Anti-pd-l1 nanobody and application thereof

Abstract

The application belongs to the technical field of biological medicine, and discloses an anti-PD-L1 nanobody and application thereof, and specifically discloses an anti-PD-L1 nanobody, and limits the specific amino acid sequence thereof. The anti-PD-L1 nanobody is obtained by immunizing a llama, the nanobody can effectively block the combination of PD1 and PDL1, has high affinity and strong specificity, can be used for immunoblotting, an enzyme-linked immunoreaction kit, flow cytometry counting, and can also be used for treating diseases such as tumors, autoimmune diseases and chronic infectious diseases, and has the potential to be developed as an immune checkpoint inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-PD-L1 nanobody and its application. Background Technology

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

[0003] Immune checkpoints are a series of molecules expressed on immune cells that regulate the level of immune activation and prevent excessive activation of the immune system. Immune checkpoints can initiate signaling pathways that suppress T cell function, while immune checkpoint inhibitors can inhibit this tumor-mediated T cell suppression. Programmed death ligand 1 (PD-L1) is an important target in tumor immune checkpoint blockade therapy, and it is expressed in a variety of cell types. Most solid tumors abnormally activate the immune checkpoint PD-1 through high expression of PD-L1, inducing tumor-specific T lymphocyte apoptosis, allowing tumor cells to evade the surveillance and killing of the immune system. Furthermore, PD-L1 can bind to tumor-infiltrating lymphocytes (TILs) to inhibit the release of various inflammatory factors such as IL-2 and IFN-γ, thereby suppressing the immune-killing activity of TILs and inducing their apoptosis. It also stimulates human peripheral blood T cells to secrete immunosuppressive cytokines such as IL-10 and TGF-β, mediating immunosuppression and enabling tumor cells to escape immunely, proliferate, and metastasize.

[0004] Currently approved by the U.S. Food and Drug Administration (FDA) for various cancers, immunotherapy, as the latest tumor treatment, activates the killing effect of immune cells on cancer cells and has become the most advanced and effective medical oncology treatment method after chemotherapy and targeted therapy in recent years. In particular, immune checkpoint therapies such as PD-1 / PD-L1 inhibitors can lead to long-term remission in about 20% of patients, 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 tumor-killing function of T cells. Currently, the industry generally believes that antibodies targeting the PD-L1 pathway will bring breakthrough progress in the treatment of various cancers: since the advent of Opdivo, the world's first PD-1 inhibitor, in 2014, several PD-1 / PD-L1 drugs have been approved for marketing. These are used to treat non-small cell lung cancer, renal cell carcinoma, ovarian cancer, and melanoma.

[0005] PD-L1 is also commonly expressed in activated cells, B cells, macrophages, dendritic cells (DCs), and neutrophils, playing a crucial role in immune homeostasis and inflammation regulation. Autoimmune diseases are a group of diseases caused by autoimmune reactions leading to organic damage and functional impairment of the body's own tissues or organs. PD-1 / PD-L1 axis dysregulation is also widespread 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 mellitus (T2DM), aplastic anemia (AA), oral lichen planus (OLP), Wegener's granulomatosis (WG), and myasthenia gravis (MG). Studies have shown that PD-1 knockout mice exhibit impaired peripheral tolerance and inhibit lymphocyte proliferation, which can induce autoimmune diseases. Furthermore, abnormalities in the PD-1 / PD-L1 signaling pathway may play a significant role in the pathogenesis of rheumatoid arthritis (RA). Matsuda K et al., through detecting the expression of PD-1 / PD-L1 on infiltrating T lymphocytes in the synovial fluid of RA patients and performing immunohistochemical staining on synovial tissue of RA patients, found that PD-1 was highly expressed on infiltrating T lymphocytes in the synovial fluid, while PD-L1 was highly expressed on synovial lining cells, and the expression levels of both were correlated with the severity of RA. Some researchers have 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 benefit from this type of treatment. PD-L1 is a key biomarker for determining whether a cancer patient is suitable for immunotherapy. Therefore, selecting appropriate patients based on biomarker detection is a rigid clinical requirement. Assessing PD-L1 expression levels in tumor tissue is currently the most clinically recognized predictive biomarker for the efficacy of PD-1 / PD-L1 inhibitors. Numerous research data show that high PD-L1 expression is detected in most tumor cell lines, including lymphoma, choriocarcinoma, melanoma, and esophageal cancer. Compared to PD-L1-negative patients, PD-L1-positive patients are more likely to benefit from immunotherapy, and the higher the PD-L1 expression level in tumor tissue, the greater the chance of benefiting from PD-1 / PD-L1 inhibitor treatment. Multiple domestic and international clinical guidelines for non-small cell lung cancer (NSCLC) recommend routine PD-L1 testing for patients with Level 1A evidence. Meanwhile, studies have shown that the expression level of PD-L1 in non-muscle-invasive bladder cancer cells is related to tumor pathological progression, and treatment plans can be formulated based on the expression of PD-L1 in patients after drug treatment. The quantification of the degree of PD-L1 expression is usually evaluated by pathologists through direct observation under a microscope, which requires high affinity and specificity of the antibody.

[0007] Nanobodies (VHHs) share the same structural domains as conventional antibodies (VHs), namely four conserved framework regions (FR1 / 2 / 3 / 4) and three complement-determining regions (CDR1 / 2 / 3). Nanobodies have a molecular weight only 10% that of traditional antibodies, retaining the complete antigen-binding capacity of HCAbs, and exhibiting high specificity, good affinity, and high stability. Because nanobodies lack an Fc fragment, they cannot produce ADCC / CDC cytotoxic effects like conventional antibodies. Therefore, VHH antibodies are often fused with an Fc fragment to construct Fc-VHH fusion proteins to increase ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) activities. Compared to conventional antibodies, these forms of nanobodies can be widely used in the treatment of various diseases. Summary of the Invention

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

[0009] A second aspect of the present invention is to provide a fusion protein.

[0010] The object of a third aspect of the present invention is to provide biomaterials 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] A fourth aspect of the present invention is to provide an immunoconjugate.

[0012] The fifth aspect of this invention is to provide a medicine.

[0013] The sixth aspect of this invention aims to provide the application of the anti-PD-L1 nanobody of the first aspect of this invention, the fusion protein of the second aspect of this invention, the biomaterial of the third aspect of this invention, the immunoconjugate of the fourth aspect of this invention, or the medicament of the fifth aspect of this invention.

[0014] The seventh aspect of this invention is to provide a reagent kit or medicine box.

[0015] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0016] In a first aspect, the present invention provides an anti-PD-L1 nanobody, the anti-PD-L1 nanobody comprising a complementarity-determining region (CDR), the CDR comprising a CDR1, a CDR2, and a CDR3, wherein...

[0017] The amino acid sequence of the complementarity-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 complementarity-determining region CDR2 is shown in SEQ ID NO4, SEQ ID NO:9, and SEQ ID NO:14.

[0019] The amino acid sequence of the complementarity-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 includes:

[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 present invention, CDR1, CDR2 and CDR3 are separated by the backbone 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 that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11 after substitution, deletion or addition of one or more amino acids.

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

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

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

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

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

[0031] Serum albumin (such as human HSA) or fragments thereof, serum albumin-binding domains (such as anti-serum albumin antibodies, including nanobodies), polyethylene glycol, polyethylene glycol-liposome complexes, or combinations thereof; and / or

[0032] Molecules that have an affinity for T cell surface molecules and / or are able to bind to surface molecules (such as CD3) present on T cells.

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

[0034] In some embodiments of the present invention, the immunoglobulin is 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 Fc region of the immunoglobulin is as shown in SEQ ID NO:16; or an amino acid sequence that is functionally identical or similar to the amino acid sequence shown in SEQ ID NO:16 after substitution, deletion or addition of one or more amino acids.

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

[0038] In some embodiments of the present invention, the first and second domains are hinged together (e.g., the anti-PD-L1 nanobody is directly fused to the hinge region of Fc) or connected via a linker peptide.

[0039] In some embodiments of the present invention, the linker 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] A third aspect of the present invention provides biomaterials 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; said biomaterials are any one of a1) to a12):

[0043] a1) A nucleic acid molecule encoding 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;

[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 molecules encoding the anti-PD-L1 nanobody of the first aspect of the present invention are shown in SEQ ID NO:2, SEQ ID NO:7, and SEQ ID NO:12.

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

[0047] In some embodiments of the present invention, the expression cassette includes a 5' transcriptional 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' transcriptional control region includes a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeper" promoter may be used), a transcription start site, an enhancer, and / or a silencing element.

[0049] In some embodiments of the invention, the 3'UTR may encode AU-rich elements, which are common regulators of mRNA stability via the 3'-5' exogenous pathway and are typically located in the 3'UTR. AU-rich elements may comprise one or more repeats of the sequence AUUUA. It may also comprise one or more so-called US2B elements having the sequence AUAUAU.

[0050] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

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

[0052] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

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

[0054] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, 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 plant or animal varieties.

[0058] In some embodiments of the present invention, the prokaryotic cells include bacteria well known in the art, such as Escherichia coli, Streptomyces, and Bacillus subtilis, which are capable of expressing the target protein.

[0059] In some embodiments of the present invention, the eukaryotic cells include at least one of yeast cells, mammalian cells, plant cells, and insect cells.

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

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

[0062] (A) 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; and

[0063] (B) Functional molecules that are connected to (A) (including but not limited to covalent connection, coupling, attachment, and adsorption).

[0064] In some embodiments of the present invention, (B) includes cytotoxins, radioisotopes, bioactive proteins, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules or detectable markers targeting immune cell surface markers, 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 the CD3ζ chain, FcεRIγ tyrosine activation motif, intracellular signaling regions of co-stimulatory signaling molecules CD27, CD28, CD137, CD134, MyD88, CD40, etc.), or combinations thereof.

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

[0066] In some embodiments of the present invention, the tumor-inhibiting molecules are anti-tumor cytokines (such as IL-12, IL-15, IFN-beta, TNFalpha) or anti-tumor toxins.

[0067] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, 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 (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, chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

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

[0070] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients.

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

[0072] In some embodiments of the present invention, the drug further includes combination drugs, including but not limited to immune effector molecules, cells, cytotoxic substances, and multi-kinase inhibitors.

[0073] A 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 medicament of the fifth aspect of the present invention in (1) or (2):

[0074] (1) To prepare preparations, kits or boxes for the diagnosis, treatment or prevention of cancer; (2) To prepare preparations or boxes for the treatment or prevention of infectious diseases or chronic inflammatory diseases.

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

[0076] In some embodiments of the present invention, the cancers include: lung cancer, melanoma, gastric cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, classical Hodgkin lymphoma, glioma, glioblastoma, squamous cell carcinoma of the skin, hematologic malignancies, head and neck cancer or nasopharyngeal carcinoma, or combinations thereof.

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

[0078] The beneficial effects of this invention are:

[0079] This invention relates to an anti-PD-L1 nanobody obtained by immunizing alpacas. This nanobody 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 assay kits, flow cytometry detection and counting, and can also be used for the treatment of diseases such as tumors, autoimmune diseases and chronic infections. It has the potential to be developed as an immune checkpoint inhibitor. Attached Figure Description

[0080] Figure 1 This is a diagram of the amino acid structure of the fusion protein.

[0081] Figure 2 Flow cytometry plot of affinity of anti-PD-L1 fusion antibody for 239T-PD-L1.

[0082] Figure 3 The results of flow cytometry analysis of the affinity of the anti-PD-L1 fusion antibody for 239T-PD-L1 are shown.

[0083] Figure 4To detect the affinity of the anti-PD-L1 fusion antibody for 239T.

[0084] Figure 5 Data on antibody concentration at each node of the clone 10 fusion antibody purification process.

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

[0086] Figure 7 Flowchart for detecting the blocking ability of fusion antibodies.

[0087] Figure 8 The results show the detection of the blocking ability of the fusion antibody.

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

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

[0090] Figure 11 Flow cytometry was used to detect the blocking ability of the clone 3 fusion antibody.

[0091] Figure 12 Flow cytometry was used to detect the blocking ability of the clone 10 fusion antibody.

[0092] Figure 13 Flow cytometry was used to detect the blocking ability of the clone 12 fusion antibody. Detailed Implementation

[0093] The present invention will be further described in detail below through specific embodiments.

[0094] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0095] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0096] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0097] Example 1: Plasmid Construction (including mutation of the target fragment)

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

[0099] (1) Preparation and procedure of single-fragment amplification reaction system

[0100] Prepare the single-fragment amplification reaction system as shown in Table 1, and carry out the reaction according to the reaction procedure shown in Table 2.

[0101] PCR amplification primers for clones 3, 10, 12, and 25 were used 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 two fragments were homologously recombinated with the large fragment 8044bp that was recovered after double digestion with NotI and NheI. The system preparation 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] Prepare the multi-fragment homologous recombination reaction system as shown in Table 3, and carry out the reaction according to the following reaction procedure: single-fragment recombination reaction, 50℃, 5min; cool to 4℃ or immediately place on ice for cooling; multi-fragment recombination reaction, 50℃, 15min; cool to 4℃ or immediately place on ice for cooling.

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

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

[0116] Example 2 Plasmid Extraction

[0117] (1) Transform the vector with the correct sequence alignment into a plasmid at 37°C for 14 hours.

[0118] (2) Pick single colonies, culture a small amount of bacterial solution (4 mL Amp+LB, 100 mg / L Amp+), at 37℃ and 220 rpm for 10 h.

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

[0120] (4) Plasmid extraction was performed using the Tiangen plasmid large-scale extraction kit:

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

[0122] b. Column equilibration procedure: 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 precipitate to resuspend the bacterial cells.

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

[0125] e. Add 10 mL of solution P4 to the centrifuge tube, gently invert 6 to 8 times to mix thoroughly, until a white, dispersed flocculent precipitate appears in the solution.

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

[0127] g. Carefully pour the supernatant lysis buffer 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 to each solution, and mix by inverting. The resulting solution will be a uniform, transparent yellow color.

[0129] i. Add 0.3 times the volume of the above filtrate in isopropanol, mix thoroughly by inverting the container, and then transfer it to the 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 plasmids, repeat step k.

[0133] m. Add 10 mL of washing buffer 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. Repeated operation steps m.

[0135] o. Place the adsorption column CP6 back into the collection tube, centrifuge at 8228×g for 5 min, then open the cap of the adsorption column CP6 and place it at room temperature for several minutes to thoroughly dry the residual rinsing 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 of the adsorption membrane, incubate at room temperature for 5 min, and then centrifuge at 8228×g for 5 min at room temperature.

[0137] q. Take 1 μL of plasmid DNA to determine the concentration (zero with Buffer TE), aliquot the plasmid DNA into 1.5 mL centrifuge tubes, and label them with the name, concentration, batch number and volume. Store at -20℃.

[0138] Example 3: Antibody Production

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

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

[0141] (3) Take the selected cells out of the incubator, pour the old culture medium in the culture flask into the waste liquid tank, add 17 mL of 10% packaged culture medium, and then put it back into the carbon dioxide incubator to adapt.

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

[0143] (5) Remove the cells obtained in step (3) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, and label the bottle with the virus name and operation date. Return the bottle to the incubator and change the medium with 5% packaged culture medium for 4-6 hours.

[0144] To prepare 5% packaged culture medium, open a new 500mL bottle of DMEM medium and add 27.5mL of FBS and 27.5mL of Gibco KnockOut. TM Serum substitute (catalog number 10828028) was added to 550 μL of 1 mol / L sodium pyruvate solution and 550 μL of 25 mmol / L chloroquine phosphate solution, and mixed well.

[0145] (6) Take out the 293T cells obtained in step (5) from the incubator, discard the old culture medium, add 25mL of 5% packaged culture medium to each bottle, and then put it back into the incubator to continue culturing. After culturing for 72 hours, 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) into a 1.5 mL centrifuge tube.

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

[0149] (3) Cell staining: Take 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 antibody supernatant from step (2), incubate at 4℃ 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, centrifuge, add flow cytometry buffer, and then perform the test.

[0152] Flow cytometry results showed that clones 3, 10, and 12 all bound to 293T-PDL1, with clone 10 showing a higher positive rate. Figures 2-3 Similarly, after co-incubation with 293T cells, secondary antibody incubation with mouse anti-human FC-PE was performed for 1 hour, followed by flow cytometry analysis. The results are as follows: Figure 4 As shown in the flow cytometry results, clones 3, 10, and 12 did not bind to 293T. These conclusions indicate that the binding of clones 3, 10, and 12 to 293T-PDL1 is specific.

[0153] The sequences of the nanobody VHH and the 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 3 is shown in SEQ ID NO:1, the nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 10 is shown in SEQ ID NO:6, the nucleotide sequence is shown in SEQ ID NO:7, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 12 is shown in SEQ ID NO:11, the nucleotide sequence is shown in SEQ ID NO:12, and the amino acid sequences of its CDR1, CDR2 and CDR3 are shown in SEQ ID NO: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 the 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 the 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 the 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 72h supernatant obtained in Example 3 was aliquoted into 50mL centrifuge tubes and centrifuged in a centrifuge with centrifugation parameters set to 4000g, 25min, and 4℃.

[0177] (2) Transfer the supernatant of the centrifuged lentivirus solution to a 022μm vacuum filtration system for filtration.

[0178] (3) Large-volume concentration was carried out using hollow fiber membrane columns / membrane packs. After concentration of 40×, it was purified by AKATA. Protein G was selected as the packing material.

[0179] (4) Small-volume antibody supernatant was purified using a gravity purification column, with Protein G and 30KD ultrafiltration tubes as the packing material for concentration.

[0180] (5) The purified antibody can be used for WB detection.

[0181] The band sizes of the fusion antibodies (clones 3, 10, and 12) were all close to 50 kDa. After the clone 10 protein was expressed in host cells, the antibody or flow-through sample retained during purification could be used for protein concentration detection. Figure 5 The purified and concentrated antibody product is then subjected to Western blotting (WB) analysis, such as... Figure 6 As shown, the size is correct, and the bands are complete, single, and without breakage, making them suitable for the subsequent large-scale antibody purification process using AKATA.

[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 72h, and detect the positive rate of RFP and EGFP by flow cytometry. Subsequent experiments can only be carried out if the positive rate reaches more than 90%.

[0184] (2) Take the above cells for cell counting;

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

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

[0187] (5) Flow cytometry testing.

[0188] A schematic diagram of the antibody blocking test experimental procedure is shown below. Figure 7 As shown. The blocking results of the anti-PD-L1 antibody during co-incubation are as follows. Figures 8 to 13 As shown, flow cytometry results for K562-PD1-RFP and K562-PD-L1 EGFP cell lines without anti-PDL1 antibody blockade showed that K562-PD1-RFP could bind to K562-PD-L1. When antibodies from clones 3, 10, and 12 were added to the mixed cell culture of K562-PD1-RFP and K562-PD-L1 EGFP, they could bind to K562-PD-L1 EGFP and prevent K562-PD1-RFP from binding to K562-PD-L1, with the antibody from clone 10 showing a stronger blocking effect.

[0189] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. An anti-PD-L1 nanobody, wherein the anti-PD-L1 nanobody comprises a complementarity-determining region (CDR1), a complementarity-determining region (CDR2), and a complementarity-determining region (CDR3), wherein, The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-PD-L1 nanobody are shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; or The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-PD-L1 nanobody are shown in SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively; or The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the anti-PD-L1 nanobody are shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15, respectively.

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 functionally identical or similar to the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11 after substitution, deletion or addition of one or more amino acids.

3. A fusion protein comprising a first domain and a second domain; The first structural domain is the anti-PD-L1 nanobody as described in claim 1 or 2; The second domain has the effect of prolonging the in vivo half-life.

4. The fusion protein according to claim 3, characterized in that, The second structural domain includes: Immunoglobulin Fc region; and / or Serum albumin or a fragment thereof, a domain that binds to serum albumin, polyethylene glycol, a polyethylene glycol-liposome complex, or a combination thereof.

5. The fusion protein according to claim 4, characterized in that, The immunoglobulin is selected from one or more combinations of IgG, IgA1, IgA2, IgD, IgE, and IgM.

6. The fusion protein according to any one of claims 3 to 5, characterized in that, The first and second domains are hinged together or connected by a linker peptide.

7. Biomaterials relating to the anti-PD-L1 nanobody of claim 1 or 2 or the fusion protein of any one of claims 3 to 6; said biomaterials being any one of a1) to a12): a1) A nucleic acid molecule encoding the anti-PD-L1 nanobody as described in claim 1 or 2 or the fusion protein as described in any one of claims 3 to 6; a2) An expression cassette containing the nucleic acid molecule described in a1); a3) A recombinant vector containing the nucleic acid molecules described in a1); a4) A recombinant vector containing the expression cassette described in a2); a5) Recombinant microorganisms containing the nucleic acid molecules described in a1); a6) Recombinant microorganisms containing the expression cassette described in a2); a7) Recombinant microorganisms containing the recombinant vector described in a3); a8) Recombinant microorganisms containing the recombinant vector described in a4); a9) Transgenic cell lines containing the nucleic acid molecules described in a1); a10) Transgenic cell lines containing the expression cassette described in a2); a11) Transgenic cell lines containing the recombinant vector described in a3); a12) Transgenic cell lines containing the recombinant vector described in a4).

8. A drug comprising the anti-PD-L1 nanobody of claim 1 or 2, the fusion protein of any one of claims 3 to 6, or the biomaterial of claim 7.

9. The use of the anti-PD-L1 nanobody of claim 1 or 2, the fusion protein of any one of claims 3 to 6, the biomaterial of claim 7, or the drug of claim 8 in the preparation of formulations, kits, or cassettes for the diagnosis, treatment, or prevention of cancer: The cancers include: Lung cancer, melanoma, stomach cancer, ovarian cancer, colon cancer, liver cancer, kidney cancer, bladder cancer, breast cancer, head and neck cancer, or nasopharyngeal cancer, or a combination thereof.

10. A kit or pharmaceutical kit comprising the anti-PD-L1 nanobody of claim 1 or 2, the fusion protein of any one of claims 3 to 6, the biomaterial of claim 7, or the drug of claim 8.

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