Anti-pd-l1 nanobodies and derivatives and uses thereof

Abstract

The present application relates to an anti-PD-L1 nanobody and derivatives and uses thereof. Specifically, the present application provides an anti-PD-L1 nanobody, which has the function of blocking the binding of PD-L1 to the receptor PD-1. Furthermore, the present application provides the gene sequence encoding the nanobody, the corresponding expression vector and the host cell capable of expressing the nanobody, and a production method of the nanobody of the present application. Meanwhile, a fusion protein having both the PD-L1 nanobody of the present application and an immunomodulatory molecule is also provided, which can inhibit the PD-1 / PD-L1 pathway on the basis of targeting and neutralizing TGF-beta in the tumor microenvironment, restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting the occurrence and development of tumors.

Description

[0001] This application is a divisional application of the invention application with application number 201910863109.0, application date of September 12, 2019, entitled "An anti-PD-L1 nanobody and its derivatives and uses". Technical Field

[0002] This invention relates to the fields of biomedicine or biopharmaceutical technology, and more specifically to an anti-PD-L1 nanobody and its derivatives and uses. Background Technology

[0003] Programmed death 1 ligand 1 (PD-L1), also known as CD274, is a member of the B7 family and is the ligand for PD-1. PD-L1 is a type I transmembrane protein, consisting of 290 amino acids, including an IgV-like region, an IgC-like region, a transmembrane hydrophobic region, and an intracellular region composed of 30 amino acids.

[0004] Unlike other B7 family molecules, PD-L1 plays a negative role in regulating immune responses. Studies have found that PD-L1 is primarily expressed in activated T cells, B cells, macrophages, and dendritic cells. Besides lymphocytes, PD-L1 is also expressed in endothelial cells of various tissues such as the thymus, heart, and placenta, as well as in various non-lymphatic cell lines 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 has a broad role in regulating autoreactive T and B cells and immune tolerance, and it plays a role in peripheral tissue T and B cell responses. High expression of PD-L1 on tumor cells is associated with poor prognosis in cancer patients.

[0005] Programmed death-1 (PD-1), also known as CD279, binds to PD-L1 and is a member of the CD28 family. Its cytoplasmic region contains two tyrosine residues: one near the N-terminus in the immunoreceptor tyrosine-based inhibitory motif (ITIM), and the other near the C-terminus in the immunoreceptor tyrosine-based switch motif (ITSM). PD-1 is primarily expressed on the surface of activated T lymphocytes, B lymphocytes, and macrophages. Under normal conditions, PD-1 inhibits T lymphocyte function and promotes Treg function, thereby suppressing autoimmune responses and preventing autoimmune diseases. However, in tumorigenesis, PD-L1 expressed by tumor cells binds to PD-1, which can promote tumor immune escape through its inhibitory effect on lymphocytes. The binding of PD-L1 to PD-1 can lead to a variety of biological changes and induce immune regulation, such as inhibiting the proliferation and activation of lymphocytes, inhibiting the differentiation of CD4+ T cells into Th1 and Th17 cells, and inhibiting the release of inflammatory cytokines.

[0006] The successful application of monoclonal antibodies in cancer detection and targeted therapy has revolutionized tumor treatment. However, traditional monoclonal antibodies (150kD) have excessively large molecular weights, making it difficult to penetrate tissues and resulting in low effective concentrations in tumor areas, leading to insufficient therapeutic effects. Traditional antibodies also exhibit high immunogenicity, while modified antibodies struggle to achieve the same affinity. Furthermore, the long development cycle, high production costs, and insufficient stability of fully humanized traditional antibodies limit their clinical application and widespread adoption.

[0007] Nanobodies are currently the smallest antibody molecules, with a molecular weight only 1 / 10 that of ordinary antibodies. In addition to possessing the antigenic reactivity of monoclonal antibodies, nanobodies also have some unique functional characteristics, such as small molecular weight, high stability, good solubility, easy expression, weak immunogenicity, strong penetration, strong targeting, simple humanization, and low preparation cost. They almost perfectly overcome the shortcomings of traditional antibodies, such as long development cycle, low stability, and harsh storage conditions.

[0008] However, there is currently a lack of satisfactory nanobodies targeting PD-L1 in this field. Therefore, there is an urgent need in this field to develop new and effective specific nanobodies targeting PD-L1. Summary of the Invention

[0009] The purpose of this invention is to provide a new type of effective specific nanobodies targeting PD-L1.

[0010] In a first aspect of the present invention, a complementarity-determining region (CDR) of a VHH chain for an anti-PD-L1 nanobody is provided, wherein the CDR of the VHH chain is composed of the following:

[0011] The amino acid sequence is CDR1 as shown in SEQ ID NO: 5n+1;

[0012] CDR2 with an amino acid sequence as shown in SEQ ID NO: 5n+2, or CDR2 with an amino acid sequence having greater than 85% sequence identity with the sequence shown in SEQ ID NO: 2; and

[0013] The amino acid sequence is CDR3 as shown in SEQ ID NO: 5n+3;

[0014] Each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0015] In another preferred embodiment, n is 0 or 1.

[0016] In another preferred embodiment, the amino acid sequence of CDR2 is as shown in SEQ ID NO: 2, 7, 81, 84, 87, 90, 93 or 96.

[0017] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0018] In a second aspect of the invention, a VHH chain for an anti-PD-L1 nanobody is provided, wherein the VHH chain of the anti-PD-L1 nanobody includes CDR1, CDR2 and CDR3 as described in the first aspect of the invention.

[0019] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 5n+4, 82, 85, 88, 91, 94 or 97;

[0020] Wherein, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15;

[0021] Among them, any of the above amino acid sequences also includes a derivative sequence which optionally has 1-8 (preferably 1-5, more preferably 1-3) amino acid residues added, deleted, modified and / or substituted, and which is able to retain the PD-L1 binding affinity of the PD-L1 nanobody.

[0022] In another preferred embodiment, n is 0 or 1.

[0023] In another preferred embodiment, the amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 4, 9, 82, 85, 88, 91, 94 or 97.

[0024] In a third aspect of the invention, an anti-PD-L1 nanobody is provided, said anti-PD-L1 nanobody being a nanobody targeting the PD-L1 epitope and having the VHH chain of the anti-PD-L1 nanobody as described in the second aspect of the invention.

[0025] In a fourth aspect of the invention, a polynucleotide is provided, the polynucleotide encoding a protein selected from the group consisting of: the CDR region of the anti-PD-L1 nanobody VHH chain as described in the first aspect of the invention, the VHH chain of the anti-PD-L1 nanobody as described in the second aspect of the invention, or the anti-PD-L1 nanobody as described in the third aspect of the invention.

[0026] In another preferred embodiment, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO: 5n, 83, 86, 89, 92, 95 or 98;

[0027] Wherein, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0028] In another preferred embodiment, the polynucleotide includes DNA or RNA.

[0029] In a fifth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the fourth aspect of the invention.

[0030] In another preferred embodiment, the expression vector further comprises a nucleotide sequence encoding the Fc segment of an immunoglobulin.

[0031] In another preferred embodiment, the immunoglobulins are IgG1, IgG2, IgG3, and IgG4.

[0032] In a sixth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fifth aspect of the invention, or having a genome containing polynucleotides as described in the fourth aspect of the invention.

[0033] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0034] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0035] In a seventh aspect of the invention, a method for generating anti-PD-L1 nanobodies is provided, comprising the steps of:

[0036] (a) Culturing host cells as described in the sixth aspect of the invention under conditions suitable for generating nanobodies, thereby obtaining a culture containing the anti-PD-L1 nanobodies; and

[0037] (b) Isolate or recover the anti-PD-L1 nanobody from the culture.

[0038] In another preferred embodiment, the anti-PD-L1 nanobody has an amino acid sequence as shown in SEQ ID NO: 5n+4, 82, 85, 88, 91, 94 or 97;

[0039] Wherein, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0040] In an eighth aspect of the invention, a nanoantibody fusion protein is provided, the nanoantibody fusion protein having a structure from the N-terminus to the C-terminus as shown in Formula I:

[0041] Z1-Z2-L-Z3 (Formula I)

[0042] In the formula,

[0043] Z1 is the VHH chain of the anti-PD-L1 nanobody as described in the second aspect of the present invention;

[0044] Z2 is the Fc segment of an immunoglobulin;

[0045] L represents the connector sequence;

[0046] Z3 is the immunomodulatory molecular component.

[0047] In another preferred embodiment, the immunoglobulins are IgG1, IgG2, IgG3, and IgG4.

[0048] In another preferred embodiment, the amino acid sequence of Z2 is shown in SEQ ID NO: 99.

[0049] In another preferred embodiment, the amino acid sequence of Z2 is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 99.

[0050] In another preferred embodiment, the L has an amino acid sequence selected from the group consisting of: GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0051] In another preferred embodiment, the amino acid sequence of L is as shown in SEQ ID NO: 100.

[0052] In another preferred embodiment, the amino acid sequence of L is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 100.

[0053] In another preferred embodiment, the immunomodulatory molecule is the extracellular domain of TGFβRII.

[0054] In another preferred embodiment, the amino acid sequence of Z3 is shown in SEQ ID NO: 101.

[0055] In another preferred embodiment, the amino acid sequence of Z3 is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 101.

[0056] In another preferred embodiment, the basic similarity means that at most 50 (preferably 1-20, more preferably 1-10, even more preferably 1-5, most preferably 1-3) amino acids are different, wherein the difference includes the substitution, deletion or addition of amino acids.

[0057] In another preferred embodiment, the substantially identical sequence means that the sequence identity of the amino acid sequence with 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%.

[0058] In another preferred embodiment, the amino acid sequence of the nanobody fusion protein is shown in SEQ ID NO: 102.

[0059] In a ninth aspect of the present invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0060] (a) The VHH chain of the anti-PD-L1 nanobody as described in the second aspect of the present invention, the anti-PD-L1 nanobody as described in the third aspect of the present invention, or the nanobody fusion protein as described in the eighth aspect of the present invention; and

[0061] (b) The coupling part selected from the group consisting of: detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0062] In another preferred embodiment, the coupling portion is a drug or toxin.

[0063] In another preferred embodiment, the coupling portion is a detectable marker.

[0064] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0065] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) VHH chain of an anti-PD-L1 nanobody as described in the second aspect of the present invention, an anti-PD-L1 nanobody as described in the third aspect of the present invention, or a nanobody fusion protein as described in the eighth aspect of the present invention.

[0066] In another preferred embodiment, the term "multivalent" means that the amino acid sequence of the immunoconjugate contains a plurality of repeating VHH chains of anti-PD-L1 nanobodies as described in the second aspect of the present invention, anti-PD-L1 nanobodies as described in the third aspect of the present invention, or nanobody fusion proteins as described in the eighth aspect of the present invention.

[0067] In a tenth aspect of the invention, the use of an anti-PD-L1 nanobody as described in the third aspect of the invention or a nanobody fusion protein as described in the eighth aspect of the invention is provided for the preparation of (a) a reagent for detecting PD-L1 molecules; and (b) a medicament for treating tumors.

[0068] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0069] In an eleventh aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0070] (i) the complementarity-determining region (CDR) of the VHH chain of the anti-PD-L1 nanobody as described in the first aspect of the present invention, the VHH chain of the anti-PD-L1 nanobody as described in the second aspect of the present invention, or the anti-PD-L1 nanobody as described in the third aspect of the present invention, the nanobody fusion protein as described in the eighth aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention; and

[0071] (ii) Pharmaceutically acceptable carriers.

[0072] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0073] In another preferred embodiment, the pharmaceutical composition is used to prepare a medicament for treating tumors, wherein the tumors are selected from the group consisting of: gastric cancer, liver cancer, leukemia, kidney tumors, lung cancer, small intestinal cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumors, bladder tumors, or combinations thereof.

[0074] In a twelfth aspect of the invention, one or more uses of an anti-PD-L1 nanobody as described in a third aspect of the invention or a nanobody fusion protein as described in an eighth aspect of the invention are provided:

[0075] (i) Used to detect human PD-L1 molecules;

[0076] (ii) Used for flow cytometry detection;

[0077] (iii) Used for cellular immunofluorescence detection;

[0078] (iv) Used to treat tumors; and

[0079] (v) Used for tumor diagnosis.

[0080] In another preferred embodiment, the use is non-diagnostic and non-therapeutic.

[0081] In a thirteenth aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0082] (i) the sequence of the heavy chain variable region VHH as described in the second aspect of the invention, or the sequence of the nanobody as described in the third aspect of the invention, or the nanobody fusion protein as described in the eighth aspect of the invention; and

[0083] (ii) Optional tag sequences to assist in expression and / or purification.

[0084] In another preferred embodiment, the tag sequence includes: 6His tag, HA tag, Flag tag, Fc tag, or a combination thereof.

[0085] In another preferred embodiment, the recombinant protein specifically binds to the PD-L1 protein.

[0086] In a fourteenth aspect of the invention, the use of the VHH chain as described in the second aspect of the invention, the nanobody as described in the third aspect of the invention, the nanobody fusion protein as described in the eighth aspect of the invention, or the immunoconjugate as described in the ninth aspect of the invention, for the preparation of pharmaceuticals, reagents, detection plates or kits is provided.

[0087] The reagents, detection plates, or kits are used to detect PD-L1 protein in samples.

[0088] The agent is used to treat or prevent tumors that express PD-L1 protein (i.e., PD-L1 positive).

[0089] 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, prostate cancer, adrenal tumor, or a combination thereof.

[0090] In a fifteenth aspect of the present invention, a method for detecting PD-L1 protein in a sample is provided, comprising the steps of:

[0091] (1) Contact the sample with the nanobody as described in the third aspect of the present invention or the nanobody fusion protein as described in the eighth aspect of the present invention;

[0092] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of PD-L1 protein in the sample.

[0093] In another preferred embodiment, the detection includes qualitative detection and quantitative detection.

[0094] In a sixteenth aspect of the invention, a method for treating a disease is provided, the method comprising administering to a desired subject a nanobody as described in a third aspect of the invention, a nanobody fusion protein as described in an eighth aspect of the invention, or an immunoconjugate as described in a ninth aspect of the invention.

[0095] In another preferred embodiment, the object includes mammals.

[0096] In another preferred embodiment, the mammal is a human.

[0097] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0098] Figure 1 The nanobodies of the present invention can bind to human PD-L1 protein on the cell surface, and the binding effect of some antibodies is similar to that of the positive control.

[0099] Figure 2 The results showed that the modified nanobodies could still bind to the human PD-L1 protein on the cell surface, and the binding effect of the antibodies was similar to that of the positive control.

[0100] Figure 3 The results showed that the modified nanobody could still block the binding of PD-L1 protein to human PD-1 protein on the cell surface, and the blocking effect of the antibody was similar to that of the positive control.

[0101] Figure 4 The nanobody of the present invention has been shown to effectively activate T cells, and the activation effect is similar to or better than that of the positive control group antibody.

[0102] Figure 5 A schematic diagram of the fusion protein structure is shown.

[0103] Figure 6 This demonstrates that the fusion protein of the present invention can bind to the human PD-L1 protein on the cell surface.

[0104] Figure 7 The fusion protein of the present invention 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.

[0105] Figure 8 The fusion protein of the present invention is shown to bind to TGFβ1, TGFβ2, and TGFβ3.

[0106] Figure 9 The fusion protein of the present invention has been shown to effectively block the TGFβ / SMAD signaling pathway.

[0107] Figure 10 The results show that the fusion protein of the present invention can effectively activate T cells, and the activation effect is similar to or better than that of the positive control group antibody.

[0108] Figure 11 The fusion protein of the present invention has been shown to effectively inhibit tumor growth in mice. Detailed Implementation

[0109] Through extensive and in-depth research and numerous screenings, the inventors have developed a new class of anti-PD-L1 nanobodies for the first time. Experimental results show that the PD-L1 nanobodies and their mutant derivatives obtained in this invention can effectively block the interaction between PD-L1 and PD-1, and exhibit good thermal stability.

[0110] Specifically, this invention utilizes human PD-L1 antigen protein to immunize llamas, obtaining a high-quality immune nanobody gene library. Unexpectedly, the inventors screened nanobody gene sequences with a high degree of humanization (sequence identity > 85%) from this immune nanobody gene library. The PD-L1 protein molecules were biotinylated and labeled, and the immune nanobody gene library was screened using yeast display technology, thereby obtaining PD-L1-specific nanobody candidate antibody genes. The obtained genes and their engineered mutants were then transformed into Expi-CHO cells for further screening based on antibody affinity, ability to block PD-L1-PD-1 binding, thermostability, and T-cell activation activity, resulting in a class of highly specific nanobody strains that can be efficiently expressed in vitro.

[0111] Furthermore, experimental results show that the fusion protein produced by fusing the nanobody sequence of the present invention (as the targeting part) with the IgG1 Fc fragment (as the linking part) and the TGFβRII extracellular domain (as the immunomodulatory molecule part) exhibits high activity with PD-L1, effectively blocking the interaction between PD-L1 and PD-1, effectively blocking the TGF-β / SMAD signaling pathway, effectively activating human T lymphocytes, and effectively inhibiting tumor growth in mice.

[0112] Based on this, the present invention was completed.

[0113] The present invention nanobody

[0114] As used herein, the terms "nanobody of the present invention," "anti-PD-L1 nanobody of the present invention," and "PD-L1 nanobody of the present invention" are used interchangeably and all refer to nanobodies that specifically recognize and bind to PD-L1 (including human PD-L1). Particularly preferred are nanobodies with VHH chain amino acid sequences as shown in SEQ ID NO: 4, 9, 82, 85, 88, 91, 94, or 97.

[0115] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among 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; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0116] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: to clone the variable region of an antibody heavy chain to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. It is the smallest antigen-binding fragment with complete function. Typically, antibodies that are naturally missing the light chain and the heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region.

[0117] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are closely packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0118] As those skilled in the art will recognize, immunoconjugates and fusion expression products include conjugates formed by binding drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules to the antibodies or fragments thereof of the present invention. The present invention also includes cell surface markers or antigens that bind to the described anti-PD-L1 protein antibody or fragments thereof.

[0119] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0120] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0121] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0122] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0123] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to the PD-L1 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0124] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0125] Generally, the antigen-binding properties of an antibody can be described by three specific regions located in the variable region of the heavy chain, called the variable region (CDR). This segment is divided into four frame regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, and are spatially close to each other through the β-sheets formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the 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 regions.

[0126] The variable regions of the heavy chains of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention includes molecules having variable regions of antibody heavy chains with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology to the CDRs identified herein.

[0127] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0128] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0129] The term "antibody of the present invention" refers to a polypeptide containing the aforementioned CDR region that has PD-L1 protein binding activity. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0130] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0131] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0132] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0133] Table A

[0134]

[0135] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0136] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0137] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0138] This invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. This invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In this invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0139] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0140] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0141] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0142] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0143] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0144] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0145] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0146] The recombinant peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, 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.

[0147] The antibodies of the present invention can be used alone or in combination or conjugated with detectable markers (for diagnostic purposes), therapeutic agents, PK (protein kinase) modified parts, or any combination of the above substances.

[0148] Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing detectable products.

[0149] Therapeutic agents that can bind to or conjugate with the antibodies of this invention include, but are not limited to: 1. radionuclides; 2. biotoxicants; 3. cytokines such as IL-2; 4. gold nanoparticles / nanorobars; 5. viral particles; 6. liposomes; 7. magnetic nanoparticles; 8. prodrug-activating enzymes (e.g., DT-cardiac flavinase (DTD) or biphenyl hydrolase-like protein (BPHL)); 10. chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0150] The fusion protein of this invention

[0151] As described herein, "fusion protein of the present invention" refers to a bifunctional fusion protein that has both the anti-PD-L1 nanobody described in the first aspect of the present invention and an immunomodulatory molecule moiety.

[0152] In this invention, a fusion protein is provided, wherein the nanoantibody fusion protein has a structure from the N-terminus to the C-terminus as shown in Formula I:

[0153] Z1-Z2-L-Z3 (Formula I)

[0154] In the formula,

[0155] Z1 is the VHH chain of the anti-PD-L1 nanobody as described in the second aspect of the present invention;

[0156] Z2 is the Fc segment of an immunoglobulin;

[0157] L represents the connector sequence;

[0158] Z3 is the immunomodulatory molecular component.

[0159] Preferably, the immunoglobulin may be IgG1, IgG2, IgG3 or IgG4, etc.

[0160] In a preferred embodiment, the immunoglobulin is IgG1, and the amino acid sequence of Z2 is as shown in SEQ ID NO: 99. In other embodiments, the amino acid sequence of Z2 is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 99.

[0161] In this invention, L is a flexible amino acid linker. Preferably, L has an amino acid sequence selected from the group consisting of: GGGGS, (GGGGS)2, (GGGGS)3, (GGGGS)4, (GGGGS)5, or a combination thereof.

[0162] In a preferred embodiment, the amino acid sequence of L is as shown in SEQ ID NO: 100. In other embodiments, the amino acid sequence of L is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 100.

[0163] In one embodiment of the present invention, the immunomodulatory molecule is the extracellular domain of TGFβRII. Preferably, the amino acid sequence of Z3 is as shown in SEQ ID NO: 101. In other embodiments, the amino acid sequence of Z3 is the same as or substantially the same as the amino acid sequence shown in SEQ ID NO: 101.

[0164] In this invention, the term "basically the same" means that at most 50 (preferably 1-20, more preferably 1-10, even more preferably 1-5, most preferably 1-3) amino acids are different, wherein the difference includes the substitution, deletion or addition of amino acids.

[0165] Preferably, the "substantially identical" means 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%.

[0166] In a preferred embodiment, the amino acid sequence of the nanoantibody fusion protein is shown in SEQ ID NO:102.

[0167] TGFβ is a key inducing factor of epithelial-mesenchymal transition (EMT). Simultaneously, TGFβ exhibits strong immunosuppressive effects in the tumor microenvironment, thereby playing a crucial regulatory role in tumorigenesis, metastasis, and drug resistance.

[0168] Therefore, in one embodiment of the present invention, TGFβ receptor II is selected as the immunomodulatory molecule in the fusion protein. The fusion protein of the present invention has the advantages of high dual-target binding affinity and strong specificity, thereby further enhancing anti-tumor immune function.

[0169] Pharmaceutical Composition

[0170] The present invention also provides a composition. Preferably, the composition is a pharmaceutical composition containing the aforementioned antibody or its active fragment or fusion protein, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0171] The pharmaceutical compositions of the present invention can be directly used to bind to PD-L1 protein molecules, and therefore can be used to treat tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0172] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described nanobody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 micrograms / kg body weight to about 50 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0173] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is between about 10 micrograms per kilogram of body weight and about 10 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0174] Labeled nanobodies

[0175] In a preferred embodiment of the invention, the nanobody carries a detectable marker. More preferably, the marker is selected from the group consisting of isotopes, colloidal gold markers, colored markers, or fluorescent markers.

[0176] Colloidal gold labeling can be performed using methods known to those skilled in the art. In a preferred embodiment of the present invention, anti-PD-L1 nanobodies are labeled with colloidal gold to obtain colloidal gold-labeled nanobodies.

[0177] The anti-PD-L1 nanobody of the present invention has excellent specificity and high potency.

[0178] Detection methods

[0179] The present invention also relates to a method for detecting PD-L1 protein. The method comprises the following steps: obtaining cell and / or tissue samples; dissolving the samples in a medium; and detecting the level of PD-L1 protein in the dissolved samples.

[0180] In the detection method of the present invention, there are no particular limitations on the samples used; a representative example is a cell-containing sample present in a cell preservation solution.

[0181] Reagent test kit

[0182] The present invention also provides a kit containing the antibody (or fragment thereof) of the present invention or a detection plate. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, buffer, etc.

[0183] This invention also provides a detection kit for detecting PD-L1 levels. The kit includes an antibody that recognizes the PD-L1 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0184] application

[0185] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of PD-L1-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of PD-L1.

[0186] The main advantages of this invention include:

[0187] 1) The nanobody of this invention has high specificity against human PD-L1 protein with the correct spatial structure.

[0188] 2) The nanobodies of this invention have strong affinity.

[0189] 3) The nanoantibodies of this invention are easy to produce.

[0190] 4) Inhibiting the PD-1 / PD-L1 pathway on the basis of targeting and neutralizing TGF-β in the tumor microenvironment can restore T cell activity, enhance immune response, and more effectively improve the effect of inhibiting tumor occurrence and development.

[0191] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0192] Example 1: Construction of a nanobody library

[0193] Animal Immunization

[0194] Two llamas were immunized with an equal volume of 1 mg human PD-L1 antigen (purchased from AcroBiosystems) and Freund's adjuvant, once a week for a total of four immunizations, stimulating B cells to express antigen-specific nanobodies. After the four immunizations, 50 ml of peripheral blood was extracted from the llamas, and lymphocytes were separated using lymphocyte separation medium. Total RNA was extracted using Trizol RNA extraction reagent (purchased from Invitrogen). Total cDNA from the llamas was obtained by reverse transcription using a cDNA synthesis kit (purchased from Invitrogen).

[0195] Nanobody gene amplification

[0196] In the first round of PCR, IgG2 and IgG3 sequences were amplified from cDNA:

[0197] Table 1. Primers for the first round of PCR

[0198]

[0199] The first-round PCR products were subjected to agarose gel electrophoresis, and the fragment at 750 bp was excised and recovered for the second-round VHH sequence amplification. The primers for the second-round PCR amplification are as follows:

[0200] Table 2. Primers for the second round of PCR

[0201]

[0202] Using the second-round PCR product as a template, a third-round PCR was performed to add a homologous arm to the VHH gene. The primers for the third-round PCR amplification are as follows:

[0203] Table 3. Primers for the third round of PCR

[0204]

[0205] The target fragment was recovered using a PCR purification kit (purchased from QIAGEN).

[0206] Library Construction

[0207] The linearized yeast display vector and the third-round PCR product were mixed and electroporated into *Saccharomyces cerevisiae* (purchased from ATCC) to construct anti-PD-L1 nanobody libraries from two animals, and the library sizes were determined to be 4.47 × 10⁻⁶. 7 and 4.14×10 7 .

[0208] Example 2: Screening of PD-L1 nanobodies

[0209] Biotinylation labeling of human PD-L1 protein

[0210] Dissolve human PD-L1 protein (purchased from AcroBiosystems) in an appropriate volume of double-distilled water. Following the instructions of the Biotin-labeled reagent kit (purchased from Thermo), dissolve biotin and mix it with the protein solution. Incubate at 4°C for 2 hours. Remove excess biotin using a desalting column (purchased from Thermo). Desalting column pretreatment and sample collection were performed according to the product instructions.

[0211] MACS enrichment of yeast that specifically binds to PD-L1

[0212] The VHH library constructed in Example 1 was inoculated into SD-CAA amplification medium (1L SD-CAA amplification medium contains 6.7g YNB, 5g casein amino acids, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4 and 2% glucose), with the number of yeast cells inoculated being >10 × library capacity (initial amplification concentration = 0.5 OD). 600 Incubate overnight at 30℃ and 225 rpm ( / ml). Take 10× library volume of yeast cells, centrifuge at 3000 rpm for 5 min (the following centrifugation operations are all the same) to remove the culture medium, resuspend the yeast cells in SD-CAA induction medium, and adjust the initial concentration to 0.5 OD. 600 / ml, induce overnight. Determine the library concentration after induction. Take yeast cells at a volume of 10× library, centrifuge to remove the culture medium. Resuspend the yeast cells in 50ml of washing buffer (PBS + 0.5% BSA + 2mM EDTA), centrifuge to remove the supernatant. Resuspend the yeast cells in 10ml of washing buffer.

[0213] Add biotin-labeled PD-L1 protein (final concentration 100 mM), incubate at room temperature for 30 min, collect yeast cells by centrifugation, and wash the yeast three times with 50 ml wash buffer. Resuspend the yeast cells in 5 ml wash buffer and add 200 μl of SA magnetic beads (purchased from Miltenyi), invert and incubate for 10 min. Wash the yeast and magnetic bead mixture three times with wash buffer, and add the mixture to an LS column (purchased from Miltenyi). Place the LS column on a magnetic rack and wash with wash buffer to remove non-specifically bound yeast cells. Remove the column from the magnetic rack and add wash buffer to elute the yeast. Centrifuge the eluted yeast and transfer it to 200 ml of SD-CAA amplification medium for amplification.

[0214] High-affinity yeast cells were obtained by flow cytometry cell sorting.

[0215] MACS-enriched yeast cells were inoculated into SD-CAA amplification medium with an initial amplification concentration of 0.5 OD. 600 / ml. Incubate overnight in shake flasks at 30℃ and 225rpm. Resuspend yeast cells in SD-CAA induction medium (1L SD-CAA induction medium contains 6.7g YNB, 5g casein amino acids, 13.62g Na2HPO4·12H2O, 7.44g NaH2PO4, 2% galactose, 2% raffinose, and 0.1% glucose) at an initial concentration of 0.5 OD. 600 / ml, induce overnight. Add 1:200 diluted anti-c-Myc mouse antibody (Thermo) and 100 nM biotin-labeled PD-L1 antigen, and incubate at room temperature for 10 min. Wash yeast three times with PBS, add 1:500 diluted goat anti-mouse IgG (H+L) Alexa Fluor Plus 488 (Invitrogen) and streptavidin APC-conjugated fluorescent antibody (Invitrogen), and incubate at 4°C in the dark for 15 min. Resuspend cells in 2 ml PBS, and sort using a BD FACSAraiII instrument to obtain yeast cells with high binding capacity to PD-L1 antigen.

[0216] Access to antibody genes of PD-L1 nanobody candidate molecules

[0217] Yeast culture with high binding capacity to PD-L1 antigen, enriched by MACS and FACS, was cultured overnight at 30°C and 225 rpm in SD-CAA amplification medium. Yeast plasmids were extracted according to the yeast plasmid extraction kit (purchased from Tiangen). The plasmids were electroporated into Top10 competent cells (purchased from Tiangen), plated on ampicillin-resistant plates, and cultured overnight at 37°C. Single clones were picked and sequenced to obtain the VHH gene sequence.

[0218] Example 3: Construction, Expression, and Purification of Heavy Chain Antibodies

[0219] Antibody gene constructed into pCDNA3.1 expression vector

[0220] The VHH gene sequence was ligated to the human IgG1 (LALA mutant) Fc segment and then digested with homologous recombinase (Vazyme) and EcoRI / Not I into the linearized pCDNA3.1 vector, following the product instructions. The homologous recombination product was transformed into Top10 competent cells, plated on ampicillin-resistant plates, incubated overnight at 37°C, and single clones were picked for sequencing.

[0221] Cell transfection

[0222] The plasmid was transfected into Expi-CHO cells using the ExpiCHO™ Expression System Kit (Thermo). Transfection was performed according to the product instructions. After 5 days of cell culture, the supernatant was collected, and the target protein was purified using protein A magnetic beads (GenScript). The magnetic beads were resuspended in an appropriate volume of binding buffer (PBS + 0.1% Tween 20, pH 7.4) (1-4 times the volume of the magnetic beads) and added to the sample to be purified. The cells were incubated at room temperature for 1 hour with gentle agitation. The sample was placed on a magnetic rack (Beaver), the supernatant was discarded, and the magnetic beads were washed three times with binding buffer. Elution buffer (0.1M sodium citrate, pH 3.2) was added at 3-5 times the volume of the magnetic beads, and the cells were agitated at room temperature for 5-10 minutes. The cells were then returned to the magnetic rack, and the elution buffer was collected and transferred to a collection tube containing neutralization buffer (1M Tris, pH 8.54).

[0223] Example 4: Purification of anti-PD-L1 antibody and binding to human PD-L1

[0224] CHO cells overexpressing human PD-L1 (CHO-hPD-L1 cells) were generated by transfecting the pCHO1.0 vector (purchased from Invitrogen) containing human PD-L1 cDNA cloned into MCS (purchased from Sino Biological). The expanded-cultured CHO-hPD-L1 cells were then adjusted to a cell density of 2 × 10⁻⁶ cells. 6 Cells / ml, 100 μl / well added to a 96-well flow cytometry plate, centrifuged and ready for use. The purified PD-L1 antibody was diluted with PBS, starting at 400 nM and 3-fold diluted 12 times. 100 μl / well of the diluted sample was added to each well of the 96-well flow cytometry plate containing cells, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of goat F(ab')2 anti-human IgG-Fc (PE) (purchased from Abcam) diluted 100-fold with PBS was added, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of cells was resuspended in PBS, and the cells were analyzed and the corresponding MFI was calculated using a CytoFlex (Bechman) flow cytometer.

[0225] In the determination experiment using the method described above, the experimental results are as follows: Figure 1 As shown, all purified samples of this invention and CHO-hPD-L1 cells have binding activity, and the binding activity of some purified samples is similar to that of the control antibody ATE (US20130034559).

[0226] Example 5: PD-L1 antibody affinity assay

[0227] ForteBio affinity assays were performed according to existing methods (Estep, P et al., high-throughput measurement of antibody-antigen affinity and epitope grading based on solution, MAbs, 2013.5(2):p.270-8). In short, the sensor was equilibrated offline in analytical buffer for 30 min, then baseline was established by online detection for 60 s, and the purified antibody obtained as described above was loaded online onto the AHQ sensor. The sensor was then incubated with 100 nM PD-L1 antigen for 5 min, followed by dissociation in PBS for 5 min. Kinetic analysis was performed using a 1:1 binding model.

[0228] Table 4. Affinity of candidate molecules

[0229]

[0230] Example 6: PD-L1 antibody gene modification

[0231] To remove potential glycosylation sites in C-Ye-18, the CDRH2 portion of the C-Ye-18 amino acid sequence was point-mutated into the six forms shown in Table 5:

[0232] Table 5. C-Ye-18 CDR region mutation sequences

[0233]

[0234] This study used IMGT (http: / / www.imgt.org) to evaluate the humanization rate of C-Ye-18 CDR region mutant sequences. The results are shown in Table 6. The humanization rate of all C-Ye-18 mutants was higher than 87%, which meets the requirements for later-stage drug development.

[0235] Table 6. C-Ye-18 CDR region mutation sequence and human homology

[0236]

[0237] The protein construction, expression, and purification methods were the same as in Example 3. Protein purity was determined using HPLC. The HPLC method was as follows: mobile phase: 150 mM Na₂HPO₄•12H₂O, pH 7.0; chromatographic conditions: detection wavelength: 280 nm, column temperature: 25℃, flow rate: 0.35 ml / min, detection time: 20 min; Zenix-C SEC-300 column (SEPAX 4.6×300 mm, 3 μm).

[0238] Table 7. Purity test results of C-Ye-10 mutant antibody

[0239]

[0240] Example 7: Binding of C-Ye-18 mutant sample to human PD-L1

[0241] This experiment tested the binding activity of the purified C-Ye-18 mutant sample with CHO-hPD-L1 cells. The experimental method was the same as in Example 4, and the results are as follows: Figure 2 As shown, the C-Ye-18 mutant sample exhibited good binding activity with CHO-hPD-L1 cells, with levels comparable to C-Ye-18 and the control antibody ATE.

[0242] Example 8: Affinity determination of C-Ye-18 mutant samples

[0243] This experiment tested the binding affinity of the purified C-Ye-18 mutant sample to human PD-L1. The experimental method was the same as in Example 5, and the experimental results are shown in Table 8. The C-Ye-18 mutant sample had good binding activity with human PD-L1 protein.

[0244] Table 8 Affinity of C-Ye-18 mutant samples

[0245]

[0246] Example 9: C-Ye-18 mutant sample blocks PD-L1 binding to PD-1

[0247] CHO cells overexpressing human PD-1 (CHO-hPD-1 cells) were generated by transfecting pCHO1.0 vector (purchased from Invitrogen) containing human PD-1 cDNA cloned into MCS (purchased from Sino Biological). The expanded-cultured CHO-hPD-1 cells were then adjusted to a cell density of 2 × 10⁻⁶ cells. 6Cells / ml, 100 μl / well added to a 96-well flow cytometry plate, centrifuged and ready for use. The purified mutant sample was diluted with PBS, starting at 400 nM and 3-fold diluted 12 times. 60 μl / well of the diluted sample was added to a 96-well sample dilution plate, along with 60 μl / well of biotinylated human PD-L1 protein (purchased from AcroBiosystems), to a final concentration of 500 ng / ml. The plates were incubated at 4°C for 30 min. 100 μl / well of the co-incubated sample was added to the 96-well flow cytometry plate containing cells, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of streptomycin R-phycoerythrin conjugate (purchased from Thermo Fisher), diluted 100-fold with PBS, was added, incubated at 4°C for 30 min, and washed twice with PBS. 100 μl / well of cells was resuspended in PBS, and the cells were analyzed and the corresponding MFI was calculated using a CytoFlex (Bechman) flow cytometer.

[0248] In the determination experiment using the method described above, the experimental results are as follows: Figure 3 As shown, all the mutant samples of this invention can block the binding of PD-L1 to PD-1, and the blocking level is comparable to that of C-Ye-18 and the control antibody ATE.

[0249] Example 10: Thermal stability of C-Ye-18 mutant samples

[0250] The thermostability of different antibodies was detected using DSC (Differential Scanning Calorimetry). Samples were concentrated and diluted to 1 mg / ml with PBS. 5000× Cypro Orange (purchased from Bio-Rad) was diluted 50-fold with ultrapure water to obtain 100× Sypro Orange. 50 μl of the 1 mg / ml sample was added to 10 μl of 100× Sypro Orange and 40 μl of ultrapure water. After mixing, 30 μl of the mixture was added to a 96-well PCR plate, with three replicates per sample. The plate was placed in a PCR instrument, and the temperature program was set as follows: 25℃ for 5 min, then increased to 99℃ at a rate of 0.5℃ / min. After the program, the temperature value of the lowest point of the "Melt Curve" plot was read, which is the Tm value of the sample. The specific results are shown in Table 9 below.

[0251] Table 9. Tm values ​​of C-Ye-18 mutant antibodies

[0252]

[0253] Example 11: Mixed Lymphocyte Reaction Experiment

[0254] This embodiment uses a mixed lymphocyte reaction (MLR) assay to detect the activity of C-Ye-18 mutant samples in activating T cells. The specific experimental method is as follows.

[0255] Resuscitate PBMCs (purchased from SAILY BIO, SLB-HPB), centrifuge, resuspend PBMCs in 10 ml of X-VIVO-15 medium (purchased from LONZA), and culture at 37°C for 2 hours. Remove non-adherent cells. Add 10 ml of DC medium: X-VIVO-15 medium with 10 ng / ml GM-CSF (purchased from R&D), 20 ng / ml IL-4, and culture for 3 days. Add 5 ml of DC medium and continue culturing until day 6. Add DC maturation medium: X-VIVO-15 medium with 1000 U / ml TNF-α (purchased from R&D), 10 ng / ml IL-6 (purchased from R&D), 5 ng / ml IL-1β (purchased from R&D), and 1 μM PGE2 (purchased from Tocris), and culture for 2 days. Collect mature DCs and adjust the cell density to 2 × 10⁶ cells / year using X-VIVO-15 medium. 5 Cells / ml.

[0256] Resuscitate PBMCs from another donor (purchased from SAILY BIO, SLB-HPB), centrifuge, and resuspend the PBMCs in 10 ml of X-VIVO-15 medium. Use CD4+... + T-cell sorting kit (purchased from Stemcell) enriches CD4 + T cells, CD4 resuspended in X-VIVO-15 + T cells, cell density adjusted to 2×10⁶ 6 cells / ml. CD4 + T cells were mixed with the collected mature DC cells at a 1:1 ratio and added to a 96-well U plate at a ratio of 100 μl / well.

[0257] The C-Ye-18 mutant sample was diluted with X-VIVO-15 medium, and 3-fold dilutions were performed starting at 200 nM for a total of 9 spots. 100 μl / well was added to the above mixed cell wells and cultured for 5 days. The supernatant was collected and the expression levels of IFN-γ and IL2 were detected by ELISA (purchased from eBioscience).

[0258] The results are as follows Figure 4 As shown, the C-Ye-18 mutant samples, C-Ye-18-1, C-Ye-18-5, and C-Ye-18-6, all exhibited good biological activity in the MLR experiment, and their activation levels were similar to or better than those of the control antibody ATE.

[0259] Example 12 Cloning and Expression of Fusion Protein PD-L1 / TGF-β Traps

[0260] In this embodiment, the extracellular domain of TGFβRII (SEQ ID NO: 101) is used as the immunomodulatory molecule in the fusion protein, and the PD-L1 antibody is used as the targeting part of the fusion protein to form a PD-L1 antibody / TGFβRII extracellular domain fusion protein (PM8001).

[0261] Using molecular cloning technology, the C-terminal amino acids of the PD-L1 single-chain antibody of this invention were linked to the extracellular region of TGFβRII via (G4S)4G, and expressed using the Expi-CHO expression system. The expression and purification methods were the same as in Example 3, yielding the structure shown below. Figure 5 The fusion protein PM8001 is shown.

[0262] Example 13: Binding of PM8001 molecule to human PD-L1

[0263] The methods for detecting the binding activity of purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824 (WO2015 / 118175 A2), and negative control IgG protein to cell surface PD-L1 were the same as in Example 4. In the assays performed using the above methods, the experimental results are as follows: Figure 6 As shown, the PM8001 molecule of this invention has binding activity with CHO-hPD-L1 cells, and the binding activity is similar to that of the positive control molecule M7824.

[0264] Example 14 PM8001 molecule blocks the binding of PD-L1 protein to PD-1 cells

[0265] The methods for detecting the blocking activity of purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824, and negative control IgG protein in inhibiting the binding activity of PD-L1 protein and PD-1 cells are the same as in Example 9. In the assays performed using the above methods, the experimental results are as follows: Figure 7 As shown, the PM8001 molecule of the present invention can block the binding of PD-L1 protein to PD-1 cells, and the blocking level is comparable to that of the positive control molecule M7824.

[0266] Example 15: ELISA assay of PM8001 molecule binding to human TGF-β family proteins.

[0267] Human TGF-β1 (acrobiosystems, TG1-H421), TGF-β2 (PeproTech, 100-35B), and TGF-β3 (PeproTech, 100-36E) proteins were diluted with ELISA coating buffer and added to ELISA plates. The plates were incubated overnight at 4°C. The coating buffer was discarded, and the plates were washed three times with 250 μl / well of PBST. The plates were then blocked with 5% BSA at room temperature for 1 hour. Purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, and positive control M7824 were serially diluted and added to the blocked ELISA plates. The plates were incubated at room temperature for 2 hours. Wash 3 times with PBST, add goat anti-human Fc-HRP (abcam, ab97225) to the wells, incubate at room temperature for 1 hour, wash 3 times with PBST, add ELISA chromogenic solution, incubate at room temperature for 3 min, add ELISA stop solution, and read the absorbance value at 450 nm.

[0268] In the determination experiment using the method described above, the experimental results are as follows: Figure 8 As shown, the PM8001 molecule of the present invention has good binding to human TGF-β1 and TGF-β3 proteins at the ELISA level, but weak binding activity to human TGF-β2 protein, and the binding level is comparable to that of the positive control molecule M7824.

[0269] Example 16: Experiment on PM8001 molecule blocking the TGF-β / SMAD signaling pathway

[0270] Seed an appropriate amount of 293-TGF-β / SMAD effector cells into 96-well white-background cell culture plates and incubate overnight at 37°C in a 5% CO2 incubator. A mixture of purified PD-L1 antibody (C-Ye-18-5), PM8001 molecules, TGF-βR2-Fc fusion protein, and positive control M7824, serially diluted, and TGF-β1 (acrobiosystems, TG1-H421) was incubated at room temperature for 30 min. The mixture was then added to the cell-containing white-background plates and incubated overnight. Bio-Glo™ reagent (Promega) was added to each well, and fluorescence signal values ​​were read using a multi-mode microplate reader.

[0271] In the determination experiment using the method described above, the experimental results are as follows: Figure 9 As shown, the PM8001 molecule of the present invention can block the TGF-β / SMAD signaling pathway in vitro, and the blocking level is comparable to that of the positive control molecule M7824.

[0272] Example 17 Mixed Lymphocyte Reaction Experiment

[0273] The method for detecting the purified PD-L1 antibody (C-Ye-18-5), PM8001 molecule, TGF-βR2-Fc fusion protein, positive control M7824, and negative control IgG protein in mixed lymphocytes to activate human T lymphocytes was the same as in Example 11. The results are as follows: Figure 10 As shown, the PM8001 molecule of the present invention exhibits good biological activity in MLR experiments, and its activation level is comparable to or better than that of the positive control molecule M7824.

[0274] Example 18 Pharmacokinetic Evaluation of PM8001 Rats

[0275] Six SD rats (half male and half female) were used in the experiment. They were kept under 12 / 12-hour light / dark cycles at a temperature of 24±2℃ and humidity of 40-70%, with free access to water and food. They were purchased from Zhejiang Vital River Experimental Technology Co., Ltd. On the day of the experiment, the SD rats were injected once via tail vein with PM8001 molecules at a dose of 10 mg / kg.

[0276] Blood collection time points: Blood was collected from the jugular vein of rats at 3 min, 4 h, 10 h, 24 h, 48 h, 72 h, 120 h, 168 h, 240 h, 336 h, 504 h, and 672 h after drug administration. Whole blood samples were incubated at 2-8℃ for 30 min, centrifuged at 12000 rpm for 5 min to collect serum. The resulting serum was then centrifuged again at 2-8℃ at 12000 rpm for 5 min and stored at -80℃. The molecular weight of free PM8001 in the serum was detected by ELISA. The results are shown in Table 10. The half-life of the free PM8001 molecule of this invention in SD rats is approximately 146 hours.

[0277] Table 10 T1 / 2 of PM8001 in SD rats

[0278]

[0279] Example 19: Study on the tumor suppressor activity of PM8001

[0280] This experiment used MC38 cells expressing human PD-L1 (h-PD-L1 KI MC38) in PD-L1 transgenic mice to determine the antitumor effect of PM8001. First, a mouse model bearing h-PD-L1 KI MC38 tumors was established via subcutaneous inoculation. The tumors were allowed to grow to an average size of 80-120 mm. 3 Mice were divided into groups and treated with different antibodies and dosages via a single intraperitoneal injection. Tumor volume and body weight changes in each group were monitored twice a week for three consecutive weeks. Dosage and administration methods are shown in Table 11. Tumor volume changes in mice are shown in Table 12. Figure 11 As shown.

[0281] Table 11 Experimental Protocol for Tumor Suppressive Activity of PM8001

[0282]

[0283] Experimental results are as follows Figure 11 As shown, after inoculation with h-PD-L1 KI MC38, the tumor volume in the negative control group continued to increase. Tumor growth was inhibited in the TGF-β R II-Fc and C-Ye-18-5 monotherapy groups, while the PM8001 group showed better control compared to the TGF-β R II-Fc and C-Ye-18-5 groups, indicating that PM8001 has a significant tumor-inhibiting effect, comparable to or even slightly better than the positive control group.

[0284] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0285] Sequence information of the present invention

[0286] SEQ ID NO: 1 C-Ye-18 CDR1 amino acid sequence

[0287] GFTFSSYWMY

[0288] SEQ ID NO: 2 C-Ye-18 CDR2 amino acid sequence

[0289] SINSSSSSTYYRDSVKG

[0290] SEQ ID NO: 3 C-Ye-18 CDR3 amino acid sequence

[0291] AKDPGGYA

[0292] SEQ ID NO: 4 C-Ye-18 VHH amino acid sequence

[0293] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0294] SEQ ID NO: 5 C-Ye-18 VHH nucleotide sequence

[0295] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0296] SEQ ID NO: 6 C-Ye-04 CDR1 amino acid sequence

[0297] SGFTFSSYWMY

[0298] SEQ ID NO: 7 C-Ye-04 CDR2 amino acid sequence

[0299] SINTSSSSTYYRDSVKG

[0300] SEQ ID NO: 8 C-Ye-04 CDR3 amino acid sequence

[0301] AKDPGGYA

[0302] SEQ ID NO: 9 C-Ye-04 VHH amino acid sequence

[0303] QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINTSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0304] SEQ ID NO: 10 C-Ye-04 VHH nucleotide sequence

[0305] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATACTAGTAGTAGTAGCA CATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0306] SEQ ID NO: 11 C-Ye-02 CDR1 amino acid sequence

[0307] GRTFNNSAMGAMG

[0308] SEQ ID NO: 12 C-Ye-02 CDR2 amino acid sequence

[0309] TITWSSGSSFYANSVKG

[0310] SEQ ID NO: 13 C-Ye-02 CDR3 amino acid sequence

[0311] ASRKLGGVVTVVTSYDF

[0312] SEQ ID NO: 14 C-Ye-02 VHH amino acid sequence

[0313] QVQLQESGGGLVQAGGSLRLSCAASGRTFNNSAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS

[0314] SEQ ID NO: 15 C-Ye-02 VHH nucleotide sequence

[0315] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAATAACTCGGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0316] SEQ ID NO: 16 Amino acid sequence of C-Ye-06 CDR1

[0317] GRTFDNYAMGAMG

[0318] SEQ ID NO: 17 Amino acid sequence of C-Ye-06 CDR2

[0319] TITWSSGSSFYANSVKG

[0320] SEQ ID NO: 18 Amino acid sequence of C-Ye-06 CDR3

[0321] ASRKLGGVVTVVTSYDF

[0322] SEQ ID NO: 19 Amino acid sequence of C-Ye-06 VHH

[0323] QVQLQESGGGLVQPGGSLRLSCAASGRTFDNYAMGAMGWFRQAPGKEREFVATITWSSGSSFYANSVKGRFTISRDNAKNTVYLQMNSLKPDDTAVYYCASRKLGGVVTVVTSYDFWGQGTQVTVSS

[0324] SEQ ID NO: 20 Nucleotide sequence of C-Ye-06 VHH

[0325] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCGATAACTATGCCATGGGCGCCATGGGATGGTTCCGCCAGGCGCCAGGGAAAGAGCGTGAGTTTGTCGCGACAATTACCTGGAGTAGTGGTAGCTCATTTTATGCAAACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGACGACACGGCCGTTTATTACTGTGCATCACGCAAATTGGGAGGGGTTGTAACGGTAGTTACTTCGTATGACTTCTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0326] SEQ ID NO: 21 C-Ye-09 CDR1 Amino Acid Sequence

[0327] GRTFSTYAVG

[0328] SEQ ID NO: 22 C-Ye-09 CDR2 Amino Acid Sequence

[0329] GRLTWSGSRTYYADSVKG

[0330] SEQ ID NO: 23 C-Ye-09 CDR3 Amino Acid Sequence

[0331] AADYRSNSTWSLQSPARYEN

[0332] SEQ ID NO: 24 C-Ye-09 VHH Amino Acid Sequence

[0333] QVQLQESGGGLVQAGDSLGLSCTASGRTFSTYAVGWFRQAPGKGREFVGRLTWSGSRTYYADSVKGRFTISRDNAKNTVYLQMNSLRPEDTAVYYCAADYRSNSTWSLQSPARYENWGQGTQVTVSS

[0334] SEQ ID NO: 25 C-Ye-09 VHH Nucleotide Sequence

[0335] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGACTCTCTGGGACTCTCCTGTACAGCCTCTGGACGCACCTTCAGTACCTATGCCGTGGGGTGGTTCCGCCAGGCTCCAGGGAAGGGGCGTGAATTTGTAGGACGTCTTACATGGAGCGGGAGTAGAACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAGACCTGAGGACACGGCCGTTTATTACTGTGCAGCCGACTACCGAAGTAACAGTACCTGGTCCCTGCAAAGCCCGGCACGTTATGAAAATTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0336] SEQ ID NO: 26 Amino acid sequence of C-Ye-10 CDR1

[0337] GRTVSNYAMG

[0338] SEQ ID NO: 27 Amino acid sequence of C-Ye-10 CDR2

[0339] RITGSGSSTFYADSVKG

[0340] SEQ ID NO: 28 Amino acid sequence of C-Ye-10 CDR3

[0341] AADRWRSMVTRSDPREYEN

[0342] SEQ ID NO: 29 Amino acid sequence of C-Ye-10 VHH

[0343] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS

[0344] SEQ ID NO: 30 Nucleotide sequence of C-Ye-10 VHH

[0345] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0346] SEQ ID NO: 31 Amino acid sequence of C-Ye-17 CDR1

[0347] NYAMG

[0348] SEQ ID NO: 32 Amino acid sequence of C-Ye-17 CDR2

[0349] RITGSGSSTFYADSVKG

[0350] SEQ ID NO: 33 Amino acid sequence of C-Ye-17 CDR3

[0351] DRWRSMVTRSDPREYEN

[0352] SEQ ID NO: 34 Amino acid sequence of C-Ye-17 VHH

[0353] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGLFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS

[0354] SEQ ID NO: 35 Nucleotide sequence of C-Ye-17 VHH

[0355] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCTATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0356] SEQ ID NO: 36 Amino acid sequence of C-Ye-20 CDR1

[0357] NYAMG

[0358] SEQ ID NO: 37 Amino acid sequence of C-Ye-20 CDR2

[0359] RITGSGSSTFYADSVKG

[0360] SEQ ID NO: 38 Amino acid sequence of C-Ye-20 CDR3

[0361] DRWRSMVTRSYPREYEN

[0362] SEQ ID NO: 39 Amino acid sequence of C-Ye-20 VHH

[0363] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRDNAKNAVYLQMNSLKPEDTAVYYCAADRWRSMVTRSYPREYENWGQGTQVTVSS

[0364] SEQ ID NO: 40 Nucleotide sequence of C-Ye-20 VHH

[0365] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACGCGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTTACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0366] SEQ ID NO: 41 C-Ye-24 CDR1 amino acid sequence

[0367] NYAMG

[0368] SEQ ID NO: 42 C-Ye-24 CDR2 amino acid sequence

[0369] RITGSGRTTYYADSVKG

[0370] SEQ ID NO: 43 C-Ye-24 CDR3 amino acid sequence

[0371] DRWRSMVTRSDPREYEN

[0372] SEQ ID NO: 44 C-Ye-24 VHH amino acid sequence

[0373] QVQLQESGGGVVQAGDSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGRTTYYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPREYENWGQGTQVTVSS

[0374] SEQ ID NO: 45 C-Ye-24 VHH nucleotide sequence

[0375] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGTGTGGTGCAGGCTGGGGACTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTCGTACCACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGAGTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0376] SEQ ID NO: 46 Amino acid sequence of C-Ye-26 CDR1

[0377] NYAMG

[0378] SEQ ID NO: 47 Amino acid sequence of C-Ye-26 CDR2

[0379] RITGSGSSTFYADSVKG

[0380] SEQ ID NO: 48 Amino acid sequence of C-Ye-26 CDR3

[0381] DRWRSMVTRSDPRDYEN

[0382] SEQ ID NO: 49 Amino acid sequence of C-Ye-26 VHH

[0383] QVQLQESGGGLVQAGGSLRLSCVASGRTVSNYAMGWFRQAPGKEREFVARITGSGSSTFYADSVKGRFTISRNNLSNTVYLQMNSLKREDTAVYYCAADRWRSMVTRSDPRDYENWGQGTQVTVSS

[0384] SEQ ID NO: 50 Nucleotide sequence of C-Ye-26 VHH

[0385] CAGGTGCAGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGTAGCCTCTGGACGCACCGTCAGTAACTATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCACGGATTACCGGGAGTGGTAGTAGCACATTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAAACAACTTGTCGAACACGGTGTATCTGCAGATGAACAGCCTGAAACGTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCGCTGGCGTTCAATGGTGACTAGATCTGACCCGAGGGATTATGAGAACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0386] SEQ ID NO: 51 C-Ye-27 CDR1 Amino Acid Sequence

[0387] GRTFSRYAVG

[0388] SEQ ID NO: 52 C-Ye-27 CDR2 Amino Acid Sequence

[0389] AITWSGGYTYYADSVKG

[0390] SEQ ID NO: 53 C-Ye-27 CDR3 Amino Acid Sequence

[0391] AVDTRNVIGPRAGDY

[0392] SEQ ID NO: 54 C-Ye-27 VHH Amino Acid Sequence

[0393] QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0394] SEQ ID NO: 55 C-Ye-27 VHH Nucleotide Sequence

[0395] CAGGTGCAGGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0396] SEQ ID NO: 56 C-Ye-30 CDR1 amino acid sequence

[0397] GSTFSRYAVG

[0398] SEQ ID NO: 57 C-Ye-30 CDR2 amino acid sequence

[0399] AITWSGGYTYYADSVKG

[0400] SEQ ID NO: 58C-Ye-30 CDR3 amino acid sequence

[0401] AVDTRNVIGPRAGDY

[0402] SEQ ID NO: 59 C-Ye-30 VHH amino acid sequence

[0403] QVQLQESGGGLVQAGGSLRLSCAASGSTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0404] SEQ ID NO: 60 C-Ye-30 VHH nucleotide sequence

[0405] CAGGTGCAGGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGAAGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0406] SEQ ID NO: 61 C-Ye-32 CDR1 amino acid sequence

[0407] GRTFSRYAVG

[0408] SEQ ID NO: 62 C-Ye-32 CDR2 amino acid sequence

[0409] AITWSGGYTYYADSVKG

[0410] SEQ ID NO: 63 C-Ye-32 CDR3 amino acid sequence

[0411] AVDTRNVIGPRAGDY

[0412] SEQ ID NO: 64 C-Ye-32 VHH amino acid sequence

[0413] QVQLQESGGGLVQAGGSLRLSCAASGRTFSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTIYLQMNSLNVEDTGVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0414] SEQ ID NO: 65 C-Ye-32 VHH nucleotide sequence

[0415] CAGGTGCAGGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCG GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATCTATCTCCAAATGAACAGCCTGAACGTTGAGGACACGGGCGTTTATTACTGCGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0416] SEQ ID NO: 66 C-Ye-34 CDR1 amino acid sequence

[0417] RFAMG

[0418] SEQ ID NO: 67 C-Ye-34 CDR2 amino acid sequence

[0419] AISWSGGMIYYTDSVKG

[0420] SEQ ID NO: 68 C-Ye-34 CDR3 amino acid sequence

[0421] DTRNVIGPRAGDY

[0422] SEQ ID NO: 69 C-Ye-34 VHH amino acid sequence

[0423] QVQLQESGGGLVQAGGSLRLSCAASGRTFSRFAMGWFRQAPGKEREFVAAISWSGGMIYYTDSVKGRFTISRDNAKNMLYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0424] SEQ ID NO: 70 C-Ye-34 VHH nucleotide sequence

[0425] CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACTTTCAGTAGGTTTGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCCGCTATTAGCTGGAGTGGTGGTATGATATACTATACA GACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACATGCTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0426] SEQ ID NO: 71 C-Ye-39 CDR1 amino acid sequence

[0427] GRAFSVYPMA

[0428] SEQ ID NO: 72 C-Ye-39 CDR2 amino acid sequence

[0429] RLTYTSNTFYADSVKG

[0430] SEQ ID NO: 73 C-Ye-39 CDR3 amino acid sequence

[0431] AVENRSSSWSLQSPARYDD

[0432] SEQ ID NO: 74 C-Ye-39 VHH amino acid sequence

[0433] QVQLQESGGGLVQAGGSLRLSCTASGRAFSVYPMAWFRQAPGKEREFIARLTYTSNTFYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVENRSSSWSLQSPARYDDWGQGTQVTVSS

[0434] SEQ ID NO: 75 C-Ye-39 VHH nucleotide sequence

[0435] CAGGTGCAGGCTGCAGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCATGTACAGCCTCTGGACGCGCCTTCAGTGTCTACCCCATGGCCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTATAGCACGTCTTACGTATACTAGTAACACATTCTATGCAGACTCCG TGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGTGTATCTGCAGATGAACAGCCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCGGTCGAGAACCGCAGTAGTAGTTGGTCCCTGCAAAGCCCGGCACGTTATGATGACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCAGT

[0436] SEQ ID NO: 76 C-Ye-42 CDR1 amino acid sequence

[0437] GRTGSRYAVG

[0438] SEQ ID NO: 77 C-Ye-42 CDR2 amino acid sequence

[0439] AITWSGGYTYYADSVKG

[0440] SEQ ID NO: 78 C-Ye-42 CDR3 amino acid sequence

[0441] AVDTRNVIGPRAGDY

[0442] SEQ ID NO: 79 C-Ye-42 VHH amino acid sequence

[0443] QVQLQESGGGLVQAGGSLRLSCAASGRTGSRYAVGWFRQAPGLGRDFVAAITWSGGYTYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAVDTRNVIGPRAGDYWGQGTQVTVSS

[0444] SEQ ID NO: 80 C-Ye-42 VHH nucleotide sequence

[0445] CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCGGCAGTAGGTATGCCGTGGGCTGGTTCCGCCAGGCTCCAGGGCTGGGGCGTGACTTTGTAGCAGCTATTACCTGGAGTGGTGGTTACACATACTATGCGGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATGTATCTGCAAATGAACAGCCTAAAACCTGAAGACACGGCCGTTTATTACTGTGCAGTCGATACGAGGAATGTAATCGGCCCAAGAGCGGGAGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0446] SEQ ID NO: 81 C-Ye-18-1 CDR2 Amino Acid Sequence

[0447] SINSGSSSTYYRDSVKG

[0448] SEQ ID NO: 82 C-Ye-18-1 VHH Amino Acid Sequence

[0449] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0450] SEQ ID NO: 83 C-Ye-18-1 VHH Nucleotide Sequence

[0451] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0452] SEQ ID NO: 84 Amino acid sequence of C-Ye-18-2 CDR2

[0453] SISSSSSSTYYRDSVKG

[0454] SEQ ID NO: 85 Amino acid sequence of C-Ye-18-2 VHH

[0455] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0456] SEQ ID NO: 86 Nucleotide sequence of C-Ye-18-2 VHH

[0457] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTAGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0458] SEQ ID NO: 87 Amino acid sequence of C-Ye-18-3 CDR2

[0459] SIGSSSSSTYYRDSVKG

[0460] SEQ ID NO: 88 Amino acid sequence of C-Ye-18-3 VHH

[0461] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIGSSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0462] SEQ ID NO: 89 Nucleotide sequence of C-Ye-18-3 VHH

[0463] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTGGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0464] SEQ ID NO: 90 C-Ye-18-4 CDR2 Amino Acid Sequence

[0465] SIYSGSSSTYYRDSVKG

[0466] SEQ ID NO: 91 C-Ye-18-4 VHH Amino Acid Sequence

[0467] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSIYSGSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0468] SEQ ID NO: 92 C-Ye-18-4 VHH Nucleotide Sequence

[0469] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTTACAGTGGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0470] SEQ ID NO: 93 C-Ye-18-5 CDR2 Amino Acid Sequence

[0471] SINSDSSSTYYRDSVKG

[0472] SEQ ID NO: 94 C-Ye-18-5 VHH Amino Acid Sequence

[0473] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0474] SEQ ID NO: 95 C-Ye-18-5 VHH Nucleotide Sequence

[0475] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAATAGTGACAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0476] SEQ ID NO: 96 C-Ye-18-6 CDR2 Amino Acid Sequence

[0477] SISGSSSSTYYRDSVKG

[0478] SEQ ID NO: 97 C-Ye-18-6 VHH Amino Acid Sequence

[0479] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSISGSSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSS

[0480] SEQ ID NO: 98 C-Ye-18-6 VHH Nucleotide Sequence

[0481] GAGGTGCAGCTGCAGGAGTCTGGAGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGCGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTACTGGATGTATTGGCTCCGTCAGGCTCCAGGGAAGGGGCTCGAGTGGGTCTCATCTATTAGTGGTAGTAGTAGTAGCACATACTATCGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTATCTGCAAATGAACAGTCTGAAATCTGAGGACACGGCCGTGTATTACTGTGCAAAAGATCCTGGTGGGTACGCCAAAGGCCAGGGGACCCAGGTCACCGTCTCCAGT

[0482] SEQ ID NO: 99 Amino acid sequence of IgG1 Fc fragment

[0483] DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0484] SEQ ID NO: 100 Amino acid sequence of fusion protein linker

[0485] GGGGSGGGGSGGGGSGGGGSG

[0486] SEQ ID NO: 101 Amino acid sequence of TGFβRII extracellular domain

[0487] IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0488] SEQ ID NO: 102 Amino acid sequence of PM8001

[0489] EVQLQESGGGLVQPGGSLRLSCAASGFTFSSYWMYWLRQAPGKGLEWVSSINSDSSSTYYRDSVKGRFTISRDNAKNTLYLQMNSLKSEDTAVYYCAKDPGGYAKGQGTQVTVSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

Claims

1. An anti-PD-L1 nanobody comprising a complementarity-determining region (CDR) of a VHH chain, characterized in that, The CDR of the VHH chain consists of the following: The amino acid sequences are CDR1 as shown in SEQ ID NO: 41, CDR2 as shown in SEQ ID NO: 42, and CDR3 as shown in SEQ ID NO:

43.

2. A VHH chain of an anti-PD-L1 nanobody, characterized in that, The VHH chain of the anti-PD-L1 nanobody includes CDR1, CDR2 and CDR3 as defined in claim 1.

3. The VHH chain of the anti-PD-L1 nanobody as described in claim 2, wherein, The amino acid sequence of the VHH chain of the anti-PD-L1 nanobody is shown in SEQ ID NO:

44.

4. An anti-PD-L1 nanobody, characterized in that, The anti-PD-L1 nanobody is a nanobody targeting the PD-L1 epitope and has the VHH chain of the anti-PD-L1 nanobody as described in claim 2 or 3.

5. A polynucleotide, characterized in that, The polynucleotide encodes a protein selected from the group consisting of: the anti-PD-L1 nanobody of claim 1, the VHH chain of the anti-PD-L1 nanobody of claim 2 or 3, or the anti-PD-L1 nanobody of claim 4.

6. An expression carrier, characterized in that, The expression vector contains the polynucleotide as described in claim 5.

7. A host cell, characterized in that, The host cell contains the expression vector as described in claim 6, or its genome is integrated with the polynucleotide as described in claim 5.

8. An immunoconjugate, characterized in that, The immunoconjugate contains: (a) The VHH chain of the anti-PD-L1 nanobody as described in claim 2 or 3, or the anti-PD-L1 nanobody as described in claim 4; and (b) Selected from the following group of coupling components: detectable markers.

9. The immunoconjugate as described in claim 8, characterized in that, The detectable marker is a radionuclide or an enzyme.

10. The immunoconjugate of claim 8 or 9, comprising: a multivalent VHH chain of the anti-PD-L1 nanobody of claim 2 or 3, or the anti-PD-L1 nanobody of claim 4.

11. The immunoconjugate of claim 8 or 9, comprising: a bivalent VHH chain of the anti-PD-L1 nanobody of claim 2 or 3, or the anti-PD-L1 nanobody of claim 4.

12. The use of the anti-PD-L1 nanobody as described in claim 4, characterized in that, Used to prepare (a) reagents for detecting PD-L1 molecules; (b) drugs for treating tumors; The tumors mentioned are selected from the following group: gastric cancer, liver cancer, kidney tumors, lung cancer, small intestine cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, adrenal tumors, bladder tumors, and any combination thereof.

13. A pharmaceutical composition, characterized in that, include: (i) the anti-PD-L1 nanobody of claim 1, the VHH chain of the anti-PD-L1 nanobody of claim 2 or 3, or the anti-PD-L1 nanobody of claim 4, or the immunoconjugate of any one of claims 8-11; and (ii) Pharmaceutically acceptable carriers.

14. The pharmaceutical composition of claim 13, wherein, The pharmaceutical composition is used to prepare a drug for treating tumors, wherein the tumors are selected from the group consisting of: gastric cancer, liver cancer, kidney tumors, lung cancer, small intestinal cancer, bone cancer, colorectal cancer, breast cancer, colon cancer, prostate cancer, cervical cancer, adrenal tumors, bladder tumors, and any combination thereof.

15. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: The sequence of the VHH chain of the anti-PD-L1 nanobody as described in claim 2 or 3, or the sequence of the nanobody as described in claim 4.

16. The recombinant protein of claim 15, further comprising a tag sequence that assists in expression and / or purification.

17. The use of the VHH chain of the anti-PD-L1 nanobody as described in claim 2 or 3, the nanobody as described in claim 4, or the immunoconjugate as described in any one of claims 8-11, characterized in that, It is used to prepare pharmaceuticals, reagents, test plates, or kits; The reagents, detection plates, or kits are used to detect PD-L1 protein in samples. The agent is used to treat or prevent PD-L1 positive tumors; the tumors are selected from the group consisting of: gastric cancer, liver cancer, kidney tumors, lung cancer, small intestine cancer, bone cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, adrenal tumors, bladder tumors, and any combination thereof.

18. A non-disease diagnostic method for detecting PD-L1 protein in a sample, characterized in that, Including the following steps: (1) Contact the sample with the nanobody as described in claim 4; (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of PD-L1 protein in the sample.

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