression on activated cells can be increased. Expression of PD-L2 is relatively limited and occurs mainly on antigen-presenting cells such as activated macrophages and dendritic cells.

PD-L1 inhibits the immune system by binding to PD-1 and B7-1. PD-L1 is expressed in many tumor cells and immune cells in the microenvironment of tumor tissue. New studies have found that high expression of the PD-L1 protein has been found in human tumor tissues, such as breast cancer, lung cancer, gastric carcinoma, colon cancer, kidney cancer, melanoma, non-small cell lung cancer, colon cancer. colon, bladder cancer, ovarian cancer, pancreatic cancer, and liver cancer, and PD-L1 expression level is closely related to the clinical situation and prognosis in patients.

Because PD-L1 plays a role in inhibiting T cell proliferation in a second signaling pathway, blocking PD-L1/PD-1 binding has become a promising new target in tumor immunotherapy.

Currently, many international pharmaceutical companies are developing monoclonal antibodies to PD-L1. By blocking PD-L1/PD-1 binding, the response of the patient's own immune system to tumors can be maximized, thereby achieving the goal of killing tumor cells. Below are the corresponding patents: WO0139722, WO2013173223, Wo2014195852, Wo2013181634, Wo2015048520, WO2015036511, US201433093, WO2014100079, Wo201405803192B1, WO2014022758, US8688, US8688, US8668, US8668, US8668, US8668.

Content of the present invention

The present invention provides a monoclonal antibody or antigen-binding fragment (also referred to as a human PD-L1 binding molecule) that binds to the amino acid sequence or three-dimensional structure of the extracellular region of PD-L1.

In some alternative embodiments, the present invention provides a monoclonal antibody, or antigen-binding fragment thereof, that binds to human PD-L1, which comprises:

(i) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 10, 12, and 13, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; where X ₁ is F or M, X ₂ is R or V, and X ₃ is N or H in HCDR2 under SEQ ID NO: 12; or

(ii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NOs: 11, 12, and 13, respectively; and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 having the amino acid sequences of SEQ ID NOs: 14, 15, and 16, respectively; where X ₁ is F or M, X ₂ is R or V, and X ₃ is N or H in HCDR2 under SEQ ID NO: 12; or

(iii) a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NOs: 20, 21 and 22, respectively; and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 23, 24 and 25, respectively; where HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, respectively, do not represent SEQ ID NOs: 30, 38, 22, 23, 40 and 25 at the same time,

where X ₄ is S or D, X ₅ is Y or K, X ₆ is H or M, X ₇ is T, S, H or G, X ₈ is S, N or G, X ₉ is is S, L or G, X ₁₀ is F, L, W or M, and X ₁₁ is A, P or T, X ₁₂ is M, V, L or S, X ₁₃ is F or Y in SEQ ID NOs: 20 and 21, and X ₁₄ is V or A, X ₁₅ is Y or N, X ₁₆ is A, L or V, and X ₁₇ is E, F, Y or A in LCDR2 under SEQ ID NO: 24.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 as defined above comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence of SEQ ID NO: 10 , HCDR2 having the amino acid sequence of SEQ ID NO: 28 or 29, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, the light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 14, 15 and 16, respectively.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 as defined above comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence of SEQ ID NO: 11 , HCDR2 having the amino acid sequence of SEQ ID NO: 28 or 29, and HCDR3 having the amino acid sequence of SEQ ID NO: 13, the light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 14, 15 and 16, respectively.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 as defined above comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence of SEQ ID NO: 30 , HCDR2 having the amino acid sequence of any of SEQ ID NO: 32-37, and HCDR3 having the amino acid sequence of SEQ ID NO: 22, the light chain variable region contains LCDR1 having the amino acid sequence of SEQ ID NO: 23, LCDR2 having the amino acid sequence of any of SEQ ID NOs: 39, 40, 41, 67 and 69, and LCDR3 having the amino acid sequence of SEQ ID NO: 25.

In some embodiments, a monoclonal antibody or antigen-binding fragment thereof that binds to human PD-L1 as defined above comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence of SEQ ID NO: 31 , HCDR2 having the amino acid sequence of any of SEQ ID NOs: 32, 33, 34, 35, 36 and 37, and HCDR3 having the amino acid sequence of SEQ ID NO: 22, the light chain variable region contains LCDR1 having the amino acid sequence of SEQ ID NO: 23, LCDR2 having the amino acid sequence of any of SEQ ID NOs: 39, 40, 41, 67 and 69, and LCDR3 having the amino acid sequence of SEQ ID NO: 25.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 28, and SEQ ID NO: 13, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 14-16, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 28, and SEQ ID NO: 13, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 14-16, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 29, and SEQ ID NO: 13, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NOs: 14-16, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 39 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 34, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 67 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 69 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 41 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 35, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 36, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the heavy chain variable region of a monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 37, and SEQ ID NO: 22, respectively; their light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively.

In some embodiments, the KD value, which characterizes the affinity of the monoclonal antibody or antigen-binding fragment thereof, as defined above, for human PD-L1 is less than 10 ^-9 M or 10 ^-10 M.

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, as defined above, is characterized by cross-linking with cynomolgus or rhesus monkey PD-L1 and/or mouse PD-L1.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 17 and a light chain variable region having the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 19.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 26 and a light chain variable region having the amino acid sequence of SEQ ID NO: 27.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof, as defined above, comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 42 and a light chain variable region having the amino acid sequence of SEQ ID NO: 45.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 and a light chain variable region having the amino acid sequence of SEQ ID NO: 45.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 44 and a light chain variable region having the amino acid sequence of SEQ ID NO: 45.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56, and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 48 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56, and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 52 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 54 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55 and 57.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody, or antigen-binding fragment thereof, as defined above, comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 48 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 52 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66 and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 55, 56, 57, 70 and 72.

In some embodiments, the monoclonal antibody or antigen-binding fragment thereof as defined above comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO: 54 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55, 57, 70 and 72.

In some embodiments, the monoclonal antibody as defined above is a full-length antibody further comprising human antibody constant regions; preferably, the heavy chain constant region of human antibody constant regions is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region of human antibody constant regions is selected from antibody κ- and λ-chain constant regions human and their traditional variants; preferably comprising a human heavy chain constant region having the amino acid sequence of SEQ ID NO: 58, 60, or 65 and a human light chain constant region having the amino acid sequence of SEQ ID NO: 59.

In some embodiments, the monoclonal antibody as defined above is a full length antibody further comprising human antibody constant regions, which comprises the human heavy chain constant region of SEQ ID NO: 58 and the human light chain constant region of SEQ ID NO: 59.

In some embodiments, the monoclonal antibody as defined above is a full-length antibody further comprising human antibody constant regions, which comprises the human heavy chain constant region of SEQ ID NO: 60 and the human light chain constant region of SEQ ID NO: 59.

In some embodiments, the monoclonal antibody as defined above is a full-length antibody further comprising human antibody constant regions, which comprises the human heavy chain constant region of SEQ ID NO: 65 and the human light chain constant region of SEQ ID NO: 59.

In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single chain variable fragment (scFv), dimerized V domain (diabody), Fv stabilized by disulfide bonds (dsFv), and peptides, containing CDRs.

In another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment as defined above, and one or more pharmaceutically acceptable carriers, diluents or excipients; the therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof is preferably a single dose of 0.1-3000 mg/kg of the monoclonal antibody or antigen-binding fragment thereof as defined above.

In some aspects, the present invention provides a nucleic acid molecule encoding a monoclonal antibody or antigennegative fragment, as defined above.

In some aspects, the present invention provides a recombinant vector containing a nucleic acid molecule as defined above.

In some aspects, the present invention provides a host cell transformed with a recombinant vector as defined above, wherein the host cell is selected from a prokaryotic cell and a eukaryotic cell, preferably a eukaryotic cell, more preferably a mammalian cell.

In some aspects, the present invention provides a method for producing a monoclonal antibody or antigen-binding fragment as defined above, wherein the method comprises culturing a host cell as defined above in a medium to produce and accumulate the monoclonal antibody or antigen-binding fragment as defined above, and harvesting the monoclonal antibody or its antigen-binding fragment from culture.

In some aspects, the present invention provides a method for immunodetection or determination of human PD-L1, where the method includes the use of a monoclonal antibody or its antigennegative fragment, as defined above.

In some aspects, the present invention provides the use of a monoclonal antibody, or antigen-binding fragment thereof, as defined above, in the preparation of an agent for diagnosing a disease associated with human PD-L1.

In some aspects, the present invention provides a method of treating diseases associated with human PD-L1, wherein the method comprises administering to a subject a pharmaceutically effective amount of a monoclonal antibody or antigen-binding fragment thereof, as defined above, or a contemplated pharmaceutical composition, as defined above, or a nucleic acid molecule as defined above. above, for the treatment of diseases associated with human PD-L1, where the disease is preferably a tumor or cancer; more preferably squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small cell lymphocytic lymphoma (SLL), T-cell/histiocyte rich large B-cell lymphoma, multiple myeloma, myeloid cell leukemia protein 1 (Mcl-1) associated disease, myelodysplastic syndrome (MDS), gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer , lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer gland, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, pleural malignant mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer and skin cancer; most preferably carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small cell lymphocytic lymphoma (SLL), large cell T cell/histiocyte rich B-cell lymphoma, multiple myeloma, myeloid cell leukemia protein 1 (Mcl-1) associated disease, myelodysplastic syndrome (MDS), gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor , neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer, and PD-L1-positive skin cancer.

In some aspects, the present invention provides the use of a monoclonal antibody or an antigen-binding fragment thereof as defined above, or a contemplated pharmaceutical composition as defined above, or a contemplated nucleic acid molecule as defined above, in the preparation of an agent for the treatment of a disease associated with human PD-L1, wherein the disease is preferably a tumor or cancer; more preferably squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small cell lymphocytic lymphoma (SLL), T-cell/histiocyte rich large B-cell lymphoma, multiple myeloma, myeloid cell leukemia protein 1 (Mcl-1) associated disease, myelodysplastic syndrome (MDS), gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer , lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer gland, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, pleural malignant mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer and skin cancer; most preferably carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small cell lymphocytic lymphoma (SLL), large cell T cell/histiocyte rich B-cell lymphoma, multiple myeloma, myeloid cell leukemia protein 1 (Mcl-1) associated disease, myelodysplastic syndrome (MDS), gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid cancer, melanoma chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor , neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer, and PD-L1-positive skin cancer.

A medicinal product based on a monoclonal antibody or antigen-binding fragment thereof, as defined above, or containing a pharmaceutical composition, as defined above, or a nucleic acid molecule as defined above.

A drug based on a monoclonal antibody or antigen-binding fragment as defined above, or containing a pharmaceutical composition as defined above, or a nucleic acid molecule as defined above, where the drug is used to treat a PD-L1 positive tumor or cancer; wherein the cancer is preferably selected from squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), primary mediastinal large B-cell lymphoma, mantle cell lymphoma (MCL), small cell lymphocytic lymphoma (SLL), T-cell/histiocyte rich large B-cell lymphoma, multiple myeloma, myeloid cell leukemia (Mcl-1) protein 1 associated disease, myelodysplastic syndrome (MDS), gastrointestinal cancer, renal cancer, cancer ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, cancer kidney, prostate cancer, thyroid cancer, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, hepatocellular carcinoma (HCC), clear cell renal cell carcinoma (RCC), head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, pleural malignant mesothelioma, systemic lung amyloidosis chains, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine neoplasm, Merkel cell carcinoma, testicular cancer, and skin cancer.

Brief description of graphic materials

Figure 1 Anti-PD-L1 promotes IFNγ secretion by cells in PBMC T-lymphocyte activation assays.

Figure 2. Comparison of the effect of ADCC in IgG1 and IgG4 forms of different antibodies to PD-L1. Figure 2A is a comparison of HRP00049 IgG1 and IgG4 forms, Figure 2B is a comparison of H5L11 IgG1 and IgG4 forms, Figure 2C is a comparison of HRP00052 IgG1 and IgG4 forms, Figure 2D is a comparison of H6L11 IgG1 and IgG4 forms, and Figure 2E is a comparison H18L61 IgG1 and IgG4 forms, Figure 2F is a comparison of H12L64 IgG1 and IgG4 forms.

Figure 3. Effect of anti-PD-L1 antibody on tumor volume in the A375 xenograft mouse model.

Figure 4. Effect of anti-PD-L1 antibody on tumor volume in a mouse model of colon cancer.

Figure 5. Effect of anti-PD-L1 antibody on tumor volume in xenograft mouse model.

Detailed Description of the Preferred Embodiment

I. Terminology

For a better understanding of the present invention, some technical and scientific terms are specifically defined below. Unless otherwise indicated in this document, all other technical and scientific terms used in this document have the same meaning as is generally understood by specialists in the field of technology to which the present invention pertains.

Three-letter codes and one-letter amino acid codes used in the present invention are described in J. Biol. Chem, 243, p3558 (1968).

The "antibody" described in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure formed by joining two identical heavy chains and two identical light chains through interchain disulfide bonds. The amino acid composition and order of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different. Accordingly, immunoglobulins can be classified into five categories called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains are μ-, δ-, γ-, α- and ε-chains, respectively. The same Ig class can be subdivided into different subclasses according to differences in the amino acid composition of the hinge region, as well as in the number and position of disulfide bonds in the heavy chain. For example, IgG can be subdivided into IgG1, IgG2, IgG3 and IgG4. The light chain is subdivided into kappa chain or lambda chain according to differences in constant regions. Each of the five Ig types can have a kappa chain or a lambda chain.

In the present invention, the light chain of the antibody described in the present invention may further comprise a light chain constant region comprising a human or mouse κ, λ chain, or variants thereof.

In the present invention, the heavy chain of the antibody described in the present invention may further comprise a heavy chain constant region comprising human or mouse IgG1, IgG2, IgG3, IgG4, or variants thereof.

The sequence of approximately 110 amino acids near the N-terminus of the heavy and light chains of an antibody varies considerably and is therefore referred to as the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are therefore referred to as the constant region. The variable region contains three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of an antibody and are also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, which are sequentially located from the amino end to the carboxy end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three light chain CDR regions are called LCDR1, LCDR2 and LCDR3; the three heavy chain CDR regions are called HCDR1, HCDR2 and HCDR3. The number and position of the CDR amino acid residues in the LCVR and HCVR of the antibody or antigen-binding fragment described in the present invention follows the known Kabat numbering rules (LCDR1-3, HCDR1-3).

Antibodies of the present invention include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.

"Monoclonal antibody" refers to an antibody derived from a population of substantially homogeneous antibodies, i. the individual antibodies that make up a population are the same and/or bind to the same epitope, with the exception of possible variants of antibodies (for example, containing naturally occurring mutations or mutations formed during the manufacture of preparations of monoclonal antibodies, and these variants are usually presented in small quantities). Unlike polyclonal antibody preparations, which typically contain different antibodies targeting different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (composition) targets a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the properties of an antibody that is derived from a population of substantially homogeneous antibodies and should not be construed as requiring the manufacture of the antibody by any particular method. For example, the monoclonal antibody of the present invention can be produced by various techniques, including, but not limited to, the hybridoma method, the recombinant DNA method, the phage display method, and the method using transgenic animals containing all or part of human immunoglobulin gene loci, such methods, as well as other illustrative methods for obtaining monoclonal antibodies are described in this document.

The term "mouse antibody" in the present invention means an anti-human PD-L1 monoclonal antibody prepared according to the knowledge and skill in the art. Test subjects are injected with PD-L1 antigen during preparation and hybridomas expressing antibodies with the desired sequence or functionality are isolated. In a preferred embodiment of the present invention, the anti-mouse PD-L1 antibody, or antigen-binding fragment thereof, may further comprise a light chain constant region comprising a mouse κ-, λ-chain or variants thereof, or further comprise a heavy chain constant region of a mouse IgG1, IgG2, IgG3 or her options.

The term "chimeric antibody" means an antibody obtained by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can attenuate the immune response initiated by the mouse antibody. To construct a chimeric antibody, first a hybridoma that secretes a specific mouse monoclonal antibody is created, then the variable region gene is cloned from mouse hybridoma cells, and then the human antibody constant region gene is cloned according to requirements. The mouse variable region gene is linked to the human constant region gene to form a chimeric gene for subsequent insertion into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system. In a preferred embodiment of the present invention, the antibody light chain in the chimeric anti-PD-L1 antibody further comprises a light chain constant region comprising a human κ, λ chain, or a variant thereof. The heavy chain of the antibody in the chimeric anti-PD-L1 antibody further comprises a human IgG1, IgG2, IgG3, IgG4 heavy chain constant region or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region or an IgG1, IgG2 or IgG4 variant with an amino acid mutation ( for example, a YTE mutation or a back mutation).

The term "humanized antibody", which is also referred to as a CDR-grafted antibody, refers to an antibody obtained by grafting a mouse CDR sequence onto a human antibody variable region framework region, that is, an antibody derived from different types of human germline antibody framework region sequences. It can prevent the heterogeneous response initiated by a chimeric antibody that carries a large amount of mouse protein components. Such framework sequences can be obtained from a publicly available DNA database containing germline antibody gene sequences or published literature. For example, germline DNA sequences for the human heavy and light chain variable region genes can be obtained from the VBase human germline gene sequence database (available at www.mrccpe.com.ac.uk/vbase) as well as from Kabat, EA, etc., 1991 Sequences of Proteins of Immunological Interest, 5th edition. To avoid a decrease in activity caused by a decrease in immunogenicity, the human antibody variable region framework sequence can be minimally reverse-mutated or reverse-mutated to maintain activity. Humanized antibodies of the present invention also include humanized antibodies with CDRs subjected to affinity maturation by phage display. In a preferred embodiment of the present invention, a human antibody variable region framework is designed and selected wherein the heavy chain FR sequence in the antibody heavy chain variable region is derived from a combined human germline heavy chain sequence of IGHV3-23*04 and hJH4.1 and a combined light chain sequence human germline type IGKV1-12*01 and hJK4.1. In order to avoid a decrease in activity caused by a decrease in immunogenicity, the variable region of a human antibody can be subjected to a minimal reverse mutation (back mutation, i.e., amino acid residues in the FR from a human antibody are mutated by substitution type with amino acid residues at the corresponding position of the original antibody) in order to keep active.

CDR transplantation can lead to a decrease in the affinity of the resulting anti-PD-L1 antibody or antigen-binding fragment for the antigen due to contact of the framework residues with the antigen. Such interactions may be due to the high frequency of mutations in somatic cells. Therefore, it may still be necessary to transfer such amino acids from the donor framework to the humanized antibody framework. Antigen-binding amino acid residues from a non-human anti-PD-L1 antibody or antigen-binding fragment thereof can be identified by examining the sequence and structure of the variable region of a mouse monoclonal antibody. Residues in the CDR donor framework that are different from the germline type can be considered related. If the closest germline type cannot be determined, the sequence can be compared to a consensus sequence for a subclass or a consensus sequence for a mouse sequence with a high percentage of similarity. The sparse framework region residues are thought to be due to the high mutation rate in somatic cells, thus playing an important role in binding.

The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more antibody fragments that retain the ability to specifically bind an antigen (eg, PD-L1). It has been shown that fragments of a full-length antibody can be used to carry out the antigen-binding function of the antibody. Examples of binding fragments referred to by the term "antigen binding fragment" of an antibody include (i) a Fab fragment, ie. a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab') ₂ fragment - a bivalent fragment containing two Fab fragments connected by a disulfide(disulfide) bridge(s) in the hinge region, (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of a VH domain and a VL domain of one antibody arm; (v) a single domain or dAb fragment (Ward et al. (1989) Nature 341: 544-546) which consists of a VH domain; and (vi) a dedicated complementarity determining region (CDR), or (vii) optionally a combination of two or more individual CDRs connected via a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these genes can be combined using a synthetic linker using recombinant methods, thereby providing a single protein chain, which is a monovalent molecule formed by pairing VL- and VH regions (called single-stranded Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879 -5883). Such scFvs are also intended to be included in the term "antigen binding fragment" of an antibody. Such antibody fragments are prepared using conventional techniques known to those skilled in the art and screened for their functionality in the same manner as intact antibodies. Antigen-binding fragments can be obtained using recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins. The antibodies may be of various isotypes, for example, IgG antibodies (eg, subtypes of IgG1, IgG2, IgG3 or IgG4), IgA1, IgA2, IgD, IgE or IgM.

The antigen binding fragments of the present invention include Fab, F(ab')2, Fab', single chain variable fragment (scFv), dimerized V domain (diabody), disulfide stabilized Fv (dsFv), CDR containing peptides, etc. .

A Fab is an antibody fragment having a molecular weight of approximately 50,000 obtained by treating an IgG antibody molecule with a protease such as papain (cleaves at the amino acid residue at position 224 of the H chain) that has antigen-binding activity of fragments where approximately half of the N-terminal parts of the H-chain and the entire L-chain are connected to each other through disulfide bonds.

The Fab of the present invention can be obtained by treating with papain a monoclonal antibody of the present invention that specifically recognizes and binds to the human PD-L1 extracellular region amino acid sequence or three-dimensional structure thereof. In addition, the Fab can be obtained by inserting the DNA encoding the Fab of the antibody into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector by transformation into a prokaryote or eukaryote to express the Fab.

F(ab')2 is an antibody fragment having a molecular weight of approximately 100,000, obtained by cleavage of the lower portions of two disulfide bonds in the hinge region of IgG with the enzyme pepsin, which has antigen-binding activity and in which two Fab regions are connected at positions in the hinge region. areas.

The F(ab')2 of the present invention can be obtained by treating with pepsin a monoclonal antibody of the present invention that specifically recognizes and binds to the human PD-L1 extracellular region amino acid sequence or its three-dimensional structure. In addition, F(ab')2 can be obtained by connecting Fab', described below, thioether(thioether) bond(s) or disulfide(disulfide) bond(s).

Fab' is an antibody fragment having a molecular weight of approximately 50,000 obtained by cleavage of a disulfide bond in the F(ab')2 hinge region described above, which has antigen-binding activity. The Fab' of the present invention can be obtained by treatment with a reducing agent such as dithiothreitol, F(ab')2 of the present invention, which specifically recognizes and binds to the amino acid sequence of the extracellular region of PD-L1 or its three-dimensional structure.

In addition, a Fab' can be obtained by inserting a DNA encoding a Fab' antibody fragment into a prokaryotic expression vector or a eukaryotic expression vector, and introducing the vector by transformation into a prokaryote or eukaryote to express the Fab'.

The term "single chain variable fragment", "single chain Fv", or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) conjugated with a linker. Such scFv molecules may have the general structure NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. Suitable linkers of the prior art consist of the GGGGS repeat amino acid sequences or variants thereof, for example using 1-4 repeat variants (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA90:6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Euro. J. Immuno l.31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

scFv of the present invention can be obtained using the following steps: obtaining a cDNA encoding VH and VL of a monoclonal antibody of the present invention that specifically recognizes and binds to the amino acid sequence of the human PD-L1 extracellular region or its three-dimensional structure, and constructing a DNA encoding scFv, inserting the DNA into a prokaryotic or eukaryotic expression vector, and then introducing the expression vector by transformation into a prokaryote or eukaryote to express the scFv.

A diabody is an antibody fragment in which scFv is dimerized and has bivalent antigen-binding activity. The two antigens for bivalent antigen-binding activity may be the same or different.

The diabody of the present invention can be prepared using the following steps: obtaining a cDNA encoding VH and VL of a monoclonal antibody of the present invention that specifically recognizes and binds to the amino acid sequence of the human PD-L1 extracellular region or its three-dimensional structure, and constructing a DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less, inserting the DNA into an expression vector in prokaryotes or eukaryotes, and then introducing the expression vector by transformation into a prokaryote or eukaryote to express the diabody.

dsFv is produced by linking a polypeptide in which one amino acid residue in each of VH and VL is replaced by a cysteine residue to form a disulfide bond between the cysteine residues. Amino acid residues to be replaced by cysteine residues can be selected in accordance with the known method (Protein Engineering, 7697(1994)) based on the prediction of the three-dimensional structure of the antibody.

dsFv of the present invention can be obtained using the following steps: obtaining a cDNA encoding VH and VL of a monoclonal antibody of the present invention that specifically recognizes and binds to the amino acid sequence of the extracellular region of human PD-L1 or its three-dimensional structure, and constructs a DNA encoding dsFv, inserting the DNA into an expression vector in prokaryotes or eukaryotes, and then introducing the expression vector by transformation into a prokaryote or eukaryote to express dsFv.

The CDR-containing peptide contains one or more CDRs derived from VH or VL. Several peptides containing CDRs can be conjugated directly or with a suitable peptide linker.

The CDR-containing peptides of the present invention can be prepared using the following steps: constructing a DNA encoding a CDR derived from the VH and VL of a monoclonal antibody of the present invention that specifically recognizes the human PD-L1 extracellular region amino acid sequence or its three-dimensional structure and binds therewith, inserting the DNA into an expression vector in prokaryotes or eukaryotes, and then introducing the expression vector by transformation into a prokaryote or eukaryote to express the peptide. The CDR-containing peptide can also be produced by a chemical synthesis method such as the Fmoc method or the tBoc method.

The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily assist in antigen binding. One of the most widely used definitions of 6 CDRs is given in Kabat E.A. et al. ((1991) Sequences of proteins of immunological interest. NIH Publication 91-3242). As used herein, the Kabat definition of CDR is applicable to CDR1, CDR2 and CDR3 (CDR L1, CDR L2, CDR L3 or L1, L2, L3) of the light chain variable domain and CDR1, CDR2 and CDR3 (CDR H1, CDR H2, CDR H3 or H2, H3) heavy chain variable domain.

The term "antibody framework region" as used herein refers to the portion of the VL or VH variable domain that serves as the framework for the antigen-binding loop (CDR) of the variable domain. In essence, it is a variable domain without a CDR.

A "traditional variant" of a human antibody heavy chain constant region and a human antibody light chain constant region refers to human-derived variants of the heavy chain constant region or light chain constant region in which the structure and function of the antibody variable region, as disclosed in the foregoing, is not altered. the level of technology. Illustrative variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that have undergone site-directed modification and amino acid substitutions in the heavy chain constant region, and such specific substitutions are known in the art: YTE mutations, L234A mutation, and/ or L235A, or an S228P mutation, or a ridge-to-bottom mutation (so that the antibody heavy chain has a combination of Fc ridges and Fc cavities), or a combination of the above mutations. It has been confirmed that such mutations confer novel properties on the antibody, but do not alter the function of the antibody's variable region.

The term "epitope" or "antigenic determinant" refers to the portion of an antigen to which an immunoglobulin or antibody specifically binds (eg, certain portions of the PD-L1 molecule). An epitope typically contains at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 consecutive or non-sequential amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, vol. 66, G. E. Morris, Ed. (1996).

The terms "specific binding", "selective binding", "binding selectively" and "binding specifically" refer to the binding of an antibody to epitopes on a predetermined antigen. Typically, antibodies bind antigens with an affinity (KD) of less than about 10 ^-8 M, such as less than about 10 ^-9 M, 10 ^-10 M, 10 ^-11 M, or less.

The term "KD" or "Kd" refers to the equilibrium dissociation constant for a specific antibody-antigen interaction. Typically, antibodies of the present invention bind PD-L1 with an equilibrium dissociation constant (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less, for example, as measured by a BIACORE instrument using Surface Plasma Resonance (SPR) technology.

The term "competition" when used in the case of antigen-binding proteins competing for the same epitope (such as neutralizing antigen-binding proteins or neutralizing antibodies) means competition between antigen-binding proteins, which is determined by the following analysis. In the assay, the antigen-binding protein (e.g., an antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) the binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., a PD-L1 antigen or its fragment). Numerous types of competitive binding assays can be used to determine if one antigen-binding protein competes with another, such as solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), competitive sandwich assay (see for example, Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct EIA using the biotin-avidin system (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), direct labeled solid phase, direct labeled sandwich solid phase (see, for example, Harlow and Lane, 1988, (Antibodies, A Laboratory Manual), Cold Spring Harbor Press); solid phase RIA with direct labeling using I-125 (see, for example, Morel et al., 1988, Molec. Immunol. 25:7-15); solid phase direct EIA using the biotin-avidin system (see e.g. Cheung, et al., 1990, Virology 176:546-552) and direct labeling RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32 :77-82). Typically, the assay involves the use of purified antigen bound to a solid surface or cell carrying an unlabeled detector antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by measuring the amount of labels that bind to a solid surface or cell in the presence of the antigen binding protein being tested. There is usually an excess of antigen-binding proteins being tested. Antigen-binding proteins identified by competitive analysis (competing antigen-binding protein) include an antigen-binding protein that binds to the same epitope as the reference antigen-binding protein and an antigen-binding protein that binds to an adjacent epitope that is sufficiently close to the epitope bound by the reference. antigen-binding protein, and at the same time each of the two epitopes spatially makes it difficult to bind to the other of them. Additional details regarding methods used to determine competitive binding are presented in the embodiments herein. Typically, if there is an excess of competing antigen-binding proteins, there will be inhibition (e.g., weakening) of the specific binding of the reference antigen-binding protein to the total antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60 -65%, 65-70%, 70-75% or 75% or more. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97% or 97% or more.

The term "nucleic acid molecule" as used herein refers to both DNA molecules and RNA molecules. The nucleic acid molecule may be single or double stranded, preferably double stranded DNA. A nucleic acid is "effectively linked" if it is operatively linked to another nucleic acid sequence. For example, if a promoter or enhancer influences the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop to which other DNA segments can be linked. In another embodiment, the vector is a viral vector in which other DNA segments can be linked to the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors with bacterial origins and episomal vectors for mammalian expression), or can be integrated into the genome of the host cell after introduction. into a host cell by transfection, thereby replicating along with the host genome (eg, non-episomal mammalian expression vectors).

Methods for preparing and purifying antibodies and antigen-binding fragments are well known in the art, such as chapters 5-8 and 15 of Using Antibodies: A Laboratory Manual published by Cold Spring Harbor. For example, a mouse can be immunized with human PD-L1 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acids sequenced using conventional methods. Antigen binding fragments can also be obtained using conventional methods. An antibody or antigen-binding fragment as defined herein is genetically engineered to include one or more human FR regions in a non-human CDR region. The human germline FR sequence can be obtained by aligning the IMGT human germline antibody variable region gene database using the MOE software on the ImMunoGeneTics (IMGT) website http://imgt.cines.fr or from the Journal of Immunoglobulins, 2001ISBN012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria that readily undergo transformation include members of Enterobacteriaceae such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary) and NS0 cells.

The engineered antibodies or antigen-binding fragments of the present invention can be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be introduced into CHO cells by stable transfection. As a more preferred prior art, mammalian cell-based expression systems can induce glycosylation of antibodies, especially in the highly conserved N-terminal portions of the Fc region. Stable clones are obtained by expression of antibodies that specifically bind to human PD-L1. Positive clones were expanded in serum-free medium in a bioreactor to obtain antibodies. The culture medium into which the antibody is secreted can be purified using conventional techniques, for example, on a Sepharose FF conjugated protein A or G column with adjusted buffer. Non-specifically bound components are removed by washing. The bound antibody was then eluted using the pH gradient method and the antibody fragments were detected by SDS-PAGE and pooled. The antibody can be concentrated by conventional filtration. Soluble mixtures and polymers can also be removed by conventional methods such as molecular sieves or ion exchange. The resulting product must be immediately frozen, such as at -70℃, or lyophilized.

When applied to an animal, human, subject, cell, tissue, organ, or biological fluid, the terms "administer" and "treat" refer to contact of an exogenous drug, therapy, diagnostic, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administer" and "treat" may refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Processing a cell includes bringing the reagent into contact with the cell and bringing the reagent into contact with a liquid, where the liquid is in contact with the cell. "Administer" and "treat" also means treating, for example, cells in vitro and ex vivo with an agent, diagnostic agent, binding composition, or other cell. "Treat" when applied to a human subject, veterinary subject, or research subject refers to therapeutic treatment, prophylactic or preventive measures, exploratory and diagnostic uses.

"Therapy" means the administration of an internal or external therapy, such as a composition containing any of the binders of the present invention, to a patient who has one or more symptoms of a disease for which the therapy is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount that provides effective relief of one or more symptoms of the disease in the patient or population being treated, inducing a reduction in the intensity of such symptoms or inhibiting the development of such symptoms to any clinically measurable degree. The amount of a therapy (also referred to as a "therapeutically effective amount") effective in relieving the symptoms of a particular disease may vary depending on a variety of factors such as the condition of the disease, the age and body weight of the patient, and the ability of the drug to be effective so as desired by the patient. Whether symptoms of a disease have improved can be assessed by any clinical testing method that a physician or other healthcare professional routinely uses to assess the severity or progression of symptoms. While embodiments of the present invention (e.g., methods or articles for treatment) may not be effective in alleviating every symptom of the target disease, they should provide symptomatic alleviation of the target disease in a statistically significant number of patients, as confirmed by any of the statistical testing methods known in the art. , such as Student's t-test, chi-square test, Mann-Whitney U-test, Kruskal-Wallis (H-test), Jonkhier-Terpstra test, and Wilcoxon test.

"Conservative modification" or "conservative substitution or substitution" refers to the replacement of amino acids in a protein with other amino acids having similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) so that it was often possible to make changes without changing the biological activity of the protein. Those skilled in the art will recognize that, as a rule, single amino acid substitutions in non-essential regions of a polypeptide do not significantly alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co. ., P224 (4th edition)). In addition, when replacing structurally or functionally similar amino acids, a violation of biological activity is unlikely.

An "effective amount" includes an amount sufficient to reduce or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to enable or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on such factors as the condition being treated, the general health of the patient, the route and dose for administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.

"Exogenous" refers to a substance which, as appropriate, is produced outside the body, cell, or human body. "Endogenous" refers to a substance that is produced in a cell, an organism, or the human body, as appropriate.

"Homology" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. If the positions in the two compared sequences are occupied by the same monomeric subunits representing a base or an amino acid, for example, if each position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared × 100. For example, in an optimal sequence comparison, if 6 out of 10 positions in two sequences are the same or homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Typically, comparisons are made by comparing two sequences to determine the highest percentage of homology.

Used in this document, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such names include their progeny. Thus, the words "transformants" and "transformed cells" include the primary test cells and cultures derived from them, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, all offspring may not have exactly the same DNA content. Included are mutant progeny that have the same functional or biological activity as the cells originally screened among the transformed cells. If different names are meant, their meanings are clearly understood from the context.

As used herein, the term "polymerase chain reaction" or "PCR" refers to a procedure or technique in which a specific amount of nucleic acid, RNA and/or DNA is amplified, as described, for example, in US patent No. 4683195. Typically, you need to get sequence information from or outside the end of the target region so that oligonucleotide primers identical or similar in sequence to the corresponding strands of the template to be amplified can be designed. The 5'-terminal nucleotides of the two primers may coincide with the end of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences derived from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA. See generally Mullis et al. (1987) Cold Spring Harbor Symp. quant. Biol. 51:263; Edited by Erlich, (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid sample to be tested, involving the use of known nucleic acids as primers and nucleic acid polymerases to amplify or obtain specific portions of nucleic acids.

"Optional" or "optional" means that the event or environment described later may or may not occur, and the description includes cases where the event or environment does or does not occur. For example, "optionally containing 1-3 antibody heavy chain variable regions" means that an antibody heavy chain variable region of a particular sequence may be present, but need not be.

"Pharmaceutical composition" means a mixture containing one or more compounds or their physiologically/pharmaceutically acceptable salt or prodrug described herein, with other chemical components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to the body, thereby facilitating the absorption of the active ingredient and displaying its biological activity.

Further, the present invention includes a medicament for treating a disease associated with PD-L1 positive cells, comprising a monoclonal antibody or an antibody fragment thereof of the present invention as an active ingredient.

There is no limitation on diseases associated with PD-L1, provided that it is a disease associated with PD-L1, for example, the therapeutic response induced by the molecules disclosed in the present invention includes binding to human PD-L1 and then blocking the binding of PD -L1 with its PD-1 ligand and B7-1 or killing tumor cells that overexpress PD-L1. Therefore, the molecules of the present invention are highly applicable to those suffering from a tumor or cancer, preferably melanoma, colon cancer, breast cancer, lung cancer, gastric carcinoma, intestinal cancer, renal cancer, non-small cell lung cancer, bladder cancer, etc. .d., in preparations and formulations suitable for therapeutic applications.

In addition, the present invention relates to a method for immunodetection or detection of PD-L1, a reagent for immunodetection or detection of PD-L1, a method for immunodetection or detection of cells expressing PD-L1, and a diagnostic tool for diagnosing diseases associated with PD-L1 positive cells. , which contains a monoclonal antibody or antibody fragment that specifically recognizes human PD-L1 and binds to the amino acid sequence of the extracellular region or its three-dimensional structure, as an active ingredient.

In the present invention, the method for detecting or quantifying PD-L1 may be any known method. For example, it includes a method for immunological detection or measurement.

An immunodetection or detection method is a method for detecting or quantifying an antibody or antigen using a labeled antigen or antibody. Examples of the immunodetection or detection method include the use of radioimmunoassay (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), fluorescent immunoassay, Western blotting, physicochemical method, etc.

The aforementioned diseases associated with PD-L1 positive cells can be diagnosed by detecting or measuring cells expressing PD-L1 using the monoclonal antibodies or antibody fragments thereof of the present invention.

A known immunodetection method, preferably an immunoprecipitation method, a fluorescent cell staining method, an immunohistochemical method, and the like can be used to detect cells expressing a polypeptide. In addition, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystems) can be used.

In the present invention, there is no particular limitation on a living sample for detecting or measuring PD-L1, as long as it is possible to include cells expressing PD-L1, such as tissue cells, blood, blood plasma, blood serum, pancreatic juice, urine, faeces, tissue fluid or culture fluid.

The diagnostic tool containing the monoclonal antibody or antibody fragment thereof of the present invention may further comprise a reagent for carrying out an antigen-antibody reaction or a reagent for testing a reaction in accordance with the diagnostic method, if necessary. Reagents for carrying out an antigen-antibody reaction include buffers, salts, and the like. Detection reagents include those commonly used in immunodetection or detection methods, such as a labeled secondary antibody that recognizes a monoclonal antibody, an antibody fragment or conjugate thereof, and a labeled substrate, and the like.

In the present invention, anti-PD-L1 antibodies with higher affinity, stronger tumor killing activity and lower immunogenicity are obtained by modification.

II. Embodiments and test cases

The present invention is further described below with reference to the embodiments, but these embodiments are not intended to limit the scope of the present invention. Experimental methods without specifying specific conditions in the embodiments of the present invention are generally carried out in accordance with conventional conditions such as those described in Using Antibodies: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; or conditions offered by producers of raw materials or consumer goods. Reagents are commercially available conventional reagents unless otherwise noted.

Embodiment 1: Construction of an Affinity Matured Anti-PD-L1 Anti-PD-L1 Antibody Library from Yeast and Library Validation

In order to obtain better anti-human PD-L1 antibodies, an affinity matured yeast scFv antibody library based on HRP00052 and HRP00049 antibodies was developed and obtained, in which novel anti-human PD-L1 antibodies were screened. All sequences of the CDRs, light chain variable regions and heavy chain variable regions of HRP00052 and HRP00049 were obtained from WO2017084495A1. Specific sequences are shown below.

HRP00049: IgG4 9-2 (H2/L10) (AA) (S228P)

Heavy chain: HRP00049 antibody heavy chain sequence: (SEQ ID NO: 1)

QVQLQESGPGLVKPSQTLSLTCTVSGGSISNDYWTWIRQHPGKGLEYIGYISYTGSTYYNPSLKSRVTISRDTSKNQFSLKLSSVTAADTAVYYCARSGGWLAPFDYWGRGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Gene sequence encoding the heavy chain of the antibody HRP00049: (SEQ ID NO: 2)

CAGGTGCAACTGCAGGAGAGCGGCCCCGGACTCGTGAAACCCTCCCAGACCCTGAGCCTGACCTGTACCGTGAGCGGCGGCAGCATCAGCAACGACTACTGGACTTGGATCAGGCAGCACCCCGGCAAAGGCCTGGAGTACATCGGCTACATCAGCTACACCGGCTCCACCTACTACAACCCCAGCCTGAAGTCCAGGGTGACCATCAGCCGGGACACCAGCAAGAACCAGTTCAGCCTGAAGCTGAGCAGCGTGACCGCTGCCGACACAGCCGTGTACTATTGTGCCAGAAGCGGCGGATGGCTGGCCCCTTTCGACTACTGGGGCAGAGGCACCCTGGTGACCGTGAGCAGC

Light chain: HRP00049 antibody light chain sequence: (SEQ ID NO: 3)

DIVMTQSPDSLAVSLGERATINCKSSQSLFYHSNQKHSLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYGYPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSKVSLSSTLSSKADYACE

Gene sequence encoding the light chain of the antibody HRP00049: (SEQ ID NO: 4)

GACATCGTGATGACCCAGAGCCCTGATAGCCTGGCTGTGAGCCTGGGCGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGTTCTACCATAGCAACCAGAAGCACAGCCTCGCCTGGTATCAGCAGAAGCCCGGCCAACCCCCCAAGCTGCTGATCTACGGCGCCAGCACAAGAGAGAGCGGAGTGCCCGATAGGTTCAGCGGCAGCGGATCCGGCACCGATTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGATGTGGCCGTGTACTACTGCCAGCAGTACTACGGCTACCCTTACACCTTCGGCGGCGGCACCAAGGTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

HRP00052: IgG4 24D5 (GF) (AA) (S228P)

Heavy chain: HRP00052 antibody heavy chain sequence: (SEQ ID NO: 5)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGRIGPNSGFTSYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Gene sequence encoding the heavy chain of the antibody HRP00052: (SEQ ID NO: 6)

CAGGTGCAACTGGTGCAGAGCGGTGCCGAGGTGAAGAAGCCTGGCGCAAGCGTGAAAGTGAGCTGCAAGGCCAGCGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAGGCAGGCCCCTGGACAGGGCCTGGAGTGGATGGGCAGGATCGGGCCCAACAGTGGTTTCACTAGCTACAATGAAAAGTTCAAGAACAGGGTAACCATGACCAGGGACACCTCCACCAGCACAGTGTATATGGAGCTGAGCAGCCTGAGGAGCGAGGACACCGCCGTGTACTACTGTGCCAGAGGCGGCAGCAGCTACGACTACTTCGACTATTGGGGCCAGGGCACCACCGTGACCGTGAGCAGT

Light chain: HRP00052 antibody light chain sequence: (SEQ ID NO: 7)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYAASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTKTLSKADYEKHKSSVY

Gene sequence encoding the light chain of the antibody HRP00052: (SEQ ID NO: 8)

GACATCGTGCTGACCCAGAGTCCCGCCTCACTTGCCGTGAGCCCCGGTCAGAGGGCCACCATCACCTGTAGGGCCAGCGAGAGCGTGAGCATCCACGGCACCCACCTGATGCACTGGTATCAACAGAAACCCGGCCAGCCCCCCAAACTGCTGATCTACGCCGCCAGCAACCTGGAGAGCGGCGTGCCCGCCAGGTTCAGCGGCTCCGGCAGCGGCACCGACTTCACCCTCACTATCAACCCCGTGGAGGCCGAGGACACCGCCAACTACTACTGCCAGCAGAGCTTCGAGGACCCCCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG CGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA

The underlined part of the above antibody sequence indicates the part of the antibody that is its variable region, and the other standardized form represents the part of the antibody that is its constant region.

Yeast Library Construction: Degenerate primers were designed and the designed mutant amino acids were introduced into the HRP00049 and HRP00052 antibody libraries by PCR. The QC libraries were then verified using a second generation sequencing method in which seven 109 capacity yeast antibody libraries were constructed based ^on the sequences HRP00049 and HRP00052.

Embodiment 2: Obtaining an antigen

Human PD-L1-Fc IgG1 fusion protein was designed and synthesized and purified on a Protein A affinity column to provide a high purity recombinant PD-L1-Fc protein to detect anti-PD-L1 antibody binding to antigen.

Human PD-L1-IgG1 Fc: (SEQ ID NO: 9)

MEFGLSWLFLVAILKGVQC FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNEREPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Note: signal peptide + extracellular domain + hIgG1Fc.

Embodiment 3 Antibody Screening

The HRP00052 library used biotinylated human PD-L1 antigen-hIgG1Fc and was subjected to two rounds of MACS screening (streptomycin magnetic beads, Invitrogen) and two rounds of FACS screening (BD FACSAria™ FUSION). Approximately 400 yeast monoclonal cultures were then selected and their expression induced. FACS (BD FACSCanto II) was used to detect the binding of yeast monoclonal antibodies to the human PD-L1 antigen-hIgG1Fc, and a yeast monoclonal antibody with higher affinity than the wild type HRP00052 antibody was selected for sequence confirmation. After comparison and analysis of sequenced clones and deletion of redundant sequences, non-redundant sequences were converted to full-length IgG (γ1, κ) for expression in mammalian cells. Full length antibodies after affinity purification were subjected to affinity determination using BIAcoreTM X-100 (GE Life Sciences).

The HRP00049 library used biotinylated human PD-L1 antigen-hIgG1Fc and biotinylated mouse PD-L1-hIgG1Fc and was subjected to three rounds of MACS screening and three rounds of FACS screening. Approximately 400 yeast monoclonal cultures were then selected and their expression induced. FACS was used to detect binding of yeast monoclonal antibodies to human PD-L1 antigen-hIgG1Fc and to mouse PD-L1 antigen-hIgG1Fc, and for sequence confirmation yeast monoclonal antibodies were selected that combine binding to human PD-L1 antigen-hIgG1Fc and antigen Mouse PD-L1-hIgG1Fc. After removal of redundant sequences, non-redundant sequences were converted to full-length IgG (γ1, κ) for expression in mammalian cells. Full length antibodies after affinity purification were subjected to affinity determination using BIAcore ™ X-100 (GE Life Sciences).

After screening, the sequence of the CDR region of the antibody was chosen.

Antibody from the library of mutant forms HRP00049

Clones selected based on sequences from the HRP00049 mutant library differ from HRP00049 in HCDR1 and HCDR2. The corresponding CDR sequences or their general formulas and their respective heavy chain variable regions are described below.

HCDR1 is DGSAYWS (SEQ ID NO: 10) or NDYWT (SEQ ID NO: 11)

HCDR2 X ₁ ISX ₂ AGSTYX ₃ TPSLKG SEQ ID NO: 12 HDDR3 SGGWLAPFDY SEQ ID NO: 13 LCDR1 KSSQSLFYHSNQKHSLA SEQ ID NO: 14 LCDR2 GASTRES SEQ ID NO: 15 LCDR3 QQYYGYPYT SEQ ID NO: 16

The resulting general formula for the corresponding heavy chain variable region sequence is shown below:

QVQLQESGPGLVKPSQTLSLTCTVSGGSIS DGSAYWS WIRQHPGKGLEYIG X ₁ ISX ₂ AGS TYX ₃ TPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS (SEQ ID NO: 17)

or

QVQLQESGPGLVKPSQTLSLTCTVSGGSIS NDYWT WIRQHPGKGLEYIG X ₁ ISX ₂ AGSTY X ₃ TPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS (SEQ ID NO: 18). X ₁ is selected from F or M, X ₂ is selected from R or V, X ₃ is selected from N or H in the above HCDR2 under SEQ ID NO: 12 and heavy chain variable region under SEQ ID NO: 17 or 18.

The resulting corresponding light chain variable region sequence is shown below: (SEQ ID NO: 19)

DIVMTQSPDSLAVSLGERATINC KSSQSLFYHSNQKHSLA WYQQKPGQPPKLLIY GASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYGYPYT FGGGTKVEIK

Antibody from the library of mutant forms HRP00052

Clones selected based on sequences from the HRP00052 mutant library differ from HRP00052 in HCDR1, HCDR2 and LCDR2. The corresponding CDR sequences or their general formulas and their respective heavy chain variable regions are described below.

HCDR1 X ₄ X ₅ WMX ₆ SEQ ID NO: 20 HCDR2 RIX ₇ PX ₈ X ₉ GX ₁₀ X ₁₁ X ₁₂ YNEKX ₁₃ KN SEQ ID NO: 21 HDDR3 GGSSYDYFDY SEQ ID NO: 22 LCDR1 RASESVSIHGTHLMH SEQ ID NO: 23 LCDR2 X ₁₄ ASX ₁₅ X ₁₆ X ₁₇ S SEQ ID NO: 24 LCDR3 QQSFEDPLT SEQ ID NO: 25

The resulting general formula of the corresponding heavy chain variable region sequence is shown below: (SEQ ID NO: 26)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT X ₄ X ₅ WMX ₆ WVRQAPGQGLEWMG RIX ₇ PX ₈ X ₉ GX ₁₀ X ₁₁ X ₁₂ YNEKX ₁₃ KN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS, in the above region HCDR1 and HCDR2 under variable chain and sub-cable SEQ ID NO: 2 NO0 under heavy chain and SEQ ID NO2: 26 X ₄ selected from S and D, X ₅ selected from Y and K, X ₆ selected from H and M, X ₇ selected from T, S, H and G, X ₈ selected from S, N and G, X ₉ selected from S, L and G, X ₁₀ is selected from F, L, W and M, X ₁₁ is selected from A, P and T, X ₁₂ is selected from M, V, L and S, X ₁₃ is selected from F and Y.

The resulting general formula of the corresponding light chain variable region sequence is shown below: (SEQ ID NO: 27)

DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY X ₁₄ ASX ₁₅ X ₁₆ X ₁₇ S GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIK where X ₁₄ is selected from V and A, X ₁₅ is selected from Y and N, X ₁₆ is selected from A, L and V, and X ₁₇ is selected from E, F, Y and A (including that X ₁₇ is selected from E, F and A and X ₁₇ is selected from Y) in LCDR2 of SEQ ID NO: 24 and the light chain variable region of SEQ ID NO. 27. Specific corresponding sequences obtained include, without limitation, those described in Table 1 and Table 2.

Table 1. Heavy chain variable region sequences determined by affinity screening

Table 2. Light chain variable region sequences determined by affinity screening

Specific sequences of antibody light chain variable regions and heavy chain variable regions obtained from the HRP00049 mutant antibody library:

Heavy chain variable region 9-2 H5 (SEQ ID NO: 42)

QVQLQESGPGLVKPSQTLSLTCTVSGGSIS DGSAYWS WIRQHPGKGLEYIG FISRAGST YNTPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS

Heavy chain variable region 9-2 H6 (SEQ ID NO: 43)

QVQLQESGPGLVKPSQTLSLTCTVSGGSIS NDYWT WIRQHPGKGLEYIG FISRAGSTYN TPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS

Heavy chain variable region 9-2 H7 (SEQ ID NO: 44)

QVQLQESGPGLVKPSQTLSLTCTVSGGSIS NDYWT WIRQHPGKGLEYIG MISVAGSTY HTPSLKG RVTISRDTSKNQFSLKLSSVTAADTAVYYCAR SGGWLAPFDY WGRGTLVTVSS

Light chain variable region 9-2 L11 (SEQ ID NO: 45)

DIVMTQSPDSLAVSLGERATINC KSSQSLFYHSNQKHSLA WYQQKPGQPPKLLIY GAST RES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYYGYPYT FGGGTKVEIK

Specific sequences of antibody light chain variable regions and heavy chain variable regions obtained from the HRP00052 mutant antibody library:

Heavy chain variable region 24D5 H12 (SEQ ID NO: 46)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RITPSSG FAMYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H13 (SEQ ID NO: 47)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RISPSLG LAVYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H14 (SEQ ID NO: 48)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RIHPSL GLPLYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H15 (SEQ ID NO: 49)

QVQLVQSGAEVKKPGASVKVSCKANGYTFT DKWMM WVRQAPGQGLEWMG RITPSS GFAMYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H16 (SEQ ID NO: 50)

QVQLVQSGAEVKKPGASMKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RISPSL GLAVYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H17 (SEQ ID NO: 51)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RIGPNL GWAMYNEKYKN RVTMTRDTSTSTVYMELSSLGSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H18 (SEQ ID NO: 52)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RISPSSG MAVYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H19 (SEQ ID NO: 53)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RISPGG GFTLYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H20 (SEQ ID NO: 54)

QVQLVQSGAEVKKPGASVKVSCKASGYTFT SYWMH WVRQAPGQGLEWMG RIGPNS GFTSYNEKFKN RVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR GGSSYDYFDY WGQGTTVTVSS

Heavy chain variable region 24D5 H21 (SEQ ID NO: 66)

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDKWMMWVRQAPGQGLEWMGRITPSS GFAMYNEKFKNRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGSSYDYFDYWGQGTTVTVSS

Light chain variable region 24D5 L64 (SEQ ID NO: 55)

DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY VASYAA S GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIK

Light chain variable region 24D5 L61 (SEQ ID NO: 56)

DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY AASNLE S GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIK

Light chain variable region 24D5 L66 (SEQ ID NO: 57)

DIVLTQSPASLAVSPGQRATITC RASESVSIHGTHLMH WYQQKPGQPPKLLIY VASNVF S GVPARFSGSGSGTDFTLTINPVEAEDTANYYC QQSFEDPLT FGQGTKLEIK

Light chain variable region 24D5 L67 (SEQ ID NO: 70)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASNVE SGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

Light chain variable region 24D5 L68 (SEQ ID NO: 71)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASNV WSGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

Light chain variable region 24D5 L69 (SEQ ID NO: 72)

DIVLTQSPASLAVSPGQRATITCRASESVSIHGTHLMHWYQQKPGQPPKLLIYVASNVY SGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQQSFEDPLTFGQGTKLEIK

For the light chain variable regions and heavy chain variable regions of the above antibodies, an IgG1 heavy chain/human kappa light chain constant region is selected and combined with each heavy chain variable region and light chain variable region to form a complete antibody heavy chain and a complete antibody light chain. The sequences of the constant region and the light chain constant region are shown below:

IgG1 heavy chain constant region (SEQ ID NO: 58)

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Kappa light chain constant region (SEQ ID NO: 59)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Table 3 Combinations of light chain variable region and heavy chain variable region of intact antibodies derived from mutant HRP00049

Table 4-1. Combinations of light chain variable region and heavy chain variable region of total antibodies derived from mutant HRP00052

Table 4-2. Combinations of light chain variable region and heavy chain variable region of total antibodies derived from mutant HRP00052

In specific embodiments of the present invention, all heavy chain variable regions and light chain variable regions derived from the HRP00049 and HRP00052 mutant antibody libraries described in Table 5-1 and Table 5-2, being associated with heavy chain constant regions and light chain constant regions to form a complete antibody, is a complete antibody formed by coupling with a human IgG1 heavy chain constant region (SEQ ID NO: 58) and a kappa light chain constant region (SEQ ID NO: 59) described above. For example, H15L61 means that the heavy chain is formed by connecting H15 to the IgG1 heavy chain constant region, and the light chain is formed by connecting L61 to the kappa light chain constant region, and the light and heavy chains are linked to form a complete antibody, and other antibodies are named after analogies.

HRP00052-IgG1 means that the full antibody is formed by replacing the heavy chain constant region (IgG4 subclass) of HRP00052 with the above IgG1 heavy chain constant region (SEQ ID NO: 58).

Table 5-1. Names of full-length antibodies obtained by combining heavy chain variable regions with IgG1 heavy chain constant region (SEQ ID NO: 58) and light chain variable regions with kappa light chain constant region (SEQ ID NO: 59)

Table 5-2. Names of full-length antibodies obtained by combining heavy chain variable regions with IgG1 heavy chain constant region (SEQ ID NO: 58) and light chain variable regions with kappa light chain constant region (SEQ ID NO: 59)

For comparison with full antibodies made by combining with a human IgG1 heavy chain constant region, in some specific embodiments, the full length antibodies of Table 6-1 and Table 6-2 are full antibodies formed by combining a heavy chain variable region and a light chain variable region. screened from libraries derived from HRP00049 and HRP00052 above, with the following human IgG4 heavy chain constant region (under SEQ ID NO: 60 containing mutations S228P and 234A235A) and kappa light chain constant region (same as SEQ ID NO: : 59) respectively. For example, H15L61-IgG4 refers to a full antibody formed by joining a heavy chain to a light chain, where the heavy chain is formed by joining H15 to an IgG4 heavy chain constant region, and the light chain is formed by joining L61 to a kappa light chain constant region, and others antibodies were named by analogy.

Human IgG4 heavy chain constant region: (SEQ ID NO: 60)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Table 6-1. Names of full-length antibodies produced by combining heavy chain variable region with IgG4 heavy chain constant region (SEQ ID NO: 60) and light chain variable region with kappa light chain constant region (SEQ ID NO: 59)

Table 6-2. Names of full-length antibodies produced by combining heavy chain variable region with IgG4 heavy chain constant region (SEQ ID NO: 60) and light chain variable region with kappa light chain constant region (SEQ ID NO: 59)

In some specific embodiments, the full length antibodies of Table 7-1 and Table 7-2 are full antibodies formed by combining the heavy chain variable region and the light chain variable region screened from libraries derived from HRP00049 and HRP00052 above, with the following constant region human IgG4 S228P heavy chain (under SEQ ID NO: 65 containing mutation S228P) and kappa light chain constant region (same as SEQ ID NO: 59), respectively. For example, H15L61-IgG4 refers to a full antibody formed by combining a heavy chain with a light chain, where the heavy chain is formed by connecting H15 to the IgG4 heavy chain constant region, and the light chain is formed by connecting L61 to the kappa light chain constant region, and others antibodies were named by analogy. The sequence of the S228P IgG4 heavy chain constant region is shown below: (SEQ ID NO: 65)

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCP P CPAPE FL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Table 7-1. Names of full-length antibodies obtained by combining the heavy chain variable region with the heavy chain constant region of IgG4 S228P (SEQ ID NO: 65) and the light chain variable region with the kappa light chain constant region (SEQ ID NO: 59)

Table 7-2. Names of full-length antibodies obtained by combining the heavy chain variable region with the heavy chain constant region of IgG4 S228P (SEQ ID NO: 65) and the light chain variable region with the kappa light chain constant region (SEQ ID NO: 59)

The specific light chain constant region and heavy chain constant region are not intended to be limiting to the antibody constant regions of the present invention, and other light chain constant regions and heavy chain constant regions and their mutant forms known in the art may also be selected to improve their functional characteristics. .

The anti-PD-L1 antibody avelumab (A09) from Merck and/or 3280A from Genentech was used herein as a positive control, where amino acid sequences based on the light chain and heavy chain amino acid sequences of A09 (obtained from US20140341917) are shown below:

> Heavy chain A09: (SEQ ID NO: 61)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYIMMWVRQAPGKGLEWVSSIYPSGGITFYADTVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARIKLGTVTTVDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

> Light chain A09: (SEQ ID NO: 62)

QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTRVFGTGTKVTVL GQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK

Amino acid sequences based on the 3280A light chain and heavy chain amino acid sequences (Genentech, atezolizumab, WHO Drug Information, Vol. 28, No. 4, 2014, P488) are shown below:

> Heavy chain 3280A: (SEQ ID NO: 63)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

> Light chain 3280A: (SEQ ID NO: 64)

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKTHNKSSVYACEVEKS

Anti-PD-L1 antibodies described above were purified using conventional methods.

All antibodies in the following examples are full length antibodies

Embodiment 4: Antibody Ligand Blocking Test

Additionally, the blocking effect of the product on PD-L1 and PD1 binding was investigated and the product was compared with similar products in clinical trials. The methods refer to Test Examples 2 and 3 of WO2017084495A1 and an anti-human PD-L1 antibody was used to block mouse PD-L1/PD-1 binding in a mouse PD-L1/PD-1 binding blocking assay. See table 8 below for details.

Table 8. Anti-PD-L1 antibody test blocking the binding of PD-L1 to its ligand

*10 µg/mL, 5 µg/mL, and 1 µg/mL are the concentrations of PD-L1 in various blocking assays.

The ligand blocking test confirms that the anti-PD-L1 antibody of the present invention can block the binding of PD-L1 and PD1 as well as the binding of PD-L1 and B7-1, and the H5L11 and H6L11 antibodies obtained by mutation from HRP00049 have cross-linking activity mouse PD-L1 and can block the ability of mouse PD-L1 to bind to PD-1.

Embodiment 5. Antibody affinity test for exemplary antibodies on BIAcore

Following the method outlined in the manual from the Human Antibody Capture Kit (p/n BR-1008-39, GE), a human capture antibody was covalently bound to a CM5 biosensor chip (p/n BR-1000-12, GE) so that some human PD-L1 (cat. no. 10084-H08H, Sino Biological), monkey PD-L1 (cat. no. 90251-C08H, Sino Biological), mouse PD-L1 (cat. no. 50010-M08H, Sino Biological) was subjected to affinity capture. The affinity of the PD-L1 antibody reactive with PD-L1 was measured using a Biacore X100 instrument, GE. In this test, 10X HBS-EP+ buffer solution (Cat. No. BR-1006-69, GE) diluted with D.I. water to 1× (pH 7.4) and also used BIAevaluation software version 4.1, GE to fit the data with a Langmuir model (1:1) to obtain an affinity value. The results are shown in Table 9.

Table 9-1. Binding affinity of HRP00049, HRP00052 and their derived mutant antibodies to different species of PD-L1 determined by Biacore (batch test)

Table 9-2. Binding affinity of HRP00052 and its derived mutant antibodies to different species of PD-L1 determined by Biacore (batch testing)

The results showed that the affinity of antibodies screened from the HRP00052 mutant antibody library (except H21L68) for human PD-L1 was higher than that of HRP00052 and both of the above two antibodies were higher than the positive control antibody, then both H5L11, H6L11 and H7L11 antibodies screened from the HRP00049 mutant antibody library showed strong cross-affinity activity for mouse PD-L1.

Embodiment 6 Cellular IFNγ Secretion by Anti-PD-L1 Antibody in PBMC T-Lymphocyte Activation Assay

In order to investigate the effect of anti-PD-L1 antibody on the function of primary human T-lymphocytes, human peripheral blood mononuclear cells (PBMC) were harvested and purified. After 5 days of in vitro stimulation with tuberculin (TB), the level of secretion of the cytokine IFNγ was determined. The experimental process is briefly described below.

PBMCs were obtained from fresh blood by Ficoll-Hypaque density gradient centrifugation (Stem Cell Technologies) and cultured in RPMI 1640 medium to which 10% (v/v) FBS was added, followed by cultivation at 37°C and 5% CO ₂ .

Freshly isolated and purified PBMCs were prepared as cell suspension at a density of 2×10 ⁶ /ml using RPMI 1640 medium, and 25 μl of tuberculin was added to each 20 ml of cell suspension, followed by cultivation in an incubator at 37°C and 5% CO ₂ for 5 days. On the sixth day, cultured cells were centrifuged, washed once in PBS, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1×10 ⁶ /ml, and plated in 96-well cell culture plates at a volume of 90 μl per well. Simultaneously, gradient dilution antibody samples (diluted with PBS) or an equal amount of IgG isotype control as a blank control were added in a volume of 10 μl per well. The cell culture plate was placed in an incubator and incubated at 37° C. and 5% CO ₂ for 3 days, and then removed and centrifuged (4000 rpm, 10 min.) to collect the cell culture supernatant. ELISA (human IFN-γ test kit, Xinbosheng, EHC102g.96) was used to determine the level of IFN-γ. For information on specific operations, refer to the reagent instructions for use.

The results (see figure 1) show that H18L61, H12L64, H5L11 and H6L11 can strongly stimulate the intracellular secretion of IFNγ. Among them, H12L64, H5L11 and H6L11 show a dose-dependent trend in promoting intracellular secretion of IFNy. All four of the above antibodies are superior to HRP00052-IgG1 at some or all doses.

Embodiment 7 Exemplary Effect of Antibody Mediated ADCC on PD-L1+ Cells

Human whole blood diluted with an equal volume of PBS buffer was added to the bottom of a SepMate tube (STEMCELL Technologies Inc., 15460) with an appropriate amount of Lymphoprep (STEMCELL Technologies Inc., 07851) and centrifuged at 1200 g for 10 minutes at room temperature. Transferring the top liquid from the tube to a new 50 ml centrifuge tube, the cells were washed with PBS buffer and centrifuged at 300 g for 8 minutes to obtain PBMC. The PBMCs were then resuspended in RPMI-1640 medium containing 5% low IgG FBS (BIOSUN, BS-0007-500) and cell counts were performed.

CHO-S/PD-L1 cells were resuspended in RPMI1640 medium containing 5% low-IgG FBS and counted, plated in 96-well plates at a density of 10,000 cells per well, and incubated with anti-PD-L1 antibody in gradient dilution for 15 minutes, then 300,000 human PBMC cells were added to each well and subjected to interaction with the antibody for 4 hours. Media controls such as spontaneous release control from CHO-S/PD-L1 cells, spontaneous release control from PBMC cells, and maximal lysis control from CHO-S/PD-L1 cells were established. After 4 hours, the contents of the 96-well plate were centrifuged, 50 µl of the supernatant from each well was transferred to another 96-well plate, 50 µl of CytoTox 96 reagent for non-radioactive cytotoxicity assay (Promega, G1780) was added to each well and incubated for 30 minutes at room temperature in the dark. A stop solution was added and the absorbance was read at 490 nm. The data was analyzed according to the following formula to calculate the lysis rate. The GraphPad Prism software was used to calculate the EC50 value for the ADCC effect of an antibody as a function of antibody concentration and corresponding lysis rate.

Lysis rate, %=(sample per well _{490 nm} - spontaneous release from PBMC cells _{490 nm} - spontaneous release from CHO-S/PD-L1 cells _{490 nm} ) / (maximum lysis of CHO-S/PD-L1 cells _{490 nm} - spontaneous release from CHO-S/PD-L1 cells _{490 nm} ) × 100

The results show (as shown in Figures 2A-2F) that all IgG1 forms of the various anti-PD-L1 antibodies HRP00049-IgG1, H5L11, HRP00052-IgG1, H6L11, H18L61 and H12L64 have strong ADCC effects, and the ADCC effects of all these molecules are significantly degree superior to the effects of the IgG4 form of the various anti-PD-L1 antibodies.

Embodiment 8 Effect of Exemplary Antibodies in A375 Human Melanoma Xenograft Model

Cultured A375 cells and PBMC were mixed and subcutaneously inoculated into NOG mice. Experiments were divided into PBMC + PBS group, PBMC + 20 mg/kg H12L64 group, PBMC + 20 mg/kg H12L64-IgG4 group, and PBMC + 20 mg/kg HRP00052 group. Six mice in each group received intraperitoneal injection once every two days for 11 consecutive times. After completion of the administration, the therapeutic effect was evaluated using the TGI tumor growth inhibition index (%) and the relative tumor growth index T/C (%).

The results are shown in Figure 3 and Table 10. The TGI in the 20 mg/kg H12L64 group was 98.15% and was significantly different from that in the PBMC + PBS group (p = 0.0018); the TGI tumor inhibition rate in the 20 mg/kg H12L64-IgG4 group was 82.15% and significantly different from that in the PBMC + PBS group (p = 0.0064); the tumor inhibition rate of TGI in the 20 mg/kg HRP00052 group was 68.84% and significantly different from that in the PBMC + PBS group (p = 0.0291). The results show that the 20mg/kg H12L64, 20mg/kg H12L64-IgG4 and 20mg/kg HRP00052 groups can effectively inhibit the growth of A375 (p < 0.05). Throughout the experiment, tumor-bearing mice treated with H12L64, H12L64-IgG4 and HRP00052 did not show weight loss or animal death, indicating that H12L64, H12L64-IgG4 and HRP00052 at this dose were well tolerated by mice.

Table 10 Efficacy of anti-PD-L1 antibodies in the humanized subcutaneous xenograft model A375

%TGI: tumor growth inhibition value; %T/C: tumor growth rate; CR: tumor regression.

Embodiment 9 Effect of Anti-PD-L1 Antibody in MC38-hPD-L1 Colon Cancer Mouse Model

100 µl of MC38-hPD-L1 cells were inoculated (hPD-L1 can be expressed on the cell surface after hPD-L1 injection by transformation into MC38 mouse colon cancer cells, 4×10 ⁵ cells) into the right rib region of 50 C57 mice, and animals with tumors that were too large or too small were excluded. According to an average tumor volume of approximately 52 mm ³ , mice were randomly divided into three separate anti-PD-L1 antibody treatment groups and a negative control group, giving a total of 4 groups of 10 mice per group. After grouping, each drug was administered intraperitoneally (mice in the control group were injected with an equal volume of PBS) three times a day for a total of 12 times. The administration period was 28 days, and monitoring of the tumor-bearing mice was terminated two days after drug cessation. The tumor volume was measured, the mass was measured, and the data were recorded twice a week. See the following table for grouping and introduction. Body weight, tumor volume and tumor weight of animals in each group were characterized using the mean ± standard deviation (mean ± SEM). GraphPad Prism 5 software and Excel were used for plotting, and Student's t-test was used for statistical analysis.

Tumor volume (TV) = 1/2 × L _long × L _short ²

Tumor proliferation rate %T/C = (T - T0) / (C - C0) × 100%

Tumor growth inhibition index %TGI = 1 - %T/C

The results of the experiment (see figure 4) show that both H6L11 and A09 can significantly inhibit the growth of subcutaneously implanted tumors from MC38-hPD-L1. Compared with the negative control group, the two groups demonstrated statistically significant differences in tumor volume 3 days after injection (p < 0.05). At the end of the experiment (27 days after injection), the values of tumor growth inhibition in the two groups were 106.74% and 103.47%, respectively, and differed from the values in the negative control group with high significance (p < 0.001). Approximately 80% - 90% of the tumors could completely regress, and H6L11 has a better tumor suppression effect than A09, but there is no statistically significant difference between the two (see Figure 4). At the end of the experiment, the tumor growth inhibition rate for HRP00052-IgG1 was 29.10%.

After the experiment, tumor-bearing mice were sacrificed, and the tumor was separated and weighed. The results of tumor weighting basically corresponded to the tumor volume: among the three drug administration groups, the average tumor mass in the H6L11 group was the smallest, followed by the A09 group, and the average tumor mass in the HRP00052-IgG1 group was the largest; there was a significant difference between each of the two H6L11 and A09 groups and the control group (p < 0.001). Tumor-bearing mice tolerated various drugs well and had no symptoms such as drug-induced weight loss.

Embodiment 10 Effect of Anti-PD-L1 Antibody in MC38-hPD-L1 Colon Cancer Mouse Model

MC38-hPD-L1 cells were inoculated subcutaneously into C57/BL-6 mice at a density of 5.8×10 ⁵ cells/100 μl/mouse. After constructing a tumor-bearing model, tumor volume was measured, and animals with too large or small body weight and tumor were excluded. Tumor-bearing mice were randomly divided into 2 groups (n=7): IgG-PBS (C25-IgG4) control group, H5L11-IgG4 S228P experimental group according to tumor size, and the date of grouping and administration was set as D0. After grouping, each drug was administered intraperitoneally three times a week for a total of 10 times. The administration period was 18 days, and monitoring of the tumor-bearing mice was terminated two days after drug cessation. The tumor volume was measured, the mass was measured, and the data were recorded twice a week. See the following table for grouping and introduction. Body weight, tumor volume and tumor weight of animals in each group were characterized using the mean ± standard deviation (mean ± SEM). GraphPad Prism 5 software and Excel were used for plotting, and Student's t-test was used for statistical analysis.

Tumor volume (TV) = 1/2 × L _long × L _short ²

Tumor proliferation rate %T/C = (T - T0) / (C - C0) × 100%

Tumor growth inhibition index %TGI = 1 - %T/C

The result, which was shown in Figure 5, was that the tumor volume in the experimental anti-PD-L1 monoclonal antibody administration group (H5L11-IgG4 S228P group), which showed cross-reactivity with mouse PD-L1, was significantly smaller than in control group, and there is a statistically significant difference between the experimental group and the control group from about one week after administration.

After the experiment, tumor-bearing mice were sacrificed, and the tumor was separated and weighed. The results of measuring tumor mass were similar to those for tumor volume. During the experiment, no significant difference in body weight was observed between the administration group and the control group, and each antibody administered was well tolerated by the mice.

Although the above invention has been described in detail using drawings and embodiments for a clear understanding, the description and embodiments are not intended to limit the scope of the present invention. Disclosures of all patents and scientific literature referenced in this document are fully and expressly incorporated by reference.

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SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO., LTD.

SHANGHAI HENGUI PHARMACEUTICAL CO.,LTD

<120> Anti-PD-L1 antibody, antigen-binding fragment thereof, and

their pharmaceutical use

<150> CN201810023267.0

<151> 2018-01-10

<160> 72

<170> SIPOSequenceListing 1.0

<210> 1

<211> 445

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Heavy chain sequence of antibody HRP00049

<400> 1

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 Gly Ser Ile Ser Asn Asp

20 25 30

Tyr Trp Thr 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 Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

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

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

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

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 2

<211> 1338

<212> DNA

<213> Artificial sequence

<220>

<221> gene

<223> Gene sequence encoding the heavy chain of the antibody HRP00049

<400> 2

caggtgcaac tgcaggagag cggccccgga ctcgtgaaac cctcccagac cctgagcctg 60

acctgtaccg tgagcggcgg cagcatcagc aacgactact ggacttggat caggcagcac 120

cccggcaaag gcctggagta catcggctac atcagctaca ccggctccac ctactacaac 180

cccagcctga agtccagggt gaccatcagc cgggacacca gcaagaacca gttcagcctg 240

aagctgagca gcgtgaccgc tgccgacaca gccgtgtact attgtgccag aagcggcgga 300

tggctggccc ctttcgacta ctggggcaga ggcaccctgg tgaccgtgag cagcgcttcc 360

accaagggcc catcggtctt ccccctggcg ccctgctcca ggagcacctc cgagagcaca 420

gccgccctgg gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac 480

tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc ctcaggactc 540

tactccctca gcagcgtggt gaccgtgccc tccagcagct tgggcacgaa gacctacacc 600

tgcaacgtag atcacaagcc cagcaacacc aaggtggaca agagagttga gtccaaatat 660

ggtcccccat gcccaccatg cccagcacct gaggctgctg ggggaccatc agtcttcctg 720

ttccccccaa aacccaagga cactctcatg atctcccggga cccctgaggt cacgtgcgtg 780

gtggtggacg tgagccagga agaccccgag gtccagttca actggtacgt ggatggcgtg 840

gaggtgcata atgccaagac aaagccgcgg gaggagcagt tcaacagcac gtaccgtgtg 900

gtcagcgtcc tcaccgtcct gcaccaggac tggctgaacg gcaaggagta caagtgcaag 960

gtctccaaca aaggcctcc gtcctccatc gagaaaacca tctccaaagc caaagggcag 1020

ccccgagagc cacaggtgta caccctgccc ccatcccagg aggagatgac caagaaccag 1080

gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag 1140

agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1200

tccttcttcc tctacagcag gctcaccgtg gacaagagca ggtggcagga ggggaatgtc 1260

ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacacagaa gagcctctcc 1320

ctgtctctgg gtaaatga 1338

<210> 3

<211> 220

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Light chain sequence of antibody HRP00049

<400> 3

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 4

<211> 663

<212> DNA

<213> Artificial sequence

<220>

<221> gene

<223> Gene sequence encoding the light chain of the antibody HRP00049

<400> 4

gacatcgtga tgacccagag ccctgatagc ctggctgtga gcctgggcga gagagccacc 60

atcaactgca agagcagcca gagcctgttc taccatagca accagaagca cagcctcgcc 120

tggtatcagc agaagcccgg ccaacccccc aagctgctga tctacggcgc cagcacaaga 180

gagagcggag tgcccgatag gttcagcggc agcggatccg gcaccgattt caccctgacc 240

atcagcagcc tgcaggccga ggatgtggcc gtgtactact gccagcagta ctacggctac 300

ccttacacct tcggcggcgg caccaaggtg gagatcaagc gtacggtggc tgcaccatct 360

gtcttcatct tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc 420

ctgctgaata acttctatcc cagaggcc aaagtacagt ggaaggtgga taacgccctc 480

caatcgggta actccgga gagtgtcaca gagcaggaca gcaaggacag cacctacagc 540

ctcagcagca ccctgacgct gagcaaagca gactacgaga aacacaaagt ctacgcctgc 600

gaagtcaccc atcagggcct gagctcgccc gtcacaaaga gcttcaacag gggagagtgt 660

tga 663

<210> 5

<211> 446

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Heavy chain sequence of antibody HRP00052

<400> 5

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

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

115 120 125

Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe

225 230 235 240

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

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val

260 265 270

Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

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

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

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

435 440 445

<210> 6

<211> 1341

<212> DNA

<213> Artificial sequence

<220>

<221> gene

<223> Gene sequence encoding the heavy chain of the antibody HRP00052

<400> 6

caggtgcaac tggtgcagag cggtgccgag gtgaagaagc ctggcgcaag cgtgaaagtg 60

agctgcaagg ccagcggcta caccttcacc agctactgga tgcactggggt gaggcaggcc 120

cctggacagg gcctggagtg gatgggcagg atcgggccca acagtggttt cactagctac 180

aatgaaaagt tcaagaacag ggtaaccatg accagggaca cctccaccag cacagtgtat 240

atggagctga gcagcctgag gagcgaggac accgccgtgt actactgtgc cagaggcggc 300

agcagctacg actacttcga ctattggggc cagggcacca ccgtgaccgt gagcagtgct 360

tccaccaagg gcccatcggt cttccccctg gcgccctgct ccaggagcac ctccgagagc 420

acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 480

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 540

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 600

660

tatggtcccc catgcccacc atgcccagca cctgaggctg ctgggggacc atcagtcttc 720

ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 780

gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 840

gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 900

gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 960

aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1020

cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat gaccaagaac 1080

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1140

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1200

ggctccttct tcctctacag caggctcacc gtggacaaga gcaggtggca ggaggggaat 1260

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1320

tccctgtctc tgggtaaatg a 1341

<210> 7

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Light chain sequence of antibody HRP00052

<400> 7

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 8

<211> 657

<212> DNA

<213> Artificial sequence

<220>

<221> gene

<223> Gene sequence encoding antibody light chain HRP00052

<400> 8

gacatcgtgc tgacccagag tcccgcctca cttgccgtga gccccggtca gagggccacc 60

atcacctgta gggccagcga gagcgtgagc atccacggca cccacctgat gcactggtat 120

caacagaaac ccggccagcc ccccaaactg ctgatctacg ccgccagcaa cctggagagc 180

ggcgtgcccg ccaggttcag cggctccggc agcggcaccg acttcaccct cactatcaac 240

cccgtggagg ccgaggacac cgccaactac tactgccagc agagcttcga ggaccccctg 300

accttcggcc agggcaccaa gctggagatc aagcgtacgg tggctgcacc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aaggcttca acaggggaga gtgttga 657

<210> 9

<211> 401

<212> PROTEIN

<213> Artificial sequence

<220>

<221> PEPTIDE

<223> Human PD-L1-Fc IgG1

<400> 9

Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Cys Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Pro Ala Gly Gln Phe Gln Thr Leu Val Glu Pro Lys Ser Ser Asp Lys

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Lys

<210> 10

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence 1 of HCDR1 antibody HRP00049

<400> 10

Asp Gly Ser Ala Tyr Trp Ser

fifteen

<210> 11

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence 2 of HCDR1 antibody HRP00049

<400> 11

Asn Asp Tyr Trp Thr

fifteen

<210> 12

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> The general sequence formula of the HCDR2 antibody obtained

from yeast antibody display library HRP00049

<220>

<221> UNDETERMINATED

<222> (1)..(1)

<223> Xaa selected from Phe or Met

<220>

<221> UNDETERMINATED

<222> (4)..(4)

<223> Xaa selected from Arg or Val

<220>

<221> UNDETERMINATED

<222> (10)..(10)

<223> Xaa selected from Asn or His

<400> 12

Xaa Ile Ser Xaa Ala Gly Ser Thr Tyr Xaa Thr Pro Ser Leu Lys Gly

1 5 10 15

<210> 13

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 antibody HRP00049

<400> 13

Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr

1 5 10

<210> 14

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 antibody HRP00049

<400> 14

Lys Ser Ser Gln Ser Leu Phe Tyr His Ser Asn Gln Lys His Ser Leu

1 5 10 15

Ala

<210> 15

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 antibody HRP00049

<400> 15

Gly Ala Ser Thr Arg Glu Ser

fifteen

<210> 16

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 antibody HRP00049

<400> 16

Gln Gln Tyr Tyr Gly Tyr Pro Tyr Thr

fifteen

<210> 17

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Generic heavy chain variable domain sequence formula

antibody derived from the HRP00049 Yeast Antibody Display Library

<220>

<221> UNDETERMINATED

<222> (52)..(52)

<223> Xaa selected from Phe or Met

<220>

<221> UNDETERMINATED

<222> (55)..(55)

<223> Xaa selected from Arg or Val

<220>

<221> UNDETERMINATED

<222> (61)..(61)

<223> Xaa selected from Asn or His

<400> 17

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 Gly Ser Ile Ser Asp Gly

20 25 30

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

35 40 45

Tyr Ile Gly Xaa Ile Ser Xaa Ala Gly Ser Thr Tyr Xaa Thr Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 18

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Generic heavy chain variable domain sequence formula

antibody derived from the HRP00049 Yeast Antibody Display Library

<220>

<221> UNDETERMINATED

<222> (50)..(50)

<223> Xaa selected from Phe or Met

<220>

<221> UNDETERMINATED

<222> (53)..(53)

<223> Xaa selected from Arg or Val

<220>

<221> UNDETERMINATED

<222> (59)..(59)

<223> Xaa selected from Asn or His

<400> 18

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 Gly Ser Ile Ser Asn Asp

20 25 30

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

35 40 45

Gly Xaa Ile Ser Xaa Ala Gly Ser Thr Tyr Xaa Thr Pro Ser Leu Lys

50 55 60

Gly 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 Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 19

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region of antibody HRP00049

<400> 19

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 20

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> The general sequence formula of the HCDR1 antibody obtained

from yeast antibody display library HRP00052

<220>

<221> UNDETERMINATED

<222> (1)..(1)

<223> Xaa selected from Ser and Asp

<220>

<221> UNDETERMINATED

<222> (2)..(2)

<223> Xaa selected from Tyr and Lys

<220>

<221> UNDETERMINATED

<222> (5)..(5)

<223> Xaa selected from His and Met

<400> 20

Xaa Xaa Trp Met Xaa

fifteen

<210> 21

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> The general sequence formula of the HCDR2 antibody obtained

from yeast antibody display library HRP00052

<220>

<221> UNDETERMINATED

<222> (3)..(3)

<223> Xaa selected from Thr，Ser，His and Gly

<220>

<221> UNDETERMINATED

<222> (5)..(5)

<223> Xaa selected from Ser，Asn and Gly

<220>

<221> UNDETERMINATED

<222>(6)..(6)

<223> Xaa selected from Ser，Leu and Gly

<220>

<221> UNDETERMINATED

<222>(8)..(8)

<223> Xaa selected from Phe，Leu，Trp and Met

<220>

<221> UNDETERMINATED

<222>(9)..(9)

<223> Xaa selected from Ala，Pro and Thr

<220>

<221> UNDETERMINATED

<222> (10)..(10)

<223> Xaa selected from Met，Val，Leu and Ser

<220>

<221> UNDETERMINATED

<222> (15)..(15)

<223> Xaa selected from Phe and Tyr

<400> 21

Arg Ile Xaa Pro Xaa Xaa Gly Xaa Xaa Xaa Tyr Asn Glu Lys Xaa Lys

1 5 10 15

Asn

<210> 22

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence of HCDR3 antibody HRP00052

<400> 22

Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr

1 5 10

<210> 23

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR1 sequence of antibody HRP00052

<400> 23

Arg Ala Ser Glu Ser Val Ser Ile His Gly Thr His Leu Met His

1 5 10 15

<210> 24

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General sequence formula of the LCDR2 antibody obtained

from yeast antibody display library HRP00052

<220>

<221> UNDETERMINATED

<222> (1)..(1)

<223> Xaa selected from Val and Ala

<220>

<221> UNDETERMINATED

<222> (4)..(4)

<223> Xaa selected from Tyr and Asn

<220>

<221> UNDETERMINATED

<222> (5)..(5)

<223> Xaa selected from Ala，Leu and Val

<220>

<221> UNDETERMINATED

<222>(6)..(6)

<223> Xaa selected from Glu，Phe，Trp, Tyr and Ala

<400> 24

Xaa Ala Ser Xaa Xaa Xaa Ser

fifteen

<210> 25

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR3 sequence of antibody HRP00052

<400> 25

Gln Gln Ser Phe Glu Asp Pro Leu Thr

fifteen

<210> 26

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Generic heavy chain variable domain sequence formula

antibody derived from the HRP00052 Yeast Antibody Display Library

<220>

<221> UNDETERMINATED

<222> (31)..(31)

<223> Xaa selected from Ser and Asp

<220>

<221> UNDETERMINATED

<222> (32)..(32)

<223> Xaa selected from Tyr and Lys

<220>

<221> UNDETERMINATED

<222> (35)..(35)

<223> Xaa selected from His and Met

<220>

<221> UNDETERMINATED

<222> (52)..(52)

<223> Xaa selected from Thr，Ser，His and Gly

<220>

<221> UNDETERMINATED

<222> (54)..(54)

<223> Xaa selected from Ser，Asn and Gly

<220>

<221> UNDETERMINATED

<222> (55)..(55)

<223> Xaa selected from Ser，Leu and Gly

<220>

<221> UNDETERMINATED

<222> (57)..(57)

<223> Xaa selected from Phe，Leu，Trp and Met

<220>

<221> UNDETERMINATED

<222> (58)..(58)

<223> Xaa selected from Ala，Pro and Thr

<220>

<221> UNDETERMINATED

<222> (59)..(59)

<223> Xaa selected from Met，Val，Leu and Ser

<220>

<221> UNDETERMINATED

<222> (64)..(64)

<223> Xaa selected from Phe and Tyr

<400> 26

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

1 5 10 15

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

20 25 30

Trp Met Xaa Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Xaa Pro Xaa Xaa Gly Xaa Xaa Xaa Tyr Asn Glu Lys Xaa

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 27

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> General formula of the light chain variable domain sequence

antibody derived from the HRP00052 Yeast Antibody Display Library

<220>

<221> UNDETERMINATED

<222> (54)..(54)

<223> Xaa selected from Val and Ala

<220>

<221> UNDETERMINATED

<222> (57)..(57)

<223> Xaa selected from Tyr and Asn

<220>

<221> UNDETERMINATED

<222> (58)..(58)

<223> Xaa selected from Ala，Leu and Val

<220>

<221> UNDETERMINATED

<222> (59)..(59)

<223> Xaa selected from Glu，Phe，Trp，Tyr and Ala

<400> 27

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Xaa Ala Ser Xaa Xaa Xaa Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 28

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 9-2 H5

<400> 28

Phe Ile Ser Arg Ala Gly Ser Thr Tyr Asn Thr Pro Ser Leu Lys Gly

1 5 10 15

<210> 29

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 9-2 H7

<400> 29

Met Ile Ser Val Ala Gly Ser Thr Tyr His Thr Pro Ser Leu Lys Gly

1 5 10 15

<210> 30

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Sequence HCDR1 24D5 H12

<400> 30

Ser Tyr Trp Met His

fifteen

<210> 31

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR1 sequence 24D5 H15

<400> 31

Asp Lys Trp Met Met

fifteen

<210> 32

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 24D5 H12

<400> 32

Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 33

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 24D5 H13

<400> 33

Arg Ile Ser Pro Ser Leu Gly Leu Ala Val Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 34

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 24D5 H14

<400> 34

Arg Ile His Pro Ser Leu Gly Leu Pro Leu Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 35

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 24D5 H17

<400> 35

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

1 5 10 15

Asn

<210> 36

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 24D5 H18

<400> 36

Arg Ile Ser Pro Ser Ser Gly Met Ala Val Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 37

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR2 sequence 24D5 H19

<400> 37

Arg Ile Ser Pro Gly Gly Gly Phe Thr Leu Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 38

<211> 17

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> HCDR3 sequence 24D5 H20

<400> 38

Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

<210> 39

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 sequence 24D5 L64

<400> 39

Val Ala Ser Tyr Ala Ala Ser

fifteen

<210> 40

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 sequence 24D5 L61

<400> 40

Ala Ala Ser Asn Leu Glu Ser

fifteen

<210> 41

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 sequence 24D5 L66

<400> 41

Val Ala Ser Asn Val Phe Ser

fifteen

<210> 42

<211> 120

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 9-2 H5

<400> 42

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 Gly Ser Ile Ser Asp Gly

20 25 30

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

35 40 45

Tyr Ile Gly Phe Ile Ser Arg Ala Gly Ser Thr Tyr Asn Thr Pro Ser

50 55 60

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

65 70 75 80

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

85 90 95

Cys Ala Arg Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 43

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 9-2 H6

<400> 43

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 Gly Ser Ile Ser Asn Asp

20 25 30

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

35 40 45

Gly Phe Ile Ser Arg Ala Gly Ser Thr Tyr Asn Thr Pro Ser Leu Lys

50 55 60

Gly 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 Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 44

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 9-2 H7

<400> 44

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 Gly Ser Ile Ser Asn Asp

20 25 30

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

35 40 45

Gly Met Ile Ser Val Ala Gly Ser Thr Tyr His Thr Pro Ser Leu Lys

50 55 60

Gly 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 Ser Gly Gly Trp Leu Ala Pro Phe Asp Tyr Trp Gly Arg Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 45

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 9-2 L11

<400> 45

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Tyr His

20 25 30

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

35 40 45

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

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210> 46

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H12

<400> 46

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 47

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H13

<400> 47

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ser Pro Ser Leu Gly Leu Ala Val Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 48

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H14

<400> 48

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile His Pro Ser Leu Gly Leu Pro Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 49

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H15

<400> 49

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

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Asn Gly Tyr Thr Phe Thr Asp Lys

20 25 30

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

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 50

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H16

<400> 50

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ser Pro Ser Leu Gly Leu Ala Val Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 51

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H17

<400> 51

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

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

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Gly Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 52

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H18

<400> 52

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ser Pro Ser Ser Gly Met Ala Val Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 53

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H19

<400> 53

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Ser Pro Gly Gly Gly Phe Thr Leu Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 54

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H20

<400> 54

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

1 5 10 15

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

20 25 30

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Arg Ile Gly Pro Asn Ser Gly Phe Thr Ser Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 55

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 24D5 L64

<400> 55

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 56

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 24D5 L61

<400> 56

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 57

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 24D5 L66

<400> 57

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 58

<211> 330

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> IgG1 heavy chain constant region

<400> 58

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

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

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

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

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

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

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

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

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 59

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Kappa light chain constant region

<400> 59

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 60

<211> 327

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Human IgG4 heavy chain constant region

<400> 60

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

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

210 215 220

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

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

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

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 61

<211> 450

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Avelumab heavy chain sequence (A09)

<400> 61

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser Ser Ile Tyr Pro Ser Gly Gly Ile Thr Phe Tyr Ala Asp Thr Val

50 55 60

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

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Gly Thr Leu 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> 62

<211> 216

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> Avelumab light chain sequence (A09)

<400> 62

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

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

20 25 30

Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Ile Tyr Asp Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Ser Thr Arg Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

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

165 170 175

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

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys Ser

210 215

<210> 63

<211> 448

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> 3280A heavy chain sequence (Genentech)

<400> 63

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

<210> 64

<211> 214

<212> PROTEIN

<213> Artificial sequence

<220>

<221> CHAIN

<223> 3280A light chain sequence (Genentech)

<400> 64

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 Val Ser Thr Ala

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr 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 Tyr Leu Tyr His Pro Ala

85 90 95

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

100 105 110

Pro Ser Val Phe Ile Phe 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> 65

<211> 327

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Human IgG4 S228P heavy chain constant region

<400> 65

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro

100 105 110

Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

115 120 125

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

130 135 140

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

145 150 155 160

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

165 170 175

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

180 185 190

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

195 200 205

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

210 215 220

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

225 230 235 240

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

245 250 255

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

260 265 270

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

275 280 285

Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser

290 295 300

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

305 310 315 320

Leu Ser Leu Ser Leu Gly Lys

325

<210> 66

<211> 119

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Heavy chain variable region 24D5 H21

<400> 66

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Arg Ile Thr Pro Ser Ser Gly Phe Ala Met Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Gly Ser Ser Tyr Asp Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 67

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 24D5 L67

<400> 67

Val Ala Ser Asn Val Glu Ser

fifteen

<210> 68

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 24D5 L68

<400> 68

Val Ala Ser Asn Val Trp Ser

fifteen

<210> 69

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> LCDR2 24D5 L69

<400> 69

Val Ala Ser Asn Val Tyr Ser

fifteen

<210> 70

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 24D5 L67

<400> 70

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 71

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 24D5 L68

<400> 71

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Trp Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 72

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<221> DOMAIN

<223> Light chain variable region 24D5 L69

<400> 72

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

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Ile His

20 25 30

Gly Thr His Leu Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Val Ala Ser Asn Val Tyr Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Ser Phe

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<---

Claims (40)

1. A monoclonal antibody or antigen-binding fragment thereof, wherein the monoclonal antibody or antigen-binding fragment thereof binds to human PD-L1 and contains a heavy chain variable region and a light chain variable region, where: the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 10, SEQ ID NO: 28, and SEQ ID NO: 13, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 28, and SEQ ID NO: 13, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 29, and SEQ ID NO: 13, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 39 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 34 and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 67 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 69 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 31, SEQ ID NO: 32, and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 41 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 35 and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 36 and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively; or the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 37 and SEQ ID NO: 22, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively, or the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 having the amino acid sequences of SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 22, respectively; the light chain variable region contains LCDR1, LCDR2 and LCDR3 having the amino acid sequences of SEQ ID NO: 23, SEQ ID NO: 40 and SEQ ID NO: 25, respectively. 2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody or antigen-binding fragment comprises: a heavy chain variable region having the amino acid sequence of SEQ ID NO: 42 and a light chain variable region having the amino acid sequence of SEQ ID NO: 45; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 43 and a light chain variable region having the amino acid sequence of SEQ ID NO: 45; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 44 and a light chain variable region having the amino acid sequence of SEQ ID NO: 45; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 46 and a light chain variable region having the amino acid sequence of any of SEQ ID NOs: 55 and 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 47 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 48 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 49 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 50 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 51 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 52 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 53 and a light chain variable region having the amino acid sequence of SEQ ID NO: 56; or a heavy chain variable region having the amino acid sequence of SEQ ID NO: 66; and a light chain variable region having the amino acid sequence of any of SEQ ID NO: 57, 70, and 72. 3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody is a full length antibody. 4. The monoclonal antibody or antigen-binding fragment thereof according to claim 3, wherein the full length antibody comprises a human heavy chain constant region of SEQ ID NO: 58, 60, or 65 and a human light chain constant region of SEQ ID NO: 59. 5. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') ₂ , single-chain variable fragment, dimerized V domain, Fv stabilized by disulfide bonds, and peptides containing CDRs. 6. Pharmaceutical composition for the treatment of diseases associated with human PD-L1, containing a therapeutically effective amount of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-5 and one or more pharmaceutically acceptable carriers, diluents or excipients. 7. A nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-5. 8. A recombinant vector containing a nucleic acid molecule according to claim 7 for the expression of a monoclonal antibody or antigen-binding fragment according to any one of claims. 1-5. 9. A host cell transformed with a recombinant vector according to claim 8 to express a monoclonal antibody or antigen-binding fragment according to any one of claims. 1-5, wherein the host cell is a prokaryotic cell or a eukaryotic cell. 10. The host cell of claim 9, wherein the host cell is a mammalian cell. 11. The method of obtaining a monoclonal antibody or antigennegative fragment according to any one of paragraphs. 1-5, where the method includes culturing a host cell according to claim 9 in an environment with the production and accumulation of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-5 and harvesting the monoclonal antibody or antigen-binding fragment thereof from the culture. 12. The use of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-5 upon receipt of an agent for diagnosing diseases associated with human PD-L1. 13. A method of treating diseases associated with human PD-L1, where the method includes administering to the subject a pharmaceutically effective amount of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-5, wherein the disease is selected from the group consisting of PD-L1 positive squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B cell lymphoma , follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histiocytes , multiple myeloma, protein 1 associated myeloid cell leukemia, myelodysplastic syndrome, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, cancer thyroid, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer , clear cell renal cell carcinoma, head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine neoplasm, carcinoma from Merkel cells, testicular cancer and skin cancer. 14. The use of a monoclonal antibody or antigen-binding fragment according to any one of paragraphs. 1-5, wherein the disease is selected from the group consisting of PD-L1 positive squamous cell carcinoma, myeloma, small cell lung cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, glioma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B cell lymphoma , follicular lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, large B-cell lymphoma rich in T-cells / histiocytes , multiple myeloma, protein 1 associated myeloid cell leukemia, myelodysplastic syndrome, gastrointestinal cancer, renal cancer, ovarian cancer, liver cancer, lymphoblastic leukemia, lymphocytic leukemia, colorectal cancer, endometrial cancer, kidney cancer, prostate cancer, cancer thyroid, melanoma, chondrosarcoma, neuroblastoma, pancreatic cancer, glioblastoma multiforme, gastric carcinoma, bone cancer, Ewing's sarcoma, cervical cancer, brain cancer, gastric carcinoma, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer , clear cell renal cell carcinoma, head and neck cancer, laryngeal cancer, liver and biliary tract cancer, central nervous system cancer, esophageal cancer, malignant pleural mesothelioma, systemic light chain amyloidosis, lymphoplasmacytic lymphoma, myelodysplastic syndrome, myeloproliferative tumor, neuroendocrine neoplasm, carcinoma from Merkel cells, testicular cancer and skin cancer.

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