WO2023125975A1 - 一种新型靶向人flt3的嵌合抗原受体修饰的t细胞的构建及应用 - Google Patents

一种新型靶向人flt3的嵌合抗原受体修饰的t细胞的构建及应用 Download PDF

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WO2023125975A1
WO2023125975A1 PCT/CN2022/144151 CN2022144151W WO2023125975A1 WO 2023125975 A1 WO2023125975 A1 WO 2023125975A1 CN 2022144151 W CN2022144151 W CN 2022144151W WO 2023125975 A1 WO2023125975 A1 WO 2023125975A1
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seq
flt3
cells
car
present
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French (fr)
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杨林
游凤涛
李亚芬
陈丹
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博生吉医药科技(苏州)有限公司
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Definitions

  • the invention belongs to the field of biomedicine and immune cell therapy, and specifically relates to a nanobody specifically targeting FLT3, and an engineered immune cell constructed therefrom targeting tumor cells with high FLT3 expression, and also relates to their use.
  • AML Acute myeloid leukemia
  • HSCT hematopoietic stem cell transplantation
  • CAR-T therapy for AML still faces great challenges.
  • Challenges it is difficult for us to find a suitable target for tumors, because most of the surface molecules expressed on AML are also expressed on the surface of hematopoietic stem/progenitor cells (HSPC), it will be on the surface of AML Cell killing can also damage hematopoietic stem cells, resulting in hemotoxicity. Therefore, it is very important to find a target that is relatively safe and has better efficacy.
  • HSPC hematopoietic stem/progenitor cells
  • FLT3 (Fms-like tyrosine kinase 3, CD135) is a protein encoded by the FLT3 gene in humans. It is a cytokine receptor and belongs to the class III receptor tyrosine kinase. It has been shown that FLT3 is expressed on about 50% of normal hematopoietic stem cells (HSCs) and some dendritic cells, but most of them are not expressed on umbilical cord blood lymphocytes. However, studies have found that FLT3 is highly expressed on the cell surface of AML patients, and about 1/3 of newly diagnosed AML patients have FLT3 activating mutations.
  • HSCs normal hematopoietic stem cells
  • dendritic cells dendritic cells
  • CAR chimeric antigen receptor
  • the object of the present invention is to provide a nanobody against FLT3, a chimeric antigen receptor immune cell constructed from the nanobody, and their preparation and application.
  • the Nanobody against FLT3 can specifically bind to FLT3.
  • the complementarity determining region CDR of the Nanobody against FLT3 is one or more selected from the following group:
  • any amino acid sequence in the above amino acid sequence also includes at least one (such as 1-3, preferably 1-2, more preferably 1) amino acid derivative sequence that can retain the ability to specifically bind to FLT3.
  • the derivative sequence that has undergone addition, deletion, modification and/or substitution of at least one amino acid and can retain the ability to specifically bind to FLT3 has a homology or sequence identity of at least 85%, At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence.
  • the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
  • the Nanobody against FLT3 further includes a framework region FR.
  • the framework region FR is derived from the amino acid sequence shown in SEQ ID NO: 7-17.
  • the framework region FR is one or more selected from the following group:
  • amino acid sequence of the VHH chain of the Nanobody against FLT3 is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or a combination thereof.
  • the nanobodies against FLT3 include humanized antibodies, camelid antibodies, and chimeric antibodies.
  • the nanobody against FLT3 is alpaca.
  • an antibody against FLT3 comprising one or more VHH chains of the Nanobody against FLT3 according to the first aspect of the present invention.
  • amino acid sequence of the VHH chain of the Nanobody against FLT3 is selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or a combination thereof.
  • the antibody against FLT3 may be a monomer, a bivalent antibody, and/or a multivalent antibody.
  • a chimeric antigen receptor CAR is provided, the CAR contains an extracellular domain, and the extracellular domain comprises the Nanobody against FLT3 as described in the first aspect of the present invention , or the antibody against FLT3 as described in the second aspect of the present invention.
  • the extracellular domain further includes a signal peptide.
  • the extracellular domain further includes a hinge region selected from the following histones: CD8, CD28, CD137, IgG, or a combination thereof.
  • the hinge region is a human IgG1 Fc hinge region.
  • the antibody contained in the extracellular domain has the amino acid sequence shown in SEQ ID NO: 7-17.
  • the amino acid sequence of the antibody contained in the extracellular domain has a homology of ⁇ 85%, preferably ⁇ 90%, and more preferably ⁇ 95%, with SEQ ID NO: 7-17, or Have 1, 2 or 3 amino acid differences compared to SEQ ID NO: 7-17.
  • the CAR has the structure shown in formula Ia:
  • L is nothing or a signal peptide sequence
  • Nb is a specific binding domain
  • H is none or hinge region
  • TM is the transmembrane domain
  • C is costimulatory signal domain
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇ (including wild type, or mutants/modifiers thereof);
  • the "-" connects a peptide or a peptide bond.
  • the Ls are respectively selected from signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or mutants/modifications thereof, or combinations thereof.
  • the Nb targets FLT3.
  • the Nb is FLT3 nanobody.
  • the H is selected from the hinge region of the following histones: CD8, CD28, CD137, IgG, or a combination thereof.
  • the H is a human IgG1 Fc hinge region.
  • the TM is selected from the transmembrane regions of the following histones: CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutants/modifications thereof, or combinations thereof.
  • the C is selected from the co-stimulatory domains of the following histones: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants/modifications thereof, or combinations thereof.
  • histones OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutants/modifications thereof, or combinations thereof.
  • the C is selected from the co-stimulatory domains of ICOS, 41BB, or combinations thereof.
  • amino acid sequence of the CAR is shown in SEQ ID NO: 1, 3, 4, 5, 6.
  • nucleotide sequence of the CAR is shown in SEQ ID NO:2.
  • a recombinant protein is provided, and the recombinant protein has:
  • the tag sequence includes Fc tag, HA tag, GGGS sequence, FLAG tag, Myc tag, 6His tag, or a combination thereof.
  • the recombinant protein specifically binds to FLT3.
  • the recombinant protein includes a fusion protein.
  • the recombinant protein is a monomer, a dimer, or a multimer.
  • the recombinant protein specifically binds to FLT3.
  • the tag sequence is an Fc tag.
  • a polynucleotide encoding a protein selected from the group consisting of the Nanobody against FLT3 as described in the first aspect of the present invention, or the nanobody as described in the second aspect of the present invention.
  • the present invention relates to a nucleic acid molecule encoding the Nanobody against FLT3 of the present invention.
  • a nucleic acid of the invention may be RNA, DNA or cDNA.
  • an expression vector containing the polynucleotide according to the fifth aspect of the present invention is provided.
  • the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.
  • the expression vector comprises a viral vector, such as lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.
  • the expression vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, pBlue and the like.
  • the expression vector is a pBlue vector.
  • the expression vector further includes a promoter, a transcriptional enhancer element WPRE, a long terminal repeat sequence LTR, etc. selected from the group.
  • a host cell containing the expression vector of the sixth aspect of the present invention, or the polynucleotide of the fifth aspect of the present invention integrated in its genome.
  • the host cells include prokaryotic cells or eukaryotic cells.
  • the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.
  • the host cells are 293F cells.
  • an engineered immune cell containing the expression vector described in the sixth aspect of the present invention or the exogenous expression vector described in the fifth aspect of the present invention integrated in the chromosome. of polynucleotides.
  • the engineered immune cells comprise the chimeric antigen receptor as described in the third aspect of the present invention.
  • the engineered immune cells are selected from the following group:
  • CAR-T cells chimeric antigen receptor ⁇ T cells
  • CAR-T cells chimeric antigen receptor ⁇ T cells
  • CAR-NKT cells chimeric antigen receptor NKT cells
  • the engineered immune cells include autologous or allogeneic ⁇ T cells, ⁇ T cells, NKT cells, NK cells, or a combination thereof.
  • the engineered immune cells are CAR-T cells.
  • a method of producing a Nanobody against FLT3 comprising the steps of:
  • step (c) Optionally, purifying and/or modifying the Nanobody against FLT3 obtained in step (b).
  • the tenth aspect of the present invention there is provided a method for preparing the engineered immune cell as described in the eighth aspect of the present invention, comprising the following steps: the polynucleotide as described in the fifth aspect of the present invention or the polynucleotide as described in the present invention
  • the expression vector described in the sixth aspect is transduced into immune cells, so as to obtain the engineered immune cells.
  • the method further includes the step of testing the function and effectiveness of the obtained engineered immune cells.
  • the method includes transducing the chimeric antigen receptor according to the third aspect of the present invention into immune cells, so as to obtain the engineered immune cells.
  • an immunoconjugate comprising:
  • a coupling moiety selected from the group consisting of detectable labels, drugs, cytokines, radionuclides, enzymes, gold nanoparticles/nanorods, nanomagnetic particles, viral coat proteins or VLPs, or combinations thereof.
  • the part (a) is coupled to the coupling part through a chemical bond or a linker.
  • the radionuclides include:
  • isotopes for diagnosis are selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y -90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166 , I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223 , Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169,
  • the coupling moiety is a drug or a toxin.
  • the drug is a drug for targeted treatment of diseases with high expression of FLT3.
  • the drug is a drug for targeted treatment of acute myeloid leukemia.
  • the drug is a cytotoxic drug.
  • the cytotoxic drugs are selected from the group consisting of anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy A sensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.
  • cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors.
  • Typical cytotoxic drugs include, for example, Auristatins, camptothecins, (Camptothecins), Duocarmycins/Duocarmycins, Etoposides, Maytansines and Maytansinoids (such as DM1 and DM4), Taxanes ( Taxanes), benzodiazepines, or benzodiazepine containing drugs (such as pyrrolo[1,4]benzodiazepines (PBDs), indoline benzodiazepines Indolinobenzodiazepines and Oxazolidinobenzodiazepines), Vinca alkaloids, or combinations thereof.
  • Auristatins camptothecins, (Camptothecins), Duocarmycins/Duocarmycins, Etoposides, Maytansines and Maytansinoids (such as DM1 and DM4), Taxanes ( Taxa
  • the toxin is selected from the group consisting of auristatins (for example, auristatin E, auristatin F, MMAE and MMAF), aureomycin, maytansinol, ricin, ricin Anesthetic toxin A-chain, combretastatin, duocarmycin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, Tenoposide, vincristine, vinblastine, colchicine, dihydroxyanthraxin diketone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, acacia Toxin, abrin A chain, lotus root toxin A chain, ⁇ -sarcinia, gelonin, Mitogellin, Retstricttocin,
  • the coupling moiety is a detectable label.
  • the coupling moiety is selected from the group consisting of fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer X-ray tomography) contrast agents, or capable of producing Detectable products of enzymes, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, virus particles, liposomes, nanomagnetic particles , prodrug-activating enzymes (eg, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), or nanoparticles in any form.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • the immunoconjugate comprises: a multivalent (eg, bivalent) VHH chain of the Nanobody against FLT3 according to the first aspect of the present invention.
  • the multivalent means that the amino acid sequence of the immunoconjugate contains multiple repetitions of the same or different VHH chains of the Nanobody against FLT3 as described in the first aspect of the present invention.
  • an active ingredient selected from the group consisting of the nanobody against FLT3 as described in the first aspect of the present invention, or the second aspect of the present invention
  • the active ingredient is selected from the group consisting of the nanobody against FLT3 as described in the first aspect of the present invention, or the antibody against FLT3 as described in the second aspect of the present invention, or as The chimeric antigen receptor according to the third aspect of the present invention, or the engineered immune cell according to the eighth aspect of the present invention, or a combination thereof.
  • the reagent shown is a diagnostic reagent, preferably, the diagnostic reagent is a detection chip or a detection plate.
  • the diagnostic reagent is used for: detecting FLT3 protein or its fragments in a sample.
  • the disease with high FLT3 expression is selected from: acute myeloid leukemia, acute lymphoblastic leukemia (Acute lymphoblastic leukemia, ALL), chronic myelogenous leukemia (Chronic myelogenous leukemia, CML), myelodysplastic syndrome (Myelodysplastic syndromes, MDS) and so on.
  • the disease with high FLT3 expression is acute myeloid leukemia.
  • a method for in vitro detection of FLT3 protein or fragments thereof in a sample comprising the steps of:
  • the detection includes diagnostic or non-diagnostic.
  • a pharmaceutical composition which contains:
  • a pharmaceutically acceptable carrier, diluent or excipient (ii) A pharmaceutically acceptable carrier, diluent or excipient.
  • the active ingredient is selected from the group consisting of the nanobody against FLT3 as described in the first aspect of the present invention, or the antibody against FLT3 as described in the second aspect of the present invention, or as The chimeric antigen receptor according to the third aspect of the present invention, or the engineered immune cell according to the eighth aspect of the present invention, or a combination thereof.
  • the dosage form of the pharmaceutical composition is selected from the group consisting of injections and freeze-dried preparations.
  • the pharmaceutical composition includes 0.01-99.99% of the nanobody against FLT3 according to the first aspect of the present invention, or the antibody against FLT3 according to the second aspect of the present invention , or the chimeric antigen receptor as described in the third aspect of the present invention, or the recombinant protein as described in the fourth aspect of the present invention, or the immunoconjugate as described in the eleventh aspect of the present invention, or a combination thereof and 0.01-99.99% of the pharmaceutical carrier, the percentage is the mass percentage of the pharmaceutical composition.
  • the concentration of the engineered immune cells in the active ingredient is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/mL, preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells /mL.
  • a kit comprising:
  • a first container containing the nanobody against FLT3 as described in the first aspect of the present invention, or the antibody against FLT3 as described in the second aspect of the present invention, or the antibody against FLT3 according to the present invention The chimeric antigen receptor as described in the third aspect, or the recombinant protein as described in the fourth aspect of the present invention, or the engineered immune cell as described in the eighth aspect of the present invention, or as described in the eleventh aspect of the present invention An immunoconjugate, or a combination thereof; and/or
  • the test kit contains a detection plate, and the detection plate includes: a substrate (support plate) and a test strip, and the test strip contains the nanobody against FLT3 as described in the first aspect of the present invention, as described in the present invention
  • the kit also contains an instruction, and according to the instruction, the kit is used to non-invasively detect the expression of FLT3 in the subject to be tested.
  • the kit is used for the detection of diseases with high FLT3 expression.
  • the disease with high FLT3 expression is selected from: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and the like.
  • the disease with high FLT3 expression is acute myeloid leukemia.
  • a method for preventing and/or treating diseases with high FLT3 expression comprising: administering the nanobody against FLT3 as described in the first aspect of the present invention to a subject in need , or the antibody against FLT3 as described in the second aspect of the present invention, or the chimeric antigen receptor as described in the third aspect of the present invention, or the recombinant protein as described in the fourth aspect of the present invention, or the recombinant protein as described in the fourth aspect of the present invention, or The engineered immune cell according to the eighth aspect, or the immunoconjugate according to the eleventh aspect of the present invention, or the pharmaceutical composition according to the fourteenth aspect of the present invention, or a combination thereof.
  • the subject includes mammals, such as humans.
  • the disease with high FLT3 expression is selected from: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and the like.
  • the disease with high FLT3 expression is acute myeloid leukemia.
  • the engineered immune cells or the CAR immune cells included in the pharmaceutical composition are cells derived from the subject (autologous cells).
  • the engineered immune cells or the CAR immune cells contained in the pharmaceutical composition are cells derived from healthy individuals (allogeneic cells).
  • the above method can be used in combination with other treatment methods.
  • the other treatment methods include chemotherapy, radiotherapy, targeted therapy and other methods.
  • a method for diagnosing diseases with high FLT3 expression comprising the steps of:
  • the sample is a blood sample or a throat swab sample, or a sample from other tissues and organs.
  • the disease with high FLT3 expression is selected from: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and the like.
  • the disease with high FLT3 expression is acute myeloid leukemia.
  • a method for preparing a recombinant polypeptide is provided, and the recombinant polypeptide is the nanobody against FLT3 as described in the first aspect of the present invention, or as described in the second aspect of the present invention.
  • the antibody against FLT3 described above, or the chimeric antigen receptor as described in the third aspect of the present invention, or the recombinant protein as described in the fourth aspect of the present invention includes:
  • Figure 1 shows the amplification results of VHH fragments.
  • Figure 2 shows the results of yeast display library diversity comparison.
  • Figure 3 shows the flow cytometry results after the first sorting.
  • Figure 4 shows the results of flow cytometry after the second sorting.
  • Figure 5 shows the results of yeast monoclonal flow cytometry detection.
  • Figure 6 shows the structure of the constructed VHH eukaryotic expression vector.
  • Figure 7 shows the results of flow cytometry detection of eukaryotic expression of the FLT3 single domain antibody.
  • Figures 8A-8K show sequence information for single domain antibodies targeting FLT3.
  • Figure 9 shows the structure diagram of the FLT3-CAR vector, in which FC is the human IgG1 Fc hinge region.
  • Figure 10 shows the titer detection results of FLT3-CAR lentiviruses with different structures.
  • Figure 11 shows the expression of FLT3 on the surface of different tumor cell lines.
  • Figure 12 shows the in vitro killing of Raji cells (FLT3-negative cell line) by FLT3-CAR-T with different structures.
  • Figure 13 shows the killing of FLT3-positive tumor cell lines in vitro by FLT3-CAR-T with different structures.
  • the target cells were MV-4-11, MOLM-13, and AML3, and different effect-to-target ratios were set.
  • Figure 14 shows the secretion of cytokine Granzyme-B in the supernatant after FLT3-CAR-T with different structures was incubated with FLT3-positive tumor cell lines.
  • the target cells were MV-4-11, MOLM-13, and AML3, and the effect-to-target ratios were 1:1.
  • Figure 15 shows the sustained killing of FLT3-CAR-T with different structures on the FLT3-positive tumor cell line MV-4-11.
  • the target cells were MV-4-11, and the effect-to-target ratios were 1:1 and 1:5, respectively. Tumor cells were added every 24h or 48h.
  • Figure 16 shows the expression of FLT3 and CD33 on the surface of human HSPC cells.
  • Figure 17 shows the release of cytokines after CD34+HSPC cells were incubated with different FLT3-CAR-T cells.
  • Figure 18 shows the colony formation after incubation of different CAR-T cells with CD34+HSPC cells.
  • BFU-E represents the number of colony formation of erythroid cells in HSPC cells
  • CFU-GM represents the colony formation of granulocytes and macrophages in HSPC cells.
  • Figure 19 shows the experimental scheme of the drug effect of TAA05-CAR-T on OCI-AML3-Luc-GFP model mice.
  • Figure 20 shows the in vivo fluorescence imaging of TAA05-CAR-T on the drug effect of AML3-Luc-GFP model mice.
  • Figure 21 shows the body weight and survival period of TAA05-CAR-T on AML3-Luc-GFP model mice.
  • mice in PBS and Mock T groups all experienced weight loss after onset, and mice in CAR-T group had a relatively stable body weight;
  • B Survival period of mice: CAR-T group The survival period of mice was significantly prolonged.
  • the inventors After extensive and in-depth research and extensive screening, the inventors first developed a new type of FLT3 nanobody, and successfully constructed FLT3-CAR-T cells targeting FLT3 based on the developed FLT3 nanobody for the treatment of refractory Treatment of patients with recurrent AML and other tumors. Through a large number of in vitro functional experiments and animal experiments, the inventors have proved that the developed FLT3-CAR-T cell product has a significant in vitro and in vivo anti-tumor effect on AML tumor cells, and proved that compared with CD33-CAR-T cells, The FLT3-CAR-T cells of the present invention have better safety. The present invention has been accomplished on this basis.
  • the novel FLT3-CAR-T targeting FLT3 of the present invention can be used as a novel therapeutic means for targeted treatment of refractory and relapsed AML.
  • the present invention takes CAR-T cells as an example to representatively describe the engineered immune cells of the present invention in detail.
  • the engineered immune cells of the present invention are not limited to the CAR-T cells described above, and the engineered immune cells of the present invention have the same or similar technical features and beneficial effects as the CAR-T cells described above.
  • immune cells express chimeric antigen receptor CAR
  • NK cells are equivalent to T cells (or T cells can be replaced by NK cells).
  • single domain antibody As used herein, the terms “single domain antibody”, “single domain antibody of the present invention”, “recombinant antibody”, “FLT3 Nanobody”, “anti-FLT3 Nanobody” are used interchangeably, and all refer to the present invention and the target protein Recombinant/single domain antibody that specifically binds FLT3.
  • Each value in the table represents the sequence number, that is, "1” means “SEQ ID NO: 1", and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the numbers of their amino acid sequences.
  • antibody or "immunoglobulin” is a heterotetrameric protein of about 150,000 Daltons with identical structural features, consisting of two identical light (L) chains and two identical heavy chains (H) Composition. Each light chain is linked to a heavy chain by one covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable region (VH) at one end followed by constant regions.
  • VH variable region
  • Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain .
  • VL variable region
  • Specific amino acid residues form the interface between the variable domains of the light and heavy chains.
  • single domain antibody As used herein, the terms “single domain antibody”, “VHH”, “Nanobody”, “single domain antibody (single domain antibody, sdAb, or nanobody nanobody)” have the same meaning and can be used interchangeably, Refers to cloning the variable region of the heavy chain of an antibody to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the heavy chain of the antibody is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.
  • VHH single domain antibody
  • variable means that certain portions of the variable regions among antibodies differ in sequence, which contribute to the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout antibody variable domains. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable domains are called the framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the variable domains of native heavy and light chains each contain four FR regions in a roughly ⁇ -sheet configuration connected by three CDRs forming connecting loops and, in some cases, partial b-sheet structures.
  • the CDRs in each chain are in close proximity through the FR regions and together with the CDRs of the other chain form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. 1, pp. 647-669 (1991)).
  • the constant regions are not directly involved in the binding of the antibody to the antigen, but they exhibit different effector functions, for example involved in the antibody-dependent cytotoxicity of the antibody.
  • immunoconjugates and fusion expression products include: drugs, toxins, cytokines (Cytokine), radionuclides, enzymes and other diagnostic or therapeutic molecules combined with antibodies or fragments thereof of the present invention to form of conjugates.
  • the present invention also includes cell surface markers or antigens that bind to the nanobody against FLT3 or a fragment thereof.
  • variable region and “complementarity determining region (CDR)” are used interchangeably.
  • the heavy chain variable region of the antibody includes three complementarity determining regions CDR1, CDR2, and CDR3.
  • the heavy chain of the antibody includes the above-mentioned heavy chain variable region and heavy chain constant region.
  • antibody of the present invention protein of the present invention
  • polypeptide of the present invention are used interchangeably, and all refer to polypeptides that specifically bind to the FLT3 protein, such as proteins or polypeptides with heavy chain variable regions . They may or may not contain starting methionine.
  • the invention also provides other proteins or fusion expression products having the antibodies of the invention.
  • the present invention includes any protein or protein conjugates and fusion expression products (i.e., immunoconjugates and fusion expression products) having a heavy chain containing a variable region, as long as the variable region is compatible with the heavy chain of the antibody of the present invention
  • the variable regions are identical or at least 90% homologous, preferably at least 95% homologous.
  • variable region which is separated into four framework regions (FR), and the amino acids of the four FR
  • FR framework regions
  • the sequence is relatively conservative and does not directly participate in the binding reaction.
  • CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure.
  • the CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antigen-binding site of the antibody.
  • Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • variable regions of the heavy chains of the antibodies of the invention are of particular interest because at least some of them are involved in binding antigen. Therefore, the present invention includes those molecules having antibody heavy chain variable regions with CDRs, as long as the CDRs have more than 90% (preferably more than 95%, most preferably more than 98%) homology to the CDRs identified herein sex.
  • the present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of said antibodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of the antibody of the present invention.
  • the polypeptide fragments, derivatives or analogs of the present invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide in combination with another compound (such as a compound that extends the half-life of the polypeptide, e.g.
  • polyethylene glycol polyethylene glycol
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • an additional amino acid sequence fused to the polypeptide sequence such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify the polypeptide, or with fusion protein formed by 6His tag.
  • the antibody of the present invention refers to a polypeptide that has FLT3 protein binding activity and includes the above-mentioned CDR region.
  • the term also includes variant forms of polypeptides comprising the above CDR regions that have the same function as the antibodies of the present invention. These variations include (but are not limited to): one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acid deletions , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the antibodies of the invention.
  • Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA hybrids that can hybridize with the DNA encoding the antibody of the present invention under high or low stringency conditions
  • the encoded protein, and the polypeptide or protein obtained by using the antiserum against the antibody of the present invention.
  • the invention also provides other polypeptides, such as fusion proteins comprising antibodies or fragments thereof.
  • the invention also includes fragments of the antibodies of the invention.
  • the fragment has at least about 50 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids of an antibody of the invention.
  • “conservative variants of the antibody of the present invention” refer to at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acid sequences compared with the amino acid sequence of the antibody of the present invention.
  • An amino acid is replaced by an amino acid with similar or similar properties to form a polypeptide.
  • These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 2.
  • the present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments or fusion proteins thereof.
  • a polynucleotide of the invention may be in the form of DNA or RNA.
  • Forms of DNA include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be either the coding strand or the non-coding strand.
  • a polynucleotide encoding a mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence for the mature polypeptide and various additional coding sequences; a coding sequence for the mature polypeptide (and optional additional coding sequences) and non-coding sequences .
  • polynucleotide encoding a polypeptide may include a polynucleotide encoding the polypeptide, or may also include additional coding and/or non-coding sequences.
  • the present invention also relates to polynucleotides which hybridize to the above-mentioned sequences and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides which are hybridizable under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only if the identity between the two sequences is at least 90%, more Preferably, hybridization occurs above 95%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • a feasible method is to use artificial synthesis to synthesize related sequences, especially when the fragment length is short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and an expression tag (such as 6His) can also be fused together to form a fusion protein.
  • biomolecules nucleic acid, protein, etc.
  • the biomolecules involved in the present invention include biomolecules in an isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragment, or its derivative) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or eg vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the present invention also relates to vectors comprising the above-mentioned appropriate DNA sequences and appropriate promoter or control sequences. These vectors can be used to transform appropriate host cells so that they express the protein.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the CaCl2 method using procedures well known in the art. Another way is to use MgCl2. Transformation can also be performed by electroporation, if desired.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional media according to the host cells used.
  • the culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.
  • the recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • the antibodies of the invention can be used alone, or combined or conjugated with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of these.
  • Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or substances capable of producing a detectable product. enzyme.
  • Therapeutic agents that can be combined or coupled with the antibody of the present invention include but are not limited to: 1. Radionuclide; 2. Biological toxicity; 3. Cytokines such as IL-2, etc.; 4. Gold nanoparticles/nanorods; 5. Viruses Particles; 6. Liposomes; 7. Nanomagnetic particles; 8. Prodrug activating enzymes (for example, DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), etc.
  • DTD DT-diaphorase
  • BPHL biphenylhydrolase-like protein
  • Nanobody refers to an antibody that naturally lacks light chains in the peripheral blood of alpacas.
  • VHH heavy chain variable region
  • CH2 and CH3 conventional constant regions
  • the lack of a VL domain also means that nanobodies have a hydrophilic side, and nanobodies are not as easy to stick to each other as artificially engineered single-chain antibody fragments (scFv), or even aggregate into clumps.
  • the VHH structure cloned and expressed separately has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest known unit that can bind the target antigen.
  • the VHH crystal is 2.5nm, 4nm long, and the molecular weight is only 15KDa.
  • Nanobodies can be described by three specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into four framework regions (FRs), the amino acid sequences of the four FRs It is relatively conservative and does not directly participate in the binding reaction. These CDRs form a ring structure, and the ⁇ sheets formed by the FRs in between are close to each other in the spatial structure, and the CDRs on the heavy chain constitute the antigen-binding site of the antibody. Which amino acids constitute FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type.
  • a chimeric immunoantigen receptor includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes an optional signal peptide and a target-specific binding domain (also known as an antigen binding domain).
  • the intracellular domain includes the co-stimulatory domain and the CD3 ⁇ chain portion.
  • the extracellular segment When CAR is expressed in T cells, the extracellular segment can recognize a specific antigen, and then transduce the signal through the intracellular domain, causing cell activation and proliferation, cytolytic toxicity and secretion of cytokines such as IL-2 and IFN- ⁇ etc., affecting tumor cells so that they do not grow, are induced to die, or are otherwise affected, and result in a reduction or elimination of the patient's tumor burden.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of the co-stimulatory molecule and the CD3zeta chain.
  • CAR-T cell As used herein, the terms “CAR-T cell”, “CAR-T”, “FLT3-CAR-T cell”, “CAR-T cell of the present invention” and the like all refer to the CAR-T described in the eighth aspect of the present invention cell.
  • the CAR-T cells of the present invention can be used to treat tumors with high expression of FLT3, such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and the like.
  • CAR-T cells have the following advantages over other T-cell-based therapies: (1) The action process of CAR-T cells is not restricted by MHC; (2) Since many tumor cells express the same tumor antigen, it can target a certain tumor Once the CAR gene construction of the antigen is completed, it can be widely used; (3) CAR can use both tumor protein antigens and glycolipid non-protein antigens, expanding the target range of tumor antigens; (4) using the patient's own The cells reduce the risk of rejection; (5) CAR-T cells have immune memory function and can survive in the body for a long time.
  • CAR-NK cell As used herein, the terms “CAR-NK cell”, “CAR-NK”, “FLT3-CAR-NK cell”, “CAR-NK cell of the present invention” and the like all refer to the CAR-NK described in the eighth aspect of the present invention cell.
  • the CAR-NK cells of the present invention can be used to treat tumors with high expression of FLT3, such as acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and the like.
  • Natural killer cells are a major type of immune effector cells, which protect the body from virus infection and tumor cell invasion through non-antigen-specific pathways. NK cells through engineering (gene modification) may obtain new functions, including the ability to specifically recognize tumor antigens and have enhanced anti-tumor cytotoxicity.
  • CAR-NK cells Compared with autologous CAR-T cells, CAR-NK cells also have the following advantages, for example: (1) directly kill tumor cells by releasing perforin and granzymes, but have no killing effect on normal cells of the body; (2) they release A very small amount of cytokines reduces the risk of cytokine storm; (3) It is very easy to expand in vitro and develop into "off-the-shelf" products. Other than that, it is similar to CAR-T cell therapy.
  • FLT3 (Fms-like tyrosine kinase, FMS-like tyrosine kinase 3) belongs to the type III receptor tyrosine kinase III (Receptor tyrosine kinase III, RTK III) family member, in recent years, many large sample studies have confirmed the activation of FLT3 Mutations play a very important pathological role in the occurrence and progression of diseases such as AML.
  • AML patients with FLT3/ITD activating mutations usually have unique clinical features such as high peripheral blood white blood cell count, poor clinical prognosis, and easy recurrence, and because the detection method of FLT3 activating mutations is simple and easy, there are more and more studies The authors are committed to developing FLT3 into a routine detection method for AML patients to guide the treatment and prognosis of AML patients and as a detection method for minimal residual leukemia, and to use it as another new target for chemotherapy drugs in leukemia patients (currently There are already drugs for the treatment of FLT3-ITD mutations).
  • the present invention also provides a composition.
  • the composition is a pharmaceutical composition, which contains the above-mentioned antibody or its active fragment or its fusion protein, and a pharmaceutically acceptable carrier.
  • these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is usually about 5-8, preferably about 6-8, although the pH value can be changed according to the Depending on the nature of the substance formulated and the condition to be treated.
  • the formulated pharmaceutical composition can be administered by conventional routes, including but not limited to: intraperitoneal, intravenous, or topical administration.
  • the pharmaceutical composition of the present invention contains a safe and effective amount (such as 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80wt%) of the above-mentioned antibody (or its conjugate) of the present invention and a pharmaceutically acceptable acceptable carrier or excipient.
  • a pharmaceutically acceptable acceptable carrier or excipient include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical formulation should match the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions are preferably produced under sterile conditions.
  • the active ingredient is administered in a therapeutically effective amount, eg, about 10 micrograms/kg to about 50 mg/kg body weight per day.
  • the polypeptides of the invention can also be used
  • a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is usually at least about 10 micrograms/kg body weight, and in most cases no more than about 50 mg/kg body weight, Preferably the dose is about 10 micrograms/kg body weight to about 10 mg/kg body weight.
  • the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.
  • the Nanobodies against FLT3 include monomers, bivalents (bivalent antibodies), tetravalents (tetravalent antibodies), and/or multivalents (multivalent antibodies).
  • the nanobody against FLT3 includes one or more of the following components such as SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO VHH chains of the amino acid sequences shown in: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17.
  • the antibody has a detectable label. More preferably, the label is selected from the group consisting of isotopes, colloidal gold labels, colored labels or fluorescent labels.
  • Colloidal gold labeling can be performed using methods known to those skilled in the art.
  • the antibody against the FLT3 protein is labeled with colloidal gold to obtain a colloidal gold-labeled antibody.
  • the nanobody against FLT3 of the present invention can effectively bind to the FLT3 protein.
  • the present invention also relates to methods for detecting FLT3 protein or fragments thereof.
  • the steps of the method are roughly as follows: obtain a cell and/or tissue sample; dissolve the sample in a medium; detect the level of FLT3 protein in the dissolved sample.
  • the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.
  • the present invention also provides a kit containing the antibody (or its fragment) or detection plate of the present invention.
  • the kit further includes a container, instructions for use, buffer and the like.
  • the present invention also provides a detection kit for detecting the level of FLT3 protein, which includes an antibody that recognizes FLT3 protein, a lysis medium for dissolving samples, and general reagents and buffers required for detection, such as various buffers, Detection label, detection substrate, etc.
  • the test kit may be an in vitro diagnostic device.
  • the present invention provides an engineered immune cell (such as CAR-T cell) according to the eighth aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/mL, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/mL.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine ; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, etc.
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as glycine
  • chelating agents such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives e.g, aluminum hydroxide
  • the antibody of the present invention has a wide range of biological and clinical application values, and its application involves the diagnosis and treatment of diseases related to FLT3 protein, basic medical research, biological research and other fields.
  • a preferred application is for clinical diagnosis, prevention and treatment of FLT3 protein.
  • the present invention also provides a method for stimulating an immune response mediated by T cells targeting mammalian tumor cell populations or tissues, comprising the following steps: administering the CAR-T cells of the present invention to mammals.
  • the present invention includes a type of cell therapy, in which a patient's own T cells (or a heterologous donor) are isolated, activated and genetically modified to produce CAR-T cells, and then injected into the same patient.
  • a patient's own T cells or a heterologous donor
  • the probability of graft-versus-host reaction is extremely low, and the antigen is recognized by T cells without MHC restriction.
  • a single CAR-T can treat all cancers that express that antigen.
  • CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.
  • the CAR-T cells of the present invention can undergo stable in vivo expansion and last for several months to several years.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-T cells can induce a specific immune response to tumor cells that overexpress the antigen recognized by the CAR antigen-binding domain.
  • the CAR-T cells of the present invention elicit a specific immune response against tumor cells with high FLT3 expression.
  • Treatable cancers include tumors that are not or substantially not vascularized, as well as vascularized tumors.
  • the types of cancer treated with the CAR of the present invention include, but are not limited to: acute myeloid leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, and the like.
  • the present invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-T cell of the present invention.
  • the CAR-T cells of the present invention can be administered alone or as a pharmaceutical composition with a diluent and/or in combination with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, or may be determined by clinical trials.
  • compositions of the invention to be administered can be determined by a physician, taking into account the patient (subject ) with individual differences in age, weight, tumor size, degree of infection or metastasis, and disease.
  • Pharmaceutical compositions comprising T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably at a dose of 10 5 to 10 7 cells/kg body weight (including all integer values within the range). T cell compositions can also be administered multiple times at these doses.
  • Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical art by monitoring the patient for signs of disease, and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection or intraperitoneally.
  • the T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by intravenous injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein, or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previously , simultaneously or subsequently) to the patient in a form of treatment including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab treatment for MS patients or erfatizumab treatment for psoriasis patients or other treatments for PML patients.
  • the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies or other immunotherapeutic agents.
  • the cell composition of the invention is administered in conjunction with (eg, before, simultaneously with, or after) bone marrow transplantation, the use of chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide patient.
  • a subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an infusion of expanded immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • Dosages administered to a patient for the above treatments will vary with the precise nature of the condition being treated and the recipient of the treatment. Dosage ratios for human administration can be implemented according to practice accepted in the art. Usually, 1 ⁇ 10 5 to 1 ⁇ 10 10 modified T cells of the present invention can be administered to the patient for each treatment or each course of treatment, for example, through intravenous infusion. Amino Acid and Nucleotide Sequence
  • SEQ ID NO: 1 PA0135-MN-CAR amino acid sequence
  • SEQ ID NO:2 PA0135-MN-CAR nucleotide sequence
  • SEQ ID NO: 4 PA0135-GH-CAR amino acid sequence
  • SEQ ID NO: 19 (CDR2 of Nanobodies C-F15-10, C-A5-11)
  • SEQ ID NO:21 CDR1 of Nanobodies C-F21-1, C-1-D3, C-1-G3, C-2-C2, C-1-E6)
  • SEQ ID NO:22 CDR2 of Nanobodies C-F21-1, C-1-G3, C-2-C2, C-1-E6, C-2-A3, C-2-C4)
  • SEQ ID NO:28 CDR3 of Nanobody C-1-G3
  • SEQ ID NO:31 CDR1 of Nanobodies C-2-D8, C-2-A3, C-2-C4
  • SEQ ID NO:36 (CDR3 of Nanobody C-2-A3)
  • SEQ ID NO:40 (FR3 of Nanobody C-F15-10)
  • SEQ ID NO:41 (FR4 of Nanobodies C-F15-10, C-F21-1)
  • SEQ ID NO:42 (FR1 of Nanobodies C-F21-1, C-1-D3, C-1-G3, C-2-C2)
  • SEQ ID NO:43 (FR2 of Nanobodies C-F21-1, C-1-D3, C-1-E6)
  • SEQ ID NO:44 (FR3 of Nanobodies C-F21-1, C-2-C2)
  • SEQ ID NO:48 (FR4 of Nanobodies C-A5-11, C-2-A5, C-2-C4)
  • SEQ ID NO:49 (FR3 of Nanobody C-1-D3)
  • SEQ ID NO:50 (FR4 of Nanobodies C-1-D3, C-2-D8)
  • SEQ ID NO:52 (FR3 of Nanobody C-1-G3)
  • SEQ ID NO:54 (FR2 of Nanobody C-2-C2)
  • SEQ ID NO:56 (FR1 of Nanobody C-1-E6)
  • SEQ ID NO:57 (FR3 of Nanobody C-1-E6)
  • SEQ ID NO:58 (FR4 of Nanobody C-1-E6)
  • SEQ ID NO:60 (FR2 of Nanobodies C-2-D8, C-2-A3, C-2-C4)
  • SEQ ID NO:62 (FR1 of Nanobodies C-2-A5, C-2-C4)
  • SEQ ID NO:65 (FR1 of Nanobody C-2-A3)
  • SEQ ID NO:66 (FR3 of Nanobody C-2-A3)
  • SEQ ID NO:68 (FR3 of Nanobodies C-2-C4)
  • the FLT3 nanobody developed by the present invention has high affinity.
  • the FLT3-CAR-T cells constructed with different FLT3 nanobody sequences have been proved to be very specific for FLT3-positive AML tumor cell lines through in vitro killing experiments.
  • the present invention successfully screened FLT3-CAR-T cells with the strongest ability to continuously kill AML tumor cells through repeated killing experiments on tumor cells. Since FLT3-CAR-T cells can replicate in vivo, they can continuously control tumors for a long time , is a good coping strategy for relapse-prone AML.
  • the FLT3-CAR-T cell therapy of the present invention can alleviate the suffering of patients and improve the prognosis of AML to a certain extent.
  • the treatment means of the present invention can be carried out in various ways such as injection, spraying, swallowing, infusion, etc. Compared with the existing chemotherapy strategies, the pain of patients receiving treatment is reduced, and the number of times required for treatment is also reduced;
  • the FLT3-CAR-T cell therapy of the present invention can control the tumor for a long time, reduce the possibility of recurrence, control the disease at a lower level, and improve the prognosis.
  • the FLT3-CAR-T cells of the present invention have a wider application range than the existing FLT3 small molecule inhibitors, and can be applied to a wider patient population.
  • FLT3-CAR-T cells are not limited to be effective only in AML patients with FLT3-ITD mutations, but also in AML patients without FLT3 mutations.
  • the FLT3-CAR-T cells of the present invention have better curative effect than the existing FLT3 small molecule inhibitors, and FLT3 is prone to new mutations in the tyrosine kinase domain, which will lead to drug resistance to FLT3 inhibitors sexuality, which limits the efficacy of FLT3 inhibitors.
  • the FLT3-CAR-T cells of the present invention are less affected by FLT3 mutations. Even if FLT3 is mutated, the FLT3-CAR-T cells still maintain their targeting and binding activity, and have little limitation on the curative effect.
  • an antigen FLT3-Fc protein expression vector was constructed.
  • the constructed FLT3-Fc protein expression vector was subjected to large-scale extraction of the plasmid, and after transiently transfecting 293 cells, cultured continuously for 8 days.
  • the culture supernatant was collected by centrifugation, filtered with a 0.45 ⁇ m filter membrane, and the filtrate was transferred to a sterile centrifuge tube , using Protein A column purification to obtain purified FLT3-Fc protein.
  • the purified FLT3-Fc protein is an antigen, also known as an immunogen or immune antigen.
  • the titer of the alpaca immune serum was tested. If the serum titer reached 1:8000 dilution after the third immunization, and the OD value was greater than 1.0, the shock immunization (the fourth immunization) could be carried out.
  • the peripheral blood of the alpaca obtained after the shock immunization was used to separate the immune serum for the construction of the subsequent yeast display library.
  • Collect 5 mL of the peripheral blood after triple immunization and/or peripheral blood after shock immunization prepared in Example 2 above place the centrifuge tube with the collected blood sample in a 37°C incubator for 1 hour, and then transfer the blood sample to 4°C overnight. Transfer the serum to a new sterile centrifuge tube, centrifuge at 5000rpm for 20min, and then use ELISA to detect the immune titer.
  • the immunized alpaca isolated serum was subjected to limiting dilution according to the dilution gradient shown in Table 4, and the ELISA experiment was performed on a 96-well plate pre-coated with FLT3 antigen, using negative serum as a negative control, PBS buffer as a blank control, and the detection results As shown in table 2.
  • the immune titer is good, and the OD value under the condition of 1:8000 has reached 2.204, which is much higher than 1.0, and the impact immunization can be carried out.
  • a shock immunization was performed, and 100 mL of peripheral blood was collected 3 days later for the construction of a yeast display library.
  • Example 1 Prepare 1 mg of the purified antigen prepared in Example 1, the buffer system is PBS, and the concentration is 1 mg/mL. Weigh NHS-biotin, dissolve it in DMSO, and prepare 10mM NHS-biotin.
  • Example 2 Collect 50 mL of peripheral blood after shock immunization prepared in Example 2, and use lymphocyte separation medium to sort PBMC.
  • Reagent Dosage The reaction solution after the above denaturation 80 ⁇ L 5 ⁇ PrimeScript II Buffer 32 ⁇ L RNase Inhibitor (40U/ ⁇ L) 4 ⁇ L PrimeScript II RTase (200U/ ⁇ L) 8 ⁇ L RNase-Free Water 36 ⁇ L
  • the PCR product was analyzed by electrophoresis using 1% agarose, and fragments with a molecular weight of about 750 bp were separated.
  • the PCR product was recovered using a gel recovery kit, and the concentration was determined with NanoDrop.
  • the PCR product was analyzed by electrophoresis using 1% agarose, and the VHH fragment with a molecular weight of about 400 bp was separated.
  • the VHH PCR product was recovered using a gel recovery kit, and the concentration was determined with a NanoDrop.
  • the peripheral blood after shock immunization prepared in Example 2 was collected, and total RNA was provided. After reverse transcription into cDNA, two rounds of PCR were performed using single-domain antibody amplification primers, and PCR products were identified by agarose gel electrophoresis.
  • PCR bands of about 1000 bp and 750 bp were obtained in the first round of PCR, and the 750 bp fragment recovered from the gel was used as a template for the second round of PCR, and the band of 450 bp obtained in the second round of PCR was VHH fragments.
  • SfiI was used to digest the pBlue vector and the product recovered from the VHH PCR gel obtained in the above-mentioned Example 5, respectively, and digest overnight at 50°C.
  • the construction of the yeast display vector was completed through the above steps, and the Escherichia coli library plasmid was obtained by electrotransformation.
  • the Escherichia coli library plasmid prepared in Example 6 was linearized with PmeI, and the enzyme digestion system was shown in Table 11:
  • Digest at 37°C for 3 hours take 5 ⁇ L for 1% agarose electrophoresis and detect, the remaining digested products are precipitated and concentrated for later use, and a total of 3 mg of plasmid is digested.
  • Competent cells are generally prepared and used immediately to ensure higher electroporation efficiency.
  • the electroporation of yeast competent cells is completed through the above steps, and the transformed strains of the yeast display library are obtained.
  • Example 8 Yeast Display Library Induced Expression and Sorting
  • yeast single clones were randomly selected for sequencing, and the diversity of the constructed yeast display library was analyzed.
  • the yeast display library has diversity, and the yeast display library has a storage capacity of 6.74 ⁇ 10 6 .
  • Biotin-FLT3-Fc was used for magnetic sorting of yeast-positive clones, and the sorting effect was better.
  • the second round of sorting was performed after the first round of sorting yeast cells were cultured to induce expression.
  • the positive rate of yeast was 91.721%, and the positive clones were significantly enriched.
  • the sorted products were directly coated on PAD plates, and single clones were selected for flow detection.
  • a part of the yeast was cultured to induce expression by flow cytometry; a part was directly coated on a PAD plate, picked a single clone for culture, inoculated into a 96-well plate, induced expression for 24 hours, and incubated with Biotin-FLT3-Fc , PE-Streptavidin was used as the secondary antibody, and flow detection was performed after incubation.
  • Example 8 According to the yeast monoclonal flow detection results in Example 8, clones with different binding abilities to the target antigen were selected, genomic DNA was extracted, PCR was performed using the universal primers of the pBlue vector, and the PCR products were sequenced to obtain the VHH antibody sequence.
  • VHH antibody sequences obtained from the analysis were separately gene-synthesized, subcloned in tandem with human IgG1 Fc into the expression vector Lenti-hIgG1-Fc2 as shown in Figure 6, and then sequenced to verify the vector.
  • the antibody expression vector Lenti-hIgG1-Fc2 was obtained, and the endotoxin-free plasmid was prepared by using the Qiagen plasmid extraction kit.
  • the recombinant antibody expressed by the antibody expression vector Lenti-hIgG1-Fc2 in Example 9 is obtained, also called single domain antibody, single domain antibody of the present invention, and FLT3 nanobody.
  • Example 11 Flow cytometric detection of the binding of the recombinant antibody to the target protein
  • Example 11 the positive recombinant antibody obtained in Example 11 was expressed and purified.
  • the FLT3-Fc recombinant protein was immobilized on the CM5 chip using 10mM Acetate buffer, and the single domain antibodies prepared in the above Examples 11 and 12 were used as the mobile phase to detect the binding ability of the candidate single domain antibody to the target protein FLT3, and the affinity detection The results are shown in Table 12.
  • test results show that the affinity of the single domain antibody of the present invention to the target protein FLT3 (very good, indicating that the single domain antibody has a good binding effect on the target protein FLT3).
  • the single-domain antibody targeting FLT3 was obtained by screening the yeast display library, and the antibody sequence information is shown in Figures 8A-8K.
  • the FR sequence (FR1-FR4) is marked with an underline
  • CDR1 is marked with a dash (.)
  • CDR2 is marked with a wavy line Marked
  • CDR3 with a line segment marked out.
  • Figures 8B-8K are represented in a similar fashion.
  • the suspension 293T lentivirus packaging system was used to package five kinds of FLT3-CAR with lentivirus, and the titer of each CAR virus stock solution was detected.
  • Example 17 Toxic effect of CAR-T cells constructed from different cloned FLT3 nanobody sequences on FLT3 positive target cells
  • the FLT3-negative target cell Raji was selected as the target cell to carry out the target cell killing experiment, and the difference in the cytotoxic effect of the CAR-T cells of the present invention and the control T cells on the negative target cells was compared.
  • the supernatant of the above killing experiment was collected, and the secretion of the cytokine Granzyme-B in the supernatant was further detected.
  • Example 18 CAR-T cells constructed from different clonal FLT3 nanobody sequences against FLT3-positive tumor cell lines Sustained lethality comparison
  • PA0135MN-CAR-T has a stronger and more durable killing ability against MV-4-11 tumor cells.
  • cytokines (Granzyme-B, IL-2, IFN-gama, TNF- ⁇ ) Release situation.
  • luciferase-labeled OCI-AML3 cells were selected to construct mouse tumor models, 15 female NCG mice were selected, and 1 ⁇ 10 6 tumor cells were inoculated into the tail vein of each mouse. On day 3, tail vein administration was performed, and a dose of 2 ⁇ 10 7 PA0135MN-CAR-T (TAA05-CAR-T) was administered. The growth of tumor cells in the mice of different administration groups was monitored every week.
  • PA0135MN-CAR-T (TAA05-CAR-T) cells can significantly inhibit the proliferation and growth of OCI-AML3 tumor cells in mice.
  • mice The body weight detection and survival curve of the mice are shown in Figure 21, and the results also showed that: compared with the control group, TAA05-CAR-T cells did not have a significant impact on the body weight of the mice, and TAA05-CAR-T cells It can significantly prolong the survival period of OCI-AML3 tumor-bearing mice, and has a significant anti-tumor effect in vivo.

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Abstract

提供了一种针对 FLT3 的纳米抗体、载体和由其构建的靶向FLT3 高表达肿瘤细胞的工程化免疫细胞，能够特异性结合人 FLT3，且具有高亲和力和显著的体内抗肿瘤作用。该免疫细胞受FLT3 突变的影响小，即使 FLT3 发生突变，也能保持其靶向性和结合活性，对疗效的限制小，可长期持续控制肿瘤，应对易复发的 AML。

Description

一种新型靶向人FLT3的嵌合抗原受体修饰的T细胞的构建及应用 技术领域
本发明属于生物医学及免疫细胞治疗领域，具体涉及一种特异性靶向FLT3的纳米抗体，和由其构建的靶向FLT3高表达肿瘤细胞的工程化免疫细胞，还涉及它们的用途。
背景技术
急性髓系白血病(Acute myeloid leukemia,AML)是一种临床预后很差的疾病。虽然大多数患者通过化疗可以达到缓解，但几乎都会复发，需要后期巩固化疗或进行造血干细胞移植(Hematopoietic stem cell transplantation,HSCT)，且大多数患者最终仍会死于疾病，5年生存率只有40-50％。因此，亟需一种治疗AML的新方法来满足现有治疗需求。
近几年，嵌合抗原受体(Chimeric antigen receptor,CAR)免疫疗法在B细胞恶性肿瘤的临床研究中展现出非常好的临床疗效，但是，针对AML的CAR-T疗法仍然面临着很大的挑战，我们很难找到一个针对肿瘤的合适靶点，因为表达在AML上的大多数表面分子同时也在造血干/祖细胞(Hematopoietic stem and progenitor cell,HSPC)表面也表达，它会在对AML细胞杀伤的同时也会损伤造血干细胞，从而造成血液毒性。因此找到一个相对安全且疗效较佳的靶点至关重要。
FLT3(Fms样酪氨酸激酶3，CD135)是人类中由FLT3基因编码的蛋白质，是一种细胞因子受体，属于受体酪氨酸激酶III类。有结果显示，FLT3在大约50％的正常造血干细胞(Hematopoietic stem cells,HSCs)和部分树突状细胞上表达，在脐血淋巴细胞上大部分不表达。但有研究发现，FLT3在AML病人细胞表面高表达，并且在初诊AML患者中，约1/3的患者存在FLT3激活突变。
靶向FLT3的药物，目前以小分子抑制剂的数量最多，进展最快，目前共有多个可作用于FLT3的小分子抑制剂药物获批上市，其中第一三共的奎扎替尼(Quizartinib)和安斯泰来的吉瑞替尼(Gilteritinib)属于第二代抑制剂，选择性较高，比第一代抑制剂效果更好，但FLT3抑制剂在临床上只对FLT3-ITD突变的AML病人有效，对FLT3未突变的AML患者无效，而且FLT3容易在酪氨酸激酶结构域部分产生新的突变而对FLT3抑制剂产生耐药性，导致疗效非常有限。
近些年来，嵌合抗原受体(CAR)T细胞在B细胞白血病和淋巴瘤患者中取得了前所未有的临床效果，提示这种新颖的治疗方式对于AML患者来说也可能是很有效的。但目前，国内外还没有靶向FLT3的CAR-T产品上市或正式获批临床。
发明内容
本发明的目的在于提供一种针对FLT3的纳米抗体、一种由所述纳米抗体构建的嵌 合抗原受体免疫细胞，以及它们的制法与应用。
在本发明的第一方面，提供了一种针对FLT3的纳米抗体。
在另一优选例中，所述针对FLT3的纳米抗体能够特异性结合FLT3。
在另一优选例中，所述针对FLT3的纳米抗体的互补决定区CDR为选自下组的一种或多种：
(1)SEQ ID NO:18所示的CDR1、SEQ ID NO:19所示的CDR2、和SEQ ID NO:20所示的CDR3；
(2)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:23所示的CDR3；
(3)SEQ ID NO:24所示的CDR1、SEQ ID NO:19所示的CDR2、和SEQ ID NO:25所示的CDR3；
(4)SEQ ID NO:21所示的CDR1、SEQ ID NO:26所示的CDR2、和SEQ ID NO:27所示的CDR3；
(5)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:28所示的CDR3；
(6)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:29所示的CDR3；
(7)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:30所示的CDR3；
(8)SEQ ID NO:31所示的CDR1、SEQ ID NO:26所示的CDR2、和SEQ ID NO:32所示的CDR3；
(9)SEQ ID NO:33所示的CDR1、SEQ ID NO:34所示的CDR2、和SEQ ID NO:35所示的CDR3；
(10)SEQ ID NO:31所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:36所示的CDR3；
(11)SEQ ID NO:31所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:37所示的CDR3。
在另一优选例中，上述氨基酸序列中任意一种氨基酸序列还包括任选地经过添加、缺失、修饰和/或取代至少一个(如1-3个，较佳地1-2个，更佳地1个)氨基酸并能保留与FLT3特异性结合能力的衍生序列。
在另一优选例中，所述的经过添加、缺失、修饰和/或取代至少一个氨基酸的，并能够保留与FLT3特异性结合能力的衍生序列为同源性或序列相同性为至少85％、至少90％、至少95％、至少96％、至少97％、至少98％或至少99％的氨基酸序列。
在另一优选例中，所述的CDR1、CDR2和CDR3由VHH链的框架区FR1、FR2、FR3 和FR4所隔开。
在另一优选例中，所述针对FLT3的纳米抗体还包括骨架区FR。
在另一优选例中，所述骨架区FR衍生自如SEQ ID NO:7-17所示的氨基酸序列。
在另一优选例中，所述的骨架区FR为选自下组的一种或多种：
(1)SEQ ID NO:38所示的FR1、SEQ ID NO:39所示的FR2、SEQ ID NO:40所示的FR3、和SEQ ID NO:41所示的FR4；
(2)SEQ ID NO:42所示的FR1、SEQ ID NO:43所示的FR2、SEQ ID NO:44所示的FR3、和SEQ ID NO:41所示的FR4；
(3)SEQ ID NO:45所示的FR1、SEQ ID NO:46所示的FR2、SEQ ID NO:47所示的FR3、和SEQ ID NO:48所示的FR4；
(4)SEQ ID NO:42所示的FR1、SEQ ID NO:43所示的FR2、SEQ ID NO:49所示的FR3、和SEQ ID NO:50所示的FR4；
(5)SEQ ID NO:42所示的FR1、SEQ ID NO:51所示的FR2、SEQ ID NO:52所示的FR3、和SEQ ID NO:53所示的FR4；
(6)SEQ ID NO:42所示的FR1、SEQ ID NO:54所示的FR2、SEQ ID NO:44所示的FR3、和SEQ ID NO:55所示的FR4；
(7)SEQ ID NO:56所示的FR1、SEQ ID NO:43所示的FR2、SEQ ID NO:57所示的FR3、和SEQ ID NO:58所示的FR4；
(8)SEQ ID NO:59所示的FR1、SEQ ID NO:60所示的FR2、SEQ ID NO:61所示的FR3、和SEQ ID NO:50所示的FR4；
(9)SEQ ID NO:62所示的FR1、SEQ ID NO:63所示的FR2、SEQ ID NO:64所示的FR3、和SEQ ID NO:48所示的FR4；
(10)SEQ ID NO:65所示的FR1、SEQ ID NO:60所示的FR2、SEQ ID NO:66所示的FR3、和SEQ ID NO:67所示的FR4；
(11)SEQ ID NO:62所示的FR1、SEQ ID NO:60所示的FR2、SEQ ID NO:68所示的FR3、和SEQ ID NO:48所示的FR4。
在另一优选例中，所述的针对FLT3的纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17，或其组合。
在另一优选例中，所述的针对FLT3的纳米抗体包括人源化抗体、骆驼源抗体、嵌合抗体。
在另一优选例中，所述的针对FLT3的纳米抗体为羊驼。
在本发明的第二方面，提供一种针对FLT3的抗体，所述抗体包括一个或多个如本 发明第一方面所述的针对FLT3的纳米抗体的VHH链。
在另一优选例中，所述的针对FLT3的纳米抗体的VHH链的氨基酸序列选自下组：SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17，或其组合。
在另一优选例中，所述的针对FLT3的抗体，可以为单体、二价抗体、和/或多价抗体。
在本发明的第三方面，提供一种嵌合抗原受体CAR，所述CAR含有一胞外结构域，所述胞外结构域包含如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体。
在另一优选例中，所述胞外结构域还包括信号肽。
在另一优选例中，所述胞外结构域还包括选自下组蛋白的铰链区：CD8、CD28、CD137、IgG，或其组合。
在另一优选例中，所述铰链区为human IgG1 Fc铰链区。
在另一优选例中，所述胞外结构域含有的抗体具有SEQ ID NO:7-17所示的氨基酸序列。
在另一优选例中，所述胞外结构域含有的抗体其氨基酸序列与SEQ ID NO:7-17的同源性≥85％，较佳地≥90％，更佳地≥95％，或与SEQ ID NO:7-17相比具有1、2或3个氨基酸的差异。
在另一优选例中，所述的CAR具有式Ia所示结构：
L1-Nb-H-TM-C-CD3ζ            (Ia)
式中，
L为无或信号肽序列；
Nb是特异性结合结构域；
H为无或铰链区；
TM为跨膜结构域；
C为共刺激信号结构域；
CD3ζ为源于CD3ζ的胞浆信号传导序列(包括野生型、或其突变体/修饰体)；
所述“-”连接肽或肽键。
在另一优选例中，所述L分别选自下组蛋白的信号肽：CD8、GM-CSF、CD4、CD28、CD137，或其突变/修饰体，或其组合。
在另一优选例中，所述Nb靶向FLT3。
在另一优选例中，所述Nb为FLT3纳米抗体。
在另一优选例中，所述H选自下组蛋白的铰链区：CD8、CD28、CD137、IgG，或其 组合。
在另一优选例中，所述H为human IgG1 Fc铰链区。
在另一优选例中，所述TM选自下组蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、CD278、CD152、CD279、CD233，或其突变/修饰体，或其组合。
在另一优选例中，所述C选自下组蛋白的共刺激结构域：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD-1、Dap10、LIGHT、NKG2C、B7-H3、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、OX40L、2B4、TLR，或其突变/修饰体，或其组合。
在另一优选例中，所述C选自ICOS、41BB，或其组合的共刺激结构域。
在另一优选例中，所述CAR的氨基酸序列如SEQ ID NO:1、3、4、5、6所示。
在另一优选例中，所述CAR的核苷酸序列如SEQ ID NO:2所示。
在本发明的第四方面，提供了一种重组蛋白，所述的重组蛋白具有：
(i)如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体；和
(ii)任选的协助表达和/或纯化的标签序列。
在另一优选例中，所述的标签序列包括Fc标签、HA标签、GGGS序列、FLAG标签、Myc标签、6His标签，或其组合。
在另一优选例中，所述的重组蛋白特异性结合FLT3。
在另一优选例中，所述的重组蛋白(或多肽)包括融合蛋白。
在另一优选例中，所述的重组蛋白为单体、二聚体，或多聚体。
在另一优选例中，所述的重组蛋白特异性结合FLT3。
在另一优选例中，所述的标签序列是Fc标签。
在本发明的第五方面，提供一种多核苷酸，所述多核苷酸编码选自下组的蛋白质：如本发明第一方面所述的针对FLT3的纳米抗体，或本发明第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白。
在另一优选例中，本发明涉及编码本发明的针对FLT3的纳米抗体的核酸分子。本发明的核酸可为RNA、DNA或cDNA。
在本发明的第六方面，提供一种表达载体，所述表达载体含有本发明的第五方面所述的多核苷酸。
在另一优选例中，所述的表达载体选自下组：DNA、RNA、病毒载体、质粒、转座 子、其他基因转移系统、或其组合。优选地，所述表达载体包括病毒载体，如慢病毒、腺病毒、AAV病毒、逆转录病毒、或其组合。
在另一优选例中，所述的表达载体选自下组：pTomo慢病毒载体、plenti、pLVTH、pLJM1、pHCMV、pLBS.CAG、pHR、pLV、pBlue等。
在另一优选例中，所述的表达载体是pBlue载体。
在另一优选例中，所述的表达载体中还包括选自下组的：启动子、转录增强元件WPRE、长末端重复序列LTR等。
在本发明的第七方面，提供一种宿主细胞，所述宿主细胞含有本发明的第六方面所述的表达载体，或其基因组中整合有本发明的第五方面所述的多核苷酸。
在另一优选例中，所述的宿主细胞包括原核细胞或真核细胞。
在另一优选例中，所述的宿主细胞选自下组：大肠杆菌、酵母细胞、哺乳动物细胞。
在另一优选例中，所述的宿主细胞为293F细胞。
在本发明的第八方面，提供一种工程化免疫细胞，所述工程化免疫细胞含有本发明的第六方面所述的表达载体或染色体中整合有外源的本发明的第五方面所述的多核苷酸。
在另一优选例中，所述工程化免疫细胞包含如本发明的第三方面所述的嵌合抗原受体。
在另一优选例中，所述工程化免疫细胞选自下组：
(i)嵌合抗原受体αβT细胞(CAR-T细胞)；
(ii)嵌合抗原受体γδT细胞(CAR-T细胞)；
(iii)嵌合抗原受体NKT细胞(CAR-NKT细胞)；
(iv)嵌合抗原受体NK细胞(CAR-NK细胞)。
在另一优选例中，所述工程化免疫细胞包括自体或异体的αβT细胞、γδT细胞、NKT细胞、NK细胞，或其组合。
在另一优选例中，所述工程化免疫细胞为CAR-T细胞。
在本发明的第九方面，提供一种产生针对FLT3的纳米抗体的方法，包括步骤：
(a)在适合产生纳米抗体的条件下，培养如本发明的第七方面所述的宿主细胞，从而获得含针对FLT3的纳米抗体的培养物；
(b)从所述培养物中分离和/或回收所述的针对FLT3的纳米抗体；和
(c)任选地，对步骤(b)获得的针对FLT3的纳米抗体进行纯化和/或修饰。
在本发明的第十方面，提供一种制备如本发明第八方面所述的工程化免疫细胞的方法，包括以下步骤：将如本发明的第五方面所述的多核苷酸或如本发明的第六方面所述的表达载体转导入免疫细胞内，从而获得所述工程化免疫细胞。
在另一优选例中，所述的方法还包括对获得的工程化免疫细胞进行功能和有效性检测的步骤。
在另一优选例中，所述的方法包括将如本发明的第三方面所述的嵌合抗原受体转导入免疫细胞内，从而获得所述工程化免疫细胞。
在本发明的第十一方面，提供一种免疫偶联物，所述免疫偶联物含有：
(a)如本发明第一方面所述的针对FLT3的纳米抗体，或如本发明第二方面所述的针对FLT3的抗体，或如本发明第四方面所述的重组蛋白；和
(b)选自下组的偶联部分：可检测标记物、药物、细胞因子、放射性核素、酶、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP，或其组合。
在另一优选例中，所述的(a)部分与所述的偶联部分通过化学键或接头进行偶联。
在另一优选例中，所述的放射性核素包括：
(i)诊断用同位素，所述的诊断用同位素选自下组：Tc-99m、Ga-68、F-18、I-123、I-125、I-131、In-111、Ga-67、Cu-64、Zr-89、C-11、Lu-177、Re-188，或其组合；和/或(ii)治疗用同位素，所述的治疗用同位素选自下组：Lu-177、Y-90、Ac-225、As-211、Bi-212、Bi-213、Cs-137、Cr-51、Co-60、Dy-165、Er-169、Fm-255、Au-198、Ho-166、I-125、I-131、Ir-192、Fe-59、Pb-212、Mo-99、Pd-103、P-32、K-42、Re-186、Re-188、Sm-153、Ra223、Ru-106、Na24、Sr89、Tb-149、Th-227、Xe-133、Yb-169、Yb-177，或其组合。
在另一优选例中，所述偶联部分为药物或毒素。
在另一优选例中，所述的药物为靶向治疗FLT3高表达疾病的药物。
在另一优选例中，所述的药物为靶向治疗急性髓系白血病的药物。
在另一优选例中，所述的药物为细胞毒性药物。
在另一优选例中，所述的细胞毒性药物选自下组：抗微管蛋白药物、DNA小沟结合试剂、DNA复制抑制剂、烷化试剂、抗生素、叶酸拮抗物、抗代谢药物、化疗增敏剂、拓扑异构酶抑制剂、长春花生物碱，或其组合。
特别有用的细胞毒性药物类的例子包括，例如，DNA小沟结合试剂、DNA烷基化试剂、和微管蛋白抑制剂、典型的细胞毒性药物包括、例如奥瑞他汀(Auristatins)、喜树碱(Camptothecins)、多卡霉素/倍癌霉素(Duocarmycins)、依托泊甙(Etoposides)、美登木素(Maytansines)和美登素类化合物(Maytansinoids)(例如DM1和DM4)、紫杉烷(Taxanes)、苯二氮卓类(Benzodiazepines)或者含有苯二氮卓的药物(Benzodiazepine containing drugs)(例如吡咯并[1,4]苯二氮卓类(PBDs)，吲哚啉苯并二氮卓类(Indolinobenzodiazepines)和噁唑烷并苯并二氮卓类(Oxazolidinobenzodiazepines))、长春花生物碱(Vinca alkaloids)，或其组合。
在另一优选例中，所述的毒素选自下组：耳他汀类(例如，耳他汀E、耳他汀F、MMAE 和MMAF)、金霉素、类美坦西醇、篦麻毒素、篦麻毒素A-链、考布他汀、多卡米星、多拉司他汀、阿霉素、柔红霉素、紫杉醇、顺铂、cc1065、溴化乙锭、丝裂霉素、依托泊甙、替诺泊甙(Tenoposide)、长春新碱、长春碱、秋水仙素、二羟基炭疽菌素二酮、放线菌素、白喉毒素、假单胞菌外毒素(PE)A、PE40、相思豆毒素、相思豆毒素A链、蒴莲根毒素A链、α-八叠球菌、白树毒素、迈托毒素(Mitogellin)、局限曲菌素(Retstrictocin)、酚霉素、依诺霉素、麻疯树毒蛋白(Curicin)、巴豆毒素、卡奇霉素、肥皂草(Sapaonaria officinalis)抑制剂、糖皮质激素，或其组合。
在另一优选例中，所述偶联部分为可检测标记物。
在另一优选例中，所述偶联部分选自下组：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂，或能够产生可检测产物的酶、放射性核素、生物毒素、细胞因子(如IL-2等)、抗体、抗体Fc片段、抗体scFv片段、金纳米颗粒/纳米棒、病毒颗粒、脂质体、纳米磁粒、前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))或任何形式的纳米颗粒。
在另一优选例中，所述免疫偶联物含有：多价(如二价)的如本发明的第一方面所述的针对FLT3的纳米抗体的VHH链。
在另一优选例中，所述多价是指在所述免疫偶联物的氨基酸序列中包含多个重复的相同或不同的如本发明的第一方面所述针对FLT3的纳米抗体VHH链。
在本发明的第十二方面，提供一种活性成分的用途，所述活性成分选自下组：如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第八方面所述的工程化免疫细胞，或如本发明第十一方面所述的免疫偶联物，或其组合，所述活性成分被用于制备：
(a)预防和/或治疗FLT3高表达疾病的药物；
(b)检测FLT3高表达疾病的试剂。
在另一优选例中，所述活性成分选自下组：如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第八方面所述的工程化免疫细胞，或其组合。
在另一优选例中，所示试剂为诊断试剂，较佳地，所述的诊断试剂为检测片或检测板。
在另一优选例中，所述诊断试剂用于：检测样品中的FLT3蛋白或其片段。
在另一优选例中，所述FLT3高表达疾病选自：急性髓系白血病、急性淋巴细胞白血病(Acute lymphoblastic leukemia,ALL)、慢性髓性白血病(Chronic myelogenous leukemia,CML)、骨髓增生异常综合征(Myelodysplastic syndromes,MDS)等。
在另一优选例中，所述FLT3高表达疾病为急性髓系白血病。
在本发明的第十三方面，提供了一种体外检测样品中FLT3蛋白或其片段的方法，所述方法包括步骤：
(1)在体外，将所述样品与如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第八方面所述的工程化免疫细胞，或如本发明第十一方面所述的免疫偶联物，或其组合接触；
(2)检测是否形成抗原-抗体复合物，其中形成复合物就表示样品中存在FLT3蛋白或其片段。
在另一优选例中，所述的检测包括诊断性的或非诊断性的。
在本发明的第十四方面，提供了一种药物组合物，所述药物组合物含有：
(i)如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第八方面所述的工程化免疫细胞，或如本发明第十一方面所述的免疫偶联物，或其组合作为活性成分；和
(ii)药学上可接受的载体、稀释剂或赋形剂。
在另一优选例中，所述活性成分选自下组：如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第八方面所述的工程化免疫细胞，或其组合。
在另一优选例中，所述药物组合物的剂型选自下组：注射剂、冻干剂。
在另一优选例中，所述的药物组合物包括0.01～99.99％的如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第十一方面所述的免疫偶联物，或其组合和0.01～99.99％的药用载体，所述百分比为占所述药物组合物的质量百分比。
在另一优选例中，所述活性成分中所述工程化的免疫细胞的浓度为1×10 3-1×10 8个细胞/mL，较佳地1×10 4-1×10 7个细胞/mL。
本发明的第十五方面，提供了一种试剂盒，所述试剂盒中包括：
(1)第一容器，所述第一容器中含有如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第八方面所述的工程化免疫细胞，或如本发明第十一方面所述的免疫偶联物，或其组合；和/或
(2)第二容器，所述第二容器中含有抗所述第一容器内容物的二抗；
或者，
所述试剂盒含有一检测板，所述检测板包括：基片(支撑板)和测试条，所述的测试条含有如本发明的第一方面所述的针对FLT3的纳米抗体、如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的重组蛋白，或如本发明第八方面所述的免疫偶联物，或其组合。
在另一优选例中，所述试剂盒中还含有一说明书，根据所述的说明书记载，所述的试剂盒用于非侵入性地检测待测对象的FLT3的表达。
在另一优选例中，所述的试剂盒用于FLT3高表达疾病的检测。
在另一优选例中，所述FLT3高表达疾病选自：急性髓系白血病、急性淋巴细胞白血病、慢性髓性白血病、骨髓增生异常综合征等。
在另一优选例中，所述FLT3高表达疾病为急性髓系白血病。
在本发明的第十六方面，提供了一种预防和/或治疗FLT3高表达疾病的方法，所述方法包括：给需要的对象施用如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第八方面所述的工程化免疫细胞，或如本发明第十一方面所述的免疫偶联物，或如本发明的第十四方面所述的药物组合物，或其组合。
在另一优选例中，所述对象包括哺乳动物，如人。
在另一优选例中，所述FLT3高表达疾病选自：急性髓系白血病、急性淋巴细胞白血病、慢性髓性白血病、骨髓增生异常综合征等。
在另一优选例中，所述FLT3高表达疾病为急性髓系白血病。
在另一优选例中，所述的工程化免疫细胞或药物组合物中所包含的CAR免疫细胞是来源于所述对象的细胞(自体细胞)。
在另一优选例中，所述的工程化免疫细胞或药物组合物中所包含的CAR免疫细胞是来源于健康个体的细胞(异体细胞)。
在另一优选例中，所述的方法可与其他治疗方法联合使用。
在另一优选例中，所述的其他治疗方法包括化疗、放疗、靶向治疗等方法。
在本发明的第十七方面，提供了一种针对FLT3高表达疾病的诊断方法，包括步骤：
(i)从诊断对象获取一样品，将所述的样品与如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，或如本发明第八方面所述的工程化免疫细胞，或如本发明第十一方面所述的免疫偶联物，或其组合接触；和
(ii)检测是否形成抗原-抗体复合物，其中形成复合物就表示所述的对象为FLT3高 表达疾病的确诊患者。
在另一优选例中，所述的样品为血液样品或咽拭子样品，或其他组织器官中的样品。
在另一优选例中，所述FLT3高表达疾病选自：急性髓系白血病、急性淋巴细胞白血病、慢性髓性白血病、骨髓增生异常综合征等。
在另一优选例中，所述FLT3高表达疾病为急性髓系白血病。
在本发明的第十八方面，提供了一种重组多肽的制备方法，所述的重组多肽是如本发明的第一方面所述的针对FLT3的纳米抗体，或如本发明的第二方面所述的针对FLT3的抗体，或如本发明第三方面所述的嵌合抗原受体，或如本发明第四方面所述的重组蛋白，所述的方法包括：
(a)在适合表达的条件下，培养如本发明第五方面所述的宿主细胞；和
(b)从培养物中分离出所述的重组多肽。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了VHH片段的扩增结果。
图2显示了酵母展示库多样性比对结果。
图3显示了第一次分选后流式检测结果。
图4显示了第二次分选后流式检测结果。
图5显示了酵母单克隆流式检测结果。
图6显示了构建的VHH真核表达载体的结构。
图7显示了FLT3单域抗体真核表达流式检测结果。
图8A-8K显示了靶向FLT3的单域抗体序列信息。
图9显示了FLT3-CAR载体结构图，其中FC为human IgG1 Fc铰链区。
图10显示了不同结构的FLT3-CAR慢病毒滴度检测结果。
图11显示了不同肿瘤细胞株表面FLT3的表达情况。
图12显示了不同结构FLT3-CAR-T对Raji细胞(FLT3阴性细胞株)的体外杀伤。
图13显示了不同结构FLT3-CAR-T对FLT3阳性肿瘤细胞株的体外杀伤。靶细胞分别为MV-4-11、MOLM-13、AML3，设置不同的效靶比。
图14显示了不同结构FLT3-CAR-T与FLT3阳性肿瘤细胞株孵育后上清中细胞因子Granzyme-B的分泌情况。靶细胞分别为MV-4-11、MOLM-13、AML3，效靶比分别为1:1。
图15显示了不同结构FLT3-CAR-T对FLT3阳性肿瘤细胞株MV-4-11的持续杀伤。靶 细胞分别为MV-4-11，效靶比分别为1:1和1:5。每隔24h或48小时补加一次肿瘤细胞。
图16显示了人HSPC细胞表面FLT3和CD33的表达情况。
图17显示了CD34+HSPC细胞分别与不同FLT3-CAR-T细胞孵育后细胞因子的释放情况。
图18显示了不同CAR-T细胞与CD34+HSPC细胞孵育后克隆形成情况。BFU-E代表HSPC细胞中红系细胞的克隆形成数量，CFU-GM代表HSPC细胞中粒系和巨噬细胞的克隆形成情况。
图19显示了TAA05-CAR-T对OCI-AML3-Luc-GFP模型鼠的药效实验方案。
图20显示了TAA05-CAR-T对AML3-Luc-GFP模型小鼠的药效实验小鼠活体荧光成像。
A.小鼠给药后不同时间的活体成像；B.D16至D23小鼠活体成像荧光值的增长情况：CAR-T组的5只小鼠中有4只出现负增长，反映出CAR-T回输后肿瘤出现了消退的现象。
图21显示了TAA05-CAR-T对AML3-Luc-GFP模型鼠的药效实验小鼠体重及生存期。
A.各组小鼠的体重变化曲线：PBS和Mock T组小鼠在发病后均出现体重下降的情况，CAR-T组小鼠体重一直比较平稳；B.小鼠生存期：CAR-T组小鼠生存期明显延长。
具体实施方式
本发明人经过广泛而深入的研究，经过大量的筛选，首次开发了一种新型FLT3纳米抗体，并基于开发的FLT3纳米抗体成功构建了靶向FLT3的FLT3-CAR-T细胞，用于治疗难治复发AML等肿瘤患者。本发明人通过大量的体外功能实验和动物实验证明了开发的FLT3-CAR-T细胞产品对AML肿瘤细胞具有显著的体外和体内抗肿瘤作用，并证明了同CD33-CAR-T细胞相比，本发明的FLT3-CAR-T细胞具有更好的安全性。在此基础上完成了本发明。本发明的靶向FLT3的新型FLT3-CAR-T可以作为靶向治疗难治复发AML的一种新型治疗手段。
本发明以CAR-T细胞为例，代表性地对本发明的工程化免疫细胞进行详细说明。本发明的工程化免疫细胞不限于上下文所述的CAR-T细胞，本发明的工程化免疫细胞具有与上下文所述的CAR-T细胞相同或类似的技术特征和有益效果。具体地，当免疫细胞表达嵌合抗原受体CAR时，NK细胞等同于T细胞(或T细胞可替换为NK细胞)。
术语
如本文所用，术语“单域抗体”、“本发明的单域抗体”、“重组抗体”、“FLT3纳米抗体”、“抗FLT3纳米抗体”可互换使用，均指本发明中与靶蛋白FLT3特异性结合的重组/单域抗体。
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。
本发明的纳米抗体的抗体编号以及对应的序列编号如下表1所示。
表1
Figure PCTCN2022144151-appb-000001
注：表中各个数值表示序列编号，即“1”表示“SEQ ID NO:1”，表中显示的CDR1、CDR2、CDR3、FR1、FR2、FR3、FR4的序列编号为其氨基酸序列的编号。
如本文所用，术语“抗体”或“免疫球蛋白”是有相同结构特征的约150000道尔顿的异四聚糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条轻链通过一个共价二硫键与重链相连，而不同免疫球蛋白同种型的重链间的二硫键数目不同。每条重链和轻链也有规则间隔的链内二硫键。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。特殊的氨基酸残基在轻链和重链的可变区之间形成界面。
如本文所用，术语“单域抗体”、“VHH”、“纳米抗体(Nanobody)”、“单域抗体(Single domain antibody,sdAb，或纳米抗体nanobody)”具有相同的含义并可互换使用，指克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)，它是具有完整功能的最小的抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体(VHH)。
如本文所用，术语“可变”表示抗体中可变区的某些部分在序列上有所不同，它形成了各种特定抗体对其特定抗原的结合和特异性。然而，可变性并不均匀地分布在整个抗体可变区中。它集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。可变区中较保守的部分称为骨架区(FR)。天然重链和轻链的可变区中各自包含四个FR区，它们大致上呈β-折叠构型，由形成连接环的三个CDR相连，在某些情况下可形成部分b折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位 (参见Kabat等，NIH Publ.No.91-3242，卷I,647-669页(1991))。恒定区不直接参与抗体与抗原的结合，但是它们表现出不同的效应功能，例如参与抗体的依赖于抗体的细胞毒性。
如本领域技术人员所知，免疫偶联物及融合表达产物包括：药物、毒素、细胞因子(Cytokine)、放射性核素、酶和其他诊断或治疗分子与本发明的抗体或其片段结合而形成的偶联物。本发明还包括与所述的针对FLT3的纳米抗体或其片段结合的细胞表面标记物或抗原。
如本文所用，术语“重链可变区”与“VH”可互换使用。
如本文所用，术语“可变区”与“互补决定区(Complementarity determining region,CDR)”可互换使用。
在本发明的一个优选的实施方式中，所述抗体的重链可变区包括三个互补决定区CDR1、CDR2、和CDR3。
在本发明的一个优选的实施方式中，所述抗体的重链包括上述重链可变区和重链恒定区。
在本发明中，术语“本发明抗体”、“本发明蛋白”、或“本发明多肽”可互换使用，都指特异性结合FLT3蛋白的多肽，例如具有重链可变区的蛋白或多肽。它们可含有或不含起始甲硫氨酸。
本发明还提供了具有本发明抗体的其他蛋白质或融合表达产物。具体地，本发明包括具有含可变区的重链的任何蛋白质或蛋白质偶联物及融合表达产物(即免疫偶联物及融合表达产物)，只要该可变区与本发明抗体的重链可变区相同或至少90％同源性，较佳地至少95％同源性。
一般，抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个骨架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相互靠近，重链上的CDR和相应轻链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
本发明抗体的重链的可变区特别令人感兴趣，因为它们中至少部分涉及结合抗原。因此，本发明包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。
本发明不仅包括完整的抗体，还包括具有免疫活性的抗体的片段或抗体与其他序列形成的融合蛋白。因此，本发明还包括所述抗体的片段、衍生物和类似物。
如本文所用，术语“片段”、“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的多肽片段、衍生物或类似物可以是(i)有一个或多个保守或非保守性氨基酸残基(优选保守性氨基酸残基)被取代的多肽，而这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的，或(ii)在一个或多个氨基酸残基中具有取代基团的多肽，或(iii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iv)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列 或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些片段、衍生物和类似物属于本领域熟练技术人员公知的范围。
本发明抗体指具有FLT3蛋白结合活性的、包括上述CDR区的多肽。该术语还包括具有与本发明抗体相同功能的、包含上述CDR区的多肽的变异形式。这些变异形式包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。该术语还包括本发明抗体的活性片段和活性衍生物。
该多肽的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。
本发明还提供了其他多肽，如包含抗体或其片段的融合蛋白。除了几乎全长的多肽外，本发明还包括了本发明抗体的片段。通常，该片段具有本发明抗体的至少约50个连续氨基酸，较佳地至少约50个连续氨基酸，更佳地至少约80个连续氨基酸，最佳地至少约100个连续氨基酸。
在本发明中，“本发明抗体的保守性变异体”指与本发明抗体的氨基酸序列相比，有至多10个，较佳地至多8个，更佳地至多5个，最佳地至多3个氨基酸被性质相似或相近的氨基酸所替换而形成多肽。这些保守性变异多肽最好根据表2进行氨基酸替换而产生。
表2
最初的残基 代表性的取代 优选的取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Lys；Arg Gln
Asp(D) Glu Glu
Cys(C) Ser Ser
Gln(Q) Asn Asn
Glu(E) Asp Asp
Gly(G) Pro；Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe Leu
Leu(L) Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Leu；Val；Ile；Ala；Tyr Leu
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala Leu
本发明还提供了编码上述抗体或其片段或其融合蛋白的多核苷酸分子。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
编码本发明的成熟多肽的多核苷酸包括：只编码成熟多肽的编码序列；成熟多肽的编码序列和各种附加编码序列；成熟多肽的编码序列(和任选的附加编码序列)以及非编码序列。
术语“编码多肽的多核苷酸”可以是包括编码此多肽的多核苷酸，也可以是还包括附加编码和/或非编码序列的多核苷酸。
本发明还涉及与上述的序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC,0.1％SDS,60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。
目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。
本发明还涉及包含上述的适当DNA序列以及适当启动子或者控制序列的载体。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细 胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl 2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的重组多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
本发明的抗体可以单独使用，也可与可检测标记物(为诊断目的)、治疗剂、PK(蛋白激酶)修饰部分或任何以上这些物质的组合结合或偶联。
用于诊断目的可检测标记物包括但不限于：荧光或发光标记物、放射性标记物、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂、或能够产生可检测产物的酶。
可与本发明抗体结合或偶联的治疗剂包括但不限于：1.放射性核素；2.生物毒；3.细胞因子如IL-2等；4.金纳米颗粒/纳米棒；5.病毒颗粒；6.脂质体；7.纳米磁粒；8.前药激活酶(例如，DT-心肌黄酶(DTD)或联苯基水解酶-样蛋白质(BPHL))等。
纳米抗体(Nb)
如本文所用，术语“纳米抗体”(Nanobody,Nb)指在羊驼外周血液中存在一种天然缺失轻链的抗体，纳米抗体和传统抗体的主要区别在于它们的结构和结构域：传统的抗体有两个可变域，称为VH和VL，它们相互提供稳定性和结合特异性；纳米抗体只包含一个重链可变区(VHH)、一个铰链区和两个常规的恒定区CH2与CH3，缺乏VL结构域，但仍然高度稳定。缺少VL结构域也意味着纳米体有亲水的一面，且纳米抗体不像人工改造的单链抗体片段(scFv)那样容易相互沾粘，甚至聚集成块。更重要的是单独克隆并表达出来的VHH结构具有与原重链抗体相当的结构稳定性以及与抗原的结合活性，是已知的可结合目标抗原的最小单位。VHH晶体为2.5nm，长4nm，分子量只有15KDa。
纳米抗体的抗原结合特性可由位于重链可变区的3个特定的区域来描述，称为可变区域(CDR)，将该段间隔成4个框架区域(FR)，4个FR的氨基酸序列相对比较保守，不直接参与结合反应。这些CDR形成环状结构，通过其间的FR形成的β折叠在空间结构上相 互靠近，重链上的CDR构成了抗体的抗原结合位点。可以通过比较同类型的抗体的氨基酸序列来确定是哪些氨基酸构成了FR或CDR区域。
嵌合抗原受体(CAR)
如本文所用，嵌合免疫抗原受体包括细胞外结构域、任选的铰链区、跨膜结构域、和细胞内结构域。胞外结构域包括任选的信号肽和靶点特异性结合结构域(也称为抗原结合结构域)。细胞内结构域包括共刺激结构域和CD3ζ链部分。CAR在T细胞中表达时，胞外段可识别一个特异的抗原，随后通过胞内结构域转导该信号，引起细胞的活化增殖、细胞溶解毒性和分泌细胞因子如IL-2和IFN-γ等，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和CD3ζ链中的一个或多个的细胞内结构域融合。
嵌合抗原受体T细胞(CAR-T细胞)
如本文所用，术语“CAR-T细胞”、“CAR-T”、“FLT3-CAR-T细胞”、“本发明的CAR-T细胞”等均指本发明第八方面所述的CAR-T细胞。本发明CAR-T细胞可用于治疗FLT3高表达的肿瘤，如急性髓系白血病、急性淋巴细胞白血病、慢性髓性白血病、骨髓增生异常综合征等。
CAR-T细胞较其它基于T细胞的治疗方式存在以下优势：(1)CAR-T细胞的作用过程不受MHC的限制；(2)鉴于很多肿瘤细胞表达相同的肿瘤抗原，针对某一种肿瘤抗原的CAR基因构建一旦完成，便可以被广泛利用；(3)CAR既可以利用肿瘤蛋白质抗原，又可利用糖脂类非蛋白质抗原，扩大了肿瘤抗原的靶点范围；(4)使用患者自体细胞降低了排异反应的风险；(5)CAR-T细胞具有免疫记忆功能，可以长期在体内存活。
嵌合抗原受体NK细胞(CAR-NK细胞)
如本文所用，术语“CAR-NK细胞”、“CAR-NK”、“FLT3-CAR-NK细胞”、“本发明的CAR-NK细胞”等均指本发明第八方面所述的CAR-NK细胞。本发明CAR-NK细胞可用于治疗FLT3高表达的肿瘤，如急性髓系白血病、急性淋巴细胞白血病、慢性髓性白血病、骨髓增生异常综合征等。
自然杀伤细胞(Natural killer cell,NK)是一类主要的免疫效应细胞，通过非抗原特异性途径去保护机体免受病毒感染和肿瘤细胞的侵袭。通过工程化(基因修饰)的NK细胞可能获得新的功能，包括特异性识别肿瘤抗原的能力及具有增强的抗肿瘤细胞毒作用。
与自体CAR-T细胞相比，CAR-NK细胞还具有以下优点，例如：(1)通过释放穿孔素和颗粒酶直接杀伤肿瘤细胞，而对机体正常的细胞没有杀伤作用；(2)它们释放很少量的细胞因子从而降低了细胞因子风暴的危险；(3)体外极易扩增及发展为“现成的”产品。除此之外，与CAR-T细胞治疗类似。
FLT3
FLT3(Fms-like tyrosine kinase,FMS样的酪氨酸激酶3)属于III型受体酪氨酸激酶(Receptor tyrosine kinase III,RTK III)家族成员，近年来，许多大样本研究已经证实FLT3的激活突变在AML等疾病的发生及进展中起到十分重要的病理作用。
具有FLT3/ITD激活突变的AML患者通常具有外周血白细胞计数高，临床预后较差，易复发等独特的临床特征，并且由于FLT3激活突变的检测方法简单易行，故有越来越多的研究者致力于将FLT3发展成为AML患者常规的检测手段用来指导AML患者的治疗和预后的判断以及作为微小残留白血病的检测手段，并将其作为白血病患者化疗药物的又一新的靶点(目前已经有针对FLT3-ITD突变进行治疗的药物)。现已证实FLT3的激活突变主要有两种：内部串联重复(Internal tandem duplication,ITD)，在AML和MDS患者中的发生率分别为15～35％和5～10％，在ALL中的发生率﹤1％，且主要见于双表型的ALL病例；活化环中的点突变(Point mutation in the activation loop,TKD点突变)，在AML、MDS和ALL患者中的发生率分别为5～10％、2～5％和1～3％。FLT3的这两种激活突变均能引起FLT3发生自动磷酸化进而导致FLT3发生配体非依赖性的组成性激活，进一步激活其下游异常的信号转导，从而起到促进增殖和抑制凋亡的作用，使得具有此突变表型的白血病患者临床预后较差
药物组合物
本发明还提供了一种组合物。优选地，所述的组合物是药物组合物，它含有上述的抗体或其活性片段或其融合蛋白，以及药学上可接受的载体。通常，可将这些物质配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地pH约为6-8，尽管pH值可随被配制物质的性质以及待治疗的病症而有所变化。配制好的药物组合物可以通过常规途径进行给药，其中包括(但并不限于)：腹膜内、静脉内、或局部给药。
本发明的药物组合物含有安全有效量(如0.001-99wt％，较佳地0.01-90wt％，更佳地0.1-80wt％)的本发明上述的抗体(或其偶联物)以及药学上可接受的载体或赋形剂。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。药物制剂应与给药方式相匹配。本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。药物组合物如针剂、溶液宜在无菌条件下制造。活性成分的给药量是治疗有效量，例如每天约10微克/千克体重至约50毫克/千克体重。此外，本发明的多肽还可与其他治疗剂一起使用。
使用药物组合物时，是将安全有效量的免疫偶联物施用于哺乳动物，其中该安全有效量通常至少约10微克/千克体重，而且在大多数情况下不超过约50毫克/千克体重，较佳地该剂量是约10微克/千克体重至约10毫克/千克体重。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。
针对FLT3的纳米抗体
在本发明中，所述针对FLT3的纳米抗体包括单体、二价体(二价抗体)、四价体(四价抗体)、和/或多价体(多价抗体)。
在本发明的一个优选例中，所述针对FLT3的纳米抗体包括一条或多条具有如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17所示的氨基酸序列的VHH链。
标记的抗体
在本发明的一个优选例中，所述抗体带有可检测标记物。更佳地，所述的标记物选自下组：同位素、胶体金标记物、有色标记物或荧光标记物。
胶体金标记可采用本领域技术人员已知的方法进行。在本发明的一个优选的方案中，针对FLT3蛋白的抗体用胶体金标记，得到胶体金标记的抗体。
本发明的针对FLT3的纳米抗体能够有效结合FLT3蛋白。
检测方法
本发明还涉及检测FLT3蛋白或其片段的方法。该方法步骤大致如下：获得细胞和/或组织样本；将样本溶解在介质中；检测在所述溶解的样本中FLT3蛋白的水平。
在本发明的检测方法中，所使用的样本没有特别限制，代表性的例子是存在于细胞保存液中的含细胞的样本。
试剂盒
本发明还提供了一种含有本发明的抗体(或其片段)或检测板的试剂盒，在本发明的一个优选例中，所述的试剂盒还包括容器、使用说明书、缓冲剂等。
本发明还提供了用于检测FLT3蛋白水平的检测试剂盒，该试剂盒包括识别FLT3蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。
制剂
本发明提供了一种含有本发明第八方面所述的工程化免疫细胞(如CAR-T细胞)，以及药学上可接受的载体、稀释剂或赋形剂。在一个实施方式中，所述制剂为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/mL，更优地1×10 4-1×10 7个细胞/mL。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等 等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的制剂优选配制用于静脉内施用。
应用
如上所述，本发明的抗体有广泛生物应用价值和临床应用价值，其应用涉及到与FLT3蛋白相关的疾病的诊断和治疗、基础医学研究、生物学研究等多个领域。一个优选的应用是用于针对FLT3蛋白的临床诊断、预防和治疗。
本发明也提供了刺激靶向哺乳动物肿瘤细胞群或组织的T细胞所介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，分离病人自体T细胞(或者异源供体)，激活并进行基因改造产生CAR-T细胞，随后注入同一病人体内。这种方式使移植物抗宿主反应的发生概率极低，抗原被T细胞以无MHC限制方式识别。此外，一种CAR-T就可以治疗表达该抗原的所有癌症。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续控制肿瘤的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳定的体内扩增并可持续数月至数年的时间。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中，CAR-T细胞可诱导对CAR抗原结合结构域所识别的抗原的高表达肿瘤细胞的特异性免疫应答。例如，本发明的CAR-T细胞引起针对FLT3高表达的肿瘤细胞的特异性免疫应答。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。用本发明的CAR治疗的癌症类型包括但不限于：急性髓系白血病、急性淋巴细胞白血病、慢性髓性白血病、骨髓增生异常综合征等。
通常地，如本文所述活化和扩增的细胞可用于治疗和预防肿瘤等疾病。因此，本发明提供了治疗癌症的方法，其包括给予给需要其的对象治疗有效量的本发明的CAR-T细胞。
本发明的CAR-T细胞可被单独给予或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由如患者的病症、和患者疾病的类型和严重度等因素确定，或可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。包括本文描述的T细胞的药物组 合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 7个细胞/kg体重的剂量(包括范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可由医学领域技术人员通过监测患者的疾病迹象容易地确定，并以此调整治疗。
对象组合物的给予可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过静脉内注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 5个至1×10 10个本发明经修饰的T细胞，通过例如静脉回输的方式，施用于患者。氨基酸及核苷酸序列
SEQ ID NO:1(PA0135-MN-CAR氨基酸序列)
Figure PCTCN2022144151-appb-000002
SEQ ID NO:2(PA0135-MN-CAR核苷酸序列)
Figure PCTCN2022144151-appb-000003
Figure PCTCN2022144151-appb-000004
SEQ ID NO:3(PA0135-EF-CAR氨基酸序列)
Figure PCTCN2022144151-appb-000005
SEQ ID NO:4(PA0135-GH-CAR氨基酸序列)
Figure PCTCN2022144151-appb-000006
SEQ ID NO:5(PA0135-KL-CAR氨基酸序列)
Figure PCTCN2022144151-appb-000007
SEQ ID NO:6(PA0135-PQ-CAR氨基酸序列)
Figure PCTCN2022144151-appb-000008
SEQ ID NO:7(纳米抗体C-F15-10)
Figure PCTCN2022144151-appb-000009
SEQ ID NO:8(纳米抗体C-F21-1)
Figure PCTCN2022144151-appb-000010
SEQ ID NO:9(纳米抗体C-A5-11)
Figure PCTCN2022144151-appb-000011
SEQ ID NO:10(纳米抗体C-1-D3)
Figure PCTCN2022144151-appb-000012
SEQ ID NO:11(纳米抗体C-1-G3)
Figure PCTCN2022144151-appb-000013
SEQ ID NO:12(纳米抗体C-2-C2)
Figure PCTCN2022144151-appb-000014
SEQ ID NO:13(纳米抗体C-1-E6)
Figure PCTCN2022144151-appb-000015
SEQ ID NO:14(纳米抗体C-2-D8)
Figure PCTCN2022144151-appb-000016
SEQ ID NO:15(纳米抗体C-2-A5)
Figure PCTCN2022144151-appb-000017
SEQ ID NO:16(纳米抗体C-2-A3)
Figure PCTCN2022144151-appb-000018
SEQ ID NO:17(纳米抗体C-2-C4)
Figure PCTCN2022144151-appb-000019
SEQ ID NO:18(纳米抗体C-F15-10的CDR1)
Figure PCTCN2022144151-appb-000020
SEQ ID NO:19(纳米抗体C-F15-10、C-A5-11的CDR2)
Figure PCTCN2022144151-appb-000021
SEQ ID NO:20(纳米抗体C-F15-10的CDR3)
Figure PCTCN2022144151-appb-000022
SEQ ID NO:21(纳米抗体C-F21-1、C-1-D3、C-1-G3、C-2-C2、C-1-E6的CDR1)
Figure PCTCN2022144151-appb-000023
SEQ ID NO:22(纳米抗体C-F21-1、C-1-G3、C-2-C2、C-1-E6、C-2-A3、C-2-C4的CDR2)
Figure PCTCN2022144151-appb-000024
SEQ ID NO:23(纳米抗体C-F21-1的CDR3)
Figure PCTCN2022144151-appb-000025
SEQ ID NO:24(纳米抗体C-A5-11的CDR1)
Figure PCTCN2022144151-appb-000026
SEQ ID NO:25(纳米抗体C-A5-11的CDR3)
Figure PCTCN2022144151-appb-000027
SEQ ID NO:26(纳米抗体C-1-D3、C-2-D8的CDR2)
Figure PCTCN2022144151-appb-000028
SEQ ID NO:27(纳米抗体C-1-D3的CDR3)
Figure PCTCN2022144151-appb-000029
SEQ ID NO:28(纳米抗体C-1-G3的CDR3)
Figure PCTCN2022144151-appb-000030
SEQ ID NO:29(纳米抗体C-2-C2的CDR3)
Figure PCTCN2022144151-appb-000031
SEQ ID NO:30(纳米抗体C-1-E6的CDR3)
Figure PCTCN2022144151-appb-000032
SEQ ID NO:31(纳米抗体C-2-D8、C-2-A3、C-2-C4的CDR1)
Figure PCTCN2022144151-appb-000033
SEQ ID NO:32(纳米抗体C-2-D8的CDR3)
Figure PCTCN2022144151-appb-000034
SEQ ID NO:33(纳米抗体C-2-A5的CDR1)
Figure PCTCN2022144151-appb-000035
SEQ ID NO:34(纳米抗体C-2-A5的CDR2)
Figure PCTCN2022144151-appb-000036
SEQ ID NO:35(纳米抗体C-2-A5的CDR3)
Figure PCTCN2022144151-appb-000037
SEQ ID NO:36(纳米抗体C-2-A3的CDR3)
Figure PCTCN2022144151-appb-000038
SEQ ID NO:37(纳米抗体C-2-C4的CDR3)
Figure PCTCN2022144151-appb-000039
SEQ ID NO:38(纳米抗体C-F15-10的FR1)
Figure PCTCN2022144151-appb-000040
SEQ ID NO:39(纳米抗体C-F15-10的FR2)
Figure PCTCN2022144151-appb-000041
SEQ ID NO:40(纳米抗体C-F15-10的FR3)
Figure PCTCN2022144151-appb-000042
SEQ ID NO:41(纳米抗体C-F15-10、C-F21-1的FR4)
Figure PCTCN2022144151-appb-000043
SEQ ID NO:42(纳米抗体C-F21-1、C-1-D3、C-1-G3、C-2-C2的FR1)
Figure PCTCN2022144151-appb-000044
SEQ ID NO:43(纳米抗体C-F21-1、C-1-D3、C-1-E6的FR2)
Figure PCTCN2022144151-appb-000045
SEQ ID NO:44(纳米抗体C-F21-1、C-2-C2的FR3)
Figure PCTCN2022144151-appb-000046
SEQ ID NO:45(纳米抗体C-A5-11的FR1)
Figure PCTCN2022144151-appb-000047
SEQ ID NO:46(纳米抗体C-A5-11的FR2)
Figure PCTCN2022144151-appb-000048
SEQ ID NO:47(纳米抗体C-A5-11的FR3)
Figure PCTCN2022144151-appb-000049
SEQ ID NO:48(纳米抗体C-A5-11、C-2-A5、C-2-C4的FR4)
Figure PCTCN2022144151-appb-000050
SEQ ID NO:49(纳米抗体C-1-D3的FR3)
Figure PCTCN2022144151-appb-000051
SEQ ID NO:50(纳米抗体C-1-D3、C-2-D8的FR4)
Figure PCTCN2022144151-appb-000052
SEQ ID NO:51(纳米抗体C-1-G3的FR2)
Figure PCTCN2022144151-appb-000053
SEQ ID NO:52(纳米抗体C-1-G3的FR3)
Figure PCTCN2022144151-appb-000054
SEQ ID NO:53(纳米抗体C-1-G3的FR4)
Figure PCTCN2022144151-appb-000055
SEQ ID NO:54(纳米抗体C-2-C2的FR2)
Figure PCTCN2022144151-appb-000056
SEQ ID NO:55(纳米抗体C-2-C2的FR4)
Figure PCTCN2022144151-appb-000057
SEQ ID NO:56(纳米抗体C-1-E6的FR1)
Figure PCTCN2022144151-appb-000058
SEQ ID NO:57(纳米抗体C-1-E6的FR3)
Figure PCTCN2022144151-appb-000059
SEQ ID NO:58(纳米抗体C-1-E6的FR4)
Figure PCTCN2022144151-appb-000060
SEQ ID NO:59(纳米抗体C-2-D8的FR1)
Figure PCTCN2022144151-appb-000061
SEQ ID NO:60(纳米抗体C-2-D8、C-2-A3、C-2-C4的FR2)
Figure PCTCN2022144151-appb-000062
SEQ ID NO:61(纳米抗体C-2-D8的FR3)
Figure PCTCN2022144151-appb-000063
SEQ ID NO:62(纳米抗体C-2-A5、C-2-C4的FR1)
Figure PCTCN2022144151-appb-000064
SEQ ID NO:63(纳米抗体C-2-A5的FR2)
Figure PCTCN2022144151-appb-000065
SEQ ID NO:64(纳米抗体C-2-A5的FR3)
Figure PCTCN2022144151-appb-000066
SEQ ID NO:65(纳米抗体C-2-A3的FR1)
Figure PCTCN2022144151-appb-000067
SEQ ID NO:66(纳米抗体C-2-A3的FR3)
Figure PCTCN2022144151-appb-000068
SEQ ID NO:67(纳米抗体C-2-A3的FR4)
Figure PCTCN2022144151-appb-000069
SEQ ID NO:68(纳米抗体C-2-C4的FR3)
Figure PCTCN2022144151-appb-000070
本发明的主要优点包括：
1.本发明开发的FLT3纳米抗体具有高亲和力，在此基础上用不同FLT3纳米抗体序列构建的FLT3-CAR-T细胞，通过体外杀伤实验证明其对FLT3阳性AML肿瘤细胞株的特异性杀伤十分显著，还对AML肿瘤模型具有显著的体内抗肿瘤作用，治疗效果佳。
2.本发明通过不同克隆FLT3纳米抗体序列构建的CAR-T细胞与正常造血干细胞孵育后的克隆形成实验，证明了本发明构建的FLT3-CAR-T细胞对正常造血干细胞不具有特异性杀伤，不会造成血液毒性，证明安全性较好。
3.本发明通过对肿瘤细胞的重复杀伤实验成功筛选到对AML肿瘤细胞持续杀伤能力最强的FLT3-CAR-T细胞，由于FLT3-CAR-T细胞能够在体内复制，因此可长期持续控制肿瘤，对于易复发的AML是良好的应对策略。
4.本发明的FLT3-CAR-T细胞疗法在一定程度上能减轻患者痛苦并改善AML的预后。一方面本发明的治疗手段可通过多种方式如注射、喷雾法、吞咽、输液等进行，相比现有的化疗策略减轻了患者接受治疗时的痛苦，也降低了进行治疗所需的次数；另一方面，本发明的FLT3-CAR-T细胞疗法可长期控制肿瘤，减少复发的可能性，将疾病控制在较低程度的水平，改善预后。
5.本发明的FLT3-CAR-T细胞相比现有FLT3小分子抑制剂的应用范围更广，能适用于更广泛的患者人群。FLT3-CAR-T细胞不限于只对FLT3-ITD突变的AML病人有效，对FLT3未突变的AML患者也有效。
6.本发明的FLT3-CAR-T细胞相比现有FLT3小分子抑制剂的疗效更佳，FLT3容易在酪氨酸激酶结构域部分产生新的突变，这会导致对FLT3抑制剂产生耐药性，限制了FLT3抑制剂的疗效。而本发明的FLT3-CAR-T细胞受FLT3突变的影响小，即使FLT3 发生突变，FLT3-CAR-T细胞依然保持其靶向性和结合活性，对疗效的限制小。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例1抗原制备方案
通过基因合成FLT3的胞外段(27AA-543AA)序列，在N端添加human IgG1 Fc标签，且FLT3和Fc之间添加肠激酶酶切位点，用于去除Fc标签；亚克隆至真核表达载体中，构建抗原FLT3-Fc蛋白表达载体。
将构建好的FLT3-Fc蛋白表达载体进行质粒大抽，瞬时转染293细胞后，连续培养8天，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化得到纯化的FLT3-Fc蛋白。
所述经纯化得到的FLT3-Fc蛋白即为抗原，也称为免疫原、免疫抗原。
实施例2抗原免疫羊驼方案
采用上述实施例1中制备的FLT3-Fc蛋白免疫1只羊驼，采用皮下多点免疫共进行4次免疫，免疫方案流程如表3所示。
表3
Figure PCTCN2022144151-appb-000071
经抗原的三次免疫后，将羊驼免疫血清进行效价检测，若三免后血清效价达到1:8000稀释条件下，OD值大于1.0以上，即可进行冲击免疫(第四次免疫)。冲击免疫后得到的羊驼外周血用于分离免疫血清，进行后续酵母展示库的构建。
实施例3免疫效价的检测
采集5mL上述实施例2中制备的三免后外周血和/或冲击免疫后外周血，将收集有血液样 本的离心管置于37℃培养箱内放置1小时，随后将血液样本转移至4℃过夜。将血清转移至一个新的无菌离心管中，5000rpm离心20min，之后采用ELISA检测免疫效价。
将免疫的羊驼分离血清按照如表4所示的稀释梯度进行有限稀释，与FLT3抗原预包被的96孔板进行ELISA实验，使用阴性血清作为阴性对照，PBS缓冲液作为空白对照，检测结果如表2所示。
表4
血清 OD值
1:1k 2.514
1:2k 2.659
1:4k 2.46
1:8k 2.204
1:16k 1.84
1:32k 1.372
1:64k 0.943
PBS 0.099
根据ELISA检测结果，经过三次免疫，免疫效价较好，1:8000条件下OD值达到了2.204，远高于1.0，可以进行冲击免疫。
进行一次冲击免疫，3天后采集100mL外周血进行酵母展示库的构建。
实施例4 Biotin偶联抗原蛋白
准备1mg的实施例1中制备的纯化抗原，缓冲体系为PBS，浓度1mg/mL。称取NHS-biotin，使用DMSO溶解，配制10mM的NHS-biotin。
向上述抗原蛋白样品中加入新鲜制备的10mM的NHS-biotin溶液，将样品管装入避光自封袋中，180rpm室温偶联30min。随后使用PBS进行置换，去除未标记的Biotin，将标记好的抗原蛋白保存于-80℃。使用ELISA方案检测Biotin偶联效果。
实施例5 PBMC分离及VHH抗体片段的获得
1.采集50mL实施例2中制备的冲击免疫后外周血，使用淋巴细胞分离液分选PBMC。
2.提取PBMC的RNA，随后使用PrimeScript TM II 1st Strand cDNA Synthesis Kit进行反转录，制备其cDNA，具体步骤如下：
(1)在200μL的PCR管中配制如表5所示的反应混合液MIX1：
表5
试剂 用量
Oligo dT Primer(50μM) 8μL
dNTP Mixture(10mM each) 8μL
总RNA样品 20μg
RNase-Free水 Up to 80μL
(2)65℃保温5min后，冰上迅速冷却。
(3)在上述PCR管中配制如表6所示反应液：
表6
试剂 用量
上述变性后的反应液 80μL
5×PrimeScript Ⅱ Buffer 32μL
RNase Inhibitor(40U/μL) 4μL
PrimeScript Ⅱ RTase(200U/μL) 8μL
RNase-Free水 36μL
(4)吹打混匀后，分装80μL/管，置于PCR仪中42℃1小时，70℃热失活15分钟，最后将cDNA样品置于冰上或-20℃长期保存。
3.VHH片段的扩增，具体步骤如下：
(1)配置如表7所示的第一轮PCR反应体系(50μL/管)：
表7
成分 用量
上游引物(5μM) 2μL
下游引物(10μM) 1μL
NuHi Power mix(2×) 25μL
cDNA模板 2μL
无菌水 20μL
配置好PCR反应体系后，按照如表8所示的程序设置PCR仪：
表8
Figure PCTCN2022144151-appb-000072
(2)PCR产物的琼脂糖电泳：
使用1％的琼脂糖进行电泳分析PCR产物，分离分子量大小为750bp左右的片段。使用胶回收试剂盒回收PCR产物，并用NanoDrop测定浓度。
(3)配置如表9所示的二轮PCR反应体系(50μL/管)：
表9
成分 用量
2 nd F引物 2μL
2 nd R引物 2μL
NuHi Power mix(2×) 25μL
一轮PCR回收产物 200ng
无菌水 补足至50μL
配置好PCR反应体系后，按照如表10所示的程序设置PCR仪：
表10
Figure PCTCN2022144151-appb-000073
(4)二轮PCR产物的琼脂糖电泳分析：
使用1％的琼脂糖进行电泳分析PCR产物，分离分子量大小为400bp左右的VHH片段。使用胶回收试剂盒回收VHH PCR产物，并用NanoDrop测定浓度。
采集实施例2中制备的冲击免疫后的外周血，提供总的RNA，逆转录为cDNA后，使用单域抗体扩增引物进行两轮PCR，使用琼脂糖凝胶电泳鉴定PCR产物。
琼脂糖凝胶电泳结果如图1所示：第一轮PCR分别获得1000bp和750bp左右的PCR条带，胶回收750bp的片段作为二轮PCR的模板，第二轮PCR获得450bp的条带即为VHH片段。
实施例6酵母展示库载体的构建及电转化
1.酵母展示载体的构建：
(1)使用SfiI分别酶切pBlue载体和上述实施例5中获得的VHH PCR胶回收产物，50℃酶切过夜。
(2)使用1％的琼脂糖凝胶分离pBlue载体片段，切取9000bp的载体片段进行胶回收。同时使用DNA片段回收试剂盒纯化PCR酶切产物，并用NanoDrop测定浓度。
(3)酶切好的pBlue载体和VHH片段使用T4ligase连接，16℃连接过夜。
2.酵母展示载体电转化获得大肠杆菌库：
(1)准备电转杯、连接产物和电转感受态置于冰上预冷。
(2)取预冷的建库连接产物加入到电转感受态中，置于冰上1min，向每个电转杯中加入70μL DNA/感受态混合物，将电转杯放于冰上。
(3)按照2500V，5ms进行电转。
(4)电击结束后，立刻加入平衡至室温的SOC培养基重悬菌体，37℃摇床培养1小时。
(5)取20μL菌液进行库容QC以及多样性QC，其余菌液接种含Amp抗性的LB培养基中，180rpm 37℃选择培养过夜。
(6)从上述过夜培养的菌液中，离心收集菌体沉淀，使用质粒大抽试剂盒进行质粒大抽。并用Nanodrop测定质粒浓度，计算获得的质粒总量。
由上述步骤完成酵母展示载体的构建，并通过电转化获得大肠杆菌库质粒。
实施例7电转化酵母感受态细胞获得酵母展示库
1.将实施例6中制备的大肠杆菌库质粒进行PmeI线性化，酶切体系如表11所示：
表11
试剂 用量
质粒 12μg
10×buffer 5μL
PmeI 1μL
无菌水 Up to 50μL
37℃酶切3h，取5μL进行1％琼脂糖电泳检测，剩余酶切产物沉淀浓缩后待用，共酶切3mg质粒。
2.制备酵母感受态细胞：
(1)从酵母甘油菌挑取酵母菌体，在YPD琼脂平板上划线，将平板置于24℃-30℃的培养箱中连续培养3-5天，直至单克隆长出。
(2)从平板上挑取酵母菌单克隆，并加入至YPD液体培养基中，置于24℃-30℃的摇床中250rpm培养1-2天。
(3)使用1L的无菌锥形瓶，加入100mL YPD培养基，将上述准备的摇菌产物加入瓶中，置于24℃-30℃的摇床中250rpm培养1-2天。
(4)取样检测OD 600直至1.3-1.5(对数生长期)。
(5)将菌液全部转移至离心管内，1500×g、4℃离心5分钟，去掉上清后，使用250mL冰上预冷的无菌水重悬菌体沉淀。
(6)再进行一轮1500×g、4℃离心5分钟，去掉上清后，使用50mL冰上预冷的无菌水重悬菌体沉淀。
(7)再进行一次1500×g、4℃离心5分钟，去掉上清后，使用10mL冰上预冷的1M山梨糖醇重悬菌体沉淀。
(8)再进行一次1500×g、4℃离心5分钟，去掉上清后，使用500μL冰上预冷的1M山梨糖醇重悬菌体沉淀。
(9)最终获得500μL酵母感受态细胞，分装为80μL/管备用，感受态细胞一般现做现用，以确保电转效率更高。
3.电转酵母感受态细胞：
(1)取80μL酵母感受态细胞，加入1μg线性化质粒DNA，充分混匀后，加入至冰上预冷的电击杯中，将电击杯置于冰上冰浴5分钟。
(2)电转化条件为：电压：1500V，时间：5ms，电击2次。
(3)电击结束后，立即加入1mL冰上预冷的YPDS培养基，轻轻吹打混匀后，将电击杯的液体全部转移到新的EP管中，30℃的培养箱中静置培养2小时。
(4)孵育结束后，取50μL电转化后产物，分别均匀的涂布在YPD选择性平板上。将平板置于28℃的培养箱中3天，直至单克隆长出。
(5)其余转化产物，转移至液体的PAD培养基中，28℃震荡培养3天。
(6)观察当白色菌体成为优势菌群后，1500×g，离心5min收集菌体，取一部分用于实施例8中酵母展示库的诱导表达。剩余加入50％无菌甘油，保存在-80摄氏度。
由上述步骤完成酵母感受态细胞的电转，并得到酵母展示库转化菌种。
实施例8酵母展示库诱导表达以及分选
1.酵母展示库诱导表达：
(1)将实施例7中获得的酵母展示库转化菌种使用PAD液体培养基培养。
(2)将PAD液体培养基选择生长的酵母菌用无菌PBS清洗一次后，重悬在BMMY培养基中，添加氨苄和卡那霉素抗生素，28℃，220rpm诱导表达24-48h。
(3)诱导前取1mL菌液，加入YPD培养基，添加氨苄和卡那霉素抗生素，28℃，220rpm震荡培养，此样品为诱导前对照。
(4)随机挑选20个酵母单克隆进行测序，分析构建酵母展示库的多样性。
根据如图2的测序结果比对显示，酵母展示库具有多样性，酵母展示库库容为6.74×10 6。
2.酵母展示库磁分选：
(1)链霉亲和素磁珠预吸附：
①取10OD诱导酵母菌，使用500μL预冷的0.5％PBSA重悬。
②取50μL链霉亲和素磁珠，充分吹打混合均匀后，加入1mL PBSA，置于磁力架上，放置3-5min，小心去除上清。
③加入1mL PBSA，重复洗涤一次磁珠。吸去上清，将步骤①中的酵母菌加入，混合均匀，4℃孵育1h。
④置于磁力架上，放置3-5min，小心吸出上清，将酵母菌转移至一新的EP管中，重复吸附一次；小心吸出上清，用于下一步实验。
(2)Biotin-Fc负分选：
①取预吸附后的酵母菌，800×g离心5min，重悬在Biotin-Fc中，4℃孵育1h。
②将孵育结束的酵母中加入1mL的PBSA，800×g离心5min，弃去上清。重复洗涤2次，最终重悬在500μL的PBS中。
③取50μL链霉亲和素磁珠，充分吹打混合均匀后，加入1mL PBSA，置于磁力架上，放置3-5min，小心去除上清。
④加入1mL PBSA，重复洗涤一次磁珠。吸去上清，将孵育Biotin-Fc的酵母菌加入，混合均匀，4℃孵育1h。
⑤置于磁力架上，放置3-5min，小心吸出上清，将酵母菌转移至一新的EP管中，重复吸附一次；小心吸出上清，800×g离心5min，收集酵母菌用于下一步实验。
(3)Biotin-FLT3-Fc磁分选：
①取Biotin-Fc负分选后的酵母菌，800×g离心5min，重悬在Biotin-FLT3-Fc中，4℃孵育1h。
②将孵育结束的酵母中加入1mL的PBSA，800×g离心5min，弃去上清。重复洗涤2次，最终重悬在500μL的PBS中。
③取100μL链霉亲和素磁珠，充分吹打混合均匀后，加入1mL PBSA，置于磁力架上，放置3-5min，小心去除上清。
④加入1mL PBSA，重复洗涤一次磁珠。吸去上清，将孵育Biotin-FLT3-Fc的酵母菌加入，混合均匀，4℃孵育1h。
⑤孵育结束后，置于磁力架上，放置3-5min，小心吸出上清；
⑥加入1mL PBSA，重复洗涤两次磁珠，使用YPD培养基重悬酵母菌，进行培养和诱导表达。分选的酵母细胞培养和诱导表达后进行流式分析，孵育Biotin-FLT3-Fc，二抗使用PE Streptavidin，孵育完成后进行流式检测。
⑦将第一轮磁分选产物，分别与Biotin-FLT3-Fc蛋白充分结合，使用链霉亲和素磁珠进行第二轮磁分选。将分选的酵母细胞一部分直接涂PAD平板，用于挑选单克隆进行验证；一部分培养进行流式分析。孵育Biotin-FLT3-Fc，二抗使用PE Streptavidin，孵育完成后进行流式检测。
根据如图3所示的第一次分选后流式检测结果，Biotin-FLT3-Fc用于酵母阳性克隆的磁分选，分选效果较好。将第一轮分选酵母细胞培养诱导表达后进行二轮分选。
如图4所示，第二次磁分选后，酵母阳性率为91.721％，阳性克隆显著富集，将分选产物直接涂PAD平板，挑选单克隆进行流式检测。
3.酵母单克隆的流式检测：
重复两轮磁分选后，一部分酵母进行培养，诱导表达流式检测；一部分直接涂PAD平板，挑取单克隆培养，接种至96孔板中，诱导表达24h后，与Biotin-FLT3-Fc孵育，二抗使用PE-Streptavidin，孵育完成后进行流式检测。
流式分析结果如图5所示，表明挑选的克隆均和Biotin-FLT3-Fc结合。
实施例9 VHH真核表达载体的构建
根据实施例8中酵母单克隆流式检测结果，选择与目的抗原不同结合能力的克隆，抽提 基因组DNA，使用pBlue载体的通用引物进行PCR，将PCR产物进行测序，获得VHH抗体序列。
将分析获得的VHH抗体序列分别进行基因合成，与human IgG1 Fc串联亚克隆至如图6所示的表达载体Lenti-hIgG1-Fc2后，测序以对载体进行验证。
载体经测序验证无误后，即得到抗体表达载体Lenti-hIgG1-Fc2，使用Qiagen质粒大抽试剂盒制备去内毒素质粒备用。
实施例10重组抗体的表达
1.从冰箱中取出LVTransm转染试剂及抗体表达载体Lenti-hIgG1-Fc2，室温解冻后，用移液枪上下吹打完全混匀。取出PBS缓冲液，温热至室温。取500μL PBS至24孔板的一个孔，加入4μg Lenti-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入12μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。此处的混合物称为DNA/LVTransm复合物。
2.将上述532μL DNA/LVTransm复合物加入到1.5mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO 2培养箱，130rpm培养6-8小时后，加入1.5mL新鲜的FreeStyle TM 293培养基，将细胞重新放回培养箱中继续培养。
3.连续培养3天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，进行后续的流式和ELISA检测。
由此步骤得到经实施例9中抗体表达载体Lenti-hIgG1-Fc2所表达的重组抗体，也称单域抗体、本发明的单域抗体、FLT3纳米抗体。
实施例11流式检测重组抗体与靶蛋白的结合
1.从液氮中复苏CHO-K1和CHO-K1-FLT3细胞株，调整细胞状态至对数生长期。
2.将两种细胞分别分为若干份，每份细胞的数量为5×10 5个细胞。
3.将表达的抗体分别孵育靶细胞，充分混匀后，室温孵育1小时。
4. 800×g室温离心5分钟，去掉含有抗体的上清，使用PBS洗涤细胞3次。
5.加入1μL PE标记的Anti-human IgG，充分混匀后，室温避光孵育30分钟。
6. 800×g室温离心5分钟，去掉含有二抗的上清，使用PBS洗涤细胞3次。
7.使用500μL PBS重悬细胞，进行流式分析。
根据如图7所示的FACS检测结果，筛选抗体均可以结合CHO-K1-FLT3重组细胞，与CHO-K1不结合，说明实施例10中制备的重组抗体与靶蛋白的结合具有特异性。
安排11个靶蛋白结合阳性抗体(SEQ ID NO:7-17)的表达纯化，同时对C-F21-1(SEQ ID NO:8)、C-A5-11(SEQ ID NO:9)和C-2-A5(SEQ ID NO:15)三个克隆的抗体进行亲和力检测。
实施例12重组抗体的表达纯化
1.从冰箱中取出LVTransm转染试剂及单链抗体表达载体，室温解冻后，用移液枪上下 吹打完全混匀。取出PBS缓冲液，温热至室温。取2mL PBS至6孔板的一个孔，分别加入130μg Lenti-hIgG1-Fc2，移液枪上下吹打充分混匀后，加入400μL LVTransm，立即用移液器上下吹打混匀，室温下静置10分钟。
2.将上述DNA/LVTransm复合物加入到50mL 293F细胞中，轻轻晃动充分混匀。将细胞置于37℃、5％CO 2培养箱，130rpm培养6-8小时后，加入50mL新鲜的FreeStyle TM 293培养基，将细胞重新放回培养箱中继续培养。
3.连续培养7天后，离心收集培养基上清，用0.45μm的滤膜过滤，滤液转至无菌离心管中，使用Protein A柱子纯化抗体。
由此步骤将实施例11中获得的阳性重组抗体进行了表达纯化。
实施例13亲和力检测
将FLT3-Fc重组蛋白使用10mM Acetate缓冲液固定在CM5芯片上，分别以上述实施例11及12中制备的单域抗体作为流动相，检测候选单域抗体与靶蛋白FLT3的结合能力，亲和力检测结果如表12所示。
表12
纳米抗体 ka(M -1s -1) kd(s -1) KD(M)
C-F21-1 4.752×10 4 0.001682 3.539×10 -8
C-A5-11 2.956×10 5 0.002313 7.824×10 -9
C-2-A5 1.411×10 5 0.001596 1.131×10 -8
检测结果显示，本发明的单域抗体与靶蛋白FLT3的亲和力(很好，说明单域抗体与靶蛋白FLT3具有良好的结合效果)。
实施例14单域抗体序列
采用酵母展示库筛选获得靶向FLT3的单域抗体，抗体序列信息如图8A-8K所示。其中，以图8A为例，FR序列(FR1-FR4)用下划线标出，CDR1用着重号(.)标出，CDR2用波浪线
Figure PCTCN2022144151-appb-000074
标出，CDR3用线段
Figure PCTCN2022144151-appb-000075
标出。图8B-8K用类似方式表示。
实施例15 FLT3-CAR载体构建
用上述开发的5个流式结果较好的FLT3纳米抗体序列(即C-F21-1，C-A5-11，C-2-D8，C-2-A5，C-2-A3)分别构建5个含有ICOS和41BB共刺激因子的三代CAR载体(即PA0135EF-CAR，PA0135GH-CAR，PA0135KL-CAR，PA0135MN-CAR，PA0135PQ-CAR)，结构如图9所示。
实施例16不同克隆FLT3纳米抗体序列构建的CAR慢病毒滴度比较
利用悬浮293T慢病毒包装系统对5种FLT3-CAR进行慢病毒包装，检测各CAR病毒原液 滴度值。
如图10结果显示原液滴度值均在1×10 7TU/mL以上，其中PA0135EF、PA0135GH、PA0135MN三个克隆的CAR慢病毒原液滴度较高。
实施例17不同克隆FLT3纳米抗体序列构建的CAR-T细胞对FLT3阳性靶细胞毒性作用
首先通过流式细胞术检测不同肿瘤细胞系表面抗原CD135(FLT3)的表达情况。
结果如图11所示，Raji和MEG01不表达FLT3，AML3，MOLM-13，MV4-11三株AML细胞株(急性髓系白血病细胞株)高表达FLT3。
选用FLT3阴性靶细胞Raji作为靶细胞进行靶细胞杀伤实验，比较本发明的CAR-T细胞与对照T细胞对阴性靶细胞毒性作用的差异。
结果如图12所示，与对照T细胞相比，PA0135EF-CAR-T、PA0135GH-CAR-T、PA0135MN-CAR-T对抗原阴性的靶细胞无明显特异性杀伤，说明本发明的CAR-T细胞对阴性靶细胞的毒性非常小，安全性高。
选取三株FLT3抗原阳性的细胞株(MV-4-11，MOLM-13，AML3)作为靶细胞进行靶细胞杀伤实验，比较PA0135EF-CAR-T、PA0135GH-CAR-T、PA0135MN-CAR-T对靶细胞的毒性作用差异。
由图13数据可知，三种CAR-T细胞对FLT3阳性细胞株都具有显著的特异性杀伤，而且PA0135MN-CAR-T对FLT3阳性肿瘤细胞的细胞毒性要优于PA0135EF-CAR-T和PA0135GH-CAR-T。
收集上述杀伤实验的上清液，进一步检测了上清中细胞因子Granzyme-B的分泌情况。
结果如图14所示，三种CAR-T细胞与靶细胞孵育后都有明显的特异性细胞因子的释放。同时，PA0135MN-CAR-T细胞具有更强的细胞因子释放能力。因此，结合PA0135EF-CAR-T、PA0135GH-CAR-T、PA0135MN-CAR-T对FLT3阳性肿瘤细胞的细胞毒性及细胞因子释放结果，三者中PA0135MN-CAR-T对阳性靶细胞具有更强的细胞毒性，杀伤效果更好。
实施例18不同克隆FLT3纳米抗体序列构建的CAR-T细胞对FLT3阳性肿瘤细胞株 的持续杀伤能力比较
选取FLT3阳性肿瘤细胞株MV-4-11作为靶细胞，比较不同克隆的FLT3-CAR-T细胞对靶细胞的持续杀伤能力，效靶比为1:1和1:5，每隔24小时或48小时补加一次肿瘤细胞。
由图15结果可知，与PA0135EF-CAR-T和PA0135GH-CAR-T相比，PA0135MN-CAR-T对MV-4-11肿瘤细胞具有更强及更持久的杀伤能力。
实施例19不同克隆FLT3纳米抗体序列构建的CAR-T细胞对正常造血干细胞的细胞毒 性
首先通过流式细胞术检测了购买的商业化人造血干细胞(HSPC)表面FLT3和CD33的表达情况。
如图16结果显示人造血干细胞表面高表达FLT3(阳性率是81.77％)和CD33(阳性率是74.47％)。
随后检测了购买的人CD34+HSPC细胞分别与3种FLT3-CAR-T细胞(CD33-CAR-T细胞为对照组)孵育后细胞因子(Granzyme-B，IL-2，IFN-gama，TNF-α)的释放情况。
结果如图17所示，CD33-CAR-T(PA033AC-CAR-T)与人CD34+造血干细胞孵育后有明显的特异性细胞因子的产生，说明CD33-CAR-T可以激活正常造血干细胞，对正常造血干细胞可能会产生细胞毒性，而3种FLT3-CAR-T与造血干细胞孵育后基本都没有特异性细胞因子的产生，说明FLT3-CAR-T细胞不会被正常造血干细胞激活，也就不会对正常造血干细胞产生特异性杀伤。尤其是PA0135MN克隆的FLT3-CAR-T细胞，细胞因子释放量最低。
最后进一步将3种FLT3-CAR-T及CD33-CAR-T细胞分别与人造血干细胞孵育后，检测造血干细胞的克隆形成情况。
结果如图18所示，与对照T细胞组相比，与CD33-CAR-T(PA033AC-CAR-T)孵育后的造血干细胞的克隆数量明显降低，说明CD33-CAR-T影响了造血干细胞正常克隆的形成，CD33-CAR-T细胞对正常造血干细胞产生了一定的细胞毒性；而3种FLT3-CAR-T细胞没有影响造血干细胞的克隆形成，说明FLT3-CAR-T细胞对正常造血干细胞没有显著的细胞毒性。
实施例20 PA0135MN-CAR-T细胞对AML细胞株OCI-AML3的体内抗肿瘤作用
如图19所示，选用荧光素酶标记的OCI-AML3细胞构建小鼠肿瘤建模，选择15只雌性NCG小鼠，每只尾静脉接种1×10 6个肿瘤细胞，在肿瘤细胞接种后第3天，进行尾静脉给药，给药剂量2×10 7的PA0135MN-CAR-T(TAA05-CAR-T)。每周监测不同给药组小鼠体内肿瘤细胞的生长情况。
如图20所示，与对照组(PBS组和Mock T组)相比，PA0135MN-CAR-T(TAA05-CAR-T)细胞可以显著抑制小鼠体内OCI-AML3肿瘤细胞的增殖和生长。
对小鼠的体重检测及生存期曲线如图21所示，结果也显示：与对照组相比，TAA05-CAR-T细胞对小鼠的体重不会产生明显的影响，TAA05-CAR-T细胞可以显著延长OCI-AML3荷瘤小鼠的生存期，具有显著的体内抗肿瘤作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种针对FLT3的纳米抗体，其特征在于，所述针对FLT3的纳米抗体的重链可变区VHH的互补决定区CDR为选自下组的一种或多种：
    (1)SEQ ID NO:18所示的CDR1、SEQ ID NO:19所示的CDR2、和SEQ ID NO:20所示的CDR3；
    (2)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:23所示的CDR3；
    (3)SEQ ID NO:24所示的CDR1、SEQ ID NO:19所示的CDR2、和SEQ ID NO:25所示的CDR3；
    (4)SEQ ID NO:21所示的CDR1、SEQ ID NO:26所示的CDR2、和SEQ ID NO:27所示的CDR3；
    (5)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:28所示的CDR3；
    (6)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:29所示的CDR3；
    (7)SEQ ID NO:21所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:30所示的CDR3；
    (8)SEQ ID NO:31所示的CDR1、SEQ ID NO:26所示的CDR2、和SEQ ID NO:32所示的CDR3；
    (9)SEQ ID NO:33所示的CDR1、SEQ ID NO:34所示的CDR2、和SEQ ID NO:35所示的CDR3；
    (10)SEQ ID NO:31所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:36所示的CDR3；
    (11)SEQ ID NO:31所示的CDR1、SEQ ID NO:22所示的CDR2、和SEQ ID NO:37所示的CDR3。
  2. 如权利要求1所述的针对FLT3的纳米抗体，其特征在于，所述针对FLT3的纳米抗体的VHH的氨基酸序列选自下组：SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17，或其组合。
  3. 一种针对FLT3的抗体，其特征在于，所述抗体包括一个或多个如权利要求2所述的针对FLT3的纳米抗体。
  4. 一种嵌合抗原受体CAR，其特征在于，所述CAR含有一胞外结构域，所述胞外结构域包含如权利要求1所述的针对FLT3的纳米抗体或如权利要求3所述的针对FLT3的抗体。
  5. 如权利要求4所述的嵌合抗原受体，其特征在于，所述CAR的氨基酸序列如SEQ ID NO:1、3、4、5、6所示。
  6. 如权利要求4或5所述的嵌合抗原受体，其特征在于，所述的CAR具有式Ia所示结构：
    L1-Nb-H-TM-C-CD3ζ  (Ia)
    式中，
    L为无或信号肽序列；
    Nb是特异性结合结构域；
    H为无或铰链区；
    TM为跨膜结构域；
    C为共刺激信号结构域；
    CD3ζ为源于CD3ζ的胞浆信号传导序列(包括野生型、或其突变体/修饰体)；
    所述“-”连接肽或肽键。
  7. 一种工程化免疫细胞，其特征在于，所述工程化免疫细胞含有如权利要求4所述的嵌合抗原受体。
  8. 一种制备如权利要求7所述的工程化免疫细胞的方法，包括以下步骤：将如权利要求4所述的嵌合抗原受体转导入免疫细胞内，从而获得所述工程化免疫细胞。
  9. 一种活性成分的用途，所述活性成分选自下组：如权利要求1所述的针对FLT3的纳米抗体，或如权利要求3所述的针对FLT3的抗体，或如权利要求4所述的嵌合抗原受体，或如权利要求7所述的工程化免疫细胞，或其组合，其特征在于，所述活性成分被用于制备：
    (a)预防和/或治疗FLT3高表达疾病的药物；
    (b)检测FLT3高表达疾病的试剂。
  10. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (i)如权利要求1所述的针对FLT3的纳米抗体，或如权利要求3所述的针对FLT3的抗体，或如权利要求4所述的嵌合抗原受体，或如权利要求7所述的工程化免疫细胞，或其组合作为活性成分；和
    (ii)药学上可接受的载体、稀释剂或赋形剂。
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