Antigen binding fragment and single-chain antibody targeting CD22 and application of antigen binding fragment and single-chain antibody

Abstract

The invention belongs to the technical field of cellular immune engineering, and particularly relates to a fully humanized CD22 targeting antigen binding fragment, a single-chain antibody and application thereof. The amino acid sequence of the antigen binding fragment and the amino acid sequence of the light chain variable region of the single-chain antibody are shown as any one of Sequence NO.1-5; the amino acid sequence of the heavy chain variable region is as shown in any one of Sequence NO.6 to Sequence NO.10. The single-chain antibody provided by the invention is good in affinity performance, can be combined with CD22 positive cells in flow cytometry, can be specifically combined with CD22 antigens in molecular interaction research, is a natural fully humanized antibody source, has a sequence completely coming from a human antibody gene pool, and has the advantages that compared with a mouse antibody, a chimeric antibody and a humanized antibody, the affinity performance of the antibody is greatly improved; the immunogenicity is greatly reduced, and the safety can be ensured to the maximum extent in clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell immune engineering, and specifically relates to a fully human antigen binding fragment and single-chain antibody targeting CD22 and applications thereof. Background Art

[0002] Conventional clinical methods for treating tumors, such as surgery, radiotherapy, and chemotherapy, all have bottlenecks such as poor specificity, great harm to normal tissues, and frequent risks of recurrence. These limitations have prompted the emergence of new treatment methods. Tumor immunotherapy has gradually emerged due to its safety, effectiveness, and low adverse reactions, becoming the fourth tumor treatment method after surgery, radiotherapy, and chemotherapy.

[0003] B cell receptor CD22, also known as sigle2, B lymphocyte adhesion molecule (BL-CAM), and T cell surface antigen Leu-14, mediates interactions between B cells and may be involved in the localization of B cells in lymphoid tissues. It is an important member of the immunoglobulin superfamily and the sialic acid-binding immunoglobulin-like lectin family. Its molecular weight is about 140kDa, and its extracellular domain contains seven Ig domains. Currently, the reported clinical indications for targeting CD22 antigen include B-cell lymphoma, diffuse large B-cell lymphoma, acute lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, systemic lupus erythematosus, etc. Because it is restrictedly expressed in mature B cells and expressed on the surface of most malignant B lymphoma cells, it has become one of the hot targets for the treatment of autoimmune diseases and B cell malignancies.

[0004] Epratuzumab, the earliest humanized anti-CD22 antibody in clinical trials (currently in Phase 3 clinical trials), has been widely used in the treatment of ALL patients through antibody-dependent cytotoxicity, CD22 phosphorylation and proliferation inhibition after cross-linking, etc. Epratuzumab has shown efficacy in a variety of hematopoietic cancers, whether used alone as a naked antibody or in combination with one or more other therapies, so antibody therapy plays an important role in tumor treatment. The first generation of monoclonal antibodies used in treatment are mouse monoclonal antibodies, but they can cause immune responses in the human body and are quickly cleared in the body, which brings certain difficulties to clinical application.

[0005] Currently, monoclonal antibodies targeting the CD22 antigen have appeared, but CD22 contains 7 different extracellular domains, and different epitopes have diverse clinical prospects. In addition, the current main target for acute lymphoblastic leukemia is CD19, which carries risks such as recurrence due to antigen loss and poor durability of CAR-T. Therefore, new anti-CD22 antibodies are still needed to be used in combination with other antibodies or drugs to find new methods for multi-dimensional tumor treatment. Summary of the invention

[0006] In view of this, the present invention aims to provide an antigen-binding fragment targeting CD22, wherein the variable region of the antigen-binding fragment is derived from a human natural antibody display library, which reduces immunogenicity and improves safety in clinical applications. The antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3;

[0007] The L-CDR1, L-CDR2 and L-CDR3 are selected from one of the following amino acid sequence combinations:

[0008] 1)QSVSSNL, GAS, QQYHSWPPLT;

[0009] 2)QSVSSTY,GAS,QQRSNWLT;

[0010] 3)QTIDNY, AAS, QQSYSTPPMYT;

[0011] 4)QSISSY, SAS, QQSFTTPFT;

[0012] 5)QSISSY, AAS, QQYSSYPHT;

[0013] The H-CDR1, H-CDR2 and H-CDR3 are selected from one of the following amino acid sequence combinations:

[0014] 1) GYTFTSYG, ISAYNGNT, ARDFQGIAVADLDY;

[0015] 2) GYTFTSY, INPSGGST, ARGVGSYAMDV;

[0016] 3)GFKFDDYP, ISWDGKTT, AKDRRSRTGWGYGGMDV;

[0017] 4) GDSVSSNSAA, TYYRSKWYN, ARSVGIARWDV;

[0018] 5) GDSVSSNSAA, TYYRSKWYN, ARATGTASGWFDP.

[0019] Furthermore, the light chain variable region includes, in addition to the above-mentioned high mutation regions of L-CDR1, L-CDR2 and L-CDR3, low mutation regions (also called skeleton regions or framework regions).

[0020] The amino acid sequence of the light chain variable region includes the sequence shown in Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4 or Sequence NO.5 or a functional variant thereof; wherein the sequence of the light chain variable region also includes a sequence with an identity of 80%, 90%, or 99% to the sequence shown in Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4 or Sequence NO.5, and the different sequences are mainly located in the low mutation region.

[0021] Further, similarly, in addition to the above-mentioned high mutation regions of H-CDR1, H-CDR2 and H-CDR3, the heavy chain variable region also includes a low mutation region, and the amino acid sequence of the heavy chain variable region includes the sequence shown in Sequence NO.6, Sequence NO.7, Sequence NO.8, Sequence NO.9 or Sequence NO.10 or its functional variant; wherein the sequence of the light chain variable region also includes a sequence with an identity of 80%, 90%, or 99% with the sequence shown in Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4 or Sequence NO.5, and the different sequences are mainly located in the low mutation region.

[0022] Furthermore, a full-length antibody targeting CD22 is provided, wherein the full-length antibody comprises the aforementioned antigen-binding fragment, and the full-length antibody can be obtained by conventional plasmid construction and expression.

[0023] The present invention aims to provide a single-chain antibody targeting CD22. Single-chain antibody (ScFv) drugs targeting tumors have broad application prospects. ScFv as a targeting molecule has the following advantages: small molecular weight, strong penetration, humanization, easy transformation, large-scale production, etc. At present, there are many ScFv drugs used in clinical practice at home and abroad. The variable region of the single-chain antibody is derived from human natural antibodies, and the sequence is completely derived from the human antibody gene library. Compared with mouse antibodies, chimeric antibodies, and humanized antibodies, its immunogenicity is greatly reduced, solving the problem of safety in clinical application.

[0024] The single-chain antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3.

[0025] The L-CDR1, L-CDR2 and L-CDR3 are selected from one of the following amino acid sequence combinations:

[0026] 1)QSVSSNL, GAS, QQYHSWPPLT;

[0027] 2)QSVSSTY,GAS,QQRSNWLT;

[0028] 3)QTIDNY, AAS, QQSYSTPPMYT;

[0029] 4)QSISSY, SAS, QQSFTTPFT;

[0030] 5)QSISSY, AAS, QQYSSYPHT;

[0031] The H-CDR1, H-CDR2 and H-CDR3 are selected from one of the following amino acid sequence combinations:

[0032] 1) GYTFTSYG, ISAYNGNT, ARDFQGIAVADLDY;

[0033] 2) GYTFTSY, INPSGGST, ARGVGSYAMDV;

[0034] 3)GFKFDDYP, ISWDGKTT, AKDRRSRTGWGYGGMDV;

[0035] 4) GDSVSSNSAA, TYYRSKWYN, ARSVGIARWDV;

[0036] 5) GDSVSSNSAA, TYYRSKWYN, ARATGTASGWFDP.

[0037] Furthermore, the light chain variable region includes, in addition to the above-mentioned high mutation regions of L-CDR1, L-CDR2 and L-CDR3, a low mutation region.

[0038] The amino acid sequence of the light chain variable region includes the sequence shown in Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4 or Sequence NO.5 or its functional variant; wherein the sequence of the light chain variable region also includes a sequence with an identity of 80%, 90%, or 99% to the sequence shown in Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4 or Sequence NO.5, and the different sequences are mainly located in the low mutation region.

[0039] Further, similarly, in addition to the above-mentioned high mutation regions of H-CDR1, H-CDR2 and H-CDR3, the heavy chain variable region also includes a low mutation region, and the amino acid sequence of the heavy chain variable region includes the sequence shown in Sequence NO.6, Sequence NO.7, Sequence NO.8, Sequence NO.9 or Sequence NO.10 or its functional variant; wherein the sequence of the light chain variable region also includes a sequence with an identity of 80%, 90%, or 99% with the sequence shown in Sequence NO.1, Sequence NO.2, Sequence NO.3, Sequence NO.4 or Sequence NO.5, and the different sequences are mainly located in the low mutation region.

[0040] Furthermore, the light chain variable region and the heavy chain variable region are connected via a linker, and the linker can be selected from any polypeptide that can have a connecting function, preferably a polypeptide whose amino acid sequence is shown in Sequence NO.11 or a functional variant thereof.

[0041] In certain specific embodiments, the single-chain antibody is one of the following light chain and heavy chain combinations:

[0042] 1) The amino acid sequence of the light chain variable region is shown in Sequence No. 1, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 6;

[0043] 2) The amino acid sequence of the light chain variable region is shown in Sequence No. 2, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 7;

[0044] 3) The amino acid sequence of the light chain variable region is shown in Sequence No. 3, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 8;

[0045] 4) The amino acid sequence of the light chain variable region is shown in Sequence No. 4, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 9;

[0046] 5) The amino acid sequence of the light chain variable region is shown in Sequence No. 5, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 10.

[0047] Furthermore, a CAR structure comprising any of the above-mentioned single-chain antibodies is also provided, and the CAR structure can be prepared into CAR-T cells after transfection of immune cells for targeted treatment of tumors with CD22 targets. The CAR structure also includes a hinge region, a transmembrane region, and an intracellular signal region. The hinge region sequence can be derived from: IgG, CD8, CD7, CD4 or other protein molecules with equivalent functions; the transmembrane region can be derived from: CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 and CD86 or other protein molecules with equivalent functions; the intracellular signal region can be derived from: CD3, CD137, CD28, CD27, OX40, ICOS, GITR, CD2, CD40, PD-1, PD1L, B7-H3, lymphocyte function-associated antigen-1 (LFA-1), ICAM-1, CD7, NKG2C, CD83, CD86 and CD127 other protein molecules with equivalent functions.

[0048] Further, the CAR structure comprises one or more components of a natural killer cell receptor (NKR), thereby forming a NKR-CAR. The NKR component can be a transmembrane domain, hinge domain or cytoplasmic domain from any of the following natural killer cell receptors: killer cell immunoglobulin-like receptors (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1 / S1, KIR3DL2, KIR3DL3, KIR2DP1 and KIR3DP1; natural cytotoxicity receptors (NCRs), e.g., NKp30, NKp44, NKp46; the signaling lymphocyte activation molecule (SLAM) family of immune cell receptors, e.g., CD48, CD229, 2B4, CD84, NTB-A, CRA, BLAME, and CD2F-10; Fc receptors (FcRs), e.g., CD16, and CD64; and Ly49 receptors, e.g., LY49A, LY49C. The NKR-CAR molecule can interact with an adapter molecule or an intracellular signaling domain (e.g., DAP12). Further, a nucleic acid sequence encoding the aforementioned single-chain antibody is provided, the single-chain antibody comprising a light chain variable region and a heavy chain variable region, and a linker connecting the light chain variable region and the heavy chain variable region, wherein the nucleotide sequence encoding the light chain is as shown in Sequence NO.12, Sequence NO.13, Sequence NO.14, Sequence NO.15, or Sequence NO.16; the nucleotide sequence encoding the heavy chain is as shown in Sequence NO.17, Sequence NO.18, Sequence NO.19, Sequence NO.20 or Sequence NO.21, and the nucleotide sequence encoding the kiner is as shown in 22 or 23.

[0049] Furthermore, a recombinant plasmid comprising the aforementioned nucleic acid sequence is also provided, wherein the recombinant plasmid also comprises an expression vector. The expression vector is any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector, and a plasmid.

[0050] In certain embodiments, the lentiviral vector is selected from the group consisting essentially of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0051] In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR contemplated herein, and a right (3') retroviral LTR.

[0052] In certain embodiments, the CAR comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

[0053] In certain embodiments, the promoter of the 5' LTR is replaced with a heterologous promoter.

[0054] In certain embodiments, the heterologous promoter is a cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter, or a Simian Virus 40 (SV40) promoter.

[0055] In certain embodiments, the 5'LTR or 3'LTR is a lentiviral LTR.

[0056] In certain embodiments, the 3'LTR is a self-inactivating (SIN) LTR.

[0057] Furthermore, an immune engineering cell is provided, which comprises the antibody binding fragment or full-length antibody described in any one of the preceding items.

[0058] Furthermore, the immune engineered cells are obtained by transfecting immune cells with any of the foregoing recombinant plasmids. Preferably, the immune cells are T cells, T cell precursors or NK cells. The immune cells are preferably T cells. The agent provides a CAR-T cell or a CAR-T cell product.

[0059] In certain embodiments, the cell may express other active agents, for example, an active agent that enhances the activity of CAR-expressing cells. An active agent may be an active agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to initiate immune effector responses in some embodiments. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as PD1 CAR.

[0060] Furthermore, there is also provided a use of the aforementioned antigen binding fragment or the aforementioned full-length antibody or the aforementioned single-chain antibody or the aforementioned CAR structure or the aforementioned nucleic acid sequence or the aforementioned recombinant plasmid or the aforementioned immune engineering cell in the preparation of anti-tumor / autoimmune inflammatory drugs. Specifically, the prepared anti-tumor drug can be used for immunotherapy of blood or solid tumors targeting CD22 targets.

[0061] Preferably, the tumor / autoimmune inflammation is B-cell lymphoma, diffuse large B-cell lymphoma, acute lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, systemic lupus erythematosus.

[0062] Furthermore, there is provided a use of the aforementioned antigen-binding fragment, the aforementioned full-length antibody or the aforementioned single-chain antibody in the preparation of a detection reagent / detection kit, wherein the detection reagent or detection kit can efficiently and accurately identify the CD22 antigen.

[0063] The present invention also aims to provide a method for preparing the aforementioned single-chain antibody, wherein a fully human single-chain antibody library is constructed by phage display technology. Compared with the conventional method of obtaining ScFv, a fully human single-chain antibody library is constructed by phage display technology, and a low-immunogenic therapeutic fully human single-chain antibody can be obtained in a shorter time.

[0064] There are currently four recombinant antibody platforms used to produce human antibodies for therapeutic purposes: (1) "humanization" of mouse monoclonal antibodies; (2) immunization of transgenic mice containing human antibody genes; (3) in vitro screening of fully human antibodies from a large number of human antibody libraries; and (4) single B cell antibody preparation technology. Phage display technology is the most mature technology for preparing monoclonal antibody drugs. It was proposed by George Smith of the University of Missouri in the United States in 1985. Its basic principle is to insert exogenous DNA into the genomic DNA of filamentous phage, fuse it with the capsid protein of the phage, and display it on the surface of the phage. Through affinity panning by appropriate methods, phages carrying a certain fragment with specific affinity are enriched, and their gene sequences can be obtained by sequencing.

[0065] In certain specific embodiments, in order to screen human antibodies targeting the CD22 antigen, a fully human single-chain antibody library was constructed. Normal human PBMC was used as the raw material, total RNA was extracted and reverse transcribed into cDNA, the antibody variable region was amplified, and the heavy chain variable region VH and the light chain variable region VL were connected by a linker to obtain the antibody sequence, which was constructed into a phagemid vector and electroporated into a display strain to construct an antibody library. The library was screened by the CD22 extracellular segment antigen, and the screened clones were identified, and finally a plurality of ScFvs with specific binding to the CD22 antigen were obtained.

[0066] In the present invention, the term "functional variant" generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the chimeric antigen receptor) and having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity therewith. In certain embodiments, the variant of the amino acid sequence has substantially the same function therewith.

[0067] The beneficial effects of the present invention are

[0068] The variable region of the single-chain antibody targeting CD22 provided by the present invention is derived from a natural fully human antibody, and the sequence is completely derived from the human antibody gene library. Compared with mouse antibodies, chimeric antibodies, and humanized antibodies, its immunogenicity is greatly reduced, and safety can be guaranteed to the greatest extent in clinical applications.

[0069] The single-chain antibody provided by the present invention can specifically recognize human CD22 antigen and can be applied to immunotherapy of blood or solid tumors targeting C22 target.

[0070] The single-chain antibody provided by the present invention has good affinity performance and can bind to CD22 positive cells in flow cytometry detection. Molecular interaction studies show that it can specifically bind to CD22 antigen and has potential clinical diagnosis and treatment uses. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Flow cytometric staining of K562 cells with m971 mouse ScFv.

[0072] Figure 2 Flow cytometric staining of K562-CD22 cells with m971 mouse ScFv.

[0073] Figure 3 Flow cytometry staining of K562 cells with SK-01-A13.

[0074] Figure 4 Flow cytometry staining of K562 cells with SK-01-A14.

[0075] Figure 5 Flow cytometry staining of K562 cells with SK-03-B09.

[0076] Figure 6 Flow cytometry staining of K562 cells with SK-04-B07.

[0077] Figure 7 Flow cytometry staining of K562 cells with SK-04-E07.

[0078] Figure 8 Flow cytometry staining of K562-CD22 cells with SK-01-A13.

[0079] Fig. 9 Flow cytometry staining of K562-CD22 cells with SK-01-A14.

[0080] Fig.10 Flow cytometry analysis of K562-CD22 cells using SK-03-B09.

[0081] Fig.11 Flow cytometric staining of K562-CD22 cells with SK-04-B07.

[0082] Fig.12 Flow cytometric staining of K562-CD22 cells with SK-04-E07.

[0083] Fig.13 This is a kinetic test of the binding between m971 and CD22 antigen.

[0084] Fig.14 This is a kinetic test of the binding between SK-01-A13 and CD22 antigen.

[0085] Fig.15 This is a kinetic test of the binding between SK-03-B09 and CD22 antigen.

[0086] Fig.16 This is a kinetic test of the binding between SK-04-B07 and CD22 antigen.

[0087] Fig.17 This is a kinetic test of the binding between SK-04-E07 and CD22 antigen. DETAILED DESCRIPTION

[0088] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0089] In the embodiment of the present invention, the specific implementation process of antigen panning is as follows:

[0090] (1) Blocking: Dissolve 5% skim milk powder in PBS, filter and use as blocking solution. Resuspend phage and CD22-protein G-coupled magnetic beads in appropriate amount of blocking solution and mix by rolling.

[0091] (2) Co-incubation: Place CD22-protein G-coupled magnetic beads on a magnetic rack, discard the supernatant, resuspend the magnetic beads with phage, and incubate at room temperature with rolling at 160 rpm for 1 h;

[0092] (3) Washing: Place the magnetic bead-phage mixture on a magnetic rack, discard the supernatant, and add an appropriate volume of 0.1% Tween-80 PBST washing solution to wash the magnetic beads. The number of washes is determined according to the number of panning rounds;

[0093] (4) Elution: Place the washed beads on a magnetic rack, aspirate the supernatant, add 400 μL of pH 2.2 Gly-HCl, mix well, incubate at room temperature, and then add about 20 μL of pH 9.0 Tris-HCl to adjust the pH. Finally, place the mixture on a magnetic rack and transfer the phage supernatant to an EP tube to complete a round of panning;

[0094] (5) Enrichment: The phages were inoculated into TGI bacterial solution for infection, centrifuged after standing at 37°C, 200 μL of culture medium was retained to resuspend the precipitate, spread on 2YTAG plates, and cultured inverted at 30°C overnight;

[0095] (6) Plate washing: The plaques on the plate cultured overnight are washed off with the culture medium and used as the seed solution for the next round of library packaging. The specific process is the same as that for the preparation of the single-chain antibody library.

[0096] In the embodiment of the present invention, the specific implementation process of ELISA detection is as follows:

[0097] (1) Coating: Dilute CD22 antigen with 50 mM carbonate coating buffer (pH 9.6), add 100 μL / well to a 96-well plate, and cover overnight;

[0098] (2) Obtaining phage samples: centrifuge the overnight cultured phage monoclonal recombinant bacterial solution at 8000 rpm at 4°C for 10 min, and take the supernatant as the test sample;

[0099] (3) Blocking: After the antigen-coated plate was washed three times with PBS on a plate washer, 200 μL of 5% skim milk powder blocking solution was added to each well and blocked at 37°C for 2 h.

[0100] (4) Antibody incubation: Wash the plate three times with PBS in a plate washer, add 100 μL of the phage monoclonal recombinant solution to be tested to each well, and incubate at 37°C for 1 h.

[0101] (5) Adding secondary antibody: Wash the plate three times with PBS in a plate washer, add 100 μL of 1:5000 Anti-M13-HRP secondary antibody to each well, and incubate at 37°C for 1 h;

[0102] (6) Color development: Wash the plate 6 times with a plate washer, add 100 μL of TMB color development solution to each well, and place at room temperature in the dark for 25 min for color development;

[0103] (7) Termination: Add 50 μL 2M H2SO4 to each well to terminate the reaction;

[0104] (8) Detection: Place the test plate in a microplate reader to detect the OD450 absorbance. Phage clones that are 2.5 times higher than the negative control are considered positive clones.

[0105] Example 1 Construction of a fully human single-chain antibody library

[0106] Ficoll separation solution is used for PBMC separation, and the Ficoll separation solution is slowly added to normal human blood so that the Ficoll separation solution and normal human blood maintain a clear separation interface. A 50mL centrifuge tube containing the blood and the separation solution is centrifuged at about 15°C for 20min. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, wherein at the junction of the upper and middle layers, there is a narrow band of white cloud layer mainly composed of PBMC, i.e., the PBMC cell layer. Carefully suck off the upper plasma mixture with a sterile Pasteur pipette, and then suck PBMC with a new sterile Pasteur pipette to obtain separated PBMC.

[0107] Total RNA was extracted by conventional methods and reverse transcribed into cDNA. Primers were then designed according to the method in Sblattero, D., Bradbury, A., (1998) A definitive set of oligonucleotide primers for amplifying human Vregions. Immunotechnology 3, 271-278, wherein VL was in front or back, VH was in back or front, and the middle was connected by a flexible Linker, and PCR was used to obtain the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody, and the ScFv nucleic acid fragment was amplified by conventional overlapping PCR (PCR method reference "Molecular Cloning: A Laboratory Manual" (third edition), Joe Sambrook, David Russell, Science Press, USA), the ScFv nucleic acid fragment was connected to the phagemid vector pComb3xss, and the product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

[0108] Example 2 Preparation of Phage Display Fully Human Single-chain Antibody Library

[0109] The library bacterial solution was added to fresh LB liquid medium for recovery, cultured to OD600≈0.5, and then VSCM13 helper phage was added at a multiplicity of infection of VCSM13: bacteria = 50:1, mixed thoroughly, and continued to be cultured in a shaker after standing. The culture was centrifuged and the supernatant was discarded. The precipitate was resuspended in ampicillin and kanamycin double resistance SOB medium and cultured overnight. The bacterial solution was centrifuged for 20 minutes, the supernatant was collected and PEG 8000 2.5mmol / LNaCl solution was added, incubated on ice for 1 hour, and then centrifuged for 30 minutes. The precipitated phage was resuspended in PBS and filtered with a filter membrane.

[0110] Example 3 Antigen panning

[0111] The CD22 protein with Fc tag is co-incubated with proteinG magnetic beads to prepare CD22-proteinG coupled magnetic beads, which are then drawn into the prepared fully human single-chain antibody library phage panning. After 3-4 rounds of co-incubation, washing and elution panning, specific monoclonal antibodies against the antigen can be enriched.

[0112] Example 4 Screening of positive clones

[0113] After the panning, the monoclonal plaques finally released from the library were picked for ELISA detection and screening to obtain phage clones that bind to the CD22 antigen. The following 5 ScFv strains were obtained by panning, all of which have the specific binding ability to the human CD22 antigen.

[0114] SK-03-B09 / D-2-13: the amino acid sequence of the light chain variable region is shown in Sequence No. 1, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 6;

[0115] SK-01-A13 / E-6-85 / D-3-368: the amino acid sequence of the light chain variable region is shown in Sequence No. 2, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 7;

[0116] SK-01-A14 / E-1-29: the amino acid sequence of the light chain variable region is shown in Sequence No. 3, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 8;

[0117] SK-04-E07 / E-1-35: the amino acid sequence of the light chain variable region is shown in Sequence No. 4, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 9;

[0118] SK-04-B07: The amino acid sequence of the light chain variable region is shown in Sequence No. 5, and the amino acid sequence of the heavy chain variable region is shown in Sequence No. 10.

[0119] Example 5 Expression and purification of ScFv

[0120] The pComb3xss plasmid of the positive phage clone was mixed with 100μL competent Rosetta gami (DE3) bacteria and placed in an ice bath, heat-shocked at 42℃, and then spread on an LB plate containing ampicillin resistance after an ice bath, and placed in a 37℃ constant temperature incubator for overnight culture; the monoclonal plaque formed was picked and placed in 4ml LB medium, and after shaking culture, the bacterial solution was transferred to the medium and cultured. When the bacterial solution OD reached 0.5-1.0, IPTG was added and the final concentration was adjusted, and the expression was induced for 12h. The bacteria were collected by centrifugation and PBS was added to resuspend the precipitate, ultrasonically broken, and the lysate was centrifuged at 12000rpm at 4℃, the precipitate was discarded, and the supernatant was collected for protein purification.

[0121] The supernatant of bacterial lysis was filtered through 0.22 μm, diluted with an equal volume of PBS, and then enriched with a GE Ni Sepharose excel purification column for ScFv. After washing with 5 column volumes of PBS, impurities were removed by washing with 5 column volumes of PBS solution containing 30 mM imidazole. The protein was eluted with a PBS solution containing 500 mM imidazole, and the washing solution was collected and concentrated with a 3 KDa ultrafiltration tube. The sample was loaded onto a GE HiLoad Superdex 16 / 600 200 pg molecular exclusion chromatography column, washed with PBS, and the ultraviolet absorption peak was collected. The ScFv purification effect was identified by SDS-PAGE, and then flow staining specificity detection was performed.

[0122] Example 6 Flow staining ability identification

[0123] K562 and K562-CD22 cells were dispensed into 1.5 mL Eppendorf tubes, 1×10 cells per tube. 6 Each cell was used as the target cell. After centrifugation at 1000g for 5min, the supernatant was discarded and resuspended with 50μL 30μg / mL m971 / SK-01-A13 / SK-01-014 / SK-03-B09 / SK-04-B07 / SK-04-E07 ScFv solution and incubated at 4℃ in the dark. After 30min, PBS was added to resuspend the cells, washed at 1000g for 5min, the supernatant was discarded, Anti-His-647 fluorescent secondary antibody for detection was added and the cells were resuspended, incubated at 4℃ in the dark; resuspended with 1mL PBS and washed twice at 1000g for 5min, the supernatant was discarded and resuspended with 200μL PBS. All cell tubes were tested for flow cytometry staining positive rate and mean fluorescence intensity (MFI), and the staining results of m971 staining group and anti-CD22 ScFv staining group obtained by panning were compared to evaluate the flow staining specificity of ScFv. The staining results of m971 ScFv on K562 are as follows Figure 1 As shown, the staining results of m971 ScFv on K562-CD22 cells are as follows Figure 2 As shown, the staining results of CD22-specific ScFv obtained by panning on K562 cells are as follows Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, the staining results of K562-CD22 are as follows Figure 8 , Fig. 9 , Fig.10 , Fig.11 and Fig.12 shown.

[0124] Example 7 Detection of Kinetic Parameters of Antigen-Antibody Binding

[0125] The SA / HIS1K biosensor was subjected to baseline1 for 60s in the analysis buffer, loading for 120s in the ScFv solution for ligand protein solidification, transferred to baseline2 in the analysis buffer for 120s, transferred to the gradient diluted analyte (CD22 monovalent antigen) solution for association for 120s, and finally dissociation for 300s in the analysis buffer. The change in the response value R is used to determine whether there is specific binding. Generally speaking, specific binding can be determined when the maximum response value Rmax after subtracting the reference is greater than 0.05nm and there is concentration dependence and dissociation. The Data Analysis software that comes with the Fortebio OctetK2 instrument can provide the kinetic parameters represented by the binding-dissociation curve, such as the binding constant (Kon), the dissociation constant (Kdis), and the equilibrium dissociation constant (KD). The unit of Kon is 1 / Ms, which is used to describe the rate of antigen-antibody binding. The higher the Kon, the faster the antibody-antigen binding to form a complex. The unit of Kdis is 1 / s, which is used to describe the rate of antigen-antibody dissociation. The higher the Kdis, the faster the antigen-antibody complex dissociates. KD is the ratio of Kdis to Kon, which is used to comprehensively describe the difficulty of antigen-antibody binding. The smaller the KD, the higher the antibody affinity. Fig.13 The kinetic parameters are shown in Table 1 below; the binding kinetics of CD22-specific ScFv obtained by panning with CD22 antigen are shown in Fig.14 , Fig.15 , Fig.16 , Fig.17 As shown, its kinetic parameters are shown in Table 2, Table 3, Table 4 and Table 5.

[0126] Table 1 Binding kinetic parameters of m971 and CD22 antigen Table 2 Kinetic parameters of binding of SK-01-A13 to CD22 antigen Table 3 Kinetic parameters of binding of SK-03-B09 to CD22 antigen Table 4 Kinetic parameters of binding of SK-04-B07 to CD22 antigen

[0127]

[0128] Table 5 Kinetic parameters of binding of SK-04-E07 to CD22 antigen

[0129]

[0130]

[0131] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An antigen-binding fragment targeting CD22, characterized in that The antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3; The L-CDR1, L-CDR2 and L-CDR3 are selected from the following amino acid sequences QSISSY, SAS, QQSFTTPFT The H-CDR1, H-CDR2 and H-CDR3 are selected from the following amino acid sequences GDSVSSNSAA,TYYRSKWYN,ARSVGIARWDV.

2. A full-length antibody targeting CD22, characterized in that: The full-length antibody comprises the antigen-binding fragment of claim 1.

3. A single-chain antibody targeting CD22, characterized in that: The single-chain antibody comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR; the L-CDR1, L-CDR2 and L-CDR3 are selected from the following amino acid sequences QSISSY, SAS, QQSFTTPFT; The H-CDR1, H-CDR2 and H-CDR are selected from the following amino acid sequences GDSVSSNSAA,TYYRSKWYN,ARSVGIARWDV.

4. The single-chain antibody according to claim 3, characterized in that The amino acid sequence of the light chain variable region is shown in Sequence NO.4 or a functional variant thereof; the amino acid sequence of the heavy chain variable region is shown in Sequence NO.9 or a functional variant thereof.

5. The single-chain antibody according to claim 3 or 4, wherein the light chain variable region and the heavy chain variable region are connected by a linker, and the amino acid sequence of the linker is as shown in Sequence NO.11 or a functional variant thereof. 6.CAR structure, characterized in that: Comprising the single-chain antibody according to any one of claims 3 to 5.

7. A nucleic acid sequence, characterized in that It comprises a nucleotide sequence encoding the single-chain antibody according to any one of claims 3 to 5, wherein the nucleotide sequence encoding the light chain is shown in Sequence No. 15; and the nucleotide sequence encoding the heavy chain is shown in Sequence No.

20.

8. A recombinant plasmid, characterized in that Comprising the nucleic acid sequence and expression vector according to claim 7.

9. An immunoengineered cell, characterized in that: The immune engineered cells are obtained by transfecting immune cells with the recombinant plasmid described in claim 8. Preferably, the immune cells are T cells, T cell precursors or NK cells.

10. Use of the antigen-binding fragment of claim 1, the full-length antibody of claim 2, the single-chain antibody of any one of claims 3-5, the CAR structure of claim 6, the nucleic acid sequence of claim 7, the recombinant plasmid of claim 8, or the immune-engineered cell of claim 9 in the preparation of anti-tumor / autoimmune inflammation drugs; preferably, the tumor / autoimmune inflammation is B-cell lymphoma, diffuse large B-cell lymphoma, acute lymphocytic leukemia, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, systemic lupus erythematosus.

11. Use of the antigen-binding fragment of claim 1, the full-length antibody of claim 2, or the single-chain antibody of any one of claims 3 to 5 in the preparation of a detection reagent / detection kit.

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