Anti-PD-L1 antibodies and their applications

Abstract

This invention provides an antibody or fragment thereof targeting PD-L1, a nucleic acid encoding the antibody or fragment thereof, and a composition comprising the antibody or fragment thereof, as well as their use in treating diseases. The antibody or fragment thereof provided by this invention can specifically bind to human PD-L1, block the binding of PD-L1 and PD-1, exhibit a strong ADCC effect on target cells, and significantly inhibit tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a novel anti-PD-L1 antibody or a functional fragment thereof. This invention also relates to the application of said antibody or its functional fragment. Background Technology

[0002] Programmed death factor ligand 1 (PD-L1), also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1), belongs to the B7 family and is encoded by the CD274 gene. The mature PD-L1 protein is 40 kDa in size and is a type I transmembrane protein composed of 272 amino acids. It is induced to be expressed on the surface of activated T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow-derived mast cells, and non-hematopoietic cells, and is widely expressed in tumor tissues, such as lung cancer, liver cancer, and bladder cancer (Dong H, Strome SE, Salomao DR et al. Tumor-associated B7-H1 promotes T-cell apoptosis: A potential mechanism of immune evasion. Nature Medicine 2002, 8(8):793–800.). It may be rapidly upregulated in tumor tissues and other tissues that respond to interferon and other inflammatory factors.

[0003] The receptor for PD-L1 is programmed death protein 1 (PD-1), also known as CD279, a member of the CD28 family of T cell receptors, expressed on the surface of various immune cells, such as activated T cells, B cells, and monocytes. Mature human PD-1 protein is a type I transmembrane protein composed of 268 amino acids, with its cytoplasmic tail region containing the immunorepressive tyrosine motif (ITIM) and the immunorepressive tyrosine switching motif (ITSM). When PD-L1 on the surface of antigen-presenting cells (APCs) binds to the receptor PD-1 on the surface of T cells, it induces phosphorylation of tyrosine residues in the ITSM domain of PD-1, thereby affecting the activation of the PI3K-AKT-mTOR and RAS-MEK-ERK pathways. Ultimately, this inhibits the proliferation of antigen-specific T cells and induces apoptosis of regulatory T cells by downregulating Bcl-2 gene expression, thus reducing the T cell-mediated immune response. Furthermore, PD-L1 can also bind to another T cell co-stimulatory molecule, B7-1 (CD80). Upon encountering PD-L1, CD80 acts as a receptor and inhibits T cell activation signaling. Additionally, PD-L1 can also act as a receptor to "reverse" transmit signals to T cells and tumor cells, thereby affecting the survival of these cells; the mechanism of this effect is not yet fully understood. Therefore, PD-L1 can perform immunomodulatory functions as both a ligand and a receptor.

[0004] Another ligand of PD-1 is PD-L2 (CD273, B7-DC). When the two bind, they also inhibit the activation and proliferation of T cells. However, compared with PD-L1, PD-L2 has a narrower expression range and is mainly expressed on antigen-presenting cells (such as macrophages and dendritic cells) and TH2 cells. The expression of PD-L2 is not as widespread among different tumor types as PD-L1, and the basal expression level of PD-L2 is low.

[0005] More importantly, multiple studies have shown a negative correlation between PD-L1 expression levels in tumors and patient survival, leading to the rapid clinical development of PD-1 / PD-L1 targeted therapy for cancer. Furthermore, inhibitors targeting PD-L1 have a potential advantage over PD-1 inhibitors: the PD-L2 signaling pathway remains unaffected. Since PD-L2 is rarely highly expressed in tumor tissues and can interact with rejection guide molecule B (RGMb), Chen et al. (Chen L, Han X. Anti–PD-1 / PD-L1 therapy of human cancer: past, present, and future. J ClinInvest. 2015, 125(9):3384–3391.) found that this interaction can reduce the incidence of severe inflammatory lung diseases such as interstitial lung disease (ILD).

[0006] In addition, antibodies targeting PD-L1 with the IgG1 subtype can theoretically not only block the interaction between PD-1 and PD-L1 on tumors, but may also mediate ADCC (antibody-dependent cell-mediated cytotoxicity) lysis of tumor cells. Furthermore, the high expression of PD-L1 on tumors makes it a potential target for the development of bispecific antibodies.

[0007] Currently, six PD-1 / PD-L1 inhibitors are available globally, including Merck's Keytruda, Bristol-Myers Squibb's Opdivo, Roche's Tecentriq, Pfizer / Merck's Bavencio, AstraZeneca's Imfinzi, and Sanofi / Regeneron's Libtayo. Among them, Roche's humanized anti-PD-L1 antibody Tecentriq (Atezolizumab) uses a weak ADCC and CDC activity IgG1 subtype and is clinically used to treat locally advanced or metastatic non-small cell lung cancer (NSCLC) and locally advanced or metastatic urothelial carcinoma (mUC). It can be combined with chemotherapy drugs (carboplatin and etoposide) for first-line treatment of extensive-stage small cell lung cancer (ES-SCLC), and with chemotherapy drug (albumin-bound paclitaxel) for first-line treatment of PD-L1-positive unresectable locally advanced or metastatic triple-negative breast cancer (TNBC). It can also be combined with bevacizumab. Paclitaxel and carboplatin are used as first-line treatments for adult metastatic non-squamous non-small cell lung cancer (NSCLC). Pfizer / Merck's fully human anti-PD-L1 antibody Bavencio (Avelumab) uses a strong ADCC-active IgG1 subtype and is clinically used to treat metastatic Merkel cell carcinoma and urothelial carcinoma. AstraZeneca's fully human anti-PD-L1 antibody Imfinzi (Durvalumab) uses a weak ADCC-active IgG1 subtype and is clinically used to treat unresectable stage III NSCLC and locally advanced or metastatic urothelial carcinoma (mUC). There are more than 10 anti-PD-L1 monoclonal antibodies in clinical trials in China, with indications covering cervical cancer, osteosarcoma, urothelial carcinoma, head and neck squamous cell carcinoma, stage IV non-squamous or squamous non-small cell lung cancer with high PD-L1 expression that has not previously received chemotherapy, stage III extensive-stage small cell lung cancer, breast cancer, primary liver cancer, gastric cancer, and prostate cancer.

[0008] Currently available PD-L1-targeting antibody drugs have limited indications, require large dosages, and have low overall response rates. Therefore, there is still room for further optimization of these antibodies, and the field still needs to develop more novel anti-PD-L1 antibodies. Summary of the Invention

[0009] This invention provides an anti-PD-L1 antibody through hybridoma screening, humanization technology, and affinity maturation. The antibody has high affinity for human PD-L1, as well as high biological activity, strong ADCC activity, and a long in vivo half-life.

[0010] The technical solution of the present invention is as follows.

[0011] On the one hand, the present invention provides an antibody or a fragment thereof, wherein the fragment of the antibody described in the present invention refers to a functional or active fragment of the antibody that binds to the antigen PD-L1.

[0012] The PD-L1 refers to programmed death factor ligand 1. Preferably, the PD-L1 is the PD-L1 of primates, more preferably the PD-L1 of humans or cynomolgus monkeys.

[0013] In this invention, the affinity of the antibody or its fragment for binding to the antigen PD-L1 can be measured by methods known in the art, and preferably by measuring K. D The dissociation constant is a value expressed as a molar concentration. Methods for measuring the binding affinity between antibodies and antigens are well-known in the art, including, for example, ELISA, flow cytometry, surface plasmon resonance, and Biacore measurements. Depending on the measurement method and its specific settings, the measured binding affinity may vary slightly or remain within an acceptable range.

[0014] According to a specific embodiment of the present invention, the K D The values ​​were measured using a Fortebio Octet QKe system instrument. See the experimental setup in the examples for details.

[0015] In this invention, preferably, the antibody or its fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) respectively comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 selected from the amino acid sequences shown in (I) to (VI) below:

[0016] (1) HCDR1: SEQ ID NO: 36 (DIYMH), SEQ ID NO: 37 (SIYMH), SEQ ID NO: 38 (SYYMH), SEQ ID NO: 39 (DIYIS), SEQ ID NO: 40 (DYYMH);

[0017] (II)HCDR2:SEQ ID NO:41(RIDPANGNTKYDPKFQD),SEQ ID NO:42(RIDAGNGNTKYDPKFQD),SEQ ID NO:43(RIDPRNGNTKYDPKFQD),SEQ ID NO:44(RIDPANANTKYDPKFQD),SEQ ID NO:45(RIDVLNANTKYDPKFQD),SEQ ID NO:46(RIDVRNGNTKYDPKFQD) NO:47(RIDPAAGNTKYDPKFQD),SEQ ID NO:48(RIDSNAGNTKYDPKFQD),SEQ ID NO:49(RIDLANANTKYDPKFQD),SEQ ID NO:50(RIDRAAGNTKYDPKFQD),SEQ ID NO:51(RIDPRNGNTKYDPKFQD);

[0018] (III)HCDR3:SEQ ID NO:52(GQLGPLGFDY),SEQ ID NO:53(GQAGSLGFDY),SEQ ID NO:54(GQLASLGFDY),SEQ ID NO:55(GQVGMLGFDY),SEQ ID NO:56(GRLGSLGFDY);

[0019] (IV)LCDR1: SEQ ID NO:57(RASQDISNYLN), SEQ ID NO:58(RASQDISYLN), SEQ ID NO:59(RASQSISSYLN), SEQ ID NO:60(RASQDISSYLN);

[0020] (V)LCDR2:SEQ ID NO:61(YTSRLHS),SEQ ID NO:62(YASRLQS),SEQ ID NO:63(YASSLQS),SEQ ID NO:64(YASNLHS),SEQ ID NO:65(YTSSLQS),SEQ ID NO:66(YTSNLHS);

[0021] (VI) LCDR3: SEQ ID NO: 67 (QQGNTLPYT), SEQ ID NO: 68 (QQGAAGPYT), SEQ ID NO: 69 (QQGFGAPYT), SEQ ID NO: 70 (QQGVGAPYT), SEQ ID NO: 71 (QQGAGRPYT), SEQ ID NO: 72 (QQGAGWPYT), SEQ ID NO:73(QQGDLRPYT), SEQ ID NO:74(QQGRLWPYT), SEQ ID NO:75(QQGVLFPYT), SEQ ID NO:76(QQGLSSPYT).

[0022] More preferably, the heavy chain variable region (VH) and the light chain variable region (VL) respectively comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 selected from the following (1) to (13):

[0023] (1) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 41 (RIDPANGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 67 (QQGNTLPYT);

[0024] (2) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 44 (RIDPANANTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 71 (QQGAGRPYT);

[0025] (3) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 41 (RIDPANGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 64 (YASNLHS), SEQ ID NO: 67 (QQGNTLPYT);

[0026] (4)SEQ ID NO:36(DIYMH)、SEQ ID NO:41(RIDPANGNTKYDPKFQD)、SEQ ID NO:52(GQLGPLGFDY)、SEQ ID NO:57(RASQDISNYLN)、SEQ ID NO:61(YTSRLHS)、SEQ ID NO:70(QQGVGAPYT);

[0027] (5)SEQ ID NO:36(DIYMH)、SEQ ID NO:41(RIDPANGNTKYDPKFQD)、SEQ ID NO:52(GQLGPLGFDY)、SEQ ID NO:57(RASQDISNYLN)、SEQ ID NO:61(YTSRLHS)、SEQ ID NO:71(QQGAGRPYT);

[0028] (6)SEQ ID NO:36(DIYMH)、SEQ ID NO:44(RIDPANANTKYDPKFQD)、SEQ ID NO:52(GQLGPLGFDY)、SEQ ID NO:57(RASQDISNYLN)、SEQ ID NO:61(YTSRLHS)、SEQ ID NO:70(QQGVGAPYT);

[0029] (7)SEQ ID NO:36(DIYMH)、SEQ ID NO:47(RIDPAAGNTKYDPKFQD)、SEQ ID NO:52(GQLGPLGFDY)、SEQ ID NO:57(RASQDISNYLN)、SEQ ID NO:66(YTSNLHS)、SEQ ID NO:67(QQGNTLPYT);

[0030] (8)SEQ ID NO:36(DIYMH)、SEQ ID NO:47(RIDPAAGNTKYDPKFQD)、SEQ ID NO:52(GQLGPLGFDY)、SEQ ID NO:57(RASQDISNYLN)、SEQ ID NO:61(YTSRLHS)、SEQ ID NO:70(QQGVGAPYT);

[0031] (9) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 47 (RIDPAAGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 67 (QQGNTLPYT);

[0032] (10) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 47 (RIDPAAGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 71 (QQGAGRPYT);

[0033] (11) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 47 (RIDPAAGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 72 (QQGAGWPYT);

[0034] (12) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 43 (RIDPRNGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 70 (QQGVGAPYT);

[0035] (13) SEQ ID NO: 36 (DIYMH), SEQ ID NO: 43 (RIDPRNGNTKYDPKFQD), SEQ ID NO: 52 (GQLGPLGFDY), SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 61 (YTSRLHS), SEQ ID NO: 71 (QQGAGRPYT).

[0036] The aforementioned heavy chain and light chain CDR combinations are derived from murine antibodies, humanized antibodies, and even affinity-matured antibodies provided by this invention. The antibodies or fragments provided by this invention can achieve K... DAffinity ≤5 nM binds to PD-L1, preferably mammalian PD-L1, more preferably primate PD-L1, and even more preferably human or cynomolgus monkey PD-L1, especially human PD-L1.

[0037] According to a specific embodiment of the present invention, the heavy chain variable region of the antibody or fragment thereof comprises a sequence selected from the following:

[0038] The amino acid sequence shown in any one of SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:18, SEQ ID NO:32 and SEQ ID NO:35 or an amino acid sequence having at least 75% identity with the shown amino acid sequence; and / or

[0039] The light chain variable region of the antibody or its fragment contains sequences selected from:

[0040] The amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:20, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33 and SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the shown amino acid sequence.

[0041] More preferably, the heavy chain variable region and light chain variable region of the antibody or fragment provided by the present invention comprise any of the following amino acid sequence combinations:

[0042] (1) The amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:2; and, the amino acid sequence shown in SEQ ID NO:4 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:4;

[0043] (2) The amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10; and, the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12;

[0044] (3) The amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18; and, the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20;

[0045] (4) The amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10; and, the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:30;

[0046] (5) The amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10; and, the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:31;

[0047] (6) The amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:10; and, the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20.

[0048] (7) The amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:18; and the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:31.

[0049] (8) The amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; and, the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:33;

[0050] (9) The amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; and the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:31.

[0051] (10) The amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; and, the amino acid sequence shown in SEQ ID NO:12 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:12;

[0052] (11) The amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; and, the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20;

[0053] (12) The amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:32; and, the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:34;

[0054] (13) The amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:35; and, the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:31;

[0055] (14) The amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:35; and the amino acid sequence shown in SEQ ID NO:20 or an amino acid sequence having at least 75% identity with the amino acid sequence shown in SEQ ID NO:20.

[0056] Generally, the antibodies or fragments provided by the present invention are in any form, such as monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single-domain antibodies, nanobodies, fully or partially humanized antibodies, or chimeric antibodies. Alternatively, the antibodies or fragments are haptens or antigen-binding fragments of haptens, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv.

[0057] Preferably, the antibody or fragment thereof further comprises a human or mouse constant region, more preferably a human or mouse constant region, such as a human or mouse light chain constant region (CL) and / or heavy chain constant region (CH);

[0058] More preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

[0059] According to a specific embodiment of the present invention, the antibody provided by the present invention is a monoclonal antibody, preferably a mouse-derived, chimeric, or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain constant region is κ type;

[0060] Preferably, the heavy chain constant region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:13 or SEQ ID NO:22, or an amino acid sequence having at least 75% identity with the amino acid sequence; preferably, the light chain constant region of the monoclonal antibody contains an amino acid sequence as shown in SEQ ID NO:16, or an amino acid sequence having at least 75% identity with the amino acid sequence.

[0061] The above-described at least 75% identity refers to any percentage of identity of at least 80%, preferably at least 85%, more preferably at least 90%, and even more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99%, etc., ≥75%.

[0062] According to a specific embodiment of the present invention, the present invention provides an antibody comprising the heavy chain variable region shown in SEQ ID NO:18, the light chain variable region shown in SEQ ID NO:20, the heavy chain constant region shown in SEQ ID NO:22, and the light chain constant region shown in SEQ ID NO:16, which is named H182-MUT4 in the present invention. Alternatively, the antibody comprises the heavy chain variable region shown in SEQ ID NO:10, the light chain variable region shown in SEQ ID NO:12, the heavy chain constant region shown in SEQ ID NO:14, and the light chain constant region shown in SEQ ID NO:16, which is named h182 in the present invention.

[0063] Based on the antibodies or fragments thereof of the present invention, the present invention also provides conjugates or fusion proteins comprising the antibodies or fragments thereof of the present invention. Such conjugates or fusion proteins may comprise other portions bound by chemical or physical methods to the antibodies or fragments thereof of the present invention, such as cell surface receptors, small molecule compounds such as amino acids and sugars, small molecule polymers, or any other portions modified to the antibodies of the present invention, or even active proteins or peptides. For example, the conjugate or fusion protein may be a bispecific antibody or a bifunctional protein comprising the antibodies or fragments thereof of the present invention.

[0064] More preferably, the bispecific antibody / bifunctional protein is a fusion protein formed by the antibody of the present invention and the extracellular region of the TGFβII receptor. For example, the extracellular region of the TGFβII receptor is fused to the C-terminus of the heavy chain of the antibody of the present invention, such as h182 or H182-MUT4, for example, via a linker sequence. The linker sequence can be a flexible linker peptide, for example, a flexible linker peptide containing one or more (GGGGS).

[0065] According to a specific embodiment of the present invention, the fusion protein is a bifunctional protein comprising an antibody having a heavy chain and a light chain, and an extracellular region sequence of a TGFβII receptor fused to the C-terminus of the constant region of the heavy chain, wherein the heavy chain of the antibody is shown in SEQ ID NO:77, the light chain is shown in SEQ ID NO:78, and the extracellular region sequence of the TGFβII receptor is a human TGFβII receptor extracellular region sequence, for example, the amino acid sequence shown in SEQ ID NO:29. Preferably, the TGFβII receptor extracellular region sequence is fused to the C-terminus via a flexible linker peptide. According to a specific embodiment of the present invention, the flexible linker peptide is shown in SEQ ID NO:79.

[0066] According to a specific embodiment of the present invention, the antibody in the bifunctional protein has two heavy chains and two light chains.

[0067] On the other hand, based on the antibody or fragment thereof of the present invention, the present invention also provides a nucleic acid molecule that encodes any antibody or fragment thereof of the present invention or encodes the heavy chain CDR, light chain CDR, light chain variable region, heavy chain variable region, heavy chain or light chain contained in the antibody or fragment thereof;

[0068] Preferably, the nucleic acid molecule comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:17 or SEQ ID NO:19.

[0069] In another aspect, the present invention provides a vector containing the nucleic acid molecule of the present invention. The vector may be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, or a bacteriophage vector, etc.

[0070] The vector or nucleic acid molecule of the present invention can be used to transform or transfect host cells or enter host cells in any way for purposes such as preserving or expressing antibodies.

[0071] Therefore, in another aspect, the present invention provides a host cell containing the nucleic acid molecules and / or vectors of the present invention, or the host cell being transformed or transfected by the nucleic acid molecules and / or vectors of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as bacterial or insect, fungal, plant, or animal cells.

[0072] Based on the disclosure of this invention, the antibodies or fragments thereof, as well as corresponding conjugates or fusion proteins, nucleic acid molecules, vectors, and / or host cells provided by this invention can be obtained using any conventional techniques known in the art. The antibodies or fragments thereof, conjugates or fusion proteins, nucleic acid molecules, vectors, and / or host cells can be included in compositions, such as pharmaceutical compositions, and more particularly in pharmaceutical formulations, for use in various purposes as needed.

[0073] Therefore, in another aspect, the present invention also provides a composition comprising the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector and / or host cell described in this invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.

[0074] The present invention also provides related applications of the above-mentioned subject matter as an antibody that binds to PD-L1 or any part thereof.

[0075] Specifically, in another aspect, the invention provides the use of the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition in the preparation of a medicament for treating diseases associated with PD-L1 positive expression or for blocking PD-L1 / PD-1 interaction;

[0076] Preferably, the disease is cancer or tumor, more preferably PD-L1 positive expression cancer or tumor; more preferably, the disease is selected from cervical cancer, osteosarcoma, urothelial carcinoma, lung cancer such as non-small cell lung cancer or small cell lung cancer, squamous cell carcinoma, ovarian cancer, colon cancer, melanoma, bladder cancer, prostate cancer, liver cancer such as primary liver cancer, gastric cancer, kidney cancer, breast cancer such as triple-negative breast cancer, head and neck cancer, lymphoma, metastatic Merkel cell carcinoma.

[0077] Furthermore, the present invention provides a method for treating diseases associated with PD-L1 positive expression, the method comprising administering the antibody or a fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition, and optionally other drugs or treatments to a subject in need. Preferably, the disease is cancer or tumor, more preferably a PD-L1 positive cancer or tumor; more preferably, the disease is selected from cervical cancer, osteosarcoma, urothelial carcinoma, lung cancer such as non-small cell lung cancer or small cell lung cancer, squamous cell carcinoma, ovarian cancer, colon cancer, melanoma, bladder cancer, prostate cancer, liver cancer such as primary liver cancer, gastric cancer, kidney cancer, breast cancer such as triple-negative breast cancer, head and neck cancer, lymphoma, and metastatic Merkel cell carcinoma. Optional other drugs or treatments refer to other immune-enhancing drugs or means that can be administered in combination with the antibody or a fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell or composition of the present invention, such as small molecule chemicals, targeted drugs, recombinant protein drugs such as antibodies, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapeutics, and radiotherapy. The combined administration of the two can be carried out in any form, such as simultaneously, continuously, or at intervals. The subject is a mammal, preferably a primate, and more preferably a human or a cynomolgus monkey, especially a human.

[0078] In another aspect, the present invention provides a kit comprising the antibody or fragment thereof, conjugate or fusion protein, nucleic acid molecule, vector, host cell and / or composition described in the present invention. The kit can be used for detection or diagnostic purposes.

[0079] Anti-PD-L1 antibodies activate T cells by blocking the binding of PD-1 to its ligand PD-L1. Although the reported antibodies can all block the interaction between PD-1 and PD-L1, they are used in large quantities clinically, and most of them are antibody subtypes with weak ADCC activity. Compared with antibody subtypes with strong ADCC activity, they have a weaker killing effect on tumor tissues that highly express PD-L1.

[0080] In contrast, this invention provides an anti-PD-L1 antibody through hybridoma screening, humanization technology, and affinity maturation. The antibody has high affinity for human PD-L1, as well as high biological activity, strong ADCC activity, and a long in vivo half-life.

[0081] Compared with the prior art, the antibody of the present invention has the following advantages:

[0082] 1. The antibody of this invention is a humanized anti-PD-L1 antibody with high affinity.

[0083] Taking the humanized anti-PD-L1 antibody H182-MUT4 of the present invention as an example, this antibody can specifically bind to human PD-L1 protein, with an affinity (KD) of 4.65E-10M, while the affinity (KD) of the control antibody Atezolizumab is 7.19E-10M and the affinity (KD) of Durvalumab is 8.24E-10M, indicating that H182-MUT4 has a high affinity for human PD-L1.

[0084] 2. The antibody of this invention has good biological activity.

[0085] Taking H182-MUT4 as an example, this antibody can effectively bind to recombinant human PD-L1 on the cell surface. The EC50 of its binding to CHO-PD-L1-CD3L cells is 0.658 nM, which is superior to the binding activity of the control antibodies Atezolizumab (EC50: 0.908 nM) and Durvalumab (EC50: 1.022 nM). It can effectively block the binding of recombinant human PD-L1 to its receptor PD-1, with an IC50 of 6.236 nM, which is similar to the blocking activity of the control antibodies Atezolizumab (IC50: 7.432 nM) and Durvalumab (IC50: 7.64 nM). Furthermore, the biological activity of H182-MUT4 was significantly better than that of the control antibodies Atezolizumab and Durvalumab, as detected by the PD-1 / PD-L1 antibody bioactivity detection system. In addition, the antibody of the present invention has good ADCC activity and can effectively kill MDA-MB-231 cells that highly express human PD-L1; and has good in vivo stability, with a half-life of about 150 hours in mice, which is consistent with the basic characteristics of monoclonal antibody drugs. Attached Figure Description

[0086] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0087] Figure 1 The first round of ELISA detection showed the binding of recombinant human PD-L1 extracellular domain protein to the supernatant of positive hybridomas.

[0088] Figure 2 The second round of ELISA detection showed the binding of recombinant human PD-L1 extracellular domain proteins to the supernatant of positive hybridomas.

[0089] Figure 3 Cross-reactivity results between ELISA-detected positive hybridoma clone supernatant and recombinant PD-L1 from different species.

[0090] Figure 4Results of ELISA detection of the inhibition of human PD-L1 binding to its receptor PD-1 by the supernatant of positive hybridoma clones.

[0091] Figure 5 FACS detection of antibody purified from the supernatant of positive hybridoma clones and its binding to PD-L1 on the surface of 293 cells resulted in screening.

[0092] Figure 6 ELISA was used to detect the binding of mouse antibodies from the supernatant of positive hybridoma clone 182 to members of the B7 and CD28 families.

[0093] Figure 7 Results of affinity comparison between antibody H182-MUT4 and known antibodies targeting the same target.

[0094] Figure 8 Results of ELISA detection of the inhibitory effect of antibody H182-MUT4 on the binding of human PD-L1 to its receptor PD-1.

[0095] Figure 9 Results of antibody H182-MUT4 binding activity to PD-L1 on CHO cell surface.

[0096] Figure 10 : Dose-response curves of antibody in vitro ADCC toxicity, where 10A: Atezolizumab; 10B: Avelumab; 10C: H182-MUT4; 10D: Durvalumab; 10E: isotype control antibody.

[0097] Figure 11 : Drug-time curves of single-dose administration of antibodies in nude mice (PD-L1 assay), where 11A: Avelumab; 11B: Atezolizumab; 11C: Durvalumab; 11D: H182-MUT4.

[0098] Figure 12 Tumor volume changes in the high-dose group of MC38-hPD-L1 tumor-bearing mouse model after H182-MUT4 administration.

[0099] Figure 13 Tumor volume changes in a PD-1 and PD-L1 humanized mouse model of subcutaneous tumor hPD-L1-MC38 after administration of H182-MUT4-TGFβRII. Detailed Implementation

[0100] The present invention will be described below with reference to specific embodiments. Those skilled in the art will understand that these embodiments are for illustrative purposes only and do not limit the scope of the invention in any way.

[0101] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all medicinal materials and reagents used in the following examples are commercially available products.

[0102] The amino acid sequence of the control antibody Atezolizumab is obtained from WHO Drug Information (Vol.29, No.3, 2015). The heavy chain amino acid sequence is shown in SEQ ID NO:23, and the light chain amino acid sequence is shown in SEQ ID NO:24.

[0103] The amino acid sequence of the control antibody Avelumab is obtained from WHO Drug Information (Vol.30, No.1, 2016). The heavy chain amino acid sequence is shown in SEQ ID NO:25, and the light chain amino acid sequence is shown in SEQ ID NO:26.

[0104] The amino acid sequence of the control antibody Durvalumab is obtained from WHO Drug Information (Vol.29, No.3, 2015). The heavy chain amino acid sequence is shown in SEQ ID NO:27, and the light chain amino acid sequence is shown in SEQ ID NO:28.

[0105] Human PD-L1, serial number: NP_054862.1, 19aa-238aa;

[0106] Human PD-1, serial number: NP_005009.2, 21aa-167aa.

[0107] Example 1: Preparation of anti-human PD-L1 antibody hybridoma cells

[0108] Immunization: Balb / c mice were immunized with human PD-L1 / mFc recombinant protein. Serum titers were detected by ELISA using 96-well microplates coated with human PD-L1-His recombinant protein. Mice with serum titers meeting the fusion requirements were used for the next step of cell fusion.

[0109] Cell fusion and hybridoma preparation: On day 67 post-primary immunization, mice with the required cell titer were selected, and their spleens were aseptically harvested. A suspension of B lymphocytes was prepared and mixed with FO myeloma cells at a 5:1 ratio. The two cell types were then fused using PEG4000. The fused cells were resuspended in HAT medium and aliquoted into 96-well cell culture plates. The plates were incubated at 37°C in a 5% CO2 incubator.

[0110] Example 2: Screening of anti-human PD-L1 antibody-positive hybridoma cell lines

[0111] 1. Screening with positive hybridoma ELISA

[0112] 10-14 days after fusion, the microplates were coated with recombinant human PD-L1-His protein (10ug / ml, pH 9.6, 0.1M NaHCO3) and incubated overnight at 4°C; blocked with 4% skim milk powder-PBS and incubated at 37°C for 2 hours; washed three times with PBST (0.05% Tween 20-PBS), and the hybridoma clone culture supernatant was added and incubated at 37°C for 1 hour. The following controls were set up: (1) Positive control (PC): serum of mice after immunization (diluted with PBS 1:1000); (2) Negative control (NC): serum of mice before immunization (diluted with PBS 1:1000); (3) Blank control: PBS. Wash three times with PBST (0.05% Tween 20-PBS), add HRP-goat anti-mouse IgG (Fcγ) diluted 1:20000, incubate at 37℃ for 1 hour; then wash five times with PBST (0.05% Tween 20-PBS), add OPD chromogenic solution, incubate in the dark for 10-15 minutes, and stop the reaction by adding 2M H2SO4; read the A492 value using a microplate reader. A positive result is defined as an A492 value in the detection well that is more than 2.1 times greater than that in the negative control well. To confirm the reliability of positive clones, a second round of screening is performed one day after the first screening medium change. After testing and identification, a total of 20 antibody-secreting positive cell lines were obtained, namely cell lines 57, 92, 143, 152, 162, 182, 189, 234, 244, 267, 301, 315, 369, 403, 453, 492, 504, 564, 582, and 651. Figure 1 , Figure 2 We will continue to screen these 20 secreted antibodies.

[0113] 2. ELISA screening for species crossover of positive hybridoma clones

[0114] Human PD-L1-His recombinant protein and cynomolgus monkey PD-L1-His recombinant protein (Cat: 90251-C08H, Beijing Yiqiao Shenzhou) were coated overnight at 4℃ with a coating concentration of 0.5 μg / mL. After washing the plate 3 times with PBS, 5% BSA PBS was added, and the plate was blocked at 37℃ for 60 min. The plate was washed 3 times with PBST. The supernatant of the above 20 hybridoma strains diluted 100 times with PBS was added. The following controls were set up: (1) Positive control (PC): 0.1 ug / ml of mouse IgG constant region form of Atezolizumab; (2) Negative control (NC): irrelevant hybridoma supernatant (diluted 1:100 with PBS); (3) Blank control: PBS. Incubate at 37℃ for 60 min, wash 4 times with PBST; add 1:5000 diluted HRP-goat anti-mouse IgG (Fcr) (Cat: 115-035-071, Jackson Immuno Research), incubate at 37℃ for 30 min, wash 4 times with PBST; add TMB substrate for color development, incubate at 37℃ for 10 min, then add 2M HCl to terminate the reaction; use 630 nm as the reference wavelength, read and record the absorbance A450nm-630nm at a wavelength of 450 nm. Except for well 189, the supernatants of the other hybridomas specifically bound to recombinant human and cynomolgus monkey PD-L1. Figure 3 ).

[0115] 3. ELISA screening of positive hybridoma clones that inhibit the binding of human PD-L1 to its receptor PD-1

[0116] Human PD-1-His, at a concentration of 0.5 μg / mL, was coated overnight at 4°C and blocked with 5% BSA in a constant temperature incubator at 37°C for 60 min. The supernatant of the above 20 hybridoma strains was diluted 100-fold with PBS, and the following controls were set up: (1) Positive control (PC): Human PD-L1 / mFc 0.1 μg / mL; (2) Negative control (NC): Irrelevant hybridoma supernatant (diluted 1:100 with PBS); (3) Blank control: PBS; (4) Control antibody control (ref mAb): Mouse IgG constant region form of Atezolizumab 0.1 μg / mL. The reaction was carried out in a constant temperature incubator at 37°C for 120 min, and simultaneously 1 μg / mL of human PD-L1 / mFc was added and co-incubated with the antibody in a constant temperature incubator at 37°C for 60 min. Then, 1:5000 diluted HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research) was added, and the reaction was allowed to proceed for 45 min. TMB (Cat: ME142, Beijing Taitianhe Biotechnology) substrate was added for color development, and the reaction was stopped with 2M HCl. The plate was then read. Using 630 nm as the reference wavelength, the absorbance values ​​(A450nm-630nm) of the wells at 450 nm were read and recorded. ELISA results showed that the supernatants from hybridomas 92, 143, 182, 267, and 453 could all block the binding of human PD-L1 to its receptor PD-1. Figure 4 ).

[0117] 4. Screening based on the binding of positive hybridomas to PD-L1 on the surface of 293 cells.

[0118] Using the full-length human PD-L1 expression plasmid (Cat.:HG10084-UT, Beijing Sinocare), and following the instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the full-length human PD-L1 expression plasmid was transfected into HEK293 cells for recombinant expression, thus constructing 293 cells expressing recombinant human PD-L1 (293 / PD-L1). Forty-eight hours after transfection, the cells were used for flow cytometry analysis (FACS).

[0119] Based on the above ELISA results, the supernatant of hybridomas from clones 92, 143, 182, 267, 453, and 492 was selected and purified by affinity. Six mouse antibodies (5 μg / ml) were obtained and incubated with a suspension of 293 cells (293 / PD-L1) expressing recombinant human PD-L1 at 37°C for 30 min. The following controls were set: (1) Positive control (PC): 5 μg / ml of mouse IgG constant region of Atezolizumab; (2) Negative control (NC): 5 μg / ml of irrelevant mouse antibody. After washing the cells three times with PBS, goat anti-mouse IgG-FITC (Cat: F9006, Sigma) diluted 1:64 was added and incubated for 30 min. After washing the cells three times with PBS, the mean fluorescence intensity (MFI) of the cells was detected by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma could bind to PD-L1 on the surface of 293 cells. Figure 5 As shown, only clone 182 showed good binding to PD-L1 on the surface of cell 293. Clone 182 was selected as a candidate clone for further screening.

[0120] 5. ELISA detection of PD-L1 family specificity by positive hybridoma clones.

[0121] Based on the above test results, clone 182 was selected for specific testing. Recombinant human CD28 (Cat: 11524-HCCH, Beijing Yiqiao Shenzhou), CTLA4-His (Cat: 11159-H08H, Beijing Yiqiao Shenzhou), B7H3-His (serial number: NP_001019907.1, 29aa-239aa), and B7H4 (serial number: NP_078902.2, 29aa-257aa) were included. ), ICOS-His (sequence number: NP_036224.1, 1aa-199aa), PD-L2-hFc (sequence number: NP_079515.2, 20aa-220aa), PD-1-His (sequence number: NP_005009.2, 21aa-167aa), human PD-L1-His recombinant protein were coated overnight at 4℃ with a coating concentration of 2μg / mL; after washing the plate 3 times with PBS, 5% BSA PBS was added, and the plate was blocked at 37℃ for 60 min, and washed 3 times with PBST; mouse antibody No. 182 (1ug / ml) was added, and the following controls were set: (1) Blank control: PBS. The plate was incubated at 37°C for 60 min, washed four times with PBST, and then incubated at 37°C for 30 min with 1:5000 diluted HRP-goat anti-mouse IgG (Fcr) (Cat: 115-035-071, Jackson Immuno Research) for 30 min. The plate was washed four times with PBST, and then TMB substrate was added for color development. After incubation at 37°C for 10 min, the reaction was terminated with 2M HCl. The absorbance (A450nm-630nm) of the plate at 450nm was read and recorded using 630nm as the reference wavelength. The experimental results showed that mouse antibody 182 specifically binds to recombinant human PD-L1, exhibits no binding activity with other B7 and CD28 family members, and does not produce cross-reactivity. Figure 6 ).

[0122] Example 3: Sequencing of murine anti-human PD-L1 antibody

[0123] Hybridoma cells 182, which secrete anti-human PD-L1 antibodies, were expanded and cultured. Subtype detection was performed using Mouse Monoclonal Antibody IgG Subclass Test Card (Cat: A12403, VicNovo) and Mouse Monoclonal Antibody Light / Heavy Chain Test Card (Cat: A12401, VicNovo) according to the reagent operation procedures. The subtype was identified as follows: heavy chain was IgG1 and light chain was Kappa chain.

[0124] Total RNA was extracted from hybridoma cells No. 182 according to the instructions of the TRIzol kit (Cat: 15596026, Invitrogen). The total RNA was reverse transcribed into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB). Degenerate primers (refer to the book [Dong Zhiwei, Wang Yan. Antibody Engineering (Second Edition). Beijing Medical University Press, 2001, 313-314]) and the Phusion kit (Cat: E0553L, NEB) were used to amplify the antibody light chain variable region IgVL(κ) and heavy chain variable region V. H Sequence; PCR amplification products were purified using a gel extraction kit (Cat: AP-GX-250, Axygen); the amplified PCR products were ligated into a T vector and transformed into competent *E. coli* cells according to the instructions of the T vector cloning kit (Cat: ZC205, Zhuangmeng Biotechnology). After amplification and plasmid extraction, DNA sequencing was performed to obtain the variable region sequence of the monoclonal antibody. Sequencing results showed that the nucleotide sequence of the 182 mouse antibody heavy chain variable region DNA from this hybridoma cell is shown in SEQ ID NO:1, and the amino acid sequence of the 182 mouse antibody heavy chain variable region deduced from this DNA sequence is shown in SEQ ID NO:2; the nucleotide sequence of the 182 mouse antibody light chain variable region DNA is shown in SEQ ID NO:3, and the amino acid sequence of the 182 mouse antibody light chain variable region deduced from this DNA sequence is shown in SEQ ID NO:4.

[0125] m182 heavy chain variable region (SEQ ID NO:2)

[0126] EVQLQQSGAELVKPGASVKLSCTVSGFNIKDIYMHWLKQRPEQGLEWIGRIDPANGNT KYDPKFQD KATMIADTSSNTAYLQLSSLTSEDTAVYYCAS GQLGPLGFDY WGQGTTLTVSS

[0127] m182 light chain variable region (SEQ ID NO:4)

[0128] DIQMTQTTSSLSASLGDRVTISC RASQDISNYLN WYQQKPDGTVKLLIY YTSRLHS GVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYT FGGGTKLEIK

[0129] Example 4: Preparation of anti-human PD-L1 chimeric antibody and control antibody

[0130] The encoding genes of the light and heavy chain amino acid sequences of the target control antibodies (Atezolizumab, Avelumab, Durvalumab) were fully synthesized and cloned into eukaryotic transient recombinant expression vectors to obtain light and heavy chain expression plasmids of the control antibodies. These plasmids were transformed into E. coli for amplification, and a large number of plasmids containing the light and heavy chains of the control antibodies were isolated. Using these plasmids, and following the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the light and heavy chain plasmids of the control antibodies were transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the control antibodies.

[0131] The murine antibody light chain variable region and heavy chain variable region genes obtained by cloning in this invention were introduced into restriction sites via PCR and cloned into eukaryotic expression vectors containing the coding genes for the human-kappa light chain constant region and the human IgG1 heavy chain constant region, respectively, to obtain human-mouse chimeric light chain (Ch182L) and human-mouse chimeric heavy chain (Ch182H) expression plasmids. These plasmids were then transformed into E. coli for amplification, and a large number of plasmids containing the human-mouse chimeric antibody light chain -Ch182L- and heavy chain -Ch182H- were isolated. Using these plasmids and following the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the light and heavy chain plasmids of the 182 chimeric antibody were transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column. The resulting 182 chimeric antibody was analyzed using a Fortebio Octet QKe system, employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. For the assay, the 182 chimeric antibody was diluted to 5 μg / mL with PBS buffer and flowed through the AHC probe (Cat: 18-5060, PALL) for 120 s. Human PD-L1-His recombinant protein was used as the mobile phase at concentrations of 50 nM and 20 nM. The binding and dissociation times were both 300 s. After the experiment, the blank control response values ​​were subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting. The results showed that the chimeric antibody Ch182 had a high affinity for human PD-L1, with an affinity (KD) of approximately 2.02E-9M, indicating that the 182 mouse antibody sequence was correctly cloned.

[0132] Example 5: Humanization and recombinant expression of anti-human PD-L1 monoclonal antibody

[0133] 1. Humanization design of mouse monoclonal antibody 182

[0134] First, a comprehensive analysis of the mouse antibody heavy chain sequence was performed to identify the antigen complementarity determinant (CDR) region for antibody-antigen binding and the framework region supporting the conserved three-dimensional conformation of the antibody. Then, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the HCDR3 sequence, CDR transplantation was performed, achieving high humanization of the 182 heavy chain variable region (VH) within the framework region. Finally, based on homology alignment results, the most similar human antibody template was searched in the human antibody germline library (http: / / www2.mrc-lmb.cam.ac.uk / vbase / alignments2.php#VHEX). Combining the full-sequence BLAST results and the LCDR3 sequence, CDR transplantation was performed, achieving full humanization of the light chain framework region. The nucleotide sequence of the humanized heavy chain variable region of the 182 antibody CDR Grafted is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8.

[0135] 2. Humanized 182 reverse mutation design

[0136] Based on the sequence characteristics of the 182 mouse antibody, reverse mutations were designed for the humanized light and heavy chain variable regions of the CDR transplantation. The reverse mutation sites are shown in Table 1 below. The nucleotide sequence of the heavy chain variable region after the 182 humanized reverse mutation is shown in SEQ ID NO:9, and the amino acid sequence is shown in SEQ ID NO:10; the nucleotide sequence of the light chain variable region after the humanized reverse mutation is shown in SEQ ID NO:11, and the amino acid sequence is shown in SEQ ID NO:12.

[0137] Table 1. Humanized Sequence Design

[0138]

[0139] Note: Y87F indicates that the 87th amino acid Y has been mutated back to F according to the Kabat numbering system.

[0140] h182_VH1(SEQ ID NO:6)

[0141] EVQLVQSGAEVKKPGATVKISCKVSGFNIK DIYMH WVQQAPGKGLEWMG RIDPANGNTKYDPKFQDRVTITADTSTDTAYMELSSLRSEDTAVYYCAT GQLGPLGFDY WGQGTTVTVSS

[0142] h182_VL1(SEQ ID NO:8)

[0143] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLLY YTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQGNTLPYT FGGGTKVEIK

[0144] h182_VH2(SEQ ID NO:10)

[0145] EVQLVQSGAEVKKPGATVKISCKVSGFNIK DIYMH WVQQAPGKGLEWMG RIDPANGNTKYDPKFQD RVTITADTSTNTAYMELSSLRSEDTAVYYCAS GQLGPLGFDY WGQGTTVTVSS

[0146] h182_VL2(SEQ ID NO:12)

[0147] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLLY YTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFC QQGNTLPYT FGGGTKVEIK

[0148] 3. Recombinant Expression of Humanized Monoclonal Antibody 182

[0149] The humanized 182 antibody light and heavy chain variable regions (h182_VL1, h182_VH2) were fully synthesized. The humanized h182_VH2 was cloned upstream of the heavy chain constant region coding gene of human IgG1 in the eukaryotic expression vector pKN041 via enzyme digestion. The nucleotide sequence of the heavy chain constant region is shown in SEQ ID NO:13, and the amino acid sequence is shown in SEQ ID NO:14. The humanized h182_VL1 was cloned upstream of the human light chain Cκ coding gene in the eukaryotic expression vector pKN019 via enzyme digestion. The nucleotide sequence of the light chain constant region is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:16. NO:16, humanized 182 light and heavy chain expression vectors were constructed to obtain light chain (pKN019-h182L1) and heavy chain (pKN041-h182H2) expression plasmids. These were transformed into *E. coli* for amplification, and the plasmids h182L1 and h182H2 of the h182 antibody light and heavy chains were isolated. Based on the reverse mutation design, the StarMut gene site-directed mutagenesis kit (Cat: T111-01, GenStar) was used to perform site-directed mutagenesis on the light chain (pKN019-h182L1) and heavy chain (pKN041-h182H2), respectively. Site-directed mutagenesis was performed on the L1 and heavy chain (pKN041-h182H2) expression plasmids, which were then transformed into E. coli for amplification to obtain the h182 antibody light and heavy chain expression plasmids h182L2 and h182H1. Using the humanized plasmids and chimeric antibody plasmids of h182, and following the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), the light and heavy chain plasmids of the h182 antibody were combined (see Table 2) and transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column. Different humanized antibodies against h182 were obtained, and antibody affinity was determined using a Fortebio Octet QKe system instrument employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. During the assay, the 182 antibody was diluted to 5 μg / mL with PBS buffer and flowed over the surface of the AHC probe (Cat: 18-5060, PALL) for 120 s. Human PD-L1-His recombinant protein was used as the mobile phase at a concentration of 100 nM. The binding time was 300 s, and the dissociation time was 300 s. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting.

[0150] Table 2. Light and heavy chain sequence combinations of antibody 182

[0151] Ch182H h182H1 h182H2 Ch182L Ch182 182-1 182-2 h182L1 182-3 182-4 182-5 h182L2 182-6 182-7 182-8

[0152] Note: This table represents the sequences obtained by various combinations of 182 light and heavy chains (the chain names are the same as the corresponding plasmids). For example, 182-1 indicates that the antibody is composed of the 182 chimeric antibody light chain Ch182L and the humanized heavy chain h182H1, and so on.

[0153] The affinity of the 182 combination antibody and the control antibody Atezolizumab to the recombinant human PD-L1-His protein was determined by ForteBio, as shown in Table 3. The optimal humanized sequence combination obtained by screening was 182-8, which had an affinity (KD) of 3.64E-9M, and showed no significant decrease compared to the affinity of Ch182 (KD: 2.14E-09M). This combination was selected as the preferred humanized sequence and named h182 for further functional verification.

[0154] Table 3 shows the affinity assay results between the antibody and the recombinant human PD-L1 extracellular domain protein, as presented in Table 2.

[0155] Antibody combination KD value (M) Atezolizumab 1.93E-10 Ch182 2.14E-09 182-1 5.82E-09 182-2 1.93E-09 182-3 4.77E-09 182-4 8.19E-09 182-5 3.43E-09 182-6 4.64E-09 182-7 5.68E-09 182-8 3.64E-09

[0156] Example 6: Affinity maturation of humanized antibody h182

[0157] To obtain antibodies with higher affinity, h182 was maturated for affinity by introducing mutations into all CDR regions of the light and heavy chains using PCR to construct a mutant library for affinity maturation of h182.

[0158] Construction of phage display mutant libraries for the heavy chain CDR region and light chain CDR region of h182

[0159] Using conventional cloning techniques, the coding gene fragments of the synthesized parental h182-Fab and six incomplete parental h182-Fab coding gene fragments containing stuffer regions were respectively ligated into phage BBTN to construct the parental expression vector BBTN-h182-Fab and six tool vectors: BBTN-h182-HCDR1, BBTN-h182-HCDR2, BBTN-h182-HCDR3, BBTN-h182-LCDR1, BBTN-h182-LCDR2, and BBTN-h182-LCDR3. The synthesized single-stranded mutant units h182-HCDR1-NNK, h182-HCDR2-NNK, h182-HCDR3-NNK, h182-LCDR1-NNK, h182-LCDR2-NNK, and h182-LCDR3-NNK were used to form double-stranded NNK fragments by PCR. Each double-stranded NNK fragment was mixed with the corresponding tool vector plasmid in a certain ratio, and IIS type endonuclease and T4 DNA ligase were added for cleavage and ligation reactions. The mixture was then electroporated into TG1 competent cells to complete the construction of six mutant libraries (HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3).

[0160] 2. Document Selection

[0161] After the phage particles for panning were rescued and packaged from the library, solid-phase panning was performed using human PD-L1-His recombinant protein. The antigen concentration was reduced in each round of panning compared to the previous round. After three rounds of panning, multiple clones were selected for phage ELISA to detect binding activity, and positive clones were sequenced.

[0162] 3. Fortebio detects affinity.

[0163] Sequencing and sequence analysis were performed. After removing redundant sequences, the non-redundant sequences (Table 4) were converted into the same IgG (γ1, κ) form as h182 and expressed in mammalian cells (293); or the light and heavy chains were recombined and expressed in mammalian cells (293). The expression supernatant was purified using a ProA affinity chromatography column. The antibody affinity was determined using a Fortebio Octet QKe system instrument, employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. During the assay, the antibody was diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat: 18-5060, PALL) for 120 s. Human PD-L1-His recombinant protein was used as the mobile phase at a concentration of 60 nM. The binding time was 300 s, and the dissociation time was 300 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated using software to fit a 1:1 Langmuir binding pattern. The affinity results are shown in Table 5. The mature antibody containing the combination h182-LCDR3-4+h182-HCDR2-3 was selected for further verification. This antibody molecule was named H182-MUT4. The nucleotide sequence of the heavy chain variable region of H182-MUT4 is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO:19, and the amino acid sequence is shown in SEQ ID NO:20.

[0164] H182-MUT4, VH (SEQ ID NO:18)

[0165] EVQLVQSGAEVKKPGATVKISCKVSGFNIKDIYMHWVQQAPGKGLEWMGRIDPANAN TKYDPKFQD RVTITADTSTNTAYMELSSLRSEDTAVYYCASGQLGPLGFDYWGQGTTVTVSS

[0166] H182-MUT4, VL (SEQ ID NO:18)

[0167] DIQMTQSPSSLSASVGDRVTITC RASQDISNYLN WYQQKPGKAPKLLLY YTSRLHS GVPSRFSGSGSGTDYTLTISSLQPEDFATYFC QQGAGRPYT FGGGTKVEIK

[0168] Table 4. Positive CDR region sequences screened from the h182 CDR region phage display mutant library.

[0169]

[0170]

[0171] Note: h182-LCDR1-1 indicates that the sequence after affinity maturation of the CDR1 region is RASQDISAYLN. The remaining sequences of the affinity maturation antibody light chain containing this light chain CDR (LCDR1) are the same as h182, and so on.

[0172] Table 5. Affinity assay results of antibodies after maturation in the h182 CDR region.

[0173]

[0174]

[0175] Example 7: Comparison of affinity between H182-MUT4 and antibodies targeting the same target

[0176] The HEK293 transient expression system was used to prepare H182-MUT4 and control antibodies Atezolizumab and Durvalumab targeting the same target. The nucleotide sequence of the heavy chain constant region of H182-MUT4 is shown in SEQ ID NO:21, and the amino acid sequence is shown in SEQ ID NO:22; the nucleotide sequence of the light chain constant region of H182-MUT4 is shown in SEQ ID NO:15, and the amino acid sequence is shown in SEQ ID NO:16. Antibody affinity was determined using a Fortebio Octet QKe system instrument, employing an anti-human antibody Fc fragment capture antibody (AHC) bioprobe to capture the antibody Fc fragment. During the assay, the antibodies (h182, H182-MUT4, and control antibodies Atezolizumab, Durvalumab) were diluted to 4 μg / mL with PBS buffer and flowed through the surface of the AHC probe (Cat: 18-5060, PALL) for 120 s. Human PD-L1-His recombinant protein was used as the mobile phase at a concentration of 60 nM. The binding time was 300 s, and the dissociation time was 300 s. After the experiment, the blank control response value was subtracted, and the antigen-antibody binding kinetic constant was calculated by fitting the 1:1 Langmuir binding pattern using software.

[0177] The results showed that the reaction curves of H182-MUT4 and the control antibodies Atezolizumab and Durvalumab with recombinant human PD-L1 protein were as follows: Figure 7As shown, the fitted curves and calculated affinity yielded the following results: H182-MUT4 affinity (KD) was 4.65E-10M, Atezolizumab affinity (KD) was 7.19E-10M, and Durvalumab affinity (KD) was 8.24-10M. Detailed kinetic parameters are shown in Table 6 below. The results indicate that matured H182-MUT4 exhibits high affinity for human PD-L1, and its dissociation value is significantly lower than that of h182.

[0178] Table 6. Affinity assay results of H182-MUT4 with marketed antibodies targeting the same target.

[0179]

[0180]

[0181] Example 8: ELISA detection of the inhibitory effect of H182-MUT4 on the binding of human PD-L1 to its receptor PD-1

[0182] Human PD-1-hFc at a concentration of 0.5 μg / mL was coated overnight at 4°C and blocked with 5% BSA in a 37°C incubator for 60 min. H182-MUT4, control antibodies Atezolizumab and Durvalumab, and isotype control NC-hIgG1 (starting concentration 4.5 μg / mL, 1.3-fold serial dilution, 12 gradients) were incubated at 37°C for 60 min. Then, 1 μg / mL of PD-L1-mFc was added and co-incubated with the antibody at 37°C for 60 min. The plate was washed four times with PBST; then, 1:5000 diluted HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research) was added, and the reaction was carried out for 45 min. TMB (Cat: ME142, Beijing Taitianhe Biotechnology) substrate was added for color development for 15 min, and the reaction was stopped with 2M HCl before reading the plate. Using 630nm as the reference wavelength, the absorbance value A450nm-630nm of the well plate at a wavelength of 450nm was read and recorded.

[0183] The results showed that H182-MUT4 effectively blocked the binding of recombinant human PD-L1 to its receptor PD-1. The competitive inhibitory effects of H182-MUT4, Atezolizumab, and Durvalumab on the binding of human PD-L1 to its receptor PD-1 were determined by ELISA, with half-maximal inhibitory concentrations (IC50) of 6.236 nM, 7.432 nM, and 7.64 nM, respectively. Figure 8H182-MUT4 showed slightly better blocking activity compared to Atezolizumab and Durvalumab.

[0184] Example 9: H182-MUT4 and CHO-PD-L1-CD3L cell binding activity analysis

[0185] CHO cells (CHO-PD-L1-CD3L, Jiangsu Taikang Biopharmaceutical Co., Ltd.) expressing recombinant human PD-L1 and anti-CD3-ScFv were used at a rate of 4 × 10⁻⁶. 5 Cells / well were inoculated into 96-well cell culture plates (Cat: 3599, Corning), and 5% BSAPBS was added. Cell surface receptors were blocked at 4°C for 30 min. H182-MUT4 and control antibodies Atezolizumab, Durvalumab, and isotype control NC-hIgG1 (starting concentration 30 μg / mL, 3-fold serial dilution, 12 gradients) were incubated with CHO-PD-L1-CD3L cell suspension at 4°C for 60 min. After washing the cells three times with PBST, 0.5 μg / mL goat anti-human IgG-FITC (Cat: F9512, Sigma) was added and incubated at 4°C for 60 min. After washing the cells twice with PBST, the mean fluorescence intensity (MFI) of the cells was measured by flow cytometry (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the test antibody could bind to PD-L1 on the surface of CHO cells. Figure 9 As shown, the EC50 values ​​of H182-MUT4, Atezolizumab, and Durvalumab for binding to CHO-PD-L1-CD3L cells were 0.658 nM, 0.908 nM, and 1.022 nM, respectively. Compared with the control antibodies Atezolizumab and Durvalumab, H182-MUT4 showed better binding activity to CHO-PD-L1-CD3L cells.

[0186] Example 10: Cellular activity observation of H182-MUT4 blocking the binding of PD-L1 to its receptor PD1

[0187] Recombinant CHO cells expressing human PD-L1 and anti-CD3-ScFv (CHO-PD-L1-CD3L, Jiangsu Taikang Biopharmaceutical Co., Ltd.) were harvested at a rate of 4 × 10⁻⁶ cells / year. 5Cells / well were inoculated into 96-well cell culture plates (Cat: 3599, Corning). After overnight culture in a cell incubator, the supernatant was discarded, and 50 μL / well of h182, H182-MUT4, and control antibodies Atezolizumab and Durvalumab (starting concentration 2 μg / mL, serially diluted 2-fold in 10 steps) and 50 μL / well of Jurkat cell suspension expressing human PD-1 and luciferase (Jurkat-PD1-NFAT, Jiangsu Taikang Biopharmaceutical Co., Ltd.) were added. The plates were incubated in a cell incubator for 6 hours. Bio-Glo was then added to the cell culture plates. TM Luciferase substrate (Cat: 7940, Promega), 100 μl / well. Place the cell culture plate in a microplate incubator and incubate at 800 rpm in the dark for 20 min. Set the multi-plate reader to Luminescence mode, select Interval 500 (instrument default value), and read the RLU.

[0188] The relative activities of other samples were calculated using Atezolizumab as a control. The EC50 and relative activities of each sample are shown in Table 7 below. The biological activity of H182-MUT4 was superior to that of the control antibodies Atezolizumab and Durvalumab; the biological activity of h182 was superior to that of the control antibody Atezolizumab.

[0189] Table 7. Detection of biological activity of H182-MUT4

[0190]

[0191] Example 11: ADCC effect of H182-mut4 on PD-L1 positive cells

[0192] Select MDA-MB-231 cells that are in the logarithmic growth phase and highly express human PD-L1. Add Calcein AM to label the live MDA-MB-231 cells. Then, mix them with serially diluted H182-MUT4 and control antibodies Atezolizumab, Durvalumab, Avelumab, and isotype control antibody (hIgG1) (starting concentration 100 ng / mL, 10-fold serial dilution, 5 gradients, 6 replicates per concentration), effector cells NK92 cells (effectant-to-target ratio 5:1), and set the following controls: (1) Positive control (PC): target cell maximum release well; (2) Negative control (NC): L15 medium (Cat: 11415064, Gibco); (3) Blank control: DPBS (Cat: 14190-144, Gibco) and incubate at 37℃ for 6 h. Add 2% Triton to the positive control. After X-100 (Cat: X100-100ML, Sigma), the mixture was incubated at 37℃ for 30 min. The wells containing spontaneous release of target cells were used as blank controls, and the wells containing the largest release of target cells were used as positive controls. The fluorescence intensity of calcein in the culture medium was detected using a microplate reader (Cat: Synergy H1, BioTek). The excitation wavelength was 490 nm, and the emission wavelength was 515 nm. The ADCC killing activity of the PD-L1 antibody was evaluated.

[0193] Kill efficiency (%) = (Fluorescence value of experimental group - Fluorescence value of spontaneous release group) / (Fluorescence value of maximum release group - Fluorescence value of spontaneous release group) × 100%

[0194] The results show ( Figure 10 Atezolizumab antibody showed no ADCC activity, while Avelumab, H182-MUT4, and Durvalumab all showed ADCC activity, with EC50 values ​​of 1.405 ng / mL, 0.9602 ng / mL, and 0.8184 ng / mL, respectively.

[0195] Example 12: Pharmacokinetics of H182-MUT4 in Mice

[0196] Healthy female 5-week-old Balb / C nude mice were divided into groups of three and administered a single dose of H182-MUT4, Atezolizumab, Durvalumab, and Avelumab via tail vein at a dose of 10 mg / kg. Blood samples were collected via tail vein at 0.5 h, 1 h, 2 h, 8 h, 24 h, 48 h, 96 h, 144 h, 216 h, and 240 h after administration. The blood samples were incubated at room temperature for at least 30 min, and serum was collected at 4000 rpm for 15 min. The serum samples were stored at -20 °C, and the last serum collection was frozen at -20 °C for at least 24 h. Three control groups were set up and administered equivalent doses (10 mg / kg) of the control products Atezolizumab, Durvalumab, and Avelumab via tail vein for comparison to observe their pharmacokinetic characteristics. After all blood collection was completed, PD-L1-His was coated onto a 96-well ELISA plate at 0.5 ug / ml, 100 ul / well, and incubated overnight at 4°C. After washing the plate 3 times with PBS, 5% BSA PBS was added, and the plate was blocked at 37°C for 60 min. The plate was washed 3 times with PBST. Serum samples to be tested (10,000, 20,000-fold dilution) were added. H182-MUT4 and control antibodies Atezolizumab, Durvalumab, and Avelumab were set up in the wells (initial concentration of 0.05 μg / mL, 2-fold serial dilution, 12 gradients). The following controls were set up: (1) Negative control (NC): blank mouse serum (10,000-fold dilution); (2) Blank control: PBS. Incubate at 37℃ for 60 min, wash 4 times with PBST; add 1:5000 diluted HRP-goat anti-human IgG (Fcr) (Cat: 109-035-098, Jackson Immuno Research), incubate at 37℃ for 40 min, wash 4 times with PBST; add TMB substrate (Cat: ME142, Beijing Taitianhe Biotechnology Co., Ltd.) for color development, incubate at 37℃ for 10 min, then add 2M HCl to terminate the reaction; use 630 nm as the reference wavelength, read and record the absorbance (A450nm-630nm) of the plate at a wavelength of 450 nm. Plot the concentration of the standard antibody on the Y-axis and the OD value on the X-axis, fit a linear curve, and substitute the OD value of the detected serum into the formula to obtain the antibody content in the serum. Then, according to the formula T... 1 / 2 =|0.693 / k|, calculate the drug half-life T 1 / 2 .

[0197] like Figure 11 As shown in Table 8, H182-MUT4 and Durvalumab maintained a good half-life in Balb / c mice. 1 / 2The in vivo activity levels reached approximately 130–150 hours, indicating that H182-MUT4 and Durvalumab antibodies did not show significant inactivation and possessed good structural stability. Their metabolism conformed to the basic characteristics of monoclonal antibody drugs. However, Atezolizumab and Avelumab showed a significant decrease in concentration at 144 hours.

[0198] Table 8. Pharmacokinetic parameters of H182-mut4 in nude mice after a single dose

[0199]

[0200] Example 13: Antitumor efficacy of H182-MUT4 in MC38-hPD-L1 tumor-bearing mice

[0201] Mouse colon cancer MC38 cells expressing human PD-L1 (MC38-hPD-L1) were used at 5 × 10⁻⁶. 5 0.1 mL of the drug was injected subcutaneously on the right side of 5-8 week old female C57BL / 6 mice until the tumor grew to approximately 100 mm. 3 Mice with tumor volumes meeting the requirements were randomly divided into four groups of six each. Each group received anti-PD-L1 antibody H182-MUT4, Atezolizumab, and Durlumab, and an isotype control antibody hIgG1 at 10 mg / kg, respectively. Dosing frequency was BIW. Mouse body weight and tumor volume were measured twice weekly during the dosing and observation periods, and the values ​​were recorded. Results are as follows: Figure 12 As shown, H182-MUT4 significantly inhibited tumor growth, and its tumor-suppressing effect was basically equivalent to that of the control antibody Atezolizumab.

[0202] Example 14: Preparation of H182-MUT4-TGFβRII bifunctional protein

[0203] The heavy chain C-terminus of the humanized anti-PD-L1 antibody H182-MUT4 obtained in this invention was linked to the extracellular region sequence of TGFβRII (SEQ ID NO. 29) via a linker peptide (SEQ ID NO. 79). After restriction enzyme digestion, it was cloned into a eukaryotic expression vector to obtain an expression plasmid containing the coding sequence of the H182-MUT4 heavy chain-TGFβRII. This plasmid was transformed into *E. coli* for amplification, and a large number of plasmids were isolated. Following the instructions of the transfection reagent 293fectin (Cat: 12347019, Gibco), this plasmid and the H182-MUT4 light chain plasmid were transformed into HEK293 cells for recombinant expression. Five to six days after cell transfection, the culture supernatant was collected and purified using a ProA affinity chromatography column to obtain the bifunctional protein H182-MUT4-TGFβRII (where the heavy chain is shown in SEQ ID NO: 77 and the light chain in SEQ ID NO: 78). Antibody affinity was determined using the Fortebio Octet QKe system with an AHC (antibody capture antibody) bioprobe to capture the Fc fragment of the anti-human antibody. For the assay, bifunctional proteins H182-MUT4-TGFβRII, H182-MUT4, and TGFβRII-hFc (Cat: CC10, Nearshore Technology) were diluted to 5 μg / mL with PBS buffer and flowed through the surface of an AHC probe (Cat: 18-5060, PALL) for 120 s. Human PD-L1-His recombinant protein and human TGFβ1 recombinant protein (Cat: CA59, Nearshore Technology) were used as mobile phases at a concentration of 60 nM. The binding time and dissociation time were 300 s and 300 s, respectively. After the experiment, the blank control response value was subtracted, and the kinetic constants of antigen-antibody binding were calculated using a 1:1 Langmuir binding model fitting. The results are shown in Table 9. Compared with H182-MUT4 and TGFβRII-hFc, the bifunctional protein H182-MUT4-TGFβRII showed no significant decrease in affinity for PD-L1 and TGFβ1.

[0204] Table 9. Results of affinity assay for the bifunctional protein H182-MUT4-TGFβRII.

[0205]

[0206] Example 15 Antitumor efficacy of bifunctional protein H182-MUT4-TGFβRII in PD-1 and PD-L1 humanized mouse tumor-bearing hPD-L1-MC38 subcutaneous tumor models

[0207] Mouse colon cancer MC38 cells expressing only human PD-L1 (MC38-hPD-L1, Cat:3111C0001CCC000667, Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) were used at a rate of 5 × 10⁻⁶ 5 0.1 mL of a single dose was injected subcutaneously on the right side of 5-8 week old PD-1 and PD-L1 humanized mice (Cat:T004022, Jicui Pharmaceutical Biotechnology). The injection was continued until the tumor grew to approximately 100 mm. 3 Eighteen mice meeting the tumor volume criteria were randomly selected and divided into three groups of six. Each group received treatment (D0), specifically anti-PD-L1 antibody H182-MUT4 (10 mg / kg), bifunctional protein H182-MUT4-TGFβRII (13.5 mg / kg), and isotype control antibody hIgG1 (10 mg / kg). Administration was done intraperitoneally (ip) at a frequency of nine times per week (tiw×9). Mouse body weight and tumor volume were measured twice weekly during the treatment and observation periods, and the values ​​were recorded. A mouse was considered considered a target if its tumor volume exceeded 3000 mm². 3 Or the average tumor volume in the treatment group exceeded 2000 mm. 3 The experiment will be terminated if the animal's weight decreases by more than 20%.

[0208] like Figure 13 As shown, both the anti-PD-L1 antibody H182-MUT4 and the bifunctional protein H182-MUT4-TGFβRII have certain tumor-suppressive effects, and the bifunctional protein composed of H182-MUT4 and TGFβRII has a better tumor-suppressive effect.

[0209] The above description of specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention, and as long as they do not depart from the spirit of the present invention, they should all fall within the scope of the appended claims. sequence list <110> Maiwei (Shanghai) Biotechnology Co., Ltd. Beijing Kenuo Xincheng Technology Co., Ltd. <120> Anti-PD-L1 antibodies and their applications <130> LC19110066 <160> 79 <170> SIPOSequenceListing 1.0 <210> 1 <211> 357 <212> DNA <213> Artificial <220> <221> gene <222> ()..() <223> m182, VH <400> 1 gaggttcagc tgcagcagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg 60 tcctgtacag tttctggctt caacattaaa gacatttata tgcactggct gaagcagagg 120 cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtaa tactaaatat 180 gacccgaagt tccaggacaa ggccactatg atagcagaca catcctccaa cacagcctac 240 ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc tagtggacag 300 ctcggccccc taggctttga ctactggggc caaggcacca ctctcacagt ctcctca 357 <210> 2 <211> [119 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> m182, VH <400> 2 Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Thr Val Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Leu Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Lys Ala Thr Met Ile Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Gly Gln Leu Gly Pro Leu Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 3 <211> 321 <212> DNA <213> artificial<​​​​​​​​​​​​​​​​aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240 gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300 gggaccaagc tggaaataaa a 321 <210> 4 <211> 107 <212> PRT <213> Artificial <220> <221> PEPTIDE <222> ()..() <223> m182, VL <400> 4 Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1 5 1​​​​​​​​​​​​​​​​​​​​​Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 357 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> h182_VH1 <400> 5 gaggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagccac cgtgaagatc 60 tcctgcaagg tgtccggctt caacatcaag gacatctaca tgcactgggt gcagcaggct 120 cctggcaagg gcctggagtg gatgggacgg atcgaccctg ccaacggcaa caccaagtac 180 gaccccaagt tccaggaccg ggtgaccatc accgctgaca cctccaccga caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgctgtgt actactgcgc caccggccag 300 ctgggacctc tgggcttcga ctactgggga cagggcacca ccgtgaccgt gtcctcc 357 <210> 6 <211> 119 <212> PRT<000065​​​​​​​​​​​​Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr[[ID=十七]] 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95<第 [[ID=二十四]]Ala Thr Gly Gln Leu Gly Pro Leu Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 7 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> h182_VL1 <400> 7 It should be noted that there seems to be some incorrect numbering in the original text (such as "十七" for ID=17), which is maintained as is in the translation for the purpose of following the rules. You may want to double-check the accuracy of the original text for a more proper translation.gacatccaga tgacccagtc tccctcctct ctgtctgcct ccgtgggcga cagagtgacc 60 atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120 ggcaaggctc ccaagctgct gctgtactac acctccaggc tgcactccgg agtgccctct 180 cggttctctg gctccggctc tggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctacta ctgccagcag ggcaacaccc tgccctacac cttcggagga 300 ggcaccaagg tggagatcaa g 321 <21​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 9 <211> 357 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> h182_VH2 <400> 9 gaggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagccac cgtgaagatc 60 tcctgcaagg tgtccggctt caacatcaag gacatctaca tgcactgggt gcagcaggct 120 cctggcaagg gcctggagtg gatgggacgg atcgaccctg ccaacggcaa caccaagtac 180 gaccccaagt tccaggaccg ggtgaccatc accgctgaca cctccaccaa caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgctgtgt actactgcgc ctctggccag 300 ctgggacctc tgggcttcga ctactgggga cagggcacca ccgtgaccgt gtcctcc 357 <210> 10 <211> 119 <212> PRT <213> artificial <220> <221> PEPTIDE​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Thr Thr Val Thr Val Ser Ser 115 <210> 11 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> h182_VL2 <400> 11 gacatccaga tgacccagtc tccctcctct ctgtctgcct ccgtgggcga cagagtgacc 60 atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120 ggcaaggctc ccaagctgct gctgtactac acctccaggc tgcactccgg agtgccctct 180 cggttctctg gctccggctc tggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctactt ctgccagcag ggcaacaccc tgccctacac cttcggagga 300 ggcaccaagg tggagatcaa g 321 <210> 12 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> h182_VL2 <400> 12 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly It should be noted that there may be some inaccuracies in the original text tags such as etc. If these are specific format requirements in a particular system, they should be maintained precisely as is. Also, the text "人工(artificial)" is translated as "artificial" here, assuming it's a direct and simple translation from the Chinese term in the context of this patent text. 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 13 <211> 330 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> h182,CH <400> 13 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 Ala 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 Glu Glu 225 230 235 240 Met 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> 14 <211> 990 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> h182,CH <400> 14 gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacgcc 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 15 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> h182 / H182‑MUT4,CL <400> 15 cggaccgtgg cggcgccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggtaccgcta gcgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t <210> 16 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> h182 / H182-MUT4,CL <400> 16 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> 17 <211> 357 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> H182-MUT4, VH <400> 17 gaggtgcagc tggtgcagtc cggagccgag gtgaagaagc ctggagccac cgtgaagatc 60 tcctgcaagg tgtccggctt caacatcaag gacatctaca tgcactgggt gcagcaggct 120 cctggcaagg gcctggagtg gatgggacgg atcgaccctg ccaacgccaa caccaagtac 180 gaccccaagt tccaggaccg ggtgaccatc accgctgaca cctccaccaa caccgcctac 240 atggagctgt cctccctgcg gtccgaggac accgctgtgt actactgcgc ctctggccag 300 ctgggacctc tgggcttcga ctactgggga cagggcacca ccgtgaccgt gtcctcc 357 <210> 18 <211> 119 <212> PRT <213> artificial​​​​​​​​​​ Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala 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 Ser Gly Gln Leu Gly Pro Leu Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 19 <211> 321 <212> DNA <213> artificial <220> <221> gene <222> ()..() <223> H182-MUT4,CL <400> 19 gacatccaga tgacccagtc tccctcctct ctgtctgcct ccgtgggcga cagagtgacc 60 atcacctgca gagcctccca ggacatctcc aactacctga actggtacca gcagaagcct 120 ggcaaggctc ccaagctgct gctgtactac acctccaggc tgcactccgg agtgccctct 180 cggttctctg gctccggctc tggcaccgac tacaccctga ccatctcctc cctgcagccc 240 gaggacttcg ccacctactt ctgccagcag ggcgctggac ggccctacac cttcggagga 300 ggcaccaagg tggagatcaa g 321 <210> 20 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> H182 - MUT4, VL <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ala Gly Arg Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 21 <211> 990 <212> DNA <213> artificial <220> <221> gene <222> ()..() ​​​​​​​​​​​​​​aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcctccatc tcgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 22 <211> 330 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> H182-MUT4, CH <400> 22 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 7 ??? 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 It seems there is an unclear "7?" in the original "65 70 75 80" which is translated as "65 7? 80" here. You may want to check and correct the original text for a more accurate translation.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> 23 <211> 448 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> atezolizumab,HC <400> 23 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> 24 <211> 214 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> atezolizumab,LC <400> 24 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 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> 25 <211> 450 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> avelumab, HC <400> 25 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​​​​​​​​​​​​​​​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> 26 <211> 216 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> avelumab,LC <400> 26 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15​​​​​​​​​​​​​​​​​​​​​​​​​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> 27 <211> 451 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> durvalumab,HC <400> 27 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 Arg Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 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 Glu Gly Gly Trp Phe Gly Glu Leu Ala Phe Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 28 <211> 215 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> durvalumab,LC <400> 28 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Arg Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Leu Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 29 <211> 136 <212> PRT <213> Homo sapiens <400> 29 Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr 1 5 10 15 Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp 20 25 30 Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys 35 40 45 Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val 50 55 60 Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp 65 70 75 80 Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro 85 90 95 Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met 100 105 110 Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu 115 120 125 Glu Tyr Asn Thr Ser Asn Pro Asp 130 135 <210> 30 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE<null>0001399<222> ()..()<null>0001400<223> VL[[ID=​​​​​​​​​​​​​​​​​​Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 31 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> VL <400> 31<​​​​​​​​​​​​​​​​​​​​Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Val Gly Ala Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 32 <211> 119 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> VH <400> 32 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Ala Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60​​​​​​​​​Ala Ser Gly Gln Leu Gly Pro Leu Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 33 <211> 107 <212> PRT <213> artificial[[ID=1​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 100 105 <210> 34 <211> 107 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> VL <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ala Gly Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100​​​​​​​​​<220> <221> PEPTIDE <222> ()..() <223> VH <400> 35 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Arg Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala 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 Ser Gly Gln Leu Gly Pro Leu Gly Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 36 <211> 5 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR1 <400> 36 Asp Ile Tyr Met His 1 5 <210> 37 <211> 5 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR1 <400> 37 Ser Ile Tyr Met His 1 5 <210> 38 <211> 5 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR1 <400> 38 Ser Tyr Tyr Met His 1 5 <210> 39 <211> 5 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR1 <400> 39 Asp Ile Tyr Ile Ser 1 5 <210> 40 <211> 5 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR1 <400> 40 Asp Tyr Tyr Met His 1 5 <210> 41 <211> 17 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR2 <400> 41 Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp[[ID=3​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<400> 43 Arg Ile Asp Pro Arg Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 44 <211> 17 <212> PRT <213> artificial <220><00016​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Arg Ile Asp Val Arg Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 47 <211> 17 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR2 <400> 47 Arg Ile Asp Pro Ala Ala Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 48 <211> 17 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..()​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Arg Ile Asp Leu Ala Asn Ala Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 50 <211> 17 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR2 <400> 50 Arg Ile Asp Arg Ala Ala Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 51 <211> 17 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR2 <400> 51 Arg Ile Asp Pro Arg Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln 1 5 10 15 Asp <210> 52 <211> 10 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR3 <400> 52 Gly Gln Leu Gly Pro Leu Gly Phe Asp Tyr 1 5 10 <210> 53 <211> 10 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR3 <400> 53 Gly Gln Ala Gly Ser Leu Gly Phe Asp Tyr 1 5 10 <210> 54 <211> 10 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR3 <400> 54 Gly Gln Leu Ala Ser Leu Gly Phe Asp Tyr 1 5 10 <210> 55 <211> 10 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR3 <400> 55 Gly Gln Val Gly Met Leu Gly Phe Asp Tyr 1 5 10 <210> 56 <211> 10 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> HCDR3 <400> 56 Gly Arg Leu Gly Ser Leu Gly Phe Asp Tyr 1 5 10 <210> 57 <211> 11 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR1 <400> 57 Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn 1 5 10 <210> 58 <211> 11 <212> PRT <213> artificial <22​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 60 <211> 11 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR1 <400> 60 Arg Ala Ser Gln Asp Ile Ser Ser Tyr Leu Asn 1 5 10 <210> 61 ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ <221> PEPTIDE <222> ()..() <223> LCDR2 <400> 63 Tyr Ala Ser Ser Leu Gln Ser 1 5 <210> 64 <211> 7 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR2 <400> 64 Tyr Ala Ser Asn Leu His Ser 1 5 <210> 65 <211> 7 <212> PRT <213> artificial <220><00018\>70<221> PEPTIDE <222> ()..() <223> LCDR2 <400> 65 Tyr Thr Ser Ser Leu Gln Ser 1 5 <210> 66<00\>1877<211> 7 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR2 <400> 66 Tyr Thr Ser Asn Leu His Ser 1 5 [[ID=7\>]]<210> 67<0001\>888<211> 9 <0\>01889<212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR3 <400> 67 Gln Gln Gly Asn Thr Leu Pro Tyr Thr 1 5 <210> 68 <211> 9 <212> PRT <213> artificial <220> <221> PEPTIDE [[ID= thirty]]<222> ()..() <223> LCDR3 <400> 68 Gln Gln Gly Ala Ala Gly Pro Tyr Thr 1 5 <210> 69 <211> 9 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR3 < forty]]<400> 69 Gln Gln Gly Phe Gly Ala Pro Tyr Thr 1 5 <210> 70 <211> 9 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR3 <400> 70<00Gln Gln Gly Val Gly Ala Pro Tyr Thr 1 5 <210> 71 <211> 9 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR3 <400> 71 Gln Gln Gly Ala Gly Arg Pro Tyr Thr 1 5 <210> 72 <211> 9 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> LCDR3 <400> 7​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<221> PEPTIDE <222> ()..() <223> LCDR3 <400> 74 Gln Gln Gly Arg Leu Trp Pro Tyr Thr 1 5 <210> 75 <211> 9 <212> PRT <213> artificial <220> <221> PEPTIDE ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Val Ser Gly Phe Asn Ile Lys Asp Ile 20 25 30 Tyr Met His Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Ala Asn Ala Asn Thr Lys Tyr Asp Pro Lys Phe 50 55 60 Gln Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asn Thr Ala 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 Ser Gly Gln Leu Gly Pro Leu Gly 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 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 <210> 78 <211> 214 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> H182‑MUT4‑TGFβRII,LC <400> 78 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Ala Gly Arg Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 79 <211> 22 <212> PRT <213> artificial <220> <221> PEPTIDE <222> ()..() <223> linker <400> 79 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 [[ID=​​

Claims

1. An antibody or antigen-binding fragment thereof that binds to PD-L1, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), said heavy chain variable region (VH) and light chain variable region (VL) comprising, respectively, HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 selected from the following (1) to (13): (1) SEQ ID NO: 36, 41, 52 and SEQ ID NO: 57, 61, 67; (2) SEQ ID NO: 36, 44, 52 and SEQ ID NO: 57, 61, 71; (3) SEQ ID NO: 36, 41, 52 and SEQ ID NO: 57, 64, 67; (4) SEQ ID NO: 36, 41, 52 and SEQ ID NO: 57, 61, 70; (5) SEQ ID NO: 36, 41, 52 and SEQ ID NO: 57, 61, 71; (6) SEQ ID NO: 36, 44, 52 and SEQ ID NO: 57, 61, 70; (7) SEQ ID NO: 36, 47, 52 and SEQ ID NO: 57, 66, 67; (8) SEQ ID NO: 36, 47, 52 and SEQ ID NO: 57, 61, 70; (9) SEQ ID NO: 36, 47, 52 and SEQ ID NO: 57, 61, 67; (10) SEQ ID NO: 36, 47, 52 and SEQ ID NO: 57, 61, 71; (11) SEQ ID NO: 36, 47, 52 and SEQ ID NO: 57, 61, 72; (12) SEQ ID NO: 36, 43, 52 and SEQ ID NO: 57, 61, 70; and (13) SEQ ID NO: 36, 43, 52 and SEQ ID NO: 57, 61, 71.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody or its antigen-binding fragment can be K D It binds to PD-L1 with an affinity of ≤5 nM.

3. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The PD-L1 mentioned is mammalian PD-L1.

4. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The PD-L1 mentioned is the primate PD-L1.

5. The antibody or its antigen-binding fragment according to claim 2, characterized in that, The PD-L1 mentioned is the human or cynomolgus monkey PD-L1.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, characterized in that, The heavy chain variable region and light chain variable region of the antibody or its antigen-binding fragment contain any of the following amino acid sequence combinations: (1) The amino acid sequence shown in SEQ ID NO: 2; and the amino acid sequence shown in SEQ ID NO: 4; (2) The amino acid sequence shown in SEQ ID NO: 10; and the amino acid sequence shown in SEQ ID NO: 12; (3) The amino acid sequence shown in SEQ ID NO: 18; and the amino acid sequence shown in SEQ ID NO: 20; (4) The amino acid sequence shown in SEQ ID NO: 10; and the amino acid sequence shown in SEQ ID NO: 30; (5) The amino acid sequence shown in SEQ ID NO: 10; and the amino acid sequence shown in SEQ ID NO: 31; (6) The amino acid sequence shown in SEQ ID NO: 10; and the amino acid sequence shown in SEQ ID NO: 20; (7) The amino acid sequence shown in SEQ ID NO: 18; and the amino acid sequence shown in SEQ ID NO: 31; (8) The amino acid sequence shown in SEQ ID NO: 32; and the amino acid sequence shown in SEQ ID NO: 33; (9) The amino acid sequence shown in SEQ ID NO: 32; and the amino acid sequence shown in SEQ ID NO: 31; (10) The amino acid sequence shown in SEQ ID NO: 32; and the amino acid sequence shown in SEQ ID NO: 12; (11) The amino acid sequence shown in SEQ ID NO: 32; and the amino acid sequence shown in SEQ ID NO: 20; (12) The amino acid sequence shown in SEQ ID NO: 32; and the amino acid sequence shown in SEQ ID NO: 34; (13) The amino acid sequence shown in SEQ ID NO: 35; and the amino acid sequence shown in SEQ ID NO: 31; or (14) The amino acid sequence shown in SEQ ID NO: 35; and the amino acid sequence shown in SEQ ID NO:

20.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody or its antigen-binding fragment is a monoclonal antibody, a single-chain antibody, a bifunctional antibody, a fully or partially humanized antibody, or a chimeric antibody; or... The antibody or its antigen-binding fragment is scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2 or Fv.

8. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody or its antigen-binding fragment also contains a human or mouse constant region.

9. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody or its antigen-binding fragment further comprises a human or mouse light chain constant region (CL) and / or heavy chain constant region (CH).

10. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody or its antigen-binding fragment further comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and / or a κ or λ type light chain constant region.

11. The antibody or its antigen-binding fragment according to claim 10, characterized in that, The antibody is a monoclonal antibody.

12. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The monoclonal antibody is a mouse-derived, chimeric, or humanized monoclonal antibody.

13. The antibody or its antigen-binding fragment according to claim 11, characterized in that, The heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, and the light chain constant region is κ type.

14. The antibody or antigen-binding fragment thereof according to any one of claims 11 to 13, characterized in that, The heavy chain constant region of the monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 13 or SEQ ID NO: 22; the light chain constant region of the monoclonal antibody contains the amino acid sequence shown in SEQ ID NO:

16.

15. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The antibody comprises the heavy chain variable region shown in SEQ ID NO: 18, the light chain variable region shown in SEQ ID NO: 20, the heavy chain constant region shown in SEQ ID NO: 22, and the light chain constant region shown in SEQ ID NO: 16; or, The antibody comprises the heavy chain variable region shown in SEQ ID NO: 10, the light chain variable region shown in SEQ ID NO: 12, the heavy chain constant region shown in SEQ ID NO: 14, and the light chain constant region shown in SEQ ID NO:

16.

16. A fusion protein comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15, wherein the fusion protein is a fusion protein formed by the antibody or antigen-binding fragment thereof and the extracellular region of a TGFβII type receptor.

17. The fusion protein according to claim 16, characterized in that, The extracellular region of the TGFβII receptor is fused to the C-terminus of the heavy chain of the antibody or its antigen-binding fragment.

18. The fusion protein according to claim 16, characterized in that, The extracellular region of the TGFβII receptor is fused to the C-terminus of the heavy chain of the antibody or its antigen-binding fragment via a linker sequence.

19. The fusion protein according to claim 18, characterized in that, The linker sequence is a flexible linker peptide.

20. The fusion protein according to claim 18, characterized in that, The linker sequence is a flexible linker peptide containing one or more GGGGS.

21. The fusion protein according to any one of claims 16 to 20, characterized in that, The fusion protein comprises an antibody having a heavy chain and a light chain and an extracellular region sequence of a TGFβII receptor fused to the C-terminus of the heavy chain constant region of the antibody, wherein the heavy chain of the antibody is shown in SEQ ID NO:77, the light chain is shown in SEQ ID NO:78, and the extracellular region sequence of the TGFβII receptor is a human TGFβII receptor extracellular region sequence.

22. The fusion protein according to claim 21, characterized in that, The extracellular region sequence of the TGFβII receptor is shown in SEQ ID NO:

29.

23. The fusion protein according to claim 21, characterized in that, The extracellular region sequence of the TGFβII receptor is fused to the C-terminus via a flexible linker peptide.

24. The fusion protein according to claim 23, characterized in that, The flexible linker peptide is shown in SEQ ID NO:

79.

25. A nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 15.

26. A vector comprising the nucleic acid molecule of claim 25.

27. A host cell comprising the nucleic acid molecule of claim 25 and / or the vector of claim 26, or the host cell being transformed or transfected by the nucleic acid molecule of claim 25 and / or the vector of claim 26.

28. A composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 15, a fusion protein as described in any one of claims 16 to 24, a nucleic acid molecule as described in claim 25, a vector as described in claim 26, and / or a host cell as described in claim 27.

29. The composition according to claim 28, characterized in that, The composition is a pharmaceutical composition and further comprises pharmaceutically acceptable excipients.

30. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the fusion protein of any one of claims 16 to 24, the nucleic acid molecule of claim 25, the vector of claim 26, the host cell of claim 27, and / or the composition of claim 28 or 29 in the preparation of a medicament for treating cancers or tumors selected from cervical cancer, osteosarcoma, urothelial carcinoma, lung cancer, squamous cell carcinoma, ovarian cancer, colon cancer, melanoma, prostate cancer, liver cancer, gastric cancer, kidney cancer, breast cancer, head and neck cancer, lymphoma, and metastatic Merkel cell carcinoma.

31. The use according to claim 30, characterized in that, The lung cancer referred to is either non-small cell lung cancer or small cell lung cancer.

32. The use according to claim 30, characterized in that, The liver cancer mentioned is primary liver cancer.

33. The use according to claim 30, characterized in that, The breast cancer mentioned is triple-negative breast cancer.

34. Use of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15, the fusion protein of any one of claims 16 to 24, the nucleic acid molecule of claim 25, the vector of claim 26, the host cell of claim 27, and / or the composition of claim 28 or 29 in the preparation of a medicament for the treatment of bladder cancer.

35. A kit comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 15, a fusion protein as described in any one of claims 16 to 24, a nucleic acid molecule as described in claim 25, a vector as described in claim 26, a host cell as described in claim 27, and / or a composition as described in claim 28 or 29.

Images