Cd20 nanobodies and methods of making and using the same

Abstract

The present application belongs to the field of biotechnology, and particularly relates to a CD20 nanobody and a preparation method and application thereof. The nanobody can specifically recognize and combine with CD20, and has good affinity therewith, and can be used for preparing products for diagnosing, preventing or treating diseases or disorders related to B cells and / or CD20, or detecting the presence or level of CD20 in a sample.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to CD20 nanobodies, their preparation methods, and applications. Background Technology

[0002] Leukocyte differentiation antigen-20, also known as Cluster of Differentiation-20 (CD20), is a non-glycosylated phosphoprotein of the MS4A family. It measures 33-37 kDa and consists of 297 amino acids. It is a transmembrane protein with a highly conserved four-transmembrane domain and an extracellular region of 44 amino acids, providing a binding site for CD20 antibodies. Three subtypes of CD20 (33, 35, and 37 kDa) have been identified, resulting from varying degrees of phosphorylation. It has been reported that CD20 phosphorylation is higher in proliferating malignant B cells than in resting B cells. As a surface antigen of B cells, CD20 is only present in the pre-B cell to mature B cell stage and is not expressed in hematopoietic stem cells, progenitor B cells, or mature plasma cells. CD20+ lymphocytes have been shown to participate in antibody-mediated humoral immunity, T lymphocyte antigen presentation, and the production of various cytokines and chemokines that play a role in immune regulation. CD20 protein acts as a link between BCR signaling and the immune microenvironment by promoting calcium ion transport, regulating cell cycle progression, and coordinating the interaction between the isotype B cell receptor (BCR) and its co-receptors. CD20 is highly expressed on the surface of B cells but absent in early Pro-B cells or plasmablasts, playing a role in their differentiation into plasma cells and activation of antigen-independent T cell responses. Furthermore, CD20 is also a target for immunotherapy in B-cell lymphomas and leukemias, as well as some myelomas, thymomas, and Hodgkin's diseases. Various pieces of evidence suggest that CD20 has a positive impact on disease treatment; CD20 therapy has been widely used to treat diseases with high CD20 antigen expression, and different CD20 monoclonal antibodies bind to unique epitopes on the CD20 surface, showing good efficacy against various cancers and diseases.

[0003] CD20 is a crucial target for monoclonal antibody drugs targeting B-cell tumors and autoimmune diseases. Currently, monoclonal antibody drugs are the most numerous in approved and investigational developments targeting CD20. Anti-CD20 antibodies exert their effects on B cells through multiple molecular mechanisms, including complement-dependent cytotoxicity (CDC), antibody-dependent cytotoxicity (ADCC), programmed cell death (PCD), and antibody-dependent phagocytosis (ADCP). Due to its broad therapeutic applications, CD20 is favored not only by developers of B-cell tumor therapies but also by developers of autoimmune drugs, and its unparalleled value ensures its continuous pursuit in drug research.

[0004] Therefore, it is necessary to develop a CD20 nanobody. Summary of the Invention

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

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

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

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

[0009] The fifth aspect of this invention aims to provide isolated nucleic acid molecules.

[0010] The sixth aspect of this invention is to provide a carrier.

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

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

[0013] The object of the ninth aspect of the present invention is to provide a coupling.

[0014] The object of the tenth aspect of this invention is to provide a pharmaceutical composition.

[0015] The object of the eleventh aspect of this invention is to provide a diagnostic or therapeutic reagent kit.

[0016] The object of the twelfth aspect of the present invention is to provide 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 carrier 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] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

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

[0019] In some embodiments, the CD20-specifically binding nanobody or its antigen-binding fragment comprises:

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

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

[0022] a1) includes the heavy chain variable region of 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) includes the heavy chain variable region of 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) includes the heavy chain variable region of 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) includes the heavy chain variable regions of 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 CD20-specifically binding nanobody or its antigen-binding fragment further includes the 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 or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

[0028] In some embodiments, the CD20-specifically binding nanobody or its antigen-binding fragment comprises:

[0029] b1) Heavy chain variable region, comprising the amino acid sequence shown in 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 with it; or

[0030] b2) Heavy chain variable region, comprising the amino acid sequence shown in 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 with it; or

[0031] b3) Heavy chain variable region, comprising the amino acid sequence shown in 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 with it; or

[0032] b4) Heavy chain variable region, comprising the amino acid sequence shown in 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 with it; or

[0033] b5) 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 with it.

[0034] In this invention, the CD20 is human CD20; more specifically, it is CD20 with an amino acid sequence as shown in SEQ ID NO:1.

[0035] A second aspect of the invention provides 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 of the first aspect of the invention.

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

[0037] A third aspect of the invention provides a chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises a nanobody or an antigen-binding fragment thereof of the first aspect of the invention, or a heavy chain antibody or an antigen-binding fragment thereof of the second aspect of the invention.

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

[0039] A fifth aspect of the invention provides isolated nucleic acid molecules comprising nucleotide sequences encoding nanobodies or antigen-binding fragments thereof of the first aspect of the invention, heavy chain antibodies or antigen-binding fragments thereof of the second aspect, chimeric antigen receptors of the third aspect, or multispecific antibodies or antigen-binding fragments thereof of the fourth aspect.

[0040] Those skilled in the art will understand that nucleotides in nucleic acid molecules can 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 its antigen-binding fragment of the first aspect of the present 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 with it.

[0042] A sixth aspect of the present invention provides a carrier 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 eukaryotic cell expression vectors and / or prokaryotic cell expression vectors. In some embodiments, the eukaryotic expression vector includes, for example, but not limited to, yeast expression vectors, mammalian expression vectors, and insect expression vectors. For example, the expression vector may include, but is not limited to, plasmids, retroviral vectors, lentiviral vectors, bacteriophage vectors, adenovirus vectors, adeno-associated vectors, or herpes simplex vectors.

[0044] In some embodiments, the carrier may be selected from nanoparticles, liposomes, exogenous bodies, microbubbles, or gene guns.

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

[0046] In some embodiments, the cells do not involve reproductive material.

[0047] In some embodiments, the cell can be a host cell conventionally used in the art, as long as the expression vector stably expresses the carried nucleic acid molecule as the nanobody or its antigen-binding fragment, heavy chain antibody or its antigen-binding fragment, chimeric antigen receptor or multispecific antibody or its antigen-binding fragment disclosed herein. In some embodiments, the host cell can be a prokaryotic cell and / or a eukaryotic cell. The prokaryotic cell may include, for example, *Escherichia coli*, and the eukaryotic cell may include, for example, CHO cells, HEK293 cells, BHK cells, NSO 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 cells may be immune cells. In some embodiments, the immune cells may include, but are not limited to, T cells, NK cells, dendritic cells (DCs), and macrophages. In these embodiments, the immune cells may express the chimeric antigen receptors described above (i.e., modified immune cells).

[0049] The eighth aspect of the present invention provides 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, obtained by culturing the cells of the seventh aspect of the present invention.

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

[0051] In some implementations, the coupling portion may include, but is not limited to, a detectable marker or a therapeutic agent.

[0052] In some embodiments, the detectable marker can be any substance detectable by means of fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, chemistry, etc. Such markers are well known in the art, and examples 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)), acridine esters, magnetic beads, calorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above markers. In some embodiments, such markers are suitable for immunological assays (e.g., enzyme-linked immunosorbent assay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable marker is selected from radioactive isotopes, fluorescent substances, luminescent substances, colored substances, or enzymes. In some embodiments, the detectable markers described above can be linked to the nanobodies or their antigen-binding fragments, or heavy chain antibodies or their antigen-binding fragments, of this disclosure using linkers of different lengths to reduce potential steric hindrance.

[0053] In some embodiments, the detectable marker may include, but is not limited to, enzymes (e.g., horseradish peroxidase), radionuclides, fluorescent dyes, luminescent substances (e.g., chemiluminescent substances), colored substances, biotin, etc.

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

[0055] In some embodiments, the coupling portion is selected from substances that can improve the biological properties of the antibody (e.g., increase serum half-life), such as chemical groups, such as polyethylene glycol (PEG), methyl, ethyl, or glycosyl groups.

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

[0057] In some embodiments, the pharmaceutical composition may also 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 diseases or conditions related to B cells and / or CD20. In some embodiments, the additional pharmaceutically active agent may be selected from, but is not limited to, tumor immunotherapy agents and / or chemotherapy drugs.

[0059] Preferably, the tumor immunotherapy agent comprises at least one of the following: monoclonal antibody, immune checkpoint inhibitor, immune cell, oncolytic virus, and 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 inhibitors acting on T cell negative co-stimulatory (co-inhibitory) molecules and / or their respective ligands comprise any one of (b1) to (b3):

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

[0065] (b2) Specific binding (neutralization) of ligand proteins or peptides that act on negative costimulatory (co-inhibitory) molecules and / or their respective ligands on T cells;

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

[0067] Preferably, the inhibitor of the ligand of the T cell negative co-stimulatory (co-inhibitory) molecule is selected from: CTLA-4 antibodies (e.g., ipilimumab, ticilimumab; CP-675,206), AAGEN-1884, ATOR-1015, MGD019 (PD-1 / CTLA-4 bispecific antibody)), PD-1 antibodies (e.g., nivolumab, pembrolizumab, tremilimumab). mab), tislelizumab (BGB-A317), spartalizumab, MEDI0680, PDR001, FAZ053, MGA012 (retifanlimab), sintilimab, toripalimab, cemiplimab, MGD019 (PD-1 / CTLA-4 bispecific antibody), MGD013 (tebotelimab,PD-1 / LAG-3 bispecific antibodies), PD-L1 antibodies (e.g., atezolizumab, camrelizumab, durvalumab, avelumab, LY3300054, CX-072 (Proclaim-CX-072), FAZ053, KN035, MDX-1105), PD-L2 antibodies, B7-1 antibodies, B7-2 antibodies, B7-H3 antibodies (e.g., enoblituzumab) (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 bispecific 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., cergutuzumabamunaleukin (RG7813),RO-6895882 or RG7802 (RO6958688)), CD47 antibodies (e.g., HuF9-G4, CC-90002, TTI-621, ALX148, NI-1701, NI-1801, SRF231 or Effi-DEM), PVRIG antibodies (e.g., COM701 (CGEN-15029)), LAIR1 antibodies, 2B4 antibodies, KIR antibodies (e.g., lirilumab (1-7F9, B)). MS-986015, IPH2101), IPH4102), CEACAM1 antibody (e.g., CM-24 (MK-6018)), GARP antibody (e.g., ARGX-115), PS antibody, CSF1R antibody (e.g., pexidartinib, LY3022855, FPA008, BLZ945), CD94 / NKG2A antibody, TDO antibody, TNFR antibody, and DcR3 antibody are selected from one or more of these.

[0068] Preferably, the immune cells comprise at least one of the following: 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 natural killer (NK) cells.

[0069] Preferably, the oncolytic virus comprises at least one of the following: alpha virus, adenovirus, vaccinia virus, Sindbis virus, Seneca Valley virus, Coxsackie virus, measles virus, reovirus, vaccinia virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, poliovirus, influenza virus, mumps virus, and parvovirus; further comprising at least one of the following: alpha virus, adenovirus, vaccinia virus, measles virus, vesicular stomatitis virus, and herpes simplex virus.

[0070] Preferably, the tumor vaccine comprises at least one of dendritic cell (DC) vaccines, nucleic acid vaccines, and peptide vaccines.

[0071] In some embodiments, the antibody or its antigen-binding fragment is provided as a separate component or as a mixed component with the additional pharmaceutically active agent.

[0072] In some embodiments, the pharmaceutical composition can be administered via, for example, parenteral, subcutaneous, sublingual, rectal, nasal, intravenous, intramuscular, oral, ocular, or topical routes.

[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 comprising: a nanobody or antigen-binding fragment thereof of the first aspect of the present invention, a heavy chain antibody or antigen-binding fragment thereof of the second aspect of the present invention, a chimeric antigen receptor thereof of the third aspect of the present invention, a multispecific antibody or antigen-binding fragment thereof of the fourth aspect of the present invention, a nucleic acid molecule thereof of the fifth aspect of the present invention, a carrier thereof of the sixth aspect of the present invention, a cell thereof of the seventh aspect of the present invention, a conjugate thereof of the ninth aspect of the present invention, or a pharmaceutical composition thereof of the tenth aspect of the present invention.

[0075] In some embodiments, the kit may also include instructions and / or a drug delivery device.

[0076] In some embodiments, the kit can be used to diagnose diseases or conditions that are associated with B cells and / or CD20.

[0077] In some embodiments, the kit can be used to prevent or treat diseases or conditions that are associated with B cells and / or CD20.

[0078] The twelfth aspect of the present invention provides the use of the nanobody or antigen-binding fragment thereof of the first aspect, 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 carrier 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, said product being used in any one of c1)-c3):

[0079] c1) Diagnose a disease or condition that is associated with B cells and / or with CD20;

[0080] c2) Prevention or treatment of diseases or conditions that are associated with B cells and / or CD20;

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

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

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

[0084] In some embodiments, the blood includes 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 excrement includes at least one of urine, feces, and tears.

[0087] In some implementations, the test subject includes mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits).

[0088] In some implementations, the subject of the test includes humans.

[0089] In this invention, the disease or symptom is a tumor. In some embodiments, the disease or symptom is a B-cell malignant tumor. In some embodiments, the B-cell malignant tumor is B-cell leukemia or 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 lymphoma, primary intraocular lymphoma, chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), hairy cell leukemia (HCL), precursor B-cell lymphoblastic leukemia, non-Hodgkin 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 diseases are associated with inappropriate or elevated B cell numbers and / or activation.

[0090] The beneficial effects of this invention are:

[0091] This invention provides a nanobody or antigen-binding fragment that specifically binds to CD20, which can specifically recognize and bind to CD20 and has good affinity for it. It can be used to prepare products for the diagnosis, prevention or treatment of diseases or conditions related to B cells and / or CD20, or for the detection of the presence or level of CD20 in a sample. Attached Figure Description

[0092] Figure 1A schematic diagram showing the affinity test results of nanobody 16G11 with the antigen is presented.

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

[0094] Figure 3 A schematic diagram showing the results of the affinity test between nanobody 16A3 and the antigen is presented.

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

[0096] Figure 5 A schematic diagram showing the results of the affinity test between nanobody 16H5 and the antigen is presented. Detailed Implementation

[0097] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0098] definition

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

[0100] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

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

[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 measures 33-37 kDa and consists of 297 amino acids. It is a transmembrane protein with a highly conserved four-transmembrane domain and an extracellular region of 44 amino acids, providing a binding site for CD20 antibodies. Currently, three isoforms of CD20 (33, 35, and 37 kDa) have been identified, resulting from different degrees of phosphorylation.

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

[0104] Nanobody Naturally occurring antibodies lacking the light chain, found in the peripheral blood of camels, contain only a single heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Unlike artificially engineered single-chain antibody fragments, they do not readily adhere to each other or aggregate. Individually cloned and expressed VHH structures exhibit structural stability and antigen-binding activity comparable to the original heavy chain antibody, representing the smallest known unit capable of binding to target antigens. VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only about 15 kDa, hence the name nanobody (Nb). Compared to traditional animals like mice and rabbits that can only recognize flat peptides on the surface of antigens, the immune system in camels can recognize the complex spatial structures on the antigen surface, producing 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 this invention utilizes antibodies produced by the alpaca's immune system, known as "nanobodies." Nanobodies are tiny antibody fragments isolated from immunoglobulins in animals such as camels. They possess the same antigen-binding ability and structural stability as intact antibodies and are the smallest existing units capable of binding target antigens, with a relative molecular mass of only about 15 kDa. Compared to traditional methods where mice and rabbits can only recognize flat polypeptides on the surface of antigens, the immune systems in animals like alpacas can recognize the complex spatial structures on the surface of antigens, enabling the production of highly specific and high-affinity nanobodies.

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

[0107] Table 1

[0108] residues Conservative replacement residues Conservative replacement 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] Furthermore, due to the degeneracy of bases, bases in polynucleotide sequences can be substituted without altering the activity or function of the polynucleotide sequence, as shown in Table 2 below:

[0110] Table 2

[0111]

[0112] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection 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 antigen

[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 culturing the cells for about 48 hours after transfection to allow CD20 to be fully expressed on the cell membrane surface;

[0116] (3) Collect approximately 4*10^8 cells, which are then subjected to ultrasonic disruption, homogenization, and ultracentrifugation. The cell membrane extract is then obtained as the suitable antigen. The preparation steps of the cell membrane extract are as follows:

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

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

[0119] 3) Homogenization: For each construct, aspirate and transfer cells to a pre-chilled DuPont homogenizer and homogenize them up and down approximately 20 times.

[0120] 4) Ultrasonic disruption: Transfer the sample to a pre-cooled 15mL tube and perform ultrasonic disruption using an ultrasonic bar of appropriate size and power.

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

[0122] 6) Resuspend in 6 mL of buffer, homogenize and grind about 20 times, centrifuge and collect the supernatant, which is recorded as supernatant 2;

[0123] 7) Repeat the previous step, and record this as Supernatant 3;

[0124] 8) Combine the supernatants 1, 2, and 3, with a total volume of approximately 15-20 mL. Centrifuge at 50000 g for 1-1.5 h.

[0125] 9) Discard the supernatant, resuspend the precipitate in an appropriate amount of PBS, and the result is the cell membrane extract.

[0126] 10) Mix all the cell membrane extracts from the construction thoroughly, with a total volume of about 4 mL. Aliquot the mixture into 1.5 mL EP tubes, divide into 4 tubes, 1 mL each, and store at -80°C.

[0127] CD20 is correctly expressed and present on the cell membrane surface, maintaining the correct assembly, folding and conformation, especially 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 Injection

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

[0134] (1) The antigen in Example 1 was divided into 4 portions, each containing about 0.5 mg; the alpacas were immunized a total of 4 times. The antigen was injected subcutaneously into the animals. The first immunization was recorded as day 1, and the subsequent immunizations were recorded as day 11, day 21, and day 31, respectively.

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

[0136] (3) On day 45, that is, 14 days after the fourth immunization, about 200 mL of alpaca peripheral blood was collected.

[0137] Compared to traditional immunization techniques using animal antibodies from mice, rabbits, etc., the technical advantage of this invention lies in the collection of a large amount of peripheral blood from alpacas, which is beneficial for subsequent screening to obtain highly diverse nanobodies.

[0138] Example 3. Construction of an alpaca nanobody library

[0139] Using two batches of alpaca peripheral blood collected in Example 2 as raw materials, a highly diverse nanobody library was constructed. The processing methods for the two batches of alpaca peripheral blood were the same, and the specific steps are as follows:

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

[0141] (2) Extract total mRNA from lymphocytes and reverse transcribe it into cDNA;

[0142] (3) Using appropriate DNA primers (see Table 3 below), the above cDNA was used as a template to amplify the VHH fragments of alpaca immunoglobulins IgG2 and IgG3 by polymerase chain reaction (PCR), which are the DNA fragments of nanobodies.

[0143] Table 3. Primers used to construct nanobody libraries

[0144]

[0145]

[0146] (4) The DNA of VHH was ligated to the phage surface display selection vector phen1 to form a VHH-pIII fusion protein expression vector plasmid library; where pIII is a protein present on the flagella of the phage surface.

[0147] (5) The DNA ligation product was transformed into TG1 competent Escherichia coli by electroporation. After appropriate culture, all colonies were collected to obtain the alpaca nanobody library.

[0148] Compared to traditional methods of isolating antibodies from the serum or lymphocytes of animals such as mice and rabbits, this invention can preserve all nanobody fragments (i.e., libraries) of alpacas for a long time, which can continuously support the subsequent screening and development of nanobodies.

[0149] Example 4. Displaying screening-specific nanobodies on the surface of bacteriophages

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

[0151] (1) Take an appropriate amount of frozen nanobody library, inoculate it into bacterial culture medium, and after appropriate culture, add an appropriate amount of helper phage (M13KO7 helper phage, NEB, N0315S) and continue to culture under appropriate conditions.

[0152] (2) The bacteriophages amplified in the bacterial culture supernatant were extracted by PEG-NaC method and frozen in an ultra-low temperature freezer at -80℃ for later 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 approximately 5*10 7 HEK293T tool cells that were not transfected with any exogenous DNA vector plasmids were thawed, and the phages were incubated with the tool cells for 2 hours. After centrifugation, the supernatant was collected.

[0155] (5) Positive screening: Collect approximately 5*10 from (3). 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, then wash the antigen cells three times with PBS buffer to wash away the phages that are not specifically bound to the antigen, and retain the phages that are specifically bound to the antigen.

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

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

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

[0160] (9) Conversion into monoclonal nanobody colonies: Take a small amount of the phage obtained in step (7) (e.g., 0.5%), dilute it, and re-infect TG1 competent E. coli, but do not add helper phages. After the phage infection is complete, spread these E. coli evenly on bacterial culture dishes and culture them under appropriate conditions to obtain monoclonal colonies containing nanobody DNA plasmids. Use these monoclonal colonies as raw materials to identify positive monoclonal nanobodies.

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

[0162] This embodiment utilizes the bacterial culture dish with monoclonal colonies obtained in step (9) of Example 4 to identify positive monoclonal nanobodies. The specific steps are as follows:

[0163] (1) Pick single colonies and culture them in microplates;

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

[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) Collect the bacterial culture supernatant containing nanobodies obtained in (2) and incubate it with the cells obtained in (3) for 2 hours. Use about 2*10^5 cells overexpressing human CD20 in each well of the 96-well plate. After incubation, centrifuge and discard the supernatant.

[0167] (5) Incubate the cells obtained in (4) above with the 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 the cells in 100 μL of PBS solution in each well of the 96-well plate.

[0169] (7) The distribution of Alexa Flour 647 fluorescence intensity on the cell surface was detected by flow cytometry to determine whether the nanobody could 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 DNA plasmids of the TG1 strain expressing the relevant monoclonal nanobodies were cultured overnight at 37°C and then Sanger sequencing was performed to obtain the nucleotide sequence of the nanobodies. After translation, the amino acid sequence of the nanobodies was obtained, as shown in Table 4-8.

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

[0172]

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

[0174]

[0175]

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

[0177]

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

[0179]

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

[0181]

[0182]

[0183] Example 6. Recombinant Expression and Purification of Small-Batch Monoclonal Nanobodies

[0184] (1) In Example 5, monoclonal nanobodies capable of specifically recognizing and binding to antigens were obtained. The DNA plasmids encoding the above nanobodies (16G11, 13G4, 16A3, 13D10, 16H5) were transformed into BL21(DE3) competent cells. The nanobodies were recombinantly expressed by Escherichia coli. The monoclonal nanobodies were obtained through bacterial lysis, histidine tag affinity chromatography, gel filtration sequence chromatography, etc., with a batch production capacity of about a few milligrams.

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

[0186] Test results as follows Figure 1-5 As shown, the affinity values ​​K for monoclonal nanobodies 16G11, 13G4, 16A3, 13D10, and 16H5 are... D The corresponding values ​​were 3.765 μM, 1.818 μM, 2.811 μM, 0.1134 μM, and 3.609 μM.

[0187] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A CD20-specifically binding nanobody or its antigen-binding fragment thereof, wherein the CD20-specifically binding nanobody or its antigen-binding fragment comprises a heavy chain variable region, the heavy chain variable region comprising: a1) CDR-H1 of the amino acid sequence shown in SEQ ID NO: 9, CDR-H2 of the amino acid sequence shown in SEQ ID NO: 10, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 11; or a2) CDR-H1 of the amino acid sequence shown in SEQ ID NO: 16, CDR-H2 of the amino acid sequence shown in SEQ ID NO: 17, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 18; or a3) CDR-H1 of the amino acid sequence shown in SEQ ID NO: 21, CDR-H2 of the amino acid sequence shown in SEQ ID NO: 22, and CDR-H3 of the amino acid sequence shown in SEQ ID NO: 23; or a4) CDR-H1 with the amino acid sequence shown in SEQ ID NO: 26, CDR-H2 with the amino acid sequence shown in SEQ ID NO: 27, and CDR-H3 with the amino acid sequence shown in SEQ ID NO:

28.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region of the specific CD20-binding nanobody or its antigen-binding fragment also includes the framework region of the heavy chain variable region.

3. The nanobody or its antigen-binding fragment according to claim 2, characterized in that, The frame region of the heavy chain variable region includes the frame region of the heavy chain variable region or a mutant thereof derived from immunoglobulins of mice, primates, cattle, horses, pigs, sheep, goats, dogs, cats, rabbits, camels, donkeys, deer, minks, chickens, ducks, or geese.

4. The nanobody or its antigen-binding fragment according to claim 3, characterized in that, The specific CD20-binding nanobody or its antigen-binding fragment includes: b1) Heavy chain variable region, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 12; or b2) Heavy chain variable region, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 14; or b3) Heavy chain variable region, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 19; or b4) Heavy chain variable region, comprising an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 24; or b5) Heavy chain variable region, which includes an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

29.

5. The nanobody or its antigen-binding fragment according to claim 4, characterized in that, The specific CD20-binding nanobody or its antigen-binding fragment includes: b1) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 12; or b2) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 14; or b3) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 19; or b4) Heavy chain variable region, comprising the amino acid sequence shown in SEQ ID NO: 24; or b5) Heavy chain variable region, which includes the amino acid sequence shown in SEQ ID NO:

29.

6. 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 as described in any one of claims 1-5.

7. A chimeric antigen receptor comprising an antigen-binding domain, a transmembrane domain, and an intracellular signal transduction domain, wherein the antigen-binding domain comprises the nanobody or its antigen-binding fragment as described in any one of claims 1-5, or the heavy chain antibody or its antigen-binding fragment as described in claim 6.

8. A multispecific antibody or an antigen-binding fragment thereof, comprising two or more antigen-binding domains, wherein one antigen-binding domain comprises the nanobody or an antigen-binding fragment thereof as described in any one of claims 1-5 or the heavy chain antibody or an antigen-binding fragment thereof as described in claim 6.

9. An isolated nucleic acid molecule, which is a nucleotide sequence encoding the nanobody or antigen-binding fragment of any one of claims 1-5, the heavy chain antibody or antigen-binding fragment of claim 6, the chimeric antigen receptor of claim 7, or the multispecific antibody or antigen-binding fragment of claim 8.

10. A carrier comprising the nucleic acid molecule of claim 9.

11. A cell comprising the nanobody or antigen-binding fragment thereof as described in any one of claims 1-5, the heavy chain antibody or antigen-binding fragment thereof as described in claim 6, the chimeric antigen receptor thereof as described in claim 7, the multispecific antibody or antigen-binding fragment thereof as described in claim 8, the nucleic acid molecule thereof as described in claim 9, or the carrier thereof as described in claim 10.

12. The method for preparing the nanobody or its antigen-binding fragment according to any one of claims 1-5, the heavy chain antibody or its antigen-binding fragment according to claim 6, the chimeric antigen receptor according to claim 7, or the multispecific antibody or its antigen-binding fragment according to claim 8, wherein the nanobody is obtained by culturing the cells according to claim 11.

13. A conjugate comprising a nanobody or an antigen-binding fragment thereof as described in any one of claims 1-5, or a heavy chain antibody or an antigen-binding fragment thereof as described in claim 6; and a conjugation portion.

14. The coupling according to claim 13, characterized in that, The coupling component includes a detectable marker or therapeutic agent.

15. The coupling according to claim 14, characterized in that, The detectable markers include enzymes, radionuclides, luminescent substances, colored substances, and / or biotin; or The therapeutic agents include chemotherapeutic agents, immunosuppressants, and / or cytotoxic drugs.

16. The coupling according to claim 15, characterized in that, The detectable markers include fluorescent dyes.

17. A pharmaceutical composition comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-5, the heavy chain antibody or its antigen-binding fragment according to claim 6, the chimeric antigen receptor according to claim 7, the multispecific antibody or its antigen-binding fragment according to claim 8, the nucleic acid molecule according to claim 9, the carrier according to claim 10, the cell according to claim 11, or the conjugate according to any one of claims 13-16; and pharmaceutically acceptable carriers.

18. The pharmaceutical composition according to claim 17, characterized in that, The pharmaceutical composition also includes additional pharmaceutically active agents.

19. A diagnostic or therapeutic reagent kit comprising: The nanobody or its antigen-binding fragment according to any one of claims 1-5, the heavy chain antibody or its antigen-binding fragment according to claim 6, the chimeric antigen receptor according to claim 7, the multispecific antibody or its antigen-binding fragment according to claim 8, the nucleic acid molecule according to claim 9, the carrier according to claim 10, the cell according to claim 11, the conjugate according to any one of claims 13-16, or the pharmaceutical composition according to any one of claims 17-18.

20. The reagent kit according to claim 19, characterized in that, The kit also includes instructions and / or a delivery device.

21. The use of the nanobody or antigen-binding fragment thereof according to any one of claims 1-5, the heavy chain antibody or antigen-binding fragment thereof according to claim 6, the chimeric antigen receptor according to claim 7, the multispecific antibody or antigen-binding fragment thereof according to claim 8, the nucleic acid molecule according to claim 9, the carrier according to claim 10, the cell according to claim 11, or the conjugate according to any one of claims 13-16 in the preparation of the product, wherein the product is used in any one of c1)-c2): c1) Diagnose a disease or condition that is associated with B cells and / or with CD20; c2) Detect the presence or level of CD20 in the sample; The disease or symptom is a tumor; or The disease or condition is an autoimmune and / or inflammatory disease.

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