Anti-CLL1 fully humanized single-chain antibody and application thereof

Abstract

The invention belongs to the technical field of cellular immune engineering, and particularly relates to a full-human-derived polypeptide for recognizing CLL-1 and application of the full-human-derived polypeptide. The polypeptide provided by the invention specifically recognizes CLL-1 and has good affinity performance, and the polypeptide specifically targeting CLL-1 provided by the invention can mark CLL-1 positive cells, can be combined with a CLL-1 antigen and the CLL-1 positive cells, and can be used as a detection reagent of CLL-1 molecules and an in-vivo tracing marking reagent. The polypeptide specifically targeting CLL-1 provided by the invention can also be used in an extracellular antigen recognition region of a chimeric antigen receptor, is used in an immune cell treatment drug targeting CLL-1, and is used for killing malignant tumors expressing CLL-1. The polypeptide for specifically recognizing the CLL-1 is a natural full-humanized antibody source, compared with a mouse antibody, a chimeric antibody and a humanized antibody, the immunogenicity of the polypeptide is greatly reduced, and the safety can be guaranteed to the maximum extent in clinical application.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor cell immunotherapy, and specifically relates to a fully human ScFv targeting CLL-1 and an application thereof. Background Art

[0002] Chimeric antigen receptor (CAR) is an artificial receptor that simulates TCR function. CAR molecules generally contain an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular signaling domain. The intracellular signaling domain is usually the CD3ζ chain or FcRγ, or is connected to one or more co-stimulatory molecules, such as 4-1BB, CD28, and ICOS (CD278).

[0003] Acute myeloid leukemia (AML) is the most common acute leukemia (AL) and the most common in adults, accounting for about 25% of adult leukemia worldwide. AML is characterized by abnormal proliferation of primitive and immature myeloid cells in the bone marrow and peripheral blood. Most cases are acute and severe, with poor prognosis. It is the disease with the lowest survival rate among all adult leukemias. The main reason is that AML is a malignant clonal disease of stem cells. The root cause of its recurrence lies in the residual leukemic stem cells (LSC). Although most AML patients achieve complete remission (CR) after standard induction chemotherapy, most patients will relapse and die after remission. Due to the lack of highly specific target antigens and the heterogeneity of AML, the progress of immunotherapy in AML is very slow. In the past few decades, many tumor antigens (such as CD33, CD123, CD44, TIM-3, CD47, etc.) have been explored as target antigens for AML treatment, but the overall treatment effect is poor.

[0004] C-type lectin-like molecule 1 (CLL1) is a type II transmembrane glycoprotein that plays an important role in immune regulation as an inhibitory receptor. Studies have found that CLL-1 is present in most CD34+ or CD34- AMLs, and is mainly expressed on myeloid and myeloid blasts in peripheral blood and bone marrow. Therefore, targeting CLL-1 has become a potential target for AML targeted therapy and is expected to bring new breakthroughs in AML treatment.

[0005] The extracellular antigen recognition domain can also be called the extracellular antigen binding domain, which can specifically recognize tumor surface antigens. The CAR molecule uses the characteristics of specific recognition of tumor antigens by the antigen binding domain to transmit immune cell activation signals through the hinge region and transmembrane region. The most common extracellular antigen recognition domain is the ScFv (single-chain antibody) that recognizes tumor antigens. The selection of ScFv is crucial for the function of CAR. Therefore, for immunotherapy targeting CLL-1, the screening of anti-CLL-1 antibodies or anti-CLL-1ScFv is very necessary. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide a fully humanized single-chain antibody targeting CLL-1 and its use in detection reagents and anti-tumor drugs. The ScFv provided by the present invention can specifically recognize CLL-1, recognize and detect CLL-1 expressed by cells and its ratio, and can be used as a detection reagent or in vivo tracer marker molecule for CLL-1; the ScFv provided by the present invention can be used as the antigen recognition domain of the chimeric antigen receptor to recognize CLL-1 on the surface of the tumor, construct a chimeric antigen receptor immune cell, and effectively inhibit the growth of tumors in vivo, and the anti-tumor effect of expressing CLL-1 targets is significant. In addition, it can be used as an effective ingredient of anti-tumor drugs for malignant tumors with high expression of CLL-1, and effectively control the proliferation of malignant tumors with high expression of CLL-1.

[0007] The antigen-binding polypeptide targeting CLL-1 comprises a heavy chain variable region and a light chain variable region, the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3; the specific CDRs are respectively a combination of any one of the following:

[0008] (1) the amino acid sequence of L-CDR1 is QSISSY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is QQSYSTPYT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY; or

[0009] (2) the amino acid sequence of L-CDR1 is QSISSY, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQSYSTPIT, the amino acid sequence of H-CDR1 is GFTFDDYA, the amino acid sequence of H-CDR2 is ISWNSGSI, and the amino acid sequence of H-CDR3 is AKDIGSSTHYGMDV; or

[0010] (3) the amino acid sequence of L-CDR1 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQSYSTPYT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY; or

[0011] (4) The amino acid sequence of L-CDR1 is QSISSY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is QQSYSTPLT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY.

[0012] The antigen-binding polypeptide targeting CLL-1, the light chain variable region amino acids of the single-chain antibody are shown in SEQ ID NO.25 or SEQ ID NO.27 or SEQ ID NO.29 or SEQ ID NO.31.

[0013] The antigen-binding polypeptide targeting CLL-1, the heavy chain variable region amino acids of the single-chain antibody are shown as SEQ ID NO.26 or SEQ ID NO.28 or SEQ ID NO.30 or SEQ ID NO.32.

[0014] The single-chain antibody that recognizes CLL-1 comprises a heavy chain variable region and a light chain variable region, comprising a sequence shown in any of the following:

[0015] (1) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.26; or

[0016] (2) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.28; or

[0017] (3) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.29, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.30; or

[0018] (4) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.31, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.32; or

[0019] (5) In some embodiments, the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.35 or SEQ ID NO.36 or SEQ ID NO.37 or SEQ ID NO.38.

[0020] Furthermore, the antigen-binding polypeptide targeting CLL-1 also comprises a Linker sequence. In some embodiments, the Linker amino acid sequence is GSTSGSGKPGSGEGSTKG or (GGGGS)n, n=1-5, and the amino acid sequence is shown in SEQ ID NO.33 or SEQ ID NO.34.

[0021] Furthermore, the antigen-binding polypeptide targeting CLL-1 also contains a Linker sequence, preferably the sequence shown in SEQ ID NO:33.

[0022] Furthermore, the sequence of the single-chain antibody targeting CLL-1 is shown in SEQ ID NO:35-SEQ ID NO:38.

[0023] Another object of the present invention is to provide a chimeric antigen receptor targeting CLL-1.

[0024] The chimeric antigen receptor comprises an extracellular antigen recognition domain that can recognize the CLL-1 antigen, and the extracellular antigen recognition domain that recognizes the CLL-1 antigen comprises the CDR sequence of SEQ ID NO:1-6 or SEQ ID NO:7-12 or SEQ ID NO:13-18 or SEQID NO:19-24; in some embodiments, the extracellular antigen recognition domain that recognizes the CLL-1 antigen comprises a sequence that may also comprise a light chain sequence of SEQ ID NO.25 or SEQ ID NO.27 or SEQ ID NO.29 or SEQ ID NO.31, and a heavy chain amino acid sequence of SEQID NO.26 or SEQ ID NO.28 or SEQ ID NO.30 or SEQ ID NO.32.

[0025] In some embodiments, the extracellular antigen recognition domain that recognizes the CLL-1 antigen comprises the amino acid sequence shown as SEQ ID NO:35-SEQ ID NO:38.

[0026] In some embodiments, the extracellular antigen recognition domain that recognizes the CLL-1 antigen comprises a Linker sequence of SEQ ID NO.33 or SEQ ID NO.34.

[0027] The chimeric antigen receptor further comprises a hinge region, a transmembrane region and an intracellular recognition domain, and the hinge can be derived from: human IgG4, human CD8, human CD4, human CD28, human CD7, etc.

[0028] The hinge region preferably comprises an amino acid sequence as shown in SEQ ID NO. 40 or a functional variant thereof.

[0029] Furthermore, the hinge region preferably comprises an amino acid sequence as shown in SEQ ID NO. 22 or a functional variant thereof.

[0030] Furthermore, the transmembrane region may be derived from: α, β or chain of T cell receptor, CD8, CD28, CD3ε, CD4, CD16, CD137, CD80 and CD86, etc.

[0031] The transmembrane region preferably comprises an amino acid sequence as shown in SEQ ID NO. 39 or a functional variant thereof.

[0032] The chimeric antigen receptor also comprises an intracellular signaling domain, which comprises a signaling region and a co-stimulatory domain; the intracellular signaling domain comprises a signaling region, which is derived from a primary cytoplasmic signaling sequence containing ITAM, including but not limited to protein molecules such as CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, ICOS (CD278), FcεRI, CD66d, DAP10 and DAP12.

[0033] The signal transduction region is derived from CD3ζ, and its amino acid sequence is shown in SEQ ID NO.42.

[0034] The chimeric antigen receptor further comprises an intracellular signaling domain, which comprises a signal transduction region and a co-stimulatory domain; the co-stimulatory domain may be derived from: CD28, 41BB, OX40, CD27, DAP10, 2B4 (SLAMF4, CD244), CD3γ, CD3δ, FcεRI, CD2, CD16, TCRζ, FcRβ, CD30, CD40, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, KIRDS2, SLAMF7, NKp80 (KLRF1), signaling lymphocyte activation molecule (SLAM protein), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, DAP12, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, LFA-1 (CD11a / CD18), GITR, BAFFR, LIGHT, HVEM (LIGHTR), etc.

[0035] Furthermore, the co-stimulatory domain comprises an amino acid sequence as shown in SEQ ID NO.41 or a functional variant thereof.

[0036] The chimeric antigen receptor described above also includes a CD8 leader peptide. The CLL-1-targeted antibody or chimeric antigen receptor is located on the cell membrane under the guidance of the CD8 leader peptide. As known to those skilled in the art, all other signal peptides that can be used in the field of CAR or antibodies can be used in the present invention. The extracellular segment generally includes a CD8α or GM-CSFRα leader peptide.

[0037] The present invention also aims to provide a nucleic acid sequence encoding the aforementioned chimeric antigen receptor.

[0038] In some embodiments, the nucleic acid sequence is combined with a carrier as a target gene and transfected into immune cells to obtain engineered immune cells.

[0039] The nucleic acid sequence of the chimeric antigen receptor comprises the nucleic acid sequence of an antigen-binding polypeptide targeting CLL-1 as shown in SEQ ID NO.43 or SEQ ID NO.44 or SEQ ID NO.45 or SEQ ID NO.46.

[0040] Another object of the present invention is to provide a multi-nucleic acid vector.

[0041] Furthermore, the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector, and a plasmid.

[0042] In certain embodiments, the lentiviral vector is selected from the group consisting essentially of human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedi virus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0043] In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract / DNA flap (cPPT / FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR encompassed by the present invention, and a right (3') retroviral LTR.

[0044] In certain embodiments, the chimeric antigen receptor or multi-target CAR targeting CLL-1 described above comprises a hepatitis B virus post-transcriptional regulatory element (HPRE) or a woodchuck post-transcriptional regulatory element (WPRE) and an optimized woodchuck post-transcriptional regulatory element (oPRE).

[0045] In certain embodiments, the nucleic acid sequence encoding the aforementioned chimeric antigen receptor comprises an optimized Kozak sequence.

[0046] In certain embodiments, the gene expression vector may comprise a secretory anti-PD-1 ScFv; in certain embodiments, the gene expression vector comprises a PD-1 conjugated transduction peptide (such as a PD-1-CD28-CD137-CD3 signal structure); in certain embodiments, the gene expression vector may comprise a plurality of CAR combinations, such as a combination of two CARs targeting different antigens or different recognition sites of the same antigen.

[0047] The present invention also aims to provide an engineered cell, wherein the cell expresses a polypeptide comprising a CDR of a sequence of SEQ ID NO: 1-6 or SEQ ID NO: 7-12 or SEQ ID NO: 13-18 or SEQ ID NO: 19-24; in some embodiments, the cell expresses a polypeptide comprising a sequence such as SEQ ID NO.25 or SEQ ID NO.27 or SEQ ID NO.29 or SEQ ID NO.31, a sequence such as SEQ ID NO.26 or SEQ ID NO.28 or SEQ ID NO.30 or SEQ ID NO.32 that recognizes a CLL-1 antigen; in some embodiments, the cell expresses a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular antigen recognition domain that can recognize a CLL-1 antigen, and the extracellular antigen recognition domain that recognizes a CLL-1 antigen comprises a CDR of a sequence of SEQ ID NO: 1-6 or SEQ ID NO: 7-12 or SEQ ID NO: 13-18 or SEQ ID NO: 19-24; in some embodiments, the extracellular antigen recognition domain that recognizes a CLL-1 antigen comprises a sequence that may also comprise SEQ ID NO.25 or SEQ ID NO.27 or SEQID NO.29 or SEQ ID NO.31, comprising a heavy chain amino acid sequence as shown in SEQ ID NO.26 or SEQ ID NO.28 or SEQ IDNO.30 or SEQ ID NO.32; in some embodiments, the amino acid sequence of the extracellular recognition domain of the chimeric antigen receptor is as shown in SEQ ID NO:35-SEQ ID NO:38; in some embodiments, the cell further comprises a nucleic acid sequence for expressing a chimeric antigen receptor, and in some embodiments, the nucleic acid sequence of the chimeric antigen receptor comprises a nucleic acid sequence of an antigen-binding polypeptide targeting CLL-1 as shown in SEQ ID NO.43 or SEQ ID NO.44 or SEQ ID NO.45 or SEQ IDNO.46.

[0048] The engineered cells are immune cells such as T cells, T cell precursors or NK cells, DC cells, macrophages, etc.

[0049] Preferably, the immune cells are T lymphocytes.

[0050] The present invention further aims to provide a cell product, which comprises any of the engineered cells described above.

[0051] Furthermore, the cell product also includes other active agents that can enhance the expression activity of CAR.

[0052] In certain embodiments, the active agent is an immunosuppressant such as cyclosporin, azathioprine, methotrexate, mycophenolate and FK506, antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapy, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines and radiation.

[0053] In certain specific embodiments, the activating agent for enhancing the activity of CAR-expressing cells can be an activating agent that blocks inhibitory molecules. Inhibitory molecules such as PD1 can reduce the ability of CAR-expressing cells to initiate immune effector responses in some embodiments. Inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, CEACAM (CEACAM-1, CEACAM-3, CEACAM-5), LAG3, VISTA, BTLA, TIG, LAIR1, CD160, 2B4, CD80, CD86, B7-H3 (CD276), B7-H4 (VTCN1), HVEM (TNFRSF14 or CD270), KIR, A2aR, MHC class I, MHC class II, GAL9, adenosine, TGFR (TGFRβ) and TGFRβ. The extracellular domain of the inhibitory molecule can be fused to a transmembrane domain and an intracellular signaling domain, such as PD1 CAR.

[0054] Further, a variety of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutic agents include PD-1 inhibitors, such as nivolumab, pembrolizumab, pidilizumab, and atezolizumab.

[0055] In particular, additional therapeutic agents suitable for use in combination with the present invention include, but are not limited to, ibrutinib, ofatumumab, rituximab, bevacizumab, trastuzumab, trastuzumab, bemtansine, imatinib, cetuximab, panitumumab, catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, trastuzumab, imatinib, cetuximab, panitumumab, catumaxomab, ibritumomab, entuximab), alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib inib), ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, and denileukin.

[0056] Furthermore, the cell product can also be used in combination with some therapeutic means, and the therapeutic means can be surgery, chemotherapy, and radiation.

[0057] The present invention further aims to provide a pharmaceutical composition, which comprises any of the above-mentioned polypeptides or single-chain antibodies, the nucleic acid sequence of a chimeric antigen receptor or any of the above-mentioned expression vectors or any of the above-mentioned engineered cells.

[0058] The object of the present invention is to provide a use of any of the above-mentioned polypeptides or single-chain antibodies, chimeric antigen receptors or nucleic acid sequences of chimeric antigen receptors or expression vectors or engineered cells or cell products or pharmaceutical compositions in drugs for tumors expressing CLL-1 targets.

[0059] Specifically, the anti-tumor drugs include but are not limited to anti-renal cancer drugs, anti-prostate cancer drugs, anti-thymic tumor drugs, anti-ovarian cancer drugs, anti-glioblastoma drugs, anti-pancreatic cancer drugs, anti-bladder cancer drugs, anti-nasopharyngeal cancer drugs, anti-gastric cancer drugs, anti-colorectal cancer drugs and other malignant solid tumor drugs.

[0060] In the present invention, the term "functional variant" generally refers to an amino acid sequence having substantially the same function (e.g., having the properties of the chimeric antigen receptor) and having at least 85% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity therewith. In certain embodiments, the variant of the amino acid sequence has substantially the same function therewith. The beneficial effects of the present invention are

[0061] The ScFv provided by the present invention can specifically recognize CLL-1, recognize and detect CLL-1 expressed by cells and its ratio, and can be used as a detection reagent for CLL-1 or an in vivo tracing marker molecule.

[0062] The ScFv provided by the present invention can be used as the antigen recognition domain of the chimeric antigen receptor to recognize CLL-1 on the surface of the tumor, construct a chimeric antigen receptor immune cell, effectively inhibit the growth of tumors in vivo, and has a significant effect on tumors expressing CLL-1 targets. In addition, it can be used as an effective ingredient of an anti-tumor drug for malignant tumors with high expression of CLL-1, and effectively control the proliferation of malignant tumors with high expression of CLL-1.

[0063] The ScFv in the CLL-1 chimeric antigen receptor provided by the present invention is a natural fully human antibody, and its sequence is completely derived from the human antibody gene library. Compared with mouse antibodies, chimeric antibodies and humanized antibodies, its immunogenicity is greatly reduced and its toxic side effects are relatively small. Its later application in clinical efficacy will be more stable and have higher affinity, and its safety and therapeutic effect can be guaranteed to the greatest extent.

[0064] The CLL-1 chimeric antigen receptor provided by the present invention can specifically recognize human CLL-1 protein and is of great significance for the treatment of acute myeloid leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1: CLL-1 antigen recognition ability of NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03; Figure 1A NK-02-G11 recognizes CLL-1 expressed by U937. Figure 1B NK-03-A02 recognizes CLL-1 expressed by U937. Figure 1C for NK-03-A09 to recognize CLL-1 expressed by U937; Figure 1D NK-03-G03 recognizes CLL-1 expressed by U937.

[0066] Figure 2: Affinity determination of NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03; Figure 2A For NK-02-G11 affinity detection, Figure 2B For NK-03-A02 affinity detection, Figure 2C For NK-03-A09 affinity detection; Figure 2D Affinity test for NK-03-G03.

[0067] Figure 3 :CLL-1-CAR-T in vitro killing data.

[0068] Figure 4 :CLL-1-CAR-T IFNγ secretion data. DETAILED DESCRIPTION

[0069] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0070] Example 1 Screening of ScFv sequences that specifically recognize CLL-1

[0071] Construction of a fully human phage antibody library

[0072] Ficoll separation solution is used to separate PBMC, and Ficoll separation solution is slowly added to normal human blood so that Ficoll separation solution and normal human blood maintain a clear separation interface. The centrifuge tube containing the blood and separation solution is centrifuged. After centrifugation, the entire liquid surface is divided into four layers, the upper layer is a plasma mixture, the lower layer is red blood cells and granulocytes, and the middle layer is Ficoll liquid, wherein there is a narrow band of white cloud layer mainly composed of PBMC at the junction of the upper and middle layers, i.e., the PBMC cell layer. Carefully suck off the plasma mixture on the upper layer with a sterile Pasteur pipette, and then suck PBMC with a new sterile Pasteur pipette to obtain separated PBMC.

[0073] Total RNA was extracted by conventional methods and reverse transcribed into cDNA, and primers were designed according to the method in Sblattero, D., Bradbury, A., (1998) A definitive set of oligonucleotide primers for amplifying human Vregions. Immunotechnology 3, 271-278, wherein VL was in front or in the back, VH was in the back or in the front, and they were connected in the middle by a flexible linker. PCR was used to obtain the heavy chain variable region gene fragment and the light chain variable region gene fragment of the antibody, and the ScFv nucleic acid fragment was amplified by conventional overlapping PCR (the PCR method refers to "Molecular Cloning: A Laboratory Manual" (3rd edition), Joe Sambrook, David Russell, Science Press, USA), the ScFv nucleic acid fragment was connected to the phagemid vector pComb3xss, and the product was transformed into the TGI strain by an electroporator to obtain a fully human single-chain antibody library.

[0074] The library bacterial solution was added to fresh LB liquid medium for recovery, cultured to OD600≈0.5, and then VSCM13 helper phage was added at a multiplicity of infection of VCSM13: bacteria = 50:1, mixed thoroughly, and continued to be cultured in a shaker after standing. The culture was centrifuged and the supernatant was discarded, and the precipitate was resuspended in ampicillin and kanamycin double resistance SOB medium and cultured overnight. The bacterial solution was centrifuged for 20 minutes, the supernatant was collected and PEG 80002.5mol / L NaCl solution was added, incubated on ice for 1 hour, and then centrifuged for 30 minutes. The precipitated phage was resuspended in PBS and filtered with a 0.22μm filter membrane to obtain the phage library.

[0075] Antigen panning

[0076] Patent 202211370708.7 introduces the steps of antigen panning into this application, based on the antibodies of the antigens described in this application:

[0077] The CLL-1 protein with Fc tag is co-incubated with proteinG magnetic beads to prepare CLL-1-proteinG coupled magnetic beads, which are then put into the prepared fully human single-chain antibody library phage panning. After 3-4 rounds of co-incubation, washing and elution panning, specific monoclonal antibodies against the antigen can be enriched.

[0078] The above-described fully human antibody preparation method was used to screen and obtain the ScFv sequences shown in Table 1 below:

[0079] Table 1 Single chain antibody targeting CLL-1: The bold and underlined sequences in the table below are the CDR sequences of the screened ScFv, and the amino acid sequences are shown in SEQ ID NO.1-24. Wherein (1) the amino acid sequence of L-CDR1 of NK-02-G11 is QSISSY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is QQSYSTPYT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY; or (2) the amino acid sequence of L-CDR1 of NK-03-A02 is QSISSY, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQSYSTPIT, the amino acid sequence of H-CDR1 is GFTFDDYA, the amino acid sequence of H-CDR2 is ISWNSGSI, and the amino acid sequence of H-CDR3 is AKDIGSSTHYGMDV; or (3) the amino acid sequence of L-CDR1 of NK-03-A09 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQSYSTPYT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY; or (4) The amino acid sequence of L-CDR1 of NK-03-G03 is QSISSY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is QQSYSTPLT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY.

[0080] After the panning, the monoclonal plaques finally released from the library were selected for chemiluminescence detection screening, and the phage clones that bind to the CLL-1 antigen were obtained for downstream research. The following 4 ScFv strains were obtained by panning, numbered NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03, which have the ability to specifically bind to the human CLL-1 antigen. The specific chemiluminescence method is as follows:

[0081] K562-CLL-1 and K562 cells were plated in 96-deep-well plates at a concentration of 1.25×10^5 / sample, and 100ul / well of phage samples were added to each well. The samples were resuspended 5 times and incubated in a 4°C refrigerator for 30min. After washing, 500ul / well of PBS was added without resuspending, centrifuged (400g, 3min), and the supernatant was discarded. This was repeated twice. HRP-anti M13 was diluted according to the ratio in the instructions (HRP-anti M13:PBS=1:10000), 100ul / well, cells were resuspended, and the samples were incubated in a 4°C refrigerator for 30min. The secondary antibody was washed, and 500ul / well of PBS was added without resuspending, centrifuged (400g, 3min), and the supernatant was discarded. This step was repeated twice. Finally, the cells were centrifuged at 250rpm for 1s, color was developed, and solution I was added in a dark place. 25ul / well, resuspend, prepare for microplate reader detection, turn on the computer, enter the program, set up the program, add solution II 25ul / well before detection, resuspend the cells for detection. Finally, the detection value of each clone on the positive cell K562-CLL-1 is divided by the detection value on the negative cell K562 to serve as the signal ratio (S / N) to evaluate the strength of the clone's specific recognition of the CLL-1 antigen on the cell surface. The results are shown in Table 2:

[0082] Table 2: Chemiluminescence detection of ScFv phage binding to membrane expressed CLL-1 antigen Detection clone name S / N value NK-02-G11 85.42 NK-03-A02 103.96 NK-03-A09 109.35 NK-03-G03 147.96 VCSM13 1

[0083] In Table 1 above, NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 are the candidate ScFv phages for final screening for chemiluminescence detection, VCSM13 is the original phage, and the S / N value represents the strength of the clone's specific binding ability to the CLL-1 protein expressed on the cell surface (S / N greater than 50 indicates that the clone to be screened has a strong binding to the CLL-1 protein on the membrane surface). It can be seen from Table 1 above that the above three candidate ScFvs can all strongly recognize the CLL-1 antigen and can be used in CLL-1 recognition reagents, compositions, and the like.

[0084] The above four candidate ScFv phage sequences were purified and expressed to obtain ScFv polypeptide proteins. After being identified as normal by SDS-PAGE electrophoresis, 5% trehalose and mannitol were added respectively and the concentrations were tested, and the proteins were frozen at -80°C.

[0085] The above ScFv was obtained using prokaryotic expression and purification methods:

[0086] The pComb3xss plasmids constructed with the above ScFv gene sequences were transformed into Rosetta gami B competent cells, spread on LB plates containing ampicillin resistance, and cultured at 37°C overnight; the next day, single clones were picked and placed in 4ml LB medium containing ampicillin resistance for 8h, and then the bacterial solution was transferred to 200mL LB medium for further culture. When the bacterial solution OD reached 0.5-1.0, IPTG with a final concentration of 1mM was added, and expression was induced at 37°C for 12h. The bacteria were collected by centrifugation and PBS was added to resuspend the precipitate, ultrasonically broken, centrifuged for 10min, the precipitate was discarded, and the supernatant was collected for protein purification, which was purified using a GE Ni Sepharose excel purification column.

[0087] Example 2: Recognition of CLL-1 antigen by ScFv protein:

[0088] The U937 cells endogenously expressing CLL-1 protein were divided into 1.5mL Ep tubes, centrifuged at 4℃300g for 5min, the supernatant was discarded, and 50μL 30ug / ml positive clone ScFv solution was added to resuspend, incubated at 4℃ for 1h, and then 1ml PBS was added to resuspend the cells, centrifuged at 4℃300g for 5min, the supernatant was discarded, 30μl Anti-His-647 fluorescent secondary antibody was added and the cells were resuspended, incubated at 4℃ in the dark for 30min, and finally resuspended with 1ml PBS and washed twice at 4℃300g for 5min, the supernatant was discarded and resuspended with 100μL PBS. All cell tubes were tested for flow staining positivity and mean fluorescence intensity (MFI). The results are shown in Figure 1. Figure 1A NK-02-G11 recognizes CLL-1 expressed by U937. Figure 1BNK-03-A02 recognizes CLL-1 expressed by U937. Figure 1C NK-03-A09 recognizes CLL-1 expressed by U937. Figure 1D NK-03-G03 recognizes CLL-1 expressed by U937; the results show that NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 can all recognize CLL-1 on the cell surface. Combined with Example 1, NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 can be used as recognition reagents for CLL-1 antigen for detection reagents or detection kits for CLL-1 antigen or in vivo tracing or protein expression detection combination products and the like.

[0089] Detection of kinetic parameters of antigen-antibody binding

[0090] The proA biosensor was baselined in the assay buffer for 60 seconds, loaded in the CLL-1-hFc solution for 180 seconds for ligand protein immobilization (one experiment for every 2 clones, the antigen immobilization concentration was 10ug / ml), transferred to the assay buffer for baseline for 120 seconds, transferred to the gradient diluted analyte solution (NK-03-H05, NK-04-D08, NK-04-D11 were diluted 2 times from 30ug / ml in sequence) for association for 120 seconds, and finally dissociated in the assay buffer for 180 seconds. By judging whether there is specific binding by the change of the response value R, the DataAnalysis analysis software that comes with the Fortebio Octet K2 instrument can be used to give the kinetic parameters represented by the binding-dissociation curve, such as the binding constant (Kon), the dissociation constant (Kdis), and the equilibrium dissociation constant (KD). The unit of Kon is M-1 / S-1, which is used to describe the rate of antigen-antibody binding. The higher the Kon, the faster the antibody and antigen bind to form a complex; the unit of Kdis is S-1, which is used to describe the dissociation rate of antigen and antibody. The higher the Kdis, the faster the antigen-antibody complex dissociates; KD is the ratio of Kdis to Kon, which is used to comprehensively describe the difficulty of antigen-antibody binding. The smaller the KD, the higher the antibody affinity is generally considered to be. The results are shown in Figure 2: Figure 2A For NK-03-H05 affinity detection, Figure 2B For NK-04-D08 affinity detection, Figure 2CAffinity test for NK-04-D11; the final KDs of NK-02-G11, NK-03-A02, NK-03-A09 and NK-03-G03 were 6.020E+08M, 5.079E+05M, 2.031E+06M and 7.075E+08M, respectively. They are all effective bindings and can be used as recognition reagents for CLL-1 antigens for detection reagents or detection kits for CLL-1 antigens or in vivo tracing or protein expression detection combination products.

[0091] Example 3 Targeting CLL-1 ScFv Function and Application Verification in CAR Structure

[0092] In order to verify the function of the screened fully human antibodies, we constructed CAR vectors to prepare CAR-T cells to verify the ability of the screened fully human antibodies to recognize CLL-1 expressed by cells and kill tumor cells expressing CLL-1.

[0093] 1) Screening for ScFv with excellent killing ability against tumor cells expressing CLL-1

[0094] Construction of CAR plasmid targeting CLL-1

[0095] The ScFv sequence was obtained by PCR amplification, and then connected to the CAR vector structure by restriction endonuclease digestion, including 8h (CD8 hinge region), 8TM (CD8 transmembrane region), BB (intracellular signaling domain of CD137), and Z (CD3ζ chain) co-stimulatory domain. The constructed lentiviral vector was successfully constructed after verification by sequencing comparison.

[0096] CAR structure: ScFv(CLL-1)-8h-8TM-BBZ

[0097] The present embodiment packages lentivirus using the calcium phosphate method to package lentivirus, referring to the Molecular Cloning Experiment Guide (3rd edition, J. Sambrook et al.), specifically: using 293T cells cultured to the best state, the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5 centrifuge tube in a certain ratio, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture medium, and the medium is changed to 10mL DMEM medium containing 10% FBS again after 3-5h, and the cell supernatant is collected after 48h or 72h, and the virus is purified. The purified virus is packaged and stored at -80℃ for standby use. The virus infects 293T cells, and the genome is extracted using the QIAamp DNA Blood Mini Kit (purchased from Qiagen, item number 511004) genome extraction kit after 72h of infection. Operate according to the instructions of the kit. qRT-PCR determines the virus titer and uses analysis software to analyze the data, calculates the virus titer according to the standard curve, and the results are expressed in TU / mL. The virus titer results of NK-03-H05, NK-04-D08, and NK-04-D11 prepared by constructing CAR structures that recognize the antigen recognition region of CLL-1 are shown in Table 3 below:

[0098] Table 3: Titers of viruses containing CLL-1-CAR gene: FWf CAR Number Virus titer (Tu / ml) NK-02-G11 CAR4 2.68E+08 NK-03-A02 CAR3 6.02E+08 NK-03-A09 CAR2 8.59E+08 NK-03-G03 CAR1 1.25E+08

[0099] From Table 3 above, it can be found that NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 can be applied to CAR structure design and CAR construction.

[0100] The lymphocytes were separated by gradient centrifugation; after centrifugation, the second layer of white lymphocytes was taken, washed with saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human PBMC cells. After the obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies for 24 hours, the viruses containing CAR structure CAR1, CAR2, CAR3, and CAR4 genes constructed by NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 infected the activated PBMCs at a certain infection multiplicity (MOI), and the positive rate of CAR-T was detected on the 8th day of virus infection. The detection method was flow cytometry, and the antibody was: Protein-L-PE, Protein-L could recognize the antibody light chain, and the light chain of the ScFv sequence in the CAR antigen recognition region could be recognized by Protein-L, so Protein-L could be used to detect the CAR positive rate and CAR expression intensity. Anti-CD3 FITC (Bio legend, Lots: B378781, Cat: 300440), Protein-L-PE (Sino Biological, Lot: HR16SE0901, Cat: 11044-H07E-P), CAR expression results in T cells are shown in Table 4 below:

[0101] Table 4: CLL-1-CAR-T positive rate test results CART Number CAR positive rate (%) CAR4 26.08 CAR3 38.53 CAR2 46.04 CAR1 25.28

[0102] It can be seen that NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 can be applied to CAR construction, and the constructed CAR structure can be normally expressed in T lymphocytes.

[0103] KG-1a-CLL-1-Luc-GFP cells were used as positive target cells, and KG-1a-Luc-GFP was used as negative target cells. CAR-T cells and target cells were plated on the target cells at a ratio of 1:2, and killing was detected by luciferase after 24 hours. Luciferase principle: During the detection, the target cells were lysed with lysis buffer, and the luciferase therein would decompose the substrate to emit fluorescence. The formula for analyzing the results using fluorescence value was: CAR-T cell killing rate = 1-(fluorescence value of experimental group ÷ fluorescence value of blank control group). Cell supernatants were collected 24 hours after killing to detect the ability of CAR-T cells to secrete IFN-γ after being stimulated by target cells. The collected supernatant was used to detect the secretion of IFN-γ using ELISA (enzyme-linked immunosorbent assay). The results are as follows Figure 3 and Figure 4 As shown, Figure 3The killing results of CAR-T cells expressing CAR structures constructed by NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 against CLL-1 positive cells: KG-1a-CLL-1-Luc-GFP and CLL-1 negative cells: KG-1a-Luc-GFP, where the horizontal axis represents different CAR structures and the vertical axis represents the killing percentage. The results show that CAR-T cells expressing CAR structures constructed by NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 have a significant killing effect on CLL-1 positive cells: KG-1a-CLL-1-Luc-GFP, but have no killing effect on CLL-1 negative cells: KG-1a-Luc-GFP. Figure 4 The cytokine secretion after killing of CLL-1 positive cells: KG-1a-CLL-1-Luc-GFP and CLL-1 negative cells: KG-1a-Luc-GFP by CAR-T cells expressing CAR structures constructed by NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03. The results showed that CAR-T cells expressing CAR structures constructed by NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03 had high IFNγ secretion after killing of CLL-1 positive cells: KG-1a-CLL-1-Luc-GFP, but no killing of CLL-1 negative cells: KG-1a-Luc-GFP was consistent with T cells (CT) that were not transduced with CAR structures. Although there were differences in the in vitro killing and cytokine secretion of CAR-T cells from different sources, such as NK-02-G11, NK-03-A02, NK-03-A09, and NK-03-G03, the in vitro killing ratio was higher than 50% at an effector-target ratio of 1:2, and IFNγ secretion was higher than 1000pg / ml, both of which were excellent efficacy results.

[0104] It should be noted that although the present invention takes T cells and CAR-T cells as examples, based on the content disclosed in the present invention, it can be clearly inferred by those skilled in the art that other immune cells such as NK cells, macrophages, DC cells and related prerequisite cells can also express the engineered immune cells of the CAR containing CLL-1 described herein, and specifically recognize CLL-1, and play a killing role against malignant tumors expressing CLL-1. The CLL-1 described in the present application as a chimeric antigen receptor constructed as an extracellular antigen recognition domain or one of the extracellular antigen recognition domains can be expressed in T cells, NK cells, macrophages, DC cells and related prerequisite cells; it can be co-expressed with fusion proteins of any structure in T cells, NK cells, macrophages, DC cells and related precursor cells, and used for drugs or drug combinations of malignant tumors expressing CLL-1 for tumor treatment.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention. The sequence table used in the present invention is as follows:

Claims

1. An antigen-binding polypeptide targeting CLL-1, characterized in that The antigen-binding polypeptide comprises a heavy chain variable region and a light chain variable region; the light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, and the heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3; the specific CDRs are respectively a combination of any one of the following: (1) the amino acid sequence of L-CDR1 is QSISSY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is QQSYSTPYT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY; or (2) the amino acid sequence of L-CDR1 is QSISSY, the amino acid sequence of L-CDR2 is DAS, the amino acid sequence of L-CDR3 is QQSYSTPIT, the amino acid sequence of H-CDR1 is GFTFDDYA, the amino acid sequence of H-CDR2 is ISWNSGSI, and the amino acid sequence of H-CDR3 is AKDIGSSTHYGMDV; or (3) the amino acid sequence of L-CDR1 is QSVSSN, the amino acid sequence of L-CDR2 is GAS, the amino acid sequence of L-CDR3 is QQSYSTPYT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, and the amino acid sequence of H-CDR3 is AKVSTGGFFDY; or (4) the amino acid sequence of L-CDR1 is QSISSY, the amino acid sequence of L-CDR2 is AAS, the amino acid sequence of L-CDR3 is QQSYSTPLT, the amino acid sequence of H-CDR1 is GGSISSSNW, the amino acid sequence of H-CDR2 is IYHSGSP, The amino acid sequence of H-CDR3 is AKVSTGGFFDY.

2. The polypeptide of claim 1, comprising a light chain variable region represented by amino acids as shown in SEQ ID NO. 25 or SEQ ID NO. 27 or SEQ ID NO. 29 or SEQ ID NO.

31.

3. The polypeptide of claim 1, comprising a heavy chain variable region having amino acids as shown in SEQ ID NO. 26, SEQ ID NO. 28, SEQ ID NO. 30, or SEQ ID NO.

32.

4. The polypeptide according to claim 1 is a single-chain antibody that recognizes CLL-1, characterized in that: Contains any of the following sequences: (1) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.25, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.26; or (2) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.27, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.28; or (3) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.29, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.30; or (4) comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the light chain variable region is as shown in SEQ ID NO.31, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO.32; or (5) The single-chain antibody, wherein the amino acid sequence is shown in SEQ ID NO.35, SEQ ID NO.36, SEQ ID NO.37 or SEQ ID NO.

38.

5. The single-chain antibody recognizing CLL-1 described in claim 4 also comprises a Linker sequence, whose amino acid sequence is shown in SEQ ID NO.33 or SEQ ID NO.

34.

6. A kit for detecting CLL-1 expression, characterized in that: Comprising the antigen-binding polypeptide of claims 1-5.

7. A chimeric antigen receptor, characterized in that It comprises an extracellular antigen recognition domain that can recognize the CLL-1 antigen, and the extracellular antigen recognition domain comprises an antigen-binding polypeptide targeting CLL-1 as described in claims 1-5.

8. The chimeric antigen receptor according to claim 7, characterized in that It also contains a hinge region, a transmembrane region and an intracellular signaling domain.

9. The chimeric antigen receptor according to claim 8, characterized in that The hinge region amino acid sequence is shown in SEQ ID NO.

40.

10. The chimeric antigen receptor according to claim 9, characterized in that It also comprises a transmembrane region, and the amino acid sequence of the transmembrane region is shown in SEQ ID NO.

39.

11. The chimeric antigen receptor of claim 10, wherein the intracellular signaling domain comprises an amino acid sequence derived from CD3ζ such as the sequence shown in SEQ ID NO.

42.

12. The chimeric antigen receptor of claim 11, wherein the intracellular signaling domain further comprises a costimulatory signaling domain having an amino acid sequence such as that shown in SEQ ID NO.

41.

13. A nucleic acid sequence, characterized in that Encoding the chimeric antigen receptor according to claim 7.

14. The nucleic acid sequence encoding the chimeric antigen receptor according to claim 13, characterized in that: The antigen recognition nucleic acid sequence is shown as SEQ ID NO.43 or SEQ ID NO.44 or SEQ ID NO.45 or SEQ ID NO.

46.

15. An expression vector comprising the nucleic acid sequence of any one of claims 13-14.

16. An engineered cell, characterized in that The cell expresses the polypeptide or single-chain antibody according to any one of claims 1 to 5, or the chimeric antigen receptor according to any one of claims 7 to 12, or the cell comprises the nucleic acid or vector according to claims 13 to 15.

17. A cell product, characterized in that The cell product comprises the engineered cells of claim 16.

18. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the polypeptide or single-chain antibody according to any one of claims 1 to 5 or the nucleic acid or vector according to claims 13 to 15, the engineered cell according to any one of claim 16 or the cell product according to claim 17.

19. Use of the polypeptide or single-chain antibody of any one of claims 1-5, the nucleic acid or vector of claims 13-15, the engineered cell of any one of claim 16, the cell product of claim 17, the pharmaceutical composition of claim 18, or the chimeric antigen receptor of claims 7-12 in the preparation of tumor drugs expressing CLL-1 targets.

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