Anti-PD-L1 nano antibody and application thereof

Abstract

The invention belongs to the technical field of biological medicine, discloses an anti-PD-L1 nano antibody and application thereof, particularly discloses an anti-PD-L1 nano antibody, and defines a specific amino acid sequence of the anti-PD-L1 nano antibody. The anti-PD-L1 nano antibody obtained by immunizing alpaca can effectively block the combination of PD1 and PDL1, has high affinity and strong specificity, can be used for immunoblotting, enzyme-linked immunosorbent assay kits and flow cytometry detection counting, can also be used for treating tumors, autoimmune diseases, chronic infectious diseases and other diseases, and has broad application prospects. The polypeptide has the potential of being developed as an immune checkpoint inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an anti-PD-L1 nanobody and applications thereof. Background Art

[0002] Malignant tumors, also known as cancer, are a serious disease caused by abnormal division and proliferation of normal cells in the human body. Once cancer occurs, normal tissues and organs around the lesion will be gradually invaded by tumor cells, and they can even use the blood system or lymphatic system to achieve the purpose of metastasis to other parts of the body. According to statistics, the number of people dying from cancer is increasing year by year around the world. In most countries, cancer ranks second among the causes of death of residents aged 30 to 69.

[0003] Immune checkpoints refer to a series of molecules expressed on immune cells that can regulate the degree of immune activation and prevent overactivation of the immune system. Immune checkpoints can initiate signaling pathways that inhibit T cell function, while immune checkpoint inhibitors can inhibit this tumor-mediated inhibition of T cell function. Programmed death ligand 1 (PD-L1) is an important target in tumor immune checkpoint blockade therapy, and it is expressed in a variety of cells. Most solid tumors abnormally activate the immune checkpoint PD-1 by highly expressing PD-L1, inducing apoptosis of tumor-specific T lymphocytes, allowing tumor cells to escape the surveillance and killing of the immune system. In addition, PD-L1 can bind to tumor infiltrating lymphocytes (TIL) to prevent the release of multiple inflammatory factors such as IL-2 and IFN-γ to inhibit the immune killing activity of TIL and induce its apoptosis, stimulate human peripheral blood T cells to secrete immunosuppressive cytokines such as IL-10 and TGF-β to mediate immunosuppression, and achieve immune escape, proliferation and metastasis of tumor cells.

[0004] Currently, it has been approved by the U.S. Food and Drug Administration (FDA) for a variety of cancers. As the latest tumor treatment method, immunotherapy has become the most advanced and effective oncology treatment method after chemotherapy and targeted therapy in recent years by activating the killing effect of immune cells on cancer cells. In particular, immune checkpoint therapy such as PD-1 / PD-L1 inhibitors can make about 20% of patients experience long-term remission, and even the possibility of tumor cure. Studies have shown that downregulating the expression of PD-L1 mRNA in lung cancer cells can enhance the killing effect of T lymphocytes on A549 cells. Blocking the interaction between PD-1 and PD-L1 can effectively restore the killing function of T cells against tumors; at present, the industry generally believes that antibodies targeting the PD-L1 pathway will bring breakthrough progress in the treatment of various tumors: since the advent of the world's first PD-1 inhibitor Opdivo in 2014, many PD-1 / PD-L1 drugs have been approved for marketing. It is used to treat non-small cell lung cancer, renal cell carcinoma, ovarian cancer, and melanoma.

[0005] PD-L1 is also often expressed in activated cells, B cells, macrophages, DC cells and neutrophils, and plays an important role in the body's immune homeostasis and inflammatory regulation. Autoimmune disease refers to a type of disease in which autoimmune reactions lead to organic damage and dysfunction of one's own tissues or organs. PD-1 / PD-L1 axis disorders are also widely present in various autoimmune diseases and chronic infectious diseases, including inflammatory bowel disease (OBD), multiple sclerosis (MS), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), type 2 diabetes (T2DM), aplastic anemia (AA), oral lichen planus (OLP), Wegener's granulomatosis (WG) and myasthenia gravis (MG). Studies have shown that peripheral tolerance of PD-1 knockout mice is destroyed, lymphocyte proliferation is inhibited, and autoimmune diseases can be induced. At the same time, abnormalities in the PD-1 / PD-L1 signaling pathway may play an important role in the pathogenesis of rheumatoid arthritis (RA). Matsuda K et al. detected the expression of PD-1 / PD-L1 on infiltrating T lymphocytes in the synovial fluid of RA patients and performed immunohistochemical staining on the synovial tissue of RA patients and found that PD-1 was highly expressed on synovial fluid infiltrating T lymphocytes, while PD-L1 was highly expressed on synovial lining cells, and the expression of both was correlated with the severity of RA. Some people also suggested that the PD-1 / PD-L1 pathway may play an important role in diseases such as ischemic stroke and multiple sclerosis.

[0006] However, not all cancer patients can benefit from this type of treatment. PD-L1 is a key biomarker for determining whether cancer patients are suitable for immunotherapy. Therefore, it is a rigid clinical need to select suitable patients with the help of biomarker detection. The evaluation of PD-L1 expression level in tumor tissue is currently the most clinically recognized marker for predicting the efficacy of PD-1 / PD-L1 inhibitors. A large amount of scientific research data shows that high expression of PD-L1 has been detected in most tumor cell lines such as lymphoma, choriocarcinoma, melanoma, and esophageal cancer. Compared with patients with negative PD-L1 expression, patients with positive PD-L1 expression are more likely to benefit from immunotherapy, and the higher the PD-L1 expression level in tumor tissue, the greater the chance that the patient will benefit from PD-1 / PD-L1 inhibitor treatment. Many clinical guidelines for non-small cell lung cancer (NSCLC) at home and abroad recommend that patients undergo routine PD-L1 testing at the 1A evidence level. At the same time, studies have shown that the expression level of PD-L1 in non-muscle invasive bladder cancer cells is correlated with the pathological progression of the tumor. The treatment plan can be formulated based on the expression of PD-L1 in the patient after drug treatment. The quantification of the expression level of PD-L1 is usually evaluated by pathologists directly observing under a microscope, so very high requirements are placed on the affinity and specificity of the antibody.

[0007] Nanobodies (VHH) have the same structural domains as ordinary antibody VH, namely 4 conserved framework regions (FR1 / 2 / 3 / 4) and 3 complementary determining regions (CDR1 / 2 / 3). The molecular weight of nanobodies is only 10% of that of traditional antibodies, and they retain the complete antigen binding ability of HCAbs, and have the characteristics of strong specificity, good affinity and high stability. Since nanobodies do not have Fc segments and cannot produce cytotoxic effects such as ADCC / CDC like traditional antibodies, VHH antibodies are often fused with Fc segments to construct Fc-VHH fusion proteins to increase ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) activity. Compared with ordinary antibodies, these forms of nanobodies can be widely used in the treatment of various diseases. Summary of the invention

[0008] The purpose of the first aspect of the present invention is to provide an anti-PD-L1 nanobody.

[0009] The second aspect of the present invention aims to provide a fusion protein.

[0010] The purpose of the third aspect of the present invention is to provide a biological material related to the anti-PD-L1 nanobody of the first aspect of the present invention or the fusion protein of the second aspect of the present invention.

[0011] The fourth aspect of the present invention aims to provide an immunoconjugate.

[0012] The fifth aspect of the present invention aims to provide a medicine.

[0013] The purpose of the sixth aspect of the present invention is to provide the use of the anti-PD-L1 nanobody of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention or the drug of the fifth aspect of the present invention.

[0014] The seventh aspect of the present invention aims to provide a test kit or a medicine box.

[0015] In order to achieve the above object, the technical solution adopted by the present invention is:

[0016] In a first aspect of the present invention, an anti-PD-L1 nanobody is provided, wherein the anti-PD-L1 nanobody comprises a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein:

[0017] The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO:3, SEQ ID NO:8, and SEQ ID NO:13;

[0018] The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:14;

[0019] The amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:15.

[0020] In some embodiments of the present invention, the CDR of the anti-PD-L1 Nanobody comprises:

[0021] CDR1 shown in SEQ ID NO:3, CDR2 shown in SEQ ID NO:4 and CDR3 shown in SEQ ID NO:5; or

[0022] CDR1 shown in SEQ ID NO:8, CDR2 shown in SEQ ID NO:9 and CDR3 shown in SEQ ID NO:10; or

[0023] CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14 and CDR3 shown in SEQ ID NO:15; or

[0024] In some embodiments of the invention, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

[0025] In some embodiments of the present invention, the amino acid sequence of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, or an amino acid sequence modified by substitution, deletion or addition of one or more amino acids with the same or similar functions as the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11.

[0026] A second aspect of the present invention provides a fusion protein comprising a first domain and a second domain;

[0027] The first domain is the anti-PD-L1 nanobody of the first aspect of the present invention;

[0028] The second domain has the effect of extending the half-life in vivo and / or has a binding effect on effector cells.

[0029] In some embodiments of the present invention, the second domain includes (but is not limited to):

[0030] Immunoglobulin Fc region (such as human immunoglobulin Fc region); and / or

[0031] Serum albumin (such as human HSA) or a fragment thereof, a domain that binds to serum albumin (such as an anti-serum albumin antibody, including a nanobody), polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or

[0032] A molecule that has affinity for a T cell surface molecule and / or is capable of binding to a surface molecule present on a T cell (eg, CD3).

[0033] In some embodiments of the present invention, the human immunoglobulin Fc region includes mutations for changing Fc-mediated effector functions, and the effector functions include a combination of one or more of CDC activity, ADCC activity, and ADCP activity.

[0034] In some embodiments of the present invention, the immunoglobulin is a combination of one or more selected from IgG, IgA1, IgA2, IgD, IgE, and IgM.

[0035] In some embodiments of the present invention, the IgG is selected from one or more combinations of IgG1, IgG2, IgG3 or IgG4 subtypes.

[0036] In some embodiments of the present invention, the amino acid sequence of the immunoglobulin Fc region is as shown in SEQ ID NO: 16; or an amino acid sequence that is modified by substitution, deletion or addition of one or more amino acids to the amino acid sequence shown in SEQ ID NO: 16 and has the same or similar function.

[0037] In some embodiments of the present invention, the first domain is connected to the N-terminus or C-terminus of the second domain.

[0038] In some embodiments of the present invention, the first domain and the second domain are hinge-connected (such as direct fusion of the anti-PD-L1 nanobody with the hinge region of Fc) or connected by a connecting peptide.

[0039] In some embodiments of the present invention, the connecting peptide is a flexible linker.

[0040] In some embodiments of the present invention, the amino acid sequence of the flexible linker includes but is not limited to GSAS, (GGCAGCGCCAGC) n 、(GGCGGCGGCAGC) n 、(GGCGGCGGCGGCAGC) n 、YAPVDV、(GGGS) n 、(GGSG) n 、(GGGGS) n 、(G) n , where 1≤n≤5, and n is an integer.

[0041] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO: 18 to SEQ ID NO: 20.

[0042] The third aspect of the present invention provides a biomaterial related to the anti-PD-L1 nanobody of the first aspect of the present invention or the fusion protein of the second aspect of the present invention; the biomaterial is any one of a1) to a12):

[0043] a1) a nucleic acid molecule encoding the anti-PD-L1 Nanobody of the first aspect of the invention or the fusion protein of the second aspect of the invention;

[0044] a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic cell line containing the nucleic acid molecule described in a1); a10) a transgenic cell line containing the expression cassette described in a2); a11) a transgenic cell line containing the recombinant vector described in a3); a12) a transgenic cell line containing the recombinant vector described in a4).

[0045] In some embodiments of the present invention, the nucleic acid molecule encoding the anti-PD-L1 nanobody of the first aspect of the present invention is shown as SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:12.

[0046] In some embodiments of the invention, the transgenic animal cell line does not contain reproductive material.

[0047] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0048] In some embodiments of the present invention, the 5' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0049] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0050] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0051] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0052] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0053] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0054] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0055] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0056] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0057] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0058] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express target proteins.

[0059] In some embodiments of the present invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell and an insect cell.

[0060] In some embodiments of the present invention, the cells include engineered cell lines such as CHO, CHO-K1, CHO-GS, lymphocyte cell lines such as T cells, NK cells, CIK cells, and the like.

[0061] The fourth aspect of the present invention provides an immunoconjugate, comprising:

[0062] (A) the anti-PD-L1 Nanobody of the first aspect of the invention or the fusion protein of the second aspect of the invention; and

[0063] (B) a functional molecule connected to (A) (including but not limited to covalently linked, coupled, attached, adsorbed).

[0064] In some embodiments of the present invention, (B) includes cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions based on chimeric antigen receptor technology, transmembrane regions (such as the transmembrane regions of CD8 or CD28) and intracellular signaling regions (such as CD3ζ chain, FcεRIγ tyrosine activation motif, intracellular signaling regions of co-stimulatory signal molecules CD27, CD28, CD137, CD134, MyD88, CD40, etc.), or a combination thereof.

[0065] In some embodiments of the present invention, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker.

[0066] In some embodiments of the present invention, the tumor-suppressing molecule is an anti-tumor cytokine (such as IL-12, IL-15, IFN-beta, TNFalpha) or an anti-tumor toxin.

[0067] In some embodiments of the present invention, the detectable marker is selected from a radioactive isotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0068] In some embodiments of the present invention, (B) is selected from: fluorescent substances, chemiluminescent markers, colored substances, radioactive isotopes, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents or enzymes capable of producing detectable products, radionuclides, biological toxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activated enzymes, chemotherapeutic agents (e.g., cisplatin) or any form of nanoparticles, etc.

[0069] The fifth aspect of the present invention provides a drug comprising the anti-PD-L1 nanobody of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, or the immunoconjugate of the fourth aspect of the present invention.

[0070] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0071] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, wetting agents, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, flavors and solvents.

[0072] In some embodiments of the present invention, the drug further comprises a combination drug, including but not limited to immune effector molecules, cells, cytotoxic substances, and multikinase inhibitors.

[0073] The sixth aspect of the present invention provides the use of the anti-PD-L1 nanobody of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention, or the drug of the fifth aspect of the present invention in (1) or (2):

[0074] (1) Preparation of preparations, test kits or medicine kits for diagnosing, treating or preventing cancer; (2) Preparation of preparations or medicine kits for treating or preventing infectious diseases or chronic inflammatory diseases.

[0075] It can also be used for targeted delivery of traditional chemotherapy drugs (traditional Chinese medicine monomers, microRNA, etc.).

[0076] In some embodiments of the invention, the cancer comprises: lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin's lymphoma, glioma, glioblastoma, squamous cell skin cancer, hematological malignancies, head and neck cancer or nasopharyngeal cancer, or a combination thereof.

[0077] The seventh aspect of the present invention provides a kit or a medicine kit, comprising the anti-PD-L1 nanobody of the first aspect of the present invention, the fusion protein of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immunoconjugate of the fourth aspect of the present invention, or the drug of the fifth aspect of the present invention.

[0078] The beneficial effects of the present invention are:

[0079] The anti-PD-L1 nanoantibody obtained by immunizing alpacas in the present invention can effectively block the binding of PD1 and PDL1, and has high affinity and strong specificity. It can be used for immunoblotting, enzyme-linked immunosorbent reaction kits, flow cytometry detection and counting, and can also be used for the treatment of tumors, autoimmune diseases, chronic infectious diseases and other diseases. It has the potential to be developed as an immune checkpoint inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 Figure 3 is the amino acid structure of the fusion protein.

[0081] Figure 2 This is the flow cytometric detection chart of the affinity of anti-PD-L1 fusion antibody to 239T-PD-L1.

[0082] Figure 3 This is the flow cytometric test result of the affinity of anti-PD-L1 fusion antibody to 239T-PD-L1.

[0083] Figure 4Affinity detection of anti-PD-L1 fusion antibody to 239T.

[0084] Figure 5 This is the antibody concentration detection data for each node of the purification of clone 10 fusion antibody.

[0085] Figure 6 These are the results of Coomassie Brilliant Blue staining after purification of clone 10 fusion antibody. The loading amounts were 10 μg, 5 μg, 2.5 μg and 1.25 μg, respectively.

[0086] Figure 7 This is a flow chart for the detection of fusion antibody blocking ability.

[0087] Figure 8 This is the result of the fusion antibody blocking ability test.

[0088] Fig. 9 The blocking ability of negative control antibody was detected by flow cytometry.

[0089] Fig.10 The blocking ability of the positive control antibody was detected by flow cytometry.

[0090] Fig.11 This is a flow cytometric test of the blocking ability of clone 3 fusion antibody.

[0091] Fig.12 This is a flow cytometric test of the blocking ability of clone 10 fusion antibody.

[0092] Fig.13 This is a flow cytometric test of the blocking ability of clone 12 fusion antibody. DETAILED DESCRIPTION

[0093] The present invention is further described in detail below through specific examples.

[0094] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0095] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0096] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0097] Example 1 Plasmid construction (including target fragment mutation)

[0098] Multiple candidate nanobody sequences were obtained by immunizing alpacas with antigen (SEQ ID NO: 25), and the VHH region nucleic acid sequence of the nanobody was connected with the human IgG Fc nucleic acid sequence using multi-fragment homologous recombination technology. The connecting region was hinge. The plasmid after homologous recombination was identified by enzyme digestion and then sequenced and analyzed. The fusion antibody sequence structure Figure 1 shown.

[0099] (1) Single fragment amplification reaction system preparation and procedure

[0100] The single fragment amplification reaction system was prepared as shown in Table 1, and the reaction was carried out according to the reaction procedure shown in Table 2.

[0101] PCR amplification primers for clones 3, 10, 12, and 25 to amplify the VHH region:

[0102] F-EF1α: CATTTCAGGTGTCGTGAAGC (SEQ ID NO: 21) (50% GC Tm=61);

[0103] R-FP: CGGTCCTCCCAGCAGCTCAG (SEQ ID NO:22) (64% GC Tm=62).

[0104] hIgG1-Fc fragment amplification primers

[0105] F-IGg1: CTGAGCTGCTGGGAGGAccg (SEQ ID NO: 23) (70% GC Tm=68);

[0106] R-IGgl:AAAAGGCGCAACCCGCTAGCtcatttacccggagacagggag (SEQ ID NO: 24) (54% GC Tm=67).

[0107] The above double fragments and the large fragment 8044 bp recovered after double digestion with NotI and NheI were subjected to homologous recombination. The system configuration is shown in Table 3.

[0108] Table 1 Single fragment amplification reaction system

[0109] 5×Q5 Reaction Buffer 10μL 10mM dNTPs 1μL Q5 High-Fidelity DNAPolymerase 0.5μL Template plasmid 0.1~2ng Primer F 1μM Primer R 1μM <![CDATA[UltraPure TM Distilled Water]]> to 50μL

[0110] Table 2 Reaction procedure

[0111]

[0112] (2) Multi-fragment homologous recombination preparation system and procedure

[0113] The multi-fragment homologous recombination reaction system was prepared as shown in Table 3, and the reaction was carried out according to the following reaction procedure: single-fragment recombination reaction, 50°C, 5 min; cool to 4°C or immediately cool on ice; multi-fragment recombination reaction, 50°C, 15 min; cool to 4°C or immediately cool on ice.

[0114] Table 3 Multi-fragment homologous recombination reaction system

[0115] Backbone plasmid digestion and purification product 50~100ng Target gene amplification recovery product 1:1 molar ratio of the same backbone plasmid digestion and purification product 2×ClonExpress Mix 5μL <![CDATA[UltraPure TM Distilled Water]]> To 10μL

[0116] Example 2 Plasmid Extraction

[0117] (1) Plasmid transformation of the vector confirmed to be correct by sequence alignment was performed at 37°C for 14 h;

[0118] (2) Pick a single clone and culture it in a small amount of culture solution, with a culture volume of 4 mL Amp+LB (100 mg / L Amp+), at 37°C and 220 rpm for 10 h.

[0119] (3) Large-scale culture of bacterial liquid: The next day, the seed liquid was inoculated into 250 mL Amp+LB (100 mg / L Amp+) and cultured at 37°C and 220 rpm for 14 h.

[0120] (4) Use Tiangen Plasmid Extraction Kit to extract plasmid:

[0121] a. Add the bacterial solution into a collection bottle (500 mL), centrifuge at 4000 rpm for 30 min, and remove the supernatant as much as possible.

[0122] b. Column equilibration step: Add 2.5 mL of equilibration solution BL to the adsorption column CP6 (the adsorption column is placed in a 50 mL collection tube).

[0123] c. Add 10 mL of solution P1 to the bacterial pellet to resuspend the bacteria.

[0124] d. Add 10 mL of solution P2 to the bacterial suspension and gently invert 6 to 8 times to fully lyse the bacteria.

[0125] e. Add 10 mL of solution P4 to the centrifuge tube and gently invert 6 to 8 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution.

[0126] f. Centrifuge the lysate at 3580×g for 30 min.

[0127] g. Carefully pour the supernatant lysate into filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50mL tube.

[0128] h. Add 3 mL of red endotoxin-free solution ER respectively, and mix by inversion until the solution becomes a uniform transparent yellow color.

[0129] i. Add 0.3 times the volume of isopropanol in the filtrate, mix by inversion, and transfer to adsorption column CP6.

[0130] j. Centrifuge at 8228×g for 2 min at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0131] k. Add 10 mL of buffer ED to the adsorption column CP6, centrifuge at 8228 × g for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0132] l. For low copy plasmid, repeat step k.

[0133] m. Add 10 mL of rinse solution PW to the adsorption column CP6, centrifuge at 8228×g for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0134] n. Repeat step m.

[0135] o. Place the adsorption column CP6 back into the collection tube, centrifuge at 8228×g for 5 min, then open the adsorption column CP6 and leave it at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material.

[0136] p. Place the adsorption column CP6 in a clean 50 mL collection tube, add 1-2 mL of elution buffer TB to the middle part of the adsorption membrane, let it stand at room temperature for 5 minutes, and then centrifuge it at 8228×g for 5 minutes.

[0137] q. Take 1 μL of plasmid DNA to determine the concentration (adjust to zero with Buffer TE), divide the plasmid DNA into 1.5 mL centrifuge tubes, mark the name, concentration, batch number and volume with a marker, and store at -20°C.

[0138] Example 3 Production of Antibodies

[0139] (1) Observe the 293T cells used for packaging under a microscope and select cells with a cell density of 80% to 95%.

[0140] (2) Prepare 10% packaging medium: open a new bottle of 500 mL DMEM medium, add 55 mL FBS, then add 550 μL 1 mol / L sodium pyruvate solution and 550 μL 25 mmol / L chloroquine phosphate solution, and mix well.

[0141] (3) Take out the selected cells from the incubator, pour the old culture medium in the culture bottle into the waste tank, add 17 mL of 10% packaging medium, and then return it to the carbon dioxide incubator to adapt.

[0142] (4) According to the number of culture bottles to be transfected, calculate the plasmid to be added (30 μg of plasmid per bottle) and 0.125 mol / L calcium chloride solution to prepare a DNA-CaCl2 mixture. Then take an equal volume of 2×HBS solution and add it dropwise to the DNA-CaCl2 mixture, vortexing the DNA-CaCl2 mixture while adding. After the addition, let it stand at room temperature for 20 minutes until a white precipitate is formed.

[0143] (5) Remove the cells obtained in step (3) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, mark the virus name and operation date on the bottle, return it to the incubator, and replace the medium with 5% packaging medium after 4 to 6 hours.

[0144] To prepare 5% packaging medium, open a new bottle of 500 mL DMEM medium and add 27.5 mL FBS and 27.5 mL gibco KnockOut TM Serum replacement (Cat. No. 10828028), add 550 μL 1 mol / L sodium pyruvate solution and 550 μL 25 mmol / L chloroquine phosphate solution, and mix well.

[0145] (6) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25 mL of 5% packaging medium to each bottle, and then return to the incubator for continued culture. After 72 hours of culture, harvest the supernatant to obtain the fusion antibody.

[0146] Example 4 Antibody supernatant specificity test

[0147] (1) Collect 1 mL of the antibody supernatant obtained 72 h after transfection (Example 3) in a 1.5 mL centrifuge tube.

[0148] (2) Centrifugation at 12000 rpm for 5 min to remove cell debris and clarify using a 0.22 μm filter for later use;

[0149] (3) Cell staining: Take the PD-L1 293T cells and 293T cells to be stained by flow cytometry, digest them with trypsin, wash them with sodium chloride / PBS, add 400 μL of the antibody supernatant in step (2), incubate at 4°C for 30 min, and protect from light;

[0150] (4) Wash the stained cells with sodium chloride, centrifuge, discard the supernatant, add anti-hunan-FC-PE flow cytometry antibody, incubate at 4°C for 30 min, and protect from light;

[0151] (5) Wash the cells to be tested with sodium chloride, add flow cytometry buffer after centrifugation, and test on the flow cytometry machine.

[0152] The flow cytometry results showed that clones 3, 10, and 12 all bound to 293T-PDL1, and clone 10 had a higher positive rate. Figure 2-3 ). Similarly, after co-incubation with 293T cells, mouse anti-human FC-PE was used for secondary antibody incubation for 1 hour before flow cytometry detection. The results are as follows Figure 4 As shown, the flow cytometry results showed that clone 3, clone 10, and clone 12 did not bind to 293T. The above conclusions indicate that the binding of clone 3, clone 10, and clone 12 to 293T-PDL1 is specific.

[0153] The sequences of the corresponding nanobody VHH and human IgG Fc in the fusion antibodies corresponding to clones 3, 10, and 12 are as follows:

[0154] The amino acid sequence of VHH of clone No. 3 is shown in SEQ ID NO: 1, the nucleotide sequence is shown in SEQ ID NO: 2, and the amino acid sequences of CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs: 3 to 5, respectively.

[0155] QLQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMAWFRQAAGKEHEFVGYINWSRGNT YYADSVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAGRVLGGTHSLQNSRGYDYWGQG TQVTVS(SEQ ID NO:1);

[0156] CAGTTTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAACTATGCCATGGCCTGGTTCCGCCAGGCTGCAGGGAAGGAGCATGAATTTGTAGGATATATTAACTGGAGCCGTGGAAACACATACTATGCAGACTCCGT GAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGCTGTTTTTGCAAATGAACAGCCTGAAACCTGAAGACACGGGCCGTGTATTACTGTGCGGGCCGGGTTCTTGGAGGTACTCACTCTCTACAAAATTCAAGGGGGTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO:2);

[0157] CDR1: GRTFSNYA (SEQ ID NO:3); CDR2: INWSRGNT (SEQ ID NO:4); CDR3: GRVLGGTHSLQNSRGYDY (SEQ ID NO:5).

[0158] The amino acid sequence of VHH of clone No. 10 is shown in SEQ ID NO: 6, the nucleotide sequence is shown in SEQ ID NO: 7, and the amino acid sequences of CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs: 8 to 10, respectively.

[0159] QVQLVESGGGLVQPGGSLRLSCASSEFTLNYYAIGWFRQAPGKGREGVSCISSSNPNTYY LDSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYYCAAAQWLPYSEYVACPPDGYDYWGQG TQVTVSS(SEQ ID NO:6);

[0160] CAGGTGCAGCTCGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCGTCCTCTGAATTCACTTTGAATTATTATGCCATAGGCTGGTTCCGCCAGGCCCCAGGGAAGGGGCGTGAGGGGGTGTCATGTATTAGTAGTAGTAATCCTAACACATACTACTTAGACTCCGTGAA GGGCCGATTCACCATCTCCAGAGACATCGCCAAGAACACGGTATATCTGCAAATGAACAGCCTGAAACCTGAGGACACAGCCGTTTATTACTGTGCAGCTGCCCAATGGCTGCCGTATAGTGAGTATGTCGCTTGCCCCCCAGATGGTTATGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA(SEQ ID NO:7);

[0161] CDR1: EFTLNYYA (SEQ ID NO:8); CDR2: ISSSNPNT (SEQ ID NO:9); CDR3: AAQWLPYSEYVACPPDGYDY (SEQ ID NO:10).

[0162] The amino acid sequence of VHH of clone No. 12 is shown in SEQ ID NO: 11, the nucleotide sequence is shown in SEQ ID NO: 12, and the amino acid sequences of CDR1, CDR2 and CDR3 thereof are shown in SEQ ID NOs: 13 to 15, respectively.

[0163] QLQLVESGGGLVQVGNSLRLSCTASGRATNMNAMGWFRQAPGKERELVAGITGTGLRTY YPDSVKGRFTVSRDNAKNTVYLQMDSLKPEDTAAYYCAASSYNVIGPKVVFDYWGQGTQVT VSS(SEQ ID NO:11);

[0164] CAGTTTGCAGCTCGTGGAGTCTGGGGGAGGATTGGTGCAGGTTGGAAACTCTCTGAGACTCTCCTGTACAGCCTCTGGACGCGCCACCAATATGAATGCCATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTGGTAGCAGGTATAACCGGCACTGGACTTAGAACTTATTATCCAGACT CCGTGAAGGGCCGATTCACCGTCTCCAGAGACAATGCCAAGAACACGGTGTATCTGCAAATGGACAGCCTGAAACCTGAGGACACGGCCGCTTATTACTGTGCAGCAAGCAGTTACAATGTGATTGGGCCTAAGGTTGTGTTTGACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA(SEQID NO:12)

[0165] CDR1: GRATNMNA (SEQ ID NO: 13); CDR2: ITGTGLRT (SEQ ID NO: 14); CDR3: ASSYNVIGPKVVFDY (SEQ ID NO: 15).

[0166] The amino acid sequence of human IgG Fc is shown in SEQ ID NO:16, and the nucleotide sequence is shown in SEQ ID NO:17.

[0167] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:16);

[0168] GAACCTAAAAGCAGCGACAAGACCCACACCTGCCCCCCTTGTCCTGCTCCTGAGCTGCTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA(SEQ ID NO:17)。

[0169] The amino acid sequence of clone 3 fusion antibody is shown in SEQ ID NO:18.

[0170] METPAQLLFLLLLLWLPDTTQLQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMAWFRQAAGKEHEFVGYINWSRGNTYYADSVKGRFTISRDNAKNTLFLQMNSLKPEDTAVYYCAGRVLGGTHSLQNSRGYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:18).

[0171] The amino acid sequence of clone 10 fusion antibody is shown in SEQ ID NO:19.

[0172] METPAQLLFLLLLLWLPDTTQVQLVESGGGLVQPGGSLRLSCASSEFTLNYYAIGWFRQAPGKGREGVSCISSSNPNTYYLDSVKGRFTISRDIAKNTVYLQMNSLKPEDTAVYYCAAAQWLPYSEYVACPPDGYDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:19).

[0173] The amino acid sequence of clone 12 fusion antibody is shown in SEQ ID NO:20.

[0174] METPAQLLFLLLLLWLPDTTQLQLVESGGGLVQVGNSLRLSCTASGRATNMNAMGWFRQAPGKERELVAGITGTGLRTYYPDSVKGRFTVSRDNAKNTVYLQMDSLKPEDTAAYYCAASSYNVIGPKVVFDYWGQGTQVTVSSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO:20).

[0175] Example 5 Antibody Concentration and Purification

[0176] (1) The 72 h supernatant obtained in Example 3 was divided into 50 mL centrifuge tubes and placed in a centrifuge for centrifugation. The centrifugation parameters were set to 4000 g, 25 min, and 4° C.

[0177] (2) The supernatant of the lentiviral solution after centrifugation was transferred to a 0.22 μm vacuum filtration system for filtration.

[0178] (3) Large volumes were concentrated using hollow fiber membrane columns / membrane packages, concentrated 40×, and purified using AKATA, with Protein G as the filler.

[0179] (4) The small volume of antibody supernatant was purified using a gravity purification column, with Protein G as the filler, and concentrated using a 30KD ultrafiltration tube.

[0180] (5) The purified antibodies can be detected by WB.

[0181] The band sizes of fusion antibodies (clone 3, clone 10, and clone 12) are all close to 50KD. After clone 10 is expressed in host cells, the antibody or flow-through sample retained during the purification process can be used for protein concentration detection, such as Figure 5 The purified and concentrated antibody product was subjected to WB detection, such as Figure 6 As shown, the size is correct, and the band is complete and single without fragmentation, which is suitable for the subsequent AKATA purification process of large quantities of antibodies.

[0182] Example 6 Antibody blocking test experiment

[0183] (1) Construction of K562-PD1-RFP and K562-PD-L1 EGFP cell lines: construct lentiviral vectors containing PD1 and PDL1 gene sequences, transduce K562 cell lines, MOI = 5, transduce for 72 hours, and detect the positive rates of RFP and EGFP by flow cytometry. Subsequent experiments can only be performed when the positive rate reaches more than 90%;

[0184] (2) taking the above cells and counting the cells;

[0185] (3) According to the counting results, the cell density was diluted to 1E6 / mL, 200 μL of each cell was taken in a 1.5 mL centrifuge tube, 1 μg of the purified antibody (Example 5) was added, RM1640 without antibody was added to the Blank group, and a blocker was added to the Control group. The control group was the PD-L1 antibody sequence obtained by literature retrieval (amino acid sequence such as SEQ ID NO: 25), and the diluent was 50 μL of 1% FBS1640 buffer.

[0186] (4) Incubate at 37°C for 1 hour.

[0187] (5) Streaming on-machine testing.

[0188] Schematic diagram of the antibody blocking test experimental process Figure 7 The blocking results of the anti-PD-L1 antibody were shown in Figure 8 to Figure 13 As shown, when the K562-PD1-RFP and K562-PD-L1 EGFP cell lines were not blocked by anti-PDL1 antibodies, the flow cytometry results showed that K562-PD1-RFP could bind to K562-PD-L1; when the antibodies of clone 3, clone 10, and clone 12 were added to the mixed cell fluid of K562-PD1-RFP and K562-PD-L1 EGFP, they were able to bind to K562-PD-L1 EGFP and prevent K562-PD1-RFP from binding to K562-PD-L1, among which the blocking effect of clone 10 antibody was stronger.

[0189] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An anti-PD-L1 nanobody, comprising a complementary determining region CDR, wherein the complementary determining region CDR comprises a complementary determining region CDR1, a complementary determining region CDR2 and a complementary determining region CDR3, wherein: The amino acid sequence of the complementary determining region CDR1 is shown in SEQ ID NO:3, SEQ ID NO:8, and SEQ ID NO:13; The amino acid sequence of the complementary determining region CDR2 is shown in SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:14; The amino acid sequence of the complementary determining region CDR3 is shown in SEQ ID NO:5, SEQ ID NO:10, and SEQ ID NO:

15.

2. The anti-PD-L1 nanobody according to claim 1, characterized in that The amino acid sequence of the anti-PD-L1 nanobody is as shown in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, or an amino acid sequence that is modified by substitution, deletion or addition of one or more amino acids with the same or similar functions as the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO:

11.

3. A fusion protein comprising a first domain and a second domain; The first domain is the anti-PD-L1 nanobody according to claim 1 or 2; The second domain has the effect of extending the half-life in vivo and / or has a binding effect on effector cells.

4. The fusion protein according to claim 3, characterized in that The second domain comprises: Immunoglobulin Fc region; and / or Serum albumin or a fragment thereof, a domain that binds serum albumin, polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof; and / or a molecule having affinity for a T cell surface molecule and / or capable of binding to a surface molecule present on a T cell; Preferably, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM; Preferably, the first domain and the second domain are connected by a hinge or by a connecting peptide.

5. A biological material related to the anti-PD-L1 nanobody according to claim 1 or 2 or the fusion protein according to claim 3 or 4; the biological material is any one of a1) to a12): a1) a nucleic acid molecule encoding the anti-PD-L1 Nanobody according to claim 1 or 2 or the fusion protein according to claim 3 or 4; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic cell line containing the nucleic acid molecule described in a1); a10) a transgenic cell line containing the expression cassette described in a2); a11) a transgenic cell line containing the recombinant vector described in a3); a12) A transgenic cell line containing the recombinant vector described in a4).

6. An immunoconjugate comprising: (A) the anti-PD-L1 Nanobody according to claim 1 or 2 or the fusion protein according to claim 3 or 4; as well as (B) Functional molecule linked to (A).

7. The immunoconjugate according to claim 6, characterized in that The (B) comprises cytotoxins, radioactive isotopes, biologically active proteins, molecules targeting tumor surface markers, tumor-suppressing molecules, molecules or detectable markers targeting surface markers of immune cells, extracellular hinge regions, transmembrane regions and intracellular signaling regions based on chimeric antigen receptor technology, or a combination thereof; Preferably, the molecule targeting a tumor surface marker is an antibody or a ligand that binds to a tumor surface marker; Preferably, the tumor-inhibiting molecule is an anti-tumor cytokine or an anti-tumor toxin.

8. A drug comprising the anti-PD-L1 nanobody according to claim 1 or 2, the fusion protein according to claim 3 or 4, the biomaterial according to claim 5, or the immunoconjugate according to claim 6 or 7.

9. Use of the anti-PD-L1 nanobody according to claim 1 or 2, the fusion protein according to claim 3 or 4, the biomaterial according to claim 5, the immunoconjugate according to claim 6 or 7, or the drug according to claim 8 in (1) or (2): (1) Preparation of preparations, test kits or pharmaceutical kits for diagnosing, treating or preventing cancer; (2) Preparation of preparations or kits for treating or preventing infectious diseases or chronic inflammatory diseases; Preferably, the cancer comprises: Lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, hematological malignancies, head and neck cancer, or nasopharyngeal cancer, or a combination thereof.

10. A kit or a medicine kit comprising the anti-PD-L1 nanobody according to claim 1 or 2, the fusion protein according to claim 3 or 4, the biomaterial according to claim 5, the immunoconjugate according to claim 6 or 7, or the drug according to claim 8.

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