A fully humanized CD19- and CD22-targeting bispecific antigen chimeric receptor and its applications

Abstract

The present invention provides a bispecific chimeric antigen receptor that targets CD19 and CD22, which comprises an extracellular antigen-binding domain comprising the heavy chain variable region and the light chain variable region of an anti-CD19 antibody and an anti-CD22 antibody.The present invention further provides a bispecific CAR-T cell that targets CD19 and CD22.

Description

[Technical Field]

[0001] This application claims priority to a Chinese patent application filed with the China Patent Office on June 30, 2020, with application number 202010618929.6, entitled "Fully humanized bispecific antigen chimeric receptor targeting CD19 and CD22 and its application," and a Chinese patent application filed with the China Patent Office on July 21, 2020, with application number 202010707612.X, entitled "Fully humanized bispecific antigen chimeric receptor targeting CD19 and CD22 and its application."

[0002] The present invention relates to bispecific chimeric antigen receptors, in particular bispecific chimeric antigen receptors that target CD19 and CD22. [Background technology]

[0003] CD19 CAR-T therapy has achieved tremendous clinical success. Currently, two CAR-T drugs (Novartis' Kymriah and Gilead / Kite's Yescarta) are approved and commercially available worldwide. The complete remission rate for B-ALL treatment with CD19 CAR-T is 65-80%, and the complete remission rate for adult lymphoma treatment is 50-60%. However, relapse remains a problem with CD19 CAR-T therapy. Approximately one-third of ALL patients experience disease relapse after CD19 CAR-T treatment due to loss of the CD19 antigen. Furthermore, the immunogenicity of mouse-derived CD19 CAR-T makes CAR-T cells less likely to persist in the human body, which is another factor contributing to relapse after CAR-T therapy.

[0004] In clinical trials of CD22 CAR-T for the treatment of B-ALL, the treatment showed favorable therapeutic effects, with a complete remission rate of 73%. However, relapse remains a problem with CD22 CAR-T therapy. Research has shown that a decrease in CD22 antigen expression density may be the cause of relapse after CD22 CAR-T therapy. Research has also shown that CD22 is a key target for CD19-CAR therapy after relapse.

[0005] Therefore, in CAR-T therapy, it is necessary to quickly solve the problem of how to overcome the aberrant recurrence of tumors due to antigen disappearance or reduction. Summary of the Invention

[0006] In one embodiment, provided herein is a bispecific chimeric antigen receptor comprising an extracellular antigen-binding domain comprising the heavy and light chain variable regions of an anti-CD19 antibody and the heavy and light chain variable regions of an anti-CD22 antibody, The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD19 antibody are A heavy chain variable region sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 2 and a light chain variable region sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 4; and a heavy chain variable region sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 8 and a light chain variable region sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 6; is selected from any combination of The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD22 antibody are A heavy chain variable region sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 10 and a light chain variable region sequence having at least 90% sequence identity with the sequence set forth in SEQ ID NO: 12; and a heavy chain variable region sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 14 and a light chain variable region sequence having at least 90% sequence identity with the sequence shown in SEQ ID NO: 16; The present invention provides a bispecific chimeric antigen receptor that targets CD19 and CD22, wherein the bispecific chimeric antigen receptor is selected from any combination of:

[0007] In some embodiments, the amino acid sequences of the heavy and light chain variable regions of the anti-CD19 antibody are: A heavy chain variable region sequence shown in SEQ ID NO: 2 and a light chain variable region sequence shown in SEQ ID NO: 4, and a heavy chain variable region sequence shown in SEQ ID NO: 8 and a light chain variable region sequence shown in SEQ ID NO: 6; is selected from any combination of The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD22 antibody are A heavy chain variable region sequence shown in SEQ ID NO: 10 and a light chain variable region sequence shown in SEQ ID NO: 12, and a heavy chain variable region sequence shown in SEQ ID NO: 14 and a light chain variable region sequence shown in SEQ ID NO: 16; The combination is selected from any one of the following:

[0008] In some embodiments, the heavy chain variable region of the anti-CD19 antibody has the sequence set forth in SEQ ID NO:2, the light chain variable region of the anti-CD19 antibody has the sequence set forth in SEQ ID NO:4, the heavy chain variable region of the anti-CD22 antibody has the sequence set forth in SEQ ID NO:10, and the light chain variable region of the anti-CD22 antibody has the sequence set forth in SEQ ID NO:12; or The heavy chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 2, the light chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 4, the heavy chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO: 14, and the light chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO: 16.

[0009] In some embodiments, the heavy chain variable region of the anti-CD19 antibody has the sequence set forth in SEQ ID NO:8, the light chain variable region of the anti-CD19 antibody has the sequence set forth in SEQ ID NO:6, the heavy chain variable region of the anti-CD22 antibody has the sequence set forth in SEQ ID NO:10, and the light chain variable region of the anti-CD22 antibody has the light chain variable region sequence set forth in SEQ ID NO:12.

[0010] In some embodiments, the heavy and light chain variable regions of the anti-CD19 antibody and the heavy and light chain variable regions of the anti-CD22 antibody are ordered from amino terminus to carboxy terminus in the extracellular antigen-binding domain. a light chain variable region of the anti-CD19 antibody, a heavy chain variable region of the anti-CD22 antibody, a light chain variable region of the anti-CD22 antibody, and a heavy chain variable region of the anti-CD19 antibody; a heavy chain variable region of the anti-CD19 antibody, a light chain variable region of the anti-CD22 antibody, a heavy chain variable region of the anti-CD22 antibody, and a light chain variable region of the anti-CD19 antibody; a light chain variable region of the anti-CD22 antibody, a heavy chain variable region of the anti-CD19 antibody, a light chain variable region of the anti-CD19 antibody, and a heavy chain variable region of the anti-CD22 antibody; or the heavy chain variable region of the anti-CD22 antibody, the light chain variable region of the anti-CD19 antibody, the heavy chain variable region of the anti-CD19 antibody, and the light chain variable region of the anti-CD22 antibody.

[0011] In some embodiments, the extracellular antigen-binding domain comprises, in order from amino terminus to carboxy terminus: a light chain variable region of the anti-CD19 antibody, a first linker, a heavy chain variable region of the anti-CD22 antibody, a second linker, a light chain variable region of the anti-CD22 antibody, a third linker, and a heavy chain variable region of the anti-CD19 antibody; a heavy chain variable region of the anti-CD19 antibody, a first linker, a light chain variable region of the anti-CD22 antibody, a second linker, a heavy chain variable region of the anti-CD22 antibody, a third linker, and a light chain variable region of the anti-CD19 antibody; a light chain variable region of the anti-CD22 antibody, a first linker, a heavy chain variable region of the anti-CD19 antibody, a second linker, a light chain variable region of the anti-CD19 antibody, a third linker, and a heavy chain variable region of the anti-CD22 antibody; or a heavy chain variable region of the anti-CD22 antibody, a first linker, a light chain variable region of the anti-CD19 antibody, a second linker, a heavy chain variable region of the anti-CD19 antibody, a third linker, and a light chain variable region of the anti-CD22 antibody; The first linker and the third linker have the amino acid sequence shown in SEQ ID NO:20, and the second linker has the amino acid sequence shown in SEQ ID NO:24.

[0012] In some embodiments, the bispecific chimeric antigen receptor comprises, in order from amino to carboxy terminus, a signal peptide sequence, the extracellular antigen-binding domain, a hinge domain, a transmembrane region, and an intracellular signaling domain, wherein the intracellular signaling domain comprises, from amino to carboxy terminus, a fragment derived from a 4-1BB molecule and a fragment derived from a CD3z molecule.

[0013] In some embodiments, the signal peptide sequence has the amino acid sequence set forth in SEQ ID NO: 36, the hinge domain has the amino acid sequence set forth in SEQ ID NO: 26, the transmembrane region has the amino acid sequence set forth in SEQ ID NO: 28, the fragment derived from the 4-1BB molecule has the amino acid sequence set forth in SEQ ID NO: 30, and the fragment derived from the CD3z molecule has the amino acid sequence set forth in SEQ ID NO: 32.

[0014] In some embodiments, the bispecific chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, or 70.

[0015] In another aspect, provided herein is a nucleic acid molecule encoding said bispecific chimeric antigen receptor.

[0016] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, or 69.

[0017] In another aspect, provided herein is an expression vector comprising the nucleic acid molecule.

[0018] In another aspect, provided herein is a host cell expressing said bispecific chimeric antigen receptor or comprising said expression vector.

[0019] In some embodiments, the host cell is an immune cell, preferably a T cell or an NK cell.

[0020] In another aspect, provided herein is the use of said bispecific chimeric antigen receptor, expression vector or host cell in the preparation of a medicament for treating cancer.

[0021] In some embodiments, the cancer is a B-cell related cancer.

[0022] In some embodiments, the cancer is B-cell non-Hodgkin's lymphoma (B-NHL) or B-cell acute lymphoblastic leukemia (B-ALL).

[0023] In some embodiments, the cancer expresses CD19 and / or CD22.

[0024] In another aspect, provided herein is a method of treating cancer in a patient, comprising administering to the patient said bispecific chimeric antigen receptor, said expression vector, or said host cell.

[0025] In some embodiments, the cancer is a B-cell related cancer.

[0026] In some embodiments, the cancer is B-NHL or B-ALL.

[0027] In some embodiments, the cancer expresses CD19 and / or CD22.

[0028] In some embodiments, the host cells are cultured at a density of 0.5×10 6 3 x 10 host cells / kg patient weight 6 The patient is administered a dose of 10 host cells / kg patient body weight.

[0029] In some embodiments, the patient is a B-NHL patient, and the host cells are administered in an amount of 1×10 63 x 10 host cells / kg patient weight 6 The patient is administered a dose of 10 host cells / kg patient body weight.

[0030] In some embodiments, the patient is a B-ALL patient, and the host cells are administered in an amount of 0.5×10 6 1 x 10 host cells / kg patient weight 6 The patient is administered a dose of 100 host cells / kg of patient body weight. The bispecific CAR-T cells (CD19x22 CAR-T) provided herein that simultaneously target CD19 and CD22 can improve the therapeutic efficacy of CAR-T and reduce the relapse rate. [Brief explanation of the drawings]

[0031] [Figure 1] Schematic diagram of the structural design of the extracellular antigen recognition region (CD19x22 scFvs) of the CD19x22 bispecific CAR molecule. [Figure 2] Schematic diagram of the structure of the CD19x22 bispecific CAR molecule. [Figure 3] 1 is a schematic diagram of the working principle of the NFAT reporter gene method. [Figure 4] Flow detection results show the expression status of CAR molecules in transiently transfected Jurkat cells and the protein binding ability of CAR molecules to CD19 and CD22. Of these, CD19-CAR and CD22-CAR are monospecific CAR molecules used as controls. Transient transfection and flow detection were performed on three batches of the 12 constructed CAR molecule structures, each containing CD19-CAR and CD22-CAR as controls. [Figure 5]The flow detection results in Figure 4 are shown as a histogram of the percentage of double-positive cells. The expression of CAR and tEGFR molecules on the surface of transiently transfected Jurkat cells was measured by simultaneously staining with EGFR antibody and CD19-FITC protein, or EGFR antibody and CD22-FITC protein, respectively, to obtain the percentage of double-positive cells. A higher percentage of double-positive cells indicates a better expression level and protein-binding ability of CAR molecules. [Figure 6] Figure 1 shows the results of NFAT reporter gene detection of bispecific CAR molecules. The reporter gene detection was performed in a total of three batches, and each batch included the results of two monospecific CARs, CD19-CAR and CD22-CAR, as controls. [Figure 7] Figure 1 shows the results of detecting CD107a degranulation function of bispecific CAR-T cells, where the scatter plot is the CD8+ cell population, the abscissa is CAR molecule expression by APC anti-EGFR antibody staining, and the ordinate is PE anti-CD107a staining. [Figure 8] The killing results of bispecific CAR-T against each target cell are shown. [Figure 9] Schematic diagram of the molecular structure of CD19×22 CAR containing antibody fragment No. 78. [Figure 10] In vitro cytotoxicity of CD19x22 CAR-T cells containing antibody fragment 78 was measured using REH (a), CD19 knockout Raji (b), and CD22 knockout Raji (c) cells as target cells, respectively. [Figure 11] Schematic diagram of the structure of bispecific CAR molecules employing different VH-VL order combinations. [Figure 12] This is a flow scatter plot of bispecific CAR molecules with different VH-VL order combinations expressed on the surface of CAR-T cells, followed by double staining with CD19-FITC or CD22-FITC protein and APC anti-EGFR antibody, respectively. [Figure 13]Figure 12 shows the mean fluorescence intensity (MFI) detected in the APC and FITC signal pathways. The MFI in the APC channel reflects the expression status of tEGFR molecules. The MFI in the FITC channel reflects the binding status of CD19 or CD22 antigen proteins with CAR molecules. Because CAR molecules and tEGFR are connected via T2A, the expression status of tEGFR molecules theoretically corresponds to the expression status of CAR molecules. [Figure 14] The degranulation activity of bispecific CAR-T cells with different VH-VL sequence combinations is shown. Flow cytometry was used to detect degranulation signals generated by stimulation of different target cells in the CD8+ / CAR+ cell population using the PE-Cy7 anti-CD107a antibody. The top table shows the MFI of the PE-Cy7 signal in the CD8+ / CAR+ cell population, reflecting the expression level of CD107a on the cell surface. The bottom table shows the CD107a positivity rate in the CD8+ / CAR+ cell population. LV60 and LV90 are controls for CD22 and CD19 monospecific CAR-Ts, respectively, and LV60 / LV90 is a mixed sample of the two monospecific CAR-Ts. [Figure 15] Figure 1 shows the cytotoxicity of different target cells by bispecific CAR-Ts with different VH-VL sequence combinations. The ordinate axis shows the fluorescence intensity (RLU) detected after 24 hours of co-incubation of luciferase-marked target cells with CAR-Ts. Lower fluorescence intensity indicates greater target cell cytotoxicity. [Figure 16] The table shows the average scores obtained by sorting the bispecific CAR-T cytotoxicity of different VH-VL ordered combinations. Calculation of the average scores: The cytotoxicity experiment was repeated several times (as shown in Figure 15), and each experimental sample was sorted to obtain a score between 0 and 1 (1 indicates the highest cytotoxicity, and 0 indicates the lowest cytotoxicity). The average of these scores is the average score in the table. [Figure 17] Figure 1 shows cytokine secretion graphs of three batches of CD19x22 CAR-T with different effector-target ratios, where the ordinate is cytokine secretion amount and the abscissa is effector-target ratio. [Figure 18]The experimental survival curves for animals in each group are shown. Vehicle = cytoprotective solution group, Mock-T = Mock-T control group, and CD19x22 CAR-T = test product group. [Figure 19] This graph shows the change in the average bioluminescence intensity of animals in each group over the experimental period. The ordinate represents the bioluminescence intensity of surviving animals, and the abscissa represents the date. Vehicle = cytoprotective solution group, Mock-T = Mock-T control group, and CD19x22 CAR-T = test article group. [Figure 20] This shows the tissue distribution of CD19×22 CAR-T in experimental animals. [Figure 21] Figure 21A shows the results of the therapeutic effect of different doses in B-NHL subjects at different time points after CAR-T cell return, and Figure 21B shows the results of the therapeutic effect of different doses in B-ALL subjects at different time points after CAR-T cell return. DETAILED DESCRIPTION OF THE INVENTION

[0032] Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0033] 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. Due to conservative differences in amino acid sequence, the heavy and light chains are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxy terminus. The heavy chain variable region (VH) and light chain variable region (VL) interact to form an antigen-binding site (Fv). In some cases, the term "antibody" also refers to antibody fragments capable of binding to antigens, such as scFv, Fab, and F(ab')2.

[0034] 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.

[0035] Chimeric antigen receptors (CARs), also known as chimeric T cell receptors or chimeric immune receptors, are engineered membrane protein receptor molecules that can confer desired specificity, such as the ability to bind to specific tumor antigens, to immune effector cells. Chimeric antigen receptors typically consist of an extracellular antigen-binding domain, a transmembrane region, 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 the CD3z molecule, which activates immune effector cells and exerts cytotoxic effects. Chimeric antigen receptors also contain a signal peptide at the amino terminus responsible for intracellular positioning of the nascent protein, and a hinge domain between the antigen-binding domain and the transmembrane region. In addition to the signaling domain, the intracellular signaling domain may also contain a costimulatory domain derived from, for example, the 4-1BB or CD28 molecule.

[0036] The term "bispecific chimeric antigen receptor" refers to a molecule that contains at least two different antigen-binding sites in the extracellular antigen-binding domain, each of which recognizes and binds to a different antigen molecule on a target cell. The bispecific chimeric antigen receptor targeting CD19 and CD22 provided herein contains one CD19-binding site (formed from the light chain variable region and heavy chain variable region of an anti-CD19 antibody) and one CD22-binding site (formed from the light chain variable region and heavy chain variable region of an anti-CD22 antibody).

[0037] When referring to a chimeric antigen receptor or its expression vector, the term "host cell" as used refers to a cell that expresses the chimeric antigen receptor or contains the expression vector, particularly an immune cell such as a T cell or an NK cell.

[0038] "CAR-T cells" refer to T cells that express a CAR molecule 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, CAR-encoding expression vectors can also be used to transform other lymphocytes, such as NK cells, to obtain target cytotoxic cells expressing the CAR.

[0039] CD19 is a B lymphocyte surface marker molecule that regulates B cell activation and development. CD19 is not only expressed on normal B cells but also on many malignant B cell tumors, laying the foundation for clinical use of CD19-targeting CAR-T for the treatment of B cell-related tumors.

[0040] "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 available in UniProt under the accession number P20273. As a transmembrane glycoprotein, its expression begins on the surface of B cells at the pre-B cell stage, is present on mature B cells, but disappears on plasma cells.

[0041] 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.

[0042] In some embodiments, the light chain variable region of an anti-CD19 antibody 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:4 or 6, 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:2 or 8.

[0043] 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: 12 or 16, 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: 10 or 14.

[0044] In some embodiments, the bispecific CAR molecules provided herein comprise 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: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, or 70.

[0045] Those skilled in the art will understand that, in addition to the specific sequences provided herein, corresponding mutants of the bispecific chimeric antigen receptors provided herein can be obtained by substituting, deleting, or adding a small number of amino acids and verifying or screening the resulting products for their ability to bind to the corresponding antigen or biological activity, and that these mutants are also within the scope of the present invention.

[0046] By using bispecific chimeric antigen receptor molecules that simultaneously target CD19 and CD22, obtained through repeated screening, target cells expressing CD19 and / or CD22 can be specifically identified and killed.

[0047] The present invention will be further described below with reference to specific examples. [Example]

[0048] Example 1 Construction of Plasmid Vectors for Bispecific CAR Molecules The extracellular antigen recognition region of bispecific CAR molecules contains four domains: CD19 VH, CD19 VL, CD22 VH, and CD22 VL. The folding and pairing of these domains significantly affect the function of bispecific CAR molecules. Therefore, it is necessary to screen linkers between each domain and the order of each domain. As shown in Figure 1, a total of 12 different structures of the CAR molecule cell recognition region were designed and used for cell recognition region structure screening. In Figure 1, 19VL represents the light chain variable region of the anti-CD19 antibody, 19VH represents the heavy chain variable region of the anti-19 antibody, 22VL represents the light chain variable region of the anti-CD22 antibody, and 22VH represents the heavy chain variable region of the anti-22 antibody. The numbers in parentheses after # represent the number of the antibody screened against the CD19 or CD22 antigen, respectively. Figure 1 is primarily used to interpret the screening of various structural combinations of extracellular antigen recognition domains. Among these, CD19 and CD22 antibodies and their combinations were some of the preferred antibodies screened. The antibody numbers in parentheses (62, 80, and 28) do not include all antibodies used in the screening study. As shown in Figure 2, these bispecific CAR molecules employed a second-generation CAR structure and co-expressed a truncated EGFR membrane protein (tEGFR) using the T2A method. The nucleic acid sequences encoding these CAR molecules were synthesized and inserted into the pLKO expression vector.

[0049] Example 2 Flow detection of CAR molecule expression and antigen-binding ability 1. Experimental Objectives and Principles Jurkat cells were transfected with a plasmid encoding a CAR molecule via electrotransduction, and the CAR molecule was transiently expressed on the cell surface. The CAR molecule and tEGFR were co-expressed via the T2A peptide (Figure 2). The expression level of the CAR molecule could be indirectly detected using an EGFR antibody via flow cytometry. At the same time, the binding ability of the bispecific CAR molecule to these two antigens could be detected via protein staining for CD19 or CD22, respectively.

[0050] 2. Operation steps i. Using the Celetrix electroporation kit, 4 μg of the plasmid encoding the CAR molecule was electroporated into 2 × 10 Jurkat cells. 6 The transfected cells were cultured at 37°C and 5% CO2 for 24 hours. ii. Electroporated Jurkat cells were stained and subjected to flow assay using APC anti-EGFR antibody and CD19-FITC protein, or APC anti-EGFR antibody and CD22-FITC protein, respectively.

[0051] 3. Screening Standards The CD19x22 CAR molecule can be normally expressed on Jurkat cells and can bind CD19 and CD22 proteins, respectively.

[0052] Example 3: Detection of the activity of CAR molecules to activate NFAT transcription factors using a reporter gene method 1. Experimental Objectives and Principles CAR-T cell activation is achieved by CD3z and costimulatory factors in the intracellular domain of the CAR molecule, and one of the prerequisites for CAR-T cell activation is that CD3z can activate the NFAT signaling pathway in cells. Therefore, the NFAT reporter gene method can be used to screen for CAR molecules that have the function of activating the NFAT signaling pathway.

[0053] In the primary screening process, Jurkat cells carrying the NFAT-RE-ffLuc (ffLuc, firefly luciferase) reporter gene were used as reporter cells (designated JLuc307, as shown in Figure 3). The CAR molecule was transiently expressed on the reporter cell surface by electroporation of the plasmid. After co-cultivating the CAR molecule-expressing reporter cells with target cells (expressing CD19 and / or CD22), the CAR molecule was specifically activated by target cell surface antigens, further activating reporter gene expression. The ability of the CAR molecule to activate the NFAT signaling pathway was then assessed by detecting luciferase activity. Since different CAR molecules exhibit different electroporation efficiencies, the electroporation efficiency can be determined using an internal reference plasmid (CMV-hRLuc, Renilla luciferase) co-localized with the CAR molecule.

[0054] 2. Operation steps i. The CAR plasmid to be measured and the internal reference plasmid were mixed in a certain ratio, and then the reporter cells were transfected by electroporation method. ii. 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. iii. 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. iv. 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.

[0055] 3. Screening Standards The bispecific CAR molecules can be activated by CD19- or CD22-positive target cells, respectively, to generate a fluorescent signal via the NFAT-RE reporter gene. Without target cell stimulation or with stimulation of CD19-CD22- target cells, there is a low level of background (tonic effects) or nonspecifically activated fluorescent signal.

[0056] Example 4 In vitro functional evaluation of bispecific CAR-T cells The in vitro function of bispecific CAR-T cells was evaluated using two main methods: the CD107a degranulation assay and the in-vitro cytotoxicity assay.

[0057] 1.CD107a degranulation experiment 1.1 Experimental Objectives and Principles 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 recovery with monesin (purchased from BioLegend) quantitatively reflects the strength of microvesicle release. 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 detection of the positivity of CD107a on the CAR-T cell surface.

[0058] 1.2 Operation steps i. Different target cells (e.g., Raji, CD19 KO Raji (CD19 knockout Raji cells), CD22 KO Raji (CD22 knockout Raji cells), K562, etc.) were centrifuged at 300g for 5 min at room temperature, the supernatant was discarded, and 2 x 10 cells were cultured in T cell medium. 5 The cells were resuspended at 100 cells / mL. ii. 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. iii. CAR-T cells to be measured (100 μL / well) and target cells (100 μL / well) were added to a U-bottom 96-well plate, mixed evenly, and then incubated in an incubator (37°C, 5% CO2) for 3 hours. iv. After the incubation was completed, the plate was centrifuged at 600 g at 4°C for 5 minutes, the supernatant was discarded, and the cells were washed twice with 200 μL / well of DPBS+1% HSA. v. 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 were added. The cells were mixed uniformly and then incubated on ice in the dark for 20 minutes. vi. 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.

[0059] 1.3 Screening Standards CD19x22 CAR can specifically identify CD19+ / CD22+ (Raji), CD19+ / CD22- (CD22 KO Raji), and CD19- / CD22+ (CD19 KO Raji) cells and effectively activate CAR-T cells (CD8+ / CAR+ cell population has a high CD107a positivity rate).

[0060] CD19x22 CAR is not activated by CD19- / CD22- (K562) cells, and the CD8+ / CAR+ cell population has a low CD107a positivity rate.

[0061] 2. In vitro cytotoxicity experiments 2.1 Experimental Objectives and Principles To evaluate the antigen-specific cytotoxicity of CAR-T cells, CD19+ / CD22+ (NALM6-ffLuc), CD19+ / CD22- (CD22 KO Raji-ffLuc), and CD19- / CD22+ (CD19 KO Raji-ffLuc) target cells were used. These target cells were cell lines stably expressing firefly luciferase via lentiviral transduction. Furthermore, cytotoxicity experiments were performed using K562, REH, and JVM2 target cells, which were also stably transduced with ffLuc via lentiviral transduction, as described below, and were measured by luciferase activity.

[0062] In in vitro cytotoxicity experiments, CAR-T cells and target cells were co-cultured at different effector-target ratios (E:T). When target cells were killed by CAR-T cells, luciferase was released and quickly inactivated (the half-life of firefly luciferase is approximately 0.5 h). 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 can be used to detect the cytotoxicity of CAR-T to target cells.

[0063] 2.2 Operation steps i. Target cells were centrifuged at 300 g for 5 min at room temperature, the supernatant was discarded, and then 2 × 10 cells were resuspended in complete T cell medium. 5 The target cells were resuspended at 100 μL / well in a clear-bottom 96-well plate. ii. 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 / well of CAR-T cells were added to each 96-well plate, mixed uniformly with the target cells, and then incubated in an incubator (37°C, 5% CO2) for 24 hours. iii. 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.). iv. For cells other than the preserved samples, luciferase activity in each well was detected using a luciferase detection kit.

[0064] 2.2 Screening Standards CD19x22 CAR-T cells can effectively kill CD19+ / CD22+ (NALM6-ffLuc), CD19+ / CD22- (CD22 KO Raji-ffLuc), and CD19- / CD22+ (CD19 KO Raji-ffLuc) cells.

[0065] Results and Analysis 1. Expression of bispecific CAR molecules and antigen binding detection The CAR plasmid vector described in Example 1 was transiently transfected into Jurkat cells by the method described in Example 2. The transiently transfected cells were co-stained with APC anti-EGFR antibody and CD19-FITC or CD22-FITC, respectively, and the flow detection results are shown in Figures 4 and 5. In Figure 5, the flow detection results in Figure 4 (EGFR) are shown in a histogram. + / CD19 + and EGFR + / CD22 + The percentages of double-positive cells are shown, and the CD19-CAR and CD22-CAR control percentages are the average of the triplicate measurements in Figure 4. The figures show that structures #9 and #10 have strong binding ability to both CD19 and CD22 proteins, respectively.

[0066] 2. NFAT activation function of bispecific CAR molecules NFAT is an important transcription factor in T cells, and activation of NFAT accompanies T cell activation. The Jurkat-luciferase cell line was a conditionally luciferase-expressing cell line constructed based on Jurkat. When CAR expressed in Jurkat cells was activated, it transmitted downstream signals, activating NFAT and promoting luciferase expression. Therefore, luciferase activity can reflect the level of T cell activation. Target cells were co-cultured with Jurkat-luciferase cells electroporated with CAR, and the level of reporter gene expression, i.e., luciferase activity, reflected the degree of activation of CAR after binding to the target protein in the target cells.

[0067] The reporter gene signals produced by the 12 different CAR molecule structures constructed in Example 1 upon activation in target cells such as Raji (CD19+CD22+), CD19 KO Raji (CD19-CD22+), CD22 KO Raji (CD19+CD22-), and K562 (CD19-CD22-) are shown in Figure 6. Among these, the CAR molecules with structures #9 and #10 exhibited high levels of NFAT activation. Furthermore, the #10 structure exhibited excellent specificity for CD19 or CD22 antigens, and the nonspecific activation signal produced by co-cultivation with K562 cells was the lowest.

[0068] 3. CD107a degranulation function of CD19×22 CAR-T cells Summarizing the above results, CAR molecules with structures #9 and #10 were selected for functional testing of CAR-T cells. CAR-T cells were prepared by lentiviral transduction. The results of detecting the CD107a degranulation function of CAR-T cells are shown in Figure 7. CAR molecules with structures #9 and #10 were activated with CD19 KO or CD22 KO target cells (Raji), respectively, and the activation rate of CD107a produced was equivalent to that of a single-specific CAR (CD19 CAR or CD22 CAR). This indicated that clones #9 and #10 could activate CAR-T cells even in the presence of CD19 or CD22 antigen shedding.

[0069] 4. In vitro cytotoxicity of CD19×22 CAR-T cells As shown in Figure 8, the bispecific CAR-T cells of the #9 and #10 structures were comparable to or better than the monospecific CAR-Ts (CD19 CAR and CD22 CAR) in their ability to cytose CD19+CD22+ target cells (JVM2 and NALM6). Furthermore, they did not cytose CD19- / CD22- cells (K562), demonstrating a favorable safety profile.

[0070] Summarizing the above results, #9 and #10 have similar structures, and CD19x22 CAR-T constructed from these structures exhibits good in vitro function. Therefore, we speculated that the CAR molecule structures of #9 and #10 may share some commonality. Therefore, instead of clone #62, we constructed a new CAR molecule using the screened anti-CD19 antibody clone #78 (shown in Figure 9), and evaluated its in vitro cytotoxicity using the method described in Example 4. As shown in Figure 10, clone #78 also exhibited good in vitro cytotoxicity, instead of clone #62.

[0071] Example 5 Structural optimization of bispecific CAR molecules Since a bispecific CAR molecule contains two pairs of VH-VL sequences, and there may be steric hindrance or mutual influence of pairing between them, the order of the two pairs of VH-VL was further changed based on the sequence structure of #9 or #10 obtained in the above examples (shown in Figure 11), and the optimal CAR molecule structure was selected.

[0072] The functions of the CAR molecules obtained by combining these different VH-VL sequences were evaluated by the method described in Example 4. The binding ability of each of these CAR molecules to the CD19 / CD22 antigen was evaluated with reference to Example 2.

[0073] Even if the same clone antibodies (No. 78 and No. 80) and the same linker sequence are used, the bispecific CAR molecules with different VH-VL sequences may have different functions. As shown in Figures 12 and 13, the bispecific CAR molecules with different VH-VL sequences had different binding abilities to CD19 and CD22 antigens. However, PXL1437 had better binding abilities to both CD19 and CD22 proteins.

[0074] Figure 14 shows the data on degranulation function of CAR-T cells co-cultured with Raji (CD19+ / CD22+), CD19KO Raji (CD19- / CD22+), CD22KO Raji (CD19+ / CD22-), and K562 (CD19- / CD22-) cells. The results show that bispecific CAR molecules with different VH-VL sequences were successfully activated by Raji, CD19KO Raji, and CD22KO Raji, resulting in degranulation, but PXL1437 had a stronger degranulation effect (higher mean fluorescence intensity (MFI) of CD107a).

[0075] As shown in Figure 15, bispecific CAR-T cells with different VH-VL sequences were all able to successfully cytosine Raji, CD19KO Raji, and CD22KO Raji target cells. However, because in vitro cytosine cytosine experiments are affected by various experimental conditions, no significant differences were observed within a single experiment. Therefore, after several in vitro cytosine cytosine experiments, the cytosine cytosine cytosine (C19KO) for each CAR molecule in each experiment was sorted and scored. The results are shown in Figure 16, among which PXL1437 showed good cytosine cytosine activity against all three target cells.

[0076] These experiments demonstrated that the specific antibody selection, the composition of different extracellular antigen-binding domains, and the order of antibody VH-VL connections all affected the antigen-binding ability, degranulation function, and in vitro cytotoxicity of bispecific CAR molecules.

[0077] Compared to monospecific CAR molecules, bispecific CAR molecules incorporate one light chain variable region and one heavy chain variable region within their extracellular antigen-binding domain (combining them to form a new antigen-binding site). These added polypeptide fragments always adversely affected the original antigen-binding fragment and its antigen binding. Alternatively, the original antigen-binding fragment adversely affected the added antigen-binding fragment and its antigen binding. Without being bound by theory, we believe that such effects are due to changes in the folding of the antigen-binding fragment, steric hindrance, or interactions between different antigen-binding fragments. After several rounds of screening, we found that by combining clones 62 and 78 (anti-CD19 antibodies) and clones 80 and 28 (anti-CD22 antibodies), we were able to obtain bispecific CAR molecules with high binding affinity to both CD19 and CD22 and significant cytotoxicity against the corresponding target cells.

[0078] In this study, we constructed a bispecific CAR-T (CD19x22 CAR-T) that simultaneously targets CD19 and CD22 using fully humanized CD19 and CD22 antibodies, improving the therapeutic efficacy of CAR-T and reducing the relapse rate. Compared to co-transfecting T cells with CD19-CAR and CD22-CAR, the bispecific CAR-T (CD19x22 CAR-T) stably targets CD19 and CD22 simultaneously, preventing antigen drift and relapse, while also providing better uniformity and easier product control. Compared to the CD19-CAR and CD22-CAR combination, the treatment time is shorter, the therapeutic effect is better, and the cost is lower.

[0079] The bispecific chimeric antigen receptors provided herein, particularly lymphocytes (e.g., CAR-T cells) modified to express the bispecific chimeric antigen receptors, are used to treat certain lymphomas and leukemias. These CAR-T cells can be formulated and administered as a pharmaceutical composition together with a pharmaceutically acceptable vector.

[0080] Example 6: Study of the ex vivo expansion ability of CD19x22 CAR-T cells Unless otherwise noted, this and the following examples were all performed using CD19x22 CAR-T expressing the PXL1437 structure (see Figure 11). Specifically, while both PXL1437 structures A and B in Figure 11 can be used to produce CD19x22 CAR-T cell therapy products, this example used the specific structure B in Figure 11.

[0081] The cell numbers of CD19x22 CAR-T cells were counted at different time points using a dual-wavelength fluorescent cell counting method, and their proliferation characteristics were analyzed. Three batches of product (HD201110-01, HD201110-02, and HD201114-01) were prepared using the same method and three experiments were performed. The results are shown in Table 1.

[0082] [Table 1] Based on the results shown in the table above, CD19x22 CAR-T showed stable in vitro amplification, with an amplification fold increase of 419.61±121.49 on day 12 and 710.90±79.08 on day 14, with a doubling time of 1.36±0.02 days. Therefore, CD19x22 CAR-T showed stable and good amplification.

[0083] Example 7 CD19x22 CAR-T in vitro cytokine release study To investigate the tumor cell damage caused by this injection, we co-cultivated CD19x22 CAR-T with target cells and investigated the expression levels of secreted interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor (TNF), interferon-γ (IFN-γ), and interleukin-17A (IL-17A).

[0084] Cytokine secretion levels in the cell supernatant were detected using the CBA (Cytometric Bead Array) method. After incubation of the positive cells with CD19x22 CAR-T, the secretion of Th1 cytokines (IL-2, TNF, IFN-γ) was observed in the single CD3 + CAR + The amount released was significant (>1.7 fg), and the single cytokines released were Th2 type cytokines (IL-4, IL-6, IL-10) and Th17 type cytokine (IL-17A). + CAR + The amount released was very low (<0.3 fg). Analysis of the results in Figure 17 revealed that CD19x22 CAR-T had stable, specific, and good cytokine secretion function. IL-4, IL-6, IL-2, TNF-α, and IFN-γ were significantly increased.

[0085] Example 8: In vivo efficacy study 8.1 Efficacy study of CD19×22 CAR-T in tumor-bearing immunodeficient mice Using mice bearing B-cell acute lymphoblastic leukemia (Nalm6) as an experimental system, the inhibitory effect of CD19×22 CAR-T on tumor cell growth in the mouse body was evaluated.

[0086] Experimental Method: 1) Screening and grouping: 0.5 × 10 per mouse for 80 female NCG mice 6 One day after intravenous inoculation of Nalm6 cells, 57 animals were screened by body weight and were assigned to a cytoprotective solution group, a mock-T control group (10 × 10 6 individual cells / head), CD19 CAR-T group (0.2×10 6 CAR-T cells / head), CD22 CAR-T group (0.2×10 6 CAR-T cells / head), experimental group low (0.2 × 10 6 pieces CAR-T cells / head), medium (1.0×10 6 pieces CAR-T cells / head), high (3.0×10 6The animals were divided into seven groups: (10 CAR-T cells / animal) dose groups, except for the CD19 CAR-T and CD22 CAR-T groups, which had 3 and 4 animals, respectively, which were too few to report as valid data groups; the remaining groups had 10 animals / group. 2) Administration method: All animals were administered a single dose via tail vein injection, and the day of the first administration was designated as D1. 3) Detection parameters: General clinical observations were performed twice daily, and the survival rate of each group was monitored until D30. Animals were weighed once before group allocation and again on D3, D7, D10, D14, D17, D21, D24, and D28 after administration. On D7, D14, D21, and D28, all animals were imaged for chemiluminescence signals using a Bruker small animal imager. Blood samples were taken from half of the animals in each group at D3, D7, and D28, except for all animals on D28, to detect lymphocyte subpopulations. Blood samples were taken from half of the animals in each group at D2, D3, D5, D7, and D28, except for all animals on D28, to detect cytokine levels of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ.

[0087] result: 1) During the general clinical observation period, animals in the cytoprotective solution group began to die from day 20. By day 21, 10 / 10 animals in the cytoprotective solution group had died. Clinical symptoms before death included mental retardation, hunchback, and rigidity, which were presumed to be related to tumor cell proliferation. By day 28, 10 / 10, 7 / 10, 0 / 10, 1 / 10, and 2 / 10 animals had died in the cytoprotective solution group, mock-T control group, and low-, medium-, and high-dose experimental groups, respectively. Compared to the cytoprotective solution group, the survival rates of animals in the low-, medium-, and high-dose experimental groups were all significantly improved (P<0.001). The survival rate curves for animals in each group during the experimental period are shown in Figure 18. During the experimental period, in the mock-T control group, except for animal Y20-6465, which died during blood collection on D5, the remaining animals began to die from D12 onwards, and by D28, 7 / 10 animals had died. Clinical symptoms prior to death included lethargy, hunchback, and rigidity, suggesting that the deaths of animals in this group were related to tumor cell proliferation. During the experimental period, 1 / 10 and 2 / 10 animals, respectively, died by D28 in the medium-dose and high-dose experimental groups. Among these, animal Y20-6509 in the medium-dose experimental group and animal Y20-6520 in the high-dose experimental group were found dead on D20, with no abnormal clinical symptoms observed prior to death. Animal Y20-6519 in the high-dose experimental group was found dead on D28, with clinical symptoms of hunchback and ragged fur before death. No tumor signals were detected in this animal during the experimental period, and its weight began to decrease from D21, dropping from 20.7g to 17.9g on D24. 2) Tumor cell proliferation (bioluminescence intensity of tumor cells): During the experimental period, the mean bioluminescence intensity of animals in the cytoprotective solution group tended to increase, with the mean bioluminescence intensity on Days 7 and 14 increasing by 6.96 x 10 9 , 1.74×10 11 During the experimental period, the mean bioluminescence intensity of the mock-T control group on days 7, 14, 21, and 28 was 6.99 × 10 9 , 1.66×10 11 , 1.55×10 11 , 6.36 x 10 10 During the experimental period, the mean bioluminescence intensity of the animals in the low-dose experimental group on D7, D14, D21, and D28 was 2.12 × 108 , 5.59 x 10 8 , 9.42 x 10 9 , 6.88×10 10 P / S. Compared to the cytoprotective solution group, the bioluminescence intensity was reduced by approximately 32-fold and 310-fold on D7 and D14, respectively, with both statistically significant differences (P<0.001). Compared to the mock-T control group, the bioluminescence intensity was reduced by approximately 32-fold and 296-fold on D7 and D14, respectively, with both statistically significant differences (P<0.001). During the experimental period, the mean bioluminescence intensity of animals in the medium-dose experimental group on D7, D14, D21, and D28 was 1.04×10 7 , 5.51 x 10 7 , 1.49×10 9 , 2.21×10 10 P / S. Compared to the cytoprotective solution group, the bioluminescence intensity was reduced by approximately 668-fold and 3157-fold on D7 and D14, respectively, with all statistically significant differences (P<0.001). Compared to the mock-T control group, the bioluminescence intensity was reduced by approximately 672-fold, 3012-fold, 104-fold, and 28-fold on D7, D14, D21, and D28, respectively, with all statistically significant differences (P<0.001). During the experimental period, in the high-dose experimental group, no tumor signal was detected on D28, but tumor signals were detected in 1 / 10, 2 / 10, and 1 / 9 animals on D7, D14, and D21, respectively, with an average bioluminescence intensity of 9.51×10 4 , 8.83×10 4 , 3.43 × 10 6 There was a statistically significant difference between the cytoprotective solution group and the mock-T control group (P<0.001). The changes in the mean bioluminescence intensity of animals in each group over the experimental period are shown in detail in Figure 19. 3) Lymphocyte subpopulation: During the experimental period, the peripheral blood of animals in the cytoprotective solution group was primarily CD45 + The mock-T control group showed sustained expansion, with no CD45 cells detected until D28. + CD3 + The percentage of CD45 T cells in the peripheral blood of animals in the low, medium, and high dose groups was 12.8±9.7%. Sustained expansion of T cells was observed in the low, medium, and high dose groups, and the expansion was dose-dependent. + CD3 +The percentages of cells were 0.2±0.5%, 23.5±30.3%, and 73.8±6.7%, respectively. 4) Cytokines: Increased IFN-γ levels are generally considered to be a major marker of T cell activation. During the experimental period, in the cytoprotective solution group, IFN-γ levels increased to the highest levels by D28 in the mock-T control group and the low, medium, and high dose experimental groups, reaching 165.26±175.17, 27.37±39.95, 48.07±75.85, and 377.53±271.88 pg / mL, respectively, with the experimental group demonstrating dose-dependence.

[0088] Under the present experimental conditions, no significant changes were observed in the levels of IL-2, IL-4, IL-6, IL-10, and TNF-α. 5) Body weight: By day 17, the body weights of the animals in the cytoprotective solution, mock-T control group, and low, medium, and high dose experimental groups were 20.2±1.2, 20.6±1.3, 20.1±1.6, 20.2±1.0, and 19.7±0.8 g, respectively, and no abnormalities were observed in the body weights of the animals in each group. From day 17 to day 28, the body weight of the mock-T control group began to decrease from day 21 and had decreased to 18.6±3.6 g by day 28, while no abnormalities were observed in the body weights of the animals in the remaining groups.

[0089] Therefore, under the present experimental conditions, the human B-cell acute lymphoblastic leukemia cell line Nalm6 was able to proliferate in the body of NCG mice after intravenous inoculation. 6 CD19x22 CAR-T cells administered intravenously in a single dose-dependent manner were able to eliminate tumor cells and prolong the survival of animals.

[0090] 8.2. Tissue distribution studies In this study, CD19x22 CAR-T was administered once to NCG tumor-bearing mice via tail vein injection in the experimental group, and its distribution in NCG tumor-bearing mice was examined to provide reference for further studies.

[0091] Research method: In the study, a total of 70 NCG mice (60 mice + 10 surrogate mice) were used, and human acute lymphocytes Nalm6 (5 × 10 5 Tumor-bearing mouse models were established by intravenous transplantation of 2 × 10 cells / head (2 × 10 cells / head) via tail vein injection. 6 All animals were euthanized at 168 h (7 d), 336 h (14 d), 672 h (28 d), 1008 h (42 d), 1344 h (56 d), and 1704 h (71 d) after administration (five animals per sex). Whole blood (EDTA-K2 anticoagulated), brain, spinal cord (cervical), femoral bone marrow, skeletal muscle, ovaries / testes, abdominal organs (stomach, small intestine, liver, kidneys, spleen), and thoracic organs (heart and lungs) were collected. Blood was collected from the orbit at 120 hours (5 days), 240 hours (10 days), 408 hours (17 days), 504 hours (21 days), 840 hours (35 days), 1176 hours (49 days), and 1272 hours (53 days) after administration. CAR-T cells (CD3 + , CD22 antibody + ) and tumor cells (CD19 + ) flow detection was performed.

[0092] Study Results and Conclusions: This report presents relevant data from the 28-day study. q-PCR was used to detect CD19x22 CAR-T DNA copy numbers in various tissues and whole blood, with the lower limit of quantification of 100 copies / μg DNA. q-PCR results showed that after a single dose of CD19x22 CAR-T was administered via tail vein injection to NCG tumor-bearing mice, the CAR-T DNA content in total tissue DNA increased over time, and the drug, CAR-T cells, was distributed throughout the body. By day 7, CAR-T cells had distributed to macroscopic tissues with high blood flow, and by 28 days after administration, CAR-T DNA was detectable in all tissues.

[0093] CAR-T cells (CD3 + , CD22 antibody + ) and tumor cells (CD19 +) in peripheral blood up to 28 days after administration. + ) was low and barely detectable, but CAR-T cells (CD3 + , CD22 antibody + ) increasingly increased over time, as shown in Figure 20.

[0094] Example 9 Exploratory Clinical Study Exploratory clinical studies are being conducted on CD19x22 CAR-T cell therapy products to investigate the safety and initial efficacy of CAR-T cells modified with this sequence in the treatment of B-cell non-Hodgkin's lymphoma (B-NHL) and B-cell acute lymphoblastic leukemia (B-ALL), as well as to explore their human pharmacokinetic (PK) characteristics. This study adhered to GCP principles in terms of the researchers and research institutions, study design, rationale review and informed consent process, subject selection, adverse event reporting and handling, study data collection and statistical analysis, etc. The effective cells of CAR-T cells are CD3 + CAR + The unit of return dose is CD3 + CAR + This study used a dose-escalation design principle, with the treatment dose of CAR-T cells ranging from 1.0 × 10 cells / kg in subjects with B-cell non-Hodgkin lymphoma (B-NHL). 6 pieces CD3 + CAR + cells / kg, 2.0×10 6 pieces CD3 + CAR + cells / kg and 3.0 × 10 6 pieces CD3 + CAR + The B-ALL subjects were divided into three dose groups of 0.5 × 10 cells / kg. 6 pieces CD3 + CAR + cells / kg and 1.0 × 10 6 pieces CD3 + CAR + The subjects were divided into two dose groups: 1000 cells / kg.

[0095] As of June 2021, 11 subjects (numbered 01-001, 01-003, 01-005, 01-006, 01-009, 02-001, 01-013, 01-014, 01-011, 01-012, and 01-016) had received CAR-T cell re-infusion, including eight subjects with B-cell non-Hodgkin's lymphoma (B-NHL) and three subjects with B-ALL. These 11 subjects achieved an overall response rate (ORR) of 81.8% (9 / 11) and a complete response (CR) rate of 72.7% (8 / 11) eight months after receiving CAR-T cell re-infusion. The results are shown in Figure 21.

[0096] Example 10: Analysis of pharmacokinetics in subjects Using the R 3.6.2 software PKNCA pack, CARs in the peripheral blood of 11 subjects who received CAR-T cell re-transfer described in Example 9 were further analyzed, and PK-related parameters were calculated. The results are shown in Table 2. max ) represents the highest blood concentration at which the DNA copy number reaches, and the peak time (T max ) indicates that the DNA copy number is C max represents the time required to reach the target, and AUC 0-28 represents the area under the concentration-time curve from day 0 to day 28, and AUC 0-last represents the area under the concentration-time curve from day 0 to the last observation time.

[0097] [Table 2] The results showed that B-NHL subjects had a 1.0 × 10 6 pieces CD3 + CAR + cells / kg, 2.0×10 6 pieces CD3 + CAR + cells / kg and 3.0 × 10 6 pieces CD3 + CAR +After receiving the cells in the cells / kg dose group, the median times to peak DNA copy numbers were 21, 12.5, and 14 days, respectively, with peak median values ​​of 70200.0 copies / μg DNA, 35087.0 copies / μg DNA, and 59875.0 copies / μg DNA, respectively, and peak geometric mean values ​​of 70200 copies / μg DNA, 34586.2 copies / μg DNA, and 50398.6 copies / μg DNA, respectively. 6 pieces CD3 + CAR + cells / kg and 1.0 × 10 6 pieces CD3 + CAR + After receiving the cells for the cells / kg dose group, the median times to peak DNA copy numbers were 21.0 days and 12.5 days, the peak median values ​​were 152,137.0 copies / μg DNA and 73,474.5 copies / μg DNA, and the peak geometric means were 152,137.0 copies / μg DNA and 68,042.6 copies / μg DNA.

[0098] Below are listed some amino acid and nucleic acid sequences that are described in the present specification and drawings. CD19-62 VH nucleic acid sequence (SEQ ID NO:1): ATGGCCGAAGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGGTCTGGATACAGCTTTACCAACTCCTGGATCGGATGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGACTCATTTACCCTGATGACTCTGATACCAGATACAG CCCATCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAGCGCCATCAACACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGCGCCAGTCTACCTACATCTACGGTGGTTACTACGATACCTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCACD19-62 VH protein sequence (SEQ ID NO: 2) MAEVQLVQSGAEVKKPGESLKISCKGSGYSFTNSWIGWVRQMPGKGLEWMGLIYPDDSDTRYSPSFQGQVTISADSAINTAYLQWSSLKASDTAMYYCARQSTYIYGGYYDTWGQGTLVTVSSCD19-62 VL nucleic acid sequence (SEQ ID NO: 3) CAGTCTGTCGTGACGCAGCCGCCCTCAGTGTCTGGGGCCCCAGGGCAGAGGGTCACCATCTCCTGCACTGGGAGCAGCTCCAACATCGGGGCAGGTTATGATGTACACTGGTACCAGCAACTTCCAGGAACAGCCCCCAAACTCCTCATCTATGAGAACACCAATCGGC CCTCAGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCACTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCCAGTCCTATGACAGCAGCCTGAGTGGTTGGAGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGT CD19-62 VL protein sequence (SEQ ID NO: 4) QSVVTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYENTNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGRVRVFGGGTKLTVLG CD19-78 VL nucleic acid sequence (SEQ ID NO: 5) CAGGCTGTGCTGACTCAGCCACCCTCGGTGTCTGAAGCCCCCAGGCAGAGGGTCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAGCTGGTACCAGCAGCTCCCAGGAAAGGCTCCCAAACTCCTCATCTATTATGATGATCTGCTCC CCTCAGGGTCTCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCAGTCTGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGGACCAAGGTCACCGTCCTAGGT CD19-78 VL protein sequence (SEQ ID NO: 6) QAVLTQPPSVSEAPRQRVTISCSSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLG CD19-78 VH nucleic acid sequence (SEQ ID NO:7) GAGGTGCAGCTGGTGCAGTCTGGAGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGGCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTGGATGGGGATCATCTATCCTGGTGACTCTGATACCAGATACA GCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGCCGACAAGTCCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCGGACACCGCCATGTATTACTGTGCGCGCTGTCTTACTCTTGGTCTTCTTGGTACTGGGATTTCTGGGGTCAAGGTACTCTGGTGACCGTCTCCTCA CD19-78 VH protein sequence (SEQ ID NO: 8) EVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSS CD22-80 VH nucleic acid sequence (SEQ ID NO: 9) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGGGACCCTGTCCCTCACCTGCGCTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGTTGGGTCCGCCAGCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCTATCATAGTGGGAGCACCAACT ACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACAAGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCGGTGTACTACTGCGCCAGACTTCCTGGATACGAGTCAGCTTTCGACATATGGGGTCAGGGTACAATGGTCACCGTCAGCTCA CD22-80 VH protein sequence (SEQ ID NO: 10) QVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSS CD22-80 VL nucleic acid sequence (SEQ ID NO: 11) GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAGCTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGGTGCATCCAGC AGGGCCACTGGCATCCCAGACAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGAAGATTTTGCAGTGTATTACTGTCAGCAGGGCCGGACTCTTCCCTTACACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA CD22-80 VL protein sequence (SEQ ID NO: 12) EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIK CD22-28 VH nucleic acid sequence (SEQ ID NO: 13) CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTACTACTGGAGCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAGTGGATTGGGCGTATCTATACCAGTGGGAGCACCAACT ACAACCCCTCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCGGTGTACTACTGCGCCAGAGACTTGTACAGAGATGGAATGGACGTATGGGGCCAGGGAACAACTGTCACCGTCAGCTCA CD22-28 VH protein sequence (SEQ ID NO: 14) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPAGKGLEWIGRIYTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDLYRDGMDVWGQGTTVTVSS CD22-28 VL nucleic acid sequence (SEQ ID NO: 15) GACATCCAGATGACCCAGTCTCCTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTTGGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTCCGATGCCTCCAGTT TGGAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTGCAACTTATTACTGCCAGCAGGCCAATACCTACTCTCCTACTTTTGGCGGAGGGACCAAGGTTGAGATCAAA CD22-28 VL protein sequence (SEQ ID NO: 16) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLISDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQANTYSPTFGGGTKVEIK Linker 1 nucleic acid sequence (SEQ ID NO: 17) GGTGGTGGTGGTAGCGGCGGCGGCGGCTCTGGTGGTGGTGGATCC Linker 1 protein sequence (SEQ ID NO: 18) GGGGSGGGGSGGGGS Linker 2 nucleic acid sequence (SEQ ID NO: 19) GGCGGAGGTGGGTCC Linker 2 protein sequence (SEQ ID NO: 20) GGGGS Linker 3 nucleic acid sequence (SEQ ID NO: 21) GGCGGAGGTGGGTCCGGTGGCGGGGGAAGCGGAGGCGGAGGGAGCGGAGGAGGGGGATCTGGAGGCGGTGGGTCT Linker 3 protein sequence (SEQ ID NO: 22) GGGGSGGGGSGGGGSGGGGSGGGGS Linker 4 nucleic acid sequence (SEQ ID NO: 23) GGCAGCACCAGCGGCTCGGGACAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGA Linker 4 protein sequence (SEQ ID NO: 24) GSTSGSGKPGSGEGSTKG CD8a hinge nucleic acid sequence (SEQ ID NO: 25) ACTACTACCCCTGCACCTAGGCCTCCCACCCCAGCCCCAACAATCGCCAGCCAGCCTCTGTCTCTGCGGCCCGAAGCCTGTAGACCTGCTGCCGGCGGAGCCGTGCACACCAGAGGCCTGGACTTCGCCTGCGAC CD8a hinge protein sequence ((SEQ ID NO: 26)) TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD CD8a TM nucleic acid sequence (SEQ ID NO: 27) ATCTACATCTGGGCCCCTCTGGCCGGCACCTGTGGCGTGCTGCTGCTGAGCCTGGTGATCACCCTGTACTGC CD8a TM protein sequence (SEQ ID NO: 28) IYIWAPLAGTCGVLLLSLVITLYC 4-1BB intracellular domain (IC) nucleic acid sequence (SEQ ID NO: 29) AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG 4-1BB intracellular domain (IC) sequence (SEQ ID NO: 30) KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD3z intracellular signaling domain nucleic acid sequence (SEQ ID NO: 31) AGAGTGAAGTTCAGCAGATCCGCCGACGCCCCTGCCTACCAGCAGGGACAGAACCAGCTGTACAACGAGCTGAACCTGGGCAGACGGGAAGAGTACGACGTGCTGGACAAGCGGAGAGGCCGGGACCCCGAGATGGGCGGAAAGCCCAGACGGAAGAACCCCCAGGAA GGCCTGTATAACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGCGAGCGGAGGCGCGGCAAGGGCCACGATGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCAGA CD3z intracellular signal domain sequence (SEQ ID NO: 32) RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR T2A nucleic acid sequence (SEQ ID NO: 33) GAGGGAAGGGGCAGCTTATTAACATGTGGCGATGTGGAAGAGAACCCCGGTCCC T2A protein sequence (SEQ ID NO: 34) EGRGSLLTCGDVEENPGP CSF2RA signal nucleic acid sequence (SEQ ID NO: 35) ATGCTGCTGCTCGTGACCTCTTTACTGTTATGTGAGCTGCCCCACCCCGCTTTTTTACTGATCCCT CSF2RA signal protein sequence (SEQ ID NO: 36) MLLLVTSLLLCELPHPAFLLIP tEGFR nucleic acid sequence (SEQ ID NO: 37) tEGFR protein sequence (SEQ ID NO: 38) RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISGDLHILPVAFRGDSFTHTPPLDPQELDILKTVKEITGFLLIQAWPENRTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITSLGLRKEISDGDVIISGNKNLCYANTINWKKLFGTSGQKTKIISNRGENSCK ATGQVCHALCSPEGCWGPEPRDCVSCRNVSRGRECVDKCNLLEGEPREFVENSECIQCHPECLPQAMNITCTGRGPDNCIQCAHYIDGPHCVKTCPAGVMGENNTLVWKYADAGHVCHLCHPNCTYGCTGPGLEGCPTNGPKIPSIATGMVGALLLLLVVALGIGLFM 1# CAR nucleic acid sequence (SEQ ID NO: 39) 1# CAR protein sequence (SEQ ID NO: 40) * 2# CAR nucleic acid sequence (SEQ ID NO: 41) 2# CAR protein sequence (SEQ ID NO: 42) * 3# CAR nucleic acid sequence (SEQ ID NO: 43) 3# CAR protein sequence (SEQ ID NO: 44) * 4# CAR nucleic acid sequence (SEQ ID NO: 45) 4# CAR protein sequence (SEQ ID NO: 46) * 5# CAR nucleic acid sequence (SEQ ID NO: 47) 5# CAR protein sequence (SEQ ID NO: 48) * 6# CAR nucleic acid sequence (SEQ ID NO: 49) 6# CAR protein sequence (SEQ ID NO: 50) * 7# CAR nucleic acid sequence (SEQ ID NO: 51) 7# CAR protein sequence (SEQ ID NO: 52) * 8# CAR nucleic acid sequence (SEQ ID NO: 53) 8# CAR protein sequence (SEQ ID NO: 54) * 9# CAR nucleic acid sequence (SEQ ID NO: 55) 9# CAR protein sequence (SEQ ID NO: 56) * 10# CAR nucleic acid sequence (SEQ ID NO: 57) 10# CAR protein sequence (SEQ ID NO: 58) * 11# CAR nucleic acid sequence (SEQ ID NO: 59) 11# CAR protein sequence (SEQ ID NO: 60) * 12# CAR nucleic acid sequence (SEQ ID NO: 61) 12# CAR protein sequence (SEQ ID NO: 62) * PXL1419 CAR nucleic acid sequence (SEQ ID NO: 63) PXL1419 CAR protein sequence (SEQ ID NO: 64) MALPVTALLLPLALLLHAARPQAVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLGGGGGSQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWI GEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSSGSTSGSGKPGSGEGSTKGEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYT FGGGTKVEIKGGGGSEVQLVQSGAEVKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR PXL1435 CAR nucleic acid sequence (SEQ ID NO: 65) PXL1435 CAR protein sequence (SEQ ID NO: 66) MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIKGSTSGSGKPGSGEGSTKGQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARL PGYESAFDIWGQGTMVTVSSGGGGSQAVLTQPPSVSEAPRQRVTISCSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLGFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR PXL1436 CAR nucleic acid sequence (SEQ ID NO: 67) PXL1436 CAR protein sequence (SEQ ID NO: 68) MALPVTALLLPLALLLHAARPEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIKGGGGSEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIY PGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARLSYSWSSWYWDFWGQGTLVTVSSGSTSGSGKPGSGEGSTKGQAVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVSWYQQLPGKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGW VFGGGTKVTVLGGGGGSQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR PXL1437 CAR nucleic acid sequence (SEQ ID NO: 69) PXL1437 CAR protein sequence (SEQ ID NO: 70) MALPVTALLLPLALLLHAARPQVQLQESGPGLVKPSGTLSLTCAVSGGSISSSNWWSWVRQPPGKGLEWIGEIYHSGSTNYNPSLKSRVTISVDKSKNQFSLKLSSVTAADTAVYYCARLPGYESAFDIWGQGTMVTVSSGGGGSQAVLTQPPSVSEAPRQRVTISSCSGSSSNIGNNAVSWYQQLP GKAPKLLIYYDDLLPSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGWVFGGGTKVTVLGGSTSGSKPGSGEGSTKGEVQLVQSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCAR LSYSWSSWYWDFWGQGTLVTVSSGGGGSEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQAGLFPYTFGGGTKVEIKFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL DFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Claims

1. A bispecific chimeric antigen receptor comprising an extracellular antigen-binding domain comprising the heavy chain variable region and light chain variable region of an anti-CD19 antibody and the heavy chain variable region and light chain variable region of an anti-CD22 antibody, The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD19 antibody are A heavy chain variable region sequence shown in SEQ ID NO: 2 and a light chain variable region sequence shown in SEQ ID NO: 4, and A heavy chain variable region sequence shown in SEQ ID NO: 8 and a light chain variable region sequence shown in SEQ ID NO: 6, is selected from any combination of The amino acid sequences of the heavy chain variable region and the light chain variable region of the anti-CD22 antibody are A heavy chain variable region sequence shown in SEQ ID NO: 10 and a light chain variable region sequence shown in SEQ ID NO: 12, and A heavy chain variable region sequence shown in SEQ ID NO: 14 and a light chain variable region sequence shown in SEQ ID NO: 16, A bispecific chimeric antigen receptor that targets CD19 and CD22, selected from any combination of:

2. the heavy chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 2, the light chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 4, the heavy chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO: 10, and the light chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO: 12; or 2. The bispecific chimeric antigen receptor of claim 1 , wherein the heavy chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 2, the light chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 4, the heavy chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO: 14, and the light chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO:

16.

3. 2. The bispecific chimeric antigen receptor of claim 1 , wherein the heavy chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 8, the light chain variable region of the anti-CD19 antibody has the sequence shown in SEQ ID NO: 6, the heavy chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO: 10, and the light chain variable region of the anti-CD22 antibody has the sequence shown in SEQ ID NO:

12.

4. The heavy and light chain variable regions of the anti-CD19 antibody and the heavy and light chain variable regions of the anti-CD22 antibody are arranged in the extracellular antigen-binding domain in the following order from amino terminus to carboxy terminus: a light chain variable region of the anti-CD19 antibody, a heavy chain variable region of the anti-CD22 antibody, a light chain variable region of the anti-CD22 antibody, and a heavy chain variable region of the anti-CD19 antibody; a heavy chain variable region of the anti-CD19 antibody, a light chain variable region of the anti-CD22 antibody, a heavy chain variable region of the anti-CD22 antibody, and a light chain variable region of the anti-CD19 antibody; a light chain variable region of the anti-CD22 antibody, a heavy chain variable region of the anti-CD19 antibody, a light chain variable region of the anti-CD19 antibody, and a heavy chain variable region of the anti-CD22 antibody; or a heavy chain variable region of the anti-CD22 antibody, a light chain variable region of the anti-CD19 antibody, a heavy chain variable region of the anti-CD19 antibody, and a light chain variable region of the anti-CD22 antibody; 4. The bispecific chimeric antigen receptor of claim 1 , wherein

5. The extracellular antigen-binding domain comprises, in order from the amino terminus to the carboxy terminus: a light chain variable region of the anti-CD19 antibody, a first linker, a heavy chain variable region of the anti-CD22 antibody, a second linker, a light chain variable region of the anti-CD22 antibody, a third linker, and a heavy chain variable region of the anti-CD19 antibody; a heavy chain variable region of the anti-CD19 antibody, a first linker, a light chain variable region of the anti-CD22 antibody, a second linker, a heavy chain variable region of the anti-CD22 antibody, a third linker, and a light chain variable region of the anti-CD19 antibody; a light chain variable region of the anti-CD22 antibody, a first linker, a heavy chain variable region of the anti-CD19 antibody, a second linker, a light chain variable region of the anti-CD19 antibody, a third linker, and a heavy chain variable region of the anti-CD22 antibody; or a heavy chain variable region of the anti-CD22 antibody, a first linker, a light chain variable region of the anti-CD19 antibody, a second linker, a heavy chain variable region of the anti-CD19 antibody, a third linker, and a light chain variable region of the anti-CD22 antibody; 5. The bispecific chimeric antigen receptor of claim 1 , wherein the first linker and the third linker have the amino acid sequence set forth in SEQ ID NO: 20, and the second linker has the amino acid sequence set forth in SEQ ID NO:

24.

6. The bispecific chimeric antigen receptor according to any one of claims 1 to 5, comprising, in order from the amino terminus to the carboxy terminus, a signal peptide sequence, the extracellular antigen-binding domain, a hinge domain, a transmembrane region, and an intracellular signaling domain, wherein the intracellular signaling domain comprises, from the amino terminus to the carboxy terminus, a fragment derived from a 4-1BB molecule and a fragment derived from a CD3z molecule.

7. the signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 36, The hinge domain has the amino acid sequence set forth in SEQ ID NO: 26, The transmembrane domain has the amino acid sequence shown in SEQ ID NO: 28, The fragment derived from the 4-1BB molecule has the amino acid sequence set forth in SEQ ID NO: 30; and The fragment derived from the CD3z molecule has the amino acid sequence set forth in SEQ ID NO:

32.

7. The bispecific chimeric antigen receptor of claim 1 .

8. 8. The bispecific chimeric antigen receptor of claim 1, comprising the amino acid sequence set forth in SEQ ID NO: 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68 or 70.

9. A nucleic acid molecule encoding the bispecific chimeric antigen receptor of any one of claims 1 to 8.

10. 10. The nucleic acid molecule of claim 9, comprising the nucleotide sequence set forth in SEQ ID NO: 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67 or 69.

11. An expression vector comprising the nucleic acid molecule of claim 9 or 10.

12. 12. A host cell expressing the bispecific chimeric antigen receptor of any one of claims 1 to 8 or comprising the expression vector of claim 11.

13. The host cell according to claim 12, which is an immune cell, preferably a T cell or an NK cell.

14. 14. Use of a bispecific chimeric antigen receptor according to any one of claims 1 to 8, an expression vector according to claim 11, or a host cell according to claim 12 or 13 in the preparation of a medicament for treating cancer.

15. 15. The use of claim 14, wherein the cancer is a B-cell related cancer.

16. The use according to claim 14 or 15, wherein the cancer is B-cell non-Hodgkin's lymphoma (B-NHL) or B-cell acute lymphoblastic leukemia (B-ALL).

17. 17. The use according to any one of claims 14 to 16, wherein the cancer expresses CD19 and / or CD22.

18. 14. The bispecific chimeric antigen receptor of any one of claims 1 to 8, the expression vector of claim 11, or the host cell of claim 12 or 13 for use in treating cancer in a patient.

19. 19. The bispecific chimeric antigen receptor, expression vector, or host cell for use according to claim 18, wherein the cancer is a B-cell related cancer.

20. The bispecific chimeric antigen receptor, expression vector, or host cell for use according to claim 18 or 19, wherein the cancer is B-NHL or B-ALL.

21. 21. The bispecific chimeric antigen receptor, expression vector, or host cell for use according to any one of claims 18 to 20, wherein the cancer expresses CD19 and / or CD22.

22. The host cells were cultured at 0.5×10 6 3 x 10 host cells / kg patient weight 6 22. The host cell for use according to any one of claims 18 to 21, administered to the patient at a dose of 1 host cell / kg patient body weight.

23. The patient is a B-NHL patient, and the host cells are added to the patient at a concentration of 1×10 6 3 x 10 host cells / kg patient weight 6 23. The host cell for use according to any one of claims 18 to 22, administered to the patient at a dose of 1 host cell / kg patient body weight.

24. The patient is a B-ALL patient, and the host cells are 0.5×10 6 1 x 10 host cells / kg patient weight 6 24. The host cell for use according to any one of claims 18 to 23, administered to the patient at a dose of 1 host cell / kg patient body weight.

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