Completely humanized antibody targeting CD22 and application thereof

Abstract

The invention relates to the field of antibodies, and provides a fully humanized antibody targeting CD22 and application thereof. The invention provides an anti-CD22 fully humanized single-domain antibody, which is combined with CD22 with high affinity and high specificity, has lower immunogenicity and side effect, and has good application potential in development of medicines for treating diseases related to CD22.

Description

Technical Field

[0001] The present invention relates to the field of antibodies, and more specifically, to fully human antibodies or single-domain antibodies or fragments thereof that specifically bind to human CD22 antigen protein and CD22 antigen in its natural state on the cell membrane surface. Background Art

[0002] Currently, nine CAR-T drugs have been approved for marketing worldwide. CD19 is one of the most popular targets for CAR-T therapy, specifically for B-cell lymphomas. Due to its proven efficacy and manageable side effects, it is the most widely used of the products currently on the market and in development. CAR-T therapy targeting CD19 is widely recognized as the perfect solution for B-cell malignancies. However, as clinical research deepens, increasing evidence indicates that approximately 50% of patients will experience relapse over time. Relapse can occur for a variety of reasons, primarily CD19-negative and CD19-positive relapses. Under the pressure of CAR-T therapy, some cancer cells that originally expressed CD19 will switch to expressing the CD22 protein.

[0003] CD22, also known as Siglec-2, is a lectin of the Siglec family, composed of seven IgG-like extracellular domains. CD22 is another member of the B cell antigen family, with a tissue distribution similar to CD19. CD22 expression on the B cell surface begins in the pre-B cell stage and continues on mature B cells. CD22 is not expressed on plasma cells. The CD22 protein is a type I transmembrane protein with a molecular weight of 140 kDa. It is also a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family and a B cell surface inhibitory coreceptor, closely associated with B cell development, differentiation, and function. CD22 is restricted to mature B cells and most malignant B lymphomas, making it a popular target for the treatment of autoimmune diseases and B cell malignancies. Currently, immunotherapies targeting CD22 include monoclonal antibodies, antibody conjugates (ADCs), and CAR-T therapies.

[0004] Although treatment outcomes for B-cell acute lymphoblastic leukemia and B-cell malignant lymphoma have greatly improved, a proportion of patients still experience relapse and refractory disease. Chimeric antigen receptor (CAR19) T-cell therapy targeting CD19 has demonstrated unprecedented therapeutic efficacy. However, CD19-negative relapse is the most common cause of CAR-T therapy failure, and effective treatment options are lacking, necessitating the search for novel treatments.

[0005] Given the effectiveness of CD22 as a therapeutic target in B-cell malignancies, there is an urgent need in the art to develop new and different forms of CD22-specific binding molecules. Summary of the Invention

[0006] The purpose of the present invention is to provide a fully human antibody targeting CD22 and its application.

[0007] In a first aspect of the present invention, an anti-CD22 single-domain antibody or antigen-binding fragment thereof is provided, wherein the single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HCDR1 as shown in SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2; and HCDR3 as shown in SEQ ID NO: 3; Alternatively, the antibody or antigen-binding fragment thereof comprises a variant of the CDR sequence, wherein the variant has at least 90% sequence identity compared to the CDR sequence, or comprises at least 1 and no more than 10, or no more than 5, 4, 3, 2 or 1 amino acid changes in the CDR sequence.

[0008] In another preferred embodiment, any one of the above amino acid sequences further comprises a derivative sequence that is optionally subjected to addition, deletion, modification and / or substitution of at least one (e.g., 1-3) amino acids and can retain the ability to specifically bind to CD22.

[0009] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 5 or has at least 90% sequence identity thereto.

[0010] In another preferred embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5.

[0011] In another preferred embodiment, the antibody or antigen-binding fragment thereof further includes a framework region FR.

[0012] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

[0013] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes a monomer, a bivalent antibody, and / or a multivalent antibody.

[0014] In another preferred embodiment, the bivalent antibody is a bispecific antibody.

[0015] In another preferred embodiment, the multivalent antibody is a multispecific antibody.

[0016] In another preferred embodiment, the antigen-binding fragment comprises a single domain fragment.

[0017] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

[0018] In another preferred embodiment, the heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0019] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has: (i) the single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and (ii) an optional tag sequence to facilitate expression and / or purification.

[0020] In another preferred example, the tag sequence includes an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

[0021] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0022] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0023] In a third aspect of the present invention, an isolated nucleic acid molecule is provided, wherein the nucleic acid molecule encodes the single domain antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or the recombinant protein as described in the second aspect of the present invention.

[0024] In another preferred embodiment, the nucleic acid molecule is selected from RNA (such as mRNA) and DNA (such as cDNA).

[0025] In another preferred embodiment, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 4.

[0026] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the nucleic acid molecule according to the third aspect of the present invention.

[0027] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0028] In another preferred embodiment, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0029] In another preferred embodiment, the expression vector is a plasmid, a retrovirus or a lentivirus vector.

[0030] In another preferred embodiment, the expression vector is selected from the following group: pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, etc.

[0031] In another preferred embodiment, the expression vector further comprises elements selected from the following group: a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, etc.

[0032] In the fifth aspect of the present invention, a host cell is provided, wherein the host cell contains the expression vector as described in the fourth aspect of the present invention, or the nucleic acid molecule as described in the third aspect of the present invention is integrated into its genome.

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

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

[0035] In the sixth aspect of the present invention, a chimeric antigen receptor CAR is provided, wherein the antigen binding region of the CAR specifically binds to CD22, and the antigen binding region comprises the single domain antibody or antigen binding fragment thereof as described in the first aspect of the present invention.

[0036] In another preferred embodiment, the antigen binding region comprises an amino acid sequence as shown in SEQ ID NO: 5 or an amino acid sequence having at least 90% sequence identity thereto.

[0037] In another preferred embodiment, the antigen binding region comprises the amino acid sequence shown in SEQ ID NO: 5.

[0038] In another preferred embodiment, the CAR further comprises a signal peptide.

[0039] In another preferred embodiment, the CAR also includes other exogenous proteins.

[0040] In another preferred embodiment, the CAR has the structure shown in Formula I: L-EB-H-TM-C-CD3ζ (I) Where, L is none or a signal peptide sequence; EB is the antigen-binding region that specifically binds to CD22; H is none or hinge region; TM is the transmembrane domain; C is the costimulatory signaling domain; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ (including wild type, or mutant / modified forms thereof); The "-" connects peptides or peptide bonds.

[0041] In another preferred embodiment, the L is selected from the signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants / modified forms thereof, or a combination thereof.

[0042] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0043] In another preferred embodiment, the TM is selected from the transmembrane region of the following histones: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants / modified forms thereof, or combinations thereof.

[0044] In another preferred embodiment, the C is selected from the costimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants / modified forms thereof, or combinations thereof.

[0045] In the seventh aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell expresses an exogenous CAR as described in the sixth aspect of the present invention.

[0046] In another preferred embodiment, the engineered immune cells are selected from the following group: (i) Chimeric antigen receptor αβ T cells (CAR-T cells); (ii) chimeric antigen receptor γδ T cells (CAR-T cells); (iii) chimeric antigen receptor NKT cells (CAR-NKT cells); (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0047] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0048] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0049] In an eighth aspect of the present invention, a method for producing an antibody or antigen-binding fragment thereof targeting CD22 is provided, comprising the steps of: (a) culturing the host cell according to the fifth aspect of the present invention under suitable conditions to obtain a culture containing an antibody or antigen-binding fragment thereof targeting CD22; (b) isolating and / or recovering the antibody or antigen-binding fragment thereof targeting CD22 from the culture; and (c) Optionally, purifying and / or modifying the CD22-targeting antibody or antigen-binding fragment thereof obtained in step (b).

[0050] In a ninth aspect of the present invention, an immunoconjugate is provided, comprising: (a) an antibody portion, said antibody portion comprising the single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention; and (b) a conjugated moiety selected from the group consisting of a detectable label, a therapeutic agent, or a combination thereof.

[0051] In another preferred embodiment, the coupling moiety is selected from the group consisting of toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, nanomagnetic particles, viral coat proteins or VLPs, or a combination thereof.

[0052] In another preferred embodiment, the (a) portion and the coupling portion are coupled via a chemical bond or a linker.

[0053] In another preferred embodiment, the radioactive nuclides include: (i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and / or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

[0054] In another preferred embodiment, the conjugated moiety may contain at least one therapeutic agent.

[0055] In another preferred embodiment, the conjugated moiety is a therapeutic agent.

[0056] In another preferred embodiment, the therapeutic agent is an immune checkpoint drug, and the immune checkpoint is selected from: PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR.

[0057] In another preferred embodiment, the immune checkpoint drug is an inhibitor or activator of the immune checkpoint.

[0058] In another preferred embodiment, the therapeutic agent is a cytotoxic drug.

[0059] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or a combination thereof.

[0060] Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposide, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0061] In another preferred embodiment, the coupling moiety is a toxin.

[0062] In another preferred embodiment, the toxin is selected from the group consisting of auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), chlortetracycline, maytansin, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxybenzoate, daptomycin, acetaminophen, chlortetracycline, chloramphenicol, chloramphenicol, daptomycin, chlortetracycline, chloramphenicol, daptomycin, chlortetracycline, chloramphenicol, chlortetracycline ... anthracnose dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, Mitogellin, Retstrictocin, phenomycin, enomycin, curcin, crotin, calicheamicin, a Sapaonaria officinalis inhibitor, a glucocorticoid, or a combination thereof.

[0063] In another preferred embodiment, the coupling moiety is a detectable label.

[0064] In another preferred embodiment, the coupling portion is selected from the following group: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)) or any form of nanoparticles.

[0065] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) single-domain antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0066] In another preferred embodiment, the multivalency refers to the presence of multiple repeats of the same or different single-domain antibodies or antigen-binding fragments thereof as described in the first aspect of the present invention in the amino acid sequence of the immunoconjugate.

[0067] In a tenth aspect of the present invention, a pharmaceutical composition is provided, comprising: (i) an active ingredient selected from the group consisting of a single domain antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a nucleic acid molecule as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, a chimeric antigen receptor CAR as described in the sixth aspect of the present invention, an engineered immune cell as described in the seventh aspect of the present invention, or an immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0068] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized preparation.

[0069] In another preferred example, the pharmaceutical composition comprises 0.01 to 99.99% of the single domain antibody or antigen binding fragment thereof as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, the nucleic acid molecule as described in the third aspect of the present invention, the expression vector as described in the fourth aspect of the present invention, the host cell as described in the fifth aspect of the present invention, the chimeric antigen receptor CAR as described in the sixth aspect of the present invention, the engineered immune cell as described in the seventh aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof and 0.01 to 99.99% of a pharmaceutically acceptable carrier, diluent or excipient, and the percentage is the mass percentage of the pharmaceutical composition.

[0070] In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1×10 3 -1×10 8 cells / mL, preferably 1×10 4 -1×10 7 cells / mL.

[0071] In the eleventh aspect of the present invention, there is provided a use of an active ingredient, wherein the active ingredient is selected from the group consisting of the single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, the nucleic acid molecule according to the third aspect of the present invention, the expression vector according to the fourth aspect of the present invention, the host cell according to the fifth aspect of the present invention, the chimeric antigen receptor CAR according to the sixth aspect of the present invention, the engineered immune cell according to the seventh aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof, wherein the active ingredient is used to prepare: (a) a drug for treating and / or preventing a CD22-associated disease or condition; and / or (b) A reagent for detecting a CD22-related disease, or a kit for detecting a CD22 protein or a fragment thereof.

[0072] In another preferred embodiment, the reagent is a diagnostic reagent; preferably, the diagnostic reagent is a detection sheet or a detection plate.

[0073] In another preferred embodiment, the diagnostic reagent is used to detect CD22 protein or a fragment thereof in a sample.

[0074] In another preferred embodiment, the disease or condition is a disease or condition in which CD22 is highly expressed or CD22 pathway is abnormal.

[0075] In another preferred embodiment, the disease or condition is selected from: cancer or autoimmune disease.

[0076] In another preferred embodiment, the disease or disorder is a tumor.

[0077] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0078] In another preferred embodiment, the tumor is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, and pancreatic cancer.

[0079] In another preferred embodiment, the cancer is B lymphocytic tumor.

[0080] In a twelfth aspect of the present invention, a method for detecting CD22 protein or a fragment thereof in a sample in vitro is provided, the method comprising the steps of: (1) contacting the sample in vitro with the single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD22 protein or its fragment in the sample.

[0081] In another preferred embodiment, the detection includes diagnostic or non-diagnostic detection.

[0082] In a thirteenth aspect of the present invention, a kit is provided, comprising: (1) a first container containing the single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof; and / or (2) a second container containing a secondary antibody against the contents of the first container; or, The kit contains a detection plate, which includes: a substrate (support plate) and a test strip, wherein the test strip contains the single-domain antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the recombinant protein as described in the second aspect of the present invention, or the immunoconjugate as described in the ninth aspect of the present invention, or a combination thereof.

[0083] In another preferred embodiment, the kit further comprises an instruction manual, according to which the kit is used for non-invasively detecting the expression of CD22 protein or a fragment thereof in a test subject.

[0084] In another preferred embodiment, the kit is used for detecting CD22-related diseases.

[0085] In the fourteenth aspect of the present invention, a method for preventing and / or treating a disease or condition is provided, the method comprising: administering to a subject in need thereof a therapeutically effective amount of a single domain antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a nucleic acid molecule as described in the third aspect of the present invention, an expression vector as described in the fourth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, a chimeric antigen receptor CAR as described in the sixth aspect of the present invention, an engineered immune cell as described in the seventh aspect of the present invention, an immunoconjugate as described in the ninth aspect of the present invention, or a pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof, to eliminate, inhibit or reduce CD22 activity, thereby preventing, alleviating, improving or inhibiting the disease or condition.

[0086] In another preferred embodiment, the subject includes mammals, such as humans.

[0087] In another preferred embodiment, the disease or condition is a disease or condition in which CD22 is highly expressed or CD22 pathway is abnormal.

[0088] In another preferred embodiment, the disease or condition is selected from: cancer or autoimmune disease.

[0089] In another preferred embodiment, the disease or disorder is a tumor.

[0090] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0091] In another preferred embodiment, the tumor is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, and pancreatic cancer.

[0092] In another preferred embodiment, the cancer is B lymphocytic tumor.

[0093] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0094] In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).

[0095] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0096] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0097] In a fifteenth aspect of the present invention, a method for diagnosing a CD22-related disease is provided, comprising the steps of: (i) obtaining a sample from a diagnostic subject, and contacting the sample with the single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the recombinant protein according to the second aspect of the present invention, or the immunoconjugate according to the ninth aspect of the present invention, or a combination thereof; and (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient of a CD22-related disease.

[0098] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

[0099] In another preferred embodiment, the disease or condition is a disease or condition in which CD22 is highly expressed or CD22 pathway is abnormal.

[0100] In another preferred embodiment, the disease or condition is selected from: cancer or autoimmune disease.

[0101] In another preferred embodiment, the disease or disorder is a tumor.

[0102] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0103] In another preferred embodiment, the tumor is selected from one or more of lung cancer, gastric cancer, liver cancer, colorectal cancer, melanoma, kidney tumor, ovarian cancer, prostate cancer, bladder cancer, breast cancer, esophageal cancer, colon cancer, nasopharyngeal cancer, brain tumor, cervical cancer, blood cancer, bone cancer, lymphoma, and pancreatic cancer.

[0104] In another preferred embodiment, the cancer is B lymphocytic tumor.

[0105] Those skilled in the art can easily discern other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application relates. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Figure 1 The general process of the present invention for screening specific antibodies targeting CD22 from a phage antibody library is shown.

[0107] Figure 2 The results of enzyme-linked immunosorbent assay (ELISA) of some selected phage monoclones with target antigen and control antigen are shown.

[0108] Figure 3 Flow cytometric analysis results of the binding of some phage monoclones to Raji and Jurkat cells are shown.

[0109] Figures 4A-4B Shown are the results of flow cytometric analysis of the binding of the screened phage monoclonals to a variety of different CD22-positive and -negative cell lines. Figure 4A To test the results of the positive control antibody using cell lines expressing CD22 antigen, Figure 4B Figure 3 is the flow cytometric analysis results of the screened phage monoclonal binding to different cell lines. NegativeControl is the negative control phage antibody clone, and FITC anti-mouse IgG Ab is the negative control with only the secondary antibody added.

[0110] Figure 5 Detailed information of Iaso-CD22(1,3,5,6,7)-bio antigen containing different domains is shown.

[0111] Figure 6Shown are the results of enzyme-linked immunosorbent assays (ELISAs) using the screened phage monoclonal clones against CD22 antigen proteins containing different domains and unrelated antigens. The negative control is a negative control phage antibody clone; the anti-M13 phage mouse Ab / anti-mouse HRP Ab are negative antibody controls using only the primary and secondary antibodies; the anti-mouse HRP Ab is negative antibody controls using only the secondary antibody; the M971 positive control Ab / anti-human IgG HRP Ab are positive antibody controls for the target antigen (CD22-Fc-Bio); the anti-human IgG-HRP Ab is the secondary antibody control for the M971 positive control antibody group; and the anti-rabbit IgG-HRP Ab / anti-human IgG-HRP Ab are positive antibody controls for detecting antigen tags. Among them, the bar graphs corresponding to each test antibody and control group represent the test results of proteins iaso-CD22-1-bio, iaso-CD22-3-bio, iaso-CD22-5-bio, iaso-CD22-6-bio, iaso-CD22-7-bio, Acro-CD22-bio, and Acro-CD19-bio from left to right.

[0112] Figure 7 This is the flow cytometric analysis result of the binding of the phage monoclonal obtained in Example 5 to the positive cell line after expressing the protein antibody. DETAILED DESCRIPTION

[0113] After extensive and in-depth research, the present inventors have developed fully human antibodies targeting CD22 and their applications. Specifically, the CD22 antibodies of the present invention bind to the CD22 antigen with high target specificity and affinity, particularly to CD22 expressed on the surface of tumor cells, while exhibiting minimal side effects. This work has led to the present invention.

[0114] the term Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0115] "Antibodies" refer to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (peptides, viruses, bacteria, etc.). These foreign substances are correspondingly called antigens. The basic structure of an antibody molecule is a tetramer composed of two identical heavy chains and two identical light chains. Based on the conservation of their amino acid sequences, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region of a heavy chain (HCVR, also known as VH) and the variable region of a light chain (LCVR, also known as VL) interact to form the antigen-binding site (Fv). Within the variable region, certain regions, called hypervariable regions (HVRs), have a more variable composition and order of amino acid residues than other regions within the variable region (framework regions, FRs). These regions are the key sites for antibody-antigen binding. Because these hypervariable regions are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Each heavy chain and light chain has three complementarity determining regions, namely HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3.

[0116] Single-chain antibodies (scFv) are composed of the variable regions of the heavy and light chains of an antibody, linked by a short peptide. Properly folded, the variable regions from the heavy and light chains interact through non-covalent bonds to form the Fv fragment, allowing scFv to retain its affinity for the antigen.

[0117] Single-domain antibodies (SDOs) are antibodies composed solely of the variable region amino acids of heavy-chain antibodies. With a molecular weight of only 12-15 kDa, they possess similar or even higher specificity and affinity than traditional antibodies. They are also attracting significant attention due to their stable physicochemical properties, high affinity, ease of recombinant expression, and ease of combination with antibodies targeting other targets or epitopes.

[0118] "Mouse antibodies" are antibodies produced by rodents against specific antigens, typically those produced by mouse B lymphocytes. In most cases, these mouse antibodies are monoclonal antibodies produced by hybridoma cells. The fully human antibodies described in this application are obtained by screening a human phage antibody library. Compared to mouse antibodies, these antibodies have reduced immunogenicity and are more suitable for therapeutic use in humans.

[0119] The "fully human antibody or single-domain antibody or fragment thereof" described in this application generally refers to any form of antigen-binding molecule that can bind to the target antigen, for example, the antigen-binding molecule can be a protein or polypeptide, including, for example, antibodies and antigen-binding fragments thereof, single-chain scFv antibodies, single-domain antibodies, various fusions and conjugates constructed based on scFv or single-domain antibodies, such as single-domain antibodies-Fc antibodies, immunoconjugates, antibody-drug conjugates (ADCs), multi / bispecific antibodies, and chimeric antigen receptors (CARs).

[0120] CD22 is restricted to mature B cells and most malignant B lymphoma cells, making it a popular target for the treatment of autoimmune diseases and B cell malignancies. There is an urgent need to develop new and diverse CD22-specific binding molecules. This application addresses this need by providing antibodies that bind to the CD22 antigen with high target specificity and affinity, particularly to CD22 expressed on the surface of tumor cells, while having minimal side effects.

[0121] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of consistency between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percentage of identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be consistent or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, the number of matching positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and / or the length of the gaps are also taken into account. For the purposes of the present invention, the publicly available alignment software BLAST (available on the webpage ncbi.nlm.nih.gov) can be used to obtain the best sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using the default settings. In some embodiments, "at least 90% sequence identity" as described herein includes, but is not limited to, at least 95%, at least 98%, at least 99% or even 100% sequence identity.

[0122] It will be understood by those skilled in the art that, based on the specific sequences provided herein, a small number of amino acids can be replaced, deleted, added, and the resulting products can be verified or screened for binding ability or biological activity to the corresponding antigen CD22, thereby obtaining corresponding variants of the CD22-targeting antibodies provided herein, and these variants should also be included within the scope of the present invention. For example, the fully human antibodies or single-domain antibodies or fragments thereof of the present application may have at least 1 and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid changes in the full length or CDR sequence.

[0123] In some embodiments, the antigen-binding molecules of the present application may further comprise post-translational modifications. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination-like, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Thus, the modified soluble polypeptide may comprise non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which binds one or more sialic acid groups to the polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins, while also reducing the potential immunogenicity of proteins. See Raju et al. Biochemistry. 2001 31; 40(30): 8868-76.

[0124] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely administered, is suitable for administration to humans and / or animals without excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent contained therein.

[0125] A "therapeutically effective amount" refers to an amount of an active compound sufficient to elicit a biological or medical response in a subject as desired by a clinician. The "therapeutically effective amount" of the antibodies of the present application can be determined by a person skilled in the art based on factors such as the route of administration, the subject's weight, age, and condition. For example, a typical daily dose range can be 0.01 mg to 100 mg of active ingredient per kg body weight. Administration of the antibodies of the present application includes, but is not limited to, injection, for example, intravenously, intramuscularly, intraarterially, subcutaneously, intraperitoneally, and the like.

[0126] "Epitope" refers to the portion of a molecule that is bound by an antigen binding protein (e.g., an antibody). An epitope can comprise non-contiguous portions of the molecule (e.g., amino acid residues in a polypeptide that are not contiguous in the primary sequence of the polypeptide but are sufficiently close to each other in the trivalent and tetravalent structures of the polypeptide to be bound by the antigen binding protein).

[0127] The present application provides a kit comprising one or more containers containing a plurality of gene constructs encoding the polypeptides of the present application and a pharmaceutically acceptable excipient. The kit may also include instructions for use. The kit may also include a notice in a form prescribed by an agency regulating the production, use, or sale of pharmaceuticals or biological products, indicating that the kit has been licensed by the agency for the production, use, or sale of pharmaceuticals for human use.

[0128] Research Overview This method uses fully human phage for antibody screening, directly obtaining fully human monoclonal antibodies. Compared with traditional hybridoma technology, this eliminates the difficult step of humanizing mouse antibodies. Furthermore, fully human antibodies have lower immunogenicity than humanized mouse antibodies, offering greater potential for applications in antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), and detection reagents.

[0129] The present invention uses an antigen protein panning method, which can efficiently enrich and simultaneously bind to recombinant CD22 and antibodies that bind to the natural structure of CD22 on the cell membrane, greatly reducing the difficulty of subsequent antibody screening and improving efficiency.

[0130] The present invention uses a large-capacity phage antibody library to screen fully human CD22-specific antibodies, and evaluates the specificity of these antibodies at the phage level through ELISA and FACS experiments. Ultimately, several fully human antibody clones with good specificity were obtained.

[0131] The present invention used different antibody libraries and panned with recombinant CD22 protein to select a total of 184 monoclonal clones for initial screening using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). Sixteen of these clones specifically bound to the CD22-Fc-Bio protein and CD22-positive Raji cells, but not to the control protein CD19-Fc-Bio or CD22-negative Jurkat cells. Sequencing yielded two distinct monoclonal sequences. Subsequently, these two antibodies were identified by flow cytometric analysis (FACS) with various CD22-positive (Raji, NALM6) and negative cell lines (Raji KO CD22, Jurkat, U266), and by enzyme-linked immunosorbent assay (ELISA) with CD22 proteins containing different domains (iaso-CD22-1-bio, iaso-CD22-3-bio, iaso-CD22-5-bio, iaso-CD22-6-bio, iaso-CD22-7-bio, Acro-CD22-bio) and non-related proteins (Acro-CD19-Bio). One of the clones showed good binding affinity and specificity on multiple cell lines and multiple protein antigens.

[0132] Subsequently, a human IgG4 antibody of this clone was prepared, and the binding ability of this antibody to the CD22-positive cell line (Raji) was further confirmed by flow cytometry at the protein level. The affinity of this clone was analyzed using Fortebio. This clone (#80) showed excellent affinity and had excellent binding ability and specificity on both antigens and cells. The acquisition of this clone laid the foundation for the subsequent development of fully human CD22 antibody drugs. The acquisition of these clones laid the foundation for the subsequent development of fully human CD22 CAR-T products or antibody drugs. The overall project process is as follows Figure 1 shown.

[0133] The sequence of SdAb 80 (Clone 80) protected by the present invention is as follows: HCDR1:GFIFTNEW (SEQ ID NO: 1); HCDR2: IRSDGGST (SEQ ID NO: 2); HCDR3: ARMYYWSLAGESYDW (SEQ ID NO: 3); VH DNA sequence (354 bp) GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTATCTTTACCAACGAATGGATGGGTTGGGTTCGTCAGGCACCTGGTAAAGGTCTGGAATGGGTTAGCAGCATCCGCAGCGATGGCGGCAGCACCTATTATGCAGA TAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAACAGCAAAAATACCCTGTACCTGCGATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCGCGCATGTACTACTGGTCTCTGGCTGGTGAATCTTACGATTGGTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA (SEQ IDNO: 4); VH amino acid sequence (118 aa) EVQLLESGGGLVQPGGSLRLSCAASGFIFTNEWMGWVRQAPGKGLEWVSSIRSDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMYYWSLAGESYDWWGQGTLVTVSS (SEQ ID NO: 5).

[0134] The present invention is described in detail below with reference to specific embodiments.

[0135] Example 1 Enrichment of specific antibody clones targeting CD22 protein from phage antibody library by affinity panning Appropriate negative and positive panning strategies are used to enrich the desired specific antibody clones from the phage antibody library.

[0136] Construction of phage antibody library: The constructed phage antibody libraries include single-domain libraries. Single-domain phage antibody libraries are composed solely of amino acids from the variable regions of heavy-chain antibodies. With a molecular weight of only 12-15 kDa, they possess similar specificity and affinity to traditional antibodies. Furthermore, single-domain antibodies have attracted considerable attention due to their stable physicochemical properties, high affinity, ease of recombinant expression, and ease of combination with antibodies targeting other targets or epitopes.

[0137] CD22 protein panning: Multiple rounds of panning were performed using CD22-Fc-Bio as the positive panning protein and CD19-Fc-Bio as the negative panning protein to obtain a phage pool enriched for the target antibody clone. The experimental steps are briefly described as follows:

[0138] 1) Block the SA magnetic beads with blocking solution for 2 hours, then bind the target antigen (CD22-Fc-Bio) to the blocked SA magnetic beads; 2) Add phage library (containing 5x10 12 phage particles) and incubated with a control antigen to subtract phage antibody clones that non-specifically bind to the Fc tag; 3) After incubation, transfer the supernatant to SA magnetic beads bound to the target antigen and continue incubation to allow the phage to bind to the target antigen; 4) Wash the magnetic beads with washing solution to remove unbound phages; 5) Elute the positive phage from the target antigen with elution buffer and neutralize with neutralization buffer; 6) Re-infect the host bacteria XL1-blue with the eluted phage and amplify the recovered phage. A small amount of sample is diluted serially and infected with the host bacteria. Plate the plate with Amp-resistant bacteria and count the recovered phage.

[0139] 7) Repeat steps 1) to 6) until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0140] The enriched phage pool can be used for subsequent single clone selection and ELISA / FACS screening.

[0141] Raji / Jurkat cell panning: Multiple rounds of panning were performed using CD22-negative Jurkat cells as negative panning cells and CD22-positive Raji cells as positive panning cells to obtain a phage pool enriched with the target antibody clone.

[0142] The brief experimental steps are as follows: 1) After protein panning, the phage pool (containing 5x10 11 phage particles) and 1x10 7 Mix the negatively selected Jurkat cells and incubate them on a rotating mixer at room temperature for 2 hours to allow the antibody clones that bind to the negatively selected cell line to fully bind to these cells;

[0143] 2) Centrifuge at 1500 rpm for 5 minutes to pellet the cells, transfer the supernatant to a new tube, and mix with 1x10 7 Raji cells (CD22 positive cells) were mixed and allowed to bind on a rotary mixer at room temperature for 2 hours; 3) Wash the cells six times with PBS, discard the supernatant each time, resuspend and centrifuge at 1500 rpm for 5 minutes to remove unbound phage; 4) Elute the positive phage from the target antigen with elution buffer and neutralize with neutralization buffer; 5) Re-infect the host bacteria with the eluted phage and amplify the recovered phage. A small amount of sample is diluted serially and infected with the host bacteria. Plate the plate with Amp-resistant bacteria and count the recovered phage.

[0144] 6) Repeat steps 1) to 5), usually 2 to 3 rounds of panning are required, until a significant increase in the phage recovery rate (number of eluted phage / number of input phage) is observed.

[0145] The enriched phage pool can be used for the next step of monoclonal selection and ELISA / FACS screening.

[0146] Main materials and reagents: Fully human phage antibody library, including natural library, semi-synthetic library and single domain library; Helper phage KO7, Thermo / Invitrogen, 18311019; Biotinylated Human Siglec-2 / CD22 Protein, Fc,Avitag™,ACRObiosystem, SI2-H82F8; Biotinylated Human CD19, Fc Tag, ultra sensitivity, ACRObiosystem, CD9-H8259; BeaverBeads™ Streptavidin, Beaver Biotech, 22307-10; High binding ELSIA plate, Costar, #3590 Blocking solution: PBS + 3% BSA Rinse buffer: PBS + 0.1% Tween20 Eluent: 0.2 M Glycine, pH 2.2 Neutralization solution: 1M Tris, pH 9.1 Experimental results: Using different antibody libraries, three rounds of protein panning were performed, and a significant increase in recovery rate was observed in each panning (Table 1), demonstrating that the antibody clones were effectively enriched.

[0147] Table 1 Protein panning experiment results

[0148] It can be seen that after 3 rounds of panning, different antibody libraries have been enriched (the recovery rate in the 3rd round was significantly improved compared to the previous round). However, in subsequent FACS experiments, the clones selected from these phage pools could not bind to the Raji cell line that highly expresses the CD22 antigen, that is, they could not recognize the CD22 antigen in the natural state on the cell surface. Therefore, in subsequent experiments, the method of alternating protein and cell panning was adopted to enrich specific antibody clones that can simultaneously bind to CD22 protein and CD22 in the natural state on the cell surface from the phage antibody library. Table 2 shows the results of the combined panning using recombinant CD22 protein and Raji / Jurkat cell line. From the recovery point of view, all 4 rounds of panning have been enriched and can be used for the next step of selecting monoclonal antibodies.

[0149] Table 2 Results of protein / cell selection experiments

[0150] Example 2 Screening of specific clones from the enriched phage pool using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS) Purpose and Principle: The phage pool enriched by affinity panning contains a variety of phage antibodies: specific clones, nonspecific clones, and negative clones. To obtain specific clones, single clones are isolated and packaged into monoclonal phage. A large number of these clones are screened using enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS). From these clones, a single clone that specifically binds to both the CD22 protein and the CD22-positive cell line Raji is selected. DNA sequencing is then performed to confirm the unique antibody sequence contained within these specific clones.

[0151] In the ELISA primary screening, the binding of streptavidin to biotin makes the biotinylated target protein (CD22-Fc-Bio) closer to the native antigen conformation in the reaction solution. Clones that only bind to CD22-Fc-Bio and not to the control antigen CD19-Fc-Bio are identified as specific clones. The FACS primary screening is performed using the CD22-positive cell line Raji, which highly expresses CD22, and the CD22-negative cell line Jurkat. Clones that only bind to Raji cells and not to Jurkat cells are identified as specific clones. Through both ELISA and FACS primary screening, candidate antibodies that can bind to recombinantly expressed CD22 protein and recognize the native CD22 molecule on the cell surface can be obtained for subsequent further screening.

[0152] Brief steps of ELISA screening experiment: 1) Cultivate and package monoclonal phage using deep-well 96-well plates; 2) Dilute Strepavidin to 2 μg / mL with PBS and add 100 μL / well to the high-binding ELISA plate. Allow to bind at room temperature for 2 h. 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C. 4) Wash the plate twice with 250 μL of rinse buffer; 5) Dilute the biotin-tagged target protein and control protein to 2 μg / mL with PBS and add 100 μg / well to the Strepavidin-precoated ELISA plate for binding at room temperature for 1 h. 6) Wash the plate twice with 250 μL of rinse buffer; 7) Add 100 μL of the phage supernatant from step 1) to the wells coated with the target antigen and allow to bind for 2 h at room temperature. 8) Wash the plate 4 times with 250 μL of rinse buffer; 9) Add 1:2000 diluted mouse anti-M13 primary antibody, 100 μL / well, and incubate at room temperature for 45 min; 10) Wash the plate 4 times with 250 μL of rinse buffer; 11) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL / well, and incubate at room temperature for 45 minutes; 12) Wash the plate 6 times with 250 μL of rinse buffer; 13) Add 100 μL TMB chromogenic substrate and develop color for 5 to 10 minutes; 14) Add 100 μL of 2M H2SO4 to terminate the reaction and read the results on a microplate reader.

[0153] Brief steps of FACS screening experiment: 1) Cultivate and package monoclonal phage using deep-well 96-well plates; 2) Wash Raji and Jurkat cells twice with PBS and resuspend in PBS to 1x10 7 / mL concentration, and 50 μL was dispensed into 96-well deep-well plates; 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and allow to bind at 4°C for 2 h. 4) Wash twice with 200 μL PBS; 5) Add 1:2000 diluted mouse anti-M13 primary antibody, 100 μL / well, mix well by pipetting, and incubate at room temperature for 45 min; 6) Wash twice with 200 μL PBS; 7) Add 1:300 diluted FITC horse anti-mouse-IgG (H+L) at 100 μL / well, mix thoroughly by pipetting, and incubate at room temperature for 45 min. 8) Wash twice with 200 μL PBS; finally, resuspend the cells in 200 μL PBS; 9) Detect the fluorescence intensity of the sample in the FITC channel on a flow cytometer and analyze the results.

[0154] Main materials and reagents: Helper phage KO7, Thermo / Invitrogen, 18311019 Streptavidin, Pierce, 21125 Biotinylated Human Siglec-2 / CD22 Protein, Fc,Avitag™,ACRObiosystem, SI2-H82F8; Biotinylated Human CD19, Fc Tag, ultra sensitivity, ACRObiosystem, CD9-H8259; High binding ELSIA plate, Costar, #3590 Corning 96 Well Clear Round Bottom TC-Treated Microplate, Costar, #3799 Blocking solution: PBS + 3% BSA Rinse buffer: PBS + 0.1% Tween20 Soluble one-component TMB substrate solution, Tiangen, PA-107-02 Anti-M13 Bacteriophage Coat Protein g8p antibody, abcam, ab9225 HRP Goat anti-mouse IgG(minimal x-reactivity) Antibody, Biolegend,405306 FITC horse anti mouse-IgG(H+L), Vector, FI2000.

[0155] Experimental results: Monoclonal clones were randomly selected from the enriched phage antibody pool, packaged into phage, and then the binding of monoclonal phage to CD22-Fc-Bio protein and control protein CD19-Fc-Bio was detected by phage ELISA to find CD22-specific phage antibody clones. The ELISA results of some clones are as follows Figure 2As shown in the figure, clones A2, A4, A6, A7, A8, and A9 bind strongly to the target antigen CD22 (CD22-Fc-Bio) and do not bind to the control antigen CD19-Fc-Bio, demonstrating good specificity. Clones A1, A3, and A5 do not bind to either the target antigen CD22 (CD22-Fc-Bio) or the control antigen CD19-Fc-Bio and are negative clones. The negative phage control is a negative control phage antibody clone that binds to neither the target antigen nor the control antigen. The anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative antibody control with only the primary and secondary antibodies added, and the anti-mouse HRP Ab is a negative antibody control with only the secondary antibody added. They do not bind to either the target antigen or the control antigen. The anti-human IgG-HRP Ab is a positive antibody control for detecting antigen tags and binds to antigens containing the Fc tag, indicating that the coated antigen has bound to the ELISA plate.

[0156] The FACS screening results of some clones are as follows Figure 3 As shown in the figure, clones A3, A8, and A9 bind to Raji cells but not to Jurkat cells and are therefore specific clones. Clones A1, A4, A5, and A7 bind weakly to Raji cells but not to Jurkat cells and are therefore weak-binding clones. Clones A2 and A6 bind to neither cell type nor to Jurkat cells and are therefore negative clones. Neither weak-binding nor negative clones meet the experimental requirements.

[0157] Through ELISA detection and FACS initial screening, a total of 16 ELISA and FACS double-positive clones with good specificity were obtained. Subsequently, the 16 double-positive clones with good specificity were sequenced, and two different monoclonal sequences were obtained after sequencing. These two monoclonal clones, Clone79 and 80 with different sequences, were then further identified by FACS identification of multiple cell lines and ELISA identification of multiple antigens to detect the binding specificity of the candidate clones.

[0158] Example 3 Identification of Monoclonal Specificity by FACS Using Multiple Cell Lines Experimental Purpose and Principle: Antibodies used for therapeutic purposes must possess excellent target specificity, binding only to the target antigen and not to any unrelated antigens. On the other hand, the amino acid sequences of the same antigen on different cell lines may differ (isomers or mutants) or bind to different ligands, so it is also necessary to examine whether the antibodies of the present invention can bind to cells positive for a variety of target proteins. To further analyze the specificity and universality of these monoclonal clones and identify the best candidate clones, the specificity of the initial screening clones was further evaluated by flow cytometry. In this experiment, multiple CD22-positive and CD22-negative cell lines were reacted with these monoclonal phage antibodies to analyze whether these clones can bind to the CD22 antigen on different cell lines and whether there is any non-specific binding to other cell lines that do not express CD22. Through this experiment, several clones with excellent specificity were obtained.

[0159] Experimental method: Same as FACS primary screening; Main samples and reagents: Raji cell line, CD22-positive cell line; NALM6 cell line, a CD22-positive cell line; Jurkat cell line, a CD22-negative cell line; U266 cell line, a CD22-negative cell line; Raji KO CD22 cell line, CD22-negative cell line; APC anti-human CD22 Antibody, Biolegend, 302510 The remaining reagents were the same as those for the FACS primary screening.

[0160] Experimental results: Antibodies used for treatment must have very good target specificity. In order to further analyze the specificity of these monoclonal antibodies, the two specific clones obtained in Example 2 were identified by enzyme-linked immunosorbent assay and flow cytometry on more antigens and cell lines. The results are shown in Figures 4A-4B middle. Figure 4A To detect the results of the assay using a positive control antibody, the cell lines expressing CD22 antigen were used: Raji and NALM6 cells were CD22 positive, while Raji KO CD22, Jurkat, and U266 did not express CD22 antigen; Figure 4BTo screen clones for binding to cell lines, a negative control phage antibody clone was used, and a FITC anti-mouse IgG antibody was used as a negative control with only the secondary antibody. Clone 79 and 80 bound to two CD22-positive cell lines, Raji and NALM6, but not to three CD22-negative cell lines: Raji KOCD22, Jurkat, and U266, demonstrating good specificity.

[0161] Example 4 Identification of monoclonal specificity by ELISA using antigens from different companies Purpose and Principle of the Experiment: Antibodies intended for therapeutic use must exhibit excellent target specificity, binding exclusively to the target antigen and not to any unrelated antigens. Furthermore, the amino acid sequences of the same antigen produced by different companies can vary (isomers or mutants), necessitating the ability of the antibodies of this invention to bind to a variety of target proteins. To further analyze the specificity and universality of these monoclonal antibodies and identify optimal candidate clones, the specificity of the initial screening clones was further evaluated using an enzyme-linked immunosorbent assay (ELISA). In this experiment, these monoclonal phage antibodies were reacted with CD22 antigens containing different structural domains and an unrelated antigen (Acro-CD19-bio) to analyze their ability to bind to CD22 antigens with distinct domains and any nonspecific binding to other unrelated CD22 antigens. This experiment resulted in the identification of several clones with excellent specificity.

[0162] Experimental method: Same as ELISA primary screening.

[0163] Table 3 Main samples and reagents

[0164] Detailed information on Iaso-CD22-(1,3,5,6,7)-bio containing different domains can be found in Figure 5 .

[0165] The remaining reagents are the same as those for the ELISA primary screening.

[0166] Experimental results: Antibodies used for treatment must have very good target specificity. To further analyze the specificity of these monoclonal antibodies, multiple clones obtained in Example 2 were identified using enzyme-linked immunosorbent assay (ELISA) on multiple antigens. The results are shown in Figure 6In the assay, the negative control is a phage antibody clone that binds neither the target nor the control antigen. The anti-M13 phage mouse Ab / anti-mouse HRP Ab are negative antibody controls with only the primary and secondary antibodies added. The anti-mouse HRP Ab is negative antibody controls with only the secondary antibody added; both bind neither the target nor the control antigen. The M971 positive control Ab / anti-human IgG HRP Ab are positive antibody controls for the target antigen (CD22-Fc-Bio), binding to the target antigen but not the control antigen. The anti-human IgG-HRP Ab is the secondary antibody control for the M971 positive control antibody experimental group, indicating that binding of the M971 antibody to the CD22 antigen is caused by the positive control antibody and that the secondary antibody does not bind nonspecifically. The anti-rabbit IgG-HRP Ab / anti-human IgG-HRP Ab are positive antibody controls for detecting antigen tags. They bind to antigens containing either the rabbit Fc tag or the human Fc tag, indicating that the coated antigen has bound to the ELISA plate. Clone 80 showed strong or weak binding to six CD22 antigens containing different structural domains and no binding to unrelated antigens, demonstrating good specificity. Clone 79 bound not only to the target antigen CD22 but also to the unrelated antigen Acro-CD19-bio, suggesting that its binding may be nonspecific.

[0167] Based on the experimental results of Example 3 and Example 4, the clone that can specifically bind is clone 80.

[0168] Example 5 Detection of the binding ability of monoclonal antibodies to high-expressing cell lines by FACS The affinity between the CD22 antibody molecule and the antigen may have a significant impact on the killing effect and survival of CAR-T or antibody drugs in the patient's body. This example uses the FACS binding method to analyze the half effective concentration (Ec50) of the antibody molecule. The FACS binding experimental steps are as follows:

[0169] (1) Preparation of antibodies at different concentrations: Anti-CD22 IgG (expressed by Clone 80) was diluted 5-fold from 300 nM to 0.00384 nM using PBS, for a total of 8 concentrations, to detect the ability of the antibody to bind to tumor-positive cells Raji; (2) Wash the Raji cells twice with PBS and resuspend them in PBS to 1×10 7 / mL concentration, and 50 μL was dispensed into 96-well deep-well plates; (3) Add 50 μL of diluted anti-CD22 IgG to each well, mix well, and allow to bind at 4°C for 2 h; (4) Wash twice with 200 μL PBS; (5) Add Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG, Fcγ fragment specific (1:300) at 100 μL / well, pipette to mix, and incubate at room temperature for 45 min. (6) Wash twice with 200 μL PBS; finally, resuspend the cells with 200 μL PBS; (7) Detect the fluorescence intensity of the sample FITC channel on a flow cytometer; (8) The binding constants were analyzed by using Graphpad Prism software.

[0170] The main samples and reagents used in this example are as follows: Raji cell line (CD22-positive cell line); Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG, Fcγ fragmentspecific, Jackson ImmunoReseach, 109-605-008.

[0171] Affinity refers to the strength of binding between a single molecule and its ligand. The binding ability to positive cell lines can usually be detected by FACS to assess the strength of the interaction between the two molecules and rank them. The smaller the Ec50 value, the greater the affinity of the antibody for its target. The results are shown in Table 4 and Figure 7 shown. Figure 7 The results of flow cytometry analysis of the protein antibody expressed by the phage monoclonal clone obtained for screening and binding to the positive cell line Raji showed that the affinity of SdAb 80 was higher than that of the control antibody M971.

[0172] Table 4 Affinity of antibodies for Raji cells

[0173] Example 6 Affinity determination of anti-CD22 monoclonal antibodies The affinity between CD22 monoclonal antibodies and antigens may significantly influence the killing effect and survival of CAR-T or antibody drugs in patients. To determine this crucial property, this example employed ForteBio's Octet molecular interaction technology. The Octet system utilizes biofilm interferometry, a label-free technique that provides real-time, high-throughput biomolecular interaction information. The instrument transmits white light onto the sensor surface and collects the reflected light. The reflected light at different frequencies is affected by the thickness of the biosensor's optical film layer, with some frequencies forming constructive interference (blue) and others destructive interference (red). This interference is detected by a spectrometer, forming an interference spectrum and displayed as the intensity of the phase shift (nm) within the interference spectrum. Therefore, as the number of molecules bound to the sensor surface increases or decreases, the spectrometer detects a shift in the interference spectrum in real time. This shift directly reflects the thickness of the biofilm on the sensor surface, enabling the acquisition of high-quality data on biomolecular interactions and the determination of interaction kinetic parameters (Kon, Kdis, and KD), providing crucial information for research and development. The experimental steps for affinity determination are as follows:

[0174] (1) Anti-CD22 IgG (composed of CD22 ScFv sequence fused to human IgG4 Fc) was diluted to 20 μg / mL with loading buffer (1× PBS, pH 7.4, 0.01% BSA and 0.02% Tween 20) and loaded on the biosensor at approximately 0.8 nM.

[0175] (2) After a 60 s equilibration phase, the binding kinetics of CD22 antigen (Acro, CD2-H52H8) were monitored at various antigen concentrations (100 to 1.563 nM). Binding was performed for 160 s and dissociation for 300 s at each concentration.

[0176] (3) Regenerate the chip by washing three times with 10 mM Glycine-HCl, pH 1.5.

[0177] (4) Binding constants were analyzed by using a 1:1 binding site model (Biacore X-100 evaluation software).

[0178] Affinity refers to the strength of binding between a single molecule and its ligand. It is typically measured and reported using the equilibrium dissociation constant (KD), which can be used to assess and rank the strength of interactions between two molecules. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is proportional to the concentration of the reactants. The smaller the KD value, the greater the affinity of the antibody for its target. The affinity test results are shown in Table 5: Clone80 IgG binds to the CD22 antigen with an affinity comparable to that of the control.

[0179] Table 5 Affinity of antibodies for CD22 antigen

[0180] In summary, the fully human CD22-targeting antibody described in the present invention has strong specificity, binding to CD22-positive cell lines but not to CD22-negative cell lines. This fully human CD22-targeting antibody specifically binds to CD22 with high affinity and exhibits lower immunogenicity compared to heterologous antibodies, suggesting significant potential for application in the development of antibody drugs and cell therapy drugs.

[0181] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-CD22 single domain antibody or an antigen-binding fragment thereof, characterized in that: The single-domain antibody or antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises: HCDR1 as shown in SEQ ID NO: 1; HCDR2 as shown in SEQ ID NO: 2; and HCDR3 as shown in SEQ ID NO:

3.

2. The single domain antibody or antigen-binding fragment thereof according to claim 1, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

5.

3. A recombinant protein, characterized in that The recombinant protein has: (i) the single domain antibody or antigen-binding fragment thereof according to claim 1; and (ii) optionally a tag sequence to facilitate expression and / or purification.

4. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule encodes the single domain antibody or antigen binding fragment thereof according to claim 1, or the recombinant protein according to claim 3.

5. An expression vector, characterized in that: The expression vector contains the nucleic acid molecule according to claim 4.

6. A host cell, characterized in that The host cell contains the expression vector according to claim 5, or the nucleic acid molecule according to claim 4 is integrated into its genome.

7. A chimeric antigen receptor CAR, characterized in that: The antigen binding region of the CAR specifically binds to CD22, and the antigen binding region comprises the single domain antibody or antigen binding fragment thereof as described in claim 1.

8. An engineered immune cell, characterized in that: The engineered immune cells express the exogenous CAR of claim 7.

9. A method for producing a single domain antibody or an antigen-binding fragment thereof targeting CD22, characterized in that: Includes steps: (a) culturing the host cell according to claim 6 under suitable conditions to obtain a culture containing a single domain antibody or an antigen-binding fragment thereof targeting CD22; (b) isolating and / or recovering the CD22-targeting single domain antibody or antigen-binding fragment thereof from the culture.

10. An immunoconjugate, characterized in that: The immunoconjugate contains: (a) an antibody portion, the antibody portion comprising the single domain antibody or antigen-binding fragment thereof as claimed in claim 1; and (b) a conjugation moiety selected from the group consisting of a detectable label, a therapeutic agent, or a combination thereof.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of: The single domain antibody or antigen binding fragment thereof according to claim 1, the recombinant protein according to claim 3, the nucleic acid molecule according to claim 4, the expression vector according to claim 5, the host cell according to claim 6, the chimeric antigen receptor CAR according to claim 7, the engineered immune cell according to claim 8, or the immunoconjugate according to claim 10, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

12. A method for detecting CD22 protein or a fragment thereof in a sample in vitro, characterized in that: The method comprises the steps of: (1) contacting the sample with the single domain antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 3, or the immunoconjugate according to claim 10, or a combination thereof in vitro; (2) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates the presence of CD22 protein or its fragment in the sample.

13. A kit, characterized in that: The kit comprises: (1) a first container, wherein the first container contains the single domain antibody or antigen-binding fragment thereof according to claim 1, the recombinant protein according to claim 3, or the immunoconjugate according to claim 10, or a combination thereof; and / or (2) a second container, wherein the second container contains a secondary antibody against the contents of the first container; or, The kit contains a test plate, which includes: a substrate and a test strip, wherein the test strip contains the single-domain antibody or its antigen-binding fragment as described in claim 1, the recombinant protein as described in claim 3, or the immunoconjugate as described in claim 10, or a combination thereof.

Images