CD20 nano antibody as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a CD20 nano antibody as well as a preparation method and application thereof. The nano antibody can specifically recognize and bind CD20, has good affinity with CD20, and can be used for preparing products for diagnosing, preventing or treating diseases or symptoms related to B cells and / or CD20, or detecting existence or level of CD20 in a sample.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a CD20 nano antibody and a preparation method and application thereof. Background Art

[0002] Leukocyte differentiation antigen-20, also known as cluster of differentiation-20 (CD20), is a non-glycosylated phosphoprotein of the MS4A family. It is 33-37 kDa in size and consists of 297 amino acids. It is a transmembrane protein with four highly conserved transmembrane domains. The extracellular region is 44 amino acids long, providing binding sites for CD20 antibodies. There are three subtypes of CD20 that have been discovered (33, 35 and 37 kDa), which are produced due to different degrees of phosphorylation. It is reported that CD20 phosphorylation in proliferating malignant B cells is higher than that in resting B cells. As a surface antigen of B cells, CD20 only appears in the pre-B cell to mature B cell stage, and is not expressed on hematopoietic stem cells, progenitor B cells and mature plasma cells. CD20+ lymphocytes have been shown to be involved in antibody-mediated humoral immunity, T lymphocyte antigen presentation, and the production of various cytokines and chemokines that play a role in immune regulation. The CD20 protein plays a role in connecting BCR signals and the immune microenvironment by promoting calcium ion transport, regulating cell cycle progression, and coordinating the interaction between the homotypic B cell receptor (BCR) and its co-receptor. CD20 is highly expressed on the surface of B cells, but is absent in early Pro-B cells or plasmablasts, and plays a role in their differentiation into plasma cells and activation of antigen-independent T cell responses. In addition, CD20 is also an immunotherapy target for B cell lymphoma and leukemia, as well as some myelomas, thymomas, and Hodgkin's disease. Various evidences show that CD20 has a positive effect on disease treatment. CD20 therapy has been widely used to treat diseases with high expression of the CD20 antigen, and different CD20 monoclonal antibodies bind to unique epitopes on the surface of CD20, with good effects on different cancers and diseases.

[0003] CD20 is a very important B-cell tumor monoclonal antibody drug target and autoimmune disease drug target. Currently, the most approved and under-development drugs for CD20 are monoclonal antibodies. Anti-CD20 antibodies act on B cells through a variety of molecular mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), programmed cell death (PCD), and antibody-dependent cellular phagocytosis (ADCP). Due to its wide range of therapeutic areas, CD20 is not only loved by B-cell tumor treatment developers, but also favored by autoimmune developers. Its unparalleled value has kept it on the road of drug research.

[0004] Therefore, there is a need to develop a CD20 nanobody. Summary of the invention

[0005] The purpose of the first aspect of the present invention is to provide a nanobody or an antigen-binding fragment thereof that specifically binds to CD20.

[0006] The second aspect of the present invention aims to provide a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to CD20.

[0007] The third aspect of the present invention aims to provide a chimeric antigen receptor.

[0008] The fourth aspect of the present invention aims to provide a multispecific antibody or an antigen-binding fragment thereof.

[0009] The fifth aspect of the present invention aims to provide an isolated nucleic acid molecule.

[0010] The sixth aspect of the present invention aims to provide a carrier.

[0011] The seventh aspect of the present invention aims to provide a cell.

[0012] The purpose of the eighth aspect of the present invention is to provide a method for preparing the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0013] The ninth aspect of the present invention aims to provide a conjugate.

[0014] The tenth aspect of the present invention aims to provide a pharmaceutical composition.

[0015] The eleventh aspect of the present invention aims to provide a diagnostic or therapeutic kit.

[0016] The object of the twelfth aspect of the present invention is to provide the use of the nanoantibody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0017] In order to achieve the above object, the technical solution adopted by the present invention is:

[0018] In a first aspect of the present invention, a Nanobody or an antigen-binding fragment thereof that specifically binds to CD20 is provided.

[0019] In some embodiments, the Nanobody or antigen-binding fragment thereof that specifically binds to CD20 comprises:

[0020] The CDR-H1, CDR-H2 and CDR-H3 included in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 12, 14, 19, 24, or 29.

[0021] A nanobody or an antigen-binding fragment thereof that specifically binds to CD20, wherein the nanobody or an antigen-binding fragment thereof that specifically binds to CD20 comprises:

[0022] a1) a heavy chain variable region comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:9, CDR-H2 having the amino acid sequence shown in SEQ ID NO:10, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:11; or

[0023] a2) a heavy chain variable region comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO: 16, CDR-H2 having the amino acid sequence shown in SEQ ID NO: 17, and CDR-H3 having the amino acid sequence shown in SEQ ID NO: 18; or

[0024] a3) a heavy chain variable region comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:21, CDR-H2 having the amino acid sequence shown in SEQ ID NO:22, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:23; or

[0025] a4) a heavy chain variable region comprising the following three CDRs: CDR-H1 having the amino acid sequence shown in SEQ ID NO:26, CDR-H2 having the amino acid sequence shown in SEQ ID NO:27, and CDR-H3 having the amino acid sequence shown in SEQ ID NO:28.

[0026] In some embodiments, the heavy chain variable region of the Nanobody or antigen-binding fragment thereof that specifically binds to CD20 also includes a framework region of the heavy chain variable region.

[0027] In some embodiments, the framework region of the heavy chain variable region includes the framework region of the heavy chain variable region of an immunoglobulin derived from mouse, primate, bovine, horse, cattle, pig, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0028] In some embodiments, the Nanobody or antigen-binding fragment thereof that specifically binds to CD20 comprises:

[0029] b1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or

[0030] b2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or

[0031] b3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or

[0032] b4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or

[0033] b5) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0034] In the present invention, the CD20 is human CD20; further, it is CD20 whose amino acid sequence is shown in SEQ ID NO: 1.

[0035] The second aspect of the present invention provides a heavy chain antibody or an antigen-binding fragment thereof that specifically binds to CD20, which comprises an immunoglobulin Fc domain and the Nanobody or an antigen-binding fragment thereof of the first aspect of the present invention.

[0036] In some embodiments, the immunoglobulin Fc domain comprises an Fc domain of an immunoglobulin from mouse, primate, bovine, horse, cattle, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof.

[0037] The third aspect of the present invention provides a chimeric antigen receptor, which includes an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain includes the nanobody or antigen binding fragment thereof of the first aspect of the present invention or the heavy chain antibody or antigen binding fragment thereof of the second aspect.

[0038] A fourth aspect of the invention provides a multispecific antibody or antigen-binding fragment thereof, which comprises two or more (e.g., three or four) antigen-binding domains, wherein one antigen-binding domain comprises the nanobody or antigen-binding fragment thereof of the first aspect of the invention or the heavy chain antibody or antigen-binding fragment thereof of the second aspect.

[0039] A fifth aspect of the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof of the first aspect of the invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect.

[0040] Those skilled in the art will appreciate that nucleotides in a nucleic acid molecule may be substituted based on codon degeneracy. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized.

[0041] In some embodiments, the nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof of the first aspect of the invention comprises: SEQ ID NO: 13, 15, 20, 25, or 30, or a nucleotide sequence having at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

[0042] The sixth aspect of the present invention provides a vector comprising the nucleic acid molecule of the fifth aspect of the present invention.

[0043] In some embodiments, the vector may be an expression vector. In some embodiments, the expression vector may include a eukaryotic expression vector and / or a prokaryotic expression vector. In some embodiments, the eukaryotic expression vector includes, for example, but is not limited to, a yeast expression vector, a mammalian expression vector, and an insect expression vector. For example, the expression vector may include, but is not limited to, a plasmid, a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

[0044] In some embodiments, the carrier can be selected from nanoparticles, liposomes, exosomes, microbubbles or gene guns.

[0045] The seventh aspect of the present invention provides a cell, which includes the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, or the vector of the sixth aspect.

[0046] In some embodiments, the cells are not involved in propagation material.

[0047] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector can stably express the carried nucleic acid molecule as the above-mentioned nano antibody or antigen-binding fragment thereof, heavy chain antibody or antigen-binding fragment thereof, chimeric antigen receptor or multispecific antibody or antigen-binding fragment thereof disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell, the prokaryotic cell can include, for example, Escherichia coli, the eukaryotic cell can include, for example, CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, HeLa cells, Vero cells, Expi293 cells, hybridoma cells, yeast cells, and insect cells.

[0048] In some embodiments, the cell can be an immune cell. In some embodiments, the immune cell can include, but is not limited to, T cells, NK cells, DC cells and macrophages. In these embodiments, the immune cell can express the above-mentioned chimeric antigen receptor of the present disclosure (i.e., a modified immune cell).

[0049] The eighth aspect of the present invention provides a method for preparing the nanoantibody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, or the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, which is obtained by culturing the cells of the seventh aspect of the present invention.

[0050] A ninth aspect of the present invention provides a conjugate comprising the Nanobody or antigen-binding fragment thereof of the first aspect of the present invention, or the heavy chain antibody or antigen-binding fragment thereof of the second aspect; and a coupling portion.

[0051] In some embodiments, the conjugated moiety may include, but is not limited to, a detectable label or a therapeutic agent.

[0052] In some embodiments, the detectable marker can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C, or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridinium ester compounds, magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned markers. In some embodiments, such labels can be suitable for immunological detection (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescent immunoassay, chemiluminescent immunoassay, etc.). In some embodiments, the detectable label is selected from a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme. In some embodiments, the detectable label as described above can be connected to the nanobody or its antigen-binding fragment, or heavy chain antibody or its antigen-binding fragment of the present disclosure by linkers of different lengths to reduce potential steric hindrance.

[0053] In some embodiments, the detectable marker may include, but is not limited to, an enzyme (eg, horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (eg, a chemiluminescent substance), a colored substance, biotin, and the like.

[0054] In some embodiments, the therapeutic agent may include, for example, but not limited to, chemotherapeutic agents, immunosuppressants, cytotoxic drugs.

[0055] In some embodiments, the coupling moiety is selected from substances that can improve the biological properties of the antibody (eg, increase serum half-life), for example, it can be a chemical group such as polyethylene glycol (PEG), methyl, ethyl or sugar group.

[0056] The tenth aspect of the present invention provides a pharmaceutical composition, which includes: the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect or the conjugate of the ninth aspect; and a pharmaceutically acceptable carrier.

[0057] In some embodiments, the pharmaceutical composition may further include additional pharmaceutically active agents.

[0058] In some embodiments, the additional pharmaceutically active agent may be a biologically active drug, such as a drug capable of treating a disease or condition associated with B cells and / or CD20. In some embodiments, the additional pharmaceutically active agent may be selected from, but not limited to, a tumor immunotherapy agent, and / or a chemotherapeutic drug.

[0059] Preferably, the tumor immunotherapeutic agent comprises: at least one of a monoclonal antibody, an immune checkpoint inhibitor, an immune cell, an oncolytic virus, and a tumor vaccine.

[0060] Preferably, the target of the monoclonal antibody is selected from one of CD20, HER2, VEGF / VEGFR, EGFR, CD19, FGL1, CD47, CD3, CD30, CD33, CD38, CD52, αVβ3, α5β1, FAP, Tenascin, CEA, EPCAM, PSMA, GAN-GD2, GAN-GD3, GM2, and IGF-IR.

[0061] Preferably, the immune checkpoint inhibitor is an inhibitor that acts on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands.

[0062] Preferably, the negative co-stimulatory (co-inhibitory) molecules acting on T cells and / or their respective ligands are selected from one or more of CTLA-4, PD-1, PD-L1, PD-L2, B7-1, B7-2, B7-H3, B7-H4, B7-H6, A2aR, IDO, TIM-3, BTLA, VISTA, TIGIT, LAG-3, CD40, CD20, CD96, CD73, CD160, STING, CEA, CD47, PVRIG, LAIR1, 2B4, KIR, CEACAM1, GARP, PS, CSF1R, CD94 / NKG2A, TDO, TNFR and DcR3.

[0063] Preferably, the inhibitor acting on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands comprises any one of (b1) to (b3):

[0064] (b1) antibodies that specifically bind to (neutralize) T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands;

[0065] (b2) ligand proteins or polypeptides that specifically bind to (neutralize) T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands;

[0066] (b3) Non-protein compounds that specifically bind to (neutralize) T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands.

[0067] Preferably, the inhibitor of the ligand of the T cell negative co-stimulatory (co-inhibitory) molecule is selected from: CTLA-4 antibodies (such as ipilimumab, ticilimumab (CP-675,206), AGEN-1884, ATOR-1015, MGD019 (PD-1 / CTLA-4 dual antibody)), PD-1 antibodies (such as nivolumab, pembrolizumab, tremelimu mab), tislelizumab (BGB-A317), spartalizumab, MEDI0680, PDR001, FAZ053, MGA012 (retifanlimab), sintilimab, toripalimab, cemiplimab, MGD019 (PD-1 / CTLA-4 dual antibody), MGD013 (tebotelimab,PD-1 / LAG-3 dual antibody), PD-L1 antibody (e.g., atezolizumab, camrelizumab, durvalumab, avelumab, LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105), PD-L2 antibody, B7-1 antibody, B7-2 antibody, B7-H3 antibody (e.g., enoblituzu mAb), MGD009, MGC018), B7-H4 antibody, B7-H6 antibody, A2aR antibody (CPI-444, PBF509), IDO antibody (e.g. GDC0919 (navoximod), epacadostat, indoximid, BMS986205), TIM-3 antibody (e.g. TSR022 (TIM-3 monoclonal antibody), MBG453 (TIM-3 monoclonal antibody)), BTLA antibody, VISTA antibody, TIGIT antibody (e.g. BMS-986207, AB 154, COM902 (CGEN-15137), OMP-313M32), LAG-3 antibodies (e.g., BMS 986016, MK-4280 (28G-10), REGN3767, GSK2831781, IMP731 (H5L7BW), BAP050, IMP-701 (LAG-5250), IMP321, TSR-033, LAG525, BI 754111, FS-118, MGD013 (tebotelimab, PD-1 / LAG-3 dual antibody)), CD40 antibodies (e.g., BMS3h-56, lucatumumab (HCD122 and CHIR-12.12), CHIR-5.9 or dacetuzumab (huS2C6, PRO 64553, RG3636, SGN 14, SGN-40)), CD20 antibodies (e.g., rituximab (RITUXAN; IDEC-102; IDEC-C2B8), ABP 798, ofatumumab or obinutuzumab), CD96 antibodies, CD73 antibodies (e.g., MEDI9447 (oleclumab)), CD160 antibodies (e.g., BY55), STING antibodies, CEA antibodies (e.g., cergutuzumab abamalamin (RG7813,RO-6895882) or RG7802(RO6958688)), CD47 antibodies (such as HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231 or Effi-DEM), PVRIG antibodies (such as COM701(CGEN-15029)), LAIR1 antibodies, 2B4 antibodies, KIR antibodies (such as lirilumab(1-7F9, B MS-986015, IPH2101), IPH4102), CEACAM1 antibody (such as CM-24 (MK-6018)), GARP antibody (such as ARGX-115), PS antibody, CSF1R antibody (such as pexidartinib, LY3022855, FPA008, BLZ945), CD94 / NKG2A antibody, TDO antibody, TNFR antibody and DcR3 antibody.

[0068] Preferably, the immune cells include: chimeric antigen receptor T cells (CAR-T), chimeric antigen receptor NK cells (CAR-NK), T cell receptor chimeric T cells (TCR-T), tumor infiltrating immune cells (TILs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, and at least one of natural killer (NK) cells.

[0069] Preferably, the oncolytic virus comprises at least one of alphavirus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, polio virus, influenza virus, mumps virus and parvovirus; further comprises at least one of alphavirus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus.

[0070] Preferably, the tumor vaccine comprises at least one of a dendritic cell (DCs) vaccine, a nucleic acid vaccine and a polypeptide vaccine.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof and the additional pharmaceutically active agent are provided as separate components or as mixed components.

[0072] In some embodiments, the pharmaceutical composition can be administered, for example, parenterally, subcutaneously, sublingually, rectally, nasally, intravenously, intramuscularly, orally, ophthalmically, topically, or the like.

[0073] In some embodiments, the pharmaceutical composition is in the form of, for example, an aqueous solution, suspension, powder, tablet, capsule, granule, powder, pill, disintegrant, syrup, spray, gel, emulsion, injection, elixir, lozenge, suppository, etc.

[0074] The eleventh aspect of the present invention provides a diagnostic or therapeutic kit, which comprises: the nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect.

[0075] In some embodiments, the kit may further include instructions and / or an administration device.

[0076] In some embodiments, the kit can be used to diagnose a disease or condition associated with B cells and / or associated with CD20.

[0077] In some embodiments, the kit can be used to prevent or treat a disease or condition associated with B cells and / or associated with CD20.

[0078] The twelfth aspect of the present invention provides the use of the Nanobody or antigen-binding fragment thereof of the first aspect of the present invention, the heavy chain antibody or antigen-binding fragment thereof of the second aspect, the chimeric antigen receptor of the third aspect, the multispecific antibody or antigen-binding fragment thereof of the fourth aspect, the nucleic acid molecule of the fifth aspect, the vector of the sixth aspect, the cell of the seventh aspect, the conjugate of the ninth aspect, or the pharmaceutical composition of the tenth aspect in the preparation of a product, wherein the product is used for any one of c1) to c3):

[0079] c1) diagnosing a disease or condition, said disease or condition being associated with B cells and / or associated with CD20;

[0080] c2) preventing or treating a disease or condition which is associated with B cells and / or associated with CD20;

[0081] c3) detecting the presence or level of CD20 in the sample.

[0082] In some embodiments, the sample is selected from at least one of body fluids, tissues, cells, and excrement of the subject to be tested.

[0083] In some embodiments, the body fluid comprises at least one of blood and lymph.

[0084] In some embodiments, the blood comprises at least one of serum, plasma, dried blood spots, and whole blood.

[0085] In some embodiments, the tissue comprises tumor tissue.

[0086] In some embodiments, the excreta comprises at least one of urine, feces, and tears.

[0087] In some embodiments, the subject to be tested comprises mammals, such as humans, non-human primates (such as gorillas, apes), rodents (such as rats, mice, guinea pigs), pets (such as cats, dogs), and livestock (such as horses, cows, sheep, pigs, rabbits).

[0088] In some embodiments, the subject comprises a human.

[0089] In the present invention, the disease or condition is a tumor. In some embodiments, the disease or condition is a B-cell malignancy. In some embodiments, the B-cell malignancy is a B-cell leukemia or a B-cell lymphoma. In some embodiments, the disease or condition is selected from the group consisting of: marginal zone lymphoma (e.g., splenic marginal zone lymphoma), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), primary central nervous system (CNS) lymphoma, primary mediastinal B-cell lymphoma (PMBL), small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia (B-PLL), follicular lymphoma (FL), Burkitt's lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B lymphoblastic leukemia, non-Hodgkin's lymphoma (NHL), high-grade B-cell lymphoma (HGBL), thymoma, Hodgkin's disease and multiple myeloma (MM). In some embodiments, the disease or condition is an autoimmune and / or inflammatory disease. In some embodiments, the autoimmune and / or inflammatory disease is associated with inappropriate or increased B cell numbers and / or activation.

[0090] The beneficial effects of the present invention are:

[0091] The present invention provides a nanobody or an antigen-binding fragment thereof that specifically binds to CD20, which can specifically recognize and bind to CD20 and has good affinity thereto, and can be used to prepare a product for diagnosing, preventing or treating a disease or condition associated with B cells and / or associated with CD20, or detecting the presence or level of CD20 in a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1A schematic diagram showing the results of affinity testing between nanobody 16G11 and antigen is shown.

[0093] Figure 2 A schematic diagram showing the results of affinity testing between nanobody 13G4 and antigen is shown.

[0094] Figure 3 A schematic diagram showing the results of affinity testing of nanobody 16A3 with antigen is shown.

[0095] Figure 4 A schematic diagram showing the results of affinity testing between nanobody 13D10 and antigen is shown.

[0096] Figure 5 A schematic diagram showing the results of affinity testing between nanobody 16H5 and antigen is shown. DETAILED DESCRIPTION

[0097] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0098] definition

[0099] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0100] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0101] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0102] CD20(Cluster of Differentiation-20):Leukocyte differentiation cluster-20, also known as leukocyte differentiation antigen-20, is a non-glycosylated phosphoprotein of the MS4A family. It is 33-37 kDa in size and consists of 297 amino acids. It is a transmembrane protein with four highly conserved transmembrane domains. The extracellular region is 44 amino acids long, providing a binding site for the CD20 antibody. There are currently three subtypes of CD20 (33, 35 and 37 kDa) that have been discovered, which are caused by different degrees of phosphorylation.

[0103] K D value :Dissociation constant (dissociation constant, K D ) is a specific type of equilibrium constant that measures the tendency of a larger object to separate (dissociate) from another smaller component. It is the reciprocal of the association constant and has units of mol / L (M) or nmol / L (nM). K D The smaller the value, the stronger the binding ability of the two substances.

[0104] Nanobodies : An antibody that naturally lacks light chains and exists in the peripheral blood of camelids. This antibody contains only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions, but it is not as easy to stick to each other or even aggregate into clumps as artificially modified single-chain antibody fragments; the VHH structure cloned and expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody, and is the smallest unit known to bind to the target antigen; the VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only about 15kD, so it is also called a nanobody (Nanobody, Nb). Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune system in camelids can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanobodies.

[0105] Unlike traditional technologies that rely on classic model animals such as mice, rabbits, monkeys, and sheep, the technical solution of the present invention relies on antibodies produced by the immune system of alpacas, which are called "nanoantibodies". Nanoantibodies are tiny antibody fragments separated from immunoglobulins in animals such as camels. They have the same antigen binding ability and structural stability as complete antibodies. They are the smallest units that can bind to target antigens, with a relative molecular mass of only about 15kD. Compared with traditional animals such as mice and rabbits that can only recognize flat polypeptides on the surface of antigens, the immune systems in animals such as alpacas can recognize the complex spatial structure of the antigen surface and can produce highly specific and high-affinity nanoantibodies.

[0106] According to the technical solution of the present invention, certain amino acids in the amino acid sequence can be conservatively substituted without changing the activity or function of the protein, as shown in Table 1 below:

[0107] Table 1

[0108] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu; Val

[0109] In addition, due to the degeneracy of the bases, the bases of the polynucleotide sequence can be substituted without changing the activity or function of the polynucleotide sequence, as shown in Table 2 below:

[0110] Table 2

[0111]

[0112] Examples and drawings are provided below to help understand the present invention. However, it should be understood that these examples and drawings are only used to illustrate the present invention, but do not constitute any limitation. The actual protection scope of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0113] Example 1. Preparation of antigens

[0114] (1) The DNA vector plasmid encoding human CD20 (pCDNA3.1) was co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher);

[0115] (2) Continue to culture cells for about 48 h after transfection to allow CD20 to be fully expressed on the cell membrane surface;

[0116] (3) Collect about 4*10^8 cells, and after ultrasonic disruption, homogenization, and ultracentrifugation, take out the cell membrane extract, which is the appropriate antigen; wherein the preparation steps of the cell membrane extract are as follows:

[0117] 1) Take out about 4*10^8 cells collected from -80℃, add appropriate amount of buffer, and let it stand on ice to thaw.

[0118] 2) Add about 6 mL of Buffer (Buffer: PBS pH 7.4, cocktail) to each construct.

[0119] 3) Homogenization: For each construction, transfer the cells to a pre-cooled Dulbecco homogenizer and grind them evenly up and down for about 20 times.

[0120] 4) Ultrasonic disruption: Transfer the sample to a pre-cooled 15 mL tube and use an ultrasonic rod of appropriate thickness and power for ultrasonic disruption.

[0121] 5) Collect the supernatant by centrifugation and record it as supernatant 1;

[0122] 6) Resuspend with 6 mL of buffer, grind the homogenate about 20 times, and collect the supernatant by centrifugation, which is recorded as supernatant 2;

[0123] 7) Repeat the previous step and record it as supernatant 3;

[0124] 8) Combine supernatants 1+2+3 to a total volume of about 15-20 mL, and ultracentrifuge at 50,000 g for 1-1.5 h.

[0125] 9) Discard the supernatant and resuspend the precipitate with an appropriate amount of PBS to obtain the cell membrane extract.

[0126] 10) Mix all the cell membrane extracts constructed uniformly, control the total volume to about 4 mL, divide into 1.5 mL EP tubes, divide into 4 tubes, 1 mL per tube, and freeze at -80°C.

[0127] CD20 can be correctly expressed and present on the cell membrane surface, maintaining the correct assembly, folding and conformational state, especially maintaining the complex spatial structure of the surface.

[0128] The amino acid sequence of fully expressed human CD20 is as follows:

[0129]

[0130] The nucleotide sequence corresponding to human CD20 is as follows:

[0131]

[0132] Example 2. Alpaca Immunization

[0133] In this example, the cell membrane extract (appropriate antigen, CD20) of Example 1 was used to immunize alpacas. The specific steps are as follows:

[0134] (1) The antigen in Example 1 was evenly divided into 4 portions, each portion was about 0.5 mg; the alpacas were immunized 4 times in total, and the antigen was injected subcutaneously into the animal body, with the first immunization being recorded as day 1, and the subsequent immunizations were recorded on days 11, 21, and 31 respectively;

[0135] (2) On day 30, before the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca;

[0136] (3) On day 45, i.e. 14 days after the fourth immunization, approximately 200 mL of peripheral venous blood was collected from the alpaca.

[0137] Compared with traditional immunization technology solutions for animal antibodies such as mice and rabbits, the technical advantage of the present invention lies in the collection of a large amount of alpaca venous peripheral blood, which is conducive to subsequent screening to obtain highly diverse nano-antibodies.

[0138] Example 3. Construction of Alpaca Nanobody Library

[0139] The two batches of alpaca venous peripheral blood collected in Example 2 were used as raw materials to construct a highly diverse nanoantibody library. The two batches of alpaca venous peripheral blood were processed in the same way, and the specific steps were as follows:

[0140] (1) Lymphocytes were isolated from alpaca venous peripheral blood using density gradient centrifugation and other methods;

[0141] (2) extracting total mRNA from lymphocytes and reverse transcribed into cDNA;

[0142] (3) using appropriate DNA primers (see Table 3 below) and the above cDNA as a template, amplifying the VHH fragments of alpaca immunoglobulins IgG2 and IgG3, i.e., the DNA fragments of the nanobody, by polymerase chain reaction (PCR);

[0143] Table 3. Primers used to construct the nanobody library

[0144]

[0145]

[0146] (4) connecting the VHH DNA to the phage surface display screening vector phen1 to form a VHH-pIII fusion protein expression vector plasmid library; wherein pIII is a protein present on the flagella on the surface of the phage;

[0147] (5) The DNA ligation product is transformed into TG1 competent Escherichia coli by electroporation. After appropriate cultivation, all colonies are collected to obtain the nanoantibody library of the alpaca.

[0148] Compared with the traditional method of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, the present invention can preserve all the nano-antibody fragments (i.e., the library) of alpacas for a long time, and can continuously support the subsequent screening and development of nano-antibodies.

[0149] Example 4. Screening of specific nanobodies by phage surface display

[0150] This example uses the nanobody library obtained in Example 3 as a source, and obtains antigen-specific nanobodies through phage surface display screening. The specific steps are as follows:

[0151] (1) Taking an appropriate amount of frozen nanobody library, inoculating it into bacterial culture medium, adding an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) after appropriate cultivation, and continuing to culture under appropriate conditions;

[0152] (2) extracting the amplified phages from the bacterial culture supernatant by the PEG-NaC method and freezing them in a -80°C ultra-low temperature freezer for future use;

[0153] (3) The DNA vector plasmid encoding human CD20 was co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher), and the cells were cultured for 24 hours after transfection;

[0154] (4) Negative screening: prepare about 5*10 7 HEK293T tool cells that have not been transfected with any exogenous DNA vector plasmids, thaw the phage, incubate the phage and tool cells for 2 hours, centrifuge and retain the supernatant;

[0155] (5) Positive screening: Collect about 5*10 7 HEK293T cells overexpressing human CD20 were incubated with the supernatant obtained after centrifugation in (4) for 2 hours.

[0156] (6) Washing: Discard the phages and rinse the antigen cells three times with PBS buffer to wash and remove the phages that non-specifically bind to the antigen and retain the phages that specifically bind to the antigen.

[0157] (7) Elution: Treat the phages that specifically bind to the antigen with an acidic glycine solution to dissociate the phages from the antigen and retain them.

[0158] At this point, phages expressing specific nanobodies have been obtained, and these phages can be used for the following technical operations:

[0159] (8) Transformation into a specific nanobody library: The phage is infected again and cultured to TG1 competent E. coli, but no helper phage is added. After the phage infection is complete, the specific nanobody exists in the E. coli in the form of a DNA plasmid. Collect all these E. coli to form an antigen-specific nanobody library. This library can be used as a raw material and return to step (1) for the next round of phage surface display screening;

[0160] (9) Transformation into monoclonal nanoantibody colonies: Take a small amount of the phage obtained in step (7) (e.g., 0.5%), dilute it, and infect and culture it again until it reaches TG1 competent E. coli, but no helper phage is added. After the phage infection is complete, the E. coli are evenly spread on a bacterial culture dish, and monoclonal colonies containing nanoantibody DNA plasmids can be obtained under appropriate conditions. These monoclonal colonies are used as raw materials to identify positive monoclonal nanoantibodies.

[0161] Example 5. Identification of positive monoclonal nanobodies and nanobody sequencing

[0162] This example uses step (9) of Example 4 to obtain a bacterial culture dish with monoclonal colonies, and conducts identification of positive monoclonal nanoantibodies. The specific steps are as follows:

[0163] (1) Pick a single clone and culture it in a microplate;

[0164] (2) adding IPTG to induce the expression of VHH-pIII (i.e., the fusion protein containing the nanobody);

[0165] (3) The DNA vector plasmid encoding human CD20 was co-transfected into HEK293T tool cells using liposome transfection reagent (Lipo3000, ThermoFisher), and the cells were cultured for 24 hours after transfection;

[0166] (4) collecting the bacterial culture supernatant containing the nanobody obtained in (2), and incubating it with the cells obtained in (3) for 2 hours, using approximately 2*10^5 cells overexpressing human CD20 in each well of a 96-well plate, and centrifuging after the incubation to discard the supernatant;

[0167] (5) Incubate the cells obtained in (4) above with His tag-specific mouse monoclonal antibody labeled with Alexa Flour 647 (brand: Research & Development, IC0501R) for 2 hours;

[0168] (6) Centrifuge, discard the supernatant, and resuspend the cells with PBS buffer. Resuspend each well of the 96-well plate with 100 μL of PBS solution;

[0169] (7) Use flow cytometry to detect the distribution of Alexa Flour 647 fluorescence intensity on the cell surface to determine whether the nanoantibody can bind to CD20 on the cell surface.

[0170] (8) For monoclonal nanobodies (16G11, 13G4, 16A3, 13D10, 16H5) that can specifically recognize and bind to antigens, the TG1 strain expressing the relevant monoclonal nanobodies was cultured at 37°C overnight, the DNA plasmid was extracted and Sanger sequencing was performed to obtain the nucleotide sequence of the nanobody, and then the amino acid sequence of the nanobody was obtained after translation, as shown in Table 4-8.

[0171] Table 4: Amino acid sequence and nucleotide sequence of 16G11

[0172]

[0173] Table 5: Amino acid sequence and nucleotide sequence of 13G4

[0174]

[0175]

[0176] Table 6: Amino acid sequence and nucleotide sequence of 16A3

[0177]

[0178] Table 7: Amino acid sequence and nucleotide sequence of 13D10

[0179]

[0180] Table 8: Amino acid sequence and nucleotide sequence of 16H5

[0181]

[0182]

[0183] Example 6. Small batch monoclonal nanobody recombinant expression and purification

[0184] (1) Example 5 obtained monoclonal nanobodies that can specifically recognize and bind to antigens. The DNA plasmids encoding the above nanobodies (16G11, 13G4, 16A3, 13D10, 16H5) were transformed into BL21 (DE3) competent cells, and the nanobodies were recombinantly expressed in Escherichia coli. Monoclonal nanobodies were obtained through bacterial lysis, histidine tag affinity chromatography, gel filtration sequence and other steps. The batch production capacity is about several milligrams.

[0185] (2) Using flow cytometry analysis, different concentrations of nanobodies were incubated, and the affinity of the nanobody to the antigen was measured based on the binding ability of the nanobody to cells overexpressing human CD20 (the construction method was the same as in Example 1).

[0186] Test results such as Figure 1-5 As shown, the affinity values ​​K of monoclonal nanoantibodies 16G11, 13G4, 16A3, 13D10, and 16H5 D They are 3.765μM, 1.818μM, 2.811μM, 0.1134μM and 3.609μM respectively.

[0187] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A nanobody or an antigen-binding fragment thereof that specifically binds to CD20.

2. The nanobody or antigen-binding fragment thereof according to claim 1, characterized in that The nanobody or antigen-binding fragment thereof that specifically binds to CD20 includes: CDR-H1, CDR-H2 and CDR-H3 included in the heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 12, 14, 19, 24, or 29; Preferably, the Nanobody or antigen-binding fragment thereof that specifically binds to CD20 comprises a heavy chain variable region, and the heavy chain variable region comprises: a1) CDR-H1 having the amino acid sequence of SEQ ID NO:9, CDR-H2 having the amino acid sequence of SEQ ID NO:10, and CDR-H3 having the amino acid sequence of SEQ ID NO:11; or a2) CDR-H1 having the amino acid sequence of SEQ ID NO: 16, CDR-H2 having the amino acid sequence of SEQ ID NO: 17, and CDR-H3 having the amino acid sequence of SEQ ID NO: 18; or a3) CDR-H1 having the amino acid sequence of SEQ ID NO:21, CDR-H2 having the amino acid sequence of SEQ ID NO:22, and CDR-H3 having the amino acid sequence of SEQ ID NO:23; or a4) CDR-H1 having the amino acid sequence of SEQ ID NO: 26, CDR-H2 having the amino acid sequence of SEQ ID NO: 27, and CDR-H3 having the amino acid sequence of SEQ ID NO: 28; Preferably, the heavy chain variable region of the Nanobody or antigen-binding fragment thereof that specifically binds to CD20 further includes a framework region of the heavy chain variable region; Preferably, the framework region of the heavy chain variable region comprises a framework region of a heavy chain variable region of an immunoglobulin derived from mouse, primate, bovine, horse, cattle, porcine, sheep, goat, dog, cat, rabbit, camel, donkey, deer, mink, chicken, duck or goose, or a mutant thereof; Preferably, the Nanobody or antigen-binding fragment thereof that specifically binds to CD20 comprises: b1) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or b2) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or b3) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or b4) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:24, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto; or b5) a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:29, or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto.

3. A heavy chain antibody or antigen-binding fragment thereof that specifically binds to CD20, comprising an immunoglobulin Fc domain and a nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2.

4. A chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and an intracellular signaling domain, wherein the antigen binding domain comprises the nanobody or antigen binding fragment thereof according to any one of claims 1 to 2 or the heavy chain antibody or antigen binding fragment thereof according to claim 3.

5. A multispecific antibody or antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2 or the heavy chain antibody or antigen-binding fragment thereof according to claim 3.

6. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, or the multispecific antibody or antigen-binding fragment thereof according to claim 5. A vector comprising the nucleic acid molecule according to claim 6.

8. A cell comprising the nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, or the vector according to claim 7.

9. A method for preparing the nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, or the multispecific antibody or antigen-binding fragment thereof according to claim 5, which is obtained by culturing the cell according to claim 8.

10. A conjugate comprising the Nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, or the heavy chain antibody or antigen-binding fragment thereof according to claim 3; and a coupling portion; Preferably, the conjugated moiety comprises a detectable label or a therapeutic agent; Preferably, the detectable marker comprises an enzyme, a radionuclide, a fluorescent dye, a luminescent substance, a colored substance, and / or biotin; Preferably, the therapeutic agent comprises a chemotherapeutic agent, an immunosuppressant and / or a cytotoxic drug.

11. A pharmaceutical composition comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8 or the conjugate according to claim 10; and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises an additional pharmaceutically active agent.

12. A diagnostic or therapeutic kit comprising: The nanobody or antigen-binding fragment thereof according to any one of claims 1 to 2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, the conjugate according to claim 10, or the pharmaceutical composition according to claim 11; Preferably, the kit further comprises instructions and / or an administration device.

13. Use of the Nanobody or antigen-binding fragment thereof according to any one of claims 1-2, the heavy chain antibody or antigen-binding fragment thereof according to claim 3, the chimeric antigen receptor according to claim 4, the multispecific antibody or antigen-binding fragment thereof according to claim 5, the nucleic acid molecule according to claim 6, the vector according to claim 7, the cell according to claim 8, the conjugate according to claim 10, or the pharmaceutical composition according to claim 11 in the preparation of a product, wherein the product is used for any one of c1)-c3): c1) diagnosing a disease or condition, said disease or condition being associated with B cells and / or associated with CD20; c2) preventing or treating a disease or condition which is associated with B cells and / or associated with CD20; c3) detecting the presence or level of CD20 in the sample; Preferably, the disease or disorder is a tumor; Preferably, the disease or disorder is an autoimmune and / or inflammatory disease.

Images