An anti-PD-L1 single domain antibody and its derivatives and uses

Abstract

The present invention provides a complementary determining region (CDR) of an anti-PD-L1 single domain antibody VHH chain, wherein the CDR of the VHH chain comprises: a CDR1 having an amino acid sequence as shown in SEQ ID NO: 5n+1; a CDR2 having an amino acid sequence as shown in SEQ ID NO: 5n+2, or a CDR2 having an amino acid sequence having a sequence identity of greater than 85% with the sequence as shown in SEQ ID NO: 2; and a CDR3 having an amino acid sequence as shown in SEQ ID NO: 5n+3; wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine or biopharmaceutical technology, and more specifically to an anti-PD-L1 single domain antibody and its derivatives and uses. Background Art

[0002] Programmed death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and a ligand for PD-1. PD-L1 is a type I transmembrane protein with 290 amino acids, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region, and an intracellular region consisting of 30 amino acids.

[0003] PD-L1 has the function of negatively regulating immune response. Studies have found that PD-L1 is mainly expressed in activated T cells, B cells, macrophages and dendritic cells. In addition to lymphocytes, PD-L1 is also expressed in endothelial cells of many other tissues such as thymus, heart, placenta, and various non-lymphoid systems such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, head and neck cancer, etc. PD-L1 has a certain degree of extensiveness in regulating autoreactive T, B cells and immune tolerance, and plays a role in T and B cell responses in peripheral tissues. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.

[0004] Programmed death-1 (PD-1) that binds to PD-L1 is also known as CD279 and is a member of the B7-CD28 superfamily. The cytoplasmic region of CD279 contains two tyrosine residues, one near the N-terminus is located in the immunoreceptor tyrosine-based inhibitory motif (ITIM), and the other near the C-terminus is located in the immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is mainly expressed on the surface of activated T lymphocytes, B lymphocytes, and macrophages. Under normal circumstances, PD-1 can inhibit the function of T lymphocytes and promote the function of Treg, thereby inhibiting autoimmune responses and preventing the occurrence of autoimmune diseases. However, in the occurrence of tumors, PD-L1 expressed by tumor cells can promote tumor immune escape by inhibiting lymphocytes after binding to PD-1. The binding of PD-L1 to PD-1 can lead to a variety of biological changes and cause immune regulation, such as inhibiting the proliferation and activation of lymphocytes, inhibiting the differentiation of CD4+T cells into Th1 and Th17 cells, and inhibiting the release of inflammatory cytokines.

[0005] The successful application of monoclonal antibodies in cancer detection and targeted therapy has brought about a revolution in tumor treatment. However, the molecular weight of traditional monoclonal antibodies (150kD) is too large and difficult to penetrate tissues, resulting in low effective concentrations in tumor areas and insufficient therapeutic effects. In addition, many factors such as the long development cycle, high production cost, and insufficient stability of fully humanized traditional antibodies limit their application and popularity in clinical practice.

[0006] Single-domain antibodies are currently the smallest antibody molecules, and their molecular weight (excluding Fc) is 1 / 10 of that of ordinary antibodies. In addition to the antigenic reactivity of monoclonal antibodies, single-domain antibodies also have some unique functional characteristics, such as small molecular weight, strong stability, good solubility, easy expression, strong penetration, simple humanization, and low preparation cost, which can overcome the defects of traditional antibodies.

[0007] However, there is currently a lack of satisfactory single-domain antibodies against PD-L1 in the art. Therefore, there is an urgent need to develop specific single-domain antibodies that are effective against PD-L1. Summary of the invention

[0008] The purpose of the present invention is to provide a class of specific single-domain antibodies that are effective against PD-L1.

[0009] In a first aspect of the present invention, a complementarity determining region (CDR) of an anti-PD-L1 single domain antibody VHH chain is provided, wherein the complementarity determining region CDR of the VHH chain consists of:

[0010] CDR1 having an amino acid sequence as shown in SEQ ID NO:5n+1;

[0011] A CDR2 having an amino acid sequence as shown in SEQ ID NO:5n+2, or a CDR2 having an amino acid sequence having a sequence identity of greater than 85% with the sequence shown in SEQ ID NO:2; and

[0012] CDR3 having an amino acid sequence as shown in SEQ ID NO:5n+3;

[0013] wherein each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0014] In another preferred embodiment, the n is 0 or 1.

[0015] In another preferred example, the amino acid sequence of the CDR2 is shown in SEQ ID NO: 2, 7, 81, 84, 87, 90, 93 or 96.

[0016] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0017] In a second aspect of the present invention, a VHH chain of an anti-PD-L1 single domain antibody is provided, wherein the VHH chain of the anti-PD-L1 single domain antibody comprises the CDR1, CDR2 and CDR3 as described in the first aspect of the present invention.

[0018] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO: 5n+4, 82, 85, 88, 91, 94 or 97;

[0019] wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0020] Among them, any one of the above amino acid sequences also includes a derivative sequence that has been added, deleted, modified and / or substituted 1-8 (preferably 1-5, more preferably 1-3) amino acid residues and can retain the PD-L1 binding affinity of the PD-L1 single domain antibody.

[0021] In another preferred embodiment, the n is 0 or 1.

[0022] In another preferred example, the amino acid sequence of the VHH chain of the anti-PD-L1 single domain antibody is as shown in SEQ ID NO:4, 9, 82, 85, 88, 91, 94 or 97.

[0023] In the third aspect of the present invention, an anti-PD-L1 single domain antibody is provided, which is a single domain antibody against the PD-L1 epitope and has the VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention.

[0024] In the fourth aspect of the present invention, a polynucleotide is provided, which encodes a protein selected from the following group: the CDR region of the anti-PD-L1 single domain antibody VHH chain as described in the first aspect of the present invention, the VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention, or the anti-PD-L1 single domain antibody as described in the third aspect of the present invention.

[0025] In another preferred embodiment, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 5n, 83, 86, 89, 92, 95 or 98;

[0026] Wherein, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0027] In another preferred embodiment, the polynucleotide includes DNA or RNA.

[0028] In the fifth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the polynucleotide as described in the fourth aspect of the present invention.

[0029] In another preferred embodiment, the expression vector further comprises a nucleotide sequence encoding the Fc segment of an immunoglobulin.

[0030] In another preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3 or IgG4.

[0031] In the sixth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector as described in the fifth aspect of the present invention, or the polynucleotide as described in the fourth aspect of the present invention is integrated into its genome.

[0032] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0033] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells and mammalian cells.

[0034] In a seventh aspect of the present invention, a method for producing an anti-PD-L1 single domain antibody is provided, comprising the steps of:

[0035] (a) culturing the host cell according to the sixth aspect of the present invention under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-PD-L1 single domain antibody; and

[0036] (b) isolating or recovering the anti-PD-L1 single domain antibody from the culture.

[0037] In another preferred embodiment, the anti-PD-L1 single domain antibody has an amino acid sequence as shown in SEQ ID NO: 5n+4, 82, 85, 88, 91, 94 or 97;

[0038] Wherein, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0039] In an eighth aspect of the present invention, a single-domain antibody fusion protein is provided, wherein the single-domain antibody fusion protein has a structure as shown in Formula I from N-terminus to C-terminus:

[0040] Z1-Z2-L-Z3 (Formula I)

[0041] In the formula,

[0042] Z1 is the VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention;

[0043] Z2 is the Fc segment of immunoglobulin;

[0044] L is the linker sequence;

[0045] Z3 is the immunomodulatory molecule.

[0046] In another preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3 or IgG4.

[0047] In another preferred embodiment, the amino acid sequence of Z2 is shown in SEQ ID NO:99.

[0048] In another preferred example, the amino acid sequence of Z2 is identical or substantially identical to the amino acid sequence shown in SEQ ID NO:99.

[0049] In another preferred embodiment, L has an amino acid sequence selected from the following group: GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0050] In another preferred embodiment, the amino acid sequence of L is shown in SEQ ID NO:100.

[0051] In another preferred example, the amino acid sequence of L is identical or substantially identical to the amino acid sequence shown in SEQ ID NO: 100.

[0052] In another preferred embodiment, the immunomodulatory molecule is the extracellular domain of TGFβRII.

[0053] In another preferred example, the amino acid sequence of Z3 is shown in SEQ ID NO:101.

[0054] In another preferred example, the amino acid sequence of Z3 is identical or substantially identical to the amino acid sequence shown in SEQ ID NO:101.

[0055] In another preferred embodiment, the basic identity means that there are at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, and most preferably 1-3) amino acid differences, wherein the differences include amino acid substitutions, deletions or additions.

[0056] In another preferred embodiment, the substantial identity means that the amino acid sequence has a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding amino acid sequence.

[0057] In another preferred example, the amino acid sequence of the single-domain antibody fusion protein is as shown in SEQ ID NO:102.

[0058] In the ninth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0059] (a) the VHH chain of the anti-PD-L1 single domain antibody according to the second aspect of the present invention, the anti-PD-L1 single domain antibody according to the third aspect of the present invention, or the single domain antibody fusion protein according to the eighth aspect of the present invention; and

[0060] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0061] In another preferred embodiment, the coupling moiety is a drug or a toxin.

[0062] In another preferred embodiment, the coupling moiety is a detectable label.

[0063] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0064] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention, the anti-PD-L1 single domain antibody as described in the third aspect of the present invention, or the single domain antibody fusion protein as described in the eighth aspect of the present invention.

[0065] In another preferred embodiment, the multivalency means that the amino acid sequence of the immunoconjugate contains multiple repeats of the VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention, the anti-PD-L1 single domain antibody as described in the third aspect of the present invention, or the single domain antibody fusion protein as described in the eighth aspect of the present invention.

[0066] In the tenth aspect of the present invention, there is provided the use of the anti-PD-L1 single domain antibody as described in the third aspect of the present invention or the single domain antibody fusion protein as described in the eighth aspect of the present invention for preparing (a) a reagent for detecting PD-L1 molecules; (b) a drug for treating tumors.

[0067] In another preferred embodiment, the detection includes flow cytometry detection and cell immunofluorescence detection.

[0068] In the eleventh aspect of the present invention, there is provided a pharmaceutical composition comprising:

[0069] (i) the complementarity determining region CDR of the anti-PD-L1 single domain antibody VHH chain as described in the first aspect of the present invention, the VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention, or the anti-PD-L1 single domain antibody as described in the third aspect of the present invention, the single domain antibody fusion protein as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention; and

[0070] (ii) a pharmaceutically acceptable carrier.

[0071] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0072] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating tumors, and the tumors are selected from the following groups: gastric cancer, liver cancer, leukemia, kidney tumors, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, lymphoma, adrenal tumors, bladder tumors, or a combination thereof.

[0073] In the twelfth aspect of the present invention, one or more uses of the anti-PD-L1 single domain antibody according to the third aspect of the present invention or the single domain antibody fusion protein according to the eighth aspect of the present invention are provided:

[0074] (i) Used to detect human PD-L1 molecules;

[0075] (ii) for flow cytometry;

[0076] (iii) used for cell immunofluorescence detection;

[0077] (iv) for tumor treatment; and

[0078] (v) Used in tumor diagnosis.

[0079] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0080] In the thirteenth aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0081] (i) the sequence of the heavy chain variable region VHH as described in the second aspect of the present invention or the sequence of the single domain antibody as described in the third aspect of the present invention, or the single domain antibody fusion protein as described in the eighth aspect of the present invention; and

[0082] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0083] In another preferred embodiment, the tag sequence includes: 6His tag, HA tag, Flag tag, Fc tag, HSA, anti-HSA antibody or single domain antibody, or a combination thereof.

[0084] In another preferred embodiment, the recombinant protein specifically binds to the PD-L1 protein.

[0085] In the fourteenth aspect of the present invention, there is provided a use of the VHH chain as described in the second aspect of the present invention, the single domain antibody as described in the third aspect of the present invention, the single domain antibody fusion protein as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention for preparing a medicament, a reagent, a detection plate or a kit;

[0086] Wherein, the reagent, detection plate or kit is used for: detecting PD-L1 protein in a sample;

[0087] The agent is used to treat or prevent tumors expressing PD-L1 protein (i.e., PD-L1 positive).

[0088] In another preferred embodiment, the tumor includes: gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, adrenal tumor, or a combination thereof.

[0089] In a fifteenth aspect of the present invention, a method for detecting PD-L1 protein in a sample is provided, comprising the steps of:

[0090] (1) contacting a sample with the single domain antibody according to the third aspect of the present invention or the single domain antibody fusion protein according to the eighth aspect of the present invention;

[0091] (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L1 protein in the sample.

[0092] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0093] In the sixteenth aspect of the present invention, a method for treating a disease is provided, the method comprising administering to a subject in need thereof a single domain antibody as described in the third aspect of the present invention, a single domain antibody fusion protein as described in the eighth aspect of the present invention, or an immunoconjugate as described in the ninth aspect of the present invention.

[0094] In another preferred embodiment, the subject includes mammals.

[0095] In another preferred embodiment, the mammal is a human.

[0096] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] Figure 1 It was shown that the single domain antibody of the present invention can bind to the human PD-L1 protein on the cell surface, and the binding effect of some antibodies is similar to that of the positive control.

[0098] Figure 2 It showed that the modified single-domain antibody could still bind to the human PD-L1 protein on the cell surface, and the binding effect of the antibody was similar to that of the positive control.

[0099] Figure 3 It was shown that the modified single-domain antibody could still block the binding of PD-L1 protein and human PD-1 protein on the cell surface, and the blocking effect of the antibody was similar to that of the positive control.

[0100] Figure 4 It is shown that the single domain antibody of the present invention can effectively activate T cells, and the activation effect is similar to or better than that of the positive control group antibody.

[0101] Figure 5 A schematic diagram of the fusion protein structure is shown.

[0102] Figure 6 It is shown that the fusion protein of the present invention can bind to the human PD-L1 protein on the cell surface.

[0103] Figure 7 It was shown that the fusion protein of the present invention can block the binding between the PD-L1 protein and the human PD-1 protein on the cell surface, and the blocking effect of the antibody is similar to that of the positive control.

[0104] Figure 8 It was shown that the fusion protein of the present invention can bind to TGFβ1, TGFβ2 and TGFβ3.

[0105] Fig. 9It is shown that the fusion protein of the present invention can effectively block the TGFβ / SMAD signaling pathway.

[0106] Fig.10 It shows that the fusion protein of the present invention can effectively activate T cells, and the activation effect is similar to or better than that of the positive control group antibody.

[0107] Fig.11 It was shown that the fusion protein of the present invention can effectively inhibit tumor growth in mice.

[0108] Fig.12 A photograph of a PD-L1-VHH crystal sample of the present invention is shown.

[0109] Fig.13 The crystal structure of the PD-L1-VHH complex of the present invention is shown (the upper structure is PD-L1, and the lower structure is VHH).

[0110] Fig.14 The hydrogen bond interaction interface of PD-L1-VHH of the present invention is shown (the upper structure is PD-L1, and the lower structure is VHH).

[0111] Fig.15 The hydrophobic interaction interface of the PD-L1-VHH of the present invention is shown (the upper structure is PD-L1, and the lower structure is VHH).

[0112] Fig.16 Shown are the effects of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0113] Fig.17 Shown are tumor images of different drug groups 27 days after inoculation.

[0114] Fig.18 The effects of each group of drugs on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors are shown.

[0115] Fig.19 Shown are the effects of different drug groups on body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0116] Fig. 20 Shown are the effects of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0117] Fig.21 Shown are the tumor images of different drug groups 28 days after inoculation.

[0118] Fig. 22The effects of each group of drugs on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors are shown.

[0119] Fig.23 Shown are the effects of different drug groups on body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. DETAILED DESCRIPTION

[0120] The inventors have developed a class of anti-PD-L1 single domain antibodies after extensive and in-depth research and a large number of screenings. The experimental results show that the PD-L1 single domain antibodies and mutant derivatives thereof obtained by the present invention can effectively block the interaction between PD-L1 and PD-1, and have good thermal stability.

[0121] Specifically, the present invention uses human PD-L1 antigen protein to immunize alpacas (Llama) to obtain a high-quality immune single-domain antibody gene library. Among them, the inventors accidentally screened single-domain antibody gene sequences with a high level of humanization (sequence identity>85%) in the immune single-domain antibody gene library. The PD-L1 protein molecule was biotinylated and labeled, and the immune single-domain antibody gene library was screened using yeast display technology to obtain PD-L1-specific single-domain antibody candidate molecule antibody genes. The obtained genes and their engineered mutants were then transferred to Expi-CHO cells, and further screening was performed from the aspects of antibody affinity, ability to block PD-L1 binding to PD-1, thermal stability, and activity of activating T cells, thereby obtaining a class of single-domain antibody strains that can be efficiently expressed in vitro and have high specificity.

[0122] In addition, the experimental results show that the fusion protein produced by fusing the single-domain antibody sequence of the present invention (as the targeting portion) with the IgG1Fc fragment (as the connecting portion) and the TGFβRII extracellular domain (as the immunomodulatory molecule portion) has high reactivity with PD-L1, can effectively block the interaction between PD-L1 and PD-1, can effectively block the TGF-β / SMAD signaling pathway, can effectively activate human T lymphocytes, and can effectively inhibit tumor growth in mice.

[0123] In addition, the experimental results show that the single domain antibody of the present invention can significantly inhibit the growth of subcutaneous transplanted tumors in mice and reduce the weight of tumors in mice. The inhibitory effect on tumor growth is higher than that of similar molecules, anti-PD-L1 single domain antibodies and TGF-βRII-Fc fusion proteins at the same molar dose, and has no obvious toxicity to disease animal models.

[0124] On this basis, the present invention has been completed.

[0125] Single domain antibodies of the present invention

[0126] As used herein, the terms "single domain antibody of the present invention", "anti-PD-L1 single domain antibody of the present invention", and "PD-L1 single domain antibody of the present invention" are used interchangeably, and all refer to single domain antibodies that specifically recognize and bind to PD-L1 (including human PD-L1). Particularly preferred are single domain antibodies whose amino acid sequences of the VHH chains are shown in SEQ ID NO: 4, 9, 82, 85, 88, 91, 94 or 97.

[0127] As used herein, the term "antibody" or "immunoglobulin" is a heterotetrameric glycoprotein of about 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains of different immunoglobulin isotypes varies. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0128] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0129] As used herein, the term "variable" means that some parts of the variable region in an antibody are different in sequence, which forms the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions (CDRs) or hypervariable regions in the variable regions of the light chain and heavy chain. The more conservative part of the variable region is called the framework region (FR). The variable regions of natural heavy and light chains each contain four FR regions, which are roughly in a β-folded configuration, connected by three CDRs forming a connecting loop, and in some cases can form a partial β-folded structure. The CDRs in each chain are closely together through the FR region and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Volume I, 647-669 pages (1991)). The constant region is not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody's antibody-dependent cytotoxicity.

[0130] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by combining drugs, toxins, cytokines, radionuclides, enzymes and other diagnostic or therapeutic molecules with the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that are combined with the anti-PD-L1 protein antibodies or fragments thereof.

[0131] As used herein, the term "heavy chain variable region" is synonymous with "V H ” can be used interchangeably.

[0132] As used herein, the term "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0133] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity determining regions: CDR1, CDR2, and CDR3.

[0134] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.

[0135] In the present invention, the terms "antibodies of the present invention", "proteins of the present invention", or "polypeptides of the present invention" are used interchangeably, and all refer to polypeptides that specifically bind to PD-L1 protein, such as proteins or polypeptides having a heavy chain variable region. They may or may not contain an initial methionine.

[0136] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, as long as the variable region is identical to or at least 90% homologous to the heavy chain variable region of the antibodies of the present invention, preferably at least 95% homologous.

[0137] In general, the antigen binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated by four framework regions (FRs). The amino acid sequences of the four FRs are relatively conservative and do not directly participate in the binding reaction. These CDRs form a ring structure, and the β-folds formed by the FRs therebetween are close to each other in spatial structure. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. The amino acids that constitute the FR or CDR region can be determined by comparing the amino acid sequences of antibodies of the same type.

[0138] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because they are at least partially involved in binding to antigen. Therefore, the present invention includes molecules having antibody heavy chain variable regions with CDRs, as long as their CDRs have more than 90% (preferably more than 95%, and most preferably more than 98%) homology with the CDRs identified herein.

[0139] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0140] As used herein, the terms "fragment", "derivative" and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) polypeptides having a substitution group in one or more amino acid residues, or (iii) polypeptides formed by fusion of a mature polypeptide with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusion of an additional amino acid sequence to the polypeptide sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). According to the teachings herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art.

[0141] The antibody of the present invention refers to a polypeptide having PD-L1 protein binding activity and including the above-mentioned CDR region. The term also includes variant forms of polypeptides having the same function as the antibody of the present invention and including the above-mentioned CDR region. These variant forms include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and optimally 1-10) amino acid deletions, insertions and / or substitutions, and addition of one or several (usually within 20, preferably within 10, and more preferably within 5) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is generally not changed. For another example, adding one or several amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the present invention.

[0142] Variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0143] The present invention also provides other polypeptides, such as fusion proteins comprising single domain antibodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of single domain antibodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0144] In the present invention, "conservative variants of the antibodies of the present invention" refer to polypeptides formed by replacing at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having similar or similar properties compared to the amino acid sequence of the antibodies of the present invention. These conservative variant polypeptides are preferably generated by amino acid substitution according to Table 1.

[0145] Table 1

[0146] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0147] The present invention also provides a polynucleotide molecule encoding the above-mentioned antibody or its fragment or its fusion protein. The polynucleotide of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0148] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only a mature polypeptide; a coding sequence of a mature polypeptide and various additional coding sequences; a coding sequence of a mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0149] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may include additional coding and / or non-coding sequences.

[0150] The present invention also relates to polynucleotides that hybridize with the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of denaturing agents during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0151] The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombination or artificial synthesis. A feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is short. Usually, a fragment with a very long sequence can be obtained by first synthesizing multiple small fragments and then connecting them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can be fused together to form a fusion protein coding sequence.

[0152] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.

[0153] At present, the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0154] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0155] Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells such as CHO, COS7, 293 cells, etc.

[0156] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method, the steps used are well known in the art. Another method is to use MgCl2. If necessary, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be selected: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0157] The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used in the culture can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0158] The recombinant polypeptide in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography techniques and combinations of these methods.

[0159] The antibodies of the invention may be used alone or in combination or conjugated to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0160] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0161] Therapeutic agents that can be combined or coupled to the antibodies of the present invention include, but are not limited to: 1. radionuclides; 2. biological toxins; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. nanomagnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0162] Fusion protein of the present invention

[0163] As described herein, the "fusion protein of the present invention" refers to a bifunctional fusion protein having both the anti-PD-L1 single domain antibody described in the first aspect of the present invention and an immunomodulatory molecule portion.

[0164] In the present invention, a fusion protein is provided, wherein the single-domain antibody fusion protein has a structure as shown in Formula I from the N-terminus to the C-terminus:

[0165] Z1-Z2-L-Z3 (Formula I)

[0166] In the formula,

[0167] Z1 is the VHH chain of the anti-PD-L1 single domain antibody as described in the second aspect of the present invention;

[0168] Z2 is the Fc segment of immunoglobulin;

[0169] L is the linker sequence;

[0170] Z3 is the immunomodulatory molecule.

[0171] Preferably, the immunoglobulin may be IgG1, IgG2, IgG3 or IgG4.

[0172] In a preferred embodiment, the immunoglobulin is IgG1, and the amino acid sequence of Z2 is as shown in SEQ ID NO: 99. In other embodiments, the amino acid sequence of Z2 is identical or substantially identical to the amino acid sequence as shown in SEQ ID NO:99.

[0173] In the present invention, L is a flexible amino acid linker. Preferably, L has an amino acid sequence selected from the group consisting of GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0174] In a preferred embodiment, the amino acid sequence of L is as shown in SEQ ID NO: 100. In other embodiments, the amino acid sequence of L is identical or substantially identical to the amino acid sequence as shown in SEQ ID NO: 100.

[0175] In one embodiment of the present invention, the immunomodulatory molecule is the extracellular domain of TGFβRII. Preferably, the amino acid sequence of Z3 is as shown in SEQ ID NO: 101. In other embodiments, the amino acid sequence of Z3 is the same or substantially the same as the amino acid sequence as shown in SEQ ID NO: 101.

[0176] In the present invention, the substantially identical sequences refer to at most 50 (preferably 1-20, more preferably 1-10, more preferably 1-5, and most preferably 1-3) amino acid differences, wherein the differences include substitution, deletion or addition of amino acids.

[0177] Preferably, the substantial identity is that the amino acid sequence has a sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the corresponding amino acid sequence.

[0178] In a preferred embodiment, the amino acid sequence of the single-domain antibody fusion protein is as shown in SEQ ID NO:102.

[0179] TGFβ is a key inducing factor of epithelial-mesenchymal transition (EMT). At the same time, TGFβ has a strong immunosuppressive effect in the tumor microenvironment, and thus plays an important regulatory role in tumor development, metastasis and drug resistance.

[0180] Therefore, in one embodiment of the present invention, TGFβ receptor II is selected as the immunomodulatory molecule in the fusion protein. The fusion protein of the present invention has the advantages of high dual-target binding affinity and strong specificity, thereby further enhancing the anti-tumor immune function.

[0181] Pharmaceutical composition

[0182] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0183] The pharmaceutical composition of the present invention can be directly used to bind to the PD-L1 protein molecule, and thus can be used to treat tumors. In addition, other therapeutic agents can also be used simultaneously.

[0184] The pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned single-domain antibody (or its conjugate) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by conventional methods using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. In addition, the polypeptide of the present invention can also be used with other therapeutic agents.

[0185] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is usually at least about 10 micrograms / kg body weight, and in most cases does not exceed about 50 milligrams / kg body weight, preferably the dosage is about 10 micrograms / kg body weight to about 10 milligrams / kg body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

[0186] Labeled single domain antibodies

[0187] In a preferred embodiment of the present invention, the single domain antibody carries a detectable marker. More preferably, the marker is selected from the group consisting of an isotope, a colloidal gold marker, a colored marker or a fluorescent marker.

[0188] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, the anti-PD-L1 single domain antibody is labeled with colloidal gold to obtain a colloidal gold-labeled single domain antibody.

[0189] The anti-PD-L1 single domain antibody of the present invention has good specificity and high titer.

[0190] Detection Methods

[0191] The present invention also relates to a method for detecting PD-L1 protein. The method generally comprises the following steps: obtaining a cell and / or tissue sample; dissolving the sample in a medium; and detecting the level of PD-L1 protein in the dissolved sample.

[0192] In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a sample containing cells in a cell storage solution.

[0193] Reagent test kit

[0194] The present invention also provides a kit containing the antibody (or fragment thereof) or the detection plate of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, and the like.

[0195] The present invention also provides a detection kit for detecting PD-L1 levels, which includes an antibody that recognizes PD-L1 protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0196] application

[0197] As described above, the single domain antibody of the present invention has a wide range of biological and clinical application values, and its application involves multiple fields such as diagnosis and treatment of diseases related to PD-L1, basic medical research, biological research, etc. A preferred application is for clinical diagnosis and targeted therapy of PD-L1.

[0198] The main advantages of the present invention include:

[0199] 1) The single-domain antibody of the present invention is highly specific to human PD-L1 protein with a correct spatial structure.

[0200] 2) The single domain antibody of the present invention has strong affinity.

[0201] 3) The single domain antibody of the present invention is easy to produce.

[0202] 4) The single-domain antibody of the present invention can inhibit the PD-1 / PD-L1 pathway on the basis of targeted neutralization of TGF-β in the tumor microenvironment, which can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting the occurrence and development of tumors.

[0203] 5) The single domain antibody of the present invention has no obvious toxicity.

[0204] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0205] Example 1: Construction of a single domain antibody library

[0206] Animal Immunization

[0207] 1 mg of human PD-L1 antigen was mixed with an equal volume of Freund's adjuvant and immunized 2 alpacas (Llama) once a week for a total of 4 times to stimulate B cells to express antigen-specific single-domain antibodies. After 4 immunizations, 50 ml of alpaca peripheral blood was extracted and lymphocytes were isolated using lymphocyte separation fluid. Total RNA was extracted using RNA extraction reagent Trizol (purchased from Invitrogen). Total cDNA of the alpaca was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0208] Single domain antibody gene amplification

[0209] In the first round of PCR, IgG2 and IgG3 sequences were amplified from cDNA:

[0210] Table 1. Primers for the first round of PCR

[0211] name Sequence (5' to 3') SEQ ID Upstream primer GTCCTGGCTGCTCTTCTACAAGG 103 Downstream primer GGTACGTGCTGTTGAACTGTTCC 104

[0212] The first round of PCR products were subjected to agarose gel electrophoresis, and the fragment at 750 bp was recovered for the second round of VHH sequence amplification. The second round of PCR amplification primers are as follows:

[0213] Table 2. Second round PCR primers

[0214] name Sequence (5' to 3') SEQ ID Upstream primer CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT 105 Downstream primer CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG 106

[0215] The second round of PCR product was used as a template for the third round of PCR to add homology arms to the VHH gene. The third round of PCR amplification primers were as follows:

[0216] Table 3. Third round PCR primers

[0217]

[0218] The target fragment was recovered using a PCR purification kit (purchased from QIAGEN).

[0219] Library construction

[0220] The linearized yeast display vector and the third round of PCR products were mixed and electroporated into Saccharomyces cerevisiae ( 20828), an anti-PD-L1 single domain antibody library from two animals was constructed and the library capacity was determined. The library capacity was 4.47×10 7 and 4.14×10 7 .

[0221] Example 2: Screening of PD-L1 single domain antibodies

[0222] Biotinylation of human PD-L1 protein

[0223] Take an appropriate volume of double distilled water to dissolve human PD-L1 protein, and according to the instructions of the biotin labeling kit (purchased from Thermo), dissolve the biotin and mix it with the protein solution, and incubate at 4°C for 2 hours. Use a desalting column (purchased from Thermo) to remove excess biotin, and the desalting column pretreatment and sample collection operations are carried out according to the steps in the product manual.

[0224] MACS enriches yeast that specifically binds to PD-L1

[0225] The VHH library constructed in Example 2 was inoculated in SD-CAA amplification medium (1L SD-CAA amplification medium containing 6.7 g YNB, 5 g casamino acids, 13.62 g Na2HPO4·12H2O, 7.44 g NaH2PO4 and 2% glucose), and the number of yeast cells inoculated was >10×library capacity (initial amplification concentration = 0.5 OD 600 / ml), 30°C, 225rpm overnight. Take 10× the yeast cell volume, centrifuge at 3000rpm×5min (the following centrifugation operations are the same) to remove the medium, resuspend the yeast cells in SD-CAA induction medium, and adjust the starting concentration to 0.5OD 600 / ml, induce overnight. Determine the library concentration after induction, take 10× the library volume of yeast cells, centrifuge to remove the culture medium. Resuspend the yeast cells with 50ml washing buffer (PBS+0.5% BSA+2mM EDTA), centrifuge to remove the supernatant. Resuspend the yeast cells with 10ml washing buffer.

[0226] Add biotin-labeled PD-L1 protein (final concentration 100mM), incubate at room temperature for 30min, collect yeast cells by centrifugation, and wash the yeast 3 times with 50ml washing buffer. Resuspend the yeast cells with 5ml washing buffer, add 200μl SA magnetic beads (purchased from Miltenyi Biotec), and incubate upside down for 10min. Wash the yeast and magnetic bead mixture 3 times with washing buffer, and add the mixture to the LS column (purchased from Miltenyi Biotec). Place the LS column on the magnetic stand and wash with washing buffer to remove non-specifically bound yeast cells. Remove the column from the magnetic stand and add washing buffer to elute the yeast. After centrifugation, the eluted yeast is transferred to 200ml SD-CAA amplification medium for amplification.

[0227] Flow cytometry sorting to obtain high affinity yeast cells

[0228] The yeast cells enriched by MACS were inoculated into SD-CAA amplification medium, and the initial amplification concentration was 0.5 OD 600 / ml. Shake the flask at 30°C and 225 rpm overnight. Resuspend the yeast cells in SD-CAA induction medium (1L SD-CAA induction medium contains 6.7g YNB, 5g casamino acids, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4, 2% galactose, 2% raffinose and 0.1% glucose) to a starting concentration of 0.5OD 600 / ml, induce overnight. Add 1:200 diluted anti-c-Myc mouse antibody (purchased from Thermo) and 100nM biotin-labeled PD-L1 antigen, incubate at room temperature for 10min. Add PBS to wash the yeast 3 times, add 1:500 diluted goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (purchased from Invitrogen) and streptavidin APC-coupled fluorescent antibody (purchased from Invitrogen), and incubate at 4°C in the dark for 15min. Add 2ml PBS to resuspend the cells, and use BD FACSAraiII instrument to sort to obtain yeast with high binding ability to PD-L1 antigen.

[0229] Retrieval of PD-L1 single domain antibody candidate molecule antibody gene

[0230] The yeast liquid with high binding ability to PD-L1 antigen obtained by MACS and FACS enrichment was cultured overnight at 30°C and 225rpm in SD-CAA amplification medium, and yeast plasmids were extracted according to the yeast plasmid extraction kit (purchased from Tiangen). The plasmid was transformed into Top10 competent cells (purchased from Tiangen) by electroporation, coated with ampicillin-resistant plates, and cultured at 37°C overnight. Single clones were selected for sequencing to obtain the VHH gene sequence.

[0231] Example 3: Construction, expression and purification of heavy chain antibodies

[0232] Antibody gene construction into pCDNA3.1 expression vector

[0233] The VHH gene sequence was linked to the Fc segment of human IgG1 (LALA mutation) and linearized into the pCDNA3.1 vector using homologous recombination enzyme (purchased from Vazyme) and EcoR I / Not I double enzyme digestion. The process was in accordance with the product instructions. The homologous recombination product was transferred into Top10 competent cells, coated with ampicillin-resistant plates, cultured at 37°C overnight, and single clones were picked for sequencing.

[0234] Cell transfection

[0235] Adopt ExpiCHO TMExpression system kit (purchased from Thermo), the plasmid was transferred into Expi-CHO cells, the transfection method was in accordance with the product manual, and the supernatant was collected after 5 days of cell culture and the target protein was purified by protein A magnetic beads (purchased from GenScript) sorting method. The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of magnetic beads) and added to the sample to be purified, incubated at room temperature for 1 hour, and gently shaken during the period. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was discarded, and the magnetic beads were washed 3 times with binding buffer. Elution buffer (0.1M sodium citrate, pH 3.2) was added according to 3-5 times the volume of the magnetic beads, and the beads were shaken at room temperature for 5-10 minutes, and then placed back on the magnetic stand, the elution buffer was collected, and transferred to a collection tube with neutralization buffer (1M Tris, pH 8.54) and mixed.

[0236] Example 4: Purification of anti-PD-L1 antibodies binding to human PD-L1

[0237] CHO cells overexpressing human PD-L1 (CHO-hPD-L1 cells) were generated by transfecting the pCHO1.0 vector (purchased from Invitrogen) cloned into the MCS human PD-L1 cDNA (purchased from Sino Biological). The cell density of the expanded cultured CHO-hPD-L1 cells was adjusted to 2×10 6 cells / ml, 100μl / well was added to a 96-well flow plate, centrifuged and set aside. The purified PD-L1 antibody was diluted with PBS, starting from 400nM and diluted 3 times for a total of 12 points. 100μl / well of the diluted sample was added to the 96-well flow plate with cells, incubated at 4°C for 30min, and washed twice with PBS. 100μl / well was added to sheep F(ab')2 anti-human IgG-Fc (PE) (purchased from Abcam) diluted 100 times with PBS, incubated at 4°C for 30min, and washed twice with PBS. 100μl / well was added to PBS to resuspend the cells, and the detection was performed on a CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated.

[0238] In the above method, the experimental results are as follows Figure 1 As shown, all purified samples of the present invention have binding activity with CHO-hPD-L1 cells, and the binding activity of some purified samples is similar to that of the control antibody. (atezolizumab) (ATE; see US20130034559, also known as 243.55.S70).

[0239] Example 5: PD-L1 antibody affinity determination

[0240] ForteBio affinity determination was performed according to existing methods (Estep, P et al., Solution-based high-throughput antibody-antigen affinity and epitope fractionation measurements, MAbs, 2013.5(2): p.270-8). In brief, the sensor was equilibrated offline in the assay buffer for 30 min, then online for 60 s to establish a baseline, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then placed in 100 nM PD-L1 antigen for 5 min, after which the sensor was transferred to PBS for dissociation for 5 min. A 1:1 binding model was used for kinetic analysis.

[0241] Table 4. Affinity of candidate molecules

[0242]

[0243]

[0244] Example 6: PD-L1 antibody gene modification

[0245] To remove the potential glycosylation sites in C-Ye-18, the CDRH2 portion of the amino acid sequence of C-Ye-18 was point mutated into the 6 forms shown in Table 5:

[0246] Table 5. C-Ye-18 CDR region mutation sequence

[0247] serial number SEQ ID CDRH2 region C-Ye-18 2 SINSSSSSTYYRDSVKG C-Ye-18-1 81 SINSGSSSTYYRDSVKG C-Ye-18-2 84 SISSSSSSTYYRDSVKG C-Ye-18-3 87 SIGSSSSSTYYRDSVKG C-Ye-18-4 90 SIYSGSSSTYYRDSVKG C-Ye-18-5 93 SINSDSSSTYYRDSVKG C-Ye-18-6 96 SISGSSSSTYYRDSVKG

[0248] In this study, IMGT (http: / / www.imgt.org) was used to evaluate the humanization level of the mutant sequences in the CDR region of C-Ye-18. The results are shown in Table 6. The humanization level of all mutants of C-Ye-18 was higher than 87%, which met the requirements for late-stage drug development.

[0249] Table 6. C-Ye-18 CDR region mutant sequences and human homology

[0250]

[0251]

[0252] The protein construction and expression purification method were the same as in Example 3, and the purity of the protein was obtained by HPLC detection. The HPLC method was as follows: mobile phase: 150 mM Na2HPO4·12H2O, pH 7.0. Chromatographic conditions: detection wavelength: 280 nm, column temperature: 25°C, flow rate: 0.35 ml / min, detection time: 20 min, Zenix-C SEC-300 chromatographic column (SEPAX 4.6×300 mm, 3 μm).

[0253] Table 7. Purity test results of C-Ye-10 mutant antibody

[0254] serial number Monomer ratio (%) C-Ye-18 90.70 C-Ye-18-1 97.40 C-Ye-18-2 96.40 C-Ye-18-3 98.50 C-Ye-18-4 73.70 C-Ye-18-5 83.00 C-Ye-18-6 95.80

[0255] Example 7: C-Ye-18 mutant sample binds to human PD-L1

[0256] This experiment detected the binding activity of the purified C-Ye-18 mutant sample with CHO-hPD-L1 cells. The experimental method was the same as in Example 4. The experimental results are shown in Figure 2 As shown, the C-Ye-18 mutant samples have good binding activity to CHO-hPD-L1 cells, and the level is comparable to that of C-Ye-18 and the control antibody ATE.

[0257] Example 8: Affinity determination of C-Ye-18 mutant samples

[0258] In this experiment, the binding affinity of the purified C-Ye-18 mutant sample to human PD-L1 was detected. The experimental method was the same as that in Example 5. The experimental results are shown in Table 8. The C-Ye-18 mutant sample had good binding activity with the human PD-L1 protein.

[0259] Table 8 Affinity of C-Ye-18 mutant samples

[0260] serial number KD(M) kon(1 / Ms) kdis(1 / s) C-Ye-18-1 4.13E-09 2.46E+05 1.02E-03 C-Ye-18-2 5.41E-09 2.34E+05 1.27E-03 C-Ye-18-3 7.03E-09 2.41E+05 1.70E-03 C-Ye-18-4 6.40E-09 2.41E+05 1.54E-03 C-Ye-18-5 4.08E-09 2.72E+05 1.11E-03 C-Ye-18-6 6.00E-09 2.32E+05 1.39E-03

[0261] Example 9: C-Ye-18 mutant sample blocks the binding of PD-L1 to PD-1

[0262] CHO cells overexpressing human PD-1 (CHO-hPD-1 cells) were generated by transfecting the pCHO1.0 vector (purchased from Invitrogen) cloned into the MCS human PD-1 cDNA (purchased from Sino Biological). The cell density of the expanded cultured CHO-hPD-1 cells was adjusted to 2×10 6cells / ml, 100μl / well was added to the 96-well flow plate, centrifuged and set aside. The purified mutant sample was diluted with PBS, 400nM was diluted 3 times for a total of 12 points, 60μl / well of the diluted sample was added to the 96-well sample dilution plate, and 60μl / well of Biotinylated human PD-L1 protein (purchased from AcroBiosystems) was added at a final concentration of 500ng / ml, and incubated with the mutant sample at 4°C for 30min. 100μl / well of the co-incubated sample was added to the above 96-well flow plate with cells, incubated at 4°C for 30min, and washed twice with PBS. 100μl / well was added to the streptomycin R-phycoerythrin conjugate (purchased from Thermo Fisher) diluted 100 times with PBS, incubated at 4°C for 30min, and washed twice with PBS. 100μl / well was added to PBS to resuspend the cells, and the detection was performed on the CytoFlex (Bechman) flow cytometer and the corresponding MFI was calculated.

[0263] In the above method, the experimental results are as follows Figure 3 As shown, all mutant samples of the present invention can block the binding of PD-L1 to PD-1, and the blocking level is comparable to that of C-Ye-18 and the control antibody ATE.

[0264] Example 10: Thermal stability of C-Ye-18 mutant samples

[0265] DSC (Differential scanning calorimetry) was used to detect the thermal stability of different antibodies. After the sample was concentrated, it was diluted to 1 mg / ml with PBS; 5000× fluorescent colorimetric agent Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100× fluorescent colorimetric agent Sypro Orange. Take 50μl of 1mg / ml sample, add 10μl 100× fluorescent colorimetric agent Sypro Orange and 40μl ultrapure water, mix well, take 30μl and add it to a 96-well PCR plate, make 3 replicates for each sample, put it into the PCR instrument, and set the heating program as follows: 25℃ constant temperature for 5min, and heat to 99℃ at a rate of 0.5℃ / min. After the program ends, read the temperature value of the lowest point of the curve in the "Melt Curve" graph, which is the Tm value of the sample. The specific results are shown in Table 9 below

[0266] Table 9 Tm values ​​of C-Ye-18 mutant antibodies

[0267] serial number Tm1(℃) Tm2(℃) Tm3(℃) C-Ye-18 53.5 67.5 82 C-Ye-18-1 60 67.5 82 C-Ye-18-2 62.5 67.5 82 C-Ye-18-3 58 67 82 C-Ye-18-4 48 66 82 C-Ye-18-5 66 66～67 82.5 C-Ye-18-6 63 67.5 82

[0268] Example 11: Mixed lymphocyte reaction assay

[0269] In this example, the activity of C-Ye-18 mutant samples in activating T cells was detected by mixed lymphocyte reaction (MLR) assay. The specific experimental method is as follows.

[0270] PBMC cells (purchased from SAILY BIO, SLB-HPB) were revived and centrifuged. PBMC were resuspended in 10 ml X-VIVO-15 medium (purchased from LONZA), cultured in a cell culture incubator at 37°C for 2 h, and non-adherent cells were removed by aspiration. 10 ml DC medium was added: X-VIVO-15 medium with 10 ng / ml GM-CSF (purchased from R&D), 20 ng / ml IL-4, cultured for 3 days, 5 ml DC medium was added, and cultured until the 6th day, DC maturation medium was added: X-VIVO-15 medium with 1000 U / ml TNF-α (purchased from R&D), 10 ng / ml IL-6 (purchased from R&D), 5 ng / ml IL-1β (purchased from R&D), 1 μM PGE2 (purchased from Tocris), cultured for 2 days, mature DC cells were collected, and the cell density was adjusted to 2×10 with X-VIVO-15 medium. 5 cells / ml.

[0271] PBMC cells from another donor (purchased from SAILY BIO, SLB-HPB) were resuscitated, centrifuged, and resuspended in 10 ml X-VIVO-15 medium. + T cell sorting kit (purchased from Stemcell) enriched CD4 + T cells, X-VIVO-15 resuspended CD4 + T cells, adjust the cell density to 2 × 10 6 cells / ml. + T cells were mixed with the mature DC cells collected above at a ratio of 1:1, and 100 μl / well was added to a 96-well U-bottom plate.

[0272] The C-Ye-18 mutant sample was diluted with X-VIVO-15 medium, starting from 200 nM and diluted 3 times for a total of 9 points, 100 μl / well was added to the above mixed cell wells, cultured for 5 days, the supernatant was collected, and the expression levels of IFN-γ and IL2 were detected by ELISA (purchased from eBioscience).

[0273] The results are as follows Figure 4 As shown, the C-Ye-18 mutant samples, C-Ye-18-1, C-Ye-18-5, and C-Ye-18-6, all showed good biological activity in the MLR experiment, and the activation levels were similar to or better than the control antibody ATE.

[0274] Example 12 Cloning and expression of fusion protein PD-L1 / TGF-b trap

[0275] In this example, the TGFβRII extracellular domain (SEQ ID NO: 101) was used as the immunomodulatory molecule part of the fusion protein, and the PD-L1 antibody (human IgG1 Fc, LALA mutation) (C-Ye-18-5, SEQ ID: 94) was used as the targeting part of the fusion protein to form the PD-L1 antibody / TGFβRII extracellular region fusion protein (PM8001, SEQ ID: 102).

[0276] The C-terminal amino acid of the PD-L1 single-chain antibody of the present invention was connected to the TGFβRII extracellular domain via (G4S)4G by molecular cloning technology, and conventional expression was performed by the Expi-CHO expression system. The expression and purification method was the same as in Example 3 to obtain a structure as shown in Figure 5 The fusion protein PM8001 is shown.

[0277] Example 13 PM8001 molecule binds to human PD-L1

[0278] The method for detecting the binding activity of purified PD-L1 antibody (C-Ye-18-5, SEQ ID: 94), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824 (WO2015 / 118175A2) and negative control IgG protein to cell surface PD-L1 is the same as in Example 4. In the above method, the experimental results are as follows Figure 6 As shown, the PM8001 molecule of the present invention has binding activity with CHO-hPD-L1 cells, and the binding activity is similar to that of the positive control molecule M7824.

[0279] Example 14 PM8001 molecule blocks the binding of PD-L1 protein to PD-1 cells

[0280] The method for detecting the blocking activity of PD-L1 protein and PD-1 cell binding by purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824 and negative control IgG protein is the same as Example 9. In the above method, the experimental results are as follows Figure 7 As shown, the PM8001 molecule of the present invention can block the binding of PD-L1 protein to PD-1 cells, and the blocking level is equivalent to that of the positive control molecule M7824.

[0281] Example 15 Binding experiment of PM8001 molecule and human TGF-β family protein at ELISA level

[0282] Human TGF-β1 (acrobiosystems, TG1-H421), TGF-β2 (PeproTech, 100-35B), and TGF-β3 (PeproTech, 100-36E) proteins were diluted with ELISA coating solution and added to the ELISA plate, and coated overnight at 4°C. The coating solution was discarded, 250 μl / well was added to PBST and washed 3 times, and blocked with 5% BSA at room temperature for 1 hour. The purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, and positive control M7824 were diluted in a gradient and added to the blocked ELISA plate, and incubated at room temperature for 2 hours. After washing three times with PBST, sheep anti-human Fc-HRP (abcam, ab97225) was added to the wells, incubated at room temperature for 1 hour, washed three times with PBST, ELISA colorimetric solution was added, and after standing at room temperature for 3 minutes, ELISA stop solution was added and the absorbance value at 450 nm was read.

[0283] In the above method, the experimental results are as follows Figure 8 As shown, PM8001 molecule of the present invention has good binding activity with human TGF-β1 and TGF-β3 proteins at ELISA level, weaker binding activity with human TGF-β2 protein, and the binding level is equivalent to that of positive control molecule M7824.

[0284] Example 16 PM8001 molecule blocks TGF-β / SMAD signaling pathway experiment

[0285] Take an appropriate amount of 293-TGF-β / SMAD effector cells and inoculate them in a 96-well cell culture white bottom plate, place it in a 37°C, 5% CO2 incubator and culture it overnight. The purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, and positive control M7824 were diluted in a gradient and mixed with TGF-β1 (acrobiosystems, TG1-H421) and incubated at room temperature for 30 minutes. The above mixture was added to the white bottom plate with cells and continued to be cultured overnight. Bio-Glo TM reagent (Promega) was added to each well, and the fluorescence signal value was read using a multifunctional microplate reader.

[0286] In the above method, the experimental results are as follows Fig. 9 As shown, the PM8001 molecule of the present invention can block the TGF-β / SMAD signaling pathway in vitro, and the blocking level is equivalent to that of the positive control molecule M7824.

[0287] Example 17 Mixed lymphocyte reaction experiment

[0288] The method for mixed lymphocyte detection of purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824 and negative control IgG protein to activate human T lymphocytes is the same as in Example 9. The results are as follows Fig.10 As shown, the PM8001 molecule of the present invention exhibits good biological activity in the MLR experiment, and the activation level is equivalent to or better than that of the positive control molecule M7824.

[0289] Example 18 PM8001 Pharmacokinetic Evaluation in Rats

[0290] Six SD rats were used in the experiment, half male and half female, with 12 / 12 hours light / dark adjustment, temperature 24±2℃, humidity 40-70%, free access to water and food. They were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, the SD rats were injected with PM8001 molecules once in the tail vein, with an injection dose of 10 mg / Kg.

[0291] Blood collection time point: 3min, 4 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, 672 hours after administration, blood was collected from the rat jugular vein. The whole blood sample was placed at 2-8°C for 30 minutes, centrifuged at 12000rpm for 5min to collect serum, and the obtained serum was centrifuged at 2-8°C, 12000rpm for 5min, and stored at -80°C. ELISA was used to detect the molecular weight of free PM8001 in serum. The results are shown in Table 10. The half-life of the free state molecule of PM8001 of the present invention in SD rats is about 146 hours.

[0292] Table 10 T1 / 2 of PM8001 in SD rats

[0293] Test drug Dosage T1 / 2 PM8001 IV 146 hours

[0294] Example 19 Study on the Tumor Suppression Activity of PM8001

[0295] In this study, MC38 cells expressing human PD-L1 (h-PD-L1 KI MC38) were used to determine the anti-tumor effect of PM8001 in PD-L1 transgenic mice. First, the h-PD-L1 KI MC38 tumor-bearing mouse model was established by subcutaneous inoculation. 3 The mice were divided into groups at different times and given different antibodies and different doses of treatment by single intraperitoneal injection. The changes in tumor volume and body weight of mice in each group were monitored. The monitoring frequency was 2 times / week for 3 consecutive weeks. The dosage and method of administration are shown in Table 11. The changes in tumor volume of mice are shown in Table 11. Fig.11 shown.

[0296] Table 11 Experimental scheme for tumor suppression activity of PM8001

[0297]

[0298]

[0299] The experimental results are as follows Fig.11 As shown. After inoculation with h-PD-L1 KI MC38, the tumor volume of the negative control group continued to increase, and the tumor growth of the TGF-βR II-Fc and C-Ye-18-5 groups was inhibited, while the PM8001 group was better controlled than the TGF-βR II-Fc and C-Ye-18-5 groups, indicating that PM8001 has a significant tumor inhibitory effect, which is equivalent to or even slightly better than the positive control group.

[0300] Example 20 Crystal structure identification of the complex of PD-L1 and single domain antibody VHH fragment

[0301] In this experiment, X-ray diffraction was used to identify the crystal structure of the complex of PD-L1 and single-domain antibody VHH fragment. Human PD-L1-His tag (SEQ ID: 109) was expressed by the Escherichia coli prokaryotic system. PD-L1 single-domain antibody fusion protein (SEQ ID: 110) was expressed by the CHO system. The PD-L1 single-domain antibody fusion protein was enzymatically cleaved and purified using IdeS enzyme and GingisKHAN enzyme in turn, and the PD-L1 single-domain antibody VHH (SEQ ID: 111) was finally obtained. PD-L1 and VHH were mixed in a molar ratio of 1:1 to prepare a complex sample for crystallization. The purified complex was enzymatically cleaved by carboxypeptidase B to remove the His tag of PD-L1. The complex (7.5 mg / mL) was mixed with the crystallization reagent in a 1:1 ratio and crystal cultured at 18°C. Three days later, crystals were observed under the INT kit culture conditions, and the crystal morphology was as follows. Fig.12 shown.

[0302] Selected single crystals were used for X-ray diffraction experiments at the Shanghai Light Source and the resolution was Diffraction data. Data processing was performed using XDS software. Crystal phase identification was performed using the molecular replacement method using the PD-L1 (PDB ID: 5jds) and VHH (PDB ID: 5m2j) structures as models. Crystal structure refinement was performed using Refmac5. Model detection, manual reconstruction, and structure verification were performed using COOT. The complex crystals belong to the P21 space group, and the unit cell parameters are: α=90.00°, β=90.02°, γ=90.00°.

[0303] The crystal structure of the PD-L1-VHH complex obtained after structural analysis is as follows Fig.13As shown. Epitope analysis showed that the main hydrogen bonding between PD-L1 and VHH is concentrated on Tyr56, Asn63, His69, Asp73, Lys75, Ser117, Gly119 and Ala121 on PD-L1 and other amino acids on PD-L1 ( Fig.14 In addition, Tyr56, His69 and Tyr123 on PD-L1 and Tyr32, Trp33, Tyr35, Leu45, Trp47, Pro100 and Tyr103 on VHH form a hydrophobic interaction interface ( Fig.15 ).

[0304] Example 21 PM8001 injection inhibits the growth of h-PD-L1 knock-in MC38 model inoculated subcutaneously in h-PD-L1 knock-in C57BL / 6 mice

[0305] This study investigated the in vivo antitumor effect of PM8001 administered by intraperitoneal injection on the h-PD-L1 knock-in MC38 mouse colon cancer tumor model inoculated subcutaneously in h-PD-L1 knock-in C57BL / 6 mice and its safety in tumor-bearing mice.

[0306] In this study, h-PD-L1 knock-in MC38 mouse colon cancer cells were subcutaneously inoculated in h-PD-L1 knock-in C57BL / 6 mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to establish a h-PD-L1 knock-in MC38 subcutaneous tumor model. Ten days after inoculation, the mice were divided into 5 groups (6 mice in each group) according to the tumor volume and intraperitoneally injected with PBS, 14.7 mg / kg PM8001, 10 mg / kg PM8001-NSD (anti-human PD-L1 VHH), 10 mg / kg PM8001-TGF-βR II, and 24.6 mg / kg M7824 (the molar dose of each group was the same) for a total of 2 times (3 days between the 2 administrations). The in vivo antitumor effects of PM8001, PM8001-NSD, PM8001-TGF-βRII, and M7824 and their safety in tumor-bearing mice were investigated.

[0307] Fig.16 Shown are the effects of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. Fig.17 The tumor images of different drug groups 27 days after inoculation are shown. The tumors of mice in the negative control PBS group grew rapidly, and the tumor volume reached 1500mm 27 days after inoculation (i.e., 17 days after administration). 3The above shows that the tumor model of this experiment was successfully established. Compared with the negative control PBS group, PM8001, PM8001-NSD, PM8001-TGF-βRII, and M7824 at the same molar dose can inhibit tumor growth to varying degrees. The TGI of the above groups on the 17th day after administration were 80%, 72%, 15%, and 53%, respectively; the PM8001 injection group had a stronger effect in inhibiting tumor growth than PM8001-NSD, PM8001-TGF-βRII, and the similar molecule group M7824. At the end of the experiment, the tumor was weighed. The tumor weight of the PM8001 injection group was less than that of PM8001-NSD, PM8001-TGF-βRII, and the similar molecule group M7824. Fig.18 The effects of each group of drugs on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors are shown.

[0308] Fig.19 The effects of different drug groups on the body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors were shown, and no abnormalities were found in the mice in each group. Compared with the control group, the body weight of mice in each drug-treated group did not decrease significantly; at the end of the experiment, no obvious lesions were found in the main organs of mice in each group of gross anatomy, indicating that the drugs in each group had no obvious toxicity to mice at the dosage used in this experiment.

[0309] Example 22 Growth inhibitory effect of different doses of PM8001 on h-PD-L1 knock-in MC38 model inoculated subcutaneously in h-PD-L1 knock-in C57BL / 6 mice

[0310] This study investigated the in vivo antitumor effects of three different doses of PM8001 injection on the h-PD-L1 knock-in MC38 mouse colon cancer tumor model inoculated subcutaneously into h-PD-L1 knock-in C57BL / 6 mice (purchased from Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) and its safety in tumor-bearing mice.

[0311] In this study, h-PD-L1 knock-in MC38 mouse colon cancer cells were subcutaneously inoculated in h-PD-L1 knock-in C57BL / 6 mice to establish a h-PD-L1 knock-in MC38 subcutaneous tumor-bearing model, which can be used to evaluate the anti-tumor effect related to the mechanism of action of the test article and the safety characteristics in the disease state. Seven days after inoculation, the mice were divided into 5 groups (6 in each group) according to the tumor volume and given a single intraperitoneal injection of PBS, different doses (0.3, 2.1, 14.7 mg / kg) of PM8001 injection, or 24.6 mg / kg M7824. The in vivo anti-tumor effect of PM8001 at different doses and its safety in tumor-bearing mice were investigated and compared with the similar molecule M7824.

[0312] Fig. 20 Shown are the effects of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. Fig.21 The tumor images of different drug groups 28 days after inoculation are shown. The tumors of mice in the negative control PBS group grew rapidly, and the tumor volume reached 1300mm 28 days after inoculation. 3 The above shows that the tumor model of this experiment was successfully established. Compared with the negative control PBS group, PM8001 can inhibit tumor growth in a dose-dependent manner. The TGI under low, medium and high doses of PM8001 are 31%, 76% and 93% respectively; at the same molar dose, the PM8001 group (14.6 mg / kg) has a stronger effect on inhibiting tumor growth than the similar molecule M7824 group (24.6 mg / kg). At the end of the experiment, the tumor was weighed and PM8001 can reduce the tumor weight in a dose-dependent manner; at the same molar dose, the tumor inhibition rate of the PM8001 group (14.6 mg / kg) is higher than that of the similar molecule M7824 group (24.6 mg / kg). Fig. 22 Shown are the effects of each group of drugs on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0313] Fig.23 The effects of different drug groups on the body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors are shown. There was no abnormality in the state of the mice in each group, and compared with the control group, the body weight of the mice in each drug-treated group did not decrease significantly; at the end of the experiment, no obvious lesions were found in the main organs of the mice in each group during gross anatomy, indicating that the drugs in each group had no obvious toxicity to the mice at the dosage used in this experiment.

[0314] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0315] Sequence information of the present invention

[0316] SEQ ID NO:1C-Ye-18 CDR1 amino acid sequence

[0317] GFTFSSYWMY

[0318] SEQ ID NO:2C-Ye-18 CDR2 amino acid sequence

[0319] SINSSSSSTYYRDSVKG

[0320] SEQ ID NO:3C-Ye-18 CDR3 amino acid sequence

[0321] AKDPGGYA

[0322] SEQ ID NO:4C-Ye-18 VHH amino acid sequence

[0323] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0324] SEQ ID NO: 5C-Ye-18 VHH nucleotide sequence

[0325] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0326] SEQ ID NO:6C-Ye-04 CDR1 amino acid sequence

[0327] SGFTFSSYWMY

[0328] SEQ ID NO:7C-Ye-04 CDR2 amino acid sequence

[0329] SINTSSSSTYYRDSVKG

[0330] SEQ ID NO:8C-Ye-04 CDR3 amino acid sequence

[0331] AKDPGGYA

[0332] SEQ ID NO:9C-Ye-04 VHH amino acid sequence

[0333] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINTSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0334] SEQ ID NO: 10C-Ye-04 VHH nucleotide sequence

[0335] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATACTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0336] SEQ ID NO:11C-Ye-02 CDR1 amino acid sequence

[0337] GRTFNNSAMGAMG

[0338] SEQ ID NO:12C-Ye-02 CDR2 amino acid sequence

[0339] TITWSSGSSFYANSVKG

[0340] SEQ ID NO:13C-Ye-02 CDR3 amino acid sequence

[0341] ASRKLGGVVTVVTSYDF

[0342] SEQ ID NO:14C-Ye-02 VHH amino acid sequence

[0343] QVQLQESGGGLVQAGGSLRLSCAASGRTFNNSAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS

[0344] SEQ ID NO:15 C-Ye-02 VHH nucleotide sequence

[0345] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAATAACTCGGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0346] SEQ ID NO:16 C-Ye-06 CDR1 amino acid sequence

[0347] GRTFDNYAMGAMG

[0348] SEQ ID NO:17 C-Ye-06 CDR2 amino acid sequence

[0349] TITWSSGSSFYANSVKG

[0350] SEQ ID NO:18 C-Ye-06 CDR3 amino acid sequence

[0351] ASRKLGGVVTVVTSYDF

[0352] SEQ ID NO:19 C-Ye-06 VHH amino acid sequence

[0353] QVQLQESGGGLVQPGGSLRLSCAASGRTFDNYAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS

[0354] SEQ ID NO:20 C-Ye-06 VHH nucleotide sequence

[0355] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCGATAACTATGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGACGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0356] SEQ ID NO:21 C-Ye-09 CDR1 amino acid sequence

[0357] GRTFSTYAVG

[0358] SEQ ID NO:22 C-Ye-09 CDR2 amino acid sequence

[0359] GRLTWSGSRTYYADSVKG

[0360] SEQ ID NO:23 C-Ye-09 CDR3 amino acid sequence

[0361] AADYRSNSTWSLQSPARYEN

[0362] SEQ ID NO:24 C-Ye-09 VHH amino acid sequence

[0363] QVQLQESGGGLVQAGDSLGLSCTASGRTFSTYAVGWFRQAPGKGREFVGRLTWSGSRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAADYRSNSTWSLQSPARYENWGQGTQVTVSS

[0364] SEQ ID NO:25 C-Ye-09 VHH nucleotide sequence

[0365] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGGGACTCTCCTGTACAGCCTCTGGACGCACCTTCAGTACCTATGCCGTGGGGTGGTTCCGCCAGGCTCCAGGGAAGGGGCGTGAATTTGTAGGACGTCTTACATGGAGCGGGAGTAGAACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCGACTACCGAAGTAACAGTACCTGGTCCCTGCAAAGCCCGGCACGTTATGAAAATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0366] SEQ ID NO:26 C-Ye-10 CDR1 amino acid sequence

[0367] GRTVSNYAMG

[0368] SEQ ID NO:27 C-Ye-10 CDR2 amino acid sequence

[0369] RITGSGSSTFYADSVKG

[0370] SEQ ID NO:28 C-Ye-10 CDR3 amino acid sequence

[0371] AADRWRSMVTRSDPREYEN

[0372] SEQ ID NO:29 C-Ye-10 VHH amino acid sequence

[0373] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS

[0374] SEQ ID NO:30 C-Ye-10 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0375] SEQ ID NO:31 C-Ye-17 CDR1 amino acid sequence

[0376] GRTVSNYAMG

[0377] SEQ ID NO:32 C-Ye-17 CDR2 amino acid sequence

[0378] RITGSGSSTFYADSVKG

[0379] SEQ ID NO:33 C-Ye-17 CDR3 amino acid sequence

[0380] AADRWRSMVTRSDPREYEN

[0381] SEQ ID NO:34 C-Ye-17 VHH amino acid sequence

[0382] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGLFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS

[0383] SEQ ID NO:35 C-Ye-17 VHH nucleotide sequence

[0384] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCTATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0385] SEQ ID NO:36 C-Ye-20 CDR1 amino acid sequence

[0386] GRTVSNYAMG

[0387] SEQ ID NO:37 C-Ye-20 CDR2 amino acid sequence

[0388] RITGSGSSTFYADSVKG

[0389] SEQ ID NO:38 C-Ye-20 CDR3 amino acid sequence

[0390] AADRWRSMVTRSYPREYEN

[0391] SEQ ID NO:39 C-Ye-20 VHH amino acid sequence

[0392] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAADRWRSMVTRSYPREYENWGQGTQVTVSS

[0393] SEQ ID NO:40 C-Ye-20 VHH nucleotide sequence

[0394] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTTACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0395] SEQ ID NO:41 C-Ye-24 CDR1 amino acid sequence

[0396] GRTVSNYAMG

[0397] SEQ ID NO:42 C-Ye-24 CDR2 amino acid sequence

[0398] RITGSGRTTYYADSVKG

[0399] SEQ ID NO:43 C-Ye-24 CDR3 amino acid sequence

[0400] AADRWRSMVTRSDPREYEN

[0401] SEQ ID NO:44 C-Ye-24 VHH amino acid sequence

[0402] QVQLQESGGGVVQAGDSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGRTTYYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS

[0403] SEQ ID NO:45 C-Ye-24 VHH nucleotide sequence

[0404] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGTGTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTCGTACCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0405] SEQ ID NO:46 C-Ye-26 CDR1 amino acid sequence

[0406] GRTVSNYAMG

[0407] SEQ ID NO:47 C-Ye-26 CDR2 amino acid sequence

[0408] RITGSGSSTFYADSVKG

[0409] SEQ ID NO:48 C-Ye-26 CDR3 amino acid sequence

[0410] AADRWRSMVTRSDPRDYEN

[0411] SEQ ID NO:49 C-Ye-26 VHH amino acid sequence

[0412] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPRDYENWGQGTQVTVSS

[0413] SEQ ID NO:50 C-Ye-26 VHH nucleotide sequence

[0414] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGATTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0415] SEQ ID NO:51 C-Ye-27 CDR1 amino acid sequence

[0416] GRTFSRYAVG

[0417] SEQ ID NO:52 C-Ye-27 CDR2 amino acid sequence

[0418] AITWSGGYTYYADSVKG

[0419] SEQ ID NO:53 C-Ye-27 CDR3 amino acid sequence

[0420] AVDTRNVIGPRAGDY

[0421] SEQ ID NO:54 C-Ye-27 VHH amino acid sequence

[0422] QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0423] SEQ ID NO:55C-Ye-27 VHH nucleotide sequence

[0424] CAGGTGCAGGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0425] SEQ ID NO:56C-Ye-30 CDR1 amino acid sequence

[0426] GSTFSRYAVG

[0427] SEQ ID NO:57C-Ye-30 CDR2 amino acid sequence

[0428] AITWSGGYTYYADSVKG

[0429] SEQ ID NO:58C-Ye-30 CDR3 amino acid sequence

[0430] AVDTRNVIGPRAGDY

[0431] SEQ ID NO:59C-Ye-30 VHH amino acid sequence

[0432] QVQLQESGGGLVQAGGSLRLSCAASGSTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0433] SEQ ID NO:60C-Ye-30 VHH nucleotide sequence

[0434] CAGGTGCAGGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0435] SEQ ID NO:61C-Ye-32 CDR1 amino acid sequence

[0436] GRTFSRYAVG

[0437] SEQ ID NO:62C-Ye-32 CDR2 amino acid sequence

[0438] AITWSGGYTYYADSVKG

[0439] SEQ ID NO:63C-Ye-32 CDR3 amino acid sequence

[0440] AVDTRNVIGPRAGDY

[0441] SEQ ID NO:64C-Ye-32 VHH amino acid sequence

[0442] QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTIYLQMNSLNVEDTGVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0443] SEQ ID NO:65C-Ye-32 VHH nucleotide sequence

[0444] CAGGTGCAGGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATCTATCTCCAAATGAACAGCCTGAACGTTGAGGACACGGGCGTTTATTACTGCGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0445] SEQ ID NO:66C-Ye-34 CDR1 amino acid sequence

[0446] AASGRTFSRFAMG

[0447] SEQ ID NO:67C-Ye-34 CDR2 amino acid sequence

[0448] AISWSGGMIYYTDSVKG

[0449] SEQ ID NO:68C-Ye-34 CDR3 amino acid sequence

[0450] AVDTRNVIGPRAGDY

[0451] SEQ ID NO:69C-Ye-34 VHH amino acid sequence

[0452] QVQLQESGGGLVQAGGSLRLSCAASGRTFSRFAMGWFRQAPGKEREFVAAISWSGGMIYYTDSVKGRFTISRDNAKNMLYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0453] SEQ ID NO:70C-Ye-34 VHH nucleotide sequence

[0454] CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACTTTCAGTAGGTTTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTAGCTGGAGTGGTGGTATGATATACTATACA GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0455] SEQ ID NO:71C-Ye-39 CDR1 amino acid sequence

[0456] GRAFSVYPMA

[0457] SEQ ID NO:72C-Ye-39 CDR2 amino acid sequence

[0458] RLTYTSNTFYADSVKG

[0459] SEQ ID NO:73C-Ye-39 CDR3 amino acid sequence

[0460] AVENRSSSWSLQSPARYDD

[0461] SEQ ID NO:74C-Ye-39 VHH amino acid sequence

[0462] QVQLQESGGGLVQAGGSLRLSCTASGRAFSVYPMAWFRQAPGKEREFIARLTYTSNTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVENRSSSWSLQSPARYDDWGQGTQVTVSS

[0463] SEQ ID NO:75C-Ye-39 VHH nucleotide sequence

[0464] CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCATGTACAGCCTCTGGACGCGCCTTCAGTGTCTACCCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTATAGCACGTCTTACGTATACTAGTAACACATTCTATGCAGACTCCG TGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGTCGAGAACCGCAGTAGTAGTTGGTCCCTGCAAAGCCCGGCACGTTATGATGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0465] SEQ ID NO:76C-Ye-42 CDR1 amino acid sequence

[0466] GRTGSRYAVG

[0467] SEQ ID NO:77C-Ye-42 CDR2 amino acid sequence

[0468] AITWSGGYTYYADSVKG

[0469] SEQ ID NO:78C-Ye-42 CDR3 amino acid sequence

[0470] AVDTRNVIGPRAGDY

[0471] SEQ ID NO:79C-Ye-42 VHH amino acid sequence

[0472] QVQLQESGGGLVQAGGSLRLSCAASGRTGSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0473] SEQ ID NO:80 C-Ye-42 VHH nucleotide sequence

[0474] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCGGCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGAACAGCCTAAAACCTGAAGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0475] SEQ ID NO:81 C-Ye-18-1 CDR2 amino acid sequence

[0476] SINSGSSSTYYRDSVKG

[0477] SEQ ID NO:82 C-Ye-18-1 VHH amino acid sequence

[0478] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0479] SEQ ID NO:83 C-Ye-18-1 VHH nucleotide sequence

[0480] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0481] SEQ ID NO:84 C - Ye - 18 - 2 CDR2 Amino Acid Sequence

[0482] SISSSSSSTYYRDSVKG

[0483] SEQ ID NO:85 C - Ye - 18 - 2 VHH Amino Acid Sequence

[0484] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0485] SEQ ID NO:86 C - Ye - 18 - 2 VHH Nucleotide Sequence

[0486] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTAGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0487] SEQ ID NO:87 C-Ye-18-3 CDR2 Amino Acid Sequence

[0488] SIGSSSSSTYYRDSVKG

[0489] SEQ ID NO:88 C-Ye-18-3 VHH Amino Acid Sequence

[0490] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIGSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0491] SEQ ID NO:89 C-Ye-18-3 VHH Nucleotide Sequence

[0492] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTGGTAGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0493] SEQ ID NO:90 C - Ye - 18 - 4 CDR2 Amino Acid Sequence

[0494] SIYSGSSSTYYRDSVKG

[0495] SEQ ID NO:91 C - Ye - 18 - 4 VHH Amino Acid Sequence

[0496] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIYSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0497] SEQ ID NO:92 C - Ye - 18 - 4 VHH Nucleotide Sequence

[0498] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTTACAGTGGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0499] SEQ ID NO:93 C-Ye-18-5 CDR2 Amino Acid Sequence

[0500] SINSDSSSTYYRDSVKG

[0501] SEQ ID NO:94 C-Ye-18-5 VHH Amino Acid Sequence

[0502] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0503] SEQ ID NO:95 C-Ye-18-5 VHH Nucleotide Sequence

[0504] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGACAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0505] SEQ ID NO:96 C-Ye-18-6 CDR2 Amino Acid Sequence

[0506] SISGSSSSTYYRDSVKG

[0507] SEQ ID NO:97 C-Ye-18-6 VHH Amino Acid Sequence

[0508] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISGSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0509] SEQ ID NO:98 C-Ye-18-6 VHH Nucleotide Sequence

[0510] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTGGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0511] SEQ ID NO:99 Amino acid sequence of IgG1 Fc fragment

[0512] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0513] SEQ ID NO:100 Amino acid sequence of linker of fusion protein

[0514] GGGGSGGGGSGGGGSGGGGSG

[0515] SEQ ID NO:101 Amino acid sequence of extracellular domain of TGFβRII

[0516] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0517] SEQ ID NO: 102PM8001 amino acid sequence

[0518] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSSDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNN DMIVTDNNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0519] SEQ ID NO: 103 First round upstream primer

[0520] GTCCTGGCTGCTCTTCTACAAGG

[0521] SEQ ID NO: 104 First round downstream primer

[0522] GGTACGTGCTGTTGAACTGTTCC

[0523] SEQ ID NO: 105 Second round upstream primer

[0524] CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT

[0525] SEQ ID NO: 106 Second round downstream primer

[0526] CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG

[0527] SEQ ID NO: 107 The third round upstream primer

[0528] ATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTAAAAGAGAGGCTGAAGCACAGGTGCAGCTGCAGGAGTCTGGRGGAGG

[0529] SEQ ID NO:108 Third-round downstream primer

[0530] AGTTGTCAGTTCCTGTGCCCCCCCTCCTCCCGCGCCACCTCCGCCCGCACCTCCGCCACCAcTGGAGACGGTGACCTGGGT

[0531] SEQ ID NO:109 Amino acid sequence of PD-L1-His tag

[0532] MFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGE

[0533] EDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYK

[0534] RITVKVNAPYHHHHHH

[0535] SEQ ID NO:110 Amino acid sequence of PD-L1 single-domain antibody fusion protein

[0536] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINS

[0537] DSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQ

[0538] GTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

[0539] HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY

[0540] KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYP

[0541] SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM

[0542] HEALHNHYTQKSLSLSPG

[0543] SEQ ID NO:111 Amino acid sequence of the PD-L1 single domain antibody

[0544] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINS

[0545] DSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQ

[0546] GTQVTVSSDK

Claims

1. An anti-PD-L1 single domain antibody comprising a complementarity determining region (CDR) of a VHH chain, wherein the CDR of the VHH chain consists of the following composition: CDR1 having an amino acid sequence as shown in SEQ ID NO: 1; A CDR2 having an amino acid sequence as shown in any one of SEQ ID NOs: 93, 81, 84, 87, 90, 96, 2; and The amino acid sequence of CDR3 is shown in SEQ ID NO:

3.

2. A VHH chain of an anti-PD-L1 single-domain antibody, wherein the VHH chain of the anti-PD-L1 single-domain antibody comprises CDR1, CDR2 and CDR3 as defined in claim 1.

3. The VHH chain of the anti-PD-L1 single domain antibody according to claim 2, in, The amino acid sequence of the VHH chain is shown in any one of SEQ ID NOs: 94, 82, 85, 88, 91, 97, and 4.

4. The VHH chain of the anti-PD-L1 single domain antibody of claim 2, wherein the amino acid sequence of the VHH chain of the anti-PD-L1 single domain antibody is shown in SEQ ID NO:

94.

5. An anti-PD-L1 single domain antibody, wherein the anti-PD-L1 single domain antibody is a single domain antibody against the PD-L1 epitope and has the VHH chain of the anti-PD-L1 single domain antibody according to any one of claims 2 to 4.

6. A polynucleotide, wherein the polynucleotide encodes a protein selected from the group consisting of the anti-PD-L1 single domain antibody according to claim 1, the VHH chain of the anti-PD-L1 single domain antibody according to any one of claims 2 to 4, or the anti-PD-L1 single domain antibody according to claim 5.

7. A single-domain antibody fusion protein, wherein the single-domain antibody fusion protein has a structure as shown in Formula I from N-terminus to C-terminus: Z1-Z2-L-Z3 (Formula I) In the formula, Z1 is the VHH chain of the anti-PD-L1 single domain antibody according to any one of claims 2 to 4; Z2 is the Fc segment of immunoglobulin; L is the linker sequence; Z3 is the immunomodulatory molecule.

8. The single-domain antibody fusion protein of claim 7, wherein the amino acid sequence of the single-domain antibody fusion protein is as shown in SEQ ID NO:

102.

9. An immunoconjugate, It is characterized in that The immunoconjugate contains: (a) the VHH chain of the anti-PD-L1 single domain antibody according to any one of claims 2 to 4, the anti-PD-L1 single domain antibody according to claim 5, or the single domain antibody fusion protein according to claim 7 or 8; and (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

10. Use of the anti-PD-L1 single domain antibody according to claim 5 or the single domain antibody fusion protein according to claim 7 or 8 for preparing (a) a reagent for detecting PD-L1 molecules or (b) a drug for treating tumors; in, The tumor is selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, and combinations thereof.

11. A pharmaceutical composition, include: (i) the anti-PD-L1 single domain antibody of claim 1, the VHH chain of the anti-PD-L1 single domain antibody of any one of claims 2 to 4, the anti-PD-L1 single domain antibody of claim 5, the single domain antibody fusion protein of claim 7 or 8, or the immunoconjugate of claim 9; and (ii) a pharmaceutically acceptable carrier.

12. Use of the VHH chain according to any one of claims 2 to 4, the single domain antibody according to claim 5, the single domain antibody fusion protein according to claim 7 or 8, or the immunoconjugate according to claim 9 for preparing a medicament, a reagent, a test plate or a kit; in, The reagent, detection plate or kit is used to: detect PD-L1 protein in a sample; Wherein, the agent is used to treat tumors expressing PD-L1 protein (i.e., PD-L1 positive); the tumor is selected from: gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, cervical cancer, lymphoma, adrenal tumor, bladder tumor, and combinations thereof.

13. A method for detecting PD-L1 protein in a sample, which is used for non-diagnostic purposes and comprises the steps of: (1) contacting a sample to be tested with the single domain antibody according to claim 5 or the single domain antibody fusion protein according to claim 7 or 8; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of PD-L1 protein in the sample.

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