Fully humanized anti-human CD22 chimeric antigen receptor and its applications

Abstract

The present invention provides anti-CD22 antibody molecules and CD22-targeting chimeric antigen receptors (CARs) constructed using the anti-CD22 antibody molecules. The present invention further provides applications of the anti-CD22 antibody molecules and the chimeric antigen receptors in the production of medicines for treating CD22-associated diseases.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application entitled "Fully humanized anti-human CD22 chimeric antigen receptor and its application" filed with the China Patent Office on April 2, 2020, application number 202010254388.3.

[0002] The present invention relates to anti-CD22 antibody molecules and chimeric antigen receptors (CARs) that target CD22, as well as applications of these antibody molecules and chimeric antigen receptors. [Background technology]

[0003] CD22 is a B-lineage differentiation antigen and a member of the Siglec lectin family, containing seven extracellular IgG-like domains. It is expressed at every stage of B-cell development, but not on plasma cells, hematopoietic stem cells, or other parenchymal cells. In many cases, CD22 expression persists during the transformation of normal B cells into tumor cells, and approximately 70% of B-cell lymphoma and leukemia cells express CD22 molecules [1].

[0004] In recent years, the development of adoptive cellular immunotherapy has provided a new approach to tumor treatment. This approach involves genetically engineered T cells to express chimeric antigen receptors on their cell surface. In a commonly employed configuration, chimeric antigen receptors combine the antigen-binding specificity of monoclonal antibodies with the effector functions of T cells, promoting the specific killing of cells expressing a particular antigen by these engineered T cells. Such chimeric antigen receptor-mediated therapy can overcome immune tolerance to autoantigens, regardless of the patient's MHC status.

[0005] Currently, there are several chimeric antigen receptors targeting CD22 in preclinical research or clinical trials, but they usually have problems such as insufficient affinity between the chimeric antigen receptor and the target antigen, and poor cytotoxicity of CAR-T cells against target cells. Summary of the Invention

[0006] In one embodiment, provided herein is an anti-CD22 antibody molecule comprising a light chain variable region and a heavy chain variable region, wherein the heavy chain variable region comprises a complementarity determining region selected from any of the following groups: HCDR1 having the sequence shown in SEQ ID NO: 4, HCDR2 having the sequence shown in SEQ ID NO: 5, and HCDR3 having the sequence shown in SEQ ID NO: 6; HCDR1 having the sequence shown in SEQ ID NO: 10, HCDR2 having the sequence shown in SEQ ID NO: 11, and HCDR3 having the sequence shown in SEQ ID NO: 12; HCDR1 having the sequence shown in SEQ ID NO: 16, HCDR2 having the sequence shown in SEQ ID NO: 17, and HCDR3 having the sequence shown in SEQ ID NO: 18.

[0007] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

[0008] In some embodiments, the anti-CD22 antibody molecule is in an IgG format and binds to CD22 with a K D or the anti-CD22 antibody molecule is in Fab format and binds to CD22 with a K value of 20 nM or less. D It has a value.

[0009] In some embodiments, the anti-CD22 antibody molecule is a fully humanized antibody molecule.

[0010] In another aspect, the present invention provides a method for the treatment of rhesus malabsorption, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a complementarity determining region selected from any of the following groups: LCDR1 having the sequence shown in SEQ ID NO: 1, LCDR2 having the sequence shown in SEQ ID NO: 2, and LCDR3 having the sequence shown in SEQ ID NO: 3; LCDR1 having the sequence shown in SEQ ID NO: 7, LCDR2 having the sequence shown in SEQ ID NO: 8 and LCDR3 having the sequence shown in SEQ ID NO: 9, LCDR1 having the sequence shown in SEQ ID NO: 13, LCDR2 having the sequence shown in SEQ ID NO: 14 and LCDR3 having the sequence shown in SEQ ID NO: 15, and The heavy chain variable region has a complementarity determining region selected from any of the following groups: HCDR1 having the sequence shown in SEQ ID NO: 4, HCDR2 having the sequence shown in SEQ ID NO: 5, and HCDR3 having the sequence shown in SEQ ID NO: 6; HCDR1 having the sequence shown in SEQ ID NO: 10, HCDR2 having the sequence shown in SEQ ID NO: 11, and HCDR3 having the sequence shown in SEQ ID NO: 12; HCDR1 having the sequence shown in SEQ ID NO: 16, HCDR2 having the sequence shown in SEQ ID NO: 17, and HCDR3 having the sequence shown in SEQ ID NO: 18; The present invention provides an anti-CD22 antibody molecule comprising:

[0011] In some embodiments, the light chain variable region comprises an LCDR1 having the sequence set forth in SEQ ID NO: 1, an LCDR2 having the sequence set forth in SEQ ID NO: 2, and an LCDR3 having the sequence set forth in SEQ ID NO: 3; and the heavy chain variable region comprises an HCDR1 having the sequence set forth in SEQ ID NO: 4, an HCDR2 having the sequence set forth in SEQ ID NO: 5, and an HCDR3 having the sequence set forth in SEQ ID NO: 6. the light chain variable region comprises an LCDR1 having the sequence set forth in SEQ ID NO:7, an LCDR2 having the sequence set forth in SEQ ID NO:8, and an LCDR3 having the sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises an HCDR1 having the sequence set forth in SEQ ID NO:10, an HCDR2 having the sequence set forth in SEQ ID NO:11, and an HCDR3 having the sequence set forth in SEQ ID NO:12, or The light chain variable region comprises an LCDR1 having the sequence shown in SEQ ID NO: 13, an LCDR2 having the sequence shown in SEQ ID NO: 14, and an LCDR3 having the sequence shown in SEQ ID NO: 15, and the heavy chain variable region comprises an HCDR1 having the sequence shown in SEQ ID NO: 16, an HCDR2 having the sequence shown in SEQ ID NO: 17, and an HCDR3 having the sequence shown in SEQ ID NO: 18.

[0012] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23.

[0013] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

[0014] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 22, or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 23, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24.

[0015] In some embodiments, the anti-CD22 antibody molecule is in an scFv format and comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

[0016] In some embodiments, the anti-CD22 antibody molecule is in an IgG format and binds to CD22 with a KD or the anti-CD22 antibody molecule is in Fab format and binds to CD22 with a K value of 20 nM or less. D It has a value.

[0017] In some embodiments, the anti-CD22 antibody molecule is a fully humanized antibody molecule.

[0018] In another embodiment, provided herein is a polypeptide comprising an antigen-binding domain that binds to CD22, comprising a light chain variable region and a heavy chain variable region, wherein said light chain variable region comprises a complementarity determining region selected from any of the following groups: LCDR1 having the sequence shown in SEQ ID NO: 1, LCDR2 having the sequence shown in SEQ ID NO: 2, and LCDR3 having the sequence shown in SEQ ID NO: 3; LCDR1 having the sequence shown in SEQ ID NO: 7, LCDR2 having the sequence shown in SEQ ID NO: 8, and LCDR3 having the sequence shown in SEQ ID NO: 9; LCDR1 having the sequence shown in SEQ ID NO: 13, LCDR2 having the sequence shown in SEQ ID NO: 14, and LCDR3 having the sequence shown in SEQ ID NO: 15; and The heavy chain variable region has a complementarity determining region selected from any of the following groups: HCDR1 having the sequence shown in SEQ ID NO: 4, HCDR2 having the sequence shown in SEQ ID NO: 5, and HCDR3 having the sequence shown in SEQ ID NO: 6; HCDR1 having the sequence shown in SEQ ID NO: 10, HCDR2 having the sequence shown in SEQ ID NO: 11, and HCDR3 having the sequence shown in SEQ ID NO: 12; HCDR1 having the sequence shown in SEQ ID NO: 16, HCDR2 having the sequence shown in SEQ ID NO: 17, and HCDR3 having the sequence shown in SEQ ID NO: 18; The present invention provides a chimeric antigen receptor that targets CD22, comprising:

[0019] In some embodiments, the light chain variable region comprises an LCDR1 having the sequence set forth in SEQ ID NO: 1, an LCDR2 having the sequence set forth in SEQ ID NO: 2, and an LCDR3 having the sequence set forth in SEQ ID NO: 3; and the heavy chain variable region comprises an HCDR1 having the sequence set forth in SEQ ID NO: 4, an HCDR2 having the sequence set forth in SEQ ID NO: 5, and an HCDR3 having the sequence set forth in SEQ ID NO: 6. the light chain variable region comprises an LCDR1 having the sequence set forth in SEQ ID NO:7, an LCDR2 having the sequence set forth in SEQ ID NO:8, and an LCDR3 having the sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises an HCDR1 having the sequence set forth in SEQ ID NO:10, an HCDR2 having the sequence set forth in SEQ ID NO:11, and an HCDR3 having the sequence set forth in SEQ ID NO:12, or The light chain variable region comprises an LCDR1 having the sequence shown in SEQ ID NO: 13, an LCDR2 having the sequence shown in SEQ ID NO: 14, and an LCDR3 having the sequence shown in SEQ ID NO: 15, and the heavy chain variable region comprises an HCDR1 having the sequence shown in SEQ ID NO: 16, an HCDR2 having the sequence shown in SEQ ID NO: 17, and an HCDR3 having the sequence shown in SEQ ID NO: 18.

[0020] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:19, SEQ ID NO:21, or SEQ ID NO:23.

[0021] In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:20, SEQ ID NO:22, or SEQ ID NO:24.

[0022] In some embodiments, the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20; the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 21, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 22; or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 23, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24.

[0023] In some embodiments, the antigen binding domain is in scFv format.

[0024] In some embodiments, the antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

[0025] In some embodiments, the chimeric antigen receptor further comprises a CD3z intracellular signaling domain and a 4-1BB costimulatory signaling domain.

[0026] In some embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a CD8α signal peptide, the antigen-binding domain, a CD8α hinge region, a transmembrane region, a 4-1BB costimulatory signaling domain, and a CD3z intracellular signaling domain.

[0027] In some embodiments, the chimeric antigen receptor comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0028] In some embodiments, the chimeric antigen receptor further comprises a self-cleaving polypeptide T2A and a tEGFR sequence at the C-terminus.

[0029] In another aspect, provided herein is a nucleic acid molecule encoding the antibody molecule or the chimeric antigen receptor.

[0030] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:38, SEQ ID NO:39, or SEQ ID NO:40.

[0031] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

[0032] In another aspect, provided herein is an expression vector comprising the above-described nucleic acid molecule.

[0033] In another aspect, provided herein is an immune cell expressing the chimeric antigen receptor.

[0034] In some embodiments, the immune cell is a T cell or an NK cell.

[0035] In another aspect, provided herein is a pharmaceutical composition comprising the antibody molecule, the chimeric antigen receptor, or the immune cell and a pharmaceutically acceptable carrier.

[0036] In another aspect, the present specification provides a use of the antibody molecule, the chimeric antigen receptor, the nucleic acid molecule, the expression vector, or the immune cell in the production of a medicament for treating a CD22-associated disease.

[0037] In some embodiments, the CD22-associated disease is B-cell leukemia or B-cell lymphoma.

[0038] In another aspect, provided herein is a method for treating a CD22-associated disease in a patient, comprising administering to said patient a therapeutically effective amount of said antibody molecule, said immune cell or said pharmaceutical composition.

[0039] In some embodiments, the CD22-associated disease is B-cell leukemia or B-cell lymphoma. [Brief explanation of the drawings]

[0040] [Figure 1] FIG. 1 is a schematic diagram showing the structure of a plasmid vector used in the present invention. [Figure 2] A schematic diagram of the working principle of the reporter gene method is shown. [Figure 3] Flow detection results of transient expression of CAR molecules with different clone numbers in reporter cells JLuc307 are shown. These were indirectly characterized using EGFR antibody (APC anti-human EGFR antibody (clone AY13)) staining, and cells electrotransfected with a plasmid encoding Renilla luciferase (Promega, pGL4.75) served as a negative control (mock). [Figure 4] Figure 4 shows the expression of CD22 antigen on target cells used in the present invention. In Figure 4A, CD22 antigen was stained on target cells using APC anti-human CD22 antibody (clone S-HCL-1). In Figure 4B, CD22 antigen was stained on target cells using FITC anti-human CD22 antibody (clone HIB22). [Figure 5] The following shows some of the detection results of the reporter gene method. In particular, the scale on the y-axis is the sample ffLuc / RLuc ratio (RLU) normalized to the ffLuc / RLuc ratio of the positive reference sample (a sample co-cultured with clone 0-2 and Raji cells). [Figure 6] Flow assay results for CAR expression status of CD22 CAR-T cell samples from donor SXW (day 6) are shown. Cell samples were stained with APC mouse anti-human CD8, PE anti-human EGFR, and FITC-CD22 protein. [Figure 7]This figure shows the data analysis process for CD107a degranulation assays of CD22 CAR-T cell samples from donor SXW. Using data from a sample co-cultured with clone 80 and REH cells as an example, we first extracted activated cells from the SSC vs. FSC scatter plot (a), then extracted monodispersed cells from the live cells (b), then extracted CD8+ cells from the monodispersed cells (c), and finally analyzed the CD107a positivity rate in the EGFR+ (i.e., CAR+) population of CD8+ cells (d). [Figure 8] Figure 1 shows the results of chemiluminescence detected after 17 hours of co-cultivation of donor SXW CD22 CAR-T cell samples with different target cells. The chemiluminescence values ​​positively correlate with the number of target cells. [Figure 9] This shows the tumor-suppressing effect of CAR-T cells produced according to the present invention on tumor-bearing NPG mice, as determined by in vivo luminescence imaging. [Figure 10] 1 shows the change curve of fluorescence intensity in tumor-bearing NPG mice. DETAILED DESCRIPTION OF THE INVENTION

[0041] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0042] An "antibody" is an immunoglobulin secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). The foreign substances are accordingly called antigens. The basic structure of a classical antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Based on conservative differences in amino acid sequence, the heavy and light chains are divided into an amino-terminal variable region (V) and a carboxy-terminal constant region (C). The variable regions of one heavy chain and one light chain interact to form an antigen-binding site (Fv). The composition and order of amino acid residues in certain regions of the variable region are more variable than those in other regions (framework regions, or FRs). These regions are called hypervariable regions (HVRs), and are essentially the key sites for antibody-antigen binding. Because these hypervariable region sequences are complementary to antigenic determinants, they are also called complementarity-determining regions (CDRs). Both the heavy chain and the light chain have three complementarity-determining regions, designated HCDR1, HCDR2, HCDR3, and LCDR1, LCDR2, LCDR3, respectively. In some cases, the term antibody also refers to an antibody fragment having antigen-binding ability, such as scFv, Fab, or F(ab')2.

[0043] A "single chain antibody (single chain fragment variable, scFv)" is composed of a single peptide chain connecting the heavy and light chain variable regions of an antibody. When properly folded, the variable regions from the heavy and light chains interact non-covalently to form the Fv fragment, allowing the scFv to retain its affinity for its antigen.

[0044] A "chimeric antigen receptor (CAR)," also known as a chimeric T cell receptor or chimeric immune receptor, is an engineered membrane protein receptor molecule that can confer desired specificity, such as the ability to bind to a specific tumor antigen, to immune effector cells. A chimeric antigen receptor typically consists of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. In some cases, the antigen-binding domain is a fragment of an scFv sequence and is responsible for recognizing and binding to a specific antigen. The intracellular signaling domain typically contains an immunoreceptor tyrosine-based activation motif (ITAM), such as a signaling domain derived from a CD3z molecule, which activates immune effector cells and exerts cytotoxic effects. A chimeric antigen receptor also contains a signal peptide at the amino terminus responsible for intracellular positioning of the nascent protein, and a hinge region between the antigen-binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also contain a costimulatory domain, such as a costimulatory domain derived from a 4-1BB or CD28 molecule. As used herein, when referring to a CAR structure, the abbreviation "bbz" refers to an intracellular signaling domain comprising 4-1BB and CD3z; for example, a CAR molecule comprising antibody clone 80 (as the antigen-binding domain) and 4-1BB and CD3z (as the intracellular signaling domain) is abbreviated as "clone 80-bbz."

[0045] "CAR-T cells" refer to T cells that express a CAR and are typically obtained by transducing T cells with an expression vector encoding a CAR. Commonly used expression vectors are viral vectors, such as lentiviral expression vectors. Chimeric antigen receptor-modified T cells (CAR-T) are not restricted by major histocompatibility complexes and possess specific target cytotoxicity and the ability to sustain proliferation. In addition to T cells, other lymphocytes, such as NK cells, can also be transformed using an expression vector encoding a CAR to obtain target cytotoxic cells expressing the CAR.

[0046] CD22 is a Siglec family lectin with seven IgG-like domains in its extramembrane region and a molecular weight of approximately 135 kD. Human CD22 and its variants are registered in UniProt under the registration number P20273. As a transmembrane glycoprotein, it begins to be expressed on the surface of B cells at the pre-B cell stage, is present on mature B cells, and is lost in plasma cells. The results of a phase I clinical trial of CD22-targeting chimeric antigen receptor T cells (CAR-T) reported by the National Cancer Institute in the United States demonstrated that CD22 CAR-T is safe and effective, achieving remission in some patients [2]. Therefore, the CD22 protein is an ideal target for B cell tumors.

[0047] The "m971 molecule" is an anti-CD22 antibody extracted from a human Fab phage library using a CD22-Fc fusion protein, which binds to a membrane-proximal epitope of the CD22 molecule. [3] CARs constructed with the m971-derived scFv have demonstrated good anti-leukemia activity in preclinical models. [4] In some examples herein, some biological activities of the CARs provided herein are evaluated using a CAR constructed with the m971 scFv (amino acid sequence SEQ ID NO: 28) as a comparison.

[0048] "K D " is the equilibrium dissociation constant and can be used to assess the strength of binding affinity between an antibody and its antigen. K D A smaller value indicates a stronger affinity.

[0049] The term "sequence identity" (also referred to as "sequence identity"), when referring to amino acid or nucleotide sequences, refers to the amount of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Typically, before calculating the percent identity between two amino acid or nucleotide sequences, alignment is first performed to introduce gaps, if any. If the amino acid residue or base in the two sequences is the same at a given alignment position, the two sequences are considered to be identical or matched at that position; if the amino acid residue or base in the two sequences is different, the two sequences are considered to be non-identical or mismatched at that position. Some algorithms calculate sequence identity by dividing the number of matching positions by the total number of positions in the alignment window. Other algorithms also consider the number and / or length of gaps. For purposes of the present invention, the publicly available alignment software BLAST (available from the ncbi.nlm.nih.gov website) can be used with default settings to obtain optimal alignment and calculate sequence identity between two amino acid or nucleotide sequences.

[0050] In some embodiments, the light chain variable region of the anti-CD22 antibody molecules provided herein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:19, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:20.

[0051] In some embodiments, the light chain variable region of the anti-CD22 antibody molecules provided herein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:21, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:22.

[0052] In some embodiments, the light chain variable region of the anti-CD22 antibody molecules provided herein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:23, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:24.

[0053] In some embodiments, the antigen-binding domain in a CAR provided herein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27.

[0054] In some embodiments, a CAR provided herein comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) to the sequence set forth in SEQ ID NO:35, SEQ ID NO:36, or SEQ ID NO:37.

[0055] Those skilled in the art will understand that, in addition to the specific sequences provided herein, corresponding mutants of the anti-CD22 antibody molecules or chimeric antigen receptors targeting CD22 provided by the present invention can be obtained by substituting, deleting, or adding a small number of amino acids and verifying or screening the resulting products for their binding ability to the corresponding antigen, CD22, or biological activity, and that these mutants are also within the scope of the present invention.

[0056] In addition to the specific heavy chain variable region sequences provided herein, those skilled in the art will understand that light chain variable regions that match the heavy chain variable regions and maintain CD22 binding ability can be obtained by screening an antibody light chain library (e.g., a humanized phage light chain library) using CD22 as an antigen. The anti-CD22 antibody molecules obtained in this manner and CD22-targeting CARs constructed using the anti-CD22 antibody molecules are also within the scope of the present invention.

[0057] When referring to pharmaceutical compositions, the term "pharmaceutically acceptable carrier" refers to a substance such as a solid or liquid diluent, filler, antioxidant, stabilizer, etc., that can be safely administered, and that is suitable for administration to humans and / or animals without causing undue adverse side effects, while maintaining the vitality of the drug or active agent therein.

[0058] A "therapeutically effective amount" refers to an amount of active compound sufficient to elicit the biological or medical response desired by a clinician in a subject. The "therapeutically effective amount" of a bispecific antibody of the present invention can be determined by one skilled in the art depending on the route of administration, the subject's weight, age, and disease state. For example, a typical daily dosage range may be 0.01 mg to 100 mg of active ingredient / kg body weight.

[0059] CAR-T cells produced using the anti-CD22 antibody molecules screened in the present invention have more favorable cytotoxic activity against target cells expressing CD22 both in vitro and in vivo, and are expected to be used in the treatment of some lymphomas and leukemias. [Example]

[0060] The present invention will be further described below with reference to specific examples.

[0061] Example 1: Generation and analysis of anti-CD22 antibody molecules We screened for fully humanized antibodies against CD22 using yeast surface display technology. The constructed scFv yeast display library was subjected to several rounds of flow selection using biotinylated CD22-llama-Fc or CD22-his proteins, and a total of 129 fully humanized antibody clones against CD22 were obtained. These were sequenced and used for subsequent in vitro and in vivo screening.

[0062] The antibodies thus produced were prepared in the form of IgG and Fab, respectively, and their binding ability to human CD22 was detected (ForteBio). Some of the results are shown in Tables 1 and 2.

[0063] [Table 1]

[0064] [Table 2]

[0065] Epitope binning analysis divided all 129 antibodies into seven Bins. Among them, Bin 3 was a competitor of m971. The antigen-binding site of Bins 4 and 5 was located in the CD22 extracellular membrane proximal region. A total of 62 antibody sequences, including all Bin 3, 4, and 5 antibodies and the remaining four Bins, were selected and used for subsequent primary screening using reporter gene assays. The antigen epitopes close to m971 binding in clones 80 and 28 both belong to Bin 3. On the other hand, the antigen epitope binding in clone 17 is different from m971 and belongs to Bin 4.

[0066] Example 2 Construction of CD22 CAR Plasmid Vector First, a nucleotide sequence was artificially synthesized (SEQ ID NO: 42), which included KOZAK (bases 1-9), CD8a signal peptide (bases 10-72; the corresponding amino acid sequence is SEQ ID NO: 30), ccdB screening gene (bases 73-428), CD8a hinge region and transmembrane region (bases 429-677; the corresponding amino acid sequence is SEQ ID NO: 31), 4-1BB costimulatory factor (bases 678-803; the corresponding amino acid sequence is SEQ ID NO: 32), CD3z intracellular signaling domain (bases 804-1139; the corresponding amino acid sequence is SEQ ID NO: 33), T2A cleavage peptide (bases 1140-1202; the corresponding amino acid sequence is SEQ ID NO: 29), and tEGFR (bases 1203-2276; the corresponding amino acid sequence is SEQ ID NO: 34). The synthetic sequence was inserted into the multiple cloning site of the lentiviral vector PLVX-EF1alpha-IRES-Puro plasmid (Clontech, Cat. No. 631988) by PCR splicing method to obtain the PXL0662 plasmid shown in FIG. 1 .

[0067] Then, nucleotide sequences encoding each scFv (e.g., nucleotide sequences such as SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41) were synthesized, and the nucleotide sequences of these scFvs were inserted into PXL0662 using the two type II endonuclease sites BsmBI (2339 and 2701 sites) in the PXL0662 plasmid to obtain each plasmid vector encoding CD22 CAR.

[0068] Example 3 Primary screening using the CD22 CAR reporter gene method Operating principle CAR-T cell activation is achieved by CD3z and costimulatory factors in the intracellular domain of the CAR molecule. CD3z activates the NFAT signaling pathway in the cell, which is a prerequisite for CAR-T cell activation. Therefore, the NFAT reporter gene method can be used to screen for CAR molecules that activate the NFAT signaling pathway [5].

[0069] In the primary screening process, Jurkat cells carrying the NFAT-RE-ffLuc reporter gene were used as reporter cells (designated JLuc307, as shown in Figure 2). The CAR molecule was transiently expressed on the surface of the reporter cells by plasmid electrotransformation. After co-cultivation of the CAR molecule-expressing reporter cells with target cells, the CAR molecule was specifically activated by target cell surface antigens, further activating the reporter gene (ffLuc, firefly luciferase). The ability of the CAR molecule to activate the NFAT signaling pathway was then assessed by detecting luciferase activity. The plasmid used in this reporter gene method also contained a sequence encoding a cleaved EGFR (tEGFR), which was used to mark cells successfully expressing the CAR when tEGFR was expressed on the cell surface. Because different CAR molecules exhibit different electrotransformation efficiencies, the electrotransformation efficiency was determined using an internal reference plasmid (CMV-hRLuc, Renilla luciferase) co-localized with the CAR molecule.

[0070] Operation steps 1) The CAR plasmid to be measured and the internal reference plasmid were mixed at a certain ratio, and then transfected into reporter cells by electrotransformation. 2) 48 h after transfection, a portion of the cells was taken and stained with PE-anti-human EGFR antibody for flow detection to evaluate the transient expression of the CAR plasmid. 3) 72 hours after transfection, reporter cells and target cells were mixed at a 1:1 ratio and then placed in a U-bottom 96-well plate and incubated for 24 hours; 3 × 10 cells per well were added. 4 Reporter cells were added, with three replicate wells per target cell. 4) After the incubation was completed, the plates were centrifuged at 1000 g for 5 minutes at 4°C, and the culture supernatant was removed. 100 μL of lysis solution was added to each well to lyse the cells, and 20 μL of the cell lysate was removed and used for detecting dual luciferase activity.

[0071] Screening Standards CD22-positive target cells can efficiently activate the NFAT-RE-ffLuc reporter gene, generating a fluorescent signal. Without target cell stimulation or with stimulation of CD22-negative target cells, there was a low level of fluorescent signal produced by background (tonic effects) or nonspecific activation.

[0072] result The primary screening was performed in six batches, and the detection for each batch included the PXL0589 (m971-bbz-T2A-tEGFR, clone 0-2) plasmid as a positive control and the pGL4.75 plasmid (number PXL0337) as a negative control (mock).

[0073] Because the 62 antibodies tested all had different koff values, and some antibodies rapidly dissociated after binding to the CD22 antigen, the transient expression of CAR molecules in the reporter cells JLuc307 was indirectly characterized via an EGFR antibody. Flow detection results showed that all 61 CAR molecules tested, except for clone 4, could be transiently expressed in JLuc307 cells. A representative flow detection diagram is shown in Figure 3.

[0074] In the primary screening process using the reporter gene method, Raji, REH, JVM2, K562, and CD22 K / O Raji cells (clone 3D11 or 3E09, our CD22 knockout / knockdown Raji cells) were used as target cells. Prior to the primary screening, CD22 antigen expression on the target cell surface was detected by flow cytometry using APC mouse anti-human CD22 antibody (clone S-HCL-1) or FITC mouse anti-human CD22 antibody (clone HIB22), respectively. The results are shown in Figure 4. Raji cells highly expressed CD22, 3E09, REH, and JVM2 cells moderately expressed CD22, and K562 and 3D11 cells were CD22 negative.

[0075] The dual-luciferase reporter gene detection kit provided two fluorescence readouts, one for firefly luciferase (ffLuc) and one for Renilla luciferase (RLuc). The Renilla luciferase readout served as an internal reference to eliminate differences in cell number or transfection efficiency. Therefore, the NFAT-RE-ffLuc transcriptional regulation level produced by each CAR sample upon activation in target cells could be characterized by the ffLuc / RLuc ratio (RLU). The results for the first batch are shown in Figure 5. Among them, clone 0-2, a control CAR sample (m971), was able to activate NFAT upon stimulation with the positive target cells Raji, 3E09, and JVM2, with signal intensity positively correlated with the density of antigen expression in the target cells. However, it did not activate NFAT and produce a fluorescent signal upon stimulation with the negative target cells 3D11 and K562, or without target cell stimulation. Compared with the results of clones 0-2, clones 28, 36, and 80 were clones that could specifically identify CD22 target cells and activate the NFAT signaling pathway, while the remaining clones were excluded.

[0076] Using reporter gene screening, a total of 10 clones were selected from the 62 clones and subjected to the following functional evaluation: Specific steps included the creation of lentiviral vectors, the creation of CAR-T cells, and the in vitro functional evaluation of CAR-T cells.

[0077] Example 4. Construction of lentiviral vectors The process for constructing lentiviral vectors corresponding to the 10 clones obtained in Example 3 is as follows.

[0078] HEK293T cells were induced and cultured in DMEM medium containing 10% FBS. After 2-3 cell expansion cultures, 6 × 10 4 pieces / cm 2 Cells were seeded into ten-layer cell factories at a density of 10 μg / mL. Plasmid transfection was performed three days after cell seeding. The plasmid transfection solution was formulated using Opti-MEM, with a final plasmid concentration of 10 μg / mL. It contained the CAR vector plasmid (T), psPAX2 plasmid (P), and pMD2.G plasmid (E) in a ratio of T:P:E = 5:3:2. PEI was further added to the plasmid transfection solution at a final concentration of 30 μg / mL, mixed thoroughly, and incubated at room temperature for 30 minutes before use. Each cell factory was transfected using 100 mL of the plasmid transfection solution.

[0079] After 72 hours, the supernatant was collected, placed in a centrifuge tube, and centrifuged at 3,000 g for 10 minutes at 4°C. The resulting supernatant was then filtered through a 0.45 μm filter. The filtered supernatant was then centrifuged at 27,000 g for 4 hours at 4°C. After centrifugation, the supernatant was discarded, and the virus was resuspended in PBS pre-cooled at 4°C. The resuspended virus was individually packaged and stored at -80°C.

[0080] Example 5. Generation of CAR-T cells In this example, CAR-T cells were produced using cells from a healthy donor and used to evaluate the function of the 10 clones obtained in Example 3. The process of producing CAR-T cells is illustrated below.

[0081] On day 1, approximately 80 mL of peripheral blood was collected from a healthy donor, separated using Ficoll to obtain PBMCs, and then selected using CD3 MicroBeads to obtain T cells. The selected T cells were activated using CD3 / CD28 Dynabeads. After activation for approximately 24 hours (day 2), the lentivirus prepared in Example 4 was added and transduced (MOI = 3), resulting in a T cell density of approximately 1.5 × 10 6 On day 3, the transduced T cells were replaced once. The cell density was then adjusted to (0.6–2.0) × 10 6 The cells were cultured to maintain a density of 10 cells / mL.

[0082] After cell culture for 6 or 7 days, the expression status of CAR molecules, tEGFR molecules, and CD8 on the cell surface was detected by flow cytometry. Here, CAR-T cells generated from peripheral blood derived from donor SXW were used as an example. After cell culture for 6 days, approximately 5 × 10 cells were obtained from each sample. 5Cells were harvested and centrifuged at 500xg to remove the medium. The cells were then washed twice with PBS + 1% HSA and resuspended in 50µL of PBS + 1% HSA. To each sample, 2µL of CD22-FITC protein (Acro Biosystems, Cat. No. SI2-HF2H6), 2µL of APC anti-human CD8 antibody (BD, Cat. No. 555369), and 2µL of PE anti-human EGFR antibody (BioLegend, Cat. No. 352904) were added. After thorough mixing, the cells were incubated at 4°C in the dark for 20 minutes. After incubation, the cells were washed twice with PBS + 1% HSA and resuspended in 200µL of PBS + 1% HSA. The cells were then loaded and detected. As shown in Figure 6 and Table 3, some of the detection results showed that all samples except for the control T cells were able to simultaneously express EGFR and CAR molecules, the CAR molecules were able to bind normally to the CD22 protein, the CAR positivity rate (CAR%) was in the range of 34.6-53.3%, and the CAR molecules were able to be expressed normally in the CD8-positive cell group.

[0083] [Table 3]

[0084] Example 6. In vitro functional evaluation of CAR-T cells The CAR-T cells prepared in Example 5 were cultured for 8-12 days and then subjected to in vitro functional evaluation using two methods: a CD107a degranulation assay and an in vitro cytotoxicity assay. The operating principles and screening criteria are as follows:

[0085] 6.1 CD107a degranulation assay Operating principle CD107a is a marker for intracellular microvesicles. After granzyme-carrying microvesicles fuse with the cell membrane, CD107a levels at the cell membrane increase. The inhibition of CD107a release by monesin (BioLegend) quantitatively reflects the strength of microvesicle release. [6] Therefore, when CAR-T cells undergo degranulation upon stimulation with target cell surface antigens, the activation status of CAR-T cells can be assessed by flow cytometry of the CD107a positivity on the CAR-T cell surface.

[0086] Operation steps 1) CD22-positive and -negative target cells were centrifuged at 300 g for 5 min at room temperature. After discarding the supernatant, 2 × 10 cells were cultured in T cell medium. 5 The cells were resuspended at 100 cells / mL. 2) Depending on the CAR-positive rate and E:T value of the CAR-T cells to be measured (usually 0.3:1), the CAR-T cells were resuspended to an appropriate density, and monensin and PE / Cy7 mouse anti-human CD107a antibody were added. 3) 100 μL / well of CAR-T cells to be measured and 100 μL / well of target cells were added to a U-bottom 96-well plate, mixed evenly, and then placed in an incubator (37°C, 5% CO2) and incubated for 3 hours. 4) After the incubation was completed, the plate was centrifuged at 4°C and 600 g for 5 minutes, the supernatant was discarded, and the cells were washed twice with 200 μL / well of DPBS+1% HSA. 5) The cells were resuspended in 20 μL / well of DPBS + 1% HSA, and APC mouse anti-human CD8 antibody and Alexa Fluor 488 anti-human EGFR antibody (or FITC-CD22 protein) were added. The cells were mixed uniformly and then incubated on ice in the dark for 20 minutes. 6) After the incubation was completed, the cells were washed three times with 200 μL / well of DPBS+1% HSA, and then resuspended in 200 μL / well of DPBS+1% HSA for flow detection.

[0087] Screening Standards CD22-positive target cells can effectively activate CAR-T cells (CD8 + / CAR + In the cell population, the percentage of CD107a positive cells was high. Without target cell stimulation, or with stimulation of CD22 negative target cells, CD8 + / CAR + The cell population has a low CD107a positivity rate.

[0088] result A CD107a degranulation assay was performed on CAR-T samples from donor SXW on day 8. CD22-positive target cells used in the CD107a degranulation assay included Raji, NALM6, REH, and JVM2. CD22-negative target cells included Jurkat, U266, HEK293, Karpas-299, K562, and 3D11, of which 3D11 was a CD22 knockout / knockdown Raji cell line. Data analysis of the CD107a degranulation assay is illustrated in Figure 7. First, viable cells were extracted from the SSC vs. FSC scatter plot (Figure 7a). Then, monodispersed cells were extracted from the FSC-H vs. FSC-A scatter plot (Figure 7b) of the viable cells. Finally, CD8-positive cells were extracted from the SSC vs. APC-CD8 scatter plot (Figure 7c) of the monodispersed cells. Finally, the CD107a positivity rate in the EGFR-positive cell group was analyzed from the PECy7-CD107a vs. AF488-EGFR scatter plot of CD8-positive cells (Figure 7d). The CD107a positivity rate was calculated from the ratio Q2 / (Q2+Q3) in Figure 7d, and the results are shown in Table 4. Note that, because T cells lack EGFR, their CD107a positivity rate was calculated from Q1 / (Q1+Q2) in Figure 7d.

[0089] Upon stimulation with CD22-positive target cells, all cloned CAR-T cells exhibited degranulation. Among them, the degranulation effects of clones 80, 28, 36, and 17 were similar to those of the control CAR (m971-bbz). However, upon stimulation with CD22-negative target cells, clone 36 exhibited significant degranulation, which may have been due to nonspecific activation, which was ruled out.

[0090] Furthermore, reduced expression of the CD22 antigen on tumor cell surfaces is one of the main causes of relapse after CD22 CAR-T cell therapy [8]. Therefore, it is desirable to develop a CAR molecule that can recognize and kill tumor cells with low CD22 expression. Because 3D11 is a CD22 knockout / knockdown Raji cell, clones 80, 28, 17, and m971 all showed a slight degranulation effect upon 3D11 stimulation, and the strength of this effect indicates the clone's ability to discriminate against low-density targets. Therefore, clone 80 may have good discrimination ability against low-density targets.

[0091] [Table 4]

[0092] 6.2 In vitro cytotoxicity experiments Operating principle To evaluate the antigen-specific cytotoxicity of CAR-T cells, NALM6-ffLuc cells were used as CD22-positive target cells, and K562-ffLuc or Jurkat-ffLuc cells were used as CD22-negative target cells, which are cell lines stably expressing firefly luciferase obtained by lentiviral transduction.

[0093] In vitro cytotoxicity experiments were performed by co-culturing CAR-T cells and target cells at different effector-target ratios (E:T). When target cells were killed by CAR-T cells, luciferase was released and rapidly inactivated (the half-life of firefly luciferase is approximately 0.5 h [7]). When target cells were not killed or suppressed by CAR-T cells, more luciferase was produced as the target cells expanded and continued to express luciferase. Therefore, luciferase activity could be used to detect CAR-T target cell damage.

[0094] Operation steps 1) NALM6-ffLuc and K562-ffLuc cells were centrifuged at 300 g for 5 minutes at room temperature, the supernatant was discarded, and then 2 × 10 cells were cultured in complete T cell medium. 5 The target cells were resuspended at 100 μL / well in a clear-bottom 96-well plate. 2) Depending on the CAR-positive rate and E:T value of the CAR-T cells to be measured (usually 2:1, 1:1, or 0.5:1), 100 μL of CAR-T cells were added to each well of a 96-well plate, mixed evenly with the target cells, and then incubated in an incubator (3°C, 5% CO2) for 24 hours. 3) After the incubation was completed, the plates were centrifuged at 800 g for 5 minutes at room temperature, and 100 μL / well of the supernatant was collected as a sample for cytokine detection (stored at −80° C.). 4) For cells other than the preserved samples, luciferase activity in each well was detected using a luciferase detection kit.

[0095] Screening Standards CAR-T cells can effectively kill CD22-positive target cells, but do not cause nonspecific damage to CD22-negative target cells.

[0096] result An in vitro cytotoxicity experiment was performed on the CAR-T sample from donor SXW on day 8. Two CD22-positive target cells, NALM6-ffLuc and REH-ffLuc, and CD22-negative target cells, K562-ffLuc, were used in the experiment.

[0097] As shown in Table 5 and Figure 8a, when the effector-to-target ratio (E:T) was 1:1 or 2:1, all CAR-T samples were able to cytosine the CD22-positive target cells NALM6-ffLuc. Among them, m971, clone 80, and clone 17 showed strong cytosine activity. The control T cell samples did not show significant nonspecific cytosine activity.

[0098] As shown in Table 6 and Figure 8b, all CAR-T samples were able to cytosine the CD22-positive target cells REH-ffLuc when the E:T ratio was 2:1. Among them, m971, clone 80, and clone 17 showed strong cytosine activity. When the E:T ratio was 1:1, m971, clone 80, clone 36, and clone 17 still had some cytosine activity. However, when the E:T ratio was 0.5:1, none of the samples were able to effectively cytosine the target cells REH-ffLuc. None of the control T cell samples showed nonspecific cytosine activity, and the target cells REH-ffLuc were rapidly amplified.

[0099] As shown in Table 7 and Figure 8c, none of the CAR-T / T samples damaged the CD22-negative target cells K562-ffLuc, and K562-ffLuc was rapidly amplified due to the MLR effect.

[0100] [Table 5]

[0101] [Table 6]

[0102] [Table 7]

[0103] The results of Example 3 (primary screening using the reporter gene method) and Example 6 (evaluation of in vitro function of CAR-T cells) suggest that CAR-T cells produced from clone 80, clone 28, and clone 17 exhibited good in vitro cell function.

[0104] Example 7. In vivo tumor suppression experiment in cancer-bearing animal models Operating principle Using immunodeficient mice (NPG) bearing Nalm6 cells, a type of human acute lymphocytic leukemia cell that specifically expresses CD22, as an experimental system, CAR-T cell samples were evaluated using the method described in Evaluation Example 5, and the pharmaceutical effects of clone 80, clone 28, and clone 17 in vivo were evaluated.

[0105] Compared with NOD / SCID mice, NPG mice have a knockout of the gamma chain of the IL-2 receptor, which is a common receptor subunit for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Knocking out this gene further reduces the immune function of mice, especially the vitality of NK cells, which is almost completely lost. Therefore, NPG mice are more suitable recipients for cell or tissue transplantation.

[0106] Nalm6 is a cell line that stably expresses firefly luciferase. After tail vein injection into mice, Nalm6 cells proliferated. D-fluorescein potassium was injected intraperitoneally, and chemiluminescent signals were imaged using a Bruker small animal imager under isoflurane anesthesia. When target cells were not killed or suppressed by CAR-T cells, more luminescent signals were detected due to target cell proliferation and sustained luciferase expression. Furthermore, specific target cell aggregation sites were also observed from the imaging location of the target cells. Therefore, the intensity of the luminescent signal can be used to detect the damage caused by CAR-T to target cells in vivo.

[0107] Operation steps 1) CAR-T cell samples containing clone 80, clone 28, and clone 17 were prepared according to the method in Example 5. 2) Female immunodeficient NPG mice, 4-5 weeks old and weighing 20±3 g, were selected. NALM6-LUC cells in the logarithmic growth phase were injected into the NPG mice via the tail vein at a concentration of 1×10 6 Two days after the tumor cell inoculation, the test product was administered at different doses via tail vein injection, and the mice were divided into groups as shown in Table 8. 3) On days 3, 7, 12, 18, and 28 after administration, D-fluorescein potassium was injected intraperitoneally, and chemiluminescence signals were captured using a Bruker small animal imager under isoflurane anesthesia to detect tumor growth inhibition by tumor imaging. 4) On days 3, 7, 14, 17, 21, and 28 after administration, mice were anesthetized with isoflurane. Once the corneal reflex disappeared, 0.1 mL of blood was collected from the orbital vein and preserved with EDTA-2K anticoagulation. CAR copy number was measured.

[0108] [Table 8]

[0109] Animal experiment results 1) Mouse imaging results As shown in Figure 9, tumor cells grew normally in the vehicle control and MockT control groups, demonstrating successful model success. After D20, mice in the vehicle control group died from tumor overload, while GvHD symptoms appeared in the MockT group before death. Clinical parameters were normal in the treated mice. Tumor growth inhibition was observed in both the low-dose and high-dose Clone 80 groups, the Clone 28 group, and the Clone 17 group. CRs appeared in each group starting on Day 12 and were maintained until D28, the final observation period. Clone 80 showed significant differences in efficacy at different doses, with a positive correlation between efficacy and dose. Clone 28 showed 3 / 6 CRs at D28. Tumor cell proliferation was observed in some Clone 17 mice at D28.

[0110] 2) Fluorescent signal of tumor cells The measured fluorescent signal intensities are shown in Table 9 below.

[0111] [Table 9]

[0112] The data in the table above was graphed, and the results are shown in Figure 10. In the vehicle control group and Mock-T group, tumor signals increased over time, with no significant difference between the two groups. Furthermore, tumor signals were observed to weaken in both the low-dose and high-dose Clone 80 groups, as well as in the Clone 28 and Clone 17 groups. In the Clone 80 group, there was a significant difference in tumor signals between the different dose groups, with the high-dose group producing significantly weaker signals than the low-dose group at the same time point. Furthermore, the Clone 28 group produced much lower tumor signals. Furthermore, in the Clone 17 group, tumor signals gradually increased after D18.

[0113] In summary, Clone 80, Clone 28, and Clone 17 all showed significant pharmaceutical effects in the Nalm6 tumor-bearing mouse model, with Clone 80 and Clone 28 showing superior tumor suppression compared to Clone 17.

[0114] Some amino acid or nucleic acid sequences described herein are as follows: SEQ ID NO: 1 (clone 17 LCDR1 amino acid sequence) RASQSISSWLA SEQ ID NO: 2 (clone 17 LCDR2 amino acid sequence) KASSLES SEQ ID NO: 3 (clone 17 LCDR3 amino acid sequence) QQYERFPWT SEQ ID NO: 4 (clone 17 HCDR1 amino acid sequence) FTFSSYAMS SEQ ID NO: 5 (clone 17 HCDR2 amino acid sequence) AISGSGGSTYYADSVKG SEQ ID NO: 6 (clone 17 HCDR3 amino acid sequence) AKVGISSLHGMDV SEQ ID NO: 7 (clone 28 LCDR1 amino acid sequence) RASQSISSWLA SEQ ID NO: 8 (clone 28 LCDR2 amino acid sequence) DASSLES SEQ ID NO: 9 (clone 28 LCDR3 amino acid sequence) QQANTYSPT SEQ ID NO: 10 (clone 28 HCDR1 amino acid sequence) GSISSYYWS SEQ ID NO: 11 (clone 28 HCDR2 amino acid sequence) RIYTSGSTNYNPSLKS SEQ ID NO: 12 (clone 28 HCDR3 amino acid sequence) ARDLYRDGMDV SEQ ID NO: 13 (clone 80 LCDR1 amino acid sequence) RASQSVSSSYLA SEQ ID NO: 14 (clone 80 LCDR2 amino acid sequence) GASSRAT SEQ ID NO: 15 (clone 80 LCDR3 amino acid sequence) QQAGLFPYT SEQ ID NO: 16 (clone 80 HCDR1 amino acid sequence) GSISSSNWWS SEQ ID NO: 17 (clone 80 HCDR2 amino acid sequence) EIYHSGSTNYNPSLKS SEQ ID NO: 18 (clone 80 HCDR3 amino acid sequence) ARLPGYESAFDI SEQ ID NO: 19 (clone 17 VL amino acid sequence) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYERFPWTFGGGTKVEIK SEQ ID NO: 20 (clone 17 VH amino acid sequence) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGISSLHGMDVWGQGTTVTVSS SEQ ID NO: 21 (clone 28 VL amino acid sequence) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLISDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQANTYSPTFGGGTKVEIK SEQ ID NO: 22 (clone 28 VH amino acid sequence) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWGQGTTVTVSS SEQ ID NO: 23 (clone 80 VL amino acid sequence) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK SEQ ID NO: 24 (clone 80 VH amino acid sequence) QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSS SEQ ID NO: 25 (clone 17 scFv amino acid sequence) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGISSLHGMDVWGQGTTVTVSSG GGGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYERFPWTFGGGTKVEIK SEQ ID NO: 26 (clone 28 scFv amino acid sequence) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWGQGTTVTVSSGG GGSGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLISDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQANTYSPTFGGGTKVEIK SEQ ID NO: 27 (clone 80 scFv amino acid sequence) QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSSGG GGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK SEQ ID NO: 28 (M971 scFv amino acid sequence) QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVS SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK SEQ ID NO: 29 (T2A amino acid sequence) EGRGSLLTCGDVEENPGP SEQ ID NO: 30 (CD8α signal peptide amino acid sequence) MALPVTALLLPLALLLHAARP SEQ ID NO: 31 (CD8α hinge and transmembrane domain amino acid sequence) FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN SEQ ID NO: 32 (4-1BB costimulatory signaling domain amino acid sequence) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL SEQ ID NO: 33 (CD3z intracellular signaling domain amino acid sequence) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 34 (tEGFR amino acid sequence) MLLLVTSLLLCELPHPAFLLIPRKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRSLKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTK IISNRGENSCKATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM SEQ ID NO: 35 (clone 17 CAR amino acid sequence) MALPVTALLLPLALLLHAARPEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVGISSLHGMDV WGQGTTVTVSSGGGGSGGGGGSGGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYKASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYERFPWTFGGGTKV EIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPG SEQ ID NO: 36 (clone 28 CAR amino acid sequence) MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWG QGTTVTVSSGGGGSGGGGSGGGSDIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLISDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQANTYSPTFGGGTKVE IKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPG SEQ ID NO: 37 (clone 80 CAR amino acid sequence) MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIW GQGTMVTVSSGGGGSGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKV EIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRV KFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGEGRGSLLTCGDVEENPG SEQ ID NO: 38 (clone 17 scFv nucleotide sequence) GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCGGTGTACTACTGCGCCAAGGTAGGAATATCCAGCTTACACGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCAGCTCAGGTGGCGGGGGCAGCGGCGGAGGCGGATCCGGAGGCGGAGGGAGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAAGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGTACGAACGCTTCCCTTGGACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA Sequence number 39 (Clone 28 scFv nucleotide sequence) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCGGTGTACTACTGCGCCAGAGACTTGTACAGAGATGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCAGCTCAGGTGGCGGGGGCAGCGGCGGAGGCGGATCCGGAGGCGGAGGGAGTGACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCCGATGCCTCCAGTTTGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGCCAATACCTACTCTCCTACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA[[ID=第1]] [[ID=第2]]Sequence number 40 (Clone 80 scFv nucleotide sequence)[[ID=第3]] CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCGGTGTACTACTGCGCCAGACTTCCTGGATACGAGTCAGCTTTCGACATATGGGGTCAGGGTACAATGGTCACCGTCAGCTCAGGTGGCGGGGGCAGCGGCGGAGGCGGATCCGGAGGCGGAGGGAGTGAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGCCGGACTCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA<> Sequence number 41 (M971 scFv nucleotide sequence)<> CAGGTGCAGCTCCAGCAGAGCGGCCCCGGCCTGGTAAAGCCCAGCCAAACCCTCTCCCTGACCTGCGCTATCAGCGGCGATTCCGTGAGCAGCAACAGCGCCGCCTGGAATTGGATCCGTCAGAGCCCCAGCAGGGGCCTGGAGTGGCTGGGGCGGACCTATTACCGGAGTAAGTGGTACAACGACTACGCCGTAAGCGTGAAGAGCCGCATCACCATTAATCCTGACACCAGCAAGAACCAGTTCAGTCTGCAGCTGAACAGCGTGACTCCCGAGGACACCGCCGTGTACTACTGCGCCCGCGAGGTGACTGGAGACCTGGAAGACGCCTTCGACATCTGGGGCCAGGGCACAATGGTGACCGTCAGCAGCGGTGGCGGGGGCAGCGGCGGAGGCGGATCCGGAGGCGGAGGGAGTGACATACAGATGACCCAGAGCCCTAGCAGCCTCTCTGCCAGCGTGGGAGACCGGGTGACCATCACCTGCCGCGCCAGTCAGACCATCTGGTCTTATCTGAACTGGTACCAGCAACGGCCCGGCAAGGCCCCTAACCTGTTGATCTACGCCGCCAGCAGTCTCCAGAGCGGCGTTCCATCTCGCTTCAGCGGCCGCGGCAGCGGCACAGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACTTCGCCACCTACTACTGCCAGCAGAGCTACAGCATCCCCCAGACTTTCGGACAGGGCACCAAGTTGGAGATCAAA Sequence number 42 (CAR partial nucleotide sequence in PXL0662) SEQ ID NO: 43 (clone 17 CAR nucleotide sequence) SEQ ID NO: 44 (clone 28 CAR nucleotide sequence) SEQ ID NO: 45 (clone 80 CAR nucleotide sequence)

[0115] References: 1. Shah, N.N., et al., Characterization of CD22 expression in acute lymphoblastic leukemia. Pediatr Blood Cancer, 2015. 62(6): p. 964-9. 2. Fry, T.J., et al., CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy. Nat Med, 2018. 24(1): p. 20-28. 3. Xiao, X., et al., Identification and characterization of fully human anti-CD22 monoclonal antibodies. MAbs, 2009. 1(3): p. 297-303. 4. Haso, W., et al., Anti-CD22-chimeric antigen receptors targeting B-cell precursor acute lymphoblastic leukemia. Blood, 2013. 121(7): p. 1165-74. 5. Rydzek, J., et al., Chimeric Antigen Receptor Library Screening Using a Novel NF-kappaB / NFAT Reporter Cell Platform. Mol Ther, 2019. 27(2): p. 287-299. 6.Alter、G.、J.M. Malenfant、and M. Altfeld、CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods、2004. 294(1-2): p. 15-22. 7.Matta、H.、et al.、Development and characterization of a novel luciferase based cytotoxicity assay. Sci Rep、2018. 8(1): p. 199. 8. Majzner、R.G. and C.L. Mackall、Tumor Antigen Escape from CAR T-cell Therapy. Cancer Discov、2018. 8(10): p. 1219-1226.

Claims

1. An anti-CD22 antibody molecule comprising a light chain variable region and a heavy chain variable region, The light chain variable region comprises an LCDR1 having the sequence shown in SEQ ID NO: 1, an LCDR2 having the sequence shown in SEQ ID NO: 2, and an LCDR3 having the sequence shown in SEQ ID NO: 3, and the heavy chain variable region comprises an HCDR1 having the sequence shown in SEQ ID NO: 4, an HCDR2 having the sequence shown in SEQ ID NO: 5, and an HCDR3 having the sequence shown in SEQ ID NO:

6. the light chain variable region comprises an LCDR1 having the sequence set forth in SEQ ID NO:7, an LCDR2 having the sequence set forth in SEQ ID NO:8, and an LCDR3 having the sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises an HCDR1 having the sequence set forth in SEQ ID NO:10, an HCDR2 having the sequence set forth in SEQ ID NO:11, and an HCDR3 having the sequence set forth in SEQ ID NO:12, or The light chain variable region comprises an LCDR1 having the sequence shown in SEQ ID NO: 13, an LCDR2 having the sequence shown in SEQ ID NO: 14, and an LCDR3 having the sequence shown in SEQ ID NO: 15, and the heavy chain variable region comprises an HCDR1 having the sequence shown in SEQ ID NO: 16, an HCDR2 having the sequence shown in SEQ ID NO: 17, and an HCDR3 having the sequence shown in SEQ ID NO:

18. Anti-CD22 antibody molecule.

2. 2. The anti-CD22 antibody molecule of claim 1, wherein the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO:

23.

3. The anti-CD22 antibody molecule of claim 1 or 2, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO:

24.

4. the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20; or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:21, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:22; or The light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 23, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

24. An anti-CD22 antibody molecule according to any one of claims 1 to 3.

5. 5. An anti-CD22 antibody molecule according to any one of claims 1 to 4, which is in scFv format and comprises an amino acid sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO:25, SEQ ID NO:26 or SEQ ID NO:

27.

6. The anti-CD22 antibody molecule is in the IgG format and binds to CD22 with a K of 2 nM or less. Ｄ or the anti-CD22 antibody molecule is in Fab format and binds to CD22 with a K of 20 nM or less. Ｄ 5. The anti-CD22 antibody molecule of claim 1 , having a value of

7. The anti-CD22 antibody molecule of any one of claims 1 to 4 and 6, wherein the anti-CD22 antibody molecule is a fully humanized antibody molecule.

8. A chimeric antigen receptor that targets CD22, comprising an antigen-binding domain that binds to CD22, wherein the antigen-binding domain comprises a light chain variable region and a heavy chain variable region; The light chain variable region comprises an LCDR1 having the sequence shown in SEQ ID NO: 1, an LCDR2 having the sequence shown in SEQ ID NO: 2, and an LCDR3 having the sequence shown in SEQ ID NO: 3, and the heavy chain variable region comprises an HCDR1 having the sequence shown in SEQ ID NO: 4, an HCDR2 having the sequence shown in SEQ ID NO: 5, and an HCDR3 having the sequence shown in SEQ ID NO:

6. the light chain variable region comprises an LCDR1 having the sequence set forth in SEQ ID NO:7, an LCDR2 having the sequence set forth in SEQ ID NO:8, and an LCDR3 having the sequence set forth in SEQ ID NO:9, and the heavy chain variable region comprises an HCDR1 having the sequence set forth in SEQ ID NO:10, an HCDR2 having the sequence set forth in SEQ ID NO:11, and an HCDR3 having the sequence set forth in SEQ ID NO:12, or The light chain variable region comprises an LCDR1 having the sequence shown in SEQ ID NO: 13, an LCDR2 having the sequence shown in SEQ ID NO: 14, and an LCDR3 having the sequence shown in SEQ ID NO: 15, and the heavy chain variable region comprises an HCDR1 having the sequence shown in SEQ ID NO: 16, an HCDR2 having the sequence shown in SEQ ID NO: 17, and an HCDR3 having the sequence shown in SEQ ID NO:

18. Chimeric antigen receptor.

9. The chimeric antigen receptor of claim 8, wherein the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO:

23.

10. The chimeric antigen receptor of claim 8 or 9, wherein the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO:

24.

11. the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 19, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 20; or the light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:21, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:22; or The light chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 23, and the heavy chain variable region comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

24. The chimeric antigen receptor of any one of claims 8 to 10.

12. The chimeric antigen receptor of any one of claims 8 to 11, wherein the antigen binding domain is in scFv format.

13. The chimeric antigen receptor of any one of claims 8 to 12, wherein the antigen-binding domain comprises an amino acid sequence having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, SEQ ID NO: 26, or SEQ ID NO:

27.

14. The chimeric antigen receptor of any one of claims 8 to 13, further comprising a CD3z intracellular signaling domain and a 4-1BB costimulatory signaling domain.

15. The chimeric antigen receptor according to any one of claims 8 to 14, comprising, from N-terminus to C-terminus, a CD8α signal peptide, the antigen-binding domain, a CD8α hinge region, a transmembrane region, a 4-1BB costimulatory signaling domain, and a CD3z intracellular signaling domain, in that order.

16. 16. The chimeric antigen receptor of claim 8, comprising an amino acid sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO:

37.

17. 17. The chimeric antigen receptor of claim 8, further comprising a self-cleaving polypeptide T2A and a tEGFR sequence at the C-terminus.

18. A nucleic acid molecule encoding an antibody molecule according to any one of claims 1 to 7 or a chimeric antigen receptor according to any one of claims 8 to 17.

19. 19. The nucleic acid molecule of claim 18, comprising the nucleotide sequence set forth in SEQ ID NO: 38, SEQ ID NO: 39 or SEQ ID NO:

40.

20. 20. The nucleic acid molecule of claim 18 or 19, comprising the nucleotide sequence set forth in SEQ ID NO: 43, SEQ ID NO: 44 or SEQ ID NO:

45.

21. 21. An expression vector comprising the nucleic acid molecule of any one of claims 18 to 20.

22. An immune cell expressing the chimeric antigen receptor of any one of claims 8 to 17.

23. The immune cell of claim 22, which is a T cell or an NK cell.

24. A pharmaceutical composition comprising an antibody molecule according to any one of claims 1 to 7, a chimeric antigen receptor according to any one of claims 8 to 17, or an immune cell according to claim 22 or 23, and a pharmaceutically acceptable carrier.

25. Use of an antibody molecule described in any one of claims 1 to 7, a chimeric antigen receptor described in any one of claims 8 to 17, a nucleic acid molecule described in any one of claims 18 to 20, an expression vector described in claim 21, or an immune cell described in claim 22 or 23 in the production of a medicament for treating a CD22-associated disease.

26. 26. The use according to claim 25, wherein the CD22-associated disease is B-cell leukemia or B-cell lymphoma.

27. 8. An antibody molecule according to any one of claims 1 to 7 for use in the treatment of a CD22-associated disease in a patient.

28. 24. The immune cell of claim 22 or 23 for use in treating a CD22-associated disease in a patient.

29. 25. The pharmaceutical composition of claim 24 for use in treating a CD22-associated disease in a patient.

30. 28. The antibody molecule of claim 27, wherein the CD22-associated disease is B-cell leukemia or B-cell lymphoma.

31. 29. The immune cell of claim 28, wherein the CD22-associated disease is B-cell leukemia or B-cell lymphoma.

32. 30. The pharmaceutical composition of claim 29, wherein the CD22-associated disease is B-cell leukemia or B-cell lymphoma.

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