WO2021136489A1

WO2021136489A1 - Anti pd-l1 antibody and use thereof

Info

Publication number: WO2021136489A1
Application number: PCT/CN2020/142038
Authority: WO
Inventors: 王双; 焦莎莎; 王荣娟; 张畅
Original assignee: 迈威(上海)生物科技股份有限公司
Priority date: 2019-12-31
Filing date: 2020-12-31
Publication date: 2021-07-08
Also published as: CN113121686A

Abstract

Provided in the present invention are an antibody against PD-L1 or a fragment thereof, a nucleic acid encoding the antibody or the fragment thereof, a composition containing the antibody or the fragment thereof and the use thereof in the treatment of diseases. The antibody or the fragment thereof provided in the present invention can specifically bind to human PD-L1, block the binding of PD-L1 and PD-1, have a strong ADCC effect on target cells and significantly inhibit tumor growth.

Description

Anti-PD-L1 antibody and its application

This patent application claims the priority rights of the Chinese invention patent application with the application number CN201911419802.5 filed on December 31, 2019, and the entire content of which is hereby incorporated by reference.

Technical field

The present invention belongs to the field of biomedicine, and relates to a new anti-PD-L1 antibody or functional fragment thereof. The invention also relates to the application of the antibody or its functional fragment.

Background technique

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

The receptor of PD-L1 is programmed death protein 1 (PD-1), also known as CD279, which is a member of the CD28 family of T cell receptors and is expressed in a variety of immune cells, such as activated T cells, B cells and Monocytes and other surfaces. The mature human PD-1 protein is a type I transmembrane protein composed of 268 amino acids. Its cytoplasmic tail contains an immunoreceptor tyrosine inhibitory motif (ITIM) and an immunoreceptor tyrosine conversion motif (ITSM). ). After PD-L1 on the surface of antigen-presenting cells (APC) binds to the receptor PD-1 on the surface of T cells, it promotes the phosphorylation of tyrosine in the ITSM domain of PD-1, which in turn affects PI3K-AKT-mTOR and The activation of the RAS-MEK-ERK pathway ultimately inhibits the proliferation of antigen-specific T cells, and induces the apoptosis of regulatory T cells by down-regulating the expression of Bcl-2 gene, thereby reducing the immune response involved in T cells. In addition, PD-L1 can also be combined with another T cell costimulatory molecule B7-1 (CD80). When PD-L1 is encountered, CD80 acts as a receptor and inhibits the transmission of T cell activation signals. In addition, PD-L1 can also act as a receptor to "reversely" transmit signals to T cells and tumor cells, thereby affecting the survival of these cells. The mechanism is still unclear. Therefore, PD-L1 can act as both a ligand and a receptor to perform immunomodulatory functions.

Another ligand of PD-1 is PD-L2 (CD273, B7-DC). The combination of the two will also inhibit the activation and proliferation of T cells, but compared with PD-L1, the expression range of PD-L2 is narrower It is mainly expressed in antigen-presenting cells (such as macrophages, dendritic cells) and TH2 cells. The expression of PD-L2 in different tumor types is not as common as PD-L1, and the basic expression level of PD-L2 is low .

More importantly, multiple studies have shown that the expression level of PD-L1 in patient tumors is negatively correlated with patient survival. Therefore, PD-1/PD-L1 targeted therapy for tumor diseases has developed rapidly in clinical practice. In addition, inhibitors targeting PD-L1 targets have a potential advantage over PD-1 target inhibitors: PD-L2 signaling pathways are not affected. Since PD-L2 is rarely highly expressed in tumor tissues, and PD-L2 can interact with repulsive targeting molecule B (RGMb), Chen et al. found (Chen L, Han X. Anti-PD-1/PD-L1 therapy of human cancer: past, present, and future. J Clin Invest. 2015, 125(9): 3384–3391.) Through this interaction, severe inflammatory pulmonary toxic diseases such as interstitial lung disease (ILD) can be reduced. occur.

In addition, an antibody targeting PD-L1 with a subtype of IgG1, in theory, in addition to blocking the interaction of PD-1 with PD-L1 on tumors, may also mediate ADCC (antibody-dependent cell-mediated) of tumor cells. Cytotoxicity, antibody-dependent cell-mediated cytotoxicity) dissolution. At the same time, due to the high expression of PD-L1 on tumors, it has become a potential target for the development of bispecific antibodies.

At present, 6 PD-1/PD-L1 inhibitors have been marketed worldwide, including Merck’s Keytruda, Bristol-Myers Squibb’s Opdivo, Roche’s Tecentriq, Pfizer/Merck’s Bavencio, and AstraZeneca’s Imfinzi. And Sanofi/Regeneron's Libtayo. Among them, the humanized antibody Tecentriq (Atezolizumab) of Roche Pharmaceuticals against human PD-L1 adopts IgG1 subtype with weak ADCC and CDC activity, and is clinically used to treat locally advanced or metastatic non-small cell lung cancer (NSCLC) and locally advanced or Metastatic urothelial carcinoma (mUC), and can be combined with chemotherapy drugs (carboplatin and etoposide) for first-line treatment of extensive-stage small cell lung cancer (ES-SCLC), combined with chemotherapy drugs (albumin paclitaxel) for first-line treatment PD-L1-positive unresectable locally advanced or metastatic triple-negative breast cancer (TNBC), combined with bevacizumab, paclitaxel and carboplatin for the first-line treatment of adult metastatic non-squamous non-small cell lung cancer (NSCLC); Pfizer/ Merck's anti-human PD-L1 fully human antibody Bavencio (Avelumab) uses a subtype of IgG1 with strong ADCC activity and is clinically used to treat metastatic Merkel cell carcinoma and urothelial carcinoma; AstraZeneca's anti-human PD -L1's fully human antibody Imfinzi (Durvalumab) adopts IgG1 subtype with weak ADCC activity and is clinically used to treat unresectable stage III non-small cell lung cancer (NSCLC) and locally advanced or metastatic urothelial carcinoma (mUC) . There are more than 10 monoclonal antibodies against human PD-L1 in the clinical stage in China, and the indications cover cervical cancer, osteosarcoma, urothelial carcinoma, head and neck squamous cell carcinoma, and PD-L1 high expression has not been accepted in the past. Stage IV non-squamous or squamous cell non-small cell lung cancer that has undergone chemotherapy, stage III extensive-stage small cell lung cancer, breast cancer, primary liver cancer, gastric cancer, and prostate cancer.

From the current market of targeted PD-L1 antibody drugs, the indication range is small, the dosage is large, and the overall response rate is not high. Therefore, there is still room for further optimization of such antibodies, and there is still a need to provide more novel anti-PD-L1 antibodies in the art.

Summary of the invention

The present invention provides an anti-PD-L1 antibody through hybridoma screening, humanization technology and affinity maturation. The antibody has high affinity to human PD-L1, high biological activity, strong ADCC activity and long in vivo half-life.

The technical scheme of the present invention is as follows.

In one aspect, the present invention provides an antibody or a fragment thereof. The antibody fragment of the present invention refers to a functional or active fragment of the antibody that binds to the antigen PD-L1.

The PD-L1 refers to the programmed death factor ligand 1. Preferably, the PD-L1 is the PD-L1 of a primate mammal, more preferably the PD-L1 of a human or a cynomolgus monkey.

In the present invention, the binding affinity of the antibody or its fragment to the antigen PD-L1 can be measured by methods known in the art, and preferably by measuring the K _D value (Dissociation constant), which is expressed as molar concentration. . Methods for measuring the binding affinity between an antibody and an antigen are well known in the art, and include, for example, ELISA, flow cytometry, surface plasmon resonance, Biacore measurement and the like. Depending on the measurement method and its specific settings, the measured binding affinity may vary slightly or be within an acceptable range.

According to a specific embodiment of the present invention, the K _D value is measured by the Octet QKe system instrument of Fortebio Company. See the experimental settings in the examples for details.

In the present invention, preferably, the antibody or fragment thereof comprises a heavy chain variable region (VH) and a light chain variable region (VL), and the heavy chain variable region (VH) and a light chain variable region ( VL) respectively comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 shown in the following amino acid sequences (I) to (VI):

(I) 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);

(II)HCDR2: SEQ ID NO: 41 (RIDPANGNTKYDPKFQD), SEQ ID NO: 42 (RIDAGNGNTKYDPKFQD), SEQ ID NO: 43 (RIDPRNGNTKYDPKFQD), SEQ ID NO: 44 (RIDPANANTKYDPKFQD), SEQ IDPANANTKYDPKFQD: 45 (DPRIDVLNANTKYDPKFQD), SEQ ID NANTKYDPKFQD: SEQ ID NO: 46 (RIDVRNGNTKYDPKFQD), SEQ ID NO: 47 (RIDPAAGNTKYDPKFQD), SEQ ID NO: 48 (RIDSNAGNTKYDPKFQD), SEQ ID NO: 49 (RIDLANANTKYDPKFQD), SEQ ID NO: 50 (RIDRIDNO QDGN) (RIDPRNGNTKYDPKFQD);

(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);

(IV) LCDR1: SEQ ID NO: 57 (RASQDISNYLN), SEQ ID NO: 58 (RASQDISAYLN), SEQ ID NO: 59 (RASQSISSYLN), SEQ ID NO: 60 (RASQDISSYLN);

(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);

(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).

Further preferably, the heavy chain variable region (VH) and the light chain variable region (VL) respectively comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 shown in (1) to (13) below. :

(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);

(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);

(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);

(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);

(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);

(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);

(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);

(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);

(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);

(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);

(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);

(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);

(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).

The above heavy chain and light chain CDR combinations are derived from the murine antibodies, humanized antibodies, and even affinity mature antibodies provided by the present invention. The antibody or fragment thereof provided by the present invention can _{bind to PD-L1 with an affinity of K D} ≤5 nM, preferably mammalian PD-L1, more preferably primate PD-L1, further preferably human or cynomolgus PD-L1, especially It is human PD-L1.

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

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 has at least 75% identity with the amino acid sequence shown The amino acid sequence of; and/or

The light chain variable region of the antibody or fragment thereof comprises a sequence selected from:

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 that has at least 75% identity with the amino acid sequence shown.

More preferably, the heavy chain variable region and light chain variable region of the antibody or its fragment provided by the present invention comprise any combination of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO: 2 or the 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;

(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;

(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;

(4) The amino acid sequence shown in SEQ ID NO: 10 or the 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;

(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;

(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;

(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;

(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;

(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;

(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;

(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;

(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;

(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;

(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.

Generally speaking, the antibodies or fragments thereof provided by the present invention are monoclonal antibodies, single-chain antibodies, bifunctional antibodies, single domain antibodies, nanobodies, fully or partially humanized antibodies or chimeric antibodies, etc., or, The antibody or fragment thereof is a half-antibody or an antigen-binding fragment of a half-antibody, such as scFv, BsFv, dsFv, (dsFv) ₂ , Fab, Fab', F(ab') ₂ or Fv;

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

More preferably, the antibody or fragment thereof comprises a heavy chain constant region selected from IgG, IgA, IgM, IgD or IgE and/or a kappa or lambda light chain constant region.

According to specific embodiments of the present invention, the antibody provided by the present invention is a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody; preferably, the heavy chain constant region of the monoclonal antibody is IgG1 or IgG4 subtype, the light chain constant region is κ type;

Preferably, the heavy chain constant region of the monoclonal antibody comprises 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 comprises the amino acid sequence shown in SEQ ID NO: 16 or an amino acid sequence having at least 75% identity with the amino acid sequence.

The above-mentioned at least 75% identity of the present invention is at least 80%, preferably at least 85%, more preferably at least 90%, further preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, or even 99% identity, etc. Any percentage of identity ≥ 75%.

According to specific embodiments of the present invention, the present invention provides the following antibody: the antibody comprises the heavy chain variable region shown in SEQ ID NO: 18, and the light chain variable region shown in SEQ ID NO: 20, SEQ ID NO: The heavy chain constant region shown in 22, the light chain constant region shown in SEQ ID NO: 16, 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, and the heavy chain constant region shown in SEQ ID NO: 14, SEQ ID NO The light chain constant region shown by :16 is named h182 in the present invention.

Based on the antibody or fragment thereof of the present invention, the present invention also provides a conjugate or fusion protein comprising the antibody or fragment thereof of the present invention. The conjugate or fusion protein may include other parts that are bound to the antibody or fragments of the present invention by chemical or physical methods, such as cell surface receptors, small molecule compounds such as amino acids and carbohydrates, small molecule polymers, or other parts of the present invention. Any other part of the antibody that is modified, or even an active protein or polypeptide. For example, the conjugate or fusion protein may be a bispecific antibody or bifunctional protein comprising the antibody or fragment thereof of the present invention.

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 TGFβ type II receptor. For example, the extracellular region of the TGFβ type II 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, can be fused to the C-terminus via a linking sequence. The linking sequence may be a flexible linking peptide, for example, a flexible linking peptide comprising one or more (GGGGS).

According to a specific embodiment of the present invention, the fusion protein is a bifunctional protein, which comprises an antibody having a heavy chain and a light chain and a TGFβ type II receptor extracellular region sequence fused to the C-terminus of the heavy chain constant region, wherein The heavy chain of the antibody is shown in SEQ ID NO: 77, and the light chain is shown in SEQ ID NO: 78. The sequence of the extracellular region of the TGFβ type II receptor is the sequence of the extracellular region of the human TGFβ type II receptor, for example, shown in SEQ ID The amino acid sequence of NO:29. Preferably, the sequence of the extracellular region of the TGFβ type II receptor is fused to the C-terminus via a flexible connecting peptide. According to a specific embodiment of the present invention, the flexible connecting peptide is shown in SEQ ID NO: 79.

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

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 and light chain CDR contained in the antibody or fragment thereof. , Light chain variable region, heavy chain variable region, heavy chain or light chain;

Preferably, the nucleic acid molecule comprises 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 The nucleotide sequence shown in SEQ ID NO: 19.

In yet another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention. The vector can be a eukaryotic expression vector, a prokaryotic expression vector, an artificial chromosome, a phage vector, and the like.

The vector or nucleic acid molecule of the present invention can be used to transform or transfect a host cell or enter the host cell in any manner for the purpose of preservation or expression of antibodies.

Therefore, in another aspect, the present invention provides a host cell comprising the nucleic acid molecule and/or vector of the present invention, or the host cell is transformed or transfected by the nucleic acid molecule and/or vector of the present invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial or insect, fungal, plant or animal cell.

Based on the disclosure of the present invention, the antibodies or fragments thereof and corresponding conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells provided by the present invention can be obtained by using any conventional technical methods known in the art. The antibody or fragments thereof, conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells can be included in a composition, such as a pharmaceutical composition, and more particularly in a pharmaceutical preparation, so as to be included in actual needs. Used for various purposes.

Therefore, in another aspect, the present invention also provides a composition comprising the antibody or fragments, conjugates or fusion proteins, nucleic acid molecules, vectors and/or host cells of the present invention. Preferably, the composition is a pharmaceutical composition, which optionally further comprises pharmaceutically acceptable excipients.

As an antibody that binds to PD-L1 or any part thereof, the present invention also provides related applications of the above-mentioned subject.

Specifically, in another aspect, the invention provides the use of the antibody or its fragment, conjugate or fusion protein, nucleic acid molecule, vector, host cell and/or composition in the preparation of a medicine, which is used for treatment Diseases related to the positive expression of PD-L1, and/or used to block PD-L1/PD-1 interaction.

Preferably, the drug is used to treat cancer or tumor, preferably a cancer or tumor that positively expresses PD-L1; more preferably, the disease is selected from cervical cancer, osteosarcoma, urothelial cancer, lung cancer such as non-small cell Lung or small cell lung cancer, squamous cell carcinoma, ovarian cancer, colon cancer, melanoma, bladder cancer, prostate cancer, liver cancer such as primary liver cancer, stomach cancer, kidney cancer, breast cancer such as triple negative breast cancer, head and neck cancer, lymph Tumor, metastatic Merkel cell carcinoma.

In addition, the present invention provides a method for treating diseases related to the positive expression of PD-L1, the method comprising administering the antibody or fragments, conjugates or fusion proteins, nucleic acid molecules, The vector, host cell and/or composition, and optionally other drugs or treatments. The treatment can be achieved by blocking the PD-L1/PD-1 interaction. Preferably, the disease is cancer or tumor, preferably PD-L1 positive expression cancer or tumor; more preferably, the disease is selected from cervical cancer, osteosarcoma, urothelial cancer, 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, stomach cancer, kidney cancer, breast cancer such as triple negative breast cancer, head and neck cancer, lymphoma, Metastatic Merkel cell carcinoma. The optional other drugs or treatment means refer to other immune enhancing drugs or means that can be administered in combination with the antibody or fragments, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells or compositions of the present invention, such as small molecules Chemical drugs, targeted drugs, antibodies and other recombinant protein drugs, vaccines, ADCs, oncolytic viruses, gene and nucleic acid therapy drugs, and radiotherapy. The combined administration of the two can be carried out in any form, for example, simultaneously, continuously or at a certain time interval. The subject is a mammal, preferably a primate, further preferably a human or cynomolgus monkey, especially a human.

In another aspect, the present invention provides a kit comprising the antibody or fragments, conjugates or fusion proteins, nucleic acid molecules, vectors, host cells and/or compositions of the present invention. The kit can be used for detection or diagnosis purposes.

Anti-PD-L1 antibodies can activate T cells by blocking the binding of PD-1 and its ligand PD-L1. Although the antibodies that have been reported so far can block the interaction between PD-1 and PD-L1, they are used in large amounts in clinical practice, and most of them are antibody subtypes with weak ADCC activity. Compared with antibody subtypes with strong ADCC activity, they have high resistance. The killing effect of tumor tissues expressing PD-L1 is weak.

In contrast, the present invention provides an anti-PD-L1 antibody through hybridoma screening, humanization technology, and affinity maturation. The antibody has high affinity to human PD-L1, and has high biological activity and strong ADCC activity. And long in vivo half-life.

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

1. The antibody of the present invention is an anti-PD-L1 humanized antibody with high affinity.

Taking the humanized anti-PD-L1 antibody H182-MUT4 of the present invention as an example, the 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, Durvalumab affinity (KD) is 8.24E-10M, indicating that H182-MUT4 has high affinity with human PD-L1.

2. The antibody of the present invention has good biological activity.

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

Description of the drawings

Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

Figure 1: The first round of ELISA to detect the binding of the supernatant of the positive hybridoma to the recombinant protein in the extracellular domain of human PD-L1.

Figure 2: The second round of ELISA to detect the binding of the supernatant of the positive hybridoma to the recombinant protein of the extracellular domain of human PD-L1.

Figure 3: ELISA test results of cross-reaction between the supernatant of positive hybridoma clones and recombinant PD-L1 of different species.

Figure 4: ELISA detection result of positive hybridoma clone supernatant on the binding of human PD-L1 to its receptor PD-1.

Figure 5: FACS detection results of binding of antibodies purified from the supernatant of positive hybridoma clones to PD-L1 on the surface of 293 cells.

Figure 6: ELISA detection of the binding of murine antibodies from the supernatant of the 182 positive hybridoma clone to members of the B7 family and CD28 family.

Figure 7: Comparison of affinity between antibody H182-MUT4 and known antibodies with the same target.

Figure 8: ELISA detection result of the inhibitory effect of antibody H182-MUT4 on the binding of human PD-L1 to its receptor PD-1.

Figure 9: Results of binding activity of antibody H182-MUT4 to PD-L1 on the surface of CHO cells.

Figure 10: The dose-effect curve of ADCC toxicity of antibody in vitro, where 10A: Atezolizumab; 10B: Avelumab; 10C: H182-MUT4; 10D: Durvalumab; 10E: isotype control antibody.

Figure 11: Antibody single administration drug-time curve in nude mice (PD-L1 detection), where 11A: Avelumab; 11B: Atezolizumab; 11C: Durvalumab; 11D: H182-MUT4.

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

Figure 13: Tumor volume changes of PD-1 and PD-L1 humanized mice bearing hPD-L1-MC38 subcutaneous tumor model after H182-MUT4-TGFβRII administration.

The best way to implement the invention

Hereinafter, the present invention will be explained with reference to specific embodiments. Those skilled in the art can understand that these embodiments are only used to illustrate the present invention, and they do not limit the scope of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are all conventional methods. The medicinal materials, reagent materials, etc. used in the following examples are all commercially available products unless otherwise specified.

The amino acid sequence of the control antibody Atezolizumab is derived 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;

The control antibody Avelumab amino acid sequence is derived 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;

The amino acid sequence of the control antibody Durvalumab is derived 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;

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

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

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

Immunization: Balb/c mice were immunized with human PD-L1/mFc recombinant protein, and the serum titer was detected by ELISA using a 96-well ELISA plate coated with human PD-L1-His recombinant protein; the serum titer reached the fusion requirement Mice are used for the next step of cell fusion.

Cell fusion and hybridoma preparation: On the 67th day after the initial immunization, select mice with the required titer, take the mouse spleen aseptically, prepare a B lymphocyte suspension, and mix it with FO myeloma cells at a ratio of 5:1. The two cells are fused under the action of PEG4000. After the fused cells were resuspended in HAT medium, they were divided into 96-well cell culture plates. Place 37 ℃, 5% CO ₂ incubator for culture.

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

1. ELISA binding screening of positive hybridomas

10-14 days after fusion, coat the ELISA plate with human PD-L1-His recombinant protein (10ug/ml, pH 9.6, 0.1M NaHCO ₃ ), 4°C, overnight; block with 4% skimmed milk powder-PBS, 37 ℃, 2h; wash with PBST (0.05% Tween20-PBS) three times, add hybridoma clone culture supernatant, 37℃, 1h. Set up the following controls: (1) Positive control (PC): mouse serum after immunization (diluted with PBS 1:1000); (2) Negative control (NC): mouse serum before immunization (diluted with PBS 1:1000); (3) Blank control: PBS. Wash three times with PBST (0.05% Tween20-PBS), add HRP-goat anti-mouse IgG (Fcγ), diluted 1:20000, 37℃, 1h; then wash five times with PBST (0.05% Tween20-PBS), add OPD color developing solution, avoid light for 10-15min, add 2M H ₂ SO _{4 to} stop the reaction; read the value of A492 with the microplate reader. The test hole A492 value is greater than the negative control hole A492 value by more than 2.1 times, it is judged as positive. In order to determine the reliability of positive clones, a second round of screening was carried out every other day after the first screening change. After testing and identification, a total of 20 antibody secretion-positive cell lines were obtained,

cell lines

57, 92, 143, 152, 162, 182, 189, 234, 244, 267, 301, 315, 369, 403, 453, 492, No. 504, 564, 582, and 651 (Figure 1, Figure 2), continue to further screen the antibodies secreted by these 20 strains.

2. Screening of positive hybridoma clones by cross-species ELISA

The recombinant human PD-L1-His protein and the recombinant cynomolgus PD-L1-His protein (Cat: 90251-C08H, Beijing Yiqiao Shenzhou) were coated overnight at 4°C, and the coating concentration was 0.5 μg/mL; the plate was washed with PBS 3 After the second, add 5% BSA PBS, block at 37°C for 60 minutes, wash the plate 3 times with PBST; add the supernatant of the above 20 hybridomas diluted 100 times in PBS, and set the following controls: (1) Positive control (PC): Atezolizumab mouse IgG constant region format is 0.1ug/ml; (2) Negative control (NC): irrelevant hybridoma supernatant (diluted 1:100 with PBS); (3) Blank control: PBS. Incubate at 37°C for 60 minutes, wash the plate with PBST 4 times; add 1:5000 diluted HRP-goat anti-mouse IgG (Fcr) (Cat: 115-035-071, Jackson Immuno Research), incubate at 37°C for 30 minutes, wash the plate with PBST 4 Times; add TMB substrate for color development, after incubating at 37°C for 10 minutes, add 2M HCl to terminate the reaction; using 630nm as the reference wavelength, read and record the absorbance of the well plate A450nm-630nm at the wavelength of 450nm. Except for No. 189, the supernatants of other hybridomas can specifically bind to recombinant human and cynomolgus PD-L1 (Figure 3).

3. ELISA screening of positive hybridoma clones inhibiting the binding of human PD-L1 to its receptor PD-1

Human PD-1-His at a concentration of 0.5 μg/mL was coated overnight at 4°C, and sealed with 5% BSA in a constant temperature incubator at 37°C for 60 minutes. Add the supernatant of the above 20 hybridomas diluted 100 times with PBS, and set the following controls: (1) Positive control (PC): human PD-L1/mFc 0.1ug/ml; (2) Negative control (NC): irrelevant hybridoma Supernatant (diluted with PBS 1:100); (3) Blank control: PBS; (4) Control antibody control (ref mAb): Atezolizumab mouse IgG constant region format 0.1ug/ml. Reaction in a 37°C constant temperature incubator for 120 minutes, while adding 1μg/mL human PD-L1/mFc to incubate with the antibody, and a 37°C constant temperature incubator for 60 minutes. Then add 1:5000 diluted HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research), react for 45 minutes, add TMB (Cat: ME142, Beijing Taitianhe Biological) substrate for 15 minutes, 2M HCl Read the plate after termination. With 630nm as the reference wavelength, read and record the absorbance value A450nm-630nm of the orifice plate at a wavelength of 450nm. ELISA results showed that the supernatants of hybridomas No. 92, 143, 182, 267, and 453 could block the binding of human PD-L1 to its receptor PD-1 (Figure 4).

4. Screening of binding between positive hybridomas and PD-L1 on the surface of 293 cells

Using the human PD-L1 full-length expression plasmid (Cat.:HG10084-UT, Beijing Yiqiao Shenzhou), and according to the operating instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the full-length human PD-L1 expression plasmid was transfected Recombinant expression into HEK293 cells to construct 293 cells (293/PD-L1) expressing human PD-L1. 48 hours after cell transfection, the cells were used for flow cytometric analysis (FACS).

According to the above ELISA results, the hybridoma supernatants of clones No. 92, 143, 182, 267, 453, 492 were selected and purified by affinity purification to obtain 6 mouse antibodies (5ug/ml) and recombinant human PD-L1 293 cells The (293/PD-L1) suspension was incubated at 37°C for 30 minutes, and the following controls were set: (1) Positive control (PC): Atezolizumab mouse IgG constant region format 5ug/ml; (2) Negative control (NC): irrelevant mouse Antibody 5ug/ml. After washing the cells 3 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 with PBS 3 times, the average fluorescence intensity (MFI) of the cells was measured by a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to verify whether the antibody secreted by the hybridoma can bind to PD-L1 on the surface of 293 cells, as shown As shown in 5, only clone No. 182 has good binding with PD-L1 on the surface of 293 cells. Select 182 as a candidate clone for the next step of screening.

5. ELISA detection of PD-L1 family specificity of positive hybridoma clones

Based on the above test results, clone 182 was selected for specific detection, and 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), B7H4 (serial number: NP_078902.2, 29aa-257aa), ICOS-His (serial number: NP_036224.1, 1aa-199aa), PD-L2-hFc (sequence No.: NP_079515.2, 20aa-220aa), PD-1-His (serial number: NP_005009.2, 21aa-167aa), human PD-L1-His recombinant protein was coated overnight at 4°C, and the coating concentration was 2μg/mL; After washing the plate 3 times with PBS, add 5% BSA PBS, block at 37°C for 60 min, wash the plate 3 times with PBST; add mouse antibody No. 182 (1ug/ml), set the following controls: (1) Blank control: PBS. Incubate at 37°C for 60 minutes, wash the plate with PBST 4 times; add 1:5000 diluted HRP-goat anti-mouse IgG (Fcr) (Cat: 115-035-071, Jackson Immuno Research), incubate at 37°C for 30 minutes, wash the plate with PBST 4 Times; add TMB substrate for color development, after incubating at 37°C for 10 minutes, add 2M HCl to terminate the reaction; using 630nm as the reference wavelength, read and record the absorbance of the well plate A450nm-630nm at the wavelength of 450nm. The experimental results show that the mouse antibody No. 182 can specifically bind to recombinant human PD-L1, and has no binding activity with other members of the B7 family and CD28 family, and does not produce cross-reactions (Figure 6).

Example 3: Sequence determination of murine anti-human PD-L1 antibody

After expanding the hybridoma cell 182 that secretes anti-human PD-L1 antibody, use Mouse Monoclonal Antibody IgG Subclass Test Card (Cat: A12403, VicNovo) and Mouse Monoclonal Antibody Light/Heavy Chain Test Card (Cat: A12401, VicNovo) The subtype was detected according to the reagent operating procedures, and the subtype was identified as: the heavy chain was IgG1, and the light chain was Kappa chain.

The 182 hybridoma cells were extracted according to the instructions of the TRIzol kit (Cat: 15596026, Invitrogen); the total RNA of the hybridoma cells was reverse-transcribed into cDNA using M-MuLV reverse transcriptase (Cat: M0253S, NEB) ; Use degenerate primers (refer to books [Dong Zhiwei, Wang Yan. Antibody Engineering (Second Edition). Beijing Medical University Press, 2001, 313-314]) and Phusion kit (Cat: E0553L, NEB) to amplify antibodies The light chain variable region IgVL (κ) and the heavy chain variable region V _H sequence; use the gel recovery kit (Cat: AP-GX-250, Axygen) to purify the PCR amplification product; follow the T vector cloning kit (Cat: ZC205, Zhuangmeng Biological) Instruction manual connect the amplified PCR product to T vector and transform E. coli competent cells, the strain is amplified, the plasmid is extracted, and then DNA sequencing is performed to obtain the monoclonal antibody variable region sequence. The sequencing results showed that the nucleotide sequence of the 182 mouse antibody heavy chain variable region DNA from the hybridoma cell is shown in SEQ ID NO:1, and the amino acid sequence of the 182 mouse antibody heavy chain variable region inferred from the 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 inferred from the DNA sequence is shown in SEQ ID NO: 4.

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

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

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

The genes encoding the light and heavy chain amino acid sequences of the same target control antibody (Atezolizumab, Avelumab, Durvalumab) were fully synthesized, and cloned into the eukaryotic transient recombinant expression vector to obtain the control antibody light chain and heavy chain expression plasmids, and then transferred to the large intestine Bacillus was amplified, and a large number of plasmids containing the light chain and heavy chain of the control antibody were obtained. Using these plasmids, and according to the operating instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), the light and heavy chain plasmids of the control antibody were transferred respectively. Recombinant expression in HEK293 cells. 5-6 days after cell transfection, the culture supernatant is taken, and the expression supernatant is purified using a ProA affinity chromatography column to obtain a control antibody.

The murine antibody light chain variable region and heavy chain variable region genes obtained by the cloning of the present invention were introduced into the restriction site by PCR, and cloned into the upstream of the human-kappa light chain constant region and human IgG1 heavy chain constant region encoding genes, respectively The human-mouse chimeric light chain (Ch182L) and human-mouse chimeric heavy chain (Ch182H) expression plasmids were obtained from the eukaryotic expression vector, and transformed into Escherichia coli for amplification, and a large number of human-mouse chimeric antibody light chains were isolated and obtained. The plasmids of the chain-Ch182L- and the heavy chain-Ch182H-, using this plasmid, and according to the operating instructions of the transfection reagent 293fectin (Cat:12347019, Gibco), transfer the light and heavy chain plasmids of the 182 chimeric antibody into HEK293 cells Recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography. The obtained 182 chimeric antibody was obtained using Fortebio's Octet QKe system instrument, and the anti-human antibody Fc segment was used. Capture antibody (AHC) biological probe captures the Fc fragment of an antibody to determine the affinity of the antibody. During the determination, the 182 chimeric antibody was diluted to 5ug/mL with PBS buffer, and passed over the surface of the AHC probe (Cat: 18-5060, PALL) for 120s. Human PD-L1-His recombinant protein was used as the mobile phase, and the concentration of PD-L1-His recombinant protein was 50 nM and 20 nM. The binding time is 300s, and the dissociation time is 300s. After the experiment is completed, the blank control response value is subtracted, and the software is used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding. The results show that the chimeric antibody Ch182 has a high affinity with human PD-L1, and its affinity (KD) is about 2.02E-9M, suggesting that the sequence of the 182 mouse antibody was cloned correctly.

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

1. Humanized design of mouse monoclonal antibody 182

First, a comprehensive analysis of the murine antibody heavy chain sequence is performed to determine the antigen complementarity determinant (CDR) region where the antibody binds to the antigen and the framework that supports the conservative three-dimensional conformation of the antibody. Then, according to the result of homology comparison, search for the most similar human antibody template 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 grafting was performed to achieve a high degree of humanization of the 182 heavy chain variable region (VH) in the framework region. According to the result of homology comparison, find the most similar human antibody template in the human antibody germline library (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php#VHEX), and combine The full sequence of blast results and LCDR3 sequence, CDR transplantation, realized the full humanization of the light chain framework region. The nucleotide sequence of the humanized heavy chain variable region of 182 antibody CDR Grafted is shown in SEQ ID NO: 5, 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, see SEQ ID NO:8 for the amino acid sequence.

2. Back mutation design of humanized 182

According to the sequence characteristics of the 182 mouse antibody, the CDR-grafted humanized light and heavy chain variable region sequence was designed for back mutations. The back mutation sites are shown in Table 1 below. 182 heavy chain variable region nucleotides after humanized back mutation See SEQ ID NO: 9 for the sequence and SEQ ID NO: 10 for the amino acid sequence; see SEQ ID NO: 11 for the nucleotide sequence of the light chain variable region after humanized back mutation, and SEQ ID NO: 12 for the amino acid sequence.

Table 1 182 humanized sequence design

Note: If Y87F is labeled according to the Kabat numbering system, change the 87th amino acid Y back to F

h182_VH1(SEQ ID NO: 6)

h182_VL1 (SEQ ID NO: 8)

h182_VH2 (SEQ ID NO: 10)

h182_VL2 (SEQ ID NO: 12)

3. Recombinant expression of humanized monoclonal antibody 182

The humanized design of the 182 antibody light and heavy chain variable region (h182_VL1, h182_VH2) sequence was fully synthesized, and the humanized h182_VH2 was cloned into the eukaryotic expression vector pKN041 of the human IgG1 heavy chain constant region coding gene by restriction enzyme digestion. Upstream, 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 into the eukaryotic expression vector pKN019 coding gene for human light chain Cκ by restriction enzyme digestion The light chain constant region nucleotide sequence is shown in SEQ ID NO: 15, and the amino acid sequence is shown in SEQ ID NO: 16. The humanized 182 light and heavy chain expression vector is constructed to obtain the light chain (pKN019-h182L1) and heavy chain (pKN041-h182H2) expression plasmid, transformed into Escherichia coli and amplified, isolated and obtained h182 antibody light chain and heavy chain plasmids h182L1, h182H2; according to the back mutation design, using the StarMut gene site-directed mutagenesis kit (Cat: T111-01, GenStar ), respectively carry out site-directed mutations on the light chain (pKN019-h182L1) and heavy chain (pKN041-h182H2) expression plasmids, and transfer them into E. coli for amplification to obtain h182 antibody light and heavy chain expression plasmids h182L2, h182H1; humans using 182 Sourceized plasmid and chimeric antibody plasmid, and according to the operating instructions of transfection reagent 293fectin (Cat:12347019, Gibco), combine the light and heavy chain plasmids of 182 antibody, see Table 2 for the combination, and transfer into HEK293 cells for recombinant expression . 5-6 days after cell transfection, take the culture supernatant, use ProA affinity chromatography column to purify the expression supernatant, obtain 182 different humanized antibodies, use Fortebio's Octet QKe system instrument, adopt anti-human Capture antibody (AHC) bioprobe for the Fc segment of the antibody. The method of capturing the Fc segment of the antibody is used to determine the affinity of the antibody. During the determination, the 182 antibody was diluted to 5ug/mL with PBS buffer, and passed through the surface of the AHC probe (Cat: 18-5060, PALL) for 120s. Human PD-L1-His recombinant protein was used as the mobile phase, and the concentration of PD-L1-His recombinant protein was 100 nM. The binding time is 300s, and the dissociation time is 300s. After the experiment, the blank control response value was subtracted, and the software was used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.

Table 2 Combinations of light and heavy chain sequences of 182 antibodies

To	Ch182HCh182H	h182H1h182H1	h182H2h182H2
Ch182LCh182L	Ch182Ch182	182-1182-1	182-2182-2
h182L1h182L1	182-3182-3	182-4182-4	182-5182-5
h182L2h182L2	182-6182-6	182-7182-7	182-8182-8

Note: This table shows the sequences obtained from various combinations of 182 light and heavy chains (the chain names are the same as the corresponding plasmids). For example, 182-1, the antibody is composed of 182 chimeric antibody light chain Ch182L and humanized heavy chain h182H1, and other analogies .

The affinity between the 182 combination antibody and the control antibody Atezolizumab and the human PD-L1-His recombinant protein determined by ForteBio is shown in Table 3. The optimal humanized sequence combination obtained by the screening is 182-8, and the antibody affinity (KD) of this combination is 3.64E-9M, the affinity of Ch182 (KD: 2.14E-09M) did not decrease significantly. This combination was regarded as the preferred sequence after humanization and named h182 for further functional verification.

Table 3 Affinity determination results of the antibodies in Table 2 and the recombinant protein of the extracellular domain of human PD-L1

抗体组合Antibody combination	KD值(M)KD value (M)
AtezolizumabAtezolizumab	1.93E-101.93E-10
Ch182Ch182	2.14E-092.14E-09
182-1182-1	5.82E-095.82E-09

182-2182-2	1.93E-091.93E-09
182-3182-3	4.77E-094.77E-09
182-4182-4	8.19E-098.19E-09
182-5182-5	3.43E-093.43E-09
182-6182-6	4.64E-094.64E-09
182-7182-7	5.68E-095.68E-09
182-8182-8	3.64E-093.64E-09

Example 6: Affinity maturation of humanized antibody h182

In order to obtain higher affinity antibodies, h182 was subjected to affinity maturation, and mutations were introduced into all CDR regions of the light and heavy chains by PCR to construct a mutation library for affinity maturation of h182.

1. Construction of phage display mutation library of h182 heavy chain CDR region and light chain CDR region

Using conventional cloning techniques, the synthesized parental h182-Fab coding gene fragments and 6 incomplete parental h182-Fab coding gene fragments containing the stuffer region were respectively connected to the phagemid BBTN to construct the parental expression vector BBTN- h182-Fab and six tool carriers: BBTN-h182-HCDR1, BBTN-h182-HCDR2, BBTN-h182-HCDR3, BBTN-h182-LCDR1, BBTN-h182-LCDR2, BBTN-h182-LCDR3. The synthesized single-stranded mutation unit h182-HCDR1-NNK, h182-HCDR2-NNK, h182-HCDR3-NNK, h182-LCDR1-NNK, h182-LCDR2-NNK, h182-LCDR3-NNK, using PCR method to form double Chain NNK fragments, each double-stranded NNK fragment and the corresponding tool vector plasmid are mixed in a certain ratio, and at the same time, type IIS endonuclease and T4 DNA ligase are added to perform a ligation reaction, and TG1 competent cells are transformed by electroporation. Complete the construction of six mutation libraries (HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3).

2. Library panning

After the library was rescued and packaged with phage particles for panning, it was panned with human PD-L1-His recombinant protein by solid phase method, and each round of screening reduced the antigen concentration compared to the previous round. After three rounds of panning, Multiple clones were selected for phage ELISA to detect the binding activity, and the positive clones were sequenced.

3.Fortebio detects affinity

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 re- Combine and express in mammalian cells (293), purify the expression supernatant with ProA affinity chromatography column, and use Fortebio's Octet QKe system instrument to obtain the antibody, and use anti-human antibody Fc segment capture antibody (AHC) The method of capturing the Fc portion of an antibody by a biological probe determines the affinity of the antibody. During the determination, the antibody was diluted to 4ug/mL with PBS buffer, and flowed over the surface of the AHC probe (Cat: 18-5060, PALL) for 120s. Human PD-L1-His recombinant protein was used as the mobile phase with a concentration of 60 nM. The binding time is 300s, and the dissociation time is 300s. After the experiment is completed, the blank control response value is subtracted, and the software is used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding. The affinity results are shown in Table 5. The affinity mature antibody containing the combination h182-LCDR3-4+h182-HCDR2-3 was selected for further verification. The antibody molecule was named H182-MUT4, and the heavy chain variable region nucleoside of H182-MUT4 The acid sequence is shown in SEQ ID NO: 17, the amino acid sequence is shown in SEQ ID NO: 18; the light chain variable region nucleotide sequence is shown in SEQ ID NO: 19, and the amino acid sequence is shown in SEQ ID NO: 20.

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

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

Table 4 Sequences of positive CDR regions screened from the h182 CDR region phage display mutation library

Note: As indicated by h182-LCDR1-1, the sequence after affinity maturation of the CDR1 region is RASQDISAYLN, and the remaining sequence of the affinity mature antibody light chain containing the light chain CDR (LCDR1) is the same as h182, and others are deduced by analogy.

Table 5 Affinity determination results of antibodies after h182 CDR region affinity maturation

Example 7: Comparison of affinity between H182-MUT4 and the same target antibody

HEK293 transient expression system respectively prepares H182-MUT4 and the same target control antibodies Atezolizumab and Durvalumab. The nucleotide sequence of the constant region of the H182-MUT4 heavy chain is shown in SEQ ID NO: 21, and the amino acid sequence is shown in SEQ ID NO: 22; H182-MUT4 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, using Fortebio's Octet QKe system instrument, using anti-human antibody Fc segment capture antibody (AHC) biological probe to capture the antibody The Fc segment method determines antibody affinity. During the determination, the antibodies (h182, H182-MUT4 and control antibodies Atezolizumab, Durvalumab) were diluted with PBS buffer to 4ug/mL, and passed through the surface of the AHC probe (Cat: 18-5060, PALL) for 120 seconds. Human PD-L1-His recombinant protein was used as the mobile phase with a concentration of 60 nM. The binding time is 300s, and the dissociation time is 300s. After the experiment is completed, the blank control response value is subtracted, and the software is used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding.

The results show that the reaction curves of H182-MUT4 and control antibodies Atezolizumab, Durvalumab and human PD-L1 recombinant protein are shown in Figure 7. The curve is fitted and the affinity is calculated. The affinity (KD) of H182-MUT4 is 4.65E-10M, and the affinity of Atezolizumab ( KD) is 7.19E-10M, and Durvalumab affinity (KD) is 8.24-10M. The detailed kinetic parameters are shown in Table 6 below. The results show that the matured H182-MUT4 has high affinity with human PD-L1, and the dissociation value is significantly lower than that of h182.

Table 6 Affinity determination results of H182-MUT4 and the same target marketed antibody

To	KD值(M)KD value (M)	kon(1/Ms)kon(1/Ms)	kdis(1/s)kdis(1/s)
h182h182	2.22E-092.22E-09	4.50E+054.50E+05	1.00E-031.00E-03
H182-MUT4H182-MUT4	4.65E-104.65E-10	4.27E+054.27E+05	1.99E-041.99E-04
AtezolizumabAtezolizumab	7.19E-107.19E-10	4.28E+054.28E+05	3.08E-043.08E-04
DurvalumabDurvalumab	8.24E-108.24E-10	4.85E+054.85E+05	3.99E-043.99E-04

Example 8: Detection of the inhibitory effect of H182-MUT4 on the binding of human PD-L1 to its receptor PD-1 by ELISA

Human PD-1-hFc, with a concentration of 0.5 μg/mL, was coated overnight at 4°C, and sealed with 5% BSA in a constant temperature incubator at 37°C for 60 minutes. H182-MUT4 and control antibodies Atezolizumab, Durvalumab, and isotype control NC-hIgG1 (initial concentration of 4.5μg/mL, 1.3 times serial dilution, 12 gradients) were reacted in a constant temperature incubator at 37°C for 60 minutes, and then 1μg/mL was added The PD-L1-mFc was incubated with the antibody and reacted in a constant temperature incubator at 37°C for 60 minutes. Wash the plate 4 times with PBST; then add 1:5000 diluted HRP-anti-mouse Fc (Cat: 115-035-071, Jackson Immuno Research), react for 45 minutes, add TMB (Cat: ME142, Beijing Taitianhe Biological) substrate The color is developed for 15 minutes, and the plate is read after 2M HCl is stopped. With 630nm as the reference wavelength, read and record the absorbance value A450nm-630nm of the orifice plate at a wavelength of 450nm.

The results show that H182-MUT4 can effectively block 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, and the IC50 values were 6.236nM, 7.432nM and 7.64nM, respectively (Figure 8) . Compared with Atezolizumab and Durvalumab, H182-MUT4 has slightly better blocking activity.

Example 9: Analysis of the binding activity of H182-MUT4 and CHO-PD-L1-CD3L cells

The recombinant human PD-L1 and anti-CD3-ScFv CHO cells (CHO-PD-L1-CD3L , Jiangsu Tai biomedical Ltd.) at 4 × 10 ⁵ cells / well in 96-well cell culture plate Access (Cat : 3599, Corning), add 5% BSA PBS, 4°C, 30min to block cell surface receptors. H182-MUT4 and control antibodies Atezolizumab, Durvalumab, and isotype control NC-hIgG1 (starting concentration 30μg/mL, 3-fold serial dilution, 12 gradients) were incubated with CHO-PD-L1-CD3L cell suspension at 4°C After washing the cells 3 times with PBST for 60 min, 0.5ug/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, use a flow cytometer (model B49007AD, SNAW31211, BECKMAN COULTER) to detect the average fluorescence intensity (MFI) of the cells to verify whether the antibody to be tested can bind to PD-L1 on the surface of CHO cells, as shown in Figure 9. As shown, the EC50 of the binding of H182-MUT4, Atezolizumab, and Durvalumab to CHO-PD-L1-CD3L cells were 0.658nM, 0.908nM, and 1.022nM, respectively, compared with the control antibodies Atezolizumab and Durvalumab, H182-MUT4 and CHO-PD-L1 -The binding activity of CD3L cells is better.

Example 10: Observation of the cytological activity of H182-MUT4 blocking the binding of PD-L1 to its receptor PD1

Take CHO cells (CHO-PD-L1-CD3L, Jiangsu Taikang Biomedicine Co., Ltd.) expressing recombinant human PD-L1 and anti-CD3-ScFv, and insert 4×10 ⁵ cells/well into a 96-well cell culture plate ( Cat: 3599, Corning), after culturing overnight in a cell incubator, discard the supernatant, add h182, H182-MUT4 and control antibodies Atezolizumab, Durvalumab (initial concentration 2μg/mL, 2-fold serial dilution, 10 gradients), 50ul/well, and 50ul of Jurkat cell (Jurkat-PD1-NFAT, Jiangsu Taikang Biomedicine Co., Ltd.) suspension that recombinantly express human PD-1 and luciferase were placed in a cell incubator and incubated for 6 hours. ^{Add Bio-Glo™} Luciferase substrate (Cat: 7940, Promega) to the cell culture plate, 100 μl/well, place the cell plate in a microplate thermostatic shaker, and incubate for 20 min at 800 rpm in the dark. Set the multi-function microplate reader to Luminescence mode, select 500 for Intergration (the default value of the instrument), and read the RLU.

Atezolizumab was used as a reference to calculate the relative activity of other samples. The EC50 and relative activity of each sample are shown in Table 7 below. The biological activity of H182-MUT4 is better than the control antibodies Atezolizumab and Durvalumab; the biological activity of h182 is better than the control antibody Atezolizumab.

Table 7 Detection of biological activity of H182-MUT4

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

Select the cells MDA-MB-231 with high expression of human PD-L1 in the logarithmic growth phase, add Calcein AM to label the live cells of MDA-MB-231, and compare it with the gradient dilution H182-MUT4 and control Antibodies Atezolizumab, Durvalumab, Avelumab and isotype control antibody (hIgG1) (initial concentration is 100ng/mL, 10-fold serial dilution, 5 gradients, each concentration is set to 6 multiple wells), effector cells NK92 cells (efficiency target ratio 5 1), and set up the following controls: (1) positive control (PC): maximum release hole of target cells; (2) negative control (NC): L15 medium (Cat: 11415064, Gibco); (3) blank control: DPBS (Cat: 14190-144, Gibco) was incubated at 37°C for 6 hours; after adding 2% Triton X-100 (Cat: X100-100ML, Sigma) to the positive control, incubate at 37°C for 30 minutes to release spontaneously as pure target cells The well was used as a blank control, and the maximum release hole of the target cell was used as a positive control. The fluorescence intensity of calcein in the culture medium was detected by a microplate reader (Cat: Synergy H1, BioTek). The excitation wavelength was 490nm and the emission wavelength was 515nm. Evaluation ADCC killing activity of PD-L1 antibody:

Killing efficiency (%) = (experimental group fluorescence value-spontaneous release group fluorescence value) / (maximum release group fluorescence value-spontaneous release group fluorescence value) × 100%

The results showed (Figure 10) that the antibody Atezolizumab had no ADCC activity. The antibodies Avelumab, H182-MUT4, and Durvalumab all had ADCC activity, with EC50 values of 1.405ng/mL, 0.9602ng/mL, 0.8184ng/mL, respectively.

Example 12: Observation of the pharmacokinetic properties of H182-MUT4 in mice

Three healthy female 5-week-old Balb/C nude mice were divided into groups, and a single single dose of H182-MUT4, Atezolizumab, Durvalumab, and Avelumab were administered to the tail vein at a dose of 10 mg/kg. After 0.5h, 1h, 2h, 8h, 24h, 48h, 96h, 144h, 216h, 240h blood is collected from the tail vein, blood samples are collected, placed at room temperature for more than 30 minutes, 4000rpm, 15min, and stored at -20℃, and the serum is collected for the last time Freeze storage at -20°C for at least 24 hours, and set up three groups of control groups. The control substances Atezolizumab, Durvalumab, and Avelumab of the same dose (10mg/kg) were injected into the tail vein to compare and observe their pharmacokinetic properties. After the blood collection is complete, coat PD-L1-His on a 96-well enzyme-linked plate, 0.5ug/ml, 100ul/well, overnight at 4°C, wash the plate 3 times with PBS, add 5% BSA PBS, and block at 37°C for 60 minutes. Wash the plate 3 times with PBST; add the serum sample to be tested (10000, 20,000-fold dilution), set H182-MUT4 and control antibodies Atezolizumab, Durvalumab, Avelumab standard curve wells (initial concentration is 0.05μg/mL, 2-fold serial dilution, 12 Gradient), and set the following controls: (1) Negative control (NC): blank mouse serum (10000 times dilution); (2) Blank control: PBS. Incubate at 37°C for 60 minutes, wash the plate 4 times with PBST; add 1:5000 diluted HRP-goat anti-human IgG (Fcr) (Cat: 109-035-098, Jackson Immuno Research), incubate at 37°C for 40 minutes, wash the plate 4 times with PBST ; Add TMB substrate (Cat: ME142, Beijing Taitianhe Biotechnology Co., Ltd.) for color development. After incubating at 37°C for 10 min, add 2M HCl to stop the reaction; take 630nm as the reference wavelength, read and record the well plate at a wavelength of 450nm Absorbance A450nm-630nm. Using the standard antibody concentration as the Y axis and the OD value as the X axis, fit a linear curve, and put the OD value of the detected serum into the formula to obtain the antibody content in the serum, and according to the formula T _1/2 =|0.693/k| , Calculate the half-life T _{1/2 of the} drug.

As shown in Figure 11 and Table 8, H182-MUT4 and Durvalumab maintained a good half-life in Balb/c mice, with T _1/2 reaching about 130-150h, suggesting that H182-MUT4 and Durvalumab antibodies have no obvious effect in vivo Deactivation phenomenon has good structural stability. Its metabolism conforms to the basic characteristics of monoclonal antibody drugs. However, the concentration of Atezolizumab and Avelumab decreased significantly at the 144th hour.

Table 8. Pharmacokinetic parameters of single administration of H182-mut4 in nude mice

Example 13: Anti-tumor efficacy of H182-MUT4 in MC38-hPD-L1 tumor-bearing mice

Mouse colon cancer MC38 cells (MC38-hPD-L1) expressing human PD-L1 were inoculated subcutaneously on the right side of 5-8 week old female C57BL/6 mice at a concentration of ^{5×10 5 cells/0.1 mL.} When the tumor grows to about 100mm ³ , the mice whose tumor volume meets the requirements are randomly divided into groups, 6 mice in each group, divided into 4 groups, respectively given anti-PD-L1 antibody H182-MUT4, Atezolizumab and Durlumab and isotype control antibody hIgG1 10mg/ kg, dosing frequency BIW, the mouse body weight and tumor volume were measured twice a week during the dosing and observation period, and the measured values were recorded. The results are shown in Figure 12, H182-MUT4 significantly inhibited tumor growth, and the tumor suppression effect was basically equivalent to that of the control antibody Atezolizumab.

Example 14: Preparation of H182-MUT4-TGFβRII bifunctional protein

The C-terminus of the heavy chain of the anti-PD-L1 humanized antibody H182-MUT4 obtained in the present invention is connected to the TGFβRII extracellular region sequence (SEQ ID NO.29) via a connecting peptide (SEQ ID NO.79), and the position is digested by restriction enzymes. Point, clone into the eukaryotic expression vector, obtain the expression plasmid containing the H182-MUT4 heavy chain-TGFβRII coding sequence, transfer it into E. coli for amplification, isolate and obtain a large number of plasmids, and according to the transfection reagent 293fectin (Cat: 12347019, Gibco) According to the operation instructions, the plasmid and H182-MUT4 light chain plasmid were transferred into HEK293 cells for recombinant expression. 5-6 days after cell transfection, the culture supernatant was taken, and the expression supernatant was purified by ProA affinity chromatography column to obtain the bifunctional protein H182-MUT4-TGFβRII (the heavy chain is shown in SEQ ID NO: 77, The light chain is shown in SEQ ID NO: 78). Using Fortebio's Octet QKe system instrument, a method of capturing the Fc segment of the antibody with a capture antibody (AHC) bioprobe of anti-human antibody Fc segment is used to determine the antibody affinity. In the determination, the bifunctional proteins H182-MUT4-TGFβRII, H182-MUT4, TGFβRII-hFc (Cat: CC10, Nearshore Technology) were diluted with PBS buffer to 5ug/mL, and then passed through the AHC probe (Cat: 18-5060, PALL) surface, time is 120s. 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 is 300s, and the dissociation time is 300s. After the experiment is completed, the blank control response value is subtracted, and the software is used to perform 1:1 Langmuir binding mode fitting to calculate the kinetic constant of antigen-antibody binding. The results are shown in Table 9. Compared with H182-MUT4 and TGFβRII-hFc, the affinity of the bifunctional protein H182-MUT4-TGFβRII to PD-L1 and TGFβ1 was not significantly reduced.

Table 9 Affinity determination results of the bifunctional protein H182-MUT4-TGFβRII

Example 15 : Anti-tumor efficacy of the bifunctional protein H182-MUT4-TGFβRII in humanized PD-1 and PD-L1 mouse hPD-L1-MC38 subcutaneous tumor models

Mouse colon cancer MC38 cells (MC38-hPD-L1, Cat: 3111C0001CCC000667, Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences) expressing only human PD-L1 were inoculated in 5 ^{×10 5 cells/0.1mL.} -8-week-old PD-1 and PD-L1 humanized mice (Cat: T004022, Jicui Yaokang Biotechnology) subcutaneously, when the tumor grows to about 100mm ³ according to the tumor volume, select 18 small mice that meet the requirements The mice were randomly divided into groups of 6 each, divided into 3 groups for administration (D0), respectively given anti-PD-L1 antibody H182-MUT4 (10mg/kg), bifunctional protein H182-MUT4-TGFβRII (13.5mg/ kg) and isotype control antibody hIgG1 10mg/kg, the route of administration ip, the frequency of administration tiw×9, the weight and tumor volume of the mice were measured twice a week during the administration and observation period, and the measured values were recorded. If the tumor volume exceeds 3000 mm ³ or the average tumor volume of the treatment group exceeds 2000 mm ³ or the animal weight is reduced by more than 20%, the experiment is terminated.

As shown in Figure 13, the anti-PD-L1 antibody H182-MUT4 and the bifunctional protein H182-MUT4-TGFβRII have a certain tumor inhibitory effect, and the bifunctional protein composed of H182-MUT4 and TGFβRII has a better anti-tumor effect.

The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications according to the present invention, as long as they do not deviate from the spirit of the present invention, they shall fall within the scope of the appended claims of the present invention.

Claims

An antibody or fragment thereof, the antibody or fragment thereof comprises a variable region of a heavy chain (VH) and a variable region of a light chain (VL), the variable region of the heavy chain (VH) and a variable region of the light chain (VL) ) Respectively comprise HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 shown in the following amino acid sequences (I) to (VI):

(I) HCDR1: SEQ ID NO: 36, 37, 38, 39, 40;

(II) HCDR2: SEQ ID NO: 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51;

(III) HCDR3: SEQ ID NO: 52, 53, 54, 55, 56;

(IV) LCDR1: SEQ ID NO: 57, 58, 59, 60;

(V) LCDR2: SEQ ID NO: 61, 62, 63, 64, 65, 66;

(VI) LCDR3: SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, 76.
The antibody or fragment thereof according to claim 1, wherein the variable region of the heavy chain (VH) and the variable region of the light chain (VL) respectively comprise those selected from the following (1) to (13) HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3:

(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;

(13) SEQ ID NO: 36, 43, 52 and SEQ ID NO: 57, 61, 71.
The antibody or fragment thereof according to claim 1 or 2, wherein the antibody or fragment thereof can bind to PD-L1 with an affinity of K D ≤ 5 nM, preferably mammalian PD-L1, more preferably primate PD-L1 is more preferably human or cynomolgus PD-L1, especially human PD-L1.
The antibody or fragment thereof according to any one of claims 1 to 3, wherein the heavy chain variable region of the antibody or fragment thereof comprises a sequence selected from:

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

The light chain variable region of the antibody or fragment thereof comprises a sequence selected from:

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

More preferably, the heavy chain variable region and the light chain variable region of the antibody or fragment thereof comprise any combination of the following amino acid sequences:

(1) The amino acid sequence shown in SEQ ID NO: 2 or the 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;

(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;

(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;

(4) The amino acid sequence shown in SEQ ID NO: 10 or the 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;

(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;

(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;

(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;

(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;

(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;

(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;

(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;

(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;

(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;

(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.
The antibody or fragment thereof according to any one of claims 1 to 4, wherein the antibody or fragment thereof is a monoclonal antibody, a single chain antibody, a bifunctional antibody, a single domain antibody, a nanobody, a complete or Partially humanized antibodies or chimeric antibodies in any form, or the antibodies or fragments thereof are half antibodies or antigen-binding fragments of half antibodies, such as scFv, BsFv, dsFv, (dsFv) 2 , Fab, Fab', F(ab') 2 or Fv;

Preferably, the antibody or fragment thereof further comprises a human or murine constant region, preferably a human or murine constant region, such as a human or murine light chain constant region (CL) and/or a heavy chain constant region (CH).
A conjugate or fusion protein comprising the antibody or fragment thereof according to any one of claims 1 to 5.
A nucleic acid molecule that encodes the antibody or fragment thereof according to any one of claims 1 to 5 or encodes the heavy chain CDR, light chain CDR, light chain variable region, and heavy chain contained in the antibody or fragment thereof Variable region, heavy chain or light chain.
A vector comprising the nucleic acid molecule of claim 7.
A host cell comprising the nucleic acid molecule of claim 7 and/or the vector of claim 8, or the host cell is composed of the nucleic acid molecule of claim 7 and/or the vector of claim 8. The vector is transformed or transfected.
A composition comprising the antibody or fragment thereof of any one of claims 1 to 5, the conjugate or fusion protein of claim 6, the nucleic acid molecule of claim 7, and the nucleic acid molecule of claim 8. The vector and/or the host cell of claim 9.
The antibody or fragment thereof according to any one of claims 1 to 5, the conjugate or fusion protein according to claim 6, the nucleic acid molecule according to claim 7, the vector according to claim 8, and claim 9 Use of the host cell and/or the composition of claim 10 in the preparation of a medicament for the treatment of diseases related to the positive expression of PD-L1, and/or the medicament for blocking PD -L1/PD-1 interaction.
A kit comprising the antibody or fragment thereof according to any one of claims 1 to 5, the conjugate or fusion protein according to claim 6, the nucleic acid molecule according to claim 7, and The vector of claim 8, the host cell of claim 9, and/or the composition of claim 10.
A method for treating diseases related to PD-L1 positive expression, the method comprising administering the antibody or fragment thereof according to any one of claims 1 to 5, and the antibody according to claim 6 to a subject in need thereof The conjugate or fusion protein of claim 7, the nucleic acid molecule of claim 7, the vector of claim 8, the host cell of claim 9 and/or the composition of claim 10, and optionally others Medication or treatment.