CN116410315A

CN116410315A - Construction and application of novel chimeric antigen receptor modified T cell targeting human FLT3

Info

Publication number: CN116410315A
Application number: CN202111672073.1A
Authority: CN
Inventors: 杨林; 游凤涛; 李亚芬; 陈丹
Original assignee: Persongen Biotherapeutics Suzhou Co ltd
Current assignee: Persongen Biotherapeutics Suzhou Co ltd
Priority date: 2021-12-31
Filing date: 2021-12-31
Publication date: 2023-07-11
Also published as: WO2023125975A1

Abstract

The invention provides a nano antibody aiming at FLT3, an engineering immune cell constructed by the nano antibody and targeting FLT3 high-expression tumor cells and application thereof. In particular, the invention provides a nanobody against FLT3 and chimeric antigen receptor CAR sequences thereof. The invention also provides an expression vector corresponding to the nanobody and the CAR, a host cell or an engineering immune cell capable of expressing the nanobody and the CAR, and a production method of the nanobody and the chimeric antigen receptor immune cell. The nano antibody and chimeric antigen receptor immune cells can specifically bind to human FLT3 and have high affinity; the chimeric antigen receptor immune cells of the invention have very obvious specific killing on FLT3 positive AML tumor cell strains, also have obvious in-vivo anti-tumor effect, have good treatment effect and improve the safety; the chimeric antigen receptor immune cells are slightly influenced by FLT3 mutation, and even if FLT3 is mutated, the chimeric antigen receptor immune cells still maintain targeting and binding activities, so that the limitation on curative effect is small; in addition, since chimeric antigen receptor immune cells can replicate in vivo, tumors can be controlled continuously for a long period of time, and thus, the chimeric antigen receptor immune cells are a good strategy for treating AML which is easy to relapse.

Description

Construction and application of novel chimeric antigen receptor modified T cell targeting human FLT3

Technical Field

The invention belongs to the fields of biomedicine and immune cell treatment, and in particular relates to a specific FLT3 targeting nanobody, an engineering immune cell constructed by the specific FLT3 targeting FLT3 high-expression tumor cell, and application of the specific FLT3 targeting nanobody.

Background

Acute myeloid leukemia (Acute myeloid leukemia, AML) is a disease with a poor clinical prognosis. Although most patients can achieve remission by chemotherapy, they almost all relapse, require late-stage consolidated chemotherapy or hematopoietic stem cell transplantation (Hematopoietic stem cell transplantation, HSCT), and most patients eventually die from the disease with a survival rate of only 40-50% for 5 years. Thus, there is a need for a new approach to treating AML that meets the existing therapeutic needs.

In recent years, chimeric antigen receptor (Chimeric antigen receptor, CAR) immunotherapy has demonstrated very good clinical efficacy in clinical studies of B-cell malignancies, but CAR-T therapy against AML still faces a great challenge, and we have difficulty finding a suitable target for tumors, because most surface molecules expressed on AML are also expressed on the surface of hematopoietic stem/progenitor cells (Hematopoietic stem and progenitor cell, HSPC), which can damage hematopoietic stem cells while killing AML cells, thus causing hematological toxicity. Therefore, it is important to find a target spot which is relatively safe and has better curative effect.

FLT3 (Fms-like tyrosine kinase 3, cd 135) is a protein encoded by FLT3 gene in humans, a cytokine receptor, belonging to the class III receptor tyrosine kinase. The results show that FLT3 is expressed on approximately 50% of normal hematopoietic stem cells (Hematopoietic stem cells, HSCs) and part of dendritic cells, and is largely not expressed on cord blood lymphocytes. However, it has been found that FLT3 is highly expressed on the cell surface of AML patients and that in initial AML patients, about 1/3 of patients have FLT3 activating mutations.

The drugs targeting FLT3 are currently marketed in the most number and most rapid progress as small molecule inhibitors, and currently share a plurality of small molecule inhibitor drugs capable of acting on FLT3, wherein the first three commonly used quezatinib (quezartiinib) and the An Si targetinib (giltidinib) are second generation inhibitors, which are more selective than the first generation inhibitors, but the FLT3 inhibitors are clinically effective only on AML patients with FLT3-ITD mutations, ineffective on AML patients with FLT3 non-mutations, and FLT3 is prone to generate new mutations in the tyrosine kinase domain portion and resistance to FLT3 inhibitors, resulting in very limited efficacy.

In recent years, chimeric Antigen Receptor (CAR) T cells have achieved unprecedented clinical effects in B-cell leukemia and lymphoma patients, suggesting that this novel therapeutic approach may also be very effective for AML patients. However, at present, CAR-T products targeting FLT3 are not marketed or formally obtained in batch clinical practice at home and abroad.

Disclosure of Invention

The invention aims to provide a nano antibody aiming at FLT3, a chimeric antigen receptor immune cell constructed by the nano antibody, and a preparation method and application thereof.

In a first aspect of the invention, there is provided a nanobody against FLT3.

In another preferred embodiment, the nanobody directed against FLT3 is capable of specifically binding FLT3.

In another preferred embodiment, the complementarity determining region CDRs of the nanobody against FLT3 are one or more selected from the group consisting of:

(1) CDR1 shown in SEQ ID NO. 18, CDR2 shown in SEQ ID NO. 19, and CDR3 shown in SEQ ID NO. 20;

(2) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 22, and CDR3 shown in SEQ ID NO. 23;

(3) CDR1 shown in SEQ ID NO. 24, CDR2 shown in SEQ ID NO. 19, and CDR3 shown in SEQ ID NO. 25;

(4) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 26, and CDR3 shown in SEQ ID NO. 27;

(5) CDR1 as shown in SEQ ID NO. 21, CDR2 as shown in SEQ ID NO. 22, and CDR3 as shown in SEQ ID NO. 28;

(6) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 22, and CDR3 shown in SEQ ID NO. 29;

(7) CDR1 shown in SEQ ID NO. 21, CDR2 shown in SEQ ID NO. 22, and CDR3 shown in SEQ ID NO. 30;

(8) CDR1 as shown in SEQ ID NO. 31, CDR2 as shown in SEQ ID NO. 26, and CDR3 as shown in SEQ ID NO. 32;

(9) CDR1 shown in SEQ ID NO. 33, CDR2 shown in SEQ ID NO. 34, and CDR3 shown in SEQ ID NO. 35;

(10) CDR1 as shown in SEQ ID NO. 31, CDR2 as shown in SEQ ID NO. 22, and CDR3 as shown in SEQ ID NO. 36;

(11) CDR1 shown in SEQ ID NO. 31, CDR2 shown in SEQ ID NO. 22, and CDR3 shown in SEQ ID NO. 37.

In another preferred embodiment, any of the above amino acid sequences further comprises a derivative sequence which is optionally added, deleted, modified and/or substituted with at least one (e.g., 1-3, preferably 1-2, more preferably 1) amino acid and which retains the ability to specifically bind to FLT 3.

In another preferred embodiment, the derivative sequence having at least one amino acid added, deleted, modified and/or substituted and capable of retaining the ability to specifically bind FLT3 is an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology or sequence identity.

In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.

In another preferred embodiment, the nanobody against FLT3 further comprises a framework region FR.

In another preferred embodiment, the framework region FR is derived from the amino acid sequence shown as SEQ ID NO. 7-17.

In another preferred embodiment, the framework region FR is one or more selected from the group consisting of:

(1) FR1 shown in SEQ ID NO. 38, FR2 shown in SEQ ID NO. 39, FR3 shown in SEQ ID NO. 40 and FR4 shown in SEQ ID NO. 41;

(2) FR1 shown in SEQ ID NO. 42, FR2 shown in SEQ ID NO. 43, FR3 shown in SEQ ID NO. 44 and FR4 shown in SEQ ID NO. 41;

(3) FR1 shown in SEQ ID NO. 45, FR2 shown in SEQ ID NO. 46, FR3 shown in SEQ ID NO. 47 and FR4 shown in SEQ ID NO. 48;

(4) FR1 shown in SEQ ID NO. 42, FR2 shown in SEQ ID NO. 43, FR3 shown in SEQ ID NO. 49 and FR4 shown in SEQ ID NO. 50;

(5) FR1 shown in SEQ ID NO. 42, FR2 shown in SEQ ID NO. 51, FR3 shown in SEQ ID NO. 52 and FR4 shown in SEQ ID NO. 53;

(6) FR1 shown in SEQ ID NO. 42, FR2 shown in SEQ ID NO. 54, FR3 shown in SEQ ID NO. 44 and FR4 shown in SEQ ID NO. 55;

(7) FR1 shown in SEQ ID NO. 56, FR2 shown in SEQ ID NO. 43, FR3 shown in SEQ ID NO. 57 and FR4 shown in SEQ ID NO. 58;

(8) FR1 shown in SEQ ID NO. 59, FR2 shown in SEQ ID NO. 60, FR3 shown in SEQ ID NO. 61 and FR4 shown in SEQ ID NO. 50;

(9) FR1 shown in SEQ ID NO. 62, FR2 shown in SEQ ID NO. 63, FR3 shown in SEQ ID NO. 64 and FR4 shown in SEQ ID NO. 48;

(10) FR1 shown in SEQ ID NO. 65, FR2 shown in SEQ ID NO. 60, FR3 shown in SEQ ID NO. 66 and FR4 shown in SEQ ID NO. 67;

(11) FR1 shown in SEQ ID NO. 62, FR2 shown in SEQ ID NO. 60, FR3 shown in SEQ ID NO. 68 and FR4 shown in SEQ ID NO. 48.

In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody directed 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.

In another preferred example, the nanobody against FLT3 includes a humanized antibody, a camelid antibody, a chimeric antibody.

In another preferred embodiment, the nanobody against FLT3 is alpaca.

In a second aspect of the invention, there is provided an antibody against FLT3 comprising one or more VHH chains of a nanobody against FLT3 according to the first aspect of the invention.

In another preferred example, the antibody against FLT3 may be a monomer, a bivalent antibody, and/or a multivalent antibody.

In a third aspect of the invention there is provided a chimeric antigen receptor CAR, the CAR comprising an extracellular domain comprising a nanobody against FLT3 as described in the first aspect of the invention, or an antibody against FLT3 as described in the second aspect of the invention.

In another preferred embodiment, the extracellular domain further comprises a signal peptide.

In another preferred embodiment, the extracellular domain further comprises a hinge region selected from the group consisting of: CD8, CD28, CD137, igG, or a combination thereof.

In another preferred embodiment, the hinge region is a human IgG1 Fc hinge region.

In another preferred embodiment, the extracellular domain comprises an antibody having the amino acid sequence shown in SEQ ID NO. 7-17.

In another preferred embodiment, the extracellular domain contains an antibody whose amino acid sequence has a homology of greater than or equal to 85%, preferably greater than or equal to 90%, more preferably greater than or equal to 95% with SEQ ID NO 7-17, or a difference of 1, 2 or 3 amino acids compared to SEQ ID NO 7-17.

In another preferred embodiment, the CAR has a structure according to formula Ia:

L1-Nb-H-TM-C-CD3ζ (Ia)

in the method, in the process of the invention,

l is a none or signal peptide sequence;

nb is a specific binding domain;

h is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling domain;

cd3ζ is a cytoplasmic signaling sequence derived from cd3ζ (including wild-type, or mutant/modification thereof);

the "-" is a connecting peptide or peptide bond.

In another preferred embodiment, the L is selected from the group consisting of signal peptides of the following histones: CD8, GM-CSF, CD4, CD28, CD137, or a mutant/modification thereof, or a combination thereof.

In another preferred embodiment, the Nb targets FLT3.

In another preferred embodiment, the Nb is FLT3 nanobody.

In another preferred embodiment, the H is selected from the group consisting of the hinge regions of: CD8, CD28, CD137, igG, or a combination thereof.

In another preferred embodiment, the H is a human IgG1 Fc hinge region.

In another preferred embodiment, the TM is selected from the transmembrane region of: CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutations/modifications thereof, or combinations thereof.

In another preferred embodiment, the C is selected from the group consisting of the costimulatory domains of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD 137), PD-1, dap10, LIGHT, NKG2C, B-H3, ICAM-1, LFA-1 (CD 11a/CD 18), ICOS (CD 278), NKG2D, GITR, OX L, 2B4, TLR, or mutations/modifications thereof, or combinations thereof.

In another preferred embodiment, the C is selected from the group consisting of ICOS, 41BB, or a co-stimulatory domain of a combination thereof.

In another preferred embodiment, the amino acid sequence of the CAR is as shown in SEQ ID NO. 1, 3, 4, 5, 6.

In another preferred embodiment, the nucleotide sequence of the CAR is set forth in SEQ ID NO. 2.

In a fourth aspect of the present invention, there is provided a recombinant protein having:

(i) Nanobodies against FLT3 as described in the first aspect of the invention, or antibodies against FLT3 as described in the second aspect of the invention, or chimeric antigen receptors as described in the third aspect of the invention; and

(ii) Optionally a tag sequence to assist expression and/or purification.

In another preferred embodiment, the tag sequence comprises an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

In another preferred embodiment, the recombinant protein specifically binds FLT3.

In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.

In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, the tag sequence is an Fc tag.

In a fifth aspect of the invention, there is provided a polynucleotide encoding a protein selected from the group consisting of: nanobody against FLT3 according to the first aspect of the invention, or antibody against FLT3 according to the second aspect of the invention, or chimeric antigen receptor according to the third aspect of the invention, or recombinant protein according to the fourth aspect of the invention.

In another preferred embodiment, the invention relates to a nucleic acid molecule encoding a nanobody of the invention against FLT 3. The nucleic acid of the invention may be RNA, DNA or cDNA.

In a sixth aspect of the invention there is provided an expression vector comprising a polynucleotide according to the fifth aspect of the invention.

In another preferred embodiment, the expression vector is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

In another preferred embodiment, the expression vector is selected from the group consisting of: pTomo lentiviral vector, plenti, pLVTH, pLJM, pHCMV, pLBS.CAG, pHR, pLV, pBlue, etc.

In another preferred embodiment, the expression vector is a pBlue vector.

In another preferred embodiment, the expression vector further comprises a gene selected from the group consisting of: promoters, transcription enhancing elements WPRE, long terminal repeat LTR, and the like.

In a seventh aspect of the invention there is provided a host cell comprising an expression vector according to the sixth aspect of the invention, or having integrated into its genome a polynucleotide according to the fifth aspect of the invention.

In another preferred embodiment, the host cell comprises a prokaryotic cell or a eukaryotic cell.

In another preferred embodiment, the host cell is selected from the group consisting of: coli, yeast cells, mammalian cells.

In another preferred embodiment, the host cell is a 293F cell.

In an eighth aspect of the invention, there is provided an engineered immune cell comprising an expression vector according to the sixth aspect of the invention or a polynucleotide according to the fifth aspect of the invention integrated with an exogenous source in a chromosome.

In another preferred embodiment, the engineered immune cell comprises a chimeric antigen receptor according to the third aspect of the invention.

In another preferred embodiment, the engineered immune cell is selected from the group consisting of:

(i) Chimeric antigen receptor alpha beta T cells (CAR-T cells);

(ii) Chimeric antigen receptor γδ T cells (CAR-T cells);

(iii) Chimeric antigen receptor NKT cells (CAR-NKT cells);

(iv) Chimeric antigen receptor NK cells (CAR-NK cells).

In another preferred embodiment, the engineered immune cells comprise autologous or allogeneic αβ T cells, γδ T cells, NKT cells, NK cells, or a combination thereof.

In another preferred embodiment, the engineered immune cell is a CAR-T cell.

In a ninth aspect of the invention, there is provided a method of producing nanobodies against FLT3, comprising the steps of:

(a) Culturing the host cell according to the seventh aspect of the invention under conditions suitable for nanobody production, thereby obtaining a culture comprising nanobodies directed against FLT 3;

(b) Isolating and/or recovering the nanobody against FLT3 from the culture; and

(c) Optionally, purifying and/or modifying the nanobody against FLT3 obtained in step (b).

In a tenth aspect of the invention, there is provided a method of preparing an engineered immune cell according to the eighth aspect of the invention, comprising the steps of: transduction of a polynucleotide according to the fifth aspect of the invention or an expression vector according to the sixth aspect of the invention into an immune cell, thereby obtaining the engineered immune cell.

In another preferred embodiment, the method further comprises the step of performing functional and validity assays on the obtained engineered immune cells.

In another preferred embodiment, the method comprises transducing a chimeric antigen receptor according to the third aspect of the invention into an immune cell, thereby obtaining the engineered immune cell.

In an eleventh aspect of the invention, there is provided an immunoconjugate comprising:

(a) Nanobody against FLT3 according to the first aspect of the invention, or antibody against FLT3 according to the second aspect of the invention, or recombinant protein according to the fourth aspect of the invention; and

(b) A coupling moiety selected from the group consisting of: a detectable label, drug, cytokine, radionuclide, enzyme, gold nanoparticle/nanorod, nanomagnetic particle, viral coat protein, or VLP, or a combination thereof.

In another preferred embodiment, said moiety (a) is coupled to said coupling moiety by a chemical bond or a linker.

In another preferred embodiment, the radionuclide comprises:

(i) A diagnostic isotope 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, yb-177, or combinations thereof.

In another preferred embodiment, the coupling moiety is a drug or a toxin.

In another preferred example, the drug is a drug targeted to treat FLT3 high expression disease.

In another preferred embodiment, the medicament is a medicament for targeted treatment of acute myeloid leukemia.

In another preferred embodiment, the drug is a cytotoxic drug.

In another preferred embodiment, the cytotoxic agent is selected from the group consisting of: an anti-tubulin drug, a DNA minor groove binding agent, a DNA replication inhibitor, an alkylating agent, an antibiotic, a folic acid antagonist, an antimetabolite, a chemosensitizer, a topoisomerase inhibitor, a vinca alkaloid, or a combination thereof.

Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors, typical cytotoxic drugs including, for example, auristatins (Auristatins), camptothecins (Camptothecins), duocarmycin/duocarmycin (Duocarmycins), etoposides (Etoposides), maytansinoids (Maytansines) and Maytansinoids (Maytansinoids) (e.g., DM1 and DM 4), taxanes (Taxanes), benzodiazepines (Benzodiazepines), or benzodiazepine-containing drugs (Benzodiazepine containing drugs) (e.g., pyrrolo [1,4] Benzodiazepines (PBDs), indoline Benzodiazepines (indomethacins) and oxazolidobenzodiazepines (oxazodiazepines)), vinca alkaloids (vilos), or combinations thereof.

In another preferred embodiment, the toxin is selected from the group consisting of: auristatins (e.g., auristatin E, auristatin F, MMAE and MMAF), aureomycin, mestaneol, ricin a-chain, combretastatin, docamicin, dolastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide (Tenoposide), vincristine, vinblastine, colchicine, dihydroxyanthrax, diketo, actinomycin, diphtheria toxin, pseudomonas Exotoxin (PE) A, PE, abrin a chain, a-sarcina, gelonin, mitogellin, restrictocin (retstricin), phenol, enomycin, curcin (curcin), crotonin, calicheamicin, saporin (Sapaonaria officinalis), a glucocorticoid, or a combination thereof.

In another preferred embodiment, the coupling moiety is a detectable label.

In another preferred embodiment, the coupling moiety is selected from the group consisting of: fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product, radionuclides, biotoxins, cytokines (e.g., IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles/nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like proteins (BPHL)) or any form of nanoparticle.

In another preferred embodiment, the immunoconjugate comprises: multivalent (e.g., bivalent) VHH chains of nanobodies against FLT3 according to the first aspect of the invention.

In another preferred embodiment, the multivalent means that a plurality of repeats of the same or different nanobody VHH chain directed against FLT3 according to the first aspect of the invention are comprised in the amino acid sequence of the immunoconjugate.

In a twelfth aspect of the invention there is provided the use of an active ingredient selected from the group consisting of: nanobody against FLT3 according to the first aspect of the invention, or antibody against FLT3 according to the second aspect of the invention, or chimeric antigen receptor according to the third aspect of the invention, or recombinant protein according to the fourth aspect of the invention, or engineered immune cell according to the eighth aspect of the invention, or immunoconjugate according to the eleventh aspect of the invention, or a combination thereof, the active ingredients are used to prepare:

(a) A medicament for preventing and/or treating FLT3 high expression disease;

(b) Reagents for detecting FLT3 high expression disease.

In another preferred embodiment, the active ingredient is selected from the group consisting of: nanobodies against FLT3 as described in the first aspect of the invention, or antibodies against FLT3 as described in the second aspect of the invention, or chimeric antigen receptors as described in the third aspect of the invention, or engineered immune cells as described in the eighth aspect of the invention, or a combination thereof.

In another preferred embodiment, the reagent is a diagnostic reagent, preferably a test strip or a test plate.

In another preferred embodiment, the diagnostic reagent is for: detecting FLT3 protein or a fragment thereof in the sample.

In another preferred embodiment, the FLT3 high expression disease is selected from: acute myelogenous leukemia, acute lymphoblastic leukemia (Acute lymphoblastic leukemia, ALL), chronic myelogenous leukemia (Chronic myelogenous leukemia, CML), myelodysplastic syndrome (Myelodysplastic syndromes, MDS), and the like.

In another preferred embodiment, the FLT3 high expressing disease is acute myeloid leukemia.

In a thirteenth aspect of the invention, there is provided a method for detecting FLT3 protein or a fragment thereof in a sample in vitro, said method comprising the steps of:

(1) Contacting the sample in vitro with a nanobody against FLT3 according to the first aspect of the invention, or an antibody against FLT3 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immunoconjugate according to the eleventh aspect of the invention, or a combination thereof;

(2) Detecting whether an antigen-antibody complex is formed, wherein the formation of a complex indicates the presence of FLT3 protein or a fragment thereof in the sample.

In another preferred embodiment, the detection comprises diagnostic or non-diagnostic.

In a fourteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:

(i) Nanobody against FLT3 according to the first aspect of the invention, or antibody against FLT3 according to the second aspect of the invention, or chimeric antigen receptor according to the third aspect of the invention, or recombinant protein according to the fourth aspect of the invention, or engineered immune cell according to the eighth aspect of the invention, or immunoconjugate according to the eleventh aspect of the invention, or a combination thereof as an active ingredient; and

(ii) A pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of: injection and freeze-dried preparation.

In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the nanobody against FLT3 according to the first aspect of the invention, or the antibody against FLT3 according to the second aspect of the invention, or the chimeric antigen receptor according to the third aspect of the invention, or the recombinant protein according to the fourth aspect of the invention, or the immunoconjugate according to the eleventh aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutically acceptable carrier, said percentages being mass percentages of the pharmaceutical composition.

In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1X 10 ³ -1×10 ⁸ Individual cells/mL, preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/mL.

In a fifteenth aspect of the present invention, there is provided a kit comprising:

(1) A first container comprising nanobodies against FLT3 according to the first aspect of the invention, or antibodies against FLT3 according to the second aspect of the invention, or chimeric antigen receptors according to the third aspect of the invention, or recombinant proteins according to the fourth aspect of the invention, or engineered immune cells according to the eighth aspect of the invention, or immunoconjugates according to the eleventh aspect of the invention, or a combination thereof; and/or

(2) A second container containing a second antibody against the contents of the first container;

or,

the kit comprises a detection plate, wherein the detection plate comprises: a substrate (support) and a test strip comprising a nanobody against FLT3 according to the first aspect of the invention, an antibody against FLT3 according to the second aspect of the invention, or a recombinant protein according to the third aspect of the invention, or an immunoconjugate according to the eighth aspect of the invention, or a combination thereof.

In another preferred embodiment, the kit further comprises a kit for non-invasively detecting the expression of FLT3 in a subject according to the instructions.

In another preferred embodiment, the kit is used for detecting FLT3 high expression diseases.

In another preferred embodiment, the FLT3 high expression disease is selected from: acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, etc.

In a sixteenth aspect of the present invention, there is provided a method for preventing and/or treating FLT3 high expressing disease, the method comprising: administering to a subject in need thereof a nanobody against FLT3 as described in the first aspect of the invention, or an antibody against FLT3 as described in the second aspect of the invention, or a chimeric antigen receptor as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention, or an engineered immune cell as described in the eighth aspect of the invention, or an immunoconjugate as described in the eleventh aspect of the invention, or a pharmaceutical composition as described in the fourteenth aspect of the invention, or a combination thereof.

In another preferred embodiment, the subject comprises a mammal, such as a human.

In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from the subject (autologous cells).

In another preferred embodiment, the CAR immune cells contained in the engineered immune cells or pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).

In another preferred embodiment, the methods described can be used in combination with other therapeutic methods.

In another preferred embodiment, the other treatment methods include chemotherapy, radiotherapy, targeted therapy, and the like.

In a seventeenth aspect of the present invention, there is provided a diagnostic method for FLT3 high expression disease, comprising the steps of:

(i) Obtaining a sample from a subject, contacting said sample with a nanobody against FLT3 according to the first aspect of the invention, or an antibody against FLT3 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immunoconjugate according to the eleventh aspect of the invention, or a combination thereof; and

(ii) Detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a patient in need of diagnosis of a FLT3 high expression disease.

In another preferred embodiment, the sample is a blood sample or a pharyngeal swab sample, or a sample in another tissue organ.

In an eighteenth aspect of the invention, there is provided a method of producing a recombinant polypeptide which is a nanobody against FLT3 as described in the first aspect of the invention, or an antibody against FLT3 as described in the second aspect of the invention, or a chimeric antigen receptor as described in the third aspect of the invention, or a recombinant protein as described in the fourth aspect of the invention, the method comprising:

(a) Culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression; and

(b) Isolating the recombinant polypeptide from the culture.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the amplification results of VHH fragments.

FIG. 2 shows the results of yeast display library diversity.

Fig. 3 shows the results of the flow-through assay after the first sort.

Fig. 4 shows the results of the flow assay after the second sort.

FIG. 5 shows the results of a yeast monoclonal flow assay.

FIG. 6 shows the structure of the constructed VHH eukaryotic expression vector.

FIG. 7 shows the results of FLT3 single domain antibody eukaryotic expression flow assay.

Figures 8A-8K show single domain antibody sequence information targeting FLT 3.

FIG. 9 shows a structural diagram of the FLT3-CAR vector, where FC is the human IgG1 Fc hinge region.

FIG. 10 shows the results of FLT3-CAR lentiviral titer assays of different structures.

FIG. 11 shows the expression of FLT3 on the surface of different tumor cell lines.

FIG. 12 shows in vitro killing of Raji cells (FLT 3 negative cell lines) by different structures of FLT 3-CAR-T.

FIG. 13 shows in vitro killing of FLT3 positive tumor cell lines by different structures of FLT 3-CAR-T. The target cells are MV-4-11, MOLM-13 and AML3 respectively, and different effective target ratios are set.

FIG. 14 shows secretion of cytokine Granzyme-B in supernatants after incubation of FLT3-CAR-T of different structures with FLT3 positive tumor cell lines. The target cells are MV-4-11, MOLM-13 and AML3 respectively, and the effective target ratio is 1:1 respectively.

FIG. 15 shows the sustained killing of FLT3-CAR-T of different structures against FLT3 positive tumor cell line MV-4-11. The target cells are MV-4-11 respectively, and the effective target ratio is 1:1 and 1:5 respectively. Tumor cells were fed every 24 hours or 48 hours.

Fig. 16 shows expression of FLT3 and CD33 on the surface of human HSPC cells.

Figure 17 shows cytokine release after incubation of cd34+ HSPC cells with different FLT3-CAR-T cells, respectively.

Figure 18 shows clonal formation following incubation of different CAR-T cells with cd34+ HSPC cells. BFU-E represents the clonal formation number of erythroid cells in HSPC cells, and CFU-GM represents the clonal formation of granulocytes and macrophages in HSPC cells.

FIG. 19 shows a pharmacodynamic protocol of TAA05-CAR-T on OCI-AML3-Luc-GFP model mice.

FIG. 20 shows in vivo fluorescence imaging of TAA05-CAR-T on AML3-Luc-GFP model mice. A. In vivo imaging at different times following administration of mice; increase in vivo imaging fluorescence values for D16 to D23 mice: negative increases in 4 out of 5 mice in the CAR-T group reflected regression of the tumor after CAR-T feedback.

FIG. 21 shows the body weight and survival of mice tested in the pharmacodynamic TAA05-CAR-T against AML3-Luc-GFP model mice. A. Body weight change curve for each group of mice: the weight of PBS and Mock T mice is reduced after the occurrence of the disease, and the weight of the CAR-T mice is stable all the time; B. survival of mice: the survival time of the CAR-T group mice is obviously prolonged.

Detailed Description

Through extensive and intensive research, the inventor develops a novel FLT3 nano antibody for the first time through a large number of screening, and successfully constructs FLT3-CAR-T cells targeting FLT3 based on the developed FLT3 nano antibody, so as to treat tumor patients with refractory recurrent AML and the like. The present inventors have demonstrated that the developed FLT3-CAR-T cell products have remarkable in vitro and in vivo anti-tumor effects on AML tumor cells through a large number of in vitro functional experiments and animal experiments, and that the FLT3-CAR-T cells of the present invention have better safety compared to CD33-CAR-T cells. The present invention has been completed on the basis of this finding. The novel FLT3-CAR-T targeting FLT3 can be used as a novel therapeutic means for targeted treatment of refractory recurrent AML.

The present invention will be described in detail with respect to the engineering immune cells of the present invention, typically by taking CAR-T cells as an example. The engineered immune cells of the invention are not limited to the CAR-T cells described in the context, but have the same or similar technical features and benefits as the CAR-T cells described in the context. Specifically, when the immune cells express the chimeric antigen receptor CAR, the NK cells are identical to T cells (or the T cells may be replaced with NK cells).

Terminology

As used herein, the terms "single domain antibody", "single domain antibody of the invention", "recombinant antibody", "FLT3 nanobody", "anti-FLT 3 nanobody" are used interchangeably and refer to a recombinant/single domain antibody of the invention that specifically binds to the target protein FLT 3.

In order that the present disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meanings given below, unless expressly specified otherwise herein.

Antibody numbers and corresponding sequence numbers of the nanobody of the invention are shown in table 1 below.

TABLE 1

Note that: each numerical value in the table indicates a sequence number, i.e. "1" indicates "SEQ ID NO:1", and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, FR4 shown in the table are the numbers of the amino acid sequences thereof.

As used herein, the term "antibody" or "immunoglobulin" is an iso-tetralin protein of about 150000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), having identical structural features. Each light chain is linked to the heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. One end of each light chain is provided with a variable region (VL) and the other end is provided with a constant region; 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. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

As used herein, the terms "single domain antibody", "VHH", "Nanobody", "single domain antibody (Single domain antibody, sdAb, or Nanobody)" have the same meaning and are used interchangeably to refer to the variable region of a cloned antibody heavy chain, constructing a single domain antibody (VHH) consisting of only one heavy chain variable region, which is the smallest antigen binding fragment with complete function. Typically, after an antibody is obtained which naturally lacks the light and heavy chain constant region 1 (CH 1), the variable region of the heavy chain of the antibody is cloned, and a single domain antibody (VHH) consisting of only one heavy chain variable region is constructed.

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region. It is concentrated in three fragments in the light and heavy chain variable regions called Complementarity Determining Regions (CDRs) or hypervariable regions. The more conserved parts of the variable region are called the Framework Regions (FR). The variable regions of the natural heavy and light chains each comprise four FR regions, which are generally in a β -sheet configuration, connected by three CDRs forming a connecting loop, which in some cases may form part of a b-sheet structure. The CDRs in each chain are held closely together by 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 publication No.91-3242, vol. I, pp. 647-669 (1991)). The constant regions are not directly involved in binding of the antibody to the antigen, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of the antibody.

Immunoconjugates and fusion expression products include, as known to those of skill in the art: conjugates of drugs, toxins, cytokines (cytokines), radionuclides, enzymes and other diagnostic or therapeutic molecules with antibodies or fragments thereof of the present invention. The invention also includes cell surface markers or antigens that bind to the nanobodies or fragments thereof against the novel coronaviruses.

As used herein, the term "heavy chain variable region" is used interchangeably with "VH".

As used herein, the term "variable region" is used interchangeably with "complementarity determining region (Complementarity determining region, CDR)".

In a preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions CDR1, CDR2, and CDR3.

In a preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and the heavy chain constant region described above.

In the present invention, the terms "antibody of the invention", "protein of the invention", or "polypeptide of the invention" are used interchangeably to refer to a polypeptide that specifically binds to FLT3 protein, such as a protein or polypeptide having a heavy chain variable region. They may or may not contain an initiating methionine.

The invention also provides other proteins or fusion expression products having the antibodies of the invention. In particular, the invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain comprising a variable region, provided that the variable region is identical or at least 90% homologous, preferably at least 95% homologous, to the heavy chain variable region of an antibody of the invention.

In general, the antigen binding properties of antibodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FRs), the amino acid sequences of the 4 FRs being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure, the β -sheets formed by the FR therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

The variable regions of the heavy chains of the antibodies of the invention are of particular interest because they are involved, at least in part, in binding to antigens. Thus, the invention includes those molecules having antibody heavy chain variable regions with CDRs, so long as the CDRs are 90% or more (preferably 95% or more, most preferably 98% or more) homologous to the CDRs identified herein.

The invention includes not only whole antibodies but also fragments of antibodies having immunological activity or fusion proteins of antibodies with other sequences. Thus, the invention also includes fragments, derivatives and analogues of said antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that retain substantially the same biological function or activity of an antibody of the invention. The polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide having one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, substituted, which may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound, such as a compound that extends the half-life of the polypeptide, for example polyethylene glycol, or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence, such as a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag. Such fragments, derivatives and analogs are within the purview of one skilled in the art and would be well known in light of the teachings herein.

The antibody of the present invention refers to a polypeptide having FLT3 protein binding activity, which includes the above CDR regions. The term also includes variants of polypeptides comprising the above-described CDR regions that have the same function as the antibodies of the invention. These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminal and/or N-terminal end. For example, in the art, substitution with amino acids of similar or similar properties does not generally alter the function of the protein. As another example, the addition of one or more amino acids at the C-terminus and/or N-terminus typically does not alter the function of the protein. The term also includes active fragments and active derivatives of the antibodies of the invention.

The variant forms of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA which hybridizes under high or low stringency conditions with the encoding DNA of an antibody of the invention, and polypeptides or proteins obtained using antisera raised against an antibody of the invention.

The invention also provides other polypeptides, such as fusion proteins comprising an antibody or fragment thereof. In addition to nearly full length polypeptides, the invention also includes fragments of the antibodies of the invention. Typically, 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 the antibody of the invention.

In the present invention, a "conservative variant of an antibody of the present invention" refers to a polypeptide in which at most 10, preferably at most 8, more preferably at most 5, and most preferably at most 3 amino acids are replaced by amino acids of similar or similar nature, as compared to the amino acid sequence of the antibody of the present invention. These conservatively variant polypeptides are preferably generated by amino acid substitutions according to Table 2.

TABLE 2

Initial residues	Representative substitution	Preferred substitution
			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

The invention also provides polynucleotide molecules encoding the antibodies or fragments thereof or fusion proteins thereof. The polynucleotides of the invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

Polynucleotides encoding the mature polypeptides of the invention include: a coding sequence encoding only the mature polypeptide; a coding sequence for a mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) of the mature polypeptide, and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may include polynucleotides encoding the polypeptide, or may include additional coding and/or non-coding sequences.

The invention also relates to polynucleotides which hybridize to the sequences described above and which have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present invention relates in particular to polynucleotides which hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" means: (1) Hybridization and elution at lower ionic strength and higher temperature, e.g., 0.2 XSSC, 0.1% SDS,60 ℃; or (2) adding denaturing agents such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll,42℃and the like during hybridization; or (3) hybridization only occurs when the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, 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 a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. One possible approach is to synthesize the sequences of interest by synthetic means, in particular with short fragment lengths. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. In addition, the heavy chain coding sequence and the expression tag (e.g., 6 His) may be fused together to form a fusion protein.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules that exist in an isolated form.

At present, it is already possible to obtain the DNA sequences encoding the proteins of the invention (or fragments or derivatives thereof) entirely by chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors, for example) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to vectors comprising the above-described suitable DNA sequences and suitable promoter or control sequences. These vectors may be used to transform an appropriate host cell to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; animal cells of CHO, COS7, 293 cells, and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl2. Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

The antibodies of the invention may be used alone or in combination or coupling with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or a combination of any of the above.

Detectable markers for diagnostic purposes include, but are not limited to: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (electronic computer tomography) contrast agents, or enzymes capable of producing a detectable product.

Therapeutic agents that may be conjugated or coupled to an antibody of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. prodrug activating enzymes (e.g., DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), and the like.

Nanometer antibody (Nb)

As used herein, the term "Nanobody" (Nb) refers to an antibody in which there is a natural deletion of the light chain in alpaca peripheral blood, the main difference between Nanobody and traditional antibody being their structure and domain: conventional antibodies have two variable domains, called VH and VL, which provide stability and binding specificity to each other; nanobodies comprise only one heavy chain variable region (VHH), one hinge region and two conventional constant regions, CH2 and CH3, lack VL domains, but are still highly stable. The lack of VL domains also means that the nanobody has a hydrophilic side and that the nanobody does not stick to each other as easily as an engineered single chain antibody fragment (scFv), or even aggregate into a block. More importantly, the VHH structure cloned and expressed alone has structural stability comparable to that of the original heavy chain antibody and binding activity to the antigen, the smallest unit known to bind the antigen of interest. The VHH crystals were 2.5nm long and 4nm long with a molecular weight of only 15kDa.

The antigen binding properties of nanobodies can be described by 3 specific regions located in the variable region of the heavy chain, called variable regions (CDRs), which are separated into 4 Framework Regions (FR), the amino acid sequences of 4 FR being relatively conserved and not directly involved in the binding reaction. These CDRs form a loop structure and the β -sheets formed by the FR therebetween are spatially close to each other, with the CDRs on the heavy chain constituting the antigen binding site of the antibody. It is possible to determine which amino acids constitute the FR or CDR regions by comparing the amino acid sequences of the same type of antibody.

Chimeric Antigen Receptor (CAR)

As used herein, a chimeric immune antigen receptor includes an extracellular domain, an optional hinge region, a transmembrane domain, and an intracellular domain. Extracellular domains include optional signal peptides and target-specific binding domains (also referred to as antigen binding domains). The intracellular domain includes a costimulatory domain and a cd3ζ chain moiety. When CAR is expressed in T cells, the extracellular segment recognizes a specific antigen, and then transduces the signal through the intracellular domain, causing activated proliferation of the cell, cytolytic toxicity, and secretion of cytokines such as IL-2 and IFN- γ, etc., affecting the tumor cells, causing the tumor cells to not grow, to be caused to die or otherwise be affected, and causing the patient's tumor burden to shrink or eliminate. The antigen binding domain is preferably fused to an intracellular domain from one or more of the costimulatory molecule and the cd3ζ chain.

Chimeric antigen receptor T cells (CAR-T cells)

As used herein, the terms "CAR-T cell", "CAR-T", "FLT3-CAR-T cell", "CAR-T cell of the invention" and the like all refer to CAR-T cells according to the eighth aspect of the invention. The CAR-T cell can be used for treating tumors with high FLT3 expression, such as acute myelogenous 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 course of action of CAR-T cells is not restricted by MHC; (2) In view of the fact that many tumor cells express the same tumor antigen, CAR gene construction for a certain tumor antigen can be widely utilized once completed; (3) The CAR can utilize not only tumor protein antigens but also glycolipid non-protein antigens, so that the target range of the tumor antigens is enlarged; (4) The use of autologous patient cells reduces the risk of rejection; (5) The CAR-T cells have an immunological memory function and can survive in vivo for a long time.

Chimeric antigen receptor NK cells (CAR-NK cells)

As used herein, the terms "CAR-NK cells", "CAR-NK", "FLT3-CAR-NK cells", "CAR-NK cells of the invention" and the like all refer to CAR-NK cells of the eighth aspect of the invention. The CAR-NK cell can be used for treating tumors with high FLT3 expression, such as acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome and the like.

Natural killer cells (Natural killer cell, NK) are a major class of immune effector cells that protect the body from viral infection and tumor cells by non-antigen specific pathways. New functions may be obtained by engineered (genetically modified) NK cells, including the ability to specifically recognize tumor antigens and enhanced anti-tumor cytotoxicity.

CAR-NK cells also have advantages over autologous CAR-T cells, such as: (1) The perforin and the granzyme are released to directly kill tumor cells, and the perforin and granzyme have no killing effect on normal cells of the organism; (2) They release very small amounts of cytokines and thus reduce the risk of cytokine storms; (3) Is easy to expand and develop into a ready-made product in vitro. In addition, similar to CAR-T cell therapy.

FLT3

FLT3 (Fms-like tyrosine kinase, fms-like tyrosine kinase 3) belongs to a family of class III receptor tyrosine kinases (Receptor tyrosine kinase III, RTK III), and in recent years, many large sample studies have demonstrated that activating mutations of FLT3 play a very important pathological role in the occurrence and progression of diseases such as AML.

AML patients with FLT3/ITD activating mutations generally have unique clinical characteristics of high peripheral blood leukocyte count, poor clinical prognosis, susceptibility to relapse, etc., and since the detection method of FLT3 activating mutations is simple and easy to implement, there is increasing effort by researchers to develop FLT3 into conventional detection means for AML patients to guide the treatment and prognosis of AML patients and as detection means for minimal residual leukemia, and as yet another new target for leukemia patients' chemotherapeutic drugs (there are currently already drugs for FLT3-ITD mutation treatment). There are two main types of activating mutations that have been demonstrated for FLT 3: internal tandem repeats (Internal tandem duplication, ITD), with incidence in AML and MDS patients of 15-35% and 5-10%, respectively, with incidence in ALL of < 1%, and seen mainly in dual-phenotype ALL cases; the incidence of point mutations in the activation loop (Point mutation in the activation loop, TKD point mutations) in AML, MDS and ALL patients was 5-10%, 2-5% and 1-3%, respectively. Both activating mutations of FLT3 can cause FLT3 to be automatically phosphorylated so as to lead to ligand independent constitutive activation of FLT3 and further activate downstream abnormal signal transduction of FLT3, thereby playing roles in promoting proliferation and inhibiting apoptosis, and leading leukemia patients with the mutant phenotype to have poorer clinical prognosis

Pharmaceutical composition

The invention also provides a composition. Preferably, the composition is a pharmaceutical composition comprising an antibody or active fragment thereof or fusion protein thereof as described above, and a pharmaceutically acceptable carrier. Typically, these materials are formulated in a nontoxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to 8, preferably about 6 to 8, although the pH may vary depending on the nature of the material being formulated and the condition being treated. The formulated pharmaceutical compositions may be administered by conventional routes including, but not limited to: intraperitoneal, intravenous, or topical administration.

The pharmaceutical compositions of the invention contain a safe and effective amount (e.g., 0.001-99wt%, preferably 0.01-90wt%, more preferably 0.1-80 wt%) of the antibodies (or conjugates thereof) of the invention as described above, and a pharmaceutically acceptable carrier or excipient. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be compatible with the mode of administration. The pharmaceutical compositions of the invention may be formulated as injectables, e.g. by conventional means using physiological saline or aqueous solutions containing glucose and other adjuvants. The pharmaceutical compositions, such as injections, solutions are preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount, for example, from about 10 micrograms per kilogram of body weight to about 50 milligrams per kilogram of body weight per day. In addition, the polypeptides of the invention may also be used with other therapeutic agents.

When a pharmaceutical composition is used, a safe and effective amount of the immunoconjugate is administered to the mammal, wherein the safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases no more than about 50 milligrams per kilogram of body weight, preferably the dose is about 10 micrograms per kilogram of body weight to about 10 milligrams per kilogram of body weight. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Nanobody against FLT3

In the present invention, the nanobody against FLT3 includes a monomer, a bivalent body (bivalent antibody), a tetravalent body (tetravalent antibody), and/or a multivalent body (multivalent antibody).

In a preferred embodiment of the invention, the nanobody against FLT3 comprises one or more VHH chains having the amino acid sequences shown as 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.

Labeled antibodies

In a preferred embodiment of the invention, the antibody is provided with a detectable label. More preferably, the marker is selected from the group consisting of: isotopes, colloidal gold labels, colored labels, or fluorescent labels.

Colloidal gold labelling can be carried out by methods known to those skilled in the art. In a preferred embodiment of the present invention, the antibody against FLT3 protein is labeled with colloidal gold, resulting in a colloidal gold-labeled antibody.

The nanobody for FLT3 can effectively bind to FLT3 protein.

Detection method

The invention also relates to methods of detecting FLT3 proteins or fragments thereof. The method comprises the following steps: obtaining a cell and/or tissue sample; dissolving a sample in a medium; detecting the level of FLT3 protein in the solubilized sample.

In the detection method of the present invention, the sample used is not particularly limited, and a representative example is a cell-containing sample present in a cell preservation solution.

Kit for detecting a substance in a sample

The invention also provides a kit comprising an antibody (or fragment thereof) or assay plate of the invention, which in a preferred embodiment of the invention further comprises a container, instructions for use, buffers, and the like.

The invention also provides a detection kit for detecting the level of FLT3 protein, which comprises an antibody for recognizing the FLT3 protein, a lysis medium for dissolving a sample, and a general reagent and a buffer solution required for detection, such as various buffers, detection markers, detection substrates and the like. The detection kit may be an in vitro diagnostic device.

Formulations

The invention provides an engineered immune cell (e.g., CAR-T cell) comprising the eighth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injection. Preferably, the concentration of said CAR-T cells in said formulation is 1 x 10 ³ -1×10 ⁸ Individual cells/mL, more preferably 1X 10 ⁴ -1×10 ⁷ Individual cells/mL.

In one embodiment, the formulation may include a buffer such as neutral buffered saline, sulfate buffered saline, or the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; a protein; polypeptides or amino acids such as glycine; an antioxidant; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and a preservative. The formulations of the present invention are preferably formulated for intravenous administration.

Application of

As described above, the antibody of the present invention has a wide range of biological and clinical applications, and its application relates to various fields such as diagnosis and treatment of diseases associated with FLT3 protein, basic medical research, biological research, etc. One preferred application is for clinical diagnosis, prevention and treatment against FLT3 protein.

The invention also provides a method of stimulating an immune response mediated by T cells targeted to a mammalian tumor cell population or tissue, comprising the steps of: administering the CAR-T cells of the invention to a mammal.

In one embodiment, the invention includes a class of cell therapies in which autologous T cells (or heterologous donors) from a patient are isolated, activated and genetically engineered to produce CAR-T cells, and subsequently injected into the same patient. This approach results in a very low probability of graft versus host response, and antigen is recognized by T cells in a non-MHC restricted manner. Furthermore, a CAR-T can treat all cancers that express this antigen. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained control of tumors.

In one embodiment, the CAR-T cells of the invention can undergo stable in vivo expansion and can last from months to years. Additionally, the CAR-mediated immune response can be part of an adoptive immunotherapy step in which the CAR-T cells can induce a specific immune response to highly expressing tumor cells of the antigen recognized by the CAR antigen binding domain. For example, the CAR-T cells of the invention elicit a specific immune response against tumor cells that are highly expressed by FLT 3.

Treatable cancers include tumors that are not vascularized or have not been substantially vascularized, as well as vascularized tumors. Types of cancers treated with the CARs of the invention include, but are not limited to: acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, etc.

In general, cells activated and expanded as described herein are useful in the treatment and prevention of diseases such as tumors. Accordingly, the 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 invention.

The CAR-T cells of the invention can be administered alone or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2, IL-17 or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the 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 may be administered in a manner suitable for the disease to be treated (or prevented). The number and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's disease, or may be determined by clinical trials.

When referring to an "immunologically effective amount", "antitumor effective amount", "tumor-inhibiting effective amount" or "therapeutic amount", the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account the age, weight, tumor size, degree of infection or metastasis and individual differences of the condition of the patient (subject). Pharmaceutical compositions comprising T cells described herein may be administered at 10 ⁴ To 10 ⁹ A dose of individual cells/kg body weight, preferably 10 ⁵ To 10 ⁷ A dose of individual cells/kg body weight (including all whole values within the range) is administered. T cell compositions may also be administered multiple times at these doses. Cells can be administered by using injection techniques well known in immunotherapy (see, e.g., 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 arts by monitoring the patient for signs of disease and adjusting the treatment accordingly.

Administration of the subject compositions may be performed in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.

In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with: such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C) or natalizumab therapy for MS patients or ertapelizumab therapy for psoriasis patients or other therapy for PML patients. In a further embodiment, the T cells of the invention may be used in combination with: chemotherapy, radiation, immunosuppressives such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to a patient in combination (e.g., before, simultaneously or after) with bone marrow transplantation, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide. For example, in one embodiment, the subject may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In some embodiments, the subject receives injection of expanded immune cells of the invention after transplantation. In an additional embodiment, the expanded cells are administered pre-operatively or post-operatively.

The dose of the above treatments administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The dosage ratio administered to humans may be carried out according to accepted practices in the art. Typically, 1X 10 will be administered per treatment or per course of treatment ⁵ Up to 1X 10 ¹⁰ The modified T cells of the invention are administered to a patient, for example, by intravenous infusion.

Amino acid and nucleotide sequence

SEQ ID NO. 1 (PA 0135-MN-CAR amino acid sequence)

MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLNLSCEVSGVIFSMLGMGWYRQAPGQERELFAAVTSGGFTSYIESVRGRFTISRDNDKRSVYLQMNNVKPEDTGVYYCNRDPVRSSDNWGQGTQVTVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 2 (PA 0135-MN-CAR nucleotide sequence)

ATGCTGCTGCTGGTGACCTCTCTGCTGCTCTGCGAACTGCCTCACCCAGCCTTTCTGCTGATCCCCCAAGTGCAACTTGTGGAATCAGGAGGAGGACTTGTGCAACCTGGAGGATCACTTAACCTTTCATGCGAAGTGTCAGGAGTGATCTTCTCCATGCTTGGAATGGGATGGTACAGACAAGCACCTGGACAAGAAAGAGAATTATTCGCCGCTGTGACATCAGGAGGATTTACATCATACATCGAATCAGTGAGAGGAAGATTTACAATCTCAAGAGATAACGATAAGCGCTCGGTGTACCTTCAAATGAACAACGTGAAACCTGAAGATACAGGAGTGTACTACTGCAACAGAGATCCTGTGAGATCATCAGATAACTGGGGGCAGGGAACACAAGTGACAGTGTCATCAGAGAGCAAATACGGCCCTCCTTGCCCTCCTTGCCCAGCCCCAGAATTTGAGGGAGGACCTAGCGTGTTCCTGTTCCCTCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCAGAAGTCACCTGCGTGGTGGTGGACGTGTCTCAGGAAGACCCCGAGGTGCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTTCCAGAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCTAGAGAGCCTCAGGTGTACACACTGCCCCCTTCTCAGGAGGAGATGACCAAGAACCAGGTGTCCCTGACTTGCCTCGTGAAGGGCTTCTACCCCAGCGATATTGCCGTGGAGTGGGAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACCCCTCCCGTGCTGGATAGCGACGGCTCTTTCTTCCTGTACAGCCGGCTGACAGTGGACAAAAGTCGCTGGCAGGAGGGCAACGTGTTCAGTTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGTCTCTGTCTCTCGGCAAGTGGCTCCCTATCGGTTGCGCCGCCTTTGTCGTCGTCTGTATCCTCGGCTGCATCCTCATCTGTTGGCTCACCAAGAAGAAGTACAGCAGCAGCGTGCACGACCCCAACGGCGAGTACATGTTCATGCGGGCCGTCAACACCGCCAAGAAGAGCAGACTGACCGACGTGACCCTGAAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGGCCAGTGCAGACAACCCAGGAGGAAGACGGCTGCTCTTGCAGATTCCCCGAGGAAGAAGAGGGCGGTTGCGAGCTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCAGCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACGACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCCCCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGTATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCACCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGGTGA

SEQ ID NO. 3 (PA 0135-EF-CAR amino acid sequence)

MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLRLSCKVSGMIFSMFGMGWYRQAPGQERELIAAITSGGFTSYVESVRGRFTISRDNAKRSVYLQMNNLKPEDTAVYYCNQDPVRSSDVWGQGTQVTVSGESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 4 (PA 0135-GH-CAR amino acid sequence)

MLLLVTSLLLCELPHPAFLLIPAVQLVESGGGLVQPGGSLRLSCVVSGTIFSMFGMGWYRQAPGHERELIAAITSGHFTSYVESVRGRFTISRDNAKRSVYLQMNGVKPEDTAVYYCNRDPIQSSDVWGQGTQVTVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 5 (PA 0135-KL-CAR amino acid sequence)

MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVQPGGSLNLTCKVSGTIFSMLGMGWYRRAPGQERELFAAITSGGFDSYVESVRGRFIISRDNDKRSVYLQMNNLKPEDTAVYYCNQDPIRFSDVWGQGTLVTVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 6 (PA 0135-PQ-CAR amino acid sequence)

MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVLPGGSLNLSCEVSGTIFSMLGMGWYRRAPGQERELFAAITSGGFTSYIESVKGRFTISRDNDKRSVYLQMNNVKPEDTAVYYCNRDPLRSSDVWGQGTQITVSSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKWLPIGCAAFVVVCILGCILICWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 7 (nanobody C-F15-10)

QVQLVESGGGLVQPGGSLRLSCAVSGTIFSMLGMGWYRRAPGQERELVAAITSGHFTSYIESVKGRFTISRDNAKRSVYLQMNSVKPEDTAVYYCNRDPVRSSDVWGQGTQVTVSG

SEQ ID NO. 8 (nanobody C-F21-1)

QVQLVESGGGLVQPGGSLRLSCKVSGMIFSMFGMGWYRQAPGQERELIAAITSGGFTSYVESVRGRFTISRDNAKRSVYLQMNNLKPEDTAVYYCNQDPVRSSDVWGQGTQVTVSG

SEQ ID NO. 9 (nanobody C-A5-11)

AVQLVESGGGLVQPGGSLRLSCVVSGTIFSMFGMGWYRQAPGHERELIAAITSGHFTSYVESVRGRFTISRDNAKRSVYLQMNGVKPEDTAVYYCNRDPIQSSDVWGQGTQVTVSS

SEQ ID NO. 10 (nanobody C-1-D3)

QVQLVESGGGLVQPGGSLRLSCKVSGMIFSMFGMGWYRQAPGQERELIAAITSGGFDSYVESVRGRFTISRDNDKRSVYLQMNNLKPEDTAVYYCNADVIRRVGSEHRGPRTIWGQGTLVTVSS

SEQ ID NO. 11 (nanobody C-1-G3)

QVQLVESGGGLVQPGGSLRLSCKVSGMIFSMFGMGWYRQTPGQERELIAAITSGGFTSYVESVRGRFTISRDNAKNTLYLQMNNIKPEDTAVYYCNGDRLARRGIWGPGTLVTVSS

SEQ ID NO. 12 (nanobody C-2-C2)

QVQLVESGGGLVQPGGSLRLSCKVSGMIFSMFGMGWYRQAPGQEREHIAAITSGGFTSYVESVRGRFTISRDNAKRSVYLQMNNLKPEDTAVYYCIRENYNNDRKDGGTQVTVSS

SEQ ID NO. 13 (nanobody C-1-E6)

QVQLVESGGGLVRPGGSLRLSCKVSGMIFSMFGMGWYRQAPGQERELIAAITSGGFTSYVESVRGRFTISRDNAKNTVYLHMNDLKPEDTAVYYCAADLWGDGTKWDRANEYDYWGGGTQVTVSS

SEQ ID NO. 14 (nanobody C-2-D8)

QVQLVESGGGLVQPGGSLNLTCKVSGTIFSMLGMGWYRRAPGQERELFAAITSGGFDSYVESVRGRFIISRDNDKRSVYLQMNNLKPEDTAVYYCNQDPIRFSDVWGQGTLVTVSS

SEQ ID NO. 15 (nanobody C-2-A5)

QVQLVESGGGLVQPGGSLNLSCEVSGVIFSMLGMGWYRQAPGQERELFAAVTSGGFTSYIESVRGRFTISRDNDKRSVYLQMNNVKPEDTGVYYCNRDPVRSSDNWGQGTQVTVSS

SEQ ID NO. 16 (nanobody C-2-A3)

QVQLVESGGGLVLPGGSLNLSCEVSGTIFSMLGMGWYRRAPGQERELFAAITSGGFTSYIESVKGRFTISRDNDKRSVYLQMNNVKPEDTAVYYCNRDPLRSSDVWGQGTQITVSS

SEQ ID NO. 17 (nanobody C-2-C4)

QVQLVESGGGLVQPGGSLNLSCEVSGTIFSMLGMGWYRRAPGQERELFAAITSGGFTSYIESVKGRFTISRDNAKRSVYLQMNNLKPEDTAVYYCNQDPVRSSDNWGQGTQVTVSS

SEQ ID NO. 18 (CDR 1 of nanobody C-F15-10)

GTIFSMLG

SEQ ID NO. 19 (nanobody C-F15-10, CDR2 of C-A5-11)

ITSGHFT

SEQ ID NO. 20 (CDR 3 of nanobody C-F15-10)

NRDPVRSSDV

SEQ ID NO. 21 (CDR 1 of nanobody C-F21-1, C-1-D3, C-1-G3, C-2-C2, C-1-E6)

GMIFSMFG

SEQ ID NO. 22 (CDR 2 of nanobody C-F21-1, C-1-G3, C-2-C2, C-1-E6, C-2-A3, C-2-C4)

ITSGGFT

SEQ ID NO. 23 (CDR 3 of nanobody C-F21-1)

NQDPVRSSDV

SEQ ID NO. 24 (CDR 1 of nanobody C-A5-11)

GTIFSMFG

SEQ ID NO. 25 (CDR 3 of nanobody C-A5-11)

NRDPIQSSDV

SEQ ID NO. 26 (CDR 2 of nanobody C-1-D3, C-2-D8)

ITSGGFD

SEQ ID NO. 27 (CDR 3 of nanobody C-1-D3)

NADVIRRVGSEHRGPRTI

SEQ ID NO. 28 (CDR 3 of nanobody C-1-G3)

NGDRLARRGI

SEQ ID NO. 29 (CDR 3 of nanobody C-2-C2)

IRENYNNDRK

SEQ ID NO. 30 (CDR 3 of nanobody C-1-E6)

AADLWGDGTKWDRANEYDY

SEQ ID NO. 31 (CDR 1 of nanobody C-2-D8, C-2-A3, C-2-C4)

GTIFSMLGMG

SEQ ID NO. 32 (CDR 3 of nanobody C-2-D8)

NQDPIRFSDV

SEQ ID NO. 33 (CDR 1 of nanobody C-2-A5)

GVIFSMLGMG

SEQ ID NO. 34 (CDR 2 of nanobody C-2-A5)

VTSGGFT

SEQ ID NO. 35 (CDR 3 of nanobody C-2-A5)

NRDPVRSSDN

SEQ ID NO. 36 (CDR 3 of nanobody C-2-A3)

NRDPLRSSDV

SEQ ID NO. 37 (CDR 3 of nanobody C-2-C4)

NQDPVRSSDN

SEQ ID NO. 38 (FR 1 of nanobody C-F15-10)

QVQLVESGGGLVQPGGSLRLSCAVS

SEQ ID NO. 39 (nanobody C-F15-10 FR 2)

MGWYRRAPGQERELVAA

SEQ ID NO. 40 (nanobody C-F15-10 FR 3)

SYIESVKGRFTISRDNAKRSVYLQMNSVKPEDTAVYYC

SEQ ID NO. 41 (nanobody C-F15-10, FR4 of C-F21-1)

WGQGTQVTVSG

SEQ ID NO. 42 (nanobody C-F21-1, C-1-D3, C-1-G3, C-2-C2 FR 1)

QVQLVESGGGLVQPGGSLRLSCKVS

SEQ ID NO. 43 (nanobody C-F21-1, C-1-D3, C-1-E6 FR 2)

MGWYRQAPGQERELIAA

SEQ ID NO. 44 (nanobody C-F21-1, FR3 of C-2-C2)

SYVESVRGRFTISRDNAKRSVYLQMNNLKPEDTAVYYC

SEQ ID NO. 45 (nanobody C-A5-11 FR 1)

AVQLVESGGGLVQPGGSLRLSCVVS

SEQ ID NO. 46 (nanobody C-A5-11 FR 2)

MGWYRQAPGHERELIAA

SEQ ID NO. 47 (nanobody C-A5-11 FR 3)

SYVESVRGRFTISRDNAKRSVYLQMNGVKPEDTAVYYC

SEQ ID NO. 48 (nanobody C-A5-11, C-2-A5, C-2-C4 FR 4)

WGQGTQVTVSS

SEQ ID NO. 49 (FR 3 of nanobody C-1-D3)

SYVESVRGRFTISRDNDKRSVYLQMNNLKPEDTAVYYC

SEQ ID NO. 50 (nanobody C-1-D3, FR4 of C-2-D8)

WGQGTLVTVSS

SEQ ID NO. 51 (nanobody C-1-G3 FR 2)

MGWYRQTPGQERELIAA

SEQ ID NO. 52 (nanobody C-1-G3 FR 3)

SYVESVRGRFTISRDNAKNTLYLQMNNIKPEDTAVYYC

SEQ ID NO. 53 (FR 4 of nanobody C-1-G3)

WGPGTLVTVSS

SEQ ID NO. 54 (nanobody C-2-C2 FR 2)

MGWYRQAPGQEREHIAA

SEQ ID NO. 55 (nanobody C-2-C2 FR 4)

DGGTQVTVSS

SEQ ID NO. 56 (FR 1 of nanobody C-1-E6)

QVQLVESGGGLVRPGGSLRLSCKVS

SEQ ID NO. 57 (nanobody C-1-E6 FR 3)

SYVESVRGRFTISRDNAKNTVYLHMNDLKPEDTAVYYC

SEQ ID NO. 58 (FR 4 of nanobody C-1-E6)

WGGGTQVTVSS

SEQ ID NO. 59 (FR 1 of nanobody C-2-D8)

QVQLVESGGGLVQPGGSLNLTCKVS

SEQ ID NO. 60 (nanobody C-2-D8, C-2-A3, C-2-C4 FR 2)

WYRRAPGQERELFAA

SEQ ID NO. 61 (FR 3 of nanobody C-2-D8)

SYVESVRGRFIISRDNDKRSVYLQMNNLKPEDTAVYYC

SEQ ID NO. 62 (nanobody C-2-A5, FR1 of C-2-C4)

QVQLVESGGGLVQPGGSLNLSCEVS

SEQ ID NO. 63 (nanobody C-2-A5 FR 2)

WYRQAPGQERELFAA

SEQ ID NO. 64 (nanobody C-2-A5 FR 3)

SYIESVRGRFTISRDNDKRSVYLQMNNVKPEDTGVYYC

SEQ ID NO. 65 (FR 1 of nanobody C-2-A3)

QVQLVESGGGLVLPGGSLNLSCEVS

SEQ ID NO. 66 (nanobody C-2-A3 FR 3)

SYIESVKGRFTISRDNDKRSVYLQMNNVKPEDTAVYYC

SEQ ID NO. 67 (nanobody C-2-A3 FR 4)

WGQGTQITVSS

SEQ ID NO. 68 (FR 3 of nanobody C-2-C4)

SYIESVKGRFTISRDNAKRSVYLQMNNLKPEDTAVYYC

The main advantages of the invention include:

1. the FLT3 nanobody developed by the invention has high affinity, and on the basis, FLT3-CAR-T cells constructed by different FLT3 nanobody sequences are proved to have very obvious specific killing on FLT3 positive AML tumor cell strains by in vitro killing experiments, and also have obvious in vivo anti-tumor effect on AML tumor models, and have good treatment effect.

2. According to the invention, through clone formation experiments after the CAR-T cells constructed by different clone FLT3 nanobody sequences are incubated with normal hematopoietic stem cells, the FLT3-CAR-T cells constructed by the invention have no specific killing to the normal hematopoietic stem cells, do not cause blood toxicity, and have better safety.

3. According to the invention, the FLT3-CAR-T cells with the strongest continuous killing capacity on the AML tumor cells are successfully screened through repeated killing experiments on the tumor cells, and the FLT3-CAR-T cells can replicate in vivo, so that tumors can be continuously controlled for a long time, and the method is a good coping strategy for the AML which is easy to relapse.

4. The FLT3-CAR-T cell therapies of the invention can alleviate patient suffering and improve AML prognosis to some extent. On one hand, the treatment means of the invention can be carried out in various modes such as injection, spray method, swallowing, transfusion and the like, and compared with the existing chemotherapy strategy, the pain of patients receiving treatment is relieved, and the frequency required for carrying out treatment is also reduced; on the other hand, the FLT3-CAR-T cell therapy can control tumors for a long time, reduce the possibility of recurrence, control diseases at a level of a lower degree and improve prognosis.

5. Compared with the existing FLT3 small molecule inhibitor, the FLT3-CAR-T cell provided by the invention has a wider application range, and can be suitable for a wider patient population. FLT3-CAR-T cells are not limited to being effective only in FLT3-ITD mutated AML patients, but also in FLT3 unmutated AML patients.

6. Compared with the existing FLT3 small molecule inhibitor, the FLT3-CAR-T cell has better curative effect, and FLT3 is easy to generate new mutation in a tyrosine kinase domain part, which can cause drug resistance to the FLT3 inhibitor and limit the curative effect of the FLT3 inhibitor. The FLT3-CAR-T cells are slightly influenced by the mutation of the FLT3, and even if the FLT3 is mutated, the FLT3-CAR-T cells still maintain the targeting and binding activities of the FLT3-CAR-T cells, so that the limitation on the curative effect is small.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Example 1 antigen preparation protocol

The extracellular segment (27 AA-543 AA) sequence of FLT3 is synthesized through genes, a human IgG1 Fc tag is added at the N end, and an enterokinase enzyme cleavage site is added between the FLT3 and the Fc for removing the Fc tag; subcloning into eukaryotic expression vector to construct antigen FLT3-Fc protein expression vector.

Carrying out plasmid large extraction on the constructed FLT3-Fc Protein expression vector, carrying out transient transfection on 293 cells, continuously culturing for 8 days, centrifugally collecting culture medium supernatant, filtering with a filter membrane with the diameter of 0.45 mu m, transferring the filtrate into a sterile centrifuge tube, and purifying by using a Protein A column to obtain the purified FLT3-Fc Protein.

The purified FLT3-Fc protein is an antigen, and is also called an immunogen or an immune antigen.

Example 2 antigen immunization protocol for alpaca

1 alpaca was immunized with the FLT3-Fc protein prepared in example 1 above, and 4 total immunizations were performed using subcutaneous multipoint immunization, and the immunization protocol is shown in table 3.

TABLE 3 Table 3

After three immunizations of antigen, the serum of alpaca immune is subjected to titer detection, and if the serum titer after three immunizations reaches 1:8000, the OD value is more than 1.0, then impact immunization (fourth immunization) can be performed. The alpaca peripheral blood obtained after impact immunization is used for separating immune serum, and the construction of a subsequent yeast display library is carried out.

EXAMPLE 3 detection of immunotitres

5mL of the post-three-phase peripheral blood and/or post-impact immune peripheral blood prepared in example 2 above was collected, and the centrifuge tube with the blood sample collected was placed in a 37℃incubator for 1 hour, followed by transferring the blood sample to 4℃overnight. The serum was transferred to a new sterile centrifuge tube, centrifuged at 5000rpm for 20min, after which the immunotiter was detected by ELISA.

The immunized alpaca-isolated serum was subjected to limiting dilution according to the dilution gradient shown in table 4, ELISA experiments were performed with FLT3 antigen pre-coated 96-well plates, using negative serum as a negative control, PBS buffer as a blank control, and the detection results are shown in table 2.

TABLE 4 Table 4

Serum	OD value
		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

According to ELISA detection results, after three times of immunization, the immunization titer is better, the OD value reaches 2.204 under the condition of 1:8000, which is far higher than 1.0, and the impact immunization can be carried out.

Performing primary impact immunization, and collecting 100mL peripheral blood after 3 days for constructing a yeast display library.

Example 4Biotin conjugated antigen protein

1mg of the purified antigen prepared in example 1 was prepared, and the buffer system was PBS at a concentration of 1mg/mL. NHS-biotin was weighed and dissolved in DMSO to prepare 10mM NHS-biotin.

Adding a freshly prepared 10mM NHS-biotin solution into the antigen protein sample, filling a sample tube into a light-proof self-sealing bag, and coupling at 180rpm for 30min at room temperature. The unlabeled Biotin was then removed by displacement with PBS and the labeled antigen protein was stored at-80 ℃. Biotin coupling effect was examined using ELISA protocol.

EXAMPLE 5PBMC isolation and acquisition of VHH antibody fragments

1. 50mL of peripheral blood after impact immunization prepared in example 2 was collected, and PBMC were sorted using lymphocyte separation liquid.

2. RNA extraction of PBMC followed by PrimeScript ^TM II 1st Strand cDNA Synthesis Kit, cDNA is prepared by reverse transcription, and the specific steps are as follows:

(1) Reaction mixture MIX1 shown in table 5 was prepared in a 200 μl PCR tube:

TABLE 5

(2) After 5min of incubation at 65℃the product was cooled rapidly on ice.

(3) The reaction solutions shown in Table 6 were prepared in the PCR tubes:

TABLE 6

Reagent(s)	Dosage of
		The denatured reaction solution	80μL
5×PrimeScriptⅡBuffer	32μL
		RNase Inhibitor(40U/μL)	4μL
PrimeScriptⅡRTase(200U/μL)	8μL
		RNase-Free water	36μL

(4) After mixing by blowing, packaging 80. Mu.L/tube, placing in a PCR instrument at 42 deg.C for 1 hr, inactivating at 70 deg.C for 15 min, and finally placing cDNA sample on ice or storing at-20 deg.C for long term.

Amplification of vhh fragments, comprising the following steps:

(1) The first round PCR reaction system (50. Mu.L/tube) as shown in Table 7 was configured:

TABLE 7

Composition of the components	Dosage of
		Upstream primer (5. Mu.M)	2μL
Downstream primer (10. Mu.M)	1μL
		NuHi Power mix(2×)	25μL
cDNA template	2μL
		Sterile water	20μL

After the PCR reaction system was configured, the PCR instrument was set up according to the procedure shown in table 8:

TABLE 8

(2) Agarose electrophoresis of PCR products:

the PCR product was analyzed by electrophoresis using 1% agarose, and a fragment having a molecular weight of about 750bp was isolated. The PCR products were recovered using a gel recovery kit and the concentration was determined using NanoDrop.

(3) The two-round PCR reaction system (50. Mu.L/tube) as shown in Table 9 was configured:

TABLE 9

After the PCR reaction system was configured, the PCR instrument was set up according to the procedure shown in table 10:

table 10

(4) Agarose electrophoresis analysis of the two rounds of PCR products:

the PCR products were analyzed by electrophoresis using 1% agarose, and VHH fragments having a molecular weight of about 400bp were isolated. VHH PCR products were recovered using a gel recovery kit and concentration was determined using NanoDrop.

Peripheral blood after impact immunization prepared in example 2 was collected, total RNA was provided, reverse transcribed into cDNA, and two rounds of PCR were performed using single domain antibody amplification primers, and PCR products were identified using agarose gel electrophoresis.

Agarose gel electrophoresis results are shown in FIG. 1: the first round of PCR respectively obtains about 1000bp and 750bp PCR bands, the gel recovers 750bp fragments as templates for the second round of PCR, and the second round of PCR obtains 450bp bands, namely VHH fragments.

EXAMPLE 6 construction of Yeast display library vector and electric transformation

1. Construction of yeast display vector:

(1) The products were recovered by digestion of the pBlue vector and the VHH PCR gel obtained in example 5 above, respectively, using SfiI, and digested overnight at 50 ℃.

(2) The pBlue vector fragment was separated using 1% agarose gel, and 9000bp of the vector fragment was excised and subjected to gel recovery. The PCR cleavage products were also purified using DNA fragment recovery kit and the concentration was determined using NanoDrop.

(3) The digested pBlue vector and VHH fragment were ligated using T4 ligase overnight at 16 ℃.

2. Electrotransformation of yeast display vectors to obtain E.coli libraries:

(1) The preparation of the electric rotating cup, the connection product and the electric rotating competence are placed on ice for precooling.

(2) Adding precooled library construction connection product into electric rotating competence, placing on ice for 1min, adding 70 mu L of DNA/competence mixture into each electric rotating cup, and placing the electric rotating cup on ice.

(3) The electrotransport was performed at 2500V,5 ms.

(4) Immediately after the completion of the electric shock, the cells were resuspended in SOC medium equilibrated to room temperature and shake-cultured at 37℃for 1 hour.

(5) 20 mu L of bacterial liquid is taken for carrying out storage capacity QC and diversity QC, the rest bacterial liquid is inoculated into LB culture medium containing Amp resistance, and the culture is selected and carried out at 180rpm and 37 ℃ for overnight.

(6) From the above overnight culture bacterial solution, the bacterial pellet was collected by centrifugation, and plasmid was extracted using a plasmid extraction kit. And the plasmid concentration was determined using Nanodrop, and the total amount of plasmid obtained was calculated.

The construction of the yeast display vector is completed by the steps, and the E.coli library plasmid is obtained through electric transformation.

EXAMPLE 7 electrotransformation of Yeast competent cells to obtain Yeast display libraries

1. The E.coli library plasmid prepared in example 6 was subjected to PmeI linearization, and the cleavage system is shown in Table 11:

TABLE 11

Reagent(s)	Dosage of
		Plasmid(s)	12μg
10×buffer	5μL
		PmeI	1μL
Sterile water	Up to 50μL

And (3) performing enzyme digestion for 3 hours at 37 ℃, taking 5 mu L of the plasmid to perform 1% agarose electrophoresis detection, and performing total enzyme digestion on 3mg of the plasmid after precipitation and concentration of the rest enzyme digestion products for later use.

2. Preparation of Yeast competent cells:

(1) Yeast cells are selected from saccharomycetes, streaked on YPD agar plates, and the plates are placed in an incubator at 24-30 ℃ for continuous culture for 3-5 days until monoclonals grow out.

(2) Yeast monoclonals are selected from the plate and added into YPD liquid culture medium, and placed in a shaking table at 24-30 ℃ for culture at 250rpm for 1-2 days.

(3) Using a 1L sterile Erlenmeyer flask, 100mL YPD medium was added, and the prepared rocking product was added to the flask and placed in a shaker at 24℃to 30℃for 1-2 days at 250 rpm.

(4) Sampling detection OD ₆₀₀ Up to 1.3-1.5 (logarithmic growth phase).

(5) All the bacterial liquid was transferred to a centrifuge tube, centrifuged at 1500 Xg at 4℃for 5 minutes, and after removing the supernatant, the bacterial pellet was resuspended using 250mL of sterile water pre-chilled on ice.

(6) A further round of centrifugation at 1500 Xg at 4℃for 5 minutes was performed, and after removal of the supernatant, the bacterial pellet was resuspended using 50mL of sterile water pre-chilled on ice.

(7) After centrifugation at 1500 Xg for 5 minutes at 4℃again, the supernatant was removed and the bacterial pellet was resuspended using 10mL of 1M sorbitol pre-chilled on ice.

(8) After centrifugation at 1500 Xg for 5 minutes at 4℃again, the supernatant was removed and the bacterial pellet was resuspended using 500. Mu.L of 1M sorbitol pre-chilled on ice.

(9) Finally, 500 mu L of yeast competent cells are obtained and are split into 80 mu L/tube for standby, and the competent cells are generally used at present so as to ensure higher electrotransformation efficiency.

3. Electrotransfer yeast competent cells:

(1) 80. Mu.L of yeast competent cells were taken, 1. Mu.g of linearized plasmid DNA was added, after thorough mixing, to a pre-chilled cuvette on ice, which was placed in an ice bath for 5 minutes.

(2) The electrotransformation conditions were: voltage: 1500V, time: 5ms, shock 2 times.

(3) After the electric shock is finished, 1mL of pre-cooled YPDS culture medium on ice is immediately added, after the mixture is gently blown and evenly mixed, all liquid of the electric shock cup is transferred into a new EP tube, and the mixture is subjected to static culture in an incubator at 30 ℃ for 2 hours.

(4) After the incubation, 50. Mu.L of the post-electric conversion product was applied uniformly to YPD selective plates. The plates were placed in an incubator at 28℃for 3 days until the monoclonal grew out.

(5) The remaining conversion product was transferred to liquid PAD medium and shake cultured at 28℃for 3 days.

(6) After observing that the white cells became dominant, 1500 Xg was centrifuged for 5min to collect the cells, and a part of the cells was used for induction expression in the yeast display library of example 8. The remainder was added with 50% sterile glycerol and stored at-80 ℃.

The electrotransformation of the yeast competent cells was completed by the above procedure, and a yeast display library transformed strain was obtained.

Example 8 Yeast display library induced expression and sorting

1. Expression was induced by yeast display library:

(1) The yeast display library transformant strain obtained in example 7 was cultured using PAD liquid medium.

(2) The PAD liquid medium is washed once by sterile PBS, resuspended in BMMY medium, and ampicillin and kanamycin antibiotics are added, and expression is induced at 28℃and 220rpm for 24-48h.

(3) 1mL of bacterial liquid is taken before induction, YPD culture medium is added, ampicillin and kanamycin antibiotics are added, shake culture is carried out at 28 ℃ and 220rpm, and the sample is used as a control before induction.

(4) Randomly selecting 20 yeast monoclonals for sequencing, and analyzing and constructing diversity of a yeast display library.

According to the sequencing result comparison as shown in FIG. 2, the yeast display library has diversity, and the yeast display library capacity is 6.7X10 ⁶ 。

2. Magnetic sorting of yeast display libraries:

(1) Streptavidin magnetic bead pre-adsorption:

(1) 10 OD-induced yeasts were resuspended with 500. Mu.L of pre-chilled 0.5% PBSA.

(2) Taking 50 mu L of streptavidin magnetic beads, fully blowing and uniformly mixing, adding 1mL of PBSA, placing on a magnetic rack, standing for 3-5min, and carefully removing the supernatant.

(3) 1mL of PBSA was added and the beads were washed once more. The supernatant was aspirated, and the yeast from step (1) was added, mixed well and incubated for 1h at 4 ℃.

(4) Placing on a magnetic rack, standing for 3-5min, carefully sucking out supernatant, transferring yeast into a new EP tube, and repeating adsorption once; the supernatant was carefully aspirated and used in the next experiment.

(2) Biotin-Fc negative sorting:

(1) taking pre-adsorbed saccharomycetes, centrifuging at 800 Xg for 5min, re-suspending in Biotin-Fc, and incubating at 4 ℃ for 1h.

(2) After incubation, 1mL of PBSA was added to the yeast, and the supernatant was discarded after centrifugation at 800 Xg for 5 min. The wash was repeated 2 times and finally resuspended in 500. Mu.L of PBS.

(3) Taking 50 mu L of streptavidin magnetic beads, fully blowing and uniformly mixing, adding 1mL of PBSA, placing on a magnetic rack, standing for 3-5min, and carefully removing the supernatant.

(4) 1mL of PBSA was added and the beads were washed once more. The supernatant was aspirated, and the yeast incubated with Biotin-Fc was added, mixed well and incubated at 4℃for 1h.

(5) Placing on a magnetic rack, standing for 3-5min, carefully sucking out supernatant, transferring yeast into a new EP tube, and repeating adsorption once; the supernatant was carefully aspirated and centrifuged at 800 Xg for 5min, and the yeast was collected for the next experiment.

(3) Biotin-FLT3-Fc magnetic sorting:

(1) taking yeast subjected to negative sorting of Biotin-Fc, centrifuging at 800 Xg for 5min, and re-suspending in Biotin-FLT3-Fc, and incubating at 4 ℃ for 1h.

(3) Taking 100 mu L of streptavidin magnetic beads, fully blowing and uniformly mixing, adding 1mL of PBSA, placing on a magnetic rack, standing for 3-5min, and carefully removing the supernatant.

(4) 1mL of PBSA was added and the beads were washed once more. The supernatant was aspirated, and the yeast incubated with Biotin-FLT3-Fc was added, mixed well and incubated at 4℃for 1h.

(5) After incubation, placing on a magnetic rack, standing for 3-5min, and carefully sucking out the supernatant;

(6) 1mL of PBSA was added, the beads were washed twice repeatedly, and the yeast was resuspended in YPD medium for cultivation and induced expression. Flow analysis is carried out after the sorted yeast cells are cultured and induced to express, biotin-FLT3-Fc is incubated, PE strepitavidin is used for secondary antibodies, and flow detection is carried out after the incubation is completed.

(7) The first round of magnetic separation products are fully combined with Biotin-FLT3-Fc protein respectively, and the second round of magnetic separation is carried out by using streptavidin magnetic beads. Directly coating a part of the sorted yeast cells on a PAD plate for selecting a monoclonal for verification; a portion of the culture was subjected to flow analysis. The Biotin-FLT3-Fc was incubated, and the secondary antibody was subjected to flow assay using PE strepitavidin after incubation was completed.

According to the flow detection result after the first sorting shown in FIG. 3, biotin-FLT3-Fc is used for magnetic sorting of yeast positive clones, and the sorting effect is good. The first round of sorting yeast cells are cultured to induce expression, and then two rounds of sorting are carried out.

As shown in fig. 4, after the second magnetic sorting, the yeast positive rate was 91.721%, positive clones were significantly enriched, the sorted products were directly plated on PAD plates, and single clones were selected for flow detection.

3. Flow detection of yeast monoclonal:

After repeating two rounds of magnetic separation, culturing a part of yeasts, and inducing expression flow detection; and (3) directly coating a PAD plate on one part, picking up monoclonal culture, inoculating to a 96-well plate, carrying out induced expression for 24 hours, incubating with Biotin-FLT3-Fc, using PE-strepitavidine for secondary antibody, and carrying out flow detection after incubation is completed.

The results of the flow assay are shown in FIG. 5, which shows that selected clones all bind to Biotin-FLT 3-Fc.

Example 9 Construction of VHH eukaryotic expression vectors

According to the results of the yeast monoclonal flow assay in example 8, clones with different binding capacities to the antigen of interest 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 VHH antibody sequences.

The VHH antibody sequences obtained by analysis were separately subjected to gene synthesis, subcloned in tandem with human IgG1 Fc into the expression vector Lenti-hIgG1-Fc2 shown in FIG. 6, and sequenced to verify the vector.

After the vector is verified by sequencing, the antibody expression vector Lenti-hIgG1-Fc2 is obtained, and the Qiagen plasmid large-drawing kit is used for preparing the endotoxin-removing plasmid for standby.

EXAMPLE 10 expression of recombinant antibodies

1. The LVTransm transfection reagent and the antibody expression vector Lenti-hIgG1-Fc2 were taken out of the refrigerator, thawed at room temperature, and thoroughly mixed by up-and-down blowing with a pipette. The PBS buffer was removed and warmed to room temperature. 500. Mu.L of PBS was added to one well of a 24-well plate, 4. Mu.g of Lenti-hIgG1-Fc2 was added, and after sufficiently mixing by pipetting up and down, 12. Mu.L of LVTransm was added, immediately mixed by pipetting up and down, and left standing at room temperature for 10 minutes. The mixture herein is referred to as a DNA/LVTransm complex.

2. The 532. Mu.L of the DNA/LVTransm complex was added to 1.5mL of 293F cells, and the mixture was thoroughly mixed with gentle shaking. The cells were exposed to 5% CO at 37 ℃ ₂ After culturing for 6-8 hours at 130rpm in an incubator, 1.5mL of fresh FreeStyle was added ^TM 293 medium, the cells were returned to the incubator for continued culture.

3. After 3 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for subsequent flow and ELISA detection.

The recombinant antibodies expressed by the antibody expression vector Lenti-hIgG1-Fc2 of example 9, also called single domain antibodies, single domain antibodies of the invention, and FLT3 nanobodies, were obtained by this procedure.

EXAMPLE 11 flow-detection of binding of recombinant antibodies to target proteins

1. The CHO-K1 and CHO-K1-FLT3 cell lines were recovered from liquid nitrogen and cell state was adjusted to logarithmic growth phase.

2. Dividing the two cells into several parts, each cell number being 5×10 ⁵ Individual cells.

3. The expressed antibodies were incubated with the target cells, respectively, and after thoroughly mixing, incubated at room temperature for 1 hour.

4.800 Xg were centrifuged at room temperature for 5 minutes, the supernatant containing the antibody was removed, and the cells were washed 3 times with PBS.

5. 1. Mu.L of PE-labeled Anti-human IgG was added, and after thoroughly mixing, incubated at room temperature for 30 minutes in the dark.

6.800 Xg were centrifuged at room temperature for 5 minutes, the supernatant containing the secondary antibody was removed, and the cells were washed 3 times with PBS.

7. Flow assays were performed using 500 μl PBS to resuspend cells.

According to the FACS detection results shown in FIG. 7, the screening antibodies all bound to CHO-K1-FLT3 recombinant cells, and did not bind to CHO-K1, indicating that the recombinant antibodies prepared in example 10 bind specifically to the target protein.

Expression purification of 11 target protein binding positive antibodies (SEQ ID NO: 7-17) was arranged, while antibodies of three clones of C-F21-1 (SEQ ID NO: 8), C-A5-11 (SEQ ID NO: 9) and C-2-A5 (SEQ ID NO: 15) were subjected to affinity detection.

EXAMPLE 12 expression purification of recombinant antibodies

1. And taking out the LVTransm transfection reagent and the single-chain antibody expression vector from the refrigerator, thawing at room temperature, and blowing up and down by a pipetting gun to completely mix uniformly. The PBS buffer was removed and warmed to room temperature. 2mL of PBS was taken into one well of a 6-well plate, 130. Mu.g of Lenti-hIgG1-Fc2 was added, and after the mixture was thoroughly mixed by pipetting up and down, 400. Mu.L of LVTransm was added, immediately mixed by pipetting up and down, and left to stand at room temperature for 10 minutes.

2. The DNA/LVTransm complex was added to 50mL of 293F cells, gently swirled and thoroughly mixed. The cells were exposed to 5% CO at 37 ℃ ₂ After culturing at 130rpm for 6-8 hours in the incubator, 50mL of fresh FreeStyle 293 medium was added and the cells were returned to the incubator for continued culturing.

3. After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube and the antibody was purified using a Protein A column.

The positive recombinant antibody obtained in example 11 was subjected to expression purification by this procedure.

Example 13 affinity detection

FLT3-Fc recombinant protein was immobilized on CM5 chip using 10mM Acetate buffer, and binding ability of the candidate single domain antibody to the target protein FLT3 was detected using the single domain antibodies prepared in examples 11 and 12 as mobile phase, respectively, and the affinity detection results are shown in Table 12.

Table 12

Nanobody	ka(M ^-1 s ^-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

The detection result shows that the single-domain antibody has affinity with the target protein FLT3 (good, the single-domain antibody has good binding effect with the target protein FLT 3).

Example 14 Single domain antibody sequences

Screening by using a yeast display library to obtain the single domain antibody targeting FLT3, wherein the sequence information of the antibody is shown in figures 8A-8K. Taking fig. 8A as an example, FR sequences (FR 1-FR 4) are underlined, CDR1 is marked with a heavy symbol (-), CDR2 is marked with a wavy line (wavy line), and CDR3 is marked with a line segment [ ] Line segment) Marking. FIGS. 8B-8K are similar formulasThe formula is shown in the specification.

Example 15 FLT3-CAR vector construction

5 three-generation CAR vectors (namely, PA0135EF-CAR, PA0135GH-CAR, PA0135KL-CAR, PA0135MN-CAR and PA0135 PQ-CAR) containing ICOS and 41BB co-stimulatory factors are respectively constructed by using 5 FLT3 nanobody sequences (namely, C-F21-1, C-A5-11, C-2-D8, C-2-A5 and C-2-A3) with better flow results, and the structures are shown in figure 9.

EXAMPLE 16 comparison of CAR lentivirus titres constructed from different cloned FLT3 nanobody sequences

The 5 FLT 3-CARs were lentivirally packaged using a suspension 293T lentiviral packaging system, and each CAR virus primary titer value was detected.

As shown in the results of FIG. 10, the raw droplet sizes were all 1X 10 ⁷ TU/mL, wherein the three cloned CAR lentivirus origins of PA0135EF, PA0135GH and PA0135MN have higher droplet sizes.

EXAMPLE 17 CAR-T cells constructed from different cloned FLT3 nanobody sequences for FLT3 positive target cytotoxicity By using

The expression of the surface antigen CD135 (FLT 3) of different tumor cell lines was first examined by flow cytometry.

As shown in FIG. 11, raji and MEG01 did not express FLT3, AML3, MOLM-13, and MV4-11 AML cell lines (acute myeloid leukemia cell lines) highly expressed FLT3.

Target cell killing experiments are carried out by selecting FLT3 negative target cells Raji as target cells, and differences of the toxic effects of the CAR-T cells and the control T cells on the negative target cells are compared.

As shown in fig. 12, PA0135EF-CAR-T, PA0135GH-CAR-T, PA0135MN-CAR-T had no apparent specific killing of antigen negative target cells compared to control T cells, demonstrating that CAR-T cells of the present invention have very little toxicity to negative target cells and high safety.

Three FLT3 antigen positive cell lines (MV-4-11, MOLM-13 and AML 3) are selected as target cells to carry out target cell killing experiments, and the toxicity effect difference of PA0135EF-CAR-T, PA0135GH-CAR-T, PA0135MN-CAR-T on the target cells is compared.

From the data in FIG. 13, it can be seen that all three CAR-T cells had significant specific killing on FLT3 positive cell lines, and that PA0135MN-CAR-T had better cytotoxicity on FLT3 positive tumor cells than PA0135EF-CAR-T and PA0135GH-CAR-T.

The supernatant from the above killing experiment was collected and the secretion of the cytokine Granzyme-B in the supernatant was further examined.

The results are shown in figure 14, where three CAR-T cells were significantly released specific cytokines after incubation with target cells. Meanwhile, the PA0135MN-CAR-T cell has stronger cytokine release capacity. Therefore, by combining the cytotoxicity of the PA0135EF-CAR-T, PA0135GH-CAR-T, PA0135MN-CAR-T on FLT3 positive tumor cells and the cytokine release result, the PA0135MN-CAR-T has stronger cytotoxicity on positive target cells and better killing effect.

EXAMPLE 18 CAR-T cells constructed from different cloned FLT3 nanobody sequences against FLT 3-positive tumor cell lines Sustained killing ability comparison

Selecting FLT3 positive tumor cell strain MV-4-11 as target cells, and comparing the continuous killing capacity of FLT3-CAR-T cells of different clones on the target cells, wherein the effective target ratio is 1:1 and 1:5, and tumor cells are supplemented every 24 hours or 48 hours.

From the results of FIG. 15, it can be seen that PA0135MN-CAR-T has a stronger and longer lasting killing capacity against MV-4-11 tumor cells than PA0135EF-CAR-T and PA0135 GH-CAR-T.

EXAMPLE 19 cell of CAR-T cells constructed from different cloned FLT3 nanobody sequences against Normal hematopoietic Stem cells Toxicity of

The expression of FLT3 and CD33 on the surface of commercially available human blood stem cells (HSPCs) was first examined by flow cytometry.

The results in FIG. 16 show that FLT3 (positive rate 81.77%) and CD33 (positive rate 74.47%) are highly expressed on the surface of human hematopoietic stem cells.

The release of cytokines (Granzyme-B, IL-2, IFN-gama, TNF- α) after incubation of purchased human CD34+ HSPC cells with 3 FLT3-CAR-T cells, respectively (CD 33-CAR-T cells as control group) was then examined.

The results are shown in FIG. 17, where CD33-CAR-T (PA 033 AC-CAR-T) produces significant specific cytokines after incubation with human CD34+ hematopoietic stem cells, indicating that CD33-CAR-T can activate, and potentially cytotoxicity, to normal hematopoietic stem cells, and that 3 FLT3-CAR-T cells are not substantially specific cytokines after incubation with hematopoietic stem cells, indicating that FLT3-CAR-T cells are not activated, i.e., not specifically killed, by normal hematopoietic stem cells. In particular, FLT3-CAR-T cells cloned by PA0135MN have the lowest cytokine release.

Finally, 3 FLT3-CAR-T cells and CD33-CAR-T cells are respectively incubated with the artificial blood stem cells, and then the colony formation of the hematopoietic stem cells is detected.

The results are shown in fig. 18, in which the number of hematopoietic stem cells after incubation with CD33-CAR-T (PA 033 AC-CAR-T) was significantly reduced compared to the control T cell group, indicating that CD33-CAR-T affects the formation of normal hematopoietic stem cell clones, and that CD33-CAR-T cells produced some cytotoxicity to normal hematopoietic stem cells; while 3 FLT3-CAR-T cells did not affect the clonal formation of hematopoietic stem cells, indicating that FLT3-CAR-T cells did not have significant cytotoxicity to normal hematopoietic stem cells.

Example 20 In vivo antitumor effect of PA0135MN-CAR-T cells on AML cell line OCI-AML3

As shown in FIG. 19, construction of mouse tumor modeling using luciferase-labeled OCI-AML3 cells, selection of 15 female NCG mice, and 1X 10 inoculation of each tail vein ⁶ Tumor cells were administered by tail vein at a dose of 2×10 on day 3 after tumor cell inoculation ⁷ Is a PA0135MN-CAR-T (TAA 05-CAR-T). Tumor cell growth was monitored weekly in mice from different dosing groups.

As shown in fig. 20, PA0135MN-CAR-T (TAA 05-CAR-T) cells significantly inhibited the proliferation and growth of OCI-AML3 tumor cells in mice compared to control groups (PBS group and Mock T group).

The body weight test and survival curves for mice are shown in fig. 21, and the results also show: compared with a control group, the TAA05-CAR-T cells have no obvious influence on the body weight of mice, and the TAA05-CAR-T cells can remarkably prolong the survival time of OCI-AML3 tumor-bearing mice and have remarkable in-vivo anti-tumor effect.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Sequence listing

<110> Bosheng Ji medicine technology (Suzhou) Co., ltd

<120> construction and application of novel chimeric antigen receptor modified T cell targeting human FLT3

<130> P2021-2598

<160> 68

<170> PatentIn version 3.5

<210> 1

<211> 579

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> chimeric antigen receptor PA0135-MN-CAR amino acid sequence

<400> 1

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Leu Val Gln Pro Gly Gly Ser Leu Asn Leu Ser Cys Glu Val Ser Gly

35 40 45

Val Ile Phe Ser Met Leu Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly

50 55 60

Gln Glu Arg Glu Leu Phe Ala Ala Val Thr Ser Gly Gly Phe Thr Ser

65 70 75 80

Tyr Ile Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp

85 90 95

Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Val Lys Pro Glu Asp Thr

100 105 110

Gly Val Tyr Tyr Cys Asn Arg Asp Pro Val Arg Ser Ser Asp Asn Trp

115 120 125

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro

130 135 140

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

145 150 155 160

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

165 170 175

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

180 185 190

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

195 200 205

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

210 215 220

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

225 230 235 240

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

245 250 255

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

260 265 270

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

275 280 285

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

290 295 300

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

305 310 315 320

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

325 330 335

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

340 345 350

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp

355 360 365

Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys

370 375 380

Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His

385 390 395 400

Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys

405 410 415

Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu

420 425 430

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

435 440 445

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

450 455 460

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

465 470 475 480

Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

485 490 495

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

500 505 510

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

515 520 525

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

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Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

545 550 555 560

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

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Pro Pro Arg

<210> 2

<211> 1740

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> chimeric antigen receptor PA0135-MN-CAR nucleotide sequence

<400> 2

atgctgctgc tggtgacctc tctgctgctc tgcgaactgc ctcacccagc ctttctgctg 60

atcccccaag tgcaacttgt ggaatcagga ggaggacttg tgcaacctgg aggatcactt 120

aacctttcat gcgaagtgtc aggagtgatc ttctccatgc ttggaatggg atggtacaga 180

caagcacctg gacaagaaag agaattattc gccgctgtga catcaggagg atttacatca 240

tacatcgaat cagtgagagg aagatttaca atctcaagag ataacgataa gcgctcggtg 300

taccttcaaa tgaacaacgt gaaacctgaa gatacaggag tgtactactg caacagagat 360

cctgtgagat catcagataa ctgggggcag ggaacacaag tgacagtgtc atcagagagc 420

aaatacggcc ctccttgccc tccttgccca gccccagaat ttgagggagg acctagcgtg 480

ttcctgttcc ctcccaagcc caaggacacc ctgatgatca gccggacccc agaagtcacc 540

tgcgtggtgg tggacgtgtc tcaggaagac cccgaggtgc agttcaattg gtacgtggac 600

ggcgtggaag tgcacaacgc caagaccaag cccagagagg agcagttcca gagcacctac 660

agagtggtgt ccgtgctgac cgtgctgcat caggattggc tgaacggcaa ggagtacaag 720

tgcaaggtgt ccaacaaggg cctgcctagc agcatcgaga agaccatcag caaggccaag 780

ggccagccta gagagcctca ggtgtacaca ctgccccctt ctcaggagga gatgaccaag 840

aaccaggtgt ccctgacttg cctcgtgaag ggcttctacc ccagcgatat tgccgtggag 900

tgggagtcta acggccagcc cgagaacaac tacaagacca cccctcccgt gctggatagc 960

gacggctctt tcttcctgta cagccggctg acagtggaca aaagtcgctg gcaggagggc 1020

aacgtgttca gttgcagcgt gatgcacgag gccctgcaca accactacac ccagaagagc 1080

ctgtctctgt ctctcggcaa gtggctccct atcggttgcg ccgcctttgt cgtcgtctgt 1140

atcctcggct gcatcctcat ctgttggctc accaagaaga agtacagcag cagcgtgcac 1200

gaccccaacg gcgagtacat gttcatgcgg gccgtcaaca ccgccaagaa gagcagactg 1260

accgacgtga ccctgaagag aggcaggaag aagctgctgt acatcttcaa gcagcccttc 1320

atgcggccag tgcagacaac ccaggaggaa gacggctgct cttgcagatt ccccgaggaa 1380

gaagagggcg gttgcgagct gcgcgtgaaa ttcagccgca gcgcagatgc tccagcctac 1440

aagcaggggc agaaccagct ctacaacgaa ctcaatcttg gtcggagaga ggagtacgac 1500

gtgctggaca agcggagagg acgggaccca gaaatgggcg ggaagccgcg cagaaagaat 1560

ccccaagagg gcctgtacaa cgagctccaa aaggataaga tggcagaagc ctatagcgag 1620

attggtatga aaggggaacg cagaagaggc aaaggccacg acggactgta ccagggactc 1680

agcaccgcca ccaaggacac ctatgacgct cttcacatgc aggccctgcc gcctcggtga 1740

<210> 3

<211> 579

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> chimeric antigen receptor PA0135-EF-CAR amino acid sequence

<400> 3

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Lys Val Ser Gly

35 40 45

Met Ile Phe Ser Met Phe Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly

50 55 60

Gln Glu Arg Glu Leu Ile Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser

65 70 75 80

Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Asn Gln Asp Pro Val Arg Ser Ser Asp Val Trp

115 120 125

Gly Gln Gly Thr Gln Val Thr Val Ser Gly Glu Ser Lys Tyr Gly Pro

130 135 140

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

145 150 155 160

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

165 170 175

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

180 185 190

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

195 200 205

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

210 215 220

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

225 230 235 240

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

245 250 255

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

260 265 270

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

275 280 285

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

290 295 300

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

305 310 315 320

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

325 330 335

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

340 345 350

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp

355 360 365

Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys

370 375 380

Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His

385 390 395 400

Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys

405 410 415

Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu

420 425 430

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

435 440 445

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

450 455 460

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

465 470 475 480

Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

485 490 495

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

500 505 510

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

515 520 525

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

530 535 540

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

545 550 555 560

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

565 570 575

Pro Pro Arg

<210> 4

<211> 579

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> chimeric antigen receptor PA0135-GH-CAR amino acid sequence

<400> 4

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Ala Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Val Ser Gly

35 40 45

Thr Ile Phe Ser Met Phe Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly

50 55 60

His Glu Arg Glu Leu Ile Ala Ala Ile Thr Ser Gly His Phe Thr Ser

65 70 75 80

Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala

85 90 95

Lys Arg Ser Val Tyr Leu Gln Met Asn Gly Val Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Asn Arg Asp Pro Ile Gln Ser Ser Asp Val Trp

115 120 125

Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro

130 135 140

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

145 150 155 160

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

165 170 175

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

180 185 190

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

195 200 205

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

210 215 220

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

225 230 235 240

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

245 250 255

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

260 265 270

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

275 280 285

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

290 295 300

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

305 310 315 320

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

325 330 335

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

340 345 350

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp

355 360 365

Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys

370 375 380

Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His

385 390 395 400

Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys

405 410 415

Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu

420 425 430

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

435 440 445

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

450 455 460

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

465 470 475 480

Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

485 490 495

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

500 505 510

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

515 520 525

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

530 535 540

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

545 550 555 560

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

565 570 575

Pro Pro Arg

<210> 5

<211> 579

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> chimeric antigen receptor PA0135-KL-CAR amino acid sequence

<400> 5

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Leu Val Gln Pro Gly Gly Ser Leu Asn Leu Thr Cys Lys Val Ser Gly

35 40 45

Thr Ile Phe Ser Met Leu Gly Met Gly Trp Tyr Arg Arg Ala Pro Gly

50 55 60

Gln Glu Arg Glu Leu Phe Ala Ala Ile Thr Ser Gly Gly Phe Asp Ser

65 70 75 80

Tyr Val Glu Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn Asp

85 90 95

Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Asn Gln Asp Pro Ile Arg Phe Ser Asp Val Trp

115 120 125

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro

130 135 140

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

145 150 155 160

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

165 170 175

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

180 185 190

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

195 200 205

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

210 215 220

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

225 230 235 240

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

245 250 255

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

260 265 270

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

275 280 285

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

290 295 300

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

305 310 315 320

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

325 330 335

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

340 345 350

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp

355 360 365

Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys

370 375 380

Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His

385 390 395 400

Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys

405 410 415

Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu

420 425 430

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

435 440 445

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

450 455 460

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

465 470 475 480

Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

485 490 495

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

500 505 510

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

515 520 525

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

530 535 540

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

545 550 555 560

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

565 570 575

Pro Pro Arg

<210> 6

<211> 579

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> chimeric antigen receptor PA0135-PQ-CAR amino acid sequence

<400> 6

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly

20 25 30

Leu Val Leu Pro Gly Gly Ser Leu Asn Leu Ser Cys Glu Val Ser Gly

35 40 45

Thr Ile Phe Ser Met Leu Gly Met Gly Trp Tyr Arg Arg Ala Pro Gly

50 55 60

Gln Glu Arg Glu Leu Phe Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser

65 70 75 80

Tyr Ile Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp

85 90 95

Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Val Lys Pro Glu Asp Thr

100 105 110

Ala Val Tyr Tyr Cys Asn Arg Asp Pro Leu Arg Ser Ser Asp Val Trp

115 120 125

Gly Gln Gly Thr Gln Ile Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro

130 135 140

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val

145 150 155 160

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

165 170 175

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

180 185 190

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

195 200 205

Thr Lys Pro Arg Glu Glu Gln Phe Gln Ser Thr Tyr Arg Val Val Ser

210 215 220

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

225 230 235 240

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

245 250 255

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

260 265 270

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

275 280 285

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

290 295 300

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

305 310 315 320

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

325 330 335

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

340 345 350

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Trp

355 360 365

Leu Pro Ile Gly Cys Ala Ala Phe Val Val Val Cys Ile Leu Gly Cys

370 375 380

Ile Leu Ile Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His

385 390 395 400

Asp Pro Asn Gly Glu Tyr Met Phe Met Arg Ala Val Asn Thr Ala Lys

405 410 415

Lys Ser Arg Leu Thr Asp Val Thr Leu Lys Arg Gly Arg Lys Lys Leu

420 425 430

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

435 440 445

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

450 455 460

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

465 470 475 480

Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

485 490 495

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

500 505 510

Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu

515 520 525

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

530 535 540

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

545 550 555 560

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

565 570 575

Pro Pro Arg

<210> 7

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-F15-10 amino acid sequence

<400> 7

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Thr Ile Phe Ser Met Leu

20 25 30

Gly Met Gly Trp Tyr Arg Arg Ala Pro Gly Gln Glu Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly His Phe Thr Ser Tyr Ile Glu Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Val Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Arg Asp Pro Val Arg Ser Ser Asp Val Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Gly

115

<210> 8

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-F21-1 amino acid sequence

<400> 8

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser Gly Met Ile Phe Ser Met Phe

20 25 30

Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Ile

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gln Asp Pro Val Arg Ser Ser Asp Val Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Gly

115

<210> 9

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-A5-11 amino acid sequence

<400> 9

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser Gly Thr Ile Phe Ser Met Phe

20 25 30

Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Arg Glu Leu Ile

35 40 45

Ala Ala Ile Thr Ser Gly His Phe Thr Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Gly Val Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Arg Asp Pro Ile Gln Ser Ser Asp Val Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 10

<211> 124

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-1-D3 amino acid sequence

<400> 10

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser Gly Met Ile Phe Ser Met Phe

20 25 30

Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Ile

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Asp Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Asp Val Ile Arg Arg Val Gly Ser Glu His Arg Gly Pro Arg Thr

100 105 110

Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 11

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-1-G3 amino acid sequence

<400> 11

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser Gly Met Ile Phe Ser Met Phe

20 25 30

Gly Met Gly Trp Tyr Arg Gln Thr Pro Gly Gln Glu Arg Glu Leu Ile

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Asn Ile Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gly Asp Arg Leu Ala Arg Arg Gly Ile Trp Gly Pro Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 12

<211> 115

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-2-C2 amino acid sequence

<400> 12

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser Gly Met Ile Phe Ser Met Phe

20 25 30

Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu His Ile

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ile

85 90 95

Arg Glu Asn Tyr Asn Asn Asp Arg Lys Asp Gly Gly Thr Gln Val Thr

100 105 110

Val Ser Ser

115

<210> 13

<211> 125

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-1-E6 amino acid sequence

<400> 13

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser Gly Met Ile Phe Ser Met Phe

20 25 30

Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Ile

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

His Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Ala Asp Leu Trp Gly Asp Gly Thr Lys Trp Asp Arg Ala Asn Glu Tyr

100 105 110

Asp Tyr Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 14

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-2-D8 amino acid sequence

<400> 14

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Thr Cys Lys Val Ser Gly Thr Ile Phe Ser Met Leu

20 25 30

Gly Met Gly Trp Tyr Arg Arg Ala Pro Gly Gln Glu Arg Glu Leu Phe

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Asp Ser Tyr Val Glu Ser Val Arg

50 55 60

Gly Arg Phe Ile Ile Ser Arg Asp Asn Asp Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gln Asp Pro Ile Arg Phe Ser Asp Val Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ser

115

<210> 15

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-2-A5 amino acid sequence

<400> 15

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Ser Cys Glu Val Ser Gly Val Ile Phe Ser Met Leu

20 25 30

Gly Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Phe

35 40 45

Ala Ala Val Thr Ser Gly Gly Phe Thr Ser Tyr Ile Glu Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Val Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Asn

85 90 95

Arg Asp Pro Val Arg Ser Ser Asp Asn Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 16

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-2-A3 amino acid sequence

<400> 16

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Leu Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Ser Cys Glu Val Ser Gly Thr Ile Phe Ser Met Leu

20 25 30

Gly Met Gly Trp Tyr Arg Arg Ala Pro Gly Gln Glu Arg Glu Leu Phe

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser Tyr Ile Glu Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Val Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Arg Asp Pro Leu Arg Ser Ser Asp Val Trp Gly Gln Gly Thr Gln Ile

100 105 110

Thr Val Ser Ser

115

<210> 17

<211> 116

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> nanobody C-2-C4 amino acid sequence

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Ser Cys Glu Val Ser Gly Thr Ile Phe Ser Met Leu

20 25 30

Gly Met Gly Trp Tyr Arg Arg Ala Pro Gly Gln Glu Arg Glu Leu Phe

35 40 45

Ala Ala Ile Thr Ser Gly Gly Phe Thr Ser Tyr Ile Glu Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Ser Val Tyr Leu

65 70 75 80

Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Gln Asp Pro Val Arg Ser Ser Asp Asn Trp Gly Gln Gly Thr Gln Val

100 105 110

Thr Val Ser Ser

115

<210> 18

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR1 of nanobody C-F15-10

<400> 18

Gly Thr Ile Phe Ser Met Leu Gly

1 5

<210> 19

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR2 of nanobody C-F15-10, C-A5-11

<400> 19

Ile Thr Ser Gly His Phe Thr

1 5

<210> 20

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-F15-10

<400> 20

Asn Arg Asp Pro Val Arg Ser Ser Asp Val

1 5 10

<210> 21

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR1 of nanobody C-F21-1, C-1-D3, C-1-G3, C-2-C2, C-1-E6

<400> 21

Gly Met Ile Phe Ser Met Phe Gly

1 5

<210> 22

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR2 of nanobody C-F21-1, C-1-G3, C-2-C2, C-1-E6, C-2-A3, C-2-C4

<400> 22

Ile Thr Ser Gly Gly Phe Thr

1 5

<210> 23

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-F21-1

<400> 23

Asn Gln Asp Pro Val Arg Ser Ser Asp Val

1 5 10

<210> 24

<211> 8

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR1 of nanobody C-A5-11

<400> 24

Gly Thr Ile Phe Ser Met Phe Gly

1 5

<210> 25

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-A5-11

<400> 25

Asn Arg Asp Pro Ile Gln Ser Ser Asp Val

1 5 10

<210> 26

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR2 of nanobody C-1-D3, C-2-D8

<400> 26

Ile Thr Ser Gly Gly Phe Asp

1 5

<210> 27

<211> 18

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-1-D3

<400> 27

Asn Ala Asp Val Ile Arg Arg Val Gly Ser Glu His Arg Gly Pro Arg

1 5 10 15

Thr Ile

<210> 28

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-1-G3

<400> 28

Asn Gly Asp Arg Leu Ala Arg Arg Gly Ile

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-2-C2

<400> 29

Ile Arg Glu Asn Tyr Asn Asn Asp Arg Lys

1 5 10

<210> 30

<211> 19

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-1-E6

<400> 30

Ala Ala Asp Leu Trp Gly Asp Gly Thr Lys Trp Asp Arg Ala Asn Glu

1 5 10 15

Tyr Asp Tyr

<210> 31

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR1 of nanobody C-2-D8, C-2-A3, C-2-C4

<400> 31

Gly Thr Ile Phe Ser Met Leu Gly Met Gly

1 5 10

<210> 32

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-2-D8

<400> 32

Asn Gln Asp Pro Ile Arg Phe Ser Asp Val

1 5 10

<210> 33

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR1 of nanobody C-2-A5

<400> 33

Gly Val Ile Phe Ser Met Leu Gly Met Gly

1 5 10

<210> 34

<211> 7

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR2 of nanobody C-2-A5

<400> 34

Val Thr Ser Gly Gly Phe Thr

1 5

<210> 35

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-2-A5

<400> 35

Asn Arg Asp Pro Val Arg Ser Ser Asp Asn

1 5 10

<210> 36

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-2-A3

<400> 36

Asn Arg Asp Pro Leu Arg Ser Ser Asp Val

1 5 10

<210> 37

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> CDR3 of nanobody C-2-C4

<400> 37

Asn Gln Asp Pro Val Arg Ser Ser Asp Asn

1 5 10

<210> 38

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-F15-10

<400> 38

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser

20 25

<210> 39

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-F15-10

<400> 39

Met Gly Trp Tyr Arg Arg Ala Pro Gly Gln Glu Arg Glu Leu Val Ala

1 5 10 15

Ala

<210> 40

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-F15-10

<400> 40

Ser Tyr Ile Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Arg Ser Val Tyr Leu Gln Met Asn Ser Val Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 41

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-F15-10, C-F21-1

<400> 41

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Gly

1 5 10

<210> 42

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-F21-1, C-1-D3, C-1-G3, C-2-C2

<400> 42

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser

20 25

<210> 43

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-F21-1, C-1-D3, C-1-E6

<400> 43

Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Ile Ala

1 5 10 15

Ala

<210> 44

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-F21-1, C-2-C2

<400> 44

Ser Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 45

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-A5-11

<400> 45

Ala Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Val Val Ser

20 25

<210> 46

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-A5-11

<400> 46

Met Gly Trp Tyr Arg Gln Ala Pro Gly His Glu Arg Glu Leu Ile Ala

1 5 10 15

Ala

<210> 47

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-A5-11

<400> 47

Ser Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Arg Ser Val Tyr Leu Gln Met Asn Gly Val Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 48

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-A5-11, C-2-A5, C-2-C4

<400> 48

Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 49

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-1-D3

<400> 49

Ser Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Asp Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 50

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-1-D3, C-2-D8

<400> 50

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 51

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-1-G3

<400> 51

Met Gly Trp Tyr Arg Gln Thr Pro Gly Gln Glu Arg Glu Leu Ile Ala

1 5 10 15

Ala

<210> 52

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-1-G3

<400> 52

Ser Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Asn Ile Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 53

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-1-G3

<400> 53

Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 54

<211> 17

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-2-C2

<400> 54

Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu His Ile Ala

1 5 10 15

Ala

<210> 55

<211> 10

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-2-C2

<400> 55

Asp Gly Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 56

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-1-E6

<400> 56

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Lys Val Ser

20 25

<210> 57

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-1-E6

<400> 57

Ser Tyr Val Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Asn Thr Val Tyr Leu His Met Asn Asp Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 58

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-1-E6

<400> 58

Trp Gly Gly Gly Thr Gln Val Thr Val Ser Ser

1 5 10

<210> 59

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-2-D8

<400> 59

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Thr Cys Lys Val Ser

20 25

<210> 60

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-2-D8, C-2-A3, C-2-C4

<400> 60

Trp Tyr Arg Arg Ala Pro Gly Gln Glu Arg Glu Leu Phe Ala Ala

1 5 10 15

<210> 61

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-2-D8

<400> 61

Ser Tyr Val Glu Ser Val Arg Gly Arg Phe Ile Ile Ser Arg Asp Asn

1 5 10 15

Asp Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 62

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-2-A5, C-2-C4

<400> 62

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Ser Cys Glu Val Ser

20 25

<210> 63

<211> 15

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR2 of nanobody C-2-A5

<400> 63

Trp Tyr Arg Gln Ala Pro Gly Gln Glu Arg Glu Leu Phe Ala Ala

1 5 10 15

<210> 64

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-2-A5

<400> 64

Ser Tyr Ile Glu Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Asp Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Val Lys Pro Glu Asp

20 25 30

Thr Gly Val Tyr Tyr Cys

35

<210> 65

<211> 25

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR1 of nanobody C-2-A3

<400> 65

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Leu Pro Gly Gly

1 5 10 15

Ser Leu Asn Leu Ser Cys Glu Val Ser

20 25

<210> 66

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-2-A3

<400> 66

Ser Tyr Ile Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Asp Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Val Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

<210> 67

<211> 11

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR4 of nanobody C-2-A3

<400> 67

Trp Gly Gln Gly Thr Gln Ile Thr Val Ser Ser

1 5 10

<210> 68

<211> 38

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<220>

<223> FR3 of nanobody C-2-C4

<400> 68

Ser Tyr Ile Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn

1 5 10 15

Ala Lys Arg Ser Val Tyr Leu Gln Met Asn Asn Leu Lys Pro Glu Asp

20 25 30

Thr Ala Val Tyr Tyr Cys

35

Claims

1. Nanobody directed against FLT3, characterized in that the complementarity determining region CDRs of the heavy chain variable region VHH of said nanobody directed against FLT3 are one or more selected from the group consisting of:

2. Nanobody against FLT3 according to claim 1, wherein the amino acid sequence of the VHH 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.

3. An antibody directed against FLT3, comprising one or more nanobodies according to claim 2 directed against FLT 3.

4. A chimeric antigen receptor CAR, comprising an extracellular domain comprising the nanobody of claim 1 against FLT3 or the antibody of claim 3 against FLT 3.

5. The chimeric antigen receptor of claim 4, wherein the amino acid sequence of the CAR is set forth in SEQ ID NOs 1, 3, 4, 5, 6.

6. The chimeric antigen receptor of claim 4 or 5, wherein the CAR has a structure according to formula Ia:

L1-Nb-H-TM-C-CD3ζ (Ia)

in the method, in the process of the invention,

l is a none or signal peptide sequence;

nb is a specific binding domain;

h is the no or hinge region;

TM is a transmembrane domain;

c is a costimulatory signaling domain;

the "-" is a connecting peptide or peptide bond.

7. An engineered immune cell comprising the chimeric antigen receptor of claim 4.

8. A method of making the engineered immune cell of claim 7, comprising the steps of: transduction of the chimeric antigen receptor of claim 4 into an immune cell, thereby obtaining the engineered immune cell.

9. Use of an active ingredient selected from the group consisting of: nanobody against FLT3 according to claim 1, or antibody against FLT3 according to claim 3, or chimeric antigen receptor according to claim 4, or engineered immune cell according to claim 7, or a combination thereof, characterized in that the active ingredient is used for the preparation of:

(a) A medicament for preventing and/or treating FLT3 high expression disease;

(b) Reagents for detecting FLT3 high expression disease.

10. A pharmaceutical composition comprising:

(i) Nanobody against FLT3 according to claim 1, or antibody against FLT3 according to claim 3, or chimeric antigen receptor according to claim 4, or engineered immune cell according to claim 7, or a combination thereof as active ingredient; and

(ii) A pharmaceutically acceptable carrier, diluent or excipient.