Anti-PD-l1 single domain antibodies as well as derivatives and uses thereof

Abstract

To provide a class of specific single-domain antibodies active against PD-L1.SOLUTION: Disclosed are complementarity determining regions (CDRs) of the VHH chain of an anti-PD-L1 single domain antibody, the CDRs of the VHH chain comprising CDR1, CDR2, and CDR3 having specific amino acid sequences. Any one of the amino acid sequences may be obtained by adding, deleting, modifying, and / or substituting 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues, and derivative sequences that retain the PD-L1 binding affinity of the anti-PD-L1 single domain antibody are also included.SELECTED DRAWING: Figure 20

Description

Detailed Description of the Invention

[0001] Technical Field This application relates to the biomedical or biopharmaceutical technical fields, and in particular to anti-PD-L1 single domain antibodies and derivatives thereof and uses thereof.

[0002] background 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 consisting of a total of 290 amino acids, including one IgV-like domain, one IgC-like domain, one transmembrane hydrophobic domain, and one intracellular domain of 30 amino acids.

[0003] PD-L1 negatively regulates immune responses. Studies have shown that PD-L1 is primarily expressed on activated T cells, B cells, macrophages, and dendritic cells. In addition to lymphocytes, PD-L1 is also expressed on endothelial cells in many other tissues, such as the thymus, heart, and placenta, as well as in various non-lymphoid cancers, such as melanoma, liver cancer, gastric cancer, renal cell carcinoma, ovarian cancer, colon cancer, breast cancer, esophageal cancer, and head and neck cancer. PD-L1 plays a role in regulating autoreactive T cells and B cells and in immune tolerance, and plays a role in T cell 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) in combination with PD-L1, also known as CD279, is a member of the B7-CD28 superfamily. The cytoplasmic domain of CD279 contains two tyrosine residues: one near the N-terminus is located within an immunoreceptor tyrosine-dependent inhibitory motif (ITIM), and the other near the C-terminus is located within an immunoreceptor tyrosine-dependent switch motif (ITSM). PD-1 is primarily expressed on the surface of activated T lymphocytes, B lymphocytes, and macrophages. Under normal circumstances, PD-1 can suppress the function of T lymphocytes and promote the function of Treg cells, thereby suppressing autoimmune responses and preventing the development of autoimmune diseases. However, during tumor development, binding of PD-L1 expressed by tumor cells to PD-1 can promote immune escape of tumors by suppressing lymphocytes. Binding of PD-L1 to PD-1 can induce various biological changes and immune regulation, such as inhibiting lymphocyte proliferation and activation, 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 diagnosis and targeted therapy has revolutionized tumor treatment. Traditional monoclonal antibodies (150 kD) have a high molecular weight that can hinder their ability to penetrate tissues, resulting in low effective concentrations in tumors and insufficient therapeutic efficacy. In addition, the long development period, high manufacturing costs, poor stability, and many other factors of traditional antibodies limit their clinical application and widespread use.

[0006] Single-domain antibodies are currently the smallest antibody molecules, with a molecular weight (without Fc) that is 1 / 10 that of conventional antibodies. In addition to the antigen reactivity of monoclonal antibodies, single-domain antibodies also have unique functional properties, such as low molecular weight, high stability, good solubility, easy expression, high tissue penetration, simple humanization, and low preparation costs, which can overcome the shortcomings of traditional antibodies.

[0007] However, there are still no satisfactory single domain antibodies against PD-L1 in the art, and therefore there is an urgent need in the field to develop effective and specific single domain antibodies against PD-L1.

[0008] overview The aim of this application is to provide a class of specific single domain antibodies that are effective against PD-L1.

[0009] In a first aspect of the application, there is provided a complementarity determining region (CDR) of the VHH chain of an anti-PD-L1 single domain antibody, wherein the CDR of the VHH chain comprises the following: CDR1 having the amino acid sequence shown in SEQ ID NO: 5n+1; a CDR2 having the amino acid sequence set forth in SEQ ID NO: 5n+2, or a CDR2 having an amino acid sequence having greater than 85% sequence identity with the sequence set forth in SEQ ID NO: 2; and CDR3 having the amino acid sequence shown in SEQ ID NO: 5n+3 It consists of:

[0010] Each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In another preferred embodiment, n is 0 or 1.

[0011] In another preferred embodiment, the amino acid sequence of CDR2 is set forth in SEQ ID NO: 2, 7, 81, 84, 87, 90, 93, or 96.

[0012] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0013] In a second aspect of the application, there is provided an anti-PD-L1 single domain antibody VHH chain, which comprises a CDR1, CDR2, and CDR3 according to the first aspect of the application.

[0014] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L1 single domain antibody is set forth in SEQ ID NO: 5n+4, 82, 85, 88, 91, 94, or 97.

[0015] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0016] Any one of the above amino acid sequences also includes derivative sequences that can be obtained by adding, deleting, modifying, and / or substituting 1 to 8 (preferably 1 to 5, more preferably 1 to 3) amino acid residues, and that retain the PD-L1 binding affinity of the anti-PD-L1 single domain antibody. In another preferred embodiment, n is 0 or 1.

[0017] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L1 single domain antibody is set forth in SEQ ID NO: 4, 9, 82, 85, 88, 91, 94, or 97.

[0018] In a third aspect of the present application, there is provided an anti-PD-L1 single domain antibody, which is a single domain antibody directed against a PD-L1 epitope and has the VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application.

[0019] In a fourth aspect of the present application, there is provided a polynucleotide encoding a protein selected from the group of proteins comprising the CDR regions of the VHH chain of an anti-PD-L1 single domain antibody according to the first aspect of the application, the VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application, or an anti-PD-L1 single domain antibody according to the third aspect of the application.

[0020] In another preferred embodiment, the polynucleotide has the amino acid sequence set forth in SEQ ID NO: 5n, 83, 86, 89, 92, 95, or 98.

[0021] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0022] In a fifth aspect of the present application, there is provided an expression vector, the expression vector containing a polynucleotide according to the fourth aspect of the present application.

[0023] In another preferred embodiment, the expression vector also contains a nucleotide sequence encoding the Fc fragment of an immunoglobulin.

[0024] In another preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

[0025] In a sixth aspect of the present application, there is provided a host cell, the host cell containing an expression vector according to the fifth aspect of the present application or wherein the genome of the host cell is integrated with a polynucleotide according to the fourth aspect of the present application.

[0026] In another preferred embodiment, the host cell comprises a prokaryotic or eukaryotic cell. In another preferred embodiment, the host cell is selected from the group of Escherichia coli, yeast cells, and mammalian cells.

[0027] In a seventh aspect of the application, there is provided a method of making an anti-PD-L1 single domain antibody, the method comprising the steps of: (a) culturing a host cell according to the sixth aspect of the application under conditions suitable for producing the single domain antibody, thereby obtaining a culture containing the anti-PD-L1 single domain antibody; and (b) isolating or recovering the anti-PD-L1 single domain antibody from the culture. In another preferred embodiment, the anti-PD-L1 single domain antibody has the amino acid sequence set forth in SEQ ID NO: 5n+4, 82, 85, 88, 91, 94, or 97.

[0028] n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0029] In an eighth aspect of the present application, there is provided a single domain antibody fusion protein, which has the structure shown in Formula I from N-terminus to C-terminus: Z1-Z2-L-Z3 (Formula I) During the ceremony, Z1 is a VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application; Z2 is an Fc fragment of an immunoglobulin; L is a linker sequence; Z3 is an immunomodulatory molecule moiety. In another preferred embodiment, the immunoglobulin is IgG1, IgG2, IgG3, or IgG4.

[0030] In another preferred embodiment, the amino acid sequence of Z2 is set forth in SEQ ID NO:99. In another preferred embodiment, the amino acid sequence of Z2 is the same as or substantially the same as the amino acid sequence set forth in SEQ ID NO:99.

[0031] In another preferred embodiment, L has an amino acid sequence selected from the group comprising GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0032] In another preferred embodiment, the amino acid sequence of L is set forth in SEQ ID NO:100. In another preferred embodiment, the amino acid sequence of L is the same as or substantially the same as the amino acid sequence set forth in SEQ ID NO:100.

[0033] In another preferred embodiment, the immunomodulatory molecule is the TGFβRII extracellular domain.

[0034] In another preferred embodiment, the amino acid sequence of Z3 is set forth in SEQ ID NO:101. In another preferred embodiment, the amino acid sequence of Z3 is the same as or substantially the same as the amino acid sequence set forth in SEQ ID NO:101.

[0035] In another preferred embodiment, being substantially the same means that at most 50 (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3) amino acids are different, and the differences include amino acid substitutions, deletions, or additions.

[0036] In another preferred embodiment, substantially the same indicates that the sequence identity between the amino acid sequence and the corresponding amino acid sequence is 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%.

[0037] In another preferred embodiment, the amino acid sequence of the single domain antibody fusion protein is set forth in SEQ ID NO:102.

[0038] In a ninth aspect of the present application, there is provided an immunoconjugate comprising: (a) a VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application, an anti-PD-L1 single domain antibody according to the third aspect of the application, or a single domain antibody fusion protein according to the eighth aspect of the application; and (b) a coupling moiety selected from the group comprising a detectable marker, a drug, a toxin, a cytokine, a radionuclide, or an enzyme; Includes.

[0039] In another preferred embodiment, the coupling moiety is a drug or toxin. In another preferred embodiment, the coupling moiety is a detectable marker.

[0040] In another preferred embodiment, the conjugate comprises a fluorescent or luminescent marker, The target substance is selected from a sex marker, an MRI (magnetic resonance imaging) or CT (electron-computed tomography) contrast agent, or an enzyme capable of producing a detectable product, a radionuclide, a biotoxin, a cytokine (e.g., IL-2 and the like), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a magnetic nanoparticle, a prodrug-activating enzyme (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), a chemotherapeutic agent (e.g., cisplatin), or any type of nanoparticle or the like.

[0041] In another preferred embodiment, the immunoconjugate comprises a multivalent (e.g. bivalent) VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application, an anti-PD-L1 single domain antibody according to the third aspect of the application, or a single domain antibody fusion protein according to the eighth aspect of the application.

[0042] In another preferred embodiment, multivalent indicates that the amino acid sequence of the immunoconjugate contains multiple repeated VHH chains of an anti-PD-L1 single domain antibody according to the second aspect of the present application, an anti-PD-L1 single domain antibody according to the third aspect of the present application, or a single domain antibody fusion protein according to the eighth aspect of the present application.

[0043] In a tenth aspect of the present application, there is provided the use of an anti-PD-L1 single domain antibody according to the third aspect of the present application or a single domain antibody fusion protein according to the eighth aspect of the present application for use in the preparation of (a) a reagent used for detecting PD-L1 molecules, and (b) a medicament used for treating tumours.

[0044] In another preferred embodiment, detection includes flow cytometry and cellular immunofluorescence detection.

[0045] In an eleventh aspect of the present application, there is provided a pharmaceutical composition comprising: (i) the CDRs of the VHH chain of an anti-PD-L1 single domain antibody according to the first aspect of the application, the VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application, an anti-PD-L1 single domain antibody according to the third aspect of the application, a single domain antibody fusion protein according to the eighth aspect of the application, or an immunoconjugate according to the ninth aspect of the application; and (ii) a pharmaceutically acceptable carrier Includes.

[0046] In another preferred embodiment, the pharmaceutical composition is in the form of an injectable solution. In another preferred embodiment, the pharmaceutical composition is used to manufacture a medicament for treating a tumor, wherein the tumor is selected from the group comprising 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, or a combination thereof.

[0047] In a twelfth aspect of the application, there is provided a use of one or more of the anti-PD-L1 single domain antibodies according to the third aspect of the application or the single domain antibody fusion proteins according to the eighth aspect of the application for: (i) for the detection of human PD-L1 molecules; (ii) for flow cytometry; (iii) for cell immunofluorescence detection; (iv) for tumor treatment; and (v) for tumor diagnosis; will be provided to. In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0048] In a thirteenth aspect of the present application, there is provided a recombinant protein, the recombinant protein comprising: (i) a sequence of a heavy chain variable region VHH according to the second aspect of the present application, a sequence of a single domain antibody according to the third aspect of the present application, or a single domain antibody fusion protein according to the eighth aspect of the present application; and (ii) an optional tag sequence to aid in expression and / or purification; Includes.

[0049] In another preferred embodiment, the tag sequence includes a 6His tag, an HA tag, a Flag tag, an Fc tag, HSA or an anti-HSA antibody or a single domain antibody, or a combination thereof.

[0050] In another preferred embodiment, the recombinant protein specifically binds to the PD-L1 protein.

[0051] In a fourteenth aspect of the present application, there is provided the use of a VHH chain according to the second aspect of the present application, a single domain antibody according to the third aspect of the present application, a single domain antibody fusion protein according to the eighth aspect of the present application, or an immunoconjugate according to the ninth aspect of the present application, for use in the manufacture of a medicament, a reagent, a detection plate, or a kit.

[0052] The reagent, detection plate, or kit is used to detect PD-L1 protein in a sample.

[0053] The medicament is used to treat or prevent tumors that express the PD-L1 protein (i.e., PD-L1 positive).

[0054] 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 gland tumor, or a combination thereof.

[0055] In a fifteenth aspect of the present application, there is provided a method for detecting PD-L1 protein in a sample, the method comprising the steps of: (1) contacting a sample with a single domain antibody according to the third aspect of the present application or a single domain antibody fusion protein according to the eighth aspect of the present application; and (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.

[0056] In another preferred embodiment, detection includes qualitative and quantitative detection.

[0057] In a sixteenth aspect of the present application, there is provided a method for treating a disease, the method comprising the step of administering to a subject in need thereof an effective amount of a single domain antibody according to the third aspect of the present application, a single domain antibody fusion protein according to the eighth aspect of the present application, or an immunoconjugate according to the ninth aspect of the present application.

[0058] In another preferred embodiment, the subject includes a mammal. In another preferred embodiment, the mammal is a human.

[0059] Within the scope of this application, the technical features of this application above and the technical features specifically described below It should be understood that the features (e.g., embodiments) may be combined with each other to form new or preferred technical solutions, and due to space limitations, more content will not be repeated here. [Brief explanation of the drawings]

[0060] [Figure 1] Figure 1 shows that the single domain antibodies of the present application can bind to human PD-L1 protein on the cell surface, and the binding effect of some of the antibodies is similar to that of the positive control. [Figure 2] Figure 10 shows that the modified single domain antibody can still bind to human PD-L1 protein on the cell surface, and the binding effect of the antibody is similar to that of the positive control. [Figure 3] Figure 10 shows that the engineered single domain antibody can further block the binding of PD-L1 protein to human PD-1 protein on the cell surface, and the blocking effect of the antibody is similar to that of the positive control. [Figure 4] FIG. 1 shows that the single domain antibodies of the present application can effectively activate T cells, and that the activation effect is similar to or better than that of a positive control antibody. [Figure 5] FIG. 1 shows a schematic structure of a fusion protein. [Figure 6] Figure 1 shows that the fusion proteins of the present application can bind to human PD-L1 protein on the cell surface. [Figure 7] Figure 1 shows that the fusion proteins of the present application can block the binding of PD-L1 protein to human PD-1 protein on the cell surface, and the blocking effect of the antibody is similar to that of the positive control. [Figure 8] FIG. 1 shows that the fusion proteins of the present application can bind to TGFβ1, TGFβ2, and TGFβ3. [Figure 9]FIG. 1 shows that the fusion protein of the present application can effectively block the TGFβ / SMAD signaling pathway. [Figure 10] FIG. 1 shows that the fusion protein of the present application can effectively activate T cells, and its activation effect is similar to or better than that of a positive control antibody. [Figure 11] FIG. 1 shows that the fusion protein of the present application can effectively inhibit tumor growth in mice. [Figure 12] Figure 1 shows a photograph of a PD-L1-VHH crystal sample of the present application. [Figure 13] Figure 1 shows the crystal structure of the PD-L1-VHH complex of the present application (the upper structure is PD-L1, and the lower structure is VHH). [Figure 14] Figure 1 shows the hydrogen-bonding interaction interface of PD-L1-VHH of the present application (the upper structure is PD-L1, and the lower structure is VHH). [Figure 15] Figure 1 shows the hydrophobic interaction interface of PD-L1-VHH of the present application (the upper structure is PD-L1, and the lower structure is VHH). [Figure 16] Figure 10 shows the effect of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. [Figure 17] Photographs of tumors in different drug groups 27 days after inoculation. [Figure 18] Figure 1 shows the effect of each drug group on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. [Figure 19] Figure 10 shows the effect of different drug groups on body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. [Figure 20] Figure 10 shows the effect of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. [Figure 21] Photographs of tumors in different drug groups 28 days after inoculation. [Figure 22] Figure 1 shows the effect of each drug group on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. [Figure 23] Figure 10 shows the effect of different drug groups on body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0061] Detailed Description After extensive and thorough research and extensive screening, the inventors have developed a class of anti-PD-L1 single domain antibodies. Experimental results show that the PD-L1 single domain antibodies and their mutant derivatives obtained in this application can effectively block the interaction between PD-L1 and PD-1, and have relatively good thermal stability.

[0062] Specifically, the present application used human-derived PD-L1 antigen protein to immunize llamas to obtain a high-quality immune library containing single-domain antibody gene sequences. The inventors screened the immune single-domain antibody gene library for single-domain antibodies with gene sequences exhibiting a relatively high level of humanization (sequence identity >85%). PD-L1 protein molecules were biotinylated, and the immune library containing the single-domain antibody sequences was screened using yeast display technology, thus obtaining genes for candidate single-domain antibodies specific to PD-L1. The obtained genes and their engineered mutants were then transfected into Expi-CHO cells and further screened in terms of antibody affinity, the ability to block PD-L1 binding to PD-1, thermostability, and T cell activation, to obtain a class and panel of single-domain antibodies that have high binding specificity to human PD-L1 antigen and can be efficiently expressed in vivo.

[0063] In addition, experimental results show that the fusion protein produced by fusing the single domain antibody of the present application (as the targeting moiety) with an IgG1 Fc fragment (as the linking moiety) and a TGFβRII extracellular domain (as the immunomodulatory molecule moiety) has high activity with PD-L1, can effectively block the interaction between PD-L1 and PD-1, effectively block the TGF-β / SMAD signaling pathway, effectively activate human T lymphocytes, and effectively inhibit tumor growth in mice.

[0064] In addition, experimental results show that the single domain antibodies of the present application can significantly inhibit the growth of subcutaneously implanted tumors in mice and reduce tumor weight, with a higher inhibitory effect on tumor growth at the same molar dose than similar molecules, anti-PD-L1 single domain antibodies, and TGF-βRII-Fc fusion proteins, and without obvious toxicity to animal models of the disease. On this basis, the present application is completed.

[0065] Single Domain Antibodies of the Present Application As used herein, the terms "single domain antibody of the present application," "anti-PD-L1 single domain antibody of the present application," and "PD-L1 single domain antibody of the present application" are used interchangeably and all refer to single domain antibodies that have the specificity to recognize and bind to PD-L1 (including human PD-L1). Particularly preferred are single domain antibodies having a VHH chain amino acid sequence set forth in SEQ ID NO: 4, 9, 82, 85, 88, 91, 94, or 97.

[0066] As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with the same structural characteristics, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain through a covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. 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; each light chain has a variable region (VL) at one end and a constant region at the other end; each light chain has a variable region (VL) at the other ... The constant region of each chain associates with the first constant region of the corresponding heavy chain, and the variable region of each light chain associates with the variable region of the corresponding heavy chain. Particular amino acid residues form an interface between the light-chain variable region and the heavy-chain variable region.

[0067] As used herein, the terms "single domain antibody (VHH)" and "nanobody" have the same meaning and refer to cloning the variable region of an antibody heavy chain to construct a single domain antibody (VHH) consisting of only one heavy chain variable region. Single domain antibodies are the smallest fully functional antigen-binding fragments. Typically, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is 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.

[0068] As used herein, the term "variable" means that certain portions of an antibody's variable region differ in sequence, resulting in a variety of specific antibody binding and specificity for a particular antigen. However, variability is not evenly distributed throughout the variable region of an antibody. Variability is concentrated in three segments in the light and heavy chain variable regions known as CDRs or hypervariable regions. The more conserved portions of the variable regions are called framework regions (FRs). Naturally occurring heavy and light chain variable regions each contain four FRs, which are roughly in a β-folded configuration and connected by three CDRs that form connecting loops, and in some cases may form a partial β-folded structure. The CDRs in each chain are tightly linked together by the FRs and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publication No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding an antibody to an antigen, but they exhibit various effector functions, such as those involved in antibody-dependent cellular cytotoxicity of the antibody.

[0069] As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates formed by attaching drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies, or fragments thereof, of the present application. The present application also includes cell surface markers or antigens attached to anti-PD-L1 single domain antibodies, or fragments thereof.

[0070] As used herein, the terms "heavy chain variable region" and "V H " are used interchangeably.

[0071] As used herein, the terms "variable region" and "complementarity determining region (CDR)" are used interchangeably.

[0072] In a preferred embodiment of the present application, the heavy chain variable region of the antibody comprises the CDRs: CDR1, CDR2, and CDR3.

[0073] In a preferred embodiment of the present application, the heavy chain of the antibody comprises the heavy chain variable region and heavy chain constant region described above.

[0074] As used herein, the terms "antibodies of the application," "proteins of the application," or "polypeptides of the application" 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, which may or may not contain an initial methionine.

[0075] The present application also provides other proteins or fusion expression products with the antibodies of the present application. Specifically, the present application includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, so long as the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the present application.

[0076] Generally, the antigen-binding properties of an antibody can be explained by three specific regions called variable regions (CDRs) located in the heavy chain variable region, separated by four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and are not directly involved in the binding reaction. These CDRs form a cyclic structure and are adjacent to each other in a spatial structure formed by a beta fold formed by the FRs between them. The CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR region.

[0077] The variable regions of the heavy chains of the antibodies of the present application are of particular interest because at least a portion of the variable region is involved in binding to the antigen. Thus, the present application includes molecules having the heavy chain variable regions of antibodies having CDRs, so long as the CDRs have 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified herein.

[0078] This application includes not only intact antibodies, but also immunologically active antibody fragments or fusion proteins formed from antibodies and other sequences. Thus, this application also includes antibody fragments, derivatives, and analogs.

[0079] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as an antibody of the present application. A polypeptide fragment, derivative, or analog of the present application may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, where such substituted amino acid residues may or may not be encoded by the genetic code; (ii) a polypeptide having a substitution within one or more amino acid residues; (iii) a polypeptide formed by fusing a mature polypeptide to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to a polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well within the skill of those in the art.

[0080] The term "antibody" as used herein refers to a polypeptide having PD-L1 protein-binding activity and the above-described CDRs. The term also encompasses variant forms of polypeptides containing the above-described CDRs and having the same functions as the antibodies of the present application. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more amino acids (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, and most preferably 1 to 10) and addition of one or more amino acids (usually 20 or less, preferably 10 or less, more preferably 5 or less) at the C-terminus and / or N-terminus. For example, when amino acids with the same or similar properties are substituted, the function of the protein generally does not change. As another example, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. The term also encompasses active fragments and active derivatives of the antibodies of the present application.

[0081] Variant forms of polypeptides include homologous sequences, conservative variants, allelic variants, natural variants, induced variants, DNA-encoded proteins, and polypeptides or proteins obtained by using antisera against the antibodies of the present application that can hybridize with the encoding DNA of the antibodies of the present application under high or low stringency conditions.

[0082] The present application also provides other polypeptides, such as fusion proteins, containing single domain antibodies or fragments thereof. In addition to nearly full-length polypeptides, the present application also includes fragments of the single domain antibodies of the present application. Generally, fragments contain at least about 50 conserved amino acids of the antibodies of the present application, preferably at least about 60 conserved amino acids, more preferably at least about 80 conserved amino acids, and most preferably at least about 100 conserved amino acids.

[0083] In the present application, a "conservative variant of an antibody of the present application" refers to a polypeptide formed by substituting at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids with amino acids having the same or similar properties compared to the amino acid sequence of an antibody of the present application. These conservative variant polypeptides are best produced through the substitution of amino acids according to Table 1-1.

[0084] [Table 1-1]

[0085] The present application also provides polynucleotide molecules encoding antibodies, or fragments thereof, or fusion proteins thereof. The polynucleotides of the present application may be in the form of DNA or RNA. DNA types include cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

[0086] Polynucleotides encoding mature polypeptides of the present application include coding sequences that only encode the mature polypeptide, coding sequences for the mature polypeptide (and optionally additional coding sequences) plus various additional coding sequences, and non-coding sequences.

[0087] The term "polynucleotide encoding a polypeptide" refers to a polynucleotide that encodes a polypeptide. The nucleic acid sequence may comprise a polynucleotide, or a polynucleotide that also contains additional coding and / or non-coding sequences.

[0088] The present application also relates to polynucleotides that hybridize to the above sequences, where the identity between the two sequences is at least 50%, preferably at least 70%, and more preferably at least 80%. The present application particularly relates to polynucleotides that can hybridize to the polynucleotides of the present application under stringent conditions. In the present application, "stringent conditions" refers to (1) hybridization and elution at relatively low ionic strength and relatively high temperature, for example, 0.2×SSC, 0.1% SDS, and 60°C; or (2) the addition of a denaturing agent during hybridization, for example, 50% (v / v) formamide, 0.1% bovine serum / 0.1% Ficoll, at 42°C or a similar temperature; or (3) hybridization that occurs only when the identity between the two sequences is at least 90%, more preferably 95%. Additionally, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0089] The full-length nucleotide sequence of the antibody of the present application or a fragment thereof can usually be obtained by PCR amplification, recombinant methods, or artificial synthesis. To synthesize a related sequence, especially when the fragment length is short, a feasible method is to use artificial synthesis. Usually, a fragment having a very long sequence can be obtained by first synthesizing multiple small fragments and then linking the fragments. In addition, the coding sequences of the heavy chain and expression tag (e.g., 6His) can be fused together to form a fusion protein.

[0090] Once the relevant sequence is obtained, recombinant methods can be used to obtain the relevant sequence in large quantities. The relevant sequence is usually cloned into a vector, then transferred into cells, and then isolated from the propagated host cells by conventional methods to obtain the relevant sequence. Biomolecules (nucleic acids, proteins, or the like) in the context of this application include biomolecules that exist in isolated form.

[0091] Currently, DNA sequences encoding the proteins of the present application (or fragments or derivatives thereof) can be obtained entirely through chemical synthesis. The DNA sequences can then be introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present application through chemical synthesis.

[0092] The present application also relates to vectors containing the appropriate DNA sequences and suitable promoter or regulatory sequences, which can be used to transform suitable host cells to express the proteins.

[0093] 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 bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COS7, and 293 cells, or the like.

[0094] Transformation of host cells with recombinant DNA can be carried out by conventional techniques well known to those skilled in the art. When the host cell is a prokaryote such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are known in the art. Another method is to use MgCl2. If necessary, transformation can also be performed by electroporation. When the host cell is a eukaryote, the following DNA transfection methods can be selected: calcium phosphate co-precipitation, microinjection, electroporation, conventional mechanical methods such as liposome packaging, or the like.

[0095] The resulting transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present application. Depending on the host cell used, the medium used for culture can be selected from a variety of conventional media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (e.g., temperature shift or chemical induction), and the cells are further cultured for a certain period of time.

[0096] The recombinant polypeptide in the above method can be expressed intracellularly or on the cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be separated and purified through various separation methods according to 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 protein precipitants (salting out), centrifugation, osmotic fungal disruption, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0097] The antibodies of the present application can be used alone, combined or coupled with a detectable marker (diagnostic), a therapeutic agent, a PK (protein kinase)-modifying moiety, or any combination of these substances.

[0098] Diagnostic detectable markers include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electron-computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0099] Therapeutic agents that can be combined or coupled with the antibodies of the present application include, but are not limited to, 1. radionuclides; 2. biotoxins; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorods; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)); 9. chemotherapeutic agents (e.g., cisplatin), or any type of nanoparticle or the like.

[0100] Fusion proteins of the present application As used herein, "the fusion protein of the application" refers to a bifunctional fusion protein comprising both an anti-PD-L1 single domain antibody moiety described in the first aspect of the application and an immunomodulatory molecule moiety.

[0101] In the present application, a fusion protein is provided, wherein the single domain antibody fusion protein has the structure as shown in Formula I from N-terminus to C-terminus: Z1-Z2-L-Z3 (Formula I) During the ceremony, Z1 is a VHH chain of an anti-PD-L1 single domain antibody according to the second aspect of the application; Z2 is an Fc fragment of an immunoglobulin; L is a linker sequence; Z3 is the immunomodulatory molecule portion.

[0102] Preferably, the immunoglobulin may be IgG1, IgG2, IgG3, or IgG4.

[0103] In a preferred embodiment, the immunoglobulin is IgG1 and the amino acid sequence of Z2 is set forth in SEQ ID NO: 99. In other embodiments, the amino acid sequence of Z2 is the same as or substantially the same as the amino acid sequence set forth in SEQ ID NO:99.

[0104] In the present application, L is a flexible amino acid linker. Preferably, L has an amino acid sequence selected from the group including GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0105] In a preferred embodiment, the amino acid sequence of L is set forth in SEQ ID NO: 100. In other embodiments, the amino acid sequence of L is the same as or substantially the same as the amino acid sequence set forth in SEQ ID NO:100.

[0106] In an embodiment of the present application, the immunomodulatory molecule is the TGFβRII extracellular domain. Preferably, the amino acid sequence of Z3 is set forth in SEQ ID NO: 101. In other embodiments, the amino acid sequence of Z3 is the same as or substantially the same as the amino acid sequence set forth in SEQ ID NO: 101.

[0107] In the present application, "substantially the same" means that at most 50 (preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, and most preferably 1 to 3) amino acids are different, and such differences include amino acid substitutions, deletions, or additions.

[0108] Preferably, substantially the same indicates that the sequence identity between the amino acid sequence and the corresponding amino acid sequence is 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%.

[0109] In a preferred embodiment, the amino acid sequence of the single domain antibody fusion protein is shown in SEQ ID NO:102.

[0110] TGFβ is a key inducer of epithelial-mesenchymal transition (EMT). At the same time, TGFβ exerts a strong immunosuppressive effect in the tumor microenvironment, thus exerting important regulatory effects on tumor development, metastasis, and drug resistance.

[0111] Therefore, in the embodiment of the present application, TGFβ receptor II is selected as the immunomodulatory molecule in the fusion protein. The fusion protein of the present application has the advantages of high dual target binding affinity and specificity, thereby further enhancing anti-tumor immune function.

[0112] Pharmaceutical Compositions The present application also provides a composition. Preferably, the composition is a pharmaceutical composition containing an antibody, or an active fragment thereof, or a fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier medium, the pH of which is usually about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the substance to be formulated and the disease state to be treated. The formulated pharmaceutical compositions can be administered by conventional routes, including, but not limited to, intratumoral, intraperitoneal, intravenous, or topical administration.

[0113] The pharmaceutical compositions of the present application can be used directly to bind PD-L1 protein molecules and thus can be used to treat tumors. In addition, other therapeutic agents can also be used simultaneously.

[0114] The pharmaceutical compositions of the present application contain a safe and effective amount (e.g., 0.001 to 99% by weight, preferably 0.01 to 90% by weight, more preferably 0.1 to 80% by weight) of the single domain antibody (or conjugate thereof) of the present application 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 be compatible with the mode of administration. The pharmaceutical compositions of the present application can be prepared into injection forms. For example, the pharmaceutical compositions are prepared by conventional methods using saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg / kg body weight to about 50 mg / kg body weight per day. In addition, the polypeptides of the present application can also be used in combination with other therapeutic agents.

[0115] When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to a mammal, and this safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases not higher than about 50 mg / kg body weight. Preferably, the dosage is about 10 μg / kg body weight to about 10 mg / kg body weight. Of course, factors such as the route of administration and the patient's health condition should also be taken into consideration for the specific dosage, and these are within the ability of a skilled physician.

[0116] Labeled single domain antibodies In a preferred embodiment of the present application, the single domain antibody contains a detectable marker. More preferably, the marker is selected from the following group: an isotope, a colloidal gold marker, a colored marker, or a fluorescent marker.

[0117] Colloidal gold labeling can be performed by methods known to those skilled in the art. In a preferred solution of the present application, an anti-PD-L1 single domain antibody is labeled with colloidal gold to obtain a colloidal gold-labeled single domain antibody. The anti-PD-L1 single domain antibodies of the present application have high specificity and potency.

[0118] Detection Method The present application also relates to a method for detecting PD-L1 protein, the steps of which generally include obtaining a cell and / or tissue sample, lysing the sample in a medium, and detecting the level of PD-L1 protein in the lysed sample.

[0119] In the detection method of the present application, the sample used is not particularly limited, and a typical example is a sample containing cells in a cell preservation solution.

[0120] kit The present application also provides a kit or detection plate containing the antibody (or fragment thereof) of the present application. In a preferred embodiment of the present application, the kit further comprises a container, instructions for use, a buffer, or the like.

[0121] The present application also provides a detection kit for detecting PD-L1 levels. The kit includes an antibody for identifying PD-L1 protein, a dissolution medium for dissolving the sample, and common reagents and buffers required for detection, such as various buffers, detection markers, detection substrates, or the like. The detection kit can be an in vitro diagnostic device.

[0122] use As described above, the single domain antibodies of the present application have high biological and clinical value, and the uses of the single domain antibodies relate to the fields of diagnosis and treatment of PD-L1-associated diseases, basic medical research, biological research, or the like. The main advantages of this application include: 1) The single domain antibodies of the present application are highly specific to the human PD-L1 protein with the correct spatial structure. 2) The single domain antibodies of the present application have high affinity. 3) The single domain antibodies of the present application are easy to make. 4) Single domain antibodies can target and inhibit the PD-1 / PD-L1 pathway based on TGF-β in the tumor microenvironment, restore T cell activity, enhance immune responses, and more effectively inhibit tumor emergence and development. 5) The single domain antibodies of the present application have no apparent toxicity. [Example]

[0123] This application is further described below with examples. It should be understood that these examples are only used to illustrate this application and are not used to limit the scope of this application. The experimental methods without specific conditions in the following examples are usually carried out according to conventional conditions, such as those in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or those recommended by manufacturers. Unless otherwise specified, percentages and parts are weight percentages and weight parts.

[0124] Example 1: Construction of a single domain antibody library animal immunity To stimulate B cells to express antigen-specific single-domain antibodies, 1 mg of human PD-L1 antigen and Freund's adjuvant were mixed in equal volumes to immunize two llamas once a week for a total of four immunizations. After four immunizations, 50 ml of llama peripheral blood was extracted and separated with lymphocyte separation solution to obtain lymphocytes. Total RNA was extracted using the RNA extraction reagent Trizol (purchased from Invitrogen). Total llama cDNA was obtained using a cDNA synthesis kit (purchased from Invitrogen) for reverse transcription.

[0125] Single domain antibody gene amplification In the first round of PCR, IgG2 and IgG3 sequences were amplified from cDNA.

[0126] [Table 1-2]

[0127] The product of the first round of PCR was subjected to agarose gel electrophoresis, and after gel cutting, a fragment at 750 bp was recovered and used for the second round of VHH sequence amplification. The primers for the second round of PCR amplification were as follows:

[0128] [Table 2]

[0129] The product of the second round of PCR was used as a template for the third round of PCR to add homologous arms to the VHH gene, and the primers for the third round of PCR were as follows:

[0130] [Table 3]

[0131] The target fragment was recovered by using a PCR purification kit (purchased from QIAGEN).

[0132] Library Construction The linearized yeast display vector and the product of the third round of PCR were mixed and electrotransformed into Saccharomyces cerevisiae (ATCC® 20828). A two-animal anti-PD-L1 single domain antibody library was constructed, with a library size of 4.47 × 1. 0 7 and 4.14 × 10 7 It was measured to be.

[0133] Example 2: Screening of PD-L1 single domain antibodies Biotinylation of human PD-L1 protein Human PD-L1 protein was dissolved in an appropriate volume of double-distilled water, and biotin was dissolved according to the biotin labeling kit (purchased from Thermo) product instructions, mixed with the protein solution, and then incubated for 2 hours at 4°C. Excess biotin was removed using a desalting column (purchased from Thermo), and both the desalting column pretreatment and sample collection procedures were performed according to the steps in the product instructions.

[0134] MACS enrichment of yeast specifically binding to PD-L1 The VHH library constructed in Example 2 was inoculated into SD-CAA amplification medium (1 L of SD-CAA amplification medium containing 6.7 g of NB, 5 g of casamino acids, 13.62 g of NaHPO 12H O, 7.44 g of NaHPO, and 2% glucose), and the number of inoculated yeast cells was more than 10 times the library volume (the initial amplification concentration was 0.5 OD 600 The yeast cells were cultured overnight at 30°C and 225 rpm. Ten times the library volume was taken and centrifuged at 3000 rpm for 5 minutes (the following centrifugation procedures were the same), the medium was removed, and the yeast cells were resuspended in SD-CAA induction medium to an initial concentration of 0.5 OD 600 The concentration of the library was adjusted to 1 / ml and induction was carried out overnight. The concentration of the library after induction was measured, and 10 times the library volume of yeast cells was taken and centrifuged to remove the medium. The yeast cells were resuspended in 50 ml of wash buffer (PBS + 0.5% BSA + 2 mM EDTA), centrifuged, and the supernatant was removed. The yeast cells were resuspended in 10 ml of wash buffer.

[0135] Biotin-labeled PD-L1 protein (final concentration 100mM) was added, incubated at room temperature for 30 minutes, and centrifuged to collect the yeast cells, which were then washed three times with 50mL of wash buffer. The yeast cells were resuspended in 5mL of wash buffer, and 200μL of SA magnetic beads (purchased from Miltenyi) were added. The cells were incubated upside down for 10 minutes. The yeast and magnetic bead mixture was washed three times with wash buffer and then applied to an LS column (purchased from Miltenyi). The LS column was placed on a magnetic stand and washed with wash buffer to remove non-specifically bound yeast cells. The column was removed from the magnetic stand, and wash buffer was added to elute the yeast. The eluted yeast was centrifuged and transferred to 200mL SD-CAA amplification medium for amplification.

[0136] Fluorescence-activated cell sorting to obtain high-affinity yeast cells The yeast cells enriched by MACS were inoculated into SD-CAA amplification medium, and the initial amplification concentration was 0.5 OD 600 The yeast cells were resuspended in SD-CAA induction medium (1 L of SD-CAA induction medium contains 6.7 g of YNB, 5 g of casamino acids, 13.62 g of NaHPO 12H O, 7.44 g of NaHPO, 2% galactose, 2% raffinose, and 0.1% glucose) to an initial concentration of 0.5 OD 600 The yeast cells were incubated overnight at 4°C for 15 minutes in the dark with 2 ml of PBS. A 1:200 dilution of anti-c-Myc mouse antibody (purchased from Thermo) and 100 nM biotin-labeled PD-L1 antigen were added and incubated at room temperature for 10 minutes. The yeast cells were washed three times with PBS, and a 1:500 dilution of goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (purchased from Invitrogen) and streptavidin-APC-conjugated fluorescent antibody (purchased from Invitrogen) were added and incubated at 4°C for 15 minutes in the dark. Two ml of PBS was added to resuspend the cells, and yeast cells with high binding affinity for PD-L1 antigen were sorted using a BD FACSAria III instrument.

[0137] Obtaining the gene sequence of PD-L1 single domain antibody candidates Yeast liquid with high binding ability to PD-L1 antigen obtained by MACS and FACS enrichment was cultured overnight at 30°C and 225 rpm in SD-CAA amplification medium. Yeast plasmids were extracted according to the instructions in the yeast plasmid extraction kit (purchased from TIANGEN). The plasmids were transformed by electrotransfection into Top10 competent cells (purchased from TIANGEN) coated on ampicillin-resistant flat plates and cultured overnight at 37°C. Single clones were picked for sequencing to obtain the VHH gene sequences.

[0138] Example 3: Construction, expression, and purification of heavy chain antibodies Construction of antibody genes into pCDNA3.1 expression vector The VHH gene sequence was ligated with a human IgG1 (LALA mutant) Fc fragment and introduced into the linearized pCDNA3.1 vector using homologous recombinase (purchased from Vazyme) and EcoR I / Not I enzymes according to the manufacturer's instructions. The homologous recombination product was transformed into Top10 competent cells coated on ampicillin-resistant flat plates and cultured overnight at 37°C. Single clones were then picked for sequencing.

[0139] Cell transfection The ExpiCHO™ Expression System Kit (purchased from Thermo) was used to transfect the plasmids into Expi-CHO cells, and the transfection method followed the manufacturer's instructions. After culturing the cells for 5 days, the supernatant was collected, and Protein A magnetic beads (purchased from GenScript) were used to purify the target protein by a sorting method. The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1 to 4 times the volume of the magnetic beads) and then added to the sample to be purified. The mixture was incubated at room temperature for 1 hour, with gentle shaking during that time. The sample was placed on a magnetic stand (purchased from Beaver), the supernatant was removed, and the magnetic beads were washed three times with binding buffer. Elution buffer (0.1 M sodium citrate, pH 3.2) was added in an amount 3–5 times the volume of magnetic beads for shaking at room temperature for 5–10 min, and the mixture was placed back on the magnetic stand to collect the elution buffer, which was then transferred to a collection tube where neutralization buffer (1 M Tris, pH 8.54) was added and mixed evenly.

[0140] Example 4: Binding of purified anti-PD-L1 single domain antibodies to human PD-L1 Human PD-L1 cDNA (purchased from Sino Biological) was cloned into the pCHO1.0 vector (purchased from Invitrogen) by transfection into MCS to generate CHO cells overexpressing human PD-L1 (CHO-hPD-L1 cells). The cell density of the expanded CHO-hPD-L1 cells was 2 x 10 6The concentration was adjusted to cells / ml, and 100μl of cells was added to each well of a 96-well flow plate and centrifuged for later use. The purified PD-L1 antibody was diluted in PBS, and 3-fold dilutions were made starting at a concentration of 400nM, for a total of 12 points. 100μl of the diluted sample was added to each well of the 96-well flow plate containing the cells, incubated at 4℃ for 30 minutes, and washed twice with PBS. 100μl of goat F(ab')2 anti-human IgG-Fc(PE) (purchased from Abcam) diluted 100-fold in PBS was added to each well, incubated at 4℃ for 30 minutes, and washed twice with PBS. 100μl of PBS was added to each well to resuspend the cells, and detection was performed using a CytoFlex (Becman) flow cytometer, and the corresponding MFI was calculated.

[0141] In the assay experiments using the above method, the experimental results are shown in Figure 1. All purified samples and CHO-hPD-L1 cells in this application had binding activity, and some purified The binding activity of the sample was similar to that of the control antibody TECENTRIQ® (atezolizumab) (ATE; documented in US20130034559 and also known as 243.55.S70).

[0142] Example 5: PD-L1 antibody affinity measurement ForteBio affinity measurements were performed according to existing methods (Estep, P. et al., Solution-based measurement of high-throughput antibody-antigen affinity and epitope classification, MAbs, 2013, Vol. 5(2):270-8). Briefly, the sensor was equilibrated offline in analysis buffer for 30 minutes and then tested online for 60 seconds 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 minutes for reaction, and then transferred to PBS for 5 minutes for dissociation. A 1:1 combination model was used for kinetic analysis.

[0143] [Table 4]

[0144] Example 6: Genetic modification of PD-L1 antibodies To remove potential glycosylation sites in C-Ye-18, the CDRH2 portion of the amino acid sequence of C-Ye-18 was point mutated into the six types in Table 5.

[0145] [Table 5]

[0146] In this study, IMGT (http: / / www.imgt.org) was used to evaluate the humanization levels of the mutant sequences of the C-Ye-18 CDR region, and the results are shown in Table 6. The humanization levels of all C-Ye-18 mutants were higher than 87%, meeting the requirements for subsequent drug development.

[0147] [Table 6]

[0148] The protein construction and expression purification methods were the same as those in Example 3, and the purity of the resulting protein was detected by HPLC. According to the HPLC method, the mobile phase was 150 mM NaHPO·12H O, pH 7.0. Chromatography conditions were: detection wavelength 280 nm, column temperature 25°C, flow rate 0.35 ml / min, detection time 20 min, and Zenix-C SEC-300 chromatography column (SEPAX 4.6 × 300 mm, 3 μm).

[0149] [Table 7]

[0150] Example 7: Binding of C-Ye-18 mutant samples to human PD-L1 This experiment detected the binding activity of purified C-Ye-18 mutant samples to CHO-hPD-L1 cells. The experimental method was the same as that in Example 4. The experimental results are shown in Figure 2. The C-Ye-18 mutant samples and CHO-hPD-L1 cells had good binding activity, which was comparable to that of C-Ye-18 and the control antibody ATE.

[0151] Example 8: Affinity measurements of C-Ye-18 mutant samples This experiment detected the binding affinity of purified C-Ye-18 mutant samples with human PD-L1. The experimental method was the same as that in Example 5. The experimental results are shown in Table 8. The C-Ye-18 mutant samples have very good binding activity with human PD-L1 protein.

[0152] [Table 8]

[0153] Example 9: Blocking of PD-L1 binding to PD-1 by C-Ye-18 mutant samples Human PD-L1 cDNA (purchased from Sino Biological) was cloned into the pCHO1.0 vector (purchased from Invitrogen) by transfection into MCS to generate CHO cells overexpressing human PD-L1 (CHO-hPD-1 cells). The cell density of the expanded CHO-hPD-1 cells was 2 × 10 6The cells were adjusted to 100 cells / ml, and 100μl of the cells were added to each well of a 96-well flow plate and centrifuged for later use. The purified mutant samples were diluted with PBS, and a 3-fold dilution was performed starting at a concentration of 400nM, for a total of 12 points. 60μl of the diluted sample was added to each well of a 96-well sample dilution plate, and 60μl of biotinylated human PD-L1 protein (purchased from AcroBiosystems) was simultaneously added to each well for a final concentration of 500ng / ml. The mutant samples were incubated at 4℃ for 30 minutes. 100μl of the co-incubation sample was added to each well of the 96-well flow plate containing the cells, incubated at 4℃ for 30 minutes, and washed twice with PBS. 100 μl of streptavidin R-phycoerythrin conjugate (purchased from Thermo Fisher Scientific), diluted 100-fold in PBS, was added to each well, incubated at 4°C for 30 minutes, and washed twice with PBS. 100 μl of PBS was added to each well to resuspend the cells, and detection was performed on a CytoFlex (Beckman) flow cytometer, and the corresponding MFI was calculated.

[0154] In the measurement experiments using the above method, the experimental results are shown in Figure 3. All of the mutant samples in this application were able to block the binding of PD-L1 to PD-1, with blocking levels comparable to those of C-Ye-18 and the control antibody ATE.

[0155] Example 10: Thermal stability of C-Ye-18 mutant samples The thermal stability of various antibodies was detected using DSC (differential scanning calorimetry). The samples were concentrated and then diluted to 1 mg / ml with PBS. 5000x fluorochrome Cypro Orange (purchased from Bio-Rad) was diluted 50 times with ultrapure water to obtain 100x fluorochrome Sypro Orange. 50 μl of each 1 mg / ml sample was taken, 10 μl of 100x fluorochrome Sypro Orange and 40 μl of ultrapure water were added, mixed uniformly, and 30 μl of each was added to a 96-well PCR plate. Each sample was added to three replicate wells and placed in a PCR machine. The temperature ramp program was set as follows: a constant temperature of 25°C was maintained for 5 minutes, and the temperature was increased to 99°C at a rate of 0.5°C / min. After the program was completed, the temperature value at the lowest point of the "melting curve" graph, i.e., the Tm value of the sample, was read. Specific results are shown in Table 9 below.

[0156] [Table 9]

[0157] Example 11: Mixed lymphocyte reaction experiments In this example, a mixed lymphocyte reaction (MLR) experiment was used to identify the C-Ye-18 mutant. The activity of the sample was detected to activate T cells. The specific experimental method was as follows.

[0158] PBMC cells (purchased from SAILY BIO, SLB-HPB) were resuscitated, centrifuged, resuspended in 10 ml of X-VIVO-15 medium (purchased from LONZA), and subjected to adherent culture in a cell incubator at 37°C for 2 hours to remove non-adherent cells. Add 10 ml of DC medium, add 10 ng / ml GM-CSF (purchased from R&D) and 20 ng / ml IL-4 to X-VIVO-15 medium for 3 days of culture, add 5 ml of DC medium, and culture the cells continuously for 6 days. Add DC maturation medium, add 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), and 1 μM PGE2 (purchased from Tocris) to X-VIVO-15 medium, culture the cells for 2 days, collect mature DC cells, and increase the cell density to 2 × 10 in X-VIVO-15 medium. 5 Adjusted to cells / ml.

[0159] PBMC cells from another donor (purchased from Saily Bio, SLB-HPB) were thawed, centrifuged, and resuspended in 10 ml of X-VIVO-15 medium. + T cells, CD4 + The cells were enriched using a T cell sorting kit (purchased from Stemcell) and resuspended in X-VIVO-15 to a cell density of 2 × 10 6 Adjust to cells / ml and CD4 + T cells were mixed with the collected mature DC cells at a 1:1 ratio, and 100 μl of the mixture was added to each well of a 96-well U-bottom plate.

[0160] The C-Ye-18 mutant samples were diluted in X-VIVO-15 medium, and diluted 3-fold for a total of 9 points at a concentration of 200 nM. 100 μl of the mixed cells was added to each well and cultured for 5 days. The supernatant was collected and the expression levels of IFN-γ and IL2 were detected using ELISA (purchased from eBioscience).

[0161] The results are shown in Figure 4, and show that the C-Ye-18 mutant samples, C-Ye-18-1, C-Ye-18-5, and C-Ye-18-6, all exhibited relatively good biological activity in the MLR experiments, with activation levels similar to or superior to those of the control antibody ATE.

[0162] Example 12: Cloning and expression of the fusion protein PD-L1 / TGFβRII In this example, the TGFβRII extracellular domain (SEQ ID NO: 101) was used as the immunomodulatory molecule portion of the fusion protein, and the PD-L1 antibody (human IgG1 Fc, LALA mutation) (C-Ye-18-5, SEQ ID NO: 94) was used as the targeting portion of the fusion protein to form a PD-L1 antibody / TGFβRII extracellular domain fusion protein (PM8001, SEQ ID NO: 102).

[0163] Using molecular cloning technology, the C-terminal amino acids of the PD-L1 single-chain antibody of the present application were linked to the extracellular domain of TGFβRII via (G4S)4G and routinely expressed in the Expi-CHO expression system. The expression and purification methods were the same as those in Example 3, and the fusion protein PM8001, with the structure shown in Figure 5, was obtained.

[0164] Example 13: Binding of PM8001 molecules to human PD-L1 The methods used to detect the binding activity of purified PD-L1 antibody (C-Ye-18-5, SEQ ID NO: 94), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824 (WO2015 / 118175 A2), and negative control IgG protein to PD-L1 on the cell surface were the same as those used in Example 4. In the measurement experiments using the above methods, the experimental results are shown in Figure 6, which show that the PM8001 molecule of the present application and CHO-hPD-L1 cells have binding activity, which is similar to that of the positive control molecule M7824. was doing.

[0165] Example 14: Blocking the binding of PD-L1 protein to PD-1 cells by PM8001 molecules The methods used to detect blocking of the binding activity of PD-L1 protein to PD-1 cells by purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824, and negative control IgG protein were the same as those used in Example 9. In the measurement experiments using the above methods, the experimental results are shown in Figure 7, which show that the PM8001 molecule of the present application was able to block the binding of PD-L1 protein to PD-1 cells, and the blocking level was comparable to that of the positive control molecule M7824.

[0166] Example 15: ELISA-level binding experiment of PM8001 molecule with human TGF-β family proteins Human TGF-β1 (acrobiosystems, TG1-H421), TGF-β2 (PeproTech, 100-35B), and TGF-β3 (PeproTech, 100-36E) proteins were diluted in ELISA coating solution and then added to the ELISA plate for overnight coating at 4°C. To remove the coating solution and wash three times, 250μl of PBST was added to each well, and the ELISA plate was blocked with 5% BSA at room temperature for 1 hour for later use. Purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, and positive control M7824 were subjected to gradient dilution and then added to the blocked ELISA plate for 2 hours of incubation at room temperature. PBST was added for three washes, and goat anti-human Fc-HRP (abcam, ab97225) was added to each well for 1 hour incubation at room temperature. After three washes and PBST, ELISA development solution was added and incubated at room temperature for 3 minutes. ELISA stop solution was added and the absorbance at 450 nm was read.

[0167] In the measurement experiment using the above method, the experimental results are shown in Figure 8, which shows that the PM8001 molecule of the present application has relatively good binding activity to human TGF-β1 and TGF-β3 proteins and relatively weak binding activity to human TGF-β2 protein at the ELISA level, and its binding level is comparable to that of the positive control molecule M7824.

[0168] Example 16: Experiments with PM8001 molecule in blocking TGF-β / SMAD signal pathway An appropriate amount of 293-TGF-β / SMAD effector cells was harvested and seeded onto a 96-well white-bottom cell culture plate and placed in a 5% CO2 incubator for overnight culture at 37°C. Purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, and positive control M7824 were subjected to gradient dilution and mixed with TGF-β1 (Acro Biosystems, TG1-H421) and incubated at room temperature for 30 minutes. The above mixture was added to the white-bottom plate containing the cells for overnight continuous culture. Bio-Glo™ reagent (Promega) was added to each well, and the fluorescent signal was read using a multifunction microplate reader.

[0169] In the measurement experiment using the above method, the experimental results are shown in Figure 9, which show that the PM8001 molecule of the present application can block the TGF-β / SMAD signal pathway in vitro, and its blocking level is comparable to that of the positive control molecule M7824.

[0170] Example 17: Mixed lymphocyte reaction experiments In the activation of human T lymphocytes, purified PD-L1 antibody (C-Ye-18-5), PM The method of using mixed lymphocytes to detect the PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824, and negative control IgG protein was the same as that in Example 9. The results are shown in Figure 10, which shows that the PM8001 molecule of the present application exhibited relatively good biological activity in the MLR experiment, and its activation level was comparable to or superior to that of the positive control molecule M7824.

[0171] Example 18: Pharmacokinetic evaluation of PM8001 in mice Six SD mice, half male and half female, were used in the experiment. Light and darkness were regulated every 12 hours, the temperature was 24±2°C, and the humidity was 40-70%. The mice had free access to water and food. Mice were purchased from Zhejiang Weitong Lihua Experimental Technology Co., Ltd. On the day of the experiment, PM8001 molecules were injected once into the tail vein of the SD mice at a dose of 10 mg / kg.

[0172] Blood was collected from the jugular vein of each mouse at 3 minutes, 4 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours, and 672 hours after administration. The whole blood samples were placed at 2-8°C for 30 minutes and centrifuged at 12,000 rpm for 5 minutes. The serum was collected, centrifuged at 12,000 rpm at 2-8°C for 5 minutes, and stored at -80°C. The molecular weight of free PM8001 in the serum was detected by ELISA. The results are shown in Table 10. The free form of PM8001 in the present application has a half-life of approximately 146 hours in SD mice.

[0173] [Table 10]

[0174] Example 19: Study on the tumor suppressor activity of PM8001 In this study, to determine the antitumor effect of PM8001 in PD-L1 transgenic mice, MC38 cells expressing human PD-L1 (h-PD-L1 KI) were cultured. First, a mouse model bearing h-PD-L1 KI MC38 tumors was established by subcutaneous inoculation. The mean tumor volume was 80-120 mm. 3 At this time, the mice were classified and given different antibodies and different doses of treatment in a single intraperitoneal injection. The tumor volume and weight changes of the mice in each group were monitored twice a week for a total of 3 weeks. The doses and modes of administration are shown in Table 11, and the tumor volume changes of the mice are shown in Figure 11.

[0175] [Table 11]

[0176] The experimental results are shown in Figure 11. After inoculation with h-PD-L1 KI MC38, the tumor volume in the negative control group increased continuously, and tumor growth in the TGF-β R II-Fc and C-Ye-18-5 single-use group was inhibited, whereas the PM8001 group produced better control of tumor growth than the TGF-β R II-Fc and C-Ye-18-5 group, indicating that PM8001 had significant tumor-inhibitory efficacy, which was comparable to or even slightly superior to that of the positive control group.

[0177] Example 20: Identification of the crystal structure of the complex between PD-L1 and a single domain antibody VHH fragment In this experiment, X-ray diffraction was used to identify the crystal structure of the complex between PD-L1 and a single domain antibody VHH fragment. Human PD-L1-His tag (SEQ ID NO: 109) was expressed in the Escherichia coli prokaryotic system. PD-L1 single domain antibody fusion protein (SEQ ID NO: 110) was expressed in the CHO system. The PD-L1 single domain antibody fusion protein was digested and purified with IdeS enzyme, followed by GingisKHAN enzyme, to finally obtain the PD-L1 single domain antibody VHH (SEQ ID NO: 111). To prepare the complex sample for crystallization, PD-L1 and VHH were mixed at a 1:1 molar ratio. The purified complex was digested with carboxypeptidase B to remove the His tag of PD-L1. The complex (7.5 mg / mL) was mixed with crystallization reagent at a 1:1 ratio and subjected to crystallization incubation at 18°C. After 3 days, crystals were observed under INT kit culture conditions, and the crystal morphology is shown in FIG.

[0178] A single crystal was selected for X-ray diffraction experiments at Shanghai Light Source, and diffraction data at 1.6 Å resolution were obtained. XDS software was used for data processing. Molecular replacement methods were employed for crystalline phase identification using the PD-L1 (PDB ID: 5jds) and VHH (PDB ID: 5m2j) structures as models, respectively. Refmac5 was used to refine the crystal structure. COOT was used for model detection, manual reconstruction, and structure validation. The complex crystal belongs to the P21 space group, with the following crystal cell parameters: a = 34.62 Å, b = 97.99 Å, c = 67.52 Å, α = 90.00°, β = 90.02°, and γ = 90.00°.

[0179] The crystal structure of the PD-L1-VHH complex obtained after structural analysis is shown in Figure 13. Epitope analysis indicates that the major hydrogen-bonding interactions between PD-L1 and VHH are concentrated at Tyr56, Asn63, His69, Asp73, Lys75, Ser117, Gly119, Ala121, and other amino acids on PD-L1 ( Figure 1 4) In addition, Tyr56, His69, and Tyr123 on PD-L1 and Tyr32, Trp33, Tyr35, Leu45, Trp47, Pro100, and Tyr103 on VHH constitute a hydrophobic interaction interface (Figure 15).

[0180] Example 21: Growth inhibitory effect of PM8001 injection on the h-PD-L1 knock-in MC38 model inoculated subcutaneously in h-PD-L1 knock-in C57BL / 6 mice This study investigated the in vivo antitumor effects of PM8001 (SEQ ID NO: 102) administered by intraperitoneal injection in a h-PD-L1 knock-in MC38 mouse colon cancer tumor-bearing model inoculated subcutaneously into h-PD-L1 knock-in C57BL / 6 mice, and its safety in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0181] In this study, a h-PD-L1 knock-in MC38 subcutaneous tumor model was established by subcutaneously inoculating h-PD-L1 knock-in MC38 mouse colon cancer cells into h-PD-L1 knock-in C57BL / 6 mice (purchased from GemPharmatech Co., Ltd.). Ten days after inoculation, the mice were divided into five groups (six mice per group) according to tumor volume and received two intraperitoneal injections of PBS, 14.7 mg / kg PM8001, 10 mg / kg PM8001-NSD (anti-human PD-L1 VHH), 10 mg / kg PM8001-TGF-βRII, and 24.6 mg / kg M7824 (the corresponding molar doses of the above treatment groups were the same). The treatments were administered twice, with an interval of three days. The antitumor effects and safety of PM8001, PM8001-NSD, PM8001-TGF-βRII, and M7824 were investigated in tumor-bearing mice.

[0182] Figure 16 shows the effect of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. Figure 17 shows photographs of tumors in the different drug groups 27 days after inoculation. Tumors in mice in the negative control PBS group grew rapidly, with tumor volumes reaching 1500 mm 27 days after inoculation (i.e., day 17 after administration). 3 The tumor growth inhibition rate reached 80% or more, indicating that the tumor model in this experiment was successfully established. Compared with the negative control PBS group, the same molar doses of PM8001, PM8001-NSD, PM8001-TGF-βRII, and M7824 were able to inhibit tumor growth to different degrees, with the TGI of the above groups on day 17 after administration being 80%, 72%, 15%, and 53%, respectively. The PM8001-injected group produced a higher tumor growth inhibition effect than PM8001-NSD, PM8001-TGF-βRII, and the similar molecule group M7824. At the end of the experiment, tumors were harvested and weighed. The tumor weight in the PM8001-injected group was 80%, 72%, 15%, and 53%, respectively. R II and that of the similar molecule M7824. Figure 18 shows the effect of each drug group on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0183] Figure 19 shows the effect of different drug groups on the body weight of h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. There were no abnormalities in the condition of the mice in each group; compared with the control group, the body weight of the mice in each treatment group was not significantly reduced; at the end of the experiment, gross anatomy of the mice in each group showed no obvious lesions in the major organs, indicating that the doses of the drugs used in this experiment did not cause obvious toxicity to the mice.

[0184] Example 22: Growth inhibitory effects of different doses of PM8001 on the h-PD-L1 knock-in MC38 model subcutaneously inoculated into h-PD-L1 knock-in C57BL / 6 mice In this study, three different doses of PM8001 were injected subcutaneously into h-PD-L1 knock-in C57BL / 6 mice (purchased from GemPharmatech Co, Ltd). We investigated the in vivo antitumor effect of h-PD-L1 knock-in MC38 mouse colon cancer tumor model inoculated with PD-L1, and safety in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0185] In this study, a h-PD-L1 knock-in MC38 subcutaneous tumor model was established by subcutaneously inoculating h-PD-L1 knock-in MC38 mouse colon carcinoma into h-PD-L1 knock-in C57BL / 6 mice. This model can be used to evaluate the mechanism-of-action-related antitumor efficacy of test products and their safety profile in disease states. Seven days after inoculation, mice were divided into five groups (six mice per group) according to tumor volume and administered a single intraperitoneal injection of PBS, different doses of PM8001 injection (0.3 mg / kg, 2.1 mg / kg, 14.7 mg / kg), or 24.6 mg / kg M7824. The antitumor efficacy and safety of different doses of PM8001 in tumor-bearing mice were investigated and compared with those of the analogous molecule M7824.

[0186] Figure 20 shows the effect of different drug groups on tumor volume in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors. Figure 21 shows tumor photographs for the different drug groups 28 days after inoculation. Tumors in mice in the negative control PBS group grew rapidly, with tumor volumes reaching 1300 mm 28 days after inoculation. 3The tumor growth inhibition rate reached or exceeded the threshold of 31%, indicating that the tumor model in this experiment was successfully established. Compared with the negative control PBS group, PM8001 inhibited tumor growth in a dose-dependent manner. The TGI values ​​at low, medium, and high doses of PM8001 were 31%, 76%, and 93%, respectively. At the same molar dose, the tumor growth inhibition effect of the PM8001 group (14.6 mg / kg) was higher than that of the analogous molecule M7824 group (24.6 mg / kg). At the end of the experiment, tumors were harvested and weighed. PM8001 reduced tumor weight in a dose-dependent manner. At the same molar dose, the tumor inhibition rate of the PM8001 group (14.6 mg / kg) was higher than that of the analogous molecule M7824 group (24.6 mg / kg). Figure 22 shows the effect of each drug group on tumor weight in h-PD-L1 knock-in mice implanted with h-PD-L1 knock-in MC38 tumors.

[0187] Figure 23 shows the effect of different drug groups on the body weight of h-PD-L1 knockin mice implanted with h-PD-L1 knockin MC38 tumors. There were no abnormalities in the condition of the mice in each group; compared with the control group, the body weight of the mice in each treatment group was not significantly reduced; at the end of the experiment, gross anatomy of the mice in each group showed no obvious lesions in the major organs, indicating that the doses of the drugs used in this experiment did not cause obvious toxicity to the mice.

[0188] All documents mentioned in this application are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teachings of this application, one skilled in the art may make various changes or modifications to the present application, and equivalent forms thereof are also within the scope defined by the appended claims of this application.

[0189] Sequence information of the present application SEQ ID NO: 1 C-Ye-18 CDR1 amino acid sequence GFTFSSYWMY SEQ ID NO: 2 C-Ye-18 CDR2 amino acid sequence SINSSSSSTYYRDSVKG SEQ ID NO: 3 C-Ye-18 CDR3 amino acid sequence AKDPGGYA SEQ ID NO: 4 C-Ye-18 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 5 C-Ye-18 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0190] SEQ ID NO: 6 C-Ye-04 CDR1 amino acid sequence SGFTFSSYWMY SEQ ID NO: 7 C-Ye-04 CDR2 amino acid sequence SINTSSSSTYYRDSVKG SEQ ID NO: 8 C-Ye-04 CDR3 amino acid sequence AKDPGGYA SEQ ID NO: 9 C-Ye-04 VHH amino acid sequence QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINTSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 10 C-Ye-04 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATACTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0191] SEQ ID NO: 11 C-Ye-02 CDR1 amino acid sequence GRTFNNSAMGAMG SEQ ID NO: 12 C-Ye-02 CDR2 amino acid sequence TITWSSGSSFYANSVKG SEQ ID NO: 13 C-Ye-02 CDR3 amino acid sequence ASRKLGGVVTVVTSYDF SEQ ID NO: 14 C-Ye-02 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLLSCAASGRTFNNSAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS SEQ ID NO: 15 C-Ye-02 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAATAACTCGGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATG CAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0192] SEQ ID NO: 16 C-Ye-06 CDR1 amino acid sequence GRTFDNYAMGAMG SEQ ID NO: 17 C-Ye-06 CDR2 amino acid sequence TITWSSGSSFYANSVKG SEQ ID NO: 18 C-Ye-06 CDR3 amino acid sequence ASRKLGGVVTVVTSYDF SEQ ID NO: 19 C-Ye-06 VHH amino acid sequence QVQLQESGGGLVQPGGSLRLSCAASGRTFDNYAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS SEQ ID NO: 20 C-Ye-06 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCGATAACTATGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATG CAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGACGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0193] SEQ ID NO: 21 C-Ye-09 CDR1 amino acid sequence GRTFSTYAVG SEQ ID NO: 22 C-Ye-09 CDR2 amino acid sequence GRLTWSGSRTYYADSVKG SEQ ID NO: 23 C-Ye-09 CDR3 amino acid sequence AADYRSNSTWSLQSPARYEN SEQ ID NO: 24 C-Ye-09 VHH amino acid sequence QVQLQESGGGLVQAGDSLGLSCTASGRTFSTYAVGWFRQAPGKGREFVGRLTWSGSRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAADYRSNSTWSLQSPARYENWGQGTQVTVSS SEQ ID NO: 25 C-Ye-09 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGGGACTCTCCTGTACAGCCTCTGGACGCACCTTCAGTACCTATGCCGTGGGGTGGTTCCGCCAGGCTCCAGGGAAGGGGCGTGAATTTGTAGGACGTCTTACATGGAGCGGGAGTAGAACATACTATGCAGACTCCG TGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCGACTACCGAAGTAACAGTACCTGGTCCCTGCAAAGCCCGGCACGTTATGAAAATTGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0194] SEQ ID NO: 26 C-Ye-10 CDR1 amino acid sequence GRTVSNYAMG SEQ ID NO: 27 C-Ye-10 CDR2 amino acid sequence RITGSGSSTFYADSVKG SEQ ID NO: 28 C-Ye-10 CDR3 amino acid sequence AADRWRSMVTRSDPREYEN SEQ ID NO: 29 C-Ye-10 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS SEQ ID NO: 30 C-Ye-10 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAGAACTGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0195] SEQ ID NO: 31 C-Ye-17 CDR1 amino acid sequence GRTVSNYAMG SEQ ID NO: 32 C-Ye-17 CDR2 amino acid sequence RITGSGSSTFYADSVKG SEQ ID NO: 33 C-Ye-17 CDR3 amino acid sequence AADRWRSMVTRSDPREYEN SEQ ID NO: 34 C-Ye-17 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGLFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS SEQ ID NO: 35 C-Ye-17 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACG CACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCTATTCACCATCTCCAGAAACAACTTGTCGA ACACGGTGTATCTGCAGATGAACAGCCTGAAACGGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0196] SEQ ID NO: 36 C-Ye-20 CDR1 amino acid sequence GRTVSNYAMG SEQ ID NO: 37 C-Ye-20 CDR2 amino acid sequence RITGSGSSTFYADSVKG SEQ ID NO: 38 C-Ye-20 CDR3 amino acid sequence AADRWRSMVTRSYPREYEN SEQ ID NO: 39 C-Ye-20 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAADRWRSMVTRSYPREYENWGQGTQVTVSS SEQ ID NO: 40 C-Ye-20 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGCGTTCAATGGTGACTAGATCTTACCCGAGGGAGTATGAGAGAACTGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0197] SEQ ID NO: 41 C-Ye-24 CDR1 amino acid sequence GRTVSNYAMG SEQ ID NO: 42 C-Ye-24 CDR2 amino acid sequence RITGSGRTTYYADSVKG SEQ ID NO: 43 C-Ye-24 CDR3 amino acid sequence AADRWRSMVTRSDPREYEN SEQ ID NO: 44 C-Ye-24 VHH amino acid sequence QVQLQESGGGVVQAGDSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGRTTYYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS SEQ ID NO: 45 C-Ye-24 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGTGTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTCGTACCACATACTATGCAGACTCC GTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAGAACTGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0198] SEQ ID NO: 46 C-Ye-26 CDR1 amino acid sequence GRTVSNYAMG SEQ ID NO: 47 C-Ye-26 CDR2 amino acid sequence RITGSGSSTFYADSVKG SEQ ID NO: 48 C-Ye-26 CDR3 amino acid sequence AADRWRSMVTRSDPRDYEN SEQ ID NO: 49 C-Ye-26 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPRDYENWGQGTQVTVSS SEQ ID NO: 50 C-Ye-26 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTAT CTGCAGATGAACAGCCTGAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGATTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0199] SEQ ID NO: 51 C-Ye-27 CDR1 amino acid sequence GRTFSRYAVG SEQ ID NO: 52 C-Ye-27 CDR2 amino acid sequence AITWSGGYTYYADSVKG SEQ ID NO: 53 C-Ye-27 CDR3 amino acid sequence AVDTRNVIGPRAGDY SEQ ID NO: 54 C-Ye-27 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS SEQ ID NO: 55 C-Ye-27 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0200] SEQ ID NO: 56 C-Ye-30 CDR1 amino acid sequence GSTFSRYAVG SEQ ID NO: 57 C-Ye-30 CDR2 amino acid sequence AITWSGGYTYYADSVKG SEQ ID NO: 58 C-Ye-30 CDR3 amino acid sequence AVDTRNVIGPRAGDY SEQ ID NO: 59 C-Ye-30 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLLSCAASGSTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS SEQ ID NO: 60 C-Ye-30 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0201] SEQ ID NO: 61 C-Ye-32 CDR1 amino acid sequence GRTFSRYAVG SEQ ID NO: 62 C-Ye-32 CDR2 amino acid sequence AITWSGGYTYYADSVKG SEQ ID NO: 63 C-Ye-32 CDR3 amino acid sequence AVDTRNVIGPRAGDY SEQ ID NO: 64 C-Ye-32 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTIYLQMNSLNVEDTGVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS SEQ ID NO: 65 C-Ye-32 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATCTTATCTCCAAATGAACAGCCTGAACGTTGAGGACACGGGCGTTTATTACTGCGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0202] SEQ ID NO: 66 C-Ye-34 CDR1 amino acid sequence AASGRTFSRFAMG SEQ ID NO: 67 C-Ye-34 CDR2 amino acid sequence AISWSGGMIYYTDSVKG SEQ ID NO: 68 C-Ye-34 CDR3 amino acid sequence AVDTRNVIGPRAGDY SEQ ID NO: 69 C-Ye-34 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLLSCAASGRTFSRFAMGWFRQAPGKEREFVAAISWSGGMIYYTDSVKGRFTISRDNAKNMLYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS SEQ ID NO: 70 C-Ye-34 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACTTTCAGTAGGTTTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTAGCTGGAGTGGTGGTATGATATACTATACA GACTCCGTGAAGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0203] SEQ ID NO: 71 C-Ye-39 CDR1 amino acid sequence GRAFSVYPMA SEQ ID NO: 72 C-Ye-39 CDR2 amino acid sequence RLTYTSNTFYADSVKG SEQ ID NO: 73 C-Ye-39 CDR3 amino acid sequence AVENRSSSWSLQSPARYDD SEQ ID NO: 74 C-Ye-39 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLSCTASGRAFSVYPMAWFRQAPGKEREFIARLTYTSNTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVENRSSSWSLQSPARYDDWGQGTQVTVSS SEQ ID NO: 75 C-Ye-39 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCATGTACAGCCTCTGGACGCGCCTTCAGGTCTACCCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTATAGCACGTCTTACGTATACTAGTAACACATTCTATGCAGACTCCG TGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGTCGAGAACCGCAGTAGTAGTTGGTCCCTGCAAAGCCCGGCACGTTATGATGACTGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0204] SEQ ID NO: 76 C-Ye-42 CDR1 amino acid sequence GRTGSRYAVG SEQ ID NO: 77 C-Ye-42 CDR2 amino acid sequence AITWSGGYTYYADSVKG SEQ ID NO: 78 C-Ye-42 CDR3 amino acid sequence AVDTRNVIGPRAGDY SEQ ID NO: 79 C-Ye-42 VHH amino acid sequence QVQLQESGGGLVQAGGSLRLLSCAASGRTGSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS SEQ ID NO: 80 C-Ye-42 VHH nucleotide sequence CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCGGCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATTCTGCAAATGAACAGCCTAAAACCTGAAGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0205] SEQ ID NO: 81 C-Ye-18-1 CDR2 amino acid sequence SINSGSSSTYYRDSVKG SEQ ID NO: 82 C-Ye-18-1 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 83 C-Ye-18-1 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0206] SEQ ID NO: 84 C-Ye-18-2 CDR2 amino acid sequence SISSSSSSTYYRDSVKG SEQ ID NO: 85 C-Ye-18-2 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 86 C-Ye-18-2 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTAGTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0207] SEQ ID NO: 87 C-Ye-18-3 CDR2 amino acid sequence SIGSSSSSTYYRDSVKG SEQ ID NO: 88 C-Ye-18-3 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIGSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 89 C-Ye-18-3 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTGGTAGTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0208] SEQ ID NO: 90 C-Ye-18-4 CDR2 amino acid sequence SIYSGSSSTYYRDSVKG SEQ ID NO: 91 C-Ye-18-4 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIYSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 92 C-Ye-18-4 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTTACAGTGGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0209] SEQ ID NO: 93 C-Ye-18-5 CDR2 amino acid sequence SINSDSSSTYYRDSVKG SEQ ID NO: 94 C-Ye-18-5 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 95 C-Ye-18-5 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGACAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0210] SEQ ID NO: 96 C-Ye-18-6 CDR2 amino acid sequence SINSGSSSTYYRDSVKG SEQ ID NO: 97 C-Ye-18-6 VHH amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISGSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS SEQ ID NO: 98 C-Ye-18-6 VHH nucleotide sequence GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTGGTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0211] SEQ ID NO: 99 IgG1 Fc fragment amino acid sequence DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 100 Fusion protein linker amino acid sequence GGGGSGGGGSGGGGSGGGGSG SEQ ID NO: 101 TGFβRII extracellular domain amino acid sequence IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD SEQ ID NO: 102 PM8001 amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSSDKTHTCPPC PAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNN DMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0212] SEQ ID NO: 103 Upstream primer for the first round GTCCTGGCTGCTCTTCTACAAGG SEQ ID NO: 104 Downstream primer for first round GGTACGTGCTGTTGAACTGTTCC SEQ ID NO: 105 Upstream primer for the second round CTAGTGCGGCCGCcTGGAGACGGTGACCTGGGT SEQ ID NO: 106 Downstream primer for second round CGCGGATCCCAGGTGCAGCTGCAGGAGTCTGGRGGAGG SEQ ID NO: 107 Upstream primer for the third round ATTTTTACTGCTGTTTTATTCGCAGCATCCTCCGCATTAGCTAAAAGAGAGGCTGAAGCACAGGTGCAGCTGCAGGAGTCTGGRGGAGG SEQ ID NO: 108 Downstream primer for the third round AGTTGTCAGTTCCTGTGCCCCCCCTCCTCCCGCGCCACCTCCGCCGCACCTCCGCCACCAcTGGAGACGGTGACCTGGGT

[0213] SEQ ID NO: 109 PD-L1-His tag amino acid sequence MFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYHHHHHH SEQ ID NO: 110 PD-L1 single domain antibody fusion protein amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 111 PD-L1 single domain antibody amino acid sequence EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSSDK

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 is one of the following: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which 1 to 3 amino acids have been added, deleted, modified and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93 or an amino acid sequence in which 1 to 3 amino acids have been added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which 1 to 3 amino acids have been added, deleted, modified and / or substituted relative to the amino acid sequence shown in SEQ ID NO: 3 An anti-PD-L1 single domain antibody consisting of:

2. The CDRs of the VHH chain are: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one or two amino acids have been added, deleted, modified and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93 or an amino acid sequence in which one or two amino acids have been added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence in which one or two amino acids are added, deleted, modified and / or substituted relative to the amino acid sequence shown in SEQ ID NO: 3 The anti-PD-L1 single domain antibody of claim 1, comprising:

3. The CDRs of the VHH chain are: a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one amino acid has been added, deleted, modified and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93 or an amino acid sequence in which one amino acid has been added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3 or an amino acid sequence in which one amino acid is added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 3 The anti-PD-L1 single domain antibody according to claim 1 or 2, comprising:

4. The CDRs of the VHH chain are: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3 The anti-PD-L1 single domain antibody according to any one of claims 1 to 3, comprising:

5. An anti-PD-L1 single domain antibody comprising a complementarity determining region (CDR) of a VHH chain, wherein the CDR of the VHH chain is selected from the following: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3 An anti-PD-L1 single domain antibody consisting of:

6. An anti-PD-L1 single domain antibody described in any one of claims 1 to 5, wherein the VHH chain comprises the amino acid sequence shown in SEQ ID NO: 94 or an amino acid sequence in which 1 to 8 amino acids have been added, deleted, modified and / or substituted relative to the amino acid sequence shown in SEQ ID NO:

94.

7. The VHH chain comprises the amino acid sequence set forth in SEQ ID NO: 94 or an amino acid sequence in which 1 to 8 amino acids have been added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 94; The CDRs of the VHH chain are: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3 The anti-PD-L1 single domain antibody according to any one of claims 1 to 6, comprising:

8. The VHH chain comprises the amino acid sequence set forth in SEQ ID NO: 94 or an amino acid sequence in which 1 to 5 amino acids have been added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 94; The CDRs of the VHH chain are: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3 The anti-PD-L1 single domain antibody according to any one of claims 1 to 7, comprising:

9. The VHH chain comprises the amino acid sequence set forth in SEQ ID NO: 94 or an amino acid sequence in which 1 to 3 amino acids have been added, deleted, modified, and / or substituted relative to the amino acid sequence set forth in SEQ ID NO: 94; The CDRs of the VHH chain are: CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; CDR2 comprising the amino acid sequence set forth in SEQ ID NO:93; and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3 The anti-PD-L1 single domain antibody according to any one of claims 1 to 8, comprising:

10. A polynucleotide encoding the anti-PD-L1 single domain antibody described in any one of claims 1 to 9.

11. The polynucleotide described in claim 10, which is DNA or RNA.

12. An expression vector comprising the polynucleotide described in claim 10 or claim 11.

13. The expression vector of claim 12, further comprising a nucleotide sequence encoding an Fc fragment of an immunoglobulin.

14. A fusion protein comprising an anti-PD-L1 single domain antibody described in any one of claims 1 to 9.

15. A fusion protein having, from N-terminus to C-terminus, the structure shown in Formula I: Z1-Z2-L-Z3 (Formula I) [In the formula, Z1 is an anti-PD-L1 single domain antibody according to any one of claims 1 to 9; Z2 is an Fc fragment of an immunoglobulin; L is a linker sequence; Z3 is an immunomodulatory molecule or a fragment thereof].

16. (a) an anti-PD-L1 single domain antibody according to any one of claims 1 to 9; and (b) a coupling moiety selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

10. An immunoconjugate comprising:

17. (a) an anti-PD-L1 single domain antibody according to any one of claims 1 to 9, a fusion protein according to claim 14 or 15, or an immunoconjugate according to claim 16; and (b) a pharmaceutically acceptable carrier 10. A pharmaceutical composition comprising:

18. The pharmaceutical composition of claim 17, suitable for injection.

19. Use of the pharmaceutical composition described in claim 17 or 18 in the manufacture of a medicament for treating a tumor expressing the PD-L1 protein.

20. The use of claim 19, wherein the tumor is selected from the group consisting of gastric cancer, lymphoma, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, bladder tumor, cervical cancer, and adrenal tumor.

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