Fully human antibodies targeting CD22 and their applications

Abstract

The present invention relates to the field of antibodies, and provides fully human antibodies targeting CD22 and their applications. The present invention provides fully human single-domain antibodies against CD22, which bind to CD22 with high affinity and high specificity, have lower immunogenicity and side effects, and have great application potential in the development of drugs for the treatment of CD22-related diseases.

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 a 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 popular targets for CAR-T therapy, and its indication is B lymphocyte tumors. Due to its established efficacy and controllable side effects, it is the most common product currently on the market and under development. CAR-T therapy targeting CD19 is recognized by the industry as a perfect solution for B-cell malignancies. However, with the deepening of clinical research, more and more evidence shows that about 50% of patients will relapse over time. There are many reasons for relapse, which are mainly divided into CD19 antigen-negative relapse and positive relapse. Some cancer cells that originally expressed CD19 will express CD22 protein under the pressure of CAR-T therapy.

[0003] CD22, also known as Siglec-2, is a lectin of the Siglec family, consisting of seven IgG-like extracellular domains. CD22 is another member of the B cell antigen family, and its tissue distribution is similar to CD19. The expression of CD22 on the surface of B cells begins in the pre-B cell stage and is continuously expressed 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. CD22 is also a member of the sialic acid-binding immunoglobulin-like lectin (Siglecs) family and one of the inhibitory co-receptors on the surface of B cells. It is closely related to the development, differentiation and function of B cells. CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, making it one of the hot targets for the treatment of autoimmune diseases and B cell malignancies. At present, the types of immunotherapy drugs targeting CD22 include monoclonal antibody drugs, antibody conjugates (ADCs) and CAR-T therapy.

[0004] Although the treatment effects of acute B-cell leukemia and B-cell malignant lymphoma have been greatly improved, there are still some patients who are refractory to relapse. Targeted CD19 chimeric antigen receptor (CAR19) T cell therapy has caused unprecedented therapeutic effects. However, CD19-negative relapse is the most common cause of CAR-T treatment failure, and there is a lack of effective treatments, so new treatments are needed.

[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 an antigen-binding fragment thereof is provided, wherein the single-domain antibody or the antigen-binding fragment thereof comprises a heavy chain variable region, wherein the heavy chain variable region comprises:

[0008] HCDR1 as shown in SEQ ID NO: 1;

[0009] HCDR2 as shown in SEQ ID NO: 2; and

[0010] HCDR3 as shown in SEQ ID NO: 3;

[0011] 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.

[0012] In another preferred embodiment, any one of the above amino acid sequences also includes 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.

[0013] 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.

[0014] In another preferred example, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5.

[0015] In another preferred embodiment, the antibody or antigen-binding fragment thereof further includes a framework region FR.

[0016] In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by framework regions FR1, FR2, FR3 and FR4.

[0017] In another preferred embodiment, the antibody or antigen-binding fragment thereof includes a monomer, a bivalent antibody, and / or a multivalent antibody.

[0018] In another preferred embodiment, the bivalent antibody is a bispecific antibody.

[0019] In another preferred embodiment, the multivalent antibody is a multispecific antibody.

[0020] In another preferred embodiment, the antigen-binding fragment comprises a single domain fragment.

[0021] In another preferred embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region.

[0022] In another preferred embodiment, the heavy chain constant region is selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4.

[0023] In a second aspect of the present invention, a recombinant protein is provided, wherein the recombinant protein has:

[0024] (i) a single domain antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0025] (ii) optionally a tag sequence to facilitate expression and / or purification.

[0026] 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.

[0027] In another preferred embodiment, the recombinant protein includes a fusion protein.

[0028] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a polymer.

[0029] In the 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.

[0030] In another preferred embodiment, the nucleic acid molecule is selected from RNA (such as mRNA) and DNA (such as cDNA).

[0031] In another preferred example, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 4.

[0032] In the fourth aspect of the present invention, an expression vector is provided, wherein the expression vector contains the nucleic acid molecule as described in the third aspect of the present invention.

[0033] 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.

[0034] In another preferred embodiment, the expression vector comprises a viral vector, such as a lentivirus, an adenovirus, an AAV virus, a retrovirus, or a combination thereof.

[0035] In another preferred embodiment, the expression vector is a plasmid, a retrovirus or a lentivirus vector.

[0036] 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.

[0037] In another preferred embodiment, the expression vector further comprises elements selected from the following group: a promoter, a transcription enhancing element WPRE, a long terminal repeat sequence LTR, etc.

[0038] 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.

[0039] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0040] In another preferred embodiment, the host cell is selected from the following group: Escherichia coli, yeast cells, and mammalian cells.

[0041] 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.

[0042] In another preferred embodiment, the antigen binding region comprises an amino acid sequence as shown in SEQ ID NO: 5 or having at least 90% sequence identity thereto.

[0043] In another preferred example, the antigen binding region comprises the amino acid sequence shown in SEQ ID NO: 5.

[0044] In another preferred embodiment, the CAR further includes a signal peptide.

[0045] In another preferred embodiment, the CAR also includes other exogenous proteins.

[0046] In another preferred embodiment, the CAR has a structure shown in Formula I:

[0047] L-EB-H-TM-C-CD3ζ (I)

[0048] In the formula,

[0049] L is none or a signal peptide sequence;

[0050] EB is the antigen binding region that specifically binds to CD22;

[0051] H is none or hinge region;

[0052] TM is the transmembrane domain;

[0053] C is the co-stimulatory signaling domain;

[0054] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ (including wild type, or mutant / modified form thereof);

[0055] The "-" connects peptides or peptide bonds.

[0056] 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.

[0057] In another preferred embodiment, the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.

[0058] In another preferred embodiment, the TM is selected from the transmembrane region of the following group of proteins: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or their mutants / modified forms, or their combinations.

[0059] In another preferred embodiment, the C is selected from the co-stimulatory 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.

[0060] 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.

[0061] In another preferred embodiment, the engineered immune cells are selected from the following group:

[0062] (i) Chimeric antigen receptor αβ T cells (CAR-T cells);

[0063] (ii) chimeric antigen receptor γδT cells (CAR-T cells);

[0064] (iii) chimeric antigen receptor NKT cells (CAR-NKT cells);

[0065] (iv) Chimeric antigen receptor NK cells (CAR-NK cells).

[0066] In another preferred embodiment, the engineered immune cells include autologous or allogeneic αβT cells, γδT cells, NKT cells, NK cells, or a combination thereof.

[0067] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0068] In an eighth aspect of the present invention, a method for producing an antibody or an antigen-binding fragment thereof targeting CD22 is provided, comprising the steps of:

[0069] (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 an antigen-binding fragment thereof targeting CD22;

[0070] (b) isolating and / or recovering the antibody or antigen-binding fragment thereof targeting CD22 from the culture; and

[0071] (c) Optionally, purifying and / or modifying the CD22-targeting antibody or antigen-binding fragment thereof obtained in step (b).

[0072] In the ninth aspect of the present invention, an immunoconjugate is provided, wherein the immunoconjugate comprises:

[0073] (a) an antibody portion, the antibody portion comprising the single domain antibody or antigen-binding fragment thereof as described in the first aspect of the present invention; and

[0074] (b) a conjugation moiety selected from the group consisting of a detectable label, a therapeutic agent, or a combination thereof.

[0075] In another preferred embodiment, the coupling part is selected from the following group: toxins, cytokines, radionuclides, enzymes, gold nanoparticles / nanorods, nanomagnetic particles, virus coat proteins or VLPs, or a combination thereof.

[0076] In another preferred embodiment, the (a) part and the coupling part are coupled via a chemical bond or a linker.

[0077] In another preferred embodiment, the radionuclide includes:

[0078] (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

[0079] (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.

[0080] In another preferred embodiment, the coupling moiety may contain at least one therapeutic agent.

[0081] In another preferred embodiment, the conjugated moiety is a therapeutic agent.

[0082] 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.

[0083] In another preferred embodiment, the immune checkpoint drug is an inhibitor or activator of the immune checkpoint.

[0084] In another preferred embodiment, the therapeutic agent is a cytotoxic drug.

[0085] In another preferred embodiment, the cytotoxic drug is selected from the following group: 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.

[0086] 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, Etoposides, 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.

[0087] In another preferred embodiment, the coupling moiety is a toxin.

[0088] 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, dihydroxyproline, succinimidyl, tadalafil, tadalafil, succinimidyl ... 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, crotonin, calicheamicin, Sapaonaria officinalis inhibitor, glucocorticoids, or a combination thereof.

[0089] In another preferred embodiment, the coupling moiety is a detectable label.

[0090] 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.

[0091] In another preferred embodiment, the immunoconjugate contains: a multivalent (eg, bivalent) single-domain antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0092] 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.

[0093] In the tenth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0094] (i) an active ingredient, wherein the active ingredient is selected from the group consisting of a single domain antibody or an 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

[0095] (ii) a pharmaceutically acceptable carrier, diluent or excipient.

[0096] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the following group: injection and lyophilized agent.

[0097] In another preferred embodiment, 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.

[0098] 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.

[0099] 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: a single domain antibody or an 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, wherein the active ingredient is used to prepare:

[0100] (a) a drug for treating and / or preventing a CD22-related disease or condition; and / or

[0101] (b) A reagent for detecting a CD22-related disease, or a kit for detecting a CD22 protein or a fragment thereof.

[0102] In another preferred embodiment, the reagent is a diagnostic reagent; preferably, the diagnostic reagent is a detection sheet or a detection plate.

[0103] In another preferred embodiment, the diagnostic reagent is used to detect CD22 protein or a fragment thereof in a sample.

[0104] In another preferred embodiment, the disease or condition is a disease or condition in which CD22 is highly expressed or CD22 pathway is abnormal.

[0105] In another preferred embodiment, the disease or condition is selected from: cancer or autoimmune disease.

[0106] In another preferred embodiment, the disease or disorder is a tumor.

[0107] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0108] 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.

[0109] In another preferred embodiment, the cancer is B lymphocytoma.

[0110] In the 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:

[0111] (1) contacting the sample in vitro with 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;

[0112] (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.

[0113] In another preferred embodiment, the detection includes diagnostic or non-diagnostic.

[0114] In the thirteenth aspect of the present invention, a kit is provided, the kit comprising:

[0115] (1) a first container, wherein the first container contains 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

[0116] (2) a second container, wherein the second container contains a secondary antibody against the contents of the first container;

[0117] or,

[0118] 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 its antigen-binding fragment 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.

[0119] In another preferred embodiment, the kit further comprises an instruction manual, and according to the instruction manual, the kit is used for non-invasively detecting the expression of CD22 protein or a fragment thereof in a test subject.

[0120] In another preferred embodiment, the kit is used for detecting CD22-related diseases.

[0121] 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 a therapeutically effective amount of a single domain antibody or an 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 a disease or condition.

[0122] In another preferred embodiment, the subject includes mammals, such as humans.

[0123] In another preferred embodiment, the disease or condition is a disease or condition in which CD22 is highly expressed or CD22 pathway is abnormal.

[0124] In another preferred embodiment, the disease or condition is selected from: cancer or autoimmune disease.

[0125] In another preferred embodiment, the disease or disorder is a tumor.

[0126] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0127] 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.

[0128] In another preferred embodiment, the cancer is B lymphocytoma.

[0129] 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).

[0130] 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).

[0131] In another preferred embodiment, the method can be used in combination with other treatment methods.

[0132] In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy and the like.

[0133] In a fifteenth aspect of the present invention, a method for diagnosing a CD22-related disease is provided, comprising the steps of:

[0134] (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

[0135] (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.

[0136] In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.

[0137] In another preferred embodiment, the disease or condition is a disease or condition in which CD22 is highly expressed or CD22 pathway is abnormal.

[0138] In another preferred embodiment, the disease or condition is selected from: cancer or autoimmune disease.

[0139] In another preferred embodiment, the disease or disorder is a tumor.

[0140] In another preferred embodiment, the tumor is a solid tumor or a hematological tumor.

[0141] 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.

[0142] In another preferred embodiment, the cancer is B lymphocytoma.

[0143] Those skilled in the art can easily perceive 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 the present 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 the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0144] Figure 1 The general process of the present invention for screening specific antibodies targeting CD22 from a phage antibody library is shown.

[0145] Figure 2The results of enzyme-linked immunosorbent assay (ELISA) of some selected phage monoclones with target antigen and control antigen are shown.

[0146] Figure 3 Flow cytometric analysis results of binding of some phage monoclones to Raji and Jurkat cells are shown.

[0147] Figure 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 assay using a positive control antibody against a cell line expressing the CD22 antigen, Figure 4B The figures are flow cytometry 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.

[0148] Figure 5 Detailed information of Iaso-CD22(1,3,5,6,7)-bio antigen containing different domains is shown.

[0149] Figure 6 The results of ELISA analysis of the screened phage monoclonals with CD22 antigen proteins containing different domains and non-related antigens are shown. Negative control is a negative control phage antibody clone, anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative antibody control with only the first antibody and the second antibody, anti-mouse HRP Ab is a negative antibody control with only the second antibody, M971 positive control Ab / anti-human IgG HRP Ab is a positive antibody control for the target antigen (CD22-Fc-Bio). Anti-human IgG-HRP Ab is the second antibody control for the M971 positive control antibody experimental group; anti-Rabbit IgG-HRP Ab / anti-human IgG-HRP Ab is a positive antibody control for detecting antigen tags. Among them, the bar graphs corresponding to each test antibody and control group represent the test results of the 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.

[0150] Figure 7This is the flow cytometry analysis result of the binding of the phage monoclonal obtained in Example 5 to the positive cell line after being expressed as protein antibody. DETAILED DESCRIPTION

[0151] After extensive and in-depth research, the 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 high affinity, especially to CD22 expressed on the surface of tumor cells, and have low side effects. On this basis, the present invention was completed.

[0152] the term

[0153] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0154] "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.). The 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 conservative differences in the amino acid sequences, the heavy chain and the light chain are divided into a variable region (V) located at the amino terminus and a constant region (C) located 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 an antigen-binding site (Fv). In the variable region, the composition and arrangement order of amino acid residues in certain regions are more variable than other regions in the variable region (framework region, FR), which are called hypervariable regions (HVR). Hypervariable regions are actually the key sites for antibody-antigen binding. Since these hypervariable region sequences are complementary to the antigenic determinants, they are also called complementarity-determining regions (C). The heavy chain and light chain each have three complementary determining regions, namely HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3.

[0155] "Single chain antibody" (single chain fragment variable, scFv) is composed of the variable region of the heavy chain and the variable region of the light chain of the antibody connected into a peptide chain by a short peptide. Through correct folding, the variable regions from the heavy chain and the light chain interact with each other through non-covalent bonds to form the Fv segment, so scFv can better retain its affinity activity for the antigen.

[0156] "Single domain antibodies" refer to antibodies composed only of amino acids in the variable region of heavy chain antibodies, with a molecular weight of only 12-15kDa, but with similar or higher specificity and affinity than traditional antibodies. In addition, single domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.

[0157] "Mouse antibodies" are antibodies produced by mice against specific antigens, usually antibodies produced by mouse B lymphocytes. In most cases, the mouse antibodies are monoclonal antibodies produced by hybridoma cells. The fully human antibodies of the present application are obtained by screening human phage antibody libraries, which have lower immunogenicity than mouse antibodies and are more conducive to therapeutic use in humans.

[0158] The "fully human antibody or single domain antibody or fragment thereof" mentioned in the present application generally refers to any form of antigen binding molecule that can bind to the target antigen, for example, the antigen binding molecule may 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 antibody-Fc antibodies, immunoconjugates, antibody drug conjugates (ADCs), multi / bispecific antibodies, and chimeric antigen receptors (CARs).

[0159] CD22 is restrictedly expressed on the surface of mature B cells and most malignant B lymphoma cells, making it one of the hot targets for the treatment of autoimmune diseases and B cell malignancies. The art is in urgent need of developing new CD22-specific binding molecules in different forms. The present application meets this demand by providing an antibody that binds to the CD22 antigen with high target specificity and high affinity, especially to CD22 expressed on the surface of tumor cells, and has low side effects.

[0160] 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. Usually, before calculating the percentage of consistency between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If the amino acid residues or bases in the two sequences are the same at a certain alignment position, 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 consistency. 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 consistency between two amino acid or nucleotide sequences using the default settings. In some embodiments, "at least 90% sequence identity" described herein includes, but is not limited to, at least 95%, at least 98%, at least 99% or even 100% sequence identity.

[0161] It will be appreciated 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 binding ability or biological activity of the resulting products with the corresponding antigen CD22 can be verified or screened, thereby obtaining the corresponding variants of the CD22-targeting antibodies provided herein, and these variants should also be included in the scope of the present invention. For example, the fully human antibody or its single domain antibody or fragment 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.

[0162] In some embodiments, the antigen binding molecules of the present application may further include post-translational modifications. Examples of post-translational protein modifications include: phosphorylation, acetylation, methylation, ADP-ribosylation, ubiquitination, glycosylation, carbonylation, ubiquitination, biotinylation, or the addition of polypeptide side chains or hydrophobic groups. Therefore, the modified soluble polypeptide may include non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation modification, which combines one or more sialic acid groups with the polypeptide. Sialic acid groups improve the solubility and serum half-life of proteins, while also reducing the possible immunogenetic properties of proteins. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

[0163] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to solid or liquid diluents, fillers, antioxidants, stabilizers and the like that can be safely administered, are suitable for administration to humans and / or animals without excessive adverse side effects, and are suitable for maintaining the activity of the drugs or active agents contained therein.

[0164] "Therapeutically effective amount" refers to the amount of active compound sufficient to cause the biological or medical response desired by the clinician in the subject. The "therapeutically effective amount" of the antibody of the present application can be determined by those 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. The administration of the antibody of the present application includes, but is not limited to, injection, such as intravenous, intramuscular, intraarterial, subcutaneous, intraperitoneal, etc.

[0165] "Epitope" refers to the portion of a molecule that is bound by an antigen binding protein (e.g., an antibody). An epitope may comprise a non-contiguous portion of the molecule (e.g., amino acid residues in a polypeptide that are not adjacent in the primary sequence of the polypeptide but are close enough to each other in the trivalent and tetravalent structures of the polypeptide to be bound by an antigen binding protein).

[0166] The present application provides a kit, comprising one or more containers, containing a large number of gene constructs encoding the polypeptide of the present application, and a pharmaceutically acceptable excipient. The kit may also include instructions for use. The kit may also have a notice in a form prescribed by an agency that manages the production, use or sale of drugs or biological products, indicating that it has been approved by the production, use or sale agency for human drugs.

[0167] Research Overview

[0168] The present invention uses fully human phages for antibody screening to directly obtain fully human monoclonal antibodies. Compared with traditional hybridoma technology, the difficult step of humanizing mouse antibodies is omitted, and fully human antibodies have lower immunogenicity than humanized mouse antibodies, and have better potential in the application of antibody drugs (including monoclonal antibodies, bispecific antibodies, ADCs, etc.), cell therapy drugs (including CAR-T, CAR-NK, etc.), detection reagents, etc.

[0169] The present invention uses an antigen protein panning method, which can efficiently enrich and simultaneously bind to recombinant CD22 and antibodies of natural structure CD22 on the cell membrane, greatly reducing the difficulty of subsequent antibody screening and improving efficiency.

[0170] 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. Finally, several fully human antibody clones with good specificity are obtained.

[0171] The present invention uses different antibody libraries, and after panning with recombinant CD22 protein, a total of 184 monoclones were selected for primary screening by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), of which 16 clones specifically bind to CD22-Fc-Bio protein and CD22-positive expression cells Raji, but not to control protein CD19-Fc-Bio and CD22-negative expression cells Jurkat. After sequencing, 2 different monoclonal sequences were obtained. 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 ability and specificity on multiple cell lines and multiple protein antigens.

[0172] Subsequently, the 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 better affinity and better 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.

[0173] The sequence of SdAb 80 (Clone 80) protected by the present invention is as follows:

[0174] HCDR1:GFIFTNEW (SEQ ID NO: 1);

[0175] HCDR2: IRSDGGST (SEQ ID NO: 2);

[0176] HCDR3: ARMYYWSLAGESYDW (SEQ ID NO: 3);

[0177] VH DNA sequence (354 bp)

[0178] GAAGTTCAGCTGCTGGAAAGCGGTGGTGGTCTGGTTCAGCCTGGTGGTAGCCTGCGTCTGAGCTGTGCAGCAAGCGGTTTTATCTTTACCAACGAATGGATGGGTTGGGTTCGTCAGGCACCTGGTAAAGGTCTGGAATGGGTTAGCAGCATCCGCAGCGATGGCGGCAGCACCTATTATGCAGA TAGCGTTAAAGGTCGTTTTACCATTAGCCGTGATAACAGCAAAAATACCCTGTACCTGCGATGAATAGTCTGCGTGCAGAGGATACCGCAGTGTATTATTGTGCGCGCATGTACTACTGGTCTCTGGCTGGTGAATCTTACGATTGGTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA (SEQ IDNO: 4);

[0179] VH amino acid sequence (118 aa)

[0180] EVQLLESGGGLVQPGGSLRLSCAASGFIFTNEWMGWVRQAPGKGLEWVSSIRSDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARMYYWSLAGESYDWWGQGTLVTVSS (SEQ ID NO: 5).

[0181] The present invention is described in detail below with reference to specific embodiments.

[0182] Example 1 Enrichment of specific antibody clones targeting CD22 protein from phage antibody library by affinity panning

[0183] Appropriate negative and positive panning strategies are used to enrich the desired specific antibody clones from the phage antibody library.

[0184] Construction of phage antibody library:

[0185] The constructed phage antibody library includes a single domain library. The single domain phage antibody library is an antibody library composed only of amino acids in the variable region of heavy chain antibodies. Its molecular weight is only 12-15 kDa, but it has similar specificity and affinity to traditional antibodies. In addition, single domain antibodies have attracted much attention due to their stable physicochemical properties, high affinity, easy recombinant expression and preparation, and easy combination with other target or epitope antibodies.

[0186] CD22 protein panning:

[0187] Multiple rounds of panning were performed using CD22-Fc-Bio as a positive panning protein and CD19-Fc-Bio as a negative panning protein to obtain a phage pool enriched with the target antibody clone. The experimental steps are briefly described as follows:

[0188] 1) Block the SA magnetic beads with blocking solution for 2 hours, and then combine the target antigen (CD22-Fc-Bio) with the blocked SA magnetic beads;

[0189] 2) Add phage library (containing 5x10 12 phage particles) and incubated with a control antigen to exclude phage antibody clones that non-specifically bind to the Fc tag;

[0190] 3) After incubation, the supernatant is transferred to the SA magnetic beads bound to the target antigen and incubated again to allow the phage to bind to the target antigen;

[0191] 4) Wash the magnetic beads with washing solution to remove unbound phages;

[0192] 5) Elute the positive phage from the target antigen with elution solution and add neutralization solution to neutralize;

[0193] 6) Re-infect the host bacteria XL1-blue with the eluted phages and amplify the recovered phages. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phages;

[0194] 7) Repeat steps 1) to 6), usually 3 rounds of panning are required until a significant increase in the phage recovery rate (number of eluted phages / number of input phages) is observed.

[0195] The enriched phage pool can be used for subsequent single clone selection and ELISA / FACS screening.

[0196] Raji / Jurkat cell panning:

[0197] 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.

[0198] The brief experimental steps are as follows:

[0199] 1) The phage pool (containing 5x10 11 phage particles) and 1x10 7 Mix the negatively selected Jurkat cells and incubate them at room temperature for 2 hours on a rotating mixer to allow the antibody clones that bind to the negatively selected cell line to fully bind to these cells;

[0200] 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 rotating mixer for 2 hours at room temperature;

[0201] 3) Wash the cells 6 times with PBS, discard the supernatant each time, resuspend and centrifuge at 1500 rpm for 5 minutes to remove unbound phages;

[0202] 4) Elute the positive phages from the target antigen with elution solution and add neutralization solution to neutralize them;

[0203] 5) Use the eluted phage to re-infect the host bacteria and amplify the recovered phage. Keep a small amount of sample for gradient dilution, infect the host bacteria, apply Amp resistance plates, and calculate the number of recovered phage;

[0204] 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 phages / number of input phages) is observed.

[0205] The enriched phage pool can be used for the next step of monoclonal selection and ELISA / FACS screening.

[0206] Main materials and reagents:

[0207] Fully human phage antibody library, including natural library, semi-synthetic library and single domain library;

[0208] Helper phage KO7, Thermo / Invitrogen, 18311019;

[0209] Biotinylated Human Siglec-2 / CD22 Protein, Fc,Avitag™,ACRObiosystem, SI2-H82F8;

[0210] Biotinylated Human CD19, Fc Tag, ultra sensitivity, ACRObiosystem, CD9-H8259;

[0211] BeaverBeads™ Streptavidin, Beaver Bio, 22307-10;

[0212] High binding ELSIA plate, Costar, #3590

[0213] Blocking solution: PBS + 3% BSA

[0214] Rinse solution: PBS + 0.1% Tween20

[0215] Eluent: 0.2M Glycine, pH 2.2

[0216] Neutralization solution: 1M Tris, pH 9.1

[0217] Experimental results:

[0218] Using different antibody libraries, three rounds of protein panning were performed, and a significant increase in the recovery rate was observed in each panning (Table 1), demonstrating that the antibody clones were effectively enriched.

[0219] Table 1 Protein panning experiment results

[0220]

[0221] 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 higher than that in 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 expressed 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 used to enrich the 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 rate point of view, all 4 rounds of panning have been enriched and can be used for the next step of selecting monoclonal clones.

[0222] Table 2 Results of protein / cell selection experiments

[0223]

[0224] Example 2 Screening of specific clones from enriched phage pools using ELISA and FACS

[0225] Purpose and Principle: The phage pool enriched by affinity panning contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. In order to obtain specific clones, it is necessary to separate monoclonal clones, package them into monoclonal phages, and screen a large number of monoclonal clones by enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS), and select monoclonal clones that specifically bind to CD22 protein and CD22-positive cell line Raji at the same time. The specific monoclonal clones are further sequenced by DNA to determine the unique antibody sequence contained therein.

[0226] In the ELISA primary screening, the biotinylated target protein (CD22-Fc-Bio) is closer to the antigen conformation in the natural state in the reaction solution through the binding of streptavidin and biotin. The clones that only bind to CD22-Fc-Bio but not to the control antigen CD19-Fc-Bio are identified as specific clones. The FACS primary screening is carried out using the positive cell line Raji with high expression of CD22 and the CD22-negative cell line Jurkat. The clones that only bind to Raji cells and not to Jurkat cells are identified as specific clones. Through the two primary screenings of ELISA and FACS, candidate antibodies that can bind to recombinantly expressed CD22 protein and recognize CD22 molecules in the natural state on the cell surface can be obtained for further screening.

[0227] Brief steps of ELISA screening experiment:

[0228] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0229] 2) Dilute Strepavidin to 2 μg / mL with PBS and add 100 μL / well to the high binding ELISA plate for binding at room temperature for 2 h;

[0230] 3) Discard the coating solution, add 250 μL of blocking solution to each well, and block overnight at 4°C;

[0231] 4) Wash the plate twice with 250 μL of rinse buffer;

[0232] 5) Dilute the biotin-tagged target protein and control protein to 2 μg / mL with PBS, add 100 μg / well to the ELISA plate pre-coated with Strepavidin, and bind at room temperature for 1 h;

[0233] 6) Wash the plate twice with 250 μL of rinse buffer;

[0234] 7) Add 100 μL of the cultured phage supernatant from step 1) to the wells coated with the target antigen and allow to bind at room temperature for 2 h;

[0235] 8) Wash the plate 4 times with 250 μL of rinse buffer;

[0236] 9) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, and incubate at room temperature for 45 min;

[0237] 10) Wash the plate 4 times with 250 μL of rinse buffer;

[0238] 11) Add 1:2000 diluted HRP Donkey anti-mouse IgG, 100 μL / well, and incubate at room temperature for 45 minutes;

[0239] 12) Wash the plate 6 times with 250 μL of rinse buffer;

[0240] 13) Add 100 μL TMB colorimetric substrate and allow color development for 5 to 10 min;

[0241] 14) Add 100 μL 2M H2SO4 to terminate the reaction and read the results on a microplate reader.

[0242] Brief steps of FACS screening experiment:

[0243] 1) Cultivate and package monoclonal phage in deep-well 96-well plates;

[0244] 2) Wash Raji and Jurkat cells twice with PBS and resuspend in PBS to 1x10 7 / mL concentration, and dispensed into 96-well deep-well plates at 50 μL;

[0245] 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and allow to bind at 4°C for 2 h;

[0246] 4) Wash twice with 200 μL PBS;

[0247] 5) Add 1:2000 diluted mouse anti M13 primary antibody, 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0248] 6) Wash twice with 200 μL PBS;

[0249] 7) Add 1:300 diluted FITC horse anti mouse-IgG (H+L), 100 μL / well, pipette to mix, and incubate at room temperature for 45 min;

[0250] 8) Wash twice with 200 μL PBS; finally resuspend the cells with 200 μL PBS;

[0251] 9) Detect the fluorescence intensity of the sample FITC channel on the flow cytometer and analyze the results.

[0252] Main materials and reagents:

[0253] Helper phage KO7, Thermo / Invitrogen, 18311019

[0254] Streptavidin, Pierce, 21125

[0255] Biotinylated Human Siglec-2 / CD22 Protein, Fc,Avitag™,ACRObiosystem, SI2-H82F8;

[0256] Biotinylated Human CD19, Fc Tag, ultra sensitivity, ACRObiosystem, CD9-H8259;

[0257] High binding ELSIA plate, Costar, #3590

[0258] Corning 96 Well Clear Round Bottom TC-Treated Microplate, Costar, #3799

[0259] Blocking solution: PBS + 3% BSA

[0260] Rinse solution: PBS + 0.1% Tween20

[0261] Soluble one-component TMB substrate solution, Tiangen, PA-107-02

[0262] Anti-M13 Bacteriophage Coat Protein g8p antibody, abcam, ab9225

[0263] HRP Goat anti-mouse IgG(minimal x-reactivity) Antibody, Biolegend,405306

[0264] FITC horse anti mouse-IgG(H+L), Vector, FI2000.

[0265] Experimental results:

[0266] 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 shown in Figure 2. Figure 2As shown in the figure. As can be seen from 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, with good specificity. Clone A1, A3, A5 do not bind to the target antigen CD22 (CD22-Fc-Bio) and the control antigen CD19-Fc-Bio, and are negative clones. Negative phage control is a negative control phage antibody clone that does not bind to either the target antigen or the control antigen. Anti-M13 phage mouse Ab / anti-mouse HRP Ab is a negative antibody control with only the first antibody and the second antibody added. Anti-mouse HRP Ab is a negative antibody control with only the second antibody added. They do not bind to either the target antigen or the control antigen. Anti-human IgG-HRP Ab is a positive antibody control for detecting antigen tags. It binds to the antigen containing the Fc tag, indicating that the coated antigen has been bound to the ELISA plate.

[0267] The initial FACS screening results of some clones are as follows Figure 3 As shown. A3, A8 and A9 clones bind to Raji cells but not to Jurkat cells, and are specific clones; A1, A4, A5 and A7 weakly bind to Raji cells but not to Jurkat cells, and are weakly binding clones; A2 and A6 clones bind to neither of the two cells, and are negative clones. Neither weakly binding clones nor negative clones meet the experimental requirements.

[0268] Through ELISA detection and FACS initial screening, a total of 16 ELISA and FACS double-positive clones with good specificity were obtained. The 16 double-positive clones with good specificity were then 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 of multiple cell lines and ELISA of multiple antigens to detect the binding specificity of the candidate clones.

[0269] Example 3 Identification of monoclonal specificity by FACS using multiple cell lines

[0270] Experimental purpose and principle: Antibodies used for treatment must have very good target specificity, only bind to the target antigen, and not bind to any unrelated antigens; on the other hand, the amino acid sequence of the same antigen on different cell lines may be different (isomers or mutants) or the bound ligands may be different, and it is also necessary to examine whether the antibodies of the present invention can bind to cells positive for various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, the specificity of the initial screening clones was further evaluated by flow cytometry. In this experiment, a variety of CD22-positive cell lines and a variety of CD22-negative cell lines were used to react 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 with other cell lines that do not express CD22. Through this experiment, several clones with excellent specificity were obtained.

[0271] Experimental method: Same as FACS initial screening;

[0272] Main samples and reagents:

[0273] Raji cell line, CD22-positive cell line;

[0274] NALM6 cell line, CD22-positive cell line;

[0275] Jurkat cell line, a CD22-negative cell line;

[0276] U266 cell line, a CD22-negative cell line;

[0277] Raji KO CD22 cell line, CD22-negative cell line;

[0278] APC anti-human CD22 Antibody, Biolegend, 302510

[0279] The remaining reagents were the same as those for the FACS primary screening.

[0280] Experimental results:

[0281] 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 Figure 4A-4B middle. Figure 4A To test the results of using positive control antibodies to detect the expression of CD22 antigen using cell lines: Raji and NALM6 cells are positive for CD22 expression, Raji KO CD22, Jurkat, and U266 do not express CD22 antigen; Figure 4BTo screen the binding of clones to cell lines, Negative Control is a negative control phage antibody clone, and FITC anti-mouse IgG Ab is a negative control with only the second antibody added. Clone79 and 80 bind to both CD22-positive cell lines Raji and NALM6, but not to three CD22-negative cell lines Raji KOCD22, Jurkat, and U266, showing good specificity.

[0282] Example 4 Identification of monoclonal specificity by ELISA using antigens from different companies

[0283] Experimental purpose and principle: Antibodies used for treatment must have very good target specificity, only bind to the target antigen, and not bind to any unrelated antigens; on the other hand, the amino acid sequences of the same antigen produced by different companies will be different (isomers or mutants), and it is also necessary to examine whether the antibodies of the present invention can bind to various target proteins. In order to further analyze the specificity and universality of these monoclonal clones and find the best candidate clones, the specificity of the initial screening clones was further evaluated by enzyme-linked immunosorbent assay (ELISA). In this experiment, CD22 antigens containing different domains and unrelated antigens (Acro-CD19-bio) were used to react with these monoclonal phage antibodies to analyze whether these clones can bind to CD22 antigens with very different domains, and whether there is any non-specific binding with other CD22 unrelated antigens. Through this experiment, several clones with excellent specificity were obtained.

[0284] Experimental method: Same as ELISA initial screening.

[0285] Table 3 Main samples and reagents

[0286]

[0287] For more information on Iaso-CD22-(1,3,5,6,7)-bio containing different domains, see Figure 5 .

[0288] The remaining reagents are the same as those for the ELISA primary screening.

[0289] Experimental results:

[0290] Antibodies used for treatment must have very good target specificity. In order 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 figure, negative control is the negative control phage antibody clone, which does not bind to the target antigen or the control antigen, anti-M13 phage mouse Ab / anti-mouse HRP Ab is the negative antibody control with only the first antibody and the second antibody, and anti-mouse HRP Ab is the negative antibody control with only the second antibody, which does not bind to the target antigen or the control antigen, M971 positive control Ab / anti-human IgG HRP Ab is the positive antibody control of the target antigen (CD22-Fc-Bio), which binds to the target antigen but not to the control antigen. Anti-human IgG-HRP Ab is the secondary antibody control of the M971 positive control antibody experimental group, indicating that the binding of M971 antibody to CD22 antigen is caused by the positive control antibody, and the secondary antibody has no non-specific binding; anti-Rabbit IgG-HRP Ab / anti-human IgG-HRP Ab is the positive antibody control for detecting antigen tags, which binds to antigens containing Rabbit Fc tags or human Fc tags, indicating that the coated antigen has been bound to the ELISA plate. Clone 80 has strong or weak binding to 6 CD22 antigens with different domains, and does not bind to non-related antigens, with good specificity. Clone 79 not only binds to the target antigen CD22, but also to the non-related antigen Acro-CD19-bio, indicating that its binding may be a non-specific clone.

[0291] Based on the experimental results of Example 3 and Example 4, the clone that can specifically bind is clone 80.

[0292] Example 5 Detection of the binding ability of monoclonal antibodies to high-expressing cell lines by FACS

[0293] The affinity between CD22 antibody molecules and antigens may have an important impact on the killing effect and survival time of CAR-T or antibody drugs in patients. This example uses the FACS binding method to analyze the half effective concentration (Ec50) of antibody molecules. The FACS binding experimental steps are as follows:

[0294] (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;

[0295] (2) Wash the Raji cells twice with PBS and resuspend them in PBS to 1×10 7 / mL concentration, and dispensed into 96-well deep-well plates at 50 μL;

[0296] (3) Add 50 μL of diluted anti-CD22 IgG to each well, mix well, and allow to bind at 4°C for 2 h.

[0297] (4) Wash twice with 200 μL PBS;

[0298] (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.

[0299] (6) Wash twice with 200 μL PBS; finally, resuspend the cells with 200 μL PBS;

[0300] (7) Detect the fluorescence intensity of the sample FITC channel on a flow cytometer;

[0301] (8) Binding constants were analyzed by using Graphpad Prism software.

[0302] The main samples and reagents used in this example are as follows:

[0303] Raji cell line (CD22-positive cell line);

[0304] Alexa Fluor® 647 AffiniPure Goat Anti-Human IgG, Fcγ fragmentspecific, Jackson ImmunoReseach, 109-605-008.

[0305] Affinity refers to the strength of binding of a single molecule to its ligand. The binding ability to positive cell lines can usually be detected by FACS to evaluate 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 flow cytometry results of the screening phage monoclonal expressed protein antibodies and then bound to the positive cell line Raji showed that the affinity of SdAb 80 was higher than that of the control antibody M971.

[0306] Table 4 Affinity of antibodies for Raji cells

[0307]

[0308] Example 6 Affinity determination of anti-CD22 monoclonal antibodies

[0309] The affinity between CD22 monoclonal antibody and antigen may have an important influence on the killing effect and survival time of CAR-T or antibody drug in the patient's body. In order to determine this important property, this embodiment uses ForteBio's Octet molecular interaction technology to measure it. The biomembrane interference technology used by the Octet system is a label-free technology that provides high-throughput biomolecular interaction information in real time. The instrument emits white light to the sensor surface and collects reflected light. The reflection spectrum of different frequencies is affected by the thickness of the optical film layer of the biosensor. Some frequencies of reflected light form constructive interference (blue), while others are destructively interfered (red). These interferences are detected by the spectrometer and form an interference spectrum, which is displayed as the phase shift intensity (nm) of the interference spectrum. Therefore, once the number of molecules bound to the sensor surface increases or decreases, the spectrometer will detect the displacement of the interference spectrum in real time, and this displacement directly reflects the thickness of the biofilm on the sensor surface, from which high-quality data of biomolecular interactions can be obtained, thereby determining the kinetic parameters of biomolecular interactions (Kon, Kdis and KD), providing important information for the research and development process. The experimental steps for affinity determination are as follows:

[0310] (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.

[0311] (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). The binding time was 160 s and the dissociation time was 300 s at each concentration.

[0312] (3) Regenerate the chip by washing three times with 10 mM Glycine-HCl, pH 1.5.

[0313] (4) Binding constants were analyzed by using a 1:1 binding site model (Biacore X-100 evaluation software).

[0314] Affinity refers to the strength of binding of a single molecule to its ligand, and is usually measured and reported by the equilibrium dissociation constant (KD), which can be used to assess and rank the strength of the interaction 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 results of the affinity test are shown in Table 5: Clone80 IgG can bind to the CD22 antigen, and the affinity of Clone80 IgG is comparable to that of the control.

[0315] Table 5 Affinity of antibodies for CD22 antigen

[0316]

[0317] In summary, the fully human antibody targeting CD22 in the present invention has strong specificity, binds to CD22-positive cell lines, and does not bind to CD22-negative cell lines. The fully human antibody targeting CD22 specifically binds to CD22 with high affinity, and compared with heterologous antibodies, the fully human antibody targeting CD22 has lower immunogenicity, and has great application potential in the development of antibody drugs and cell therapy drugs.

[0318] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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) Tag sequences that 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 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.

8. 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 conjugated moiety, wherein the conjugated moiety is a detectable label.

9. 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, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

10. 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 8, 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.

11. 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 8, 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 8, or a combination thereof.

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