An antibody against cd66e and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to an anti-CD66e antibody and application thereof, the anti-CD66e antibody has the following technical features: the heavy chain variable region includes CDR-H1 with the amino acid sequence as shown in SEQ ID No. 1 or 2, CDR-H2 as shown in SEQ ID No. 3 or 4 and CDR-H3 as shown in SEQ ID No. 5 or 6. The anti-CD66e antibody has higher affinity, is used as an antigen binding domain to construct a chimeric antigen receptor, and utilizes the chimeric antigen receptor to prepare a CAR-T cell, the CAR-T cell has killing activity to tumor cells positive to CD66e, and after being co-cultured with CD66e positive cells, can efficiently secrete a cytokine IFN-gamma, the nanobody can be effectively applied to immunotherapy, and has important significance for developing tumor treatment drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an antibody against CD66e and use thereof. BACKGROUND

[0002] CD66e (also known as CEACAM5) was first discovered in colon cancer and embryonic tissue. Subsequent studies have shown that it is present in various digestive system tumors, such as gastric cancer, liver cancer, pancreatic cancer and colon cancer, and is also elevated in the serum of malignant tumors such as breast cancer and lung cancer. It is a widely present tumor marker.

[0003] CD66e belongs to the immunoglobulin (Ig) superfamily and is bound to the membrane through glycosylphosphatidylinositol (GPI). It has seven immunoglobulin-like domains in the extracellular region, including one variable IgV-like domain (N domain) and six IgC-like domains. In addition, there is a signal peptide of 34 amino acids in front of the Ig domain. The human CD66e gene is located on chromosome 19q13.2, with a length of about 21 kb, consisting of 9 exons and 3 non-coding exons. The CD66e protein is composed of 642 amino acids (molecular weight about 70 kDa), with 28 potential N-linked glycosylation sites. One CD66e protein molecule contains 24-26 asparagine-linked sugar chains, with a final molecular weight of about 180 kDa.

[0004] In normal adult tissues, the expression of CD66e is limited, but when tumor occurs, its expression in tumor cells changes, making it a potential target for immunotherapy. Adoptive immunotherapy, especially CAR-T cell therapy, can eliminate cancer cells by recognizing tumor-associated antigens, and has shown good efficacy in hematological tumors and is being explored in solid tumor treatment.

[0005] The high expression of CD66e in tumor cells and its unique expression pattern make it a promising target for CAR-T cell therapy. Clinical trials have shown that CD66e CAR-T cell therapy has good tolerability in patients with colon cancer and has observed therapeutic effects in some patients. These findings suggest that CD66e can be used as a target for CAR-T cell therapy, providing a new strategy for the treatment of solid tumors.

[0006] Therefore, the development of antibodies with high specificity and high affinity against CD66e has important clinical significance for the treatment of malignant tumors. SUMMARY

[0007] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide an antibody against CD66e and use thereof to solve the problems in the prior art.

[0008] To achieve the above object and other related objects, the present application provides an anti-CD66e antibody, which comprises a heavy chain variable region, wherein the anti-CD66e antibody heavy chain variable region has one or more of the following technical features:

[0009] <1> comprising a CDR-H1 with an amino acid sequence as shown in SEQ ID No. 1 or 2;

[0010] <2> comprising a CDR-H2 with an amino acid sequence as shown in SEQ ID No. 3 or 4;

[0011] <3> comprising a CDR-H3 with an amino acid sequence as shown in SEQ ID No. 5 or 6.

[0012] The present application also provides an isolated polypeptide, which comprises a transmembrane domain, an intracellular domain and an extracellular domain, wherein the extracellular domain comprises the anti-CD66e antibody.

[0013] The present application also provides an isolated polynucleotide encoding the anti-CD66e antibody or the polypeptide.

[0014] The present application also provides a nucleic acid construct comprising the isolated polynucleotide.

[0015] The present application also provides a lentivirus, which is packaged from the nucleic acid construct.

[0016] The present application also provides a lentivirus vector system, which comprises the nucleic acid construct and a helper plasmid or a host cell.

[0017] The present application also provides a chimeric antigen receptor immune cell, which expresses the membrane-bound isolated polypeptide.

[0018] As described above, the anti-CD66e antibody and its use of the present application have the following beneficial effects:

[0019] (1) The present application immunizes a naive llama with CD66e recombinant protein and CD66e overexpressing cells, constructs a phage display nanobody library, and screens the anti-CD66e antibody according to the phage display nanobody library, so that the obtained nanobody can specifically bind to the CD66e antigen, and has good affinity;

[0020] (2) The anti-CD66e nanobody provided by the application has high affinity, and is used as an antigen binding domain to construct a chimeric antigen receptor, and the CAR-T cell prepared by using the chimeric antigen receptor has killing activity on tumor cells positive for CD66e, and can efficiently secrete the cytokine IFN-γ after being co-cultured with CD66e positive cells, so that the nanobody can be effectively applied to immunotherapy, and has important significance for developing tumor treatment drugs. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 It is a FACS detection result graph of the anti-CD66e nanobody recognizing the CD66e antigen in Example 3.

[0022] Figure 2 It is an EC50 detection result graph of the anti-CD66e nanobody binding to the CD66e antigen in Example 4.

[0023] Figure 3 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9.

[0024] Figure 4 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9.

[0025] Figure 5 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9.

[0026] Figure 6 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9.

[0027] Figure 7 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9.

[0028] Figure 8 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9.

[0029] Figure 9 It is a CAR-T cell killing effect curve graph of the CAR-T cell on the CD66e positive cell human gastric cancer cell N87-CD66e in Example 9. DETAILED DESCRIPTION

[0030] The application provides an anti-CD66e antibody, which comprises a heavy chain variable region, and the anti-CD66e antibody heavy chain variable region has one or more of the following technical features.

[0031] <1> a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID No. 1 or 2;

[0032] <2> a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID No. 3 or 4;

[0033] <3> a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID No. 5 or 6;

[0034] GRTFNNYA (SEQ ID No. 1)

[0035] GRTFDSYA (SEQ ID No. 2)

[0036] INWGGDWT (SEQ ID No. 3)

[0037] INWRGDWT (SEQ ID No. 4)

[0038] AADEDYSGSTAHEYAY (SEQ ID No. 5)

[0039] AADEDYSGPTAHEYAY (SEQ ID No. 6).

[0040] The numbering scheme and definition scheme are for labeling CDR and FR regions by the method of IMGT.

[0041] CDR (complementarity determining region) generally refers to the region in an antibody that can form complementarity with an antigenic determinant in the spatial structure. The variability in the antibody is usually not uniformly distributed in the entire variable region of the antibody, and the heavy chain variable region of the monoclonal antibody usually has 3 hypervariable regions (HVR), which can usually form complementarity with the antigenic determinant in the spatial structure, so the hypervariable region is also called complementarity determining region (complementarity determining region, CDR), that is, the heavy chain variable region usually includes three complementarity determining regions, namely HCDR1, HCDR2 and HCDR3.

[0042] In some embodiments of the present application, the anti-CD66e antibody is coded as VHH-A062 or anti-CD66e-A062, and the heavy chain variable region thereof includes a CDR-H1 comprising an amino acid sequence as set forth in SEQ ID No. 1, a CDR-H2 comprising an amino acid sequence as set forth in SEQ ID No. 3, and a CDR-H3 comprising an amino acid sequence as set forth in SEQ ID No. 5.

[0043] In some embodiments of the present application, the anti-CD66e antibody is designated as VHH-A108 or anti-CD66e-A108, and the heavy chain variable region thereof comprises a CDR-H1 having an amino acid sequence as set forth in SEQ ID No. 2, a CDR-H2 having an amino acid sequence as set forth in SEQ ID No. 4, and a CDR-H3 having an amino acid sequence as set forth in SEQ ID No. 6.

[0044] The anti-CD66e antibody is an antibody fragment and / or a monoclonal antibody.

[0045] The "antibody fragment" comprises a portion of the intact antibody, preferably comprises the antigen binding region or variable region thereof. For example, the antibody fragment includes a nanobody (VHH), a single-chain antibody (scFv), a Fab, a Fab', a F(ab') or a F(ab')2.

[0046] In the present application, a naive alpaca is immunized with a CD66e recombinant protein and a cell strain overexpressing CD66e, a phage display nanobody library is constructed, and a monoclonal antibody capable of specifically binding to the CD66e antigen is obtained by screening the phage display nanobody library.

[0047] In some embodiments of the present application, the anti-CD66e antibody is a nanobody (Nb), i.e., a heavy-chain single-domain antibody VHH (variable domain of heavy chain of heavy-chain antibody). The nanobody only comprises a heavy chain variable region (VHH) and CH2 and CH3 regions, and the light chain of the nanobody is naturally absent compared with other antibodies. The nanobody has a crystal diameter of about 2.5 nm and a length of about 4 nm, and is the smallest fragment naturally existing and capable of binding to an antigen. The anti-CD66e antibody of the present application only comprises a heavy chain variable region, has high affinity and specificity, can efficiently target the CD66e antigen, has a simple structure, is easy to prepare, and has important application value in the field of preparing drugs targeting CD66e.

[0048] In some embodiments of the present application, the heavy chain variable region can further comprise a framework region, which can be located between the complementarity determining regions or at both ends of the complementarity determining regions. In some specific embodiments of the present application, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence obtained by substituting, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the framework region sequence of a mouse monoclonal antibody variable region, and the framework region sequence can have a homology of 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more with the framework region sequence of the human monoclonal antibody variable region.

[0049] In some embodiments of the present application, the heavy chain variable region further comprises a framework region. The framework region comprises framework regions FR1-FR4. The amino acid sequences of the framework regions FR1-FR4 are selected from any of the sequences shown in SEQ ID No. 7-13.

[0050] Preferably, the amino acid sequence of the FR1 is shown in SEQ ID No. 7 or 8:

[0051] QVQLVESGGGLVQAGGSLKLSCVAS (SEQ ID No. 7).

[0052] EVQLQESGGGLVQAGGSLKLSCAAS (SEQ ID No. 8)

[0053] Preferably, the amino acid sequence of the FR2 is shown in SEQ ID No. 9 or 10:

[0054] FGWFRQAPGKERAFVVA (SEQ ID No. 9)

[0055] FGWFRQAPGKERAFVAA (SEQ ID No. 10)

[0056] Preferably, the amino acid sequence of the FR3 is shown in SEQ ID No. 11 or 12:

[0057] YYGDSVKGRFTISRDNEKNTVYLQMNSLKPEDTAVYYC (SEQ ID No. 11)

[0058] YYGGSVEGRFSISRDNGKNTVFLQMNSLKPEDTAVYYC (SEQ ID No. 12)

[0059] Preferably, the amino acid sequence of the FR4 is shown in SEQ ID No. 13:

[0060] WGLGTQVTVSS (SEQ ID No. 13)

[0061] In some embodiments of the present application, the anti-CD66e antibody VHH-A062 heavy chain variable region comprises a CDR-H1 with an amino acid sequence shown in SEQ ID No. 1, a CDR-H2 with an amino acid sequence shown in SEQ ID No. 3, a CDR-H3 with an amino acid sequence shown in SEQ ID No. 5, and framework regions FR1-FR4 with amino acid sequences shown in SEQ ID No. 7, 9, 11, and 13, respectively.

[0062] Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD66e antibody VHH-A062 is shown as SEQ ID No. 14:

[0063] QVQLVESGGGLVQAGGSLKLSCVASGRTFNNYAFGWFRQAPGKERAFVVAINWGGD WTYYGDSVKGRFTISRDNEKNTVYLQMNSLKPEDTAVYYCAADEDYSGSTAHEYAYWG QGTQVTVSS (SEQ ID No. 14)

[0064] In some embodiments of the present application, the anti-CD66e antibody VHH-A062 is obtained by screening from a phage antibody library, and the nucleotide sequence of the heavy chain variable region thereof is shown as SEQ ID No. 15:

[0065] CAAGTGCAGCTGGTGGAATCCGGCGGGGGCCTGGTCCAAGCCGGCGGCAGCCTGAAGCTGAGCTGCGTGGCTAGCGGCAGAACCTTCAACAACTACGCCTTCGGCTGGTTCAGACAAGCCCCCGGCAAGGAGAGAGCCTTCGTGGTGGCCATCAACTGGGGCGGCGACTGGACCTACTACGGCGACAGCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGAGAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGAGGACTACAGCGGCAGCACCGCCCACGAGTACGCCTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGC (SEQ ID No. 15)

[0066] In some embodiments of the present application, the heavy chain variable region of the anti-CD66e antibody VHH-A108 comprises CDR-H1 with the amino acid sequence shown as SEQ ID No. 2, CDR-H2 with the amino acid sequence shown as SEQ ID No. 4, CDR-H3 with the amino acid sequence shown as SEQ ID No. 6, and the amino acid sequences of the framework regions FR1 to FR4 are shown as SEQ ID Nos. 8, 10, 12 and 13, respectively.

[0067] Preferably, the amino acid sequence of the heavy chain variable region of the anti-CD66e antibody VHH-A108 is shown as SEQ ID No. 16.

[0068] EVQLQESGGGLVQAGGSLKLSCAASGRTFDSYAFGWFRQAPGKERAFVAAINWRGD WTYYGGSVEGRFSISRDNGKNTVFLQMNSLKPEDTAVYYCAADEDYSGPTAHEYAYWGL GTQVTVSS (SEQ ID No. 16)

[0069] In some embodiments of the present application, the anti-CD66e antibody VHH-A108 is obtained by screening from a phage antibody library, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No. 17:

[0070] GAAGTGCAGCTCCAAGAATCCGGCGGGGGCCTCGTGCAAGCCGGCGGGAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCAGAACCTTCGACAGCTACGCCTTCGGCTGGTTCAGACAAGCCCCCGGCAAGGAGAGAGCCTTCGTGGCCGCCATCAACTGGAGAGGCGACTGGACCTACTACGGCGGCAGCGTGGAGGGCAGATTCAGCATCAGCAGAGACAACGGCAAGAACACCGTGTTCCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGAGGACTACAGCGGCCCCACCGCCCACGAGTACGCCTACTGGGGCCTGGGCACCCAAGTGACCGTGAGCAGC (SEQ ID No. 17)

[0071] In another aspect of the present application, an isolated polynucleotide encoding the anti-CD66e antibody is provided.

[0072] In some embodiments of the present application, the sequence of the polynucleotide comprises the sequence shown in SEQ ID No. 15 or 17.

[0073] In another aspect of the present application, the use of the anti-CD66e antibody in the preparation or screening of a therapeutic drug, or in the preparation of a diagnostic drug is provided.

[0074] The therapeutic drug can be a drug that targets CD66e antigen, binds to or acts on the CD66e antigen, thereby treating and / or preventing indications.

[0075] In some embodiments of the present application, the therapeutic drug can be a tumor therapeutic drug. The tumor can be a CD66e-expressing tumor. The tumor therapeutic drug can be a drug that targets, binds to or acts on CD66e antigen on the functional surface of tumor cells, thereby treating and / or preventing the tumor. The tumor can be a CD66e-expressing tumor such as colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, ovarian cancer, lung cancer, gastric cancer, gallbladder cancer, etc.

[0076] In some embodiments of the present application, the therapeutic drug is a chimeric antigen receptor cell.

[0077] The chimeric antigen receptor cell therapeutic drug generally includes a chimeric antigen receptor cell, which can be a chimeric antigen receptor T cell, a chimeric antigen receptor NK cell, etc. The chimeric antigen receptor T cell generally includes a T lymphocyte, which further includes a chimeric antigen receptor. The chimeric antigen receptor NK cell generally includes an NK cell, which further includes a chimeric antigen receptor. The chimeric antigen receptor includes a transmembrane domain, an intracellular domain and an extracellular domain. In some embodiments of the present application, the extracellular domain includes the anti-CD66e antibody, i.e., the chimeric antigen receptor cell can express the anti-CD66e antibody on the cell surface, thereby enabling the cell to act on cells expressing CD66e antigen (e.g., tumor cells). The action on cells expressing CD66e antigen can be killing cells expressing CD66e antigen, etc.

[0078] The diagnostic drug specifically refers to an agent for diagnosis against the action target CD66e antigen, taking CD66e antigen as a biomarker.

[0079] Another aspect of the present application provides an isolated polypeptide, which includes a transmembrane domain, an intracellular domain and an extracellular domain, and the extracellular domain includes the anti-CD66e antibody.

[0080] In some embodiments of the present application, the polypeptide is a chimeric antigen receptor. In the present application, the anti-CD66e antibody is used to construct a chimeric antigen receptor, which can efficiently target CD66e.

[0081] In some embodiments of the present application, the transmembrane domain can be selected from any one or more transmembrane domains in CD8a transmembrane region, CD28 transmembrane region, DAP10 transmembrane region, etc.

[0082] For example, the sequence of CD8a can refer to NM_001145873, the sequence of CD28 can refer to NM_006139, and the sequence of DAP10 can refer to NM_014266.

[0083] In some embodiments of the application, the intracellular domain can comprise a signal transduction domain.

[0084] The signal transduction domain comprises an immunoreceptor tyrosine-based activation motif. The immunoreceptor tyrosine-based activation motif can be selected from CD3 zeta.

[0085] Preferably, the signal transduction domain further comprises a costimulatory molecule. For example, the costimulatory molecule can be selected from any one or a combination of at least two of 4-1BB, CD28, OX40, ICOS, DAP 10, etc. For another example, the amino acid sequence of 4-1BB can comprise the following:

[0086] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID No. 18)

[0087] For another example, the sequence of 4-1BB can refer to NM_001561, the sequence of CD28 can refer to NM_006139, the sequence of OX40 can refer to NM_003327, the sequence of ICOS can refer to NM_012092, the sequence of CD3 zeta can refer to NM_198053, and the sequence of DAP 10 can refer to NM_014266.

[0088] In one embodiment of the application, the intracellular domain comprises 4-1BB and CD3 zeta from N-terminus to C-terminus.

[0089] In some embodiments of the application, the extracellular domain can comprise a signal peptide, an anti-CD66e antibody, a hinge region.

[0090] In some embodiments of the application, the signal peptide comprises a CD8a signal peptide.

[0091] In some embodiments of the application, the hinge region is selected from a CD8a hinge region.

[0092] In some embodiments of the application, the polypeptide comprises, from N-terminus to C-terminus, a CD8a signal peptide, the anti-CD66e antibody, a transmembrane domain, an intracellular domain.

[0093] In some embodiments of the application, the polypeptide comprises, from N-terminus to C-terminus, a CD8a signal peptide, an anti-CD66e nanobody, a CD8a hinge region, a CD8a transmembrane region, a costimulatory molecule, CD3 zeta.

[0094] In some embodiments of the application, the polypeptide comprises, from N-terminus to C-terminus, a CD8a signal peptide, an anti-CD66e nanobody, a CD8a hinge region, a CD8a transmembrane region, 4-1BB, CD3 zeta.

[0095] In some embodiments of the application, the polynucleotide sequence encoding the isolated polypeptide of VHH-A062 is set forth in SEQ ID No. 19:

[0096]

[0097] In some embodiments of the application, the polynucleotide sequence encoding the isolated polypeptide of VHH-A108 is set forth in SEQ ID No. 20:

[0098]

[0099] The present application also provides a nucleic acid construct comprising a polynucleotide encoding the isolated polypeptide.

[0100] The nucleic acid construct can be a lentiviral vector, a retroviral vector or an adeno-associated viral vector. Taking the lentiviral vector as an example, the lentiviral vector comprises a vector backbone, i.e. an empty vector, and an expression framework. That is, the nucleic acid construct is a vector comprising a coding gene of the chimeric antigen receptor.

[0101] The term "nucleic acid construct" or "vector" refers to a nucleic acid fragment or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into or to a target cell or tissue. Typically, a vector is used to introduce foreign DNA into another cell or tissue. The vector can comprise a bacterial resistance gene for growth in bacteria and a promoter for expression of a protein of interest in an organism. DNA can be generated in vitro by PCR or any other suitable technique known to those skilled in the art.

[0102] The term "expression framework" refers to a sequence with the potential to encode a protein.

[0103] The present application also provides a lentivirus, which is packaged from the nucleic acid construct. The lentivirus comprises the nucleic acid construct.

[0104] The present application also provides a lentiviral vector system, which comprises the nucleic acid construct and a helper plasmid.

[0105] Further, the helper plasmid encodes one or more nucleotide sequences of gag, pol, Rev, VSVg protein and other necessary viral packaging component nucleotide sequences. The helper plasmid can be obtained commercially, for example, addgene catalog numbers 14887 and 8454.

[0106] Further, the lentiviral vector system further comprises a host cell, which can be a cell for producing lentivirus, for example, a mammalian cell, specifically, a 293T cell.

[0107] The lentivirus can be obtained by transfecting the host cell with the nucleic acid construct and the helper plasmid in the lentiviral vector system. The host cell can be a mammalian cell.

[0108] Another aspect of the present application provides a chimeric antigen receptor immune cell expressing the membrane-bound isolated polypeptide.

[0109] Preferably, the chimeric antigen receptor immune cell comprises the nucleic acid construct and / or the lentivirus.

[0110] The immune cell is selected from any one of T lymphocyte, B lymphocyte, NK cell, mast cell or macrophage.

[0111] In another embodiment of the present application, the chimeric antigen receptor immune cell is a T lymphocyte.

[0112] The T lymphocyte can generally express the polypeptide, which can generally bind to CD66e antigen, more particularly bind to CD66e antigen through the extracellular domain comprising the anti-CD66e antibody, and the T lymphocyte can generally be activated and / or stimulated to proliferate when the polypeptide binds to the CD66e antigen. In some embodiments of the present application, the T lymphocyte, i.e. chimeric antigen receptor T cell, can express the anti-CD66e antibody on the surface of the T lymphocyte, thereby guiding the T lymphocyte to act on cells expressing CD66e antigen (e.g. tumor cells), and the action can be killing cells expressing CD66e antigen, etc.

[0113] In another embodiment of the present application, the chimeric antigen receptor immune cell is an NK cell.

[0114] The NK cell can generally express the polypeptide, which can generally bind to CD66e antigen, more particularly bind to CD66e antigen through the extracellular domain comprising the anti-CD66e antibody, and the NK cell can generally be activated and / or stimulated to proliferate when the polypeptide binds to the CD66e antigen. In some embodiments of the present application, the NK cell, i.e. chimeric antigen receptor NK cell, can express the anti-CD66e antibody on the surface of the NK cell, thereby guiding the NK cell to act on cells expressing CD66e antigen (e.g. tumor cells), and the action can be killing cells expressing CD66e antigen, etc.

[0115] In another aspect of the present application, the use of the isolated polypeptide, the isolated polynucleotide, the nucleic acid construct, the lentivirus, the chimeric antigen receptor immune cell in the preparation or screening of a therapeutic drug, or in the preparation of a diagnostic drug is provided.

[0116] The therapeutic or diagnostic drug can be a drug that targets CD66e antigen, binds to or acts on CD66e antigen, thereby treating and / or preventing indications.

[0117] In some embodiments of the present application, the therapeutic drug can be a tumor therapeutic drug. The tumor therapeutic drug can be a drug that targets, binds to, or acts on CD66e antigen functionally expressed on the surface of tumor cells, thereby treating and / or preventing tumors. The tumor can be a CD66e-expressing tumor such as gastric cancer, lung cancer, pancreatic cancer, intestinal cancer, etc.

[0118] The present application provides a pharmaceutical composition comprising the chimeric antigen receptor immune cell and / or the anti-CD66e antibody and / or the isolated polypeptide.

[0119] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0120] "Pharmaceutically acceptable" means they are not deleterious, allergenic, or otherwise undesirable when the drug is properly administered to an animal or human.

[0121] A "pharmaceutically acceptable carrier or excipient" should be compatible with the active ingredient, i.e., it can be blended with the active ingredient without substantially diminishing the effect of the drug under normal conditions. The pharmaceutically acceptable carrier or excipient is selected from any one or a combination of at least two of a surfactant, a disintegrant, a coating material, an excipient, a solubilizer, a diluent, a pH adjuster, a binder, a wetting agent, a colorant, an emulsifier, a bacteriostatic agent, a solubilizer, an osmotic pressure adjuster, a filler, an antioxidant, or a buffer. Specific examples can be sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methyl cellulose, ethyl cellulose, and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavorings; tabletting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or smell in the case of oral administration.

[0122] The embodiments of the present application are illustrated by specific examples below, and other advantages and effects of the present application can be easily understood by those skilled in the art from the disclosure herein. The present application can also be implemented or applied by other different specific embodiments, and various modifications or changes can be made to the details in the specification based on different perspectives and applications without departing from the spirit of the present application.

[0123] Before particular embodiments of the present application are described, it is to be understood that the application is not limited to the particular specific embodiments described; it is also to be understood that the terminology used in the description is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present application; and that the use herein of "including," "comprising," "having," and variations thereof, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0124] When a numerical range is given in the embodiments, it is understood that, unless otherwise specified by the present application, each numerical range has two endpoints and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, devices, materials described in the embodiments, any methods, devices and materials similar or equivalent to those described in the embodiments of the present application can also be used to implement the present application according to the knowledge of the prior art and the description of the present application by those skilled in the art.

[0125] The specific techniques or conditions not specified in the embodiments are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not specified by the manufacturer are all conventional products that can be purchased through regular channels.

[0126] Example 1

[0127] This embodiment constructs a phage nanobody library and screening, and uses ELISA for preliminary screening, the specific steps are as follows:

[0128] (1) Construction of phage nanobody library

[0129] After immunizing the alpaca with human CD66e recombinant protein and CD66e overexpressing cell strain, and detecting the serum titer by ELISA, peripheral blood is extracted; lymphocytes are separated, total RNA is extracted, and then cDNA is reverse transcribed, and then VHH gene is amplified by nested PCR; the VHH gene is inserted into the PhD-dAb-1 vector, and the SS320 competent cells are electrotransformed, and after amplification, the phage is separated and purified to obtain the antibody library; adjust the concentration, and store at -80℃ refrigerator for standby use;

[0130] (2) Screening of phage nanobody library

[0131] First, the phage antibody library is screened by liquid phase and solid phase two rounds respectively, and then the screened antibody library is incubated with cells AsPC-1, Colo205 and SNU5 for negative screening, and then incubated with cells AsPC-1-CD66e, Colo205-CD66e / GFP for positive screening;

[0132] The phage library was separated and purified, and the next round of ELISA screening was performed. After enrichment, the obtained phage was used as a template to amplify the VHH region, and second-generation sequencing was performed to obtain two anti-CD66e nanobodies. The amino acid sequence of one is shown in SEQ ID NO. 14, and it is named VHH-A062. The amino acid sequence of the other is shown in SEQ ID NO. 16, and it is named VHH-A108.

[0133] Example 2

[0134] In order to further identify the antibody, the VHH Fc nanobody expression, purification and antibody affinity determination were performed in this embodiment.

[0135] The obtained antibody quality detection results are shown in Table 1.

[0136] Table 1

[0137]

[0138] Example 3

[0139] In this embodiment, the flow assay of the anti-CD66e nanobody of Example 1 was performed.

[0140] HGC-27 and HGC-27-CD66e cells (prepared in Example 7) were mixed with purified anti-CD66e nanobodies, incubated in an ice bath for 30 min, then incubated with APC-labeled anti-human IgG antibody for 30 min, and detected by flow cytometry. The results are shown in Figure 1 It is shown that the anti-CD66e nanobody of the present application can recognize the CD66e antigen on the surface of the cells.

[0141] Example 4

[0142] In this embodiment, the ELISA detection of the anti-CD66e nanobody of Example 1 was performed to determine the EC50 value of its binding with the CD66e protein.

[0143] First, two clean ELISA empty plates were taken, washed with PBS once, and the PBS was discarded. The plates were dried on a blotting paper. Human CD66e-His (acro, cat# CE5-H5226) labeled antigen was diluted with PBS to 1 μg / mL, 100 μL of CD66e-His labeled antigen was added to each well of one plate, and the plate was sealed and placed in a 4°C refrigerator overnight for coating.

[0144] After overnight, the coated ELISA plate in the refrigerator was taken out, the liquid was discarded, 300 μL PBS was added to each well, the supernatant was discarded, and the plate was dried on the absorbent paper. 300 μL of 1xNAP blocking solution was added to each well, and the plate was incubated at room temperature for 1 hour. The supernatant was discarded, and the plate was dried on the absorbent paper. The primary antibody was diluted with 1xNAP to 4 μg / mL, and 11 concentration gradients were obtained by 4-fold gradient dilution. 50 μL was added to each well. The same volume of 1xNAP was added to the blank well as a control. The plate was sealed with a sealing film and incubated at room temperature for 1 hour. The supernatant was discarded, and the plate was dried on the absorbent paper. The plate was washed with 300 μL of TBST for 30 s, the liquid was discarded, and the plate was dried on the absorbent paper. The washing was repeated 4-5 times. The secondary antibody (0.4 μg / ml, Goat Anti-Human IgG HRP H&L, Cat#ab6858) was diluted with 1xNAP and added to the reaction well, 50 μL per well. Incubate at room temperature for 1 hour. Discard the supernatant, wash each well with 300 μL of TBST, soak for 30 s, discard the liquid, and dry the plate on the absorbent paper. Repeat the washing 4-5 times. Add 50 μL of HRP substrate to each well, and incubate at room temperature for 5-30 minutes in the dark. Add 50 μL of 1N HCL to stop the substrate reaction. Measure the absorbance at 450 nm with a microplate reader.

[0145] The results are shown in Figure 2 CD66e nanobody VHH-A062 binds to CD66e protein with an EC50 of 0.0099 μg / mL, and CD66e nanobody VHH-A108 binds to CD66e protein with an EC50 of 0.0444 μg / mL, both in nanomolar level, indicating that they can bind to CD66e protein well.

[0146] Example 5

[0147] In this embodiment, a lentiviral vector expressing a chimeric antigen receptor (CD66e CAR) targeting CD66e was prepared.

[0148] First, a lentiviral vector HD SIN03 anti-CD66e VHH-41BBz carrying a CD66e CAR chimeric antigen receptor was constructed, and the vector map is shown in Figure 3 The schematic diagram of the chimeric antigen receptor is shown in Figure 4 It includes CD8α signal peptide, anti-CD66e nanobody (anti-CD66e VHH), CD8α hinge region, transmembrane region, costimulatory molecule and immunoreceptor tyrosine-based activation motif (CD3ζ).

[0149] The amino acid sequence of the signal peptide (SEQ ID NO. 21) is:

[0150] MALPVTALLLPLALLLHAARP.

[0151] The amino acid sequence of the anti-CD66e VHH is shown in SEQ ID NO. 14 or 16.

[0152] The amino acid sequence of the CD8a hinge and transmembrane region (SEQ ID NO. 22) is:

[0153] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0154] The amino acid sequence of the 4-1BB co-stimulatory molecule (SEQ ID NO. 18) is:

[0155] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0156] The amino acid sequence of the CD3ζ (SEQ ID NO. 23) is:

[0157] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0158] The specific preparation method is as follows:

[0159] (1) The PCR reaction system was prepared according to Table 2 (the reagents in the table are from TOYOBO Inc.), and each anti-CD66e nanobody fragment was amplified, and the PCR program shown in Table 3 was used for reaction, and the primer sequence was as follows:

[0160] CEACAM5-A062-F (SEQ ID NO. 24):

[0161] CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAAGTGCAGCTGGTGGAATC CEACAM5-A108-F (SEQ ID NO. 25):

[0162] CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAAGTGCAGCTCCAAGAATC CEACAM5-A062 / A108-R (SEQ ID NO. 26):

[0163] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACTTGG.

[0164] Table 2

[0165] Reagent Volume (μL) 10× PCR Buffer 5 2 mM dNTP 5 25 mM MgSO4 3 10 μM Upstream primer 2 10 μM Downstream primer 2 Template DNA (cDNA clone) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo high fidelity PCR enzyme 1

[0166] Table 3

[0167]

[0168] After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and a fragment of about 400 bp was recovered and quantified by UV absorption;

[0169] (2) The PCR reaction system was prepared according to Table 4, the CD8α signal peptide was added before the amplification product obtained in step (1), and the PCR reaction was carried out according to the PCR program shown in Table 3, and the primers were as follows:

[0170] BamH-CD8αsig-F (SEQ ID NO. 27):

[0171] GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC.

[0172] CD66e-A062 / A108-R (SEQ ID NO. 26):

[0173] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACTTGG.

[0174] Table 4

[0175] Reagent Volume (μL) 10× PCR Buffer 5 2 mM dNTP 5 25 mM MgSO4 3 10 μM Upstream primer 2 10 μM Downstream primer 2 Template DNA (VHH fragment PCR reaction solution) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo high fidelity PCR enzyme 1

[0176] After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis, and a fragment of about 490 bp was recovered and quantified by UV absorption;

[0177] (3) The PCR reaction system was prepared according to Table 5, and after the preparation was completed, the PCR reaction was carried out according to Table 3, and the CD8α hinge-TM-41BB-CD3Z fragment was amplified, and the primers were as follows:

[0178] CD8αH-F (SEQ ID NO. 28): ACCACGACGCCAGCGCCGCGAC.

[0179] Vector-R (SEQ ID NO. 29): TCGATAAGCTTGATATCG.

[0180] Table 5

[0181] Reagent Volume (μL) 10× PCR Buffer 5 2 mM dNTP 5 25 mM MgSO4 3 10 μM Upstream primer CD8a H-F 2 10 μM Downstream primer Vector-R 2 Template DNA (HD CD66e CAR) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo high fidelity PCR enzyme 1

[0182] After PCR, 1% agarose gel electrophoresis was performed, and fragments of about 680 bp were recovered by ultraviolet absorption method for quantification;

[0183] (4) 25 μg of the laboratory constructed HD SIN03 GUCY2C A3-41BBz plasmid was subjected to BamHI and EcoRI double enzyme digestion, and after 37°C water bath reaction for 1 h, the vector was recovered;

[0184] The two recovered fragments and the vector backbone were ligated with recombinase, and the recombination reaction system is shown in Table 6. After 37°C water bath reaction for 0.5 h, the conventional method was used to transform into E. coli Stbl3 competent cells.

[0185] Table 6

[0186] Reagent Usage HD CD66e CAR skeleton 154.2 ng CD8a singal CD66e VHH 10 ng CD8a hinge-TM-41BB-CD3Z 13.6 ng 5× CE Buffer 2.4 μL Exnase MultiS 1.2 μL Sterile deionized water (PCR grade water) Make up to 12 μL

[0187] Single clones were selected from the solid culture medium for sequencing identification, and the results were as expected. The sequencing primer sequences are as follows:

[0188] LV-F2 (SEQ ID NO. 30): TCTTGGTTCATTCTCAAGCCTC.

[0189] LV-R (SEQ ID NO. 31): GCAACATAGTTAAGAATACC.

[0190] Example 6

[0191] In this example, the lentivirus vector HD SIN03 anti-CD66e VHH-41BBz prepared in Example 5 was subjected to lentivirus packaging, concentration and titer detection, including the following steps:

[0192] (1) Lentivirus packaging

[0193] Suitable concentration of 293T cells was inoculated in a 15 cm culture dish, and 293T cells were cultured at 37°C, 5% CO2 overnight to prepare virus packaging. The culture medium was DMEM containing 10% fetal bovine serum; 14.5 μg of lentivirus vector HD SIN03 anti-CD66e VHH-41BBz, 16.7 μg of helper plasmid pMDLg-RRE (Addgene, 12251), 16.7 μg of helper plasmid pRSV-REV (Addgene, 12253) and 6.5 μg of envelope plasmid VSVg (Addgene, 8454) were dissolved in 2 mL of serum-free DMEM medium, and mixed well;

[0194] Dissolve 163.2 μg PEI (1 μg / μL) in 2 mL serum-free DMEM medium, vortex at 1000 rpm for 5 s, and incubate at 25 °C for 5 min; add the PEI mixture to the DNA mixture, mix gently, and incubate at 25 °C for 20 min to form a transfection complex; then add 4 mL of the transfection complex to 18 mL of DMEM medium containing 293T cells, and replace the medium with fresh medium after 6 h; collect the virus supernatant after 48 h;

[0195] (2) Lentivirus titer detection

[0196] Seed 500 μL of Jurkat (1 × 10 5 cells into a 24-well plate, add the collected virus supernatant to the cell suspension at a volume of 1 μL, 5 μL, and 25 μL, respectively, and add polybrene to a final concentration of 8 μg / mL; after incubation at 37 °C and 5% CO2 for 72 h, centrifuge at 500 g for 5 min, discard the supernatant, resuspend the cells in 100 μL of PBS + 2% FBS, add 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody, and incubate on ice for 30 min; after washing once with flow buffer (PBS containing 2% FBS), resuspend the cells in 300 μL of flow buffer, and detect the infection efficiency using a flow cytometer; calculate the titer, and the titer calculation formula is as follows: titer (TU / mL) = cell number × positive rate / virus volume (mL). According to the titer formula, the VHH-A062 virus titer is calculated to be 1.37E+07 TU / ml, and the VHH-A108 virus titer is calculated to be 6.48E+06 TU / ml.

[0197] Example 7 Construction of overexpression cell strain

[0198] The lentivirus overexpressing CD66e is obtained by co-transfecting CD66e-puro plasmid, pMGlg-RRE, pRSV-REV, and VSVg plasmid, and the steps are the same as in Example 6.

[0199] Seed 1 × 10 6 HGC-27 cells and N87 cells into a 6-well plate, respectively, and add 1 mL of the above-obtained CD66e-puro lentivirus to obtain HGC-27-CD66e and N87-CD66e overexpressing CD66e protein.

[0200] Example 8

[0201] This example uses lentivirus prepared in Example 6 to transduce T lymphocytes, including the following steps:

[0202] (1) Adjust human PBMC to a density of 1 x 10 6 / mL with T cell medium (X-VIVO + 10% FBS + 300 U / mL IL-2), add 1 / 100 volume of T Cell TransAct (commercially available magnetic beads coupled with CD3 and CD28) and activate for 24 h;

[0203] (2) Collect the activated T cells, adjust the cell density to 1 x 10 6 / mL, add lentivirus at a multiplicity of infection (MOI) of 10, and add polybrene to a final concentration of 8 μg / mL; replace the fresh medium after incubation at 37°C in a 5% CO2 environment overnight, and subculture every 3 days;

[0204] (3) After 8 days of T cell infection, take 3 x 10 5 T cells, centrifuge at 4°C and 500 g for 5 min, discard the supernatant, and wash once with flow buffer; resuspend the cells with 50 μL of buffer, add 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody, and incubate on ice for 30 min; wash once with buffer, resuspend the cells with 300 μL of buffer, and analyze by flow cytometry.

[0205] The chimeric antigen receptor expression rate of T lymphocytes was detected by flow cytometry, and the results are shown in Figure 5 . The infection efficiency of CAR-T cells in each group was 78.8% and 67.8%, respectively, indicating that CAR-T cells were successfully constructed.

[0206] Example 9

[0207] In this example, CAR-T cell in vitro toxicity experiment was performed, including the following steps:

[0208] (1) Target cell inoculation

[0209] 293T-GPF-luci (CD66e - ), N87-GFP-luci-CD66e (CD66e + ), and BxPC3-GFP-luci (CD66e + ) were used as target cells, and the target cell concentration was adjusted to 2 x 10 5 / mL, and 50 μL was inoculated into a white opaque 96-well plate;

[0210] (2) Effect cell inoculation

[0211] CAR-T targeting CD66e and control T cells as effector cells, CAR-T cells and control T cells were added to 96-well plates at effector-target ratios of 0.3:1, 1:1 and 3:1;

[0212] (3) Each group has 2 duplicate wells, wherein each experimental group and each control group is as follows:

[0213] Experimental group: each target cell + T / CAR-T; control group: only inoculate target cells.

[0214] (5) Detection method:

[0215] After the effector cells and target cells were co-cultured for 18h, 70ul Steady-Lumi TM Firefly luciferase reporter gene detection reagent (Bi Yun Tian, item number: RG058M), reaction for 5min, and multifunctional enzyme label instrument was used to detect bioluminescence signal.

[0216] (5) The CAR-T killing efficiency calculation formula is: killing efficiency % = (1-experimental group / control group) x 100%.

[0217] The results are shown in the following table: Figure 6 to Figure 8 As shown in the table, the CAR-T cells constructed in the application have no killing effect on CD66e negative 293T cells, and have killing activity on CD66e positive tumor cells, indicating that the CAR-T cells constructed in the application not only have high tumor killing ability, but also have high specificity.

[0218] Example 10

[0219] In this embodiment, the secretion of CAR-T cell factor IFN-γ is detected, and the kits used are Human IFN-γ ELISA Kit (Lianke Biology, item number: EK180-96).

[0220] 1. Cell culture supernatant

[0221] Centrifuge 400g of cell culture with effector-target ratio of 1:1 for 10min to remove precipitate, and store the supernatant at-80℃ for detection.

[0222] 2. Reagent preparation

[0223] Before detection, all reagents and samples are restored to 25℃, if the concentrated reagent appears crystallization, 37℃ incubation until all crystallization is dissolved, according to the instruction to prepare 1x washing solution, 1x detection buffer.

[0224] 3. Preparation of standard and sample

[0225] Standard: 2-fold dilution of standard stock solution with 5% FBS 1640 medium, a total of 8 dilution gradients, including zero concentration.

[0226] Sample: Dilute the sample with 5% FBS 1640 medium.

[0227] 4. Detection step

[0228] (1) Soak the enzyme-labeled plate: add 300 μL of 1x washing solution and soak for 30 s, discard the washing solution, and pat the microplate on the absorbent paper;

[0229] (2) Add standard: add 100 μL of 2-fold dilution of standard to the standard well, and add 100 μL of standard dilution to the blank well;

[0230] (3) Add sample: add 100 μL of cell culture supernatant to the sample well;

[0231] (4) Add detection antibody: add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0232] (5) Incubate: seal the plate with a sealing film, shake at 300 rpm, and incubate at 25°C for 2 h;

[0233] (6) Wash: discard the liquid, add 300 μL of washing solution to each well, and wash the plate 6 times, each time washing the plate and patting it on the absorbent paper;

[0234] (7) Add enzyme incubation: add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0235] (8) Incubate: seal the plate with a new sealing film, shake at 300 rpm, and incubate at 25°C for 45 min, then wash;

[0236] (9) Add substrate for color development: add 100 μL of color development substrate TMB to each well, avoid light, and incubate at 25°C for 20 min;

[0237] (10) Add stop solution: add 100 μL of stop solution to each well;

[0238] (11) Detect the reading: within 30 min, use an enzyme-labeled instrument to detect the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength; the calibrated OD value is the measured value at 450 nm minus the measured value at the reference wavelength.

[0239] The results of IFN-γ factor secretion are as follows: Figure 9As shown in the figure, the spontaneous MOCK group is a single CAR-T cell group, and the cytokine release is basically not detected; the CAR-T cell co-culture group with 293T cells also basically does not detect the cytokine; after co-culture with CD66e positive target cells, the CAR-T cell secretes IFN-γ of more than 500 pg / mL. The CAR-T cell constructed by the application releases cytokines to CD66e positive tumor cells, and has no obvious cytokine secretion to CD66e negative cells.

[0240] In summary, the anti-CD66e nanobody with high affinity screened and prepared by the application can specifically bind to CD66e with high efficiency, and is used as an antigen binding domain to construct a chimeric antigen receptor and a CAR-T cell. The obtained CAR-T cell has obvious killing activity and specificity to CD66e positive tumor cells, and can secrete a tumor killing cytokine, indicating that the nanobody of the application can be effectively applied to immunotherapy, and has important significance for developing tumor treatment drugs.

[0241] The above examples are intended to illustrate the embodiments disclosed in the application, and should not be construed as limiting the application. In addition, various modifications listed herein and changes in the method of the application are obvious to those skilled in the art without departing from the scope and spirit of the application. Although the application has been specifically described in conjunction with various specific preferred embodiments thereof, it should be understood that the application should not be limited to these specific embodiments. In fact, various modifications as described above to obtain the application which are obvious to those skilled in the art should be included within the scope of the application.

Claims

1. An anti-CD66e antibody, characterized in that, The anti-CD66e antibody is a nanobody, the anti-CD66e antibody comprises a heavy chain variable region, the heavy chain variable region of the anti-CD66e antibody comprises a CDR-H1 with an amino acid sequence as shown in SEQ ID No. 1, a CDR-H2 with an amino acid sequence as shown in SEQ ID No. 3, and a CDR-H3 with an amino acid sequence as shown in SEQ ID No.

5.

2. The anti-CD66e antibody according to claim 1, characterized in that The heavy chain variable region further comprises a framework region, the framework region is FR1~FR4, and the amino acid sequences of the FR1~FR4 are shown in SEQ ID No. 7, 9, 11, and 13 in sequence.

3. The anti-CD66e antibody according to claim 1, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

14.

4. An anti-CD66e antibody, characterized in that The anti-CD66e antibody is a nanobody, the anti-CD66e antibody comprises a heavy chain variable region, the heavy chain variable region of the anti-CD66e antibody comprises a CDR-H1 with an amino acid sequence as shown in SEQ ID No. 2, a CDR-H2 with an amino acid sequence as shown in SEQ ID No. 4, and a CDR-H3 with an amino acid sequence as shown in SEQ ID No.

6.

5. The anti-CD66e antibody according to claim 4, characterized in that The heavy chain variable region further comprises a framework region, the framework region is FR1~FR4, and the amino acid sequences of the FR1~FR4 are shown in SEQ ID No. 8, 10, 12, and 13 in sequence.

6. The anti-CD66e antibody according to claim 4, characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.

16.

7. An isolated polypeptide, comprising, The polypeptide is a chimeric antigen receptor, the polypeptide comprises, from N-terminus to C-terminus, a CD8 alpha signal peptide, an anti-CD66e antibody, a CD8 alpha hinge region, a CD8 alpha transmembrane region, a costimulatory molecule, and a CD3 zeta signaling domain, the amino acid sequence of the CD8 alpha signal peptide is shown in SEQ ID No. 21, the amino acid sequence of the anti-CD66e antibody is shown in SEQ ID No. 14 or 16, the amino acid sequences of the CD8 alpha hinge region and the CD8 alpha transmembrane region are shown in SEQ ID No. 22, the amino acid sequence of the costimulatory molecule is shown in SEQ ID No. 18, and the amino acid sequence of the CD3 zeta signaling domain is shown in SEQ ID No.

23.

8. An isolated polynucleotide, comprising: The polypeptide of claim 7.

9. A nucleic acid construct, characterized in that, The polypeptide of claim 7.

10. The nucleic acid construct of claim 9, wherein, The nucleic acid construct is any one of a lentiviral vector, a retroviral vector, or an adeno-associated viral vector.

11. A lentivirus, characterized in that, The lentivirus is packaged from the nucleic acid construct of claim 9 or 10.

12. A lentiviral vector system, characterized in that, The lentiviral vector system comprises the nucleic acid construct of claim 9 or 10 and a helper plasmid or a host cell.

13. A chimeric antigen receptor immune cell, comprising: The chimeric antigen receptor immune cell expresses the membrane-bound polypeptide of claim 7.

14. The chimeric antigen receptor immune cell of claim 13, wherein, The immune cell is any one selected from a T lymphocyte, a B lymphocyte, an NK cell, a mast cell, or a macrophage.

15. Use of the anti-CD66e antibody according to any one of claims 1 to 3, the anti-CD66e antibody according to any one of claims 4 to 6, the isolated polypeptide according to claim 7, the chimeric antigen receptor immune cell according to any one of claims 13 to 14 for the manufacture of a medicament for the treatment of a tumor selected from the group consisting of pancreatic cancer or gastric cancer.

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