Preparation method and application of targeted FLT3 gene editing universal CAR-T cell

Abstract

The invention discloses a preparation method and application of a targeted FLT3 gene editing universal type CAR-T cell. The anti-FLT3 antibody or the antigen binding fragment thereof provided by the invention comprises CDRs in a heavy chain variable region, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 6, 12 or 17, and the amino acid sequence of a light chain variable region is as shown in SEQ ID NO: 7. The target FLT3 antibody or the antigen binding fragment thereof is used as an antigen binding structural domain to construct a chimeric antigen receptor, and a universal CAR-T cell prepared from T cells separated from healthy donor peripheral blood has obvious killing activity on FLT3 positive tumors such as acute myelogenous leukemia, and can be applied to preparation of drugs for preventing and treating the FLT3 positive tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly, to a method for preparing and applying a universal CAR-T cell targeting the FLT3 gene editing. Background Art

[0002] FLT3 (Fms-like tyrosine kinase 3) is a receptor tyrosine kinase highly expressed in various hematological malignancies, especially playing a key role in acute myeloid leukemia (AML). FLT3 mutations, especially FLT3-ITD (intron insertion mutation), have a relatively high incidence in AML patients and are associated with poor prognosis. Therefore, FLT3 is considered an important target in AML treatment. Treatment methods targeting FLT3 include small molecule inhibitors, monoclonal antibodies, and immunotherapy, etc.

[0003] CAR-T cell therapy (chimeric antigen receptor T cell therapy), as a revolutionary cell immunotherapy, has made remarkable progress in the field of tumor treatment. CAR-T cells can be engineered to express specific antigen receptors on T cells, thereby precisely recognizing and killing tumor cells. However, traditional autologous CAR-T cell therapy still has many limitations, such as high preparation cost, fewer T cells in AML patients with multi-line treatment or decreased quality due to impaired T cell function after chemotherapy, T cell dysfunction in patients, and a long manufacturing cycle, which causes patients to miss the best treatment time. With the rapid development of various gene editing technologies such as ZFN, TALEN, and CRISPR / Cas9, gene editing technologies are used to knockout T cell receptors (TCR), HLA molecule β2-microglobulin (B2M), and CD52 on the surface of CAR-T cells to minimize the risk of GVHD. The development of this universal CAR-T (UCAR-T) cell therapy can overcome most of the disadvantages of autologous CAR-T cell therapy, such as large-scale and industrial manufacturing with standardized processes, and cost reduction. A large number of CAR-T cells can be prepared from a single donor. Allogeneic CAR-T cells can be cryopreserved, enabling patients to receive treatment immediately without missing the best treatment opportunity. It simplifies the process of introducing multiple modifications in a single cell product and the standardization of CAR-T cell products based on donor selection and processing.

[0004] Currently, there is a lack of high-affinity antibodies against FLT3 and universal CAR-T cells targeting the FLT3 gene with high anti-tumor activity in the market.

[0005] In view of this, the present invention is specifically proposed. Summary of the Invention

[0006] The object of the present invention is to provide a method for preparing a universal CAR-T cell targeting the FLT3 gene and its application.

[0007] The present invention is implemented as follows:

[0008] In a first aspect, an embodiment of the present invention provides an antibody or an antigen-binding fragment thereof, which includes: HCDR1, HCDR2, and HCDR3 in the heavy-chain variable region and / or LCDR1, LCDR2, and LCDR3 in the light-chain variable region;

[0009] The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO: 6, 12, or 17, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO: 7.

[0010] In a second aspect, an embodiment of the present invention provides an isolated nucleic acid encoding the anti-FLT3 antibody or an antigen-binding fragment thereof as described in any of the foregoing embodiments.

[0011] In a third aspect, an embodiment of the present invention provides a recombinant vector containing the isolated nucleic acid as described in the foregoing embodiments.

[0012] In a fourth aspect, an embodiment of the present invention provides a host cell containing the recombinant vector as described in the foregoing embodiments.

[0013] In a fifth aspect, an embodiment of the present invention provides a method for preparing an anti-FLT3 antibody or an antigen-binding fragment thereof, which includes: culturing the host cell as described in the foregoing embodiments to obtain the anti-FLT3 antibody or an antigen-binding fragment thereof.

[0014] In a sixth aspect, an embodiment of the present invention provides a chimeric antigen receptor, and the antigen-binding domain of the chimeric antigen receptor includes the anti-FLT3 antibody or an antigen-binding fragment thereof as described in the foregoing embodiments.

[0015] In a seventh aspect, an embodiment of the present invention provides a CAR-T cell, which includes the chimeric antigen receptor as described in the foregoing embodiments.

[0016] In an eighth aspect, an embodiment of the present invention provides the application of the anti-FLT3 antibody or an antigen-binding fragment thereof as described in the foregoing embodiments, or the isolated nucleic acid as described in the foregoing embodiments, or the recombinant vector as described in the foregoing embodiments, or the host cell as described in the foregoing embodiments, or the chimeric antigen receptor as described in the foregoing embodiments, or the CAR-T cell as described in the foregoing embodiments in the preparation of products for preventing or treating tumors.

[0017] In a ninth aspect, an embodiment of the present invention provides a cell injection solution, which includes an anti-FLT3 antibody or its antigen-binding fragment as described in the foregoing embodiments, or the isolated nucleic acid as described in the foregoing embodiments, or the recombinant vector as described in the foregoing embodiments, or the host cell as described in the foregoing embodiments, or the chimeric antigen receptor as described in the foregoing embodiments, or the CAR-T cell as described in the foregoing embodiments.

[0018] The present invention has the following beneficial effects:

[0019] The anti-FLT3 antibody or its antigen-binding fragment screened by the mouse monoclonal antibody platform of the present invention can specifically bind to the FLT3 antigen and has good affinity.

[0020] In addition, the screened antibody or its antigen-binding fragment is used as an antigen-binding domain to construct a universal chimeric antigen receptor, and allogeneic FLT3-CAR-T cells are isolated and prepared from the peripheral blood of healthy donors, which have obvious killing activity against FLT3-positive tumor cell lines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0022] Figure 1 This is for detecting the binding of hybridoma supernatant to FLT3 antigen by ELISA in the embodiments of the present invention;

[0023] Figure 2 This is for identifying the reactivity of the recombinant scFv single-chain antibody with FLT3 antigen by ELISA in the embodiments of the present invention;

[0024] Figure 3 This is for detecting the binding effect of the recombinant scFv single-chain antibody to HeLa cells overexpressing FLT3 by immunofluorescence (IFA) in the embodiments of the present invention;

[0025] Figure 4 This is for identifying the binding of the recombinant scFv single-chain antibody to FLT3-positive tumor cells THP-1 by flow cytometry in the embodiments of the present invention;

[0026] Figure 5 This is for evaluating the in vivo anti-tumor activity of FLT3-CAR-γδT cells using a NCG mouse xenograft model. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Those not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments not indicated with the manufacturer are all conventional products that can be obtained by purchasing in the market.

[0028] On the one hand, embodiments of the present invention provide an anti-FLT3 antibody or an antigen-binding fragment thereof, which includes: HCDR1, HCDR2, and HCDR3 in the heavy-chain variable region and / or LCDR1, LCDR2, and LCDR3 in the light-chain variable region;

[0029] The amino acid sequence of the heavy-chain variable region is as shown in SEQ ID NO: 6, 12, or 17, and the amino acid sequence of the light-chain variable region is as shown in SEQ ID NO: 7.

[0030] In some embodiments, the HCDR1, HCDR2, and HCDR3 and / or LCDR1, LCDR2, and LCDR3 are defined by any one of the Kabat, Chothia, AbM, Contact, and IMGT schemes or a combination of multiple schemes.

[0031] In some embodiments, the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are successively as shown in SEQ ID No. 1-3, 9-11, or 14-16, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are successively as shown in SEQ ID No. 4, AAS, and SEQ ID No. 5.

[0032] In some embodiments, the heavy-chain variable region and the light-chain variable region further include framework regions.

[0033] In the present invention, the "framework region" or "FR" region refers to the region other than the CDR in the heavy-chain variable region of the antibody; the heavy-chain framework region can be further divided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs. Among them, the heavy-chain framework region can be further divided into adjacent regions separated by CDRs, including the HFR1, HFR2, HFR3, and HFR4 framework regions. The heavy-chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (ranging from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4.

[0034] In some embodiments, the amino acid sequences of the heavy-chain variable region and the light-chain variable region are successively as shown in SEQ IDNO: 6-7;

[0035] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are as shown in SEQ ID NO: 12 and 7 in sequence;

[0036] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are as shown in SEQ ID NO: 17 and 7 in sequence;

[0037] In some embodiments, the antibody or its antigen-binding fragment further comprises a constant region.

[0038] In some embodiments, the constant region is selected from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

[0039] In some embodiments, the species origin of the constant region is bovine, equine, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

[0040] In some embodiments, the antibody is selected from any one of monoclonal antibody, polyclonal antibody, multispecific antibody, murine antibody, chimeric antibody and full-length antibody.

[0041] In some embodiments, the antigen-binding fragment is selected from any one of F(ab’) 2 , Fab’, Fab, Fv and scFv of the antibody; an “antigen-binding fragment” is a part of a complete antibody, and the part specifically binds to the antigen bound by the complete antibody. Those skilled in the art can easily understand from the content recorded in the present invention that antigen-binding fragments can be prepared by methods known in the art, for example, by enzymatic digestion methods (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds, and can also be obtained by recombinant genetic techniques or by an automated peptide synthesizer (such as an automated peptide synthesizer of Applied BioSystems).

[0042] In some embodiments, when the antigen-binding fragment is scFv, the amino acid sequence of the scFv is as shown in SEQ ID NO: 8, 13 or 18.

[0043] On the other hand, an embodiment of the present invention provides an isolated nucleic acid encoding the anti-FLT3 antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0044] On the other hand, an embodiment of the present invention provides a recombinant vector containing the isolated nucleic acid as described in any of the foregoing embodiments.

[0045] The recombinant vector is an expression vector or a cloning vector, preferably an expression vector, and can refer to any recombinant polynucleotide construct that can directly or indirectly (such as packaged into a virus) introduce a target DNA fragment into a host cell by means of transformation, transfection or transduction for the expression of a target gene. One type of vector is a plasmid, that is, a circular double-stranded DNA molecule, to which a target DNA fragment can be ligated into the plasmid ring. Another type of vector is a viral vector, which can ligate and package a target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, lentivirus, oncolytic virus). After these vectors enter the host cell, the expression of the target gene can be carried out.

[0046] On the other hand, embodiments of the present invention provide a host cell containing the recombinant vector as described in any of the foregoing embodiments.

[0047] Specifically, the host cell includes at least one of a prokaryotic host cell, a eukaryotic host cell, and a bacteriophage. The prokaryotic host cell can be Escherichia coli, Streptomyces, or Bacillus subtilis, etc. The eukaryotic host cell can be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6, Saccharomyces cerevisiae, Pichia pastoris, Hansenula yeast, Candida yeast, some insect cells, and plant cells. 293 series cells, Per6 cells, and CHO cells are common mammalian cells used for the production of antibodies or recombinant proteins and are well-known to those of ordinary skill in the art.

[0048] On the other hand, embodiments of the present invention provide a method for preparing an anti-FLT3 antibody or an antigen-binding fragment thereof, which includes: culturing the host cell as described in any of the foregoing embodiments to obtain the anti-FLT3 antibody or an antigen-binding fragment thereof.

[0049] Specifically, the present invention does not specifically limit the culture conditions of the host cell, and culture conditions that can enable the host cell to express and produce the anti-FLT3 antibody or an antigen-binding fragment thereof can be obtained based on conventional technical knowledge.

[0050] On the other hand, embodiments of the present invention provide a chimeric antigen receptor, and the antigen-binding domain of the chimeric antigen receptor includes the anti-FLT3 antibody or an antigen-binding fragment thereof as described in any of the foregoing embodiments.

[0051] In some embodiments, the chimeric antigen receptor further includes a signal peptide, a hinge region, a transmembrane region, and a signal transduction domain.

[0052] In some embodiments, the signal transduction domain includes CD3ζ.

[0053] In some embodiments, the signal transduction domain further includes the intracellular region of 4-1BB.

[0054] On the other hand, embodiments of the present invention provide a CAR-T cell, which comprises a chimeric antigen receptor as described in any of the foregoing embodiments.

[0055] In some embodiments, the CAR-T cell comprises a universal allogeneic CAR-T cell.

[0056] Autologous CAR-T cell therapy has advantages such as no immune rejection reaction and can persist in the body for a long time. However, autologous CAR-T cell therapy has some limitations, such as high preparation cost, small number of T cells in AML patients with multi-line treatment or impaired T cell function after chemotherapy leading to a decline in its quality, and a long manufacturing cycle, which causes patients to miss the best treatment time. With the rapid development of universal allogeneic cell therapy technology, the development of this universal cell therapy can overcome most of the disadvantages of autologous CAR-T cell therapy, such as large-scale and industrialized manufacturing of standardized processes and reduced costs, and a large number of CAR-T cells can be prepared from a single donor.

[0057] On the other hand, embodiments of the present invention also provide the use of an anti-FLT3 antibody or an antigen-binding fragment thereof as described in any of the foregoing embodiments or a separated nucleic acid as described in any of the foregoing embodiments or a recombinant vector as described in any of the foregoing embodiments or a host cell as described in any of the foregoing embodiments or a chimeric antigen receptor as described in any of the foregoing embodiments or a CAR-T cell as described in any of the foregoing embodiments in the preparation of a product for preventing or treating tumors.

[0058] In some embodiments, the product comprises at least one of immune cells, reagents, reagent kits, drugs, and pharmaceutical compositions;

[0059] In some embodiments, the tumor comprises an FLT3-positive tumor.

[0060] In some embodiments, the tumor comprises FLT3-positive acute myeloid leukemia.

[0061] In addition, embodiments of the present invention also provide a cell injection solution, which comprises an anti-FLT3 antibody or an antigen-binding fragment thereof as described in any of the foregoing embodiments or a separated nucleic acid as described in any of the foregoing embodiments or a recombinant vector as described in any of the foregoing embodiments or a host cell as described in any of the foregoing embodiments or a chimeric antigen receptor as described in any of the foregoing embodiments or a CAR-T cell as described in any of the foregoing embodiments.

[0062] "Treatment" in the present invention includes preventing or alleviating a certain condition, reducing the rate of onset or development of a certain condition, reducing the risk of developing a certain condition, preventing or delaying the development of symptoms associated with a certain condition, reducing or terminating the symptoms associated with a certain condition, producing a complete or partial reversal of a certain condition, curing a certain condition, or a combination of the above.

[0063] For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination of the above.

[0064] The features and properties of the present invention will be further described in detail below in conjunction with embodiments.

[0065] Example 1 Preparation of Recombinant FLT3 Protein

[0066] Using the full-length FLT3 gene (NM_004119.2) as a template, selecting the extracellular region of the FLT3 gene as the immunotargeting fragment, and cloning it into the pcDNA3.1 expression vector carrying a His tag at the C-terminus. Then, it is expressed through the 293 cell expression system, and the recombinant FLT3 protein is purified using the His tag subsequently.

[0067] Example 2 Cell Fusion and Hybridoma Screening

[0068] (1) Animal Immunization

[0069] Using 5-6-week-old female Balb / c mice as the immunized animals, the immunization dose is 100 μg / mouse. For the first immunization, 100 μl of Freund's complete adjuvant (Sigma) is mixed with an equal volume of recombinant FLT3 protein, fully emulsified, and then injected subcutaneously at multiple points. After a 2-week interval, an equal volume of Freund's incomplete adjuvant (Sigma) is mixed with the recombinant protein, fully emulsified, and then injected subcutaneously at multiple points. The booster immunization is carried out 4 times in total. On the 10th day after the last booster immunization, blood is collected to detect the antibody titer of the mice. Three days before cell fusion, 100 μg of recombinant protein is given as an intraperitoneal boost.

[0070] (2) Cell Fusion and Hybridoma Screening

[0071] Under sterile conditions, take the spleen of the mice, prepare a suspension rich in B cells, and perform cell fusion with SP2 / 0 according to the classical PEG (Sigma) method. The fused cells are resuspended in HAT medium for culture. On the 5th and 10th days after fusion, semi-medium replacement culture is carried out using fresh HAT medium. From the 11th to 15th days after fusion, ELISA is used to screen for positive clones.

[0072] The specific steps are as follows: ELISA screening was carried out using a 96-well plate. The FLT3 recombinant protein was coated onto the bottom of the well at a dose of 100 ng / well overnight at 4 °C. The HRP-conjugated anti-mouse IgG antibody and chemiluminescent reagent (Beyotime Biotechnology Company) were used for color development, and the absorbance was read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0073] According to the above ELISA analysis results, three optimal hybridoma clones (named 8#, 9#, and 11# respectively) Figure 1 ) were finally identified for subsequent experiments such as sequence cloning and affinity analysis.

[0074] (3) Sequencing analysis of specific antibodies

[0075] Cloning of the variable region sequences of hybridoma antibodies: The optimal hybridoma clone cells in the logarithmic growth phase were collected, and RNA was extracted using Trizol (Invitrogen) and reverse-transcribed (PrimeScriptTM Reverse Transcriptase, Takara). The cDNA obtained by reverse transcription was amplified by PCR using the mouse Ig-Primer Set (Novagen) and then sequenced, and finally the variable region sequences of the heavy and light chains were obtained. The CDR sequences of the variable regions of the heavy and light chains and the single-chain antibody sequence (scFv) are shown in Table 1.

[0076] Table 1 CDR sequences and single-chain antibody sequences (scFv) contained in the variable regions of the heavy and light chains of murine monoclonal antibodies

[0077]

[0078]

[0079] Example 3 Expression, purification of single-chain antibody (scFv) and reactivity with antigen

[0080] A single-chain antibody (scFv) expression platform was constructed based on a eukaryotic expression vector. The VH and VL gene fragments of hybridoma clones 8#, 9#, and 11# were respectively linked in series using (G4S)3 linker and constructed into the pcDNA3.1 vector. The light chain sequences VL of the three clones were identical. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE identification showed that the size of the single-chain antibody (scFv) was consistent with the theoretical molecular weight, and the sequence of the single-chain antibody is shown in Table 2.

[0081] Table 2 Nucleotide sequences encoding single-chain antibodies

[0082]

[0083]

[0084] To identify the reactivity of single-chain antibodies (scFv) with antigens, 200 ng / well of FLT3 recombinant protein was pre-coated on an enzyme-linked immunosorbent assay (ELISA) plate, and the plate was blocked after overnight incubation at 4°C. Different amounts of recombinant antibodies (dilution: 10 2 ~10 -5 μg / mL) were added. After adding the secondary antibody, washing, color development, and termination of the reaction, the optical density (OD450) at 450 nm was measured using an ELISA reader, and the binding ability was determined by fitting with a four-parameter non-linear regression curve. The results showed that three single-chain antibodies (scFv) of FLT3 had high specific binding to the FLT3 recombinant protein ( Figure 2 ).

[0085] To identify the reactivity of single-chain antibodies (scFv) with antigens, FLT3 + -HeLa cells were plated in a 96-well plate and cultured overnight. The next day, purified single-chain antibodies (scFv) were added and incubated for 1 h. After washing with PBS, 594-fluorescent-labeled goat anti-human secondary antibody was added and incubated for 1 h. After washing, the results were observed using a fluorescence microscope. The results showed that three single-chain antibodies (scFv) of FLT3 had good specific binding to FLT3 + -HeLa ( Figure 3 ).

[0086] Example 4: Binding analysis of antibodies to endogenously expressed FLT3 in cells

[0087] Flow cytometry analysis was performed on THP-1 human acute myeloid leukemia cell lines that were positive for FLT3 expression. The results showed that THP-1 cells with positive FLT3 expression could be stained almost 100% positive with four FLT3 single-chain antibodies (scFv). This indicated that three FLT3 single-chain antibodies (scFv) could specifically recognize the endogenously expressed FLT3 molecule ( Figure 4 ).

[0088] Example 5: Determination of the affinity of FLT3 antibodies

[0089] The affinity of single-chain antibodies (scFv) was verified by surface plasmon resonance, and it was determined that the one with the highest binding was 11 # scFv (1.66×10 -13 ), followed by 9 # scFv (4.7×10 -12 ), and 8 # scFv (1.32×10 -11 ). Moreover, the dissociation rates of the three antibodies were also slow. In summary, Biacore data indicated that three single-chain antibodies (scFv) of FLT3 could bind to the human FLT3 antigen, and the specific data detected are shown in Table 3.

[0090] Summary Table of Affinity Data of Antibodies

[0091] Antibody Ka (1 / Ms) kd (1 / s) KD (M) <![CDATA[8 # scFv]]> <![CDATA[5.69×10 7 > <![CDATA[7.53×10 -4 > <![CDATA[1.32×10 -11 > <![CDATA[9 # scFv]]> <![CDATA[7.83×10 5 > <![CDATA[3.68×10 -6 > <![CDATA[4.7×10 -12 > <![CDATA[11 # scFv]]> <![CDATA[2.56×10 9 > <![CDATA[4.25×10 -4 > <![CDATA[1.66×10 -13 >

[0092] Example 6 Construction of Universal CAR-T (UCAR-T) Cells Targeting FLT3

[0093] According to Example 5, an antibody sequence was selected to construct a CAR with the following structure: CD8α signal-peptide-scFv(FLT3)-CD8α hinge-CD28α Tm-4-1BB-CD3ζ-P2A-EGFP. HEK293T cells were used as the cells for lentivirus packaging, and the three-plasmid packaging system (psPAX2, pMD2.G, CAR-T vector) was used for lentivirus packaging. After 48 h, the supernatant virus solution was collected. After ultracentrifugation and concentration, RetroNectin protein was pre-coated and the concentrated virus was added to infect T cells. After 48 h of infection, the transfection efficiency of CAR-T cells was evaluated by flow cytometry to be above 80.0%.

[0094] Using the CRISPR-Cas9 / RNPs electroporation method, sgRNAs targeting TRAC and B2M and Cas9 protein were electroporated into CAR-T cells to prepare universal FLT3 UCAR-T cells. After 72 h of amplification, flow cytometry was used to detect TCR and β-2-Microglobulin expressed by CAR-T cells. The flow cytometry results showed that T cells with the TRAC gene knocked out reached 60.2%, and B2M also reached 58.3%. Subsequently, through magnetic bead sorting, TRAC - / B2M - negative CAR-T cells were obtained.

[0095] Example 7 Anti-Tumor Experiment in a Xenograft Mouse Model

[0096] The in vivo anti-tumor activity of universal UCAR-T targeting FLT3 gene editing (FLT3 UCAR-T cells in Example 6) was evaluated through a xenograft mouse model. The NCG mouse FLT3-positive acute myeloid leukemia model established using the THP-1 cell line was used for evaluation.

[0097] A mouse FLT3-positive AML xenograft tumor model was established by subcutaneous injection of 1×10 6 THP-1-mCherry.ffLuc cells into the right hind limb of NCG mice to verify the in vivo effect of FLT3 UCAR-T. On the 5th day after inoculation, NCG mice were randomly divided into 5 groups of 5 each, and 1×10 were injected via the tail vein respectively7 Mock T cells, 1×10 7 FLT3 UCAR-T (8 # , 9 # , 11 # ) cells, and a control group injected with PBS was set up. The growth status and survival of the mice were observed every day.

[0098] The experimental results showed that the survival of the mice in the three FLT3 UCAR-T groups was significantly higher than that in the Mock T and PBS groups. One-way ANOVA analysis was used for data analysis, where ns indicates no statistical difference, and **** indicates P < 0.0001 ( Figure 5 ).

[0099] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anti-FLT3 antibody or an antigen-binding fragment thereof, characterized in that: It includes: HCDR1, HCDR2 and HCDR3 in the heavy chain variable region and / or LCDR1, LCDR2 and LCDR3 in the light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:6, 12 or 17, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The HCDR1, HCDR2 and HCDR3 and / or LCDR1, LCDR2 and LCDR3 are defined by any one of Kabat, Chothia, AbM, Contact and IMGT or a combination of multiple schemes; Optionally, the amino acid sequences of the HCDR1, HCDR2 and HCDR3 are shown in SEQ ID No. 1 to 3 or 9 to 11 or 14 to 16, and the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No. 4, AAS and SEQ ID No. 5, respectively; Optionally, the heavy chain variable region and the light chain variable region further comprise a framework region; Optionally, the antibody or antigen-binding fragment thereof further comprises a constant region; Optionally, the constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD; Optionally, the species of origin of the constant region is cattle, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human; Optionally, the antibody is selected from any one of a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a murine antibody, a chimeric antibody and a full-length antibody; Optionally, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of an antibody; Optionally, when the antigen-binding fragment is scFv, the amino acid sequence of the scFv is as shown in SEQ ID NO:8, 13 or 18.

3. An isolated nucleic acid, characterized in that It encodes the anti-FLT3 antibody or antigen-binding fragment thereof according to any one of claims 1 or 2.

4. A recombinant vector, characterized in that: It contains the isolated nucleic acid as claimed in claim 3.

5. A host cell, characterized in that It contains the recombinant vector as claimed in claim 4.

6. A method for preparing an anti-FLT3 antibody or an antigen-binding fragment thereof, characterized in that: It includes: Cultivate the host cell according to claim 5 to obtain the anti-FLT3 antibody or antigen-binding fragment thereof.

7. A chimeric antigen receptor, characterized in that The antigen binding domain of the chimeric antigen receptor comprises the anti-FLT3 antibody or antigen binding fragment thereof according to claim 1 or 2; Optionally, the chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region and a signal transduction domain; Optionally, the signal transduction domain comprises CD3ζ; Optionally, the signal transduction domain also includes a 4-1BB intracellular region.

8. A CAR-T cell, characterized in that: It comprises the chimeric antigen receptor as claimed in claim 7.

9. Use of the anti-FLT3 antibody or antigen-binding fragment thereof according to claim 1 or 2, the isolated nucleic acid according to claim 3, the recombinant vector according to claim 4, the host cell according to claim 5, the chimeric antigen receptor according to claim 7, or the CAR-T cell according to claim 8 in the preparation of a product for preventing or treating tumors; Optionally, the product comprises: at least one of an immune cell, a reagent, a kit, a drug, and a pharmaceutical composition; Optionally, the tumor comprises a FLT3-positive tumor; Optionally, the tumor comprises FLT3-positive acute myeloid leukemia.

10. A cell injection solution, characterized in that: It includes the anti-FLT3 antibody or antigen-binding fragment thereof as described in claim 1 or 2, the isolated nucleic acid as described in claim 3, the recombinant vector as described in claim 4, the host cell as described in claim 5, the chimeric antigen receptor as described in claim 7, or the CAR-T cell as described in claim 8.

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