Preparation method and application of universal CAR-T cells targeting FLT3 gene editing

Abstract

The present invention discloses a preparation method and application of a universal CAR-T cell targeting FLT3 gene editing. The anti-FLT3 antibody or antigen-binding fragment thereof provided by the present invention includes CDRs in the heavy 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. The chimeric antigen receptor is constructed using the FLT3-targeting antibody or antigen-binding fragment thereof as the antigen-binding domain, and the universal CAR-T cell prepared using T cells isolated from the peripheral blood of healthy donors has significant killing activity against FLT3-positive tumors such as acute myeloid leukemia, and can be used to prepare drugs for the prevention and treatment of FLT3-positive tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a preparation method and application of universal CAR-T cells targeted to the FLT3 gene editing. Background Art

[0002] FLT3 (Fms-like tyrosine kinase 3) is a receptor tyrosine kinase that is highly expressed in a variety of hematologic malignancies and plays a key role in acute myeloid leukemia (AML). FLT3 mutations, particularly FLT3-ITD (intron insertion mutation), are highly prevalent in AML patients and are associated with a poor prognosis. Therefore, FLT3 is considered an important target in AML treatment. Treatment options targeting FLT3 include small molecule inhibitors, monoclonal antibodies, and immunotherapy.

[0003] CAR-T cell therapy (chimeric antigen receptor T cell therapy), a revolutionary cellular immunotherapy, has made significant progress in cancer treatment. CAR-T cells engineer T cells to express specific antigen receptors, enabling them to precisely recognize and kill tumor cells. However, traditional autologous CAR-T cell therapy still has many limitations, such as high production costs, low T cell numbers in AML patients undergoing multiple lines of treatment, impaired T cell function after chemotherapy, poor quality, and long manufacturing cycles, which can delay optimal treatment. With the rapid development of various gene editing technologies, such as ZFNs, TALENs, and CRISPR / Cas9, gene editing techniques are being used to knock out T cell receptors (TCRs), HLA molecules like β2-microglobulin (B2M), and CD52 on the surface of CAR-T cells to minimize the risk of GVHD. The development of universal CAR-T (UCAR-T) cell therapy could overcome most of the shortcomings of autologous CAR-T cell therapy, including standardized, scalable, and industrialized manufacturing processes, reduced costs, and the ability to produce large numbers of CAR-T cells from a single donor. Allogeneic CAR-T cells can be cryopreserved, allowing patients to receive treatment immediately without missing the optimal treatment window. This simplifies the process of introducing multiple modifications into a single cell product, as well as standardizing donor selection and processing of CAR-T cell products.

[0004] Currently, the market lacks high-affinity antibodies against FLT3 and universal CAR-T cells that target the FLT3 gene and have high anti-tumor activity.

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

[0006] The purpose of the present invention is to provide a preparation method and application of universal CAR-T cells targeting FLT3 gene editing.

[0007] The present invention is achieved in that:

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

[0009] 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.

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

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

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

[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, comprising: culturing the host cell as described in the above embodiment to obtain the anti-FLT3 antibody or the antigen-binding fragment thereof.

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

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

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

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

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

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

[0020] In addition, the screened antibodies or their antigen-binding fragments were used as antigen-binding domains to construct universal chimeric antigen receptors, and allogeneic FLT3-CAR-T cells were prepared using peripheral blood isolated from healthy donors, which had significant 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 briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 The binding between hybridoma supernatant and FLT3 antigen was detected by ELISA in the embodiment of the present invention;

[0023] Figure 2 In the examples of the present invention, ELISA was used to identify the reactivity of the recombinant scFv single-chain antibody with the FLT3 antigen;

[0024] Figure 3 The binding effect of the recombinant scFv single-chain antibody and HeLa cells overexpressing FLT3 was detected by immunofluorescence assay (IFA) in the examples of the present invention;

[0025] Figure 4 In the examples of the present invention, flow cytometry was used to identify the binding of the recombinant scFv single-chain antibody to the FLT3-positive tumor cell THP-1;

[0026] Figure 5 To evaluate the in vivo anti-tumor activity of FLT3-CAR-γδT cells using the NCG mouse xenograft model. DETAILED DESCRIPTION

[0027] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0028] In one aspect, an embodiment of the present invention provides an anti-FLT3 antibody or antigen-binding fragment thereof, comprising: 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 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.

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

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

[0032] In some embodiments, the heavy chain variable region and light chain variable region further comprise a framework region.

[0033] As used herein, the term "framework region" or "FR" refers to the region of the antibody heavy chain variable region excluding the CDRs. The heavy chain framework region can be further subdivided into contiguous regions separated by CDRs (FR1, FR2, FR3, and FR4). The heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, comprising the HFR1, HFR2, HFR3, and HFR4 framework regions. The heavy chain variable region is formed by the following numbered CDRs and FRs (arranged from amino-terminus to 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 shown in SEQ ID NOs: 6 to 7, respectively;

[0035] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 12 and 7, respectively;

[0036] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region are shown in SEQ ID NOs: 17 and 7, respectively;

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

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

[0039] In some embodiments, the species of origin of the constant region is cow, horse, pig, sheep, 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 a monoclonal antibody, a polyclonal antibody, a multispecific antibody, a murine antibody, a chimeric antibody, and a 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 an antibody; an "antigen-binding fragment" is a portion of an intact antibody that specifically binds to the antigen bound by the intact antibody. A person skilled in the art will readily appreciate, based on the disclosure herein, that antigen-binding fragments can be prepared by methods known in the art, such as enzymatic digestion (including pepsin or papain) and / or chemical reduction to cleave disulfide bonds, or synthesized by recombinant genetics or using an automated peptide synthesizer (e.g., an Applied BioSystems automated peptide synthesizer).

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

[0043] In another aspect, embodiments of the present invention provide an isolated nucleic acid encoding the anti-FLT3 antibody or antigen-binding fragment thereof according to any of the preceding embodiments.

[0044] In another aspect, an embodiment of the present invention provides a recombinant vector comprising the isolated nucleic acid according to any of the preceding 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, which can be directly or indirectly (such as packaged into a virus) introduced into a host cell by means of transformation, transfection or transduction to express the target gene. One type of vector is a plasmid, i.e., a circular double-stranded DNA molecule, which can connect the target DNA fragment to a plasmid ring. Another type of vector is a viral vector, which can connect and package the target DNA fragment into a viral genome (such as adenovirus, adeno-associated virus, retrovirus, slow virus, oncolytic virus). After these vectors enter the host cell, the expression of the target gene can be performed.

[0046] In another aspect, an embodiment of the present invention provides a host cell comprising the recombinant vector according to any of the preceding 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 may be Escherichia coli, Streptomyces, or Bacillus subtilis. The eukaryotic host cell may be 293 cells, 293T cells, 293FT cells, CHO cells, COS cells, Per6 cells, Saccharomyces cerevisiae, Pichia pastoris, Hansenula, Candida, some insect cells, and plant cells. 293 series cells, Per6 cells, and CHO cells are commonly used mammalian cells for producing antibodies or recombinant proteins and are well known to those skilled in the art.

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

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

[0050] On the other hand, an embodiment of the present invention provides a chimeric antigen receptor, wherein the antigen binding domain of the chimeric antigen receptor comprises the anti-FLT3 antibody or antigen-binding fragment thereof according to any of the aforementioned embodiments.

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

[0052] In some embodiments, the signaling domain comprises CD3ζ.

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

[0054] On the other hand, an embodiment of the present invention provides a CAR-T cell comprising the chimeric antigen receptor as described in any of the aforementioned embodiments.

[0055] In some embodiments, the CAR-T cells comprise universal allogeneic CAR-T cells.

[0056] Autologous CAR-T cell therapy has advantages such as no immune rejection and the ability to persist in the body for extended periods of time. However, autologous CAR-T cell therapy has some limitations, such as high production costs, low numbers of T cells in AML patients undergoing multiple lines of treatment, or impaired T cell function after chemotherapy, which leads to decreased quality, and long manufacturing cycles, which can cause patients to miss the optimal treatment window. With the rapid development of universal allogeneic cell therapy technology, the development of this universal cell therapy can overcome most of the shortcomings of autologous CAR-T cell therapy, such as standardized, scaled-up, and industrialized manufacturing, reducing costs and enabling the preparation of large numbers of CAR-T cells from a single donor.

[0057] On the other hand, embodiments of the present invention further provide the use of an anti-FLT3 antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments, or an isolated 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: an immune cell, a reagent, a kit, a drug, and a pharmaceutical composition;

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

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

[0061] In addition, an embodiment of the present invention further provides a cell injection solution, comprising an anti-FLT3 antibody or antigen-binding fragment thereof as described in any of the foregoing embodiments, or an isolated 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] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or progresses, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination thereof.

[0063] For cancer, "treating" can mean inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treating" includes eliminating all or part of a tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the progression of a tumor, or some combination thereof.

[0064] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0065] Example 1 Preparation of FLT3 recombinant protein

[0066] The full-length FLT3 gene (NM_004119.2) was used as a template, and the extracellular region of the FLT3 gene was selected as the immune targeting fragment and cloned into the pcDNA3.1 expression vector carrying a His tag at the C-terminus. The recombinant FLT3 protein was then expressed in a 293 cell expression system, and the His tag was subsequently used to purify the recombinant FLT3 protein.

[0067] Example 2 Cell fusion and hybridoma screening

[0068] (1) Animal immunization

[0069] Balb / c female mice aged 5-6 weeks were used as immunized animals, and the immunization dose was 100 μg / mouse. For the first immunization, 100 μl of Freund's complete adjuvant (Sigma) was mixed with an equal volume of recombinant FLT3 protein, and after sufficient emulsification, multiple subcutaneous injections were performed. After an interval of 2 weeks, an equal volume of Freund's incomplete adjuvant (Sigma) was mixed with the recombinant protein, and after sufficient emulsification, multiple subcutaneous injections were performed. Booster immunizations were performed 4 times in total. On the 10th day after the last booster immunization, blood was collected to detect the antibody titer of the mice. Three days before cell fusion, 100 μg of recombinant protein was injected into the abdominal cavity once.

[0070] (2) Cell fusion and hybridoma screening

[0071] Under sterile conditions, mouse spleens were removed and a B cell-rich suspension was prepared. Cells were then fused with SP2 / 0 cells using the classic PEG (Sigma) method. The fused cells were resuspended in HAT medium and cultured. Half-changes of the medium were performed with fresh HAT medium on days 5 and 10 after fusion. Positive clones were screened by ELISA on days 11-15 after fusion.

[0072] The specific steps are as follows: ELISA screening was performed using a 96-well plate. FLT3 recombinant protein was coated onto the bottom of the plate at 100 ng / well at 4°C overnight. HRP-conjugated anti-mouse IgG antibody and chemiluminescence reagent (Beyotime Biotech) were used for color development, and the values ​​were read on a microplate reader at a wavelength of 450 nm.

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

[0074] (3) Sequencing analysis of specific antibodies

[0075] Hybridoma antibody variable region sequence cloning: The best hybridoma clones in the logarithmic growth phase were harvested, and RNA was extracted using Trizol (Invitrogen) and reverse transcribed using PrimeScript™ Reverse Transcriptase, Takara. The reverse-transcribed cDNA was amplified by PCR using the Mouse Ig-Primer Set (Novagen) and then sequenced to obtain the heavy and light chain variable region sequences. The heavy and light chain variable region CDR sequences and the single-chain antibody (scFv) sequence are shown in Table 1.

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

[0077]

[0078]

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

[0080] A single-chain antibody (scFv) expression platform was constructed based on a eukaryotic expression vector. The VH and VL gene segments of hybridoma clones 8#, 9#, and 11# were tandemly linked to the pcDNA3.1 vector using a (G4S) 3-linker. The light chain sequences of the three clones were identical. The constructed expression vector was expressed and purified using the HEK293T eukaryotic protein expression system. SDS-PAGE analysis showed that the size of the single-chain antibody (scFv) was consistent with the theoretical molecular weight. 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 FLT3 antibody (scFv) with antigen, 200 ng / well of FLT3 recombinant protein was coated on the ELISA plate in advance, the plate was blocked after overnight at 4°C, and different amounts of recombinant antibody (dilution: 10 2 ~10 -5 μg / mL), added secondary antibody for washing, color development, terminated reaction, and measured the optical density (OD450) at 450nm using a microplate reader. The binding capacity was determined using a four-parameter nonlinear regression curve fitting. The results showed that the three single-chain antibodies (scFv) of FLT3 had high specificity binding to FLT3 recombinant protein ( Figure 2 ).

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

[0086] Example 4 Analysis of Antibody Binding to Endogenously Expressed FLT3

[0087] The FLT3-positive THP-1 human acute myeloid leukemia cell line was selected for flow cytometry analysis. The results showed that the FLT3-positive THP-1 cells could be stained almost 100% positive with the four FLT3 single-chain antibodies (scFv). This indicates that the three FLT3 single-chain antibodies (scFv) can specifically recognize the endogenously expressed FLT3 molecule ( Figure 4 ).

[0088] Example 5 FLT3 Antibody Affinity Determination

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

[0090] Table 3 Summary of antibody affinity data

[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: CD8αsignal-peptide-scFv(FLT3)-CD8αhinge-CD28αTm-4-1BB-CD3ζ-P2A-EG FP. HEK293T cells were used as lentiviral packaging cells, and lentiviral packaging was performed using a three-plasmid packaging system (psPAX2, pMD2.G, CAR-T vector). After 48 hours, the supernatant virus solution was collected and concentrated by ultracentrifugation. The supernatant was pre-coated with RetroNectin protein and the concentrated virus was added to infect T cells. 48 hours after infection, the transfection efficiency of the CAR-T cells was evaluated by flow cytometry to be above 80.0%.

[0094] Using the CRISPR-Cas9 / RNPs electroporation method, sgRNA targeting TRAC and B2M and Cas9 protein were electroporated into CAR-T cells to prepare universal FLT3 UCAR-T cells. After 72 hours of amplification, the TCR and β-2-Microglobulin expressed by CAR-T cells were detected by flow cytometry. The flow cytometry results showed that the T cells with TRAC gene knockout reached 60.2%, and B2M also reached 58.3%. TRAC was subsequently obtained by magnetic bead sorting. - / B2M - Negative CAR-T cells.

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

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

[0097] 1 × 10 6 A mouse FLT3-positive AML xenograft tumor model was established with THP-1-mCherry.ffLuc cells to verify the in vivo efficacy of FLT3 UCAR-T. On the 5th day after inoculation, NCG mice were randomly divided into 5 groups, 5 mice in each group, and injected with 1×107 Mock T cells, 1×10 7 FLT3UCAR-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 rate of mice in the three FLT3 UCAR-T groups was significantly higher than that in the Mock T and PBS groups. The data were analyzed using one-way ANOVA. ns indicates no statistical difference, **** indicates P < 0.0001 ( Figure 5 ).

[0099] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection 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 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; The HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by any one of the schemes of Kabat, Chothia, AbM, Contact and IMGT.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein 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, respectively; the amino acid sequences of the LCDR1, LCDR2 and LCDR3 are shown in SEQ ID No. 4, AAS and SEQ ID No. 5, respectively.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The heavy chain variable region and light chain variable region also include framework regions.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof further comprises a constant region.

5. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that The constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD.

6. The antibody or antigen-binding fragment thereof according to claim 4, characterized in that The species origin of the constant region is cow, horse, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody is selected from any one of a monoclonal antibody, a murine antibody, a chimeric antibody and a full-length antibody.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of an antibody.

9. The antibody or antigen-binding fragment thereof according to claim 8, characterized in that When the antigen-binding fragment is scFv, the amino acid sequence of the scFv is shown in SEQ ID NO: 8, 13 or 18.

10. 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 to 9.

11. A recombinant vector, characterized in that It contains the isolated nucleic acid according to claim 10.

12. A host cell, characterized in that It contains the recombinant vector as claimed in claim 11.

13. 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 12 to obtain the anti-FLT3 antibody or antigen-binding fragment thereof.

14. 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 any one of claims 1 to 9.

15. The chimeric antigen receptor according to claim 14, characterized in that The chimeric antigen receptor further comprises a signal peptide, a hinge region, a transmembrane region and a signal transduction domain.

16. The chimeric antigen receptor according to claim 15, characterized in that The signal transduction domain includes CD3ζ.

17. The chimeric antigen receptor according to claim 15, characterized in that The signal transduction domain also includes the 4-1BB intracellular region.

18. A CAR-T cell, characterized in that: It comprises the chimeric antigen receptor according to any one of claims 14 to 17.

19. Use of the anti-FLT3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid according to claim 10, the recombinant vector according to claim 11, the host cell according to claim 12, the chimeric antigen receptor according to any one of claims 14 to 17, or the CAR-T cell according to claim 18 in the preparation of a product for preventing or treating tumors; the tumor is FLT3-positive acute myeloid leukemia.

20. The use according to claim 19, characterized in that The product includes at least one of immune cells, reagents, and kits.

21. The use according to claim 19, characterized in that The product is a drug.

22. The use according to claim 19, characterized in that The product is a pharmaceutical composition.

23. A cell injection solution, characterized in that: The method comprises the anti-FLT3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, the isolated nucleic acid according to claim 10, the recombinant vector according to claim 11, the host cell according to claim 12, the chimeric antigen receptor according to any one of claims 14 to 17, or the CAR-T cell according to claim 18.

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