Anti-CD66e antibody and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an anti-CD66e antibody and application thereof, and the anti-CD66e antibody has the following technical characteristics: a heavy chain variable region comprises CDR-H1 with an amino acid sequence shown as SEQ ID No.1 or 2, CDR-H2 with an amino acid sequence shown as SEQ ID No.3 or 4 and CDR-H3 with an amino acid sequence shown as SEQ ID No.5 or 6. The anti-CD66e antibody has high affinity, the anti-CD66e antibody is used as an antigen binding structural domain to construct a chimeric antigen receptor, the chimeric antigen receptor is used for preparing a CAR-T cell, the CAR-T cell has killing activity on CD66e positive tumor cells, after the CAR-T cell and the CD66e positive cells are co-cultured, cell factors IFN-gamma are efficiently secreted, and the CAR-T cell can be used for killing the CD66e positive tumor cells. The nano antibody can be effectively applied to immunotherapy, and has important significance for developing tumor treatment drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to an anti-CD66e antibody and uses thereof. Background Art

[0002] CD66e (also known as CEACAM5) was first discovered in colon cancer and embryonic tissue. Later studies have shown that it exists in a variety of digestive system tumors, such as gastric cancer, liver cancer, pancreatic cancer and colon cancer. It 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 binds to the membrane through glycosylphosphatidylinositol (GPI). It has a total of 7 immunoglobulin-like domains outside the cell, including a variable lgV-like domain (N domain) and six lgC-like domains. In addition, there is a 34-amino acid signal peptide before the lg domain. The human CD66e gene is located on chromosome 19q13.2, with a gene length of approximately 21kb, consisting of 9 exons and 3 non-coding exons. The CD66e protein consists of 642 amino acids (molecular weight of approximately 70kDa) and has 28 potential N-linked glycosylation sites. A CD66e protein molecule contains 24-26 asparagine-linked sugar chains, and its final molecular weight is approximately 180kDa.

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

[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 is well tolerated in patients with colon cancer, and therapeutic effects have been observed 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 highly specific and high-affinity antibodies against CD66e has important clinical significance for the treatment of malignant tumors. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, an object of the present invention is to provide an anti-CD66e antibody and use thereof, so as to solve the problems in the prior art.

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

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

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

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

[0012] The present invention also provides a separated 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 invention also provides an isolated polynucleotide encoding the anti-CD66e antibody or the polypeptide.

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

[0015] The present invention also provides a lentivirus, which is formed by viral packaging of the nucleic acid construct.

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

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

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

[0019] (1) The present invention immunizes unimmunized alpacas with CD66e recombinant protein and cells overexpressing CD66e to construct a phage-displayed nanoantibody library, and screens anti-CD66e antibodies based on the phage-displayed nanoantibody library. The obtained nanoantibodies can specifically bind to the CD66e antigen with good affinity;

[0020] (2) The anti-CD66e nanobody provided by the present invention has a high affinity. It is used as an antigen binding domain to construct a chimeric antigen receptor, and the chimeric antigen receptor is used to prepare CAR-T cells. The CAR-T cells have killing activity against CD66e-positive tumor cells, and after co-culture with CD66e-positive cells, they efficiently secrete the cytokine IFN-γ. The nanobody can be effectively used in immunotherapy, which is of great significance for the development of tumor treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a diagram showing the FACS detection results of the anti-CD66e nanobody recognizing the CD66e antigen in Example 3;

[0022] Figure 2 This is a graph showing the EC50 test results of the anti-CD66e nanobody binding to the CD66e antigen in Example 4;

[0023] Figure 3 This is the plasmid map of the chimeric antigen receptor lentiviral vector targeting CD66e in Example 5;

[0024] Figure 4 This is a schematic diagram of the chimeric antigen receptor structure expressing CD66e in Example 5;

[0025] Figure 5 This is a flow cytometry result diagram of the chimeric antigen receptor expression rate of T lymphocytes in Example 8;

[0026] Figure 6 This is a graph showing the killing effect of CAR-T cells on CD66e negative human embryonic kidney cells 293T in Example 9;

[0027] Figure 7 This is a graph showing the killing effect of CAR-T cells on CD66e-positive human gastric cancer cells N87-CD66e in Example 9;

[0028] Figure 8 This is a graph showing the killing effect of CAR-T cells on CD66e-positive human in situ pancreatic adenocarcinoma cells BxPC3 in Example 9;

[0029] Fig. 9 This is a bar graph of IFN-γ secretion by CAR-T cells in Example 10. DETAILED DESCRIPTION

[0030] The present invention provides an anti-CD66e antibody, wherein the anti-CD66e antibody 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> comprising a CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1 or 2;

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

[0033] <3> comprising a CDR-H3 having an amino acid sequence as shown 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 and definition scheme uses the IMGT method to annotate CDR and FR regions.

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

[0042] In certain embodiments of the present invention, the anti-CD66e antibody is coded as VHH-A062 or anti-CD66e-A062, and its heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.1, CDR-H2 with an amino acid sequence as shown in SEQ ID No.3, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.5.

[0043] In certain embodiments of the present invention, the anti-CD66e antibody is coded as VHH-A108 or anti-CD66e-A108, and its heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.2, CDR-H2 with an amino acid sequence as shown in SEQ ID No.4, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.6.

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

[0045] "Antibody fragments" include a portion of an intact antibody, preferably its antigen binding region or variable region. For example, antibody fragments include nanobodies (VHH), single-chain antibodies (scFv), Fab, Fab', F(ab') or F(ab')2.

[0046] In the present invention, an unimmunized alpaca is immunized with CD66e recombinant protein and a cell line overexpressing CD66e, a phage display nanoantibody library is constructed, and CD66e antibodies are screened based on the phage display nanoantibody library to obtain a monoclonal antibody that can specifically bind to the CD66e antigen.

[0047] In certain embodiments of the present invention, 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). Nanobodies only contain a heavy chain variable region (VHH) and CH2, CH3 regions. Compared with other antibodies, nanobody light chains are naturally absent. The nanobody crystal has a diameter of about 2.5nm and a length of about 4nm. It is the smallest naturally occurring fragment that can bind to an antigen. The anti-CD66e antibody of the present invention includes only 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 certain embodiments of the present invention, the heavy chain variable region may further include a framework region, which may be located between the complementary determining regions or at both ends of the complementary determining regions. In certain specific embodiments of the present invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence of a mouse monoclonal antibody variable region obtained by replacing, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence and the framework region sequence of the human monoclonal antibody variable region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology.

[0049] In certain embodiments of the present invention, the heavy chain variable region further comprises a framework region. The framework region comprises framework regions FR1 to FR4. The amino acid sequence of the framework regions FR1 to FR4 is selected from any sequence shown in SEQ ID No. 7 to 13.

[0050] Preferably, the amino acid sequence of FR1 is as 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 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 FR3 is as 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 FR4 is as shown in SEQ ID No.13:

[0060] WGLGTQVTVSS (SEQ ID No. 13)

[0061] In certain embodiments of the present invention, the heavy chain variable region of the anti-CD66e antibody VHH-A062 includes 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, a CDR-H3 with an amino acid sequence as shown in SEQ ID No.5, and the amino acid sequences of the framework regions FR1 to FR4 are shown in SEQ ID Nos.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 as shown in SEQ ID No.14:

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

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

[0065] CAAGTGCAGCTGGTGGAATCCGGCGGGCCTGGTCCAAGCCGGCGGCAGCCTGAAGCTGAGCTGCGTGGCTAGCGGCAGAACCTTCAACAACTACGCCTTCGGCTGGTTCAGACAAGCCCCCGGCAAGGAGAGAGCCTTCGTGGTGGCCATCAACTGGGCGGCCGACTGGACCTACTACGGCGACA GCGTGAAGGGCAGATTCACCATCAGCAGAGACAACGAGAAGAACACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGAGGACTACAGCGGCAGCACCGCCCACGAGTACGCCTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGC(SEQID No.15)

[0066] In certain embodiments of the present invention, the heavy chain variable region of the anti-CD66e antibody VHH-A108 includes 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, a CDR-H3 with an amino acid sequence as shown in SEQ ID No. 6, and the amino acid sequences of the framework regions FR1 to FR4 are shown in 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 certain embodiments of the present invention, the anti-CD66e antibody VHH-A108 is obtained by screening from a phage antibody library, and the nucleotide sequence of its heavy chain variable region is shown in SEQ ID No. 17:

[0070] GAAGTGCAGCTCCAAGAATCCGGCGGGGGCCTCGTGCAAGCCGGCGGGAGCCTGAAGCTGAGCTGCGCCGCTAGCGGCAGAACCTTCGACAGCTACGCCTTCGGCTGGTTCAGACAAGCCCCCGGCAAGGAGAGAGCCTTCGTGGCCGCCATCAACTGGAGAGGCGACTGGACCTACTACGGCGGCA GCGTGGAGGGCAGATTCAGCATCAGCAGAGACAACGGCAAGAACACCGTGTTCCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGAGGACTACAGCGGCCCCACCGCCCACGAGTACGCCTACTGGGGCCTGGGCACCCAAGTGACCGTGAGCAGC(SEQID No.17)

[0071] Another aspect of the present invention provides an isolated polynucleotide encoding the anti-CD66e antibody.

[0072] In certain embodiments of the present invention, the sequence of the polynucleotide includes the sequence shown in SEQ ID No.15 or 17.

[0073] Another aspect of the present invention provides the use of the anti-CD66e antibody in preparing or screening therapeutic drugs, or in preparing diagnostic drugs.

[0074] The therapeutic drug may be a drug that uses the CD66e antigen as a target, binds to or acts on the CD66e antigen, and thereby treats and / or prevents the indication.

[0075] In certain embodiments of the present invention, the therapeutic drug may be a tumor therapeutic drug. The tumor is a tumor expressing CD66e. The tumor therapeutic drug may be a drug that targets the CD66e antigen on the functional surface of tumor cells, binds to or acts on the CD66e antigen, and thus treats and / or prevents tumors. The tumor may be a tumor that is positive for CD66e expression, such as colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, ovarian cancer, lung cancer, gastric cancer, gallbladder cancer, etc.

[0076] In certain embodiments of the present invention, the therapeutic agent is a chimeric antigen receptor cell.

[0077] The chimeric antigen receptor cell therapy drug generally includes a chimeric antigen receptor cell, and the chimeric antigen receptor cell 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 also includes a chimeric antigen receptor. The chimeric antigen receptor NK cell generally includes an NK cell, which also includes a chimeric antigen receptor. The chimeric antigen receptor includes a transmembrane domain, an intracellular domain, and an extracellular domain. In certain embodiments of the present invention, the extracellular domain includes the anti-CD66e antibody, that is, the chimeric antigen receptor cell can express the anti-CD66e antibody on the cell surface, thereby guiding the cell to act on cells expressing the CD66e antigen (e.g., tumor cells). The action on cells expressing the CD66e antigen can be killing cells expressing the CD66e antigen, etc.

[0078] The diagnostic drug specifically refers to a reagent that targets the target CD66e antigen and uses the CD66e antigen as a biomarker for diagnosis.

[0079] Another aspect of the present invention 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.

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

[0081] In certain embodiments of the present invention, the transmembrane domain can be selected from any one or more transmembrane domains of CD8α transmembrane region, CD28 transmembrane region, DAP 10 transmembrane region, etc.

[0082] For another example, the sequence of CD8α can be referenced to NM_001145873, the sequence of CD28 can be referenced to NM_006139, and the sequence of DAP10 can be referenced to NM_014266.

[0083] In certain embodiments of the invention, the intracellular domain may include a signal transduction domain.

[0084] The signal transduction domain includes an immunoreceptor tyrosine-based activation motif. The immunoreceptor tyrosine-based activation motif can be selected from CD3ζ.

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

[0086] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.18)

[0087] For another example, the sequence of 4-1BB can be referred to NM_001561, the sequence of CD28 can be referred to NM_006139, the sequence of OX40 can be referred to NM_003327, the sequence of ICOS can be referred to NM_012092, the sequence of CD3 zeta can be referred to NM_198053, and the sequence of DAP10 can be referred to NM_014266.

[0088] In a specific embodiment of the present invention, the intracellular domain includes 4-1BB and CD3 zeta in sequence from the N-terminus to the C-terminus.

[0089] In certain embodiments of the present invention, the extracellular domain may include a signal peptide, an anti-CD66e antibody, and a hinge region.

[0090] In certain embodiments of the present invention, the signal peptide comprises a CD8α signal peptide.

[0091] In certain embodiments of the present invention, the hinge region is selected from the CD8α hinge region.

[0092] In certain embodiments of the present invention, the polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, the anti-CD66e antibody, a transmembrane domain, and an intracellular domain.

[0093] In some specific embodiments of the present invention, the polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, an anti-CD66e nanobody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule, and CD3 zeta.

[0094] In some specific embodiments of the present invention, the polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, an anti-CD66e nanobody, a CD8α hinge region, a CD8α transmembrane region, 4-1BB, and CD3 zeta.

[0095] In certain embodiments of the present invention, the polynucleotide sequence encoding the isolated polypeptide of VHH-A062 is shown in SEQ ID No. 19:

[0096]

[0097] In certain embodiments of the present invention, the polynucleotide sequence encoding the isolated polypeptide of VHH-A108 is shown in SEQ ID No. 20:

[0098]

[0099] The present invention 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 a lentiviral vector as an example, the lentiviral vector includes a vector skeleton, i.e., an empty vector and an expression framework. That is, the nucleic acid construct is a vector containing the 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 a target cell or tissue. Typically, a vector is used to introduce exogenous DNA into another cell or tissue. The vector may contain bacterial resistance genes for growth in bacteria and promoters for expressing a target protein in an organism. DNA may be produced in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.

[0102] The term "expression frame" refers to a sequence that has the potential to encode a protein.

[0103] The present invention also provides a lentivirus, wherein the lentivirus is formed by viral packaging of the nucleic acid construct and contains the nucleic acid construct.

[0104] The present invention also provides a lentiviral vector system, characterized in that the lentiviral vector system comprises the nucleic acid construct and a helper plasmid.

[0105] Furthermore, the auxiliary plasmid encodes one or more nucleotide sequences of gag, pol, Rev, VSVg protein and other necessary viral packaging component nucleotide sequences. The auxiliary plasmids can be purchased commercially, for example, addgene item numbers 14887 and 8454.

[0106] Furthermore, the lentiviral vector system also includes a host cell, and the host cell can be a cell for producing lentivirus, for example, a mammalian cell, specifically a 293T cell.

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

[0108] Another aspect of the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the isolated polypeptide bound to a membrane.

[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 lymphocytes, B lymphocytes, NK cells, mast cells or macrophages.

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

[0112] The T lymphocytes can usually express the polypeptide, which can usually bind to the CD66e antigen, and more specifically can bind to the CD66e antigen by comprising the extracellular domain of the anti-CD66e antibody. When the polypeptide binds to the CD66e antigen, the T lymphocytes can usually be activated and / or stimulated to proliferate. In certain embodiments of the present invention, the T lymphocytes, i.e., chimeric antigen receptor T cells, can express the anti-CD66e antibody on the surface of the T lymphocytes, thereby guiding the T lymphocytes to act on cells expressing the CD66e antigen (e.g., tumor cells), and the action can be to kill cells expressing the CD66e antigen, etc.

[0113] In another specific embodiment of the present invention, the chimeric antigen receptor immune cell is a NK cell.

[0114] The NK cell can usually express the polypeptide and can usually bind to the CD66e antigen, more specifically, can bind to the CD66e antigen by comprising the extracellular domain of the anti-CD66e antibody, and when the polypeptide binds to the antigen, the NK cell can usually be activated and / or stimulated to proliferate. In certain embodiments of the present invention, the NK cell, i.e., the 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 the CD66e antigen (e.g., tumor cells), and the action can be to kill cells expressing the CD66e antigen, etc.

[0115] Another aspect of the present invention provides the use of the isolated polypeptide, isolated polynucleotide, nucleic acid construct, lentivirus, chimeric antigen receptor immune cell in the preparation or screening of therapeutic drugs, or in the preparation of diagnostic drugs.

[0116] The therapeutic or diagnostic drug may be a drug that uses the CD66e antigen as a target, binds to or acts on the CD66e antigen, and thereby treats and / or prevents the indication.

[0117] In certain embodiments of the present invention, the therapeutic drug may be a tumor therapeutic drug. The tumor therapeutic drug may be a drug that targets the CD66e antigen functionally expressed on the surface of tumor cells, binds to or acts on the CD66e antigen, and thereby treats and / or prevents tumors. The tumor may be a CD66e-positive tumor such as gastric cancer, lung cancer, pancreatic cancer, or intestinal cancer.

[0118] The present invention provides a pharmaceutical composition, which comprises 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 that the drugs do not produce adverse, allergic or other untoward reactions when they are properly administered to animals or humans.

[0121] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the effect of the drug under normal circumstances. Pharmaceutically acceptable carriers or excipients are selected from any one or a combination of at least two of surfactants, disintegrants, coating materials, excipients, solubilizers, diluents, pH regulators, binders, wetting agents, colorants, emulsifiers, antibacterial agents, cosolvents, osmotic pressure regulators, fillers, antioxidants or buffers. Specific examples may be sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose and methylcellulose; 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; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; 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 following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0123] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0124] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0125] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0126] Example 1

[0127] In this example, a phage nanobody library was constructed and panned, and ELISA was used for preliminary screening. The specific steps are as follows:

[0128] (1) Construction of phage nanoantibody library

[0129] Alpacas were immunized with human CD66e recombinant protein and cell lines overexpressing CD66e. After the serum titer was detected by ELISA, peripheral blood was drawn. Lymphocytes were separated, total RNA was extracted, and then reverse transcribed into cDNA. Then, VHH genes were amplified by nested PCR. VHH genes were inserted into PhD-dAb-1 vectors, electroporated into SS320 competent cells, and after amplification, phages were separated and purified to obtain antibody libraries. The concentration was adjusted, and the cells were aliquoted and frozen in a -80℃ refrigerator for later use.

[0130] (2) Screening of phage nanoantibody library

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

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

[0133] Example 2

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

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

[0136] Table 1

[0137]

[0138] Example 3

[0139] In this example, the anti-CD66e nanobody of Example 1 was subjected to flow cytometry.

[0140] HGC-27 and HGC-27-CD66e cells (prepared in Example 7) were mixed with purified anti-CD66e nanobodies, incubated for 30 min in an ice bath, and then incubated with APC-labeled anti-human IgG antibody for 30 min. Flow cytometry was used to detect the results. Figure 1 As shown, it is demonstrated that the anti-CD66e Nanobody of the present invention can recognize the CD66e antigen on the cell surface.

[0141] Example 4

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

[0143] First, take two clean ELISA blank plates, wash them with PBS, discard the PBS, and pat dry on absorbent paper. Dilute the Human CD66e-His (acro, cat#CE5-H5226) tag antigen to 1 μg / mL with PBS, add 100 μL of CD66e-His tag antigen to each well of one plate, seal the plate, and place it in a 4°C refrigerator overnight for coating.

[0144] After overnight, take out the coated ELISA plate from the refrigerator, discard the liquid, add 300μL PBS to each well, pour out the supernatant, and pat dry on absorbent paper. Add 300μL 1×NAP blocking solution to each well, incubate at room temperature for 1 hour, pour out the supernatant, and pat dry on absorbent paper. Dilute the primary antibody to 4μg / mL with 1×NAP, and dilute it 4 times in sequence, a total of 11 concentration gradients, 50μL per well. Add an equal volume of 1×NAP to the blank well as a control, seal the plate with a sealing film, incubate at room temperature for 1 hour, pour out the supernatant, and pat dry on absorbent paper. Add 300μL TBST to each well for washing, soak for 30s, pour out the liquid, pat dry on absorbent paper, and repeat washing 4 to 5 times. Dilute the secondary antibody (0.4μg / ml, Goat Anti-Human IgG HRP H&L, Cat#ab6858) with 1×NAP and add it to the reaction well, 50μL per well. Incubate at room temperature for 1 hour. Discard the supernatant, add 300μL TBST to each well for washing, soak for 30 seconds, pour out the liquid, pat dry on absorbent paper, and repeat washing 4 to 5 times. Then add 50μL HRP substrate to each well and incubate at room temperature in the dark for 5 to 30 minutes. Add 50μL 1N HCL to each well to terminate the substrate reaction. Measure the absorbance at 450nm with an enzyme reader.

[0145] The results are as follows Figure 2 As shown, the EC50 of CD66e nanobody VHH-A062 binding to CD66e protein is 0.0099 μg / mL, and the EC50 of CD66e nanobody VHH-A108 binding to CD66e protein is 0.0444 μg / mL, both of which are nanomolar level, indicating that they can bind to CD66e protein well.

[0146] Example 5

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

[0148] First, construct a lentiviral vector HD SIN03 anti-CD66eVHH-41BBz carrying a CD66e CAR chimeric antigen receptor. The vector map is as follows Figure 3 A schematic diagram of the chimeric antigen receptor is shown in Figure 4 As shown, it includes CD8α signal peptide, anti-CD66e nanobody (anti-CD66e VHH), CD8α hinge region, transmembrane region, co-stimulatory molecules and immunoreceptor tyrosine activation motif (CD3ζ).

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

[0150] MALPVTALLLPLALLLHAARP.

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

[0152] The amino acid sequence of the hinge region and transmembrane region of CD8α (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 CD3ζ (SEQ ID NO.23) is:

[0157] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0158] The specific preparation method is as follows:

[0159] (1) Prepare the PCR reaction system according to Table 2 (the reagents in the table are from TOYOBO Inc.), amplify each anti-CD66e nanobody fragment, and react according to the PCR program shown in Table 3. The primer sequences are:

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

[0161] CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGCAAGTGCAGCTGGTGGAATCCEACAM5-A108-F (SEQ ID NO.25):

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

[0163] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACTTGG.

[0164] Table 2

[0165] Reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO 4 ]]> 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 products were subjected to 1% agarose gel electrophoresis, and fragments of about 400 bp were recovered and quantified by UV absorption;

[0169] (2) Prepare the PCR reaction system according to Table 4, add the CD8α signal peptide before the amplified product obtained in step (1), and perform the reaction according to the PCR program shown in Table 3. The primers are:

[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] Reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 25mM 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 products were subjected to 1% agarose gel electrophoresis, and the fragment of about 490 bp was recovered and quantified by UV absorption method;

[0177] (3) Prepare a PCR reaction system according to Table 5. After the preparation, perform a PCR reaction according to Table 3 to amplify the CD8αhinge-TM-41BB-CD3Z fragment. The primers used are as follows:

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

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

[0180] Table 5

[0181] Reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 25mM MgSO4 3 10 μM upstream primer CD8αH-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 to recover the fragment of about 680 bp and quantify it by UV absorption method;

[0183] (4) 25 μg of the HD SIN03 GUCY2C A3-41BBz plasmid constructed in the laboratory was double-digested with BamHI and EcoRI, reacted in a 37°C water bath for 1 h, and then the vector was recovered;

[0184] The two fragments recovered above and the vector backbone were connected with the recombinase. The recombination reaction system was shown in Table 6. After the preparation, the reaction was carried out in a water bath at 37° C. for 0.5 h and transformed into Escherichia coli Stbl3 competent cells according to the conventional method.

[0185] Table 6

[0186] Reagents Usage HD CD66e CAR backbone 154.2ng CD8αsingal CD66e VHH 10ng CD8αhinge-TM-41BB-CD3Z 13.6ng 5×CE Buffer 2.4μL Exnase MultiS 1.2μL Sterile deionized water (PCR grade water) Make up to 12 μL

[0187] Single clones were selected from the solid culture medium for sequencing and identification, and the results were consistent with expectations. 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 lentiviral vector HD SIN03 anti-CD66e VHH-41BBz prepared in Example 5 was packaged, concentrated and titered, including the following steps:

[0192] (1) Lentiviral packaging

[0193] 293T cells of appropriate concentration were inoculated into a 15 cm culture dish and incubated at 37°C and 5% CO. 2 Culture overnight to prepare for virus packaging, the culture medium is DMEM containing 10% fetal bovine serum; dissolve 14.5 μg HD SIN03 anti-CD66eVHH-41BBz, 16.7 μg auxiliary plasmid pMDLg-RRE (Addgene, 12251), 16.7 μg auxiliary plasmid pRSV-REV (Addgene, 12253) and 6.5 μg envelope plasmid VSVg (Addgene, 8454) in 2 mL serum-free DMEM culture medium and mix well;

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

[0195] (2) Lentiviral titer detection

[0196] 500 μL Jurkat (1×10 5 The cells were inoculated into 24-well culture plates, and the collected virus supernatant was added to the cell suspension in volumes of 1 μL, 5 μL, and 25 μL, respectively. Polybrene was added to a final concentration of 8 μg / mL and the culture medium was incubated at 37°C and 5% CO. 2 After 72 hours of culture, centrifuge at 500g for 5 minutes, discard the supernatant to collect the cells, add 100μL PBS+2% FBS to resuspend the cells, add 0.5μg of Rabbit Anti-Camelid VHH (iFluor488) antibody, and incubate on ice for 30 minutes; after washing once with flow buffer (PBS containing 2% FBS), add 300μL flow buffer to resuspend the cells, and use flow cytometry to detect infection efficiency; calculate the titer, and the titer calculation formula is as follows: titer (TU / mL) = number of cells × positive rate / virus volume (mL). According to the titer formula, the VHH-A062 virus titer is 1.37E+07TU / ml; the VHH-A108 virus titer is 6.48E+06TU / ml.

[0197] Example 7 Construction of overexpression cell lines

[0198] The lentivirus overexpressing CD66e was obtained by co-transfection of CD66e-puro plasmid, pMGlg-RRE, pRSV-REV and VSVg plasmid, and the steps were the same as those in Example 6;

[0199] Take 1×10 6 HGC-27 cells and N87 cells were seeded into 6-well plates, and 1 mL of the CD66e-puro lentivirus obtained above was added to obtain HGC-27-CD66e and N87-CD66e overexpressing CD66e protein.

[0200] Example 8

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

[0202] (1) Human PBMCs were adjusted to a density of 1×10 using T cell culture medium (X-VIVO + 10% FBS + 300 U / mL IL-2). 6 / mL, add 1 / 100 volume of T Cell TransAct (commercial magnetic beads coupled to CD3 and CD28) and activate for 24h;

[0203] (2) Collect activated T cells and adjust the cell density to 1×10 6 / mL, add lentivirus at a multiplicity of infection (MOI) of 10, add polybrene to a final concentration of 8 μg / mL; incubate at 37°C, 5% CO 2 After overnight culture in the environment, fresh medium was replaced and subcultured every 3 days;

[0204] (3) 8 days after T cell infection, 3×10 5 T cells were centrifuged at 4°C and 500 g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to resuspend the cells, 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody was added, and the cells were incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells;

[0205] Flow cytometry was used to detect the expression rate of chimeric antigen receptors on T lymphocytes. Figure 5 As shown, 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, the in vitro toxicity experiment of CAR-T cells 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 + ) as target cells and adjust the target cell concentration to 2×10 5 / mL, take 50μL and inoculate into a white opaque 96-well plate;

[0210] (2) Effector cell inoculation

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

[0212] (3) Each group has two replicate wells, and each experimental group and each control group are as follows:

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

[0214] (5) Detection method:

[0215] After the effector cells and target cells were co-cultured for 18 h, 70 μL of Steady-Lumi TM Firefly luciferase reporter gene detection reagent (Biyuntian, catalog number: RG058M) was used for reaction for 5 min, and the bioluminescent signal was detected using a multifunctional microplate reader.

[0216] (5) The formula for calculating the killing efficiency of CAR-T cells is: killing efficiency % = (1-experimental group / control group) × 100%.

[0217] The results are as follows Figures 6 to 8 As shown, the CAR-T cells constructed by the present invention have no killing effect on CD66e-negative 293T cells, but have killing activity on CD66e-positive tumor cells, indicating that the CAR-T cells constructed by the present invention not only have efficient tumor killing ability, but also have high specificity.

[0218] Example 10

[0219] In this example, the secretion of CAR-T cell factor IFN-γ was detected, and the kits used were Human IFN-γ ELISA Kit (Lianke Bio, catalog number: EK180-96).

[0220] 1. Cell culture supernatant

[0221] The cell culture with an effector-target ratio of 1:1 was centrifuged at 400 g for 10 min to remove the precipitate, and the supernatant was stored at -80°C for testing.

[0222] 2. Reagent preparation

[0223] Before testing, restore all reagents and samples to 25°C. If concentrated reagents are crystallized, incubate at 37°C until all crystals are dissolved. Prepare 1× washing solution and 1× detection buffer according to the instructions.

[0224] 3. Preparation of standards and samples

[0225] Standards: Use 5% FBS1640 medium to dilute the standard stock solution 2-fold, with a total of 8 dilution gradients, including zero concentration.

[0226] Sample: Dilute the sample using 5% FBS1640 medium.

[0227] 4. Detection steps

[0228] (1) Soaking the microplate: Add 300 μL of 1× washing solution and let it soak for 30 seconds. Discard the washing solution and pat the microplate dry on absorbent paper.

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

[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) Incubation: Seal the plate with a sealing film, shake at 300 rpm, and incubate at 25°C for 2 h;

[0233] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well, wash 6 times, and pat dry on absorbent paper after each wash;

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

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

[0236] (9) Add substrate for color development: Add 100 μL of color development substrate TMB to each well, protect from 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) Detection reading: Within 30 min, dual-wavelength detection was performed using an ELISA reader to measure the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength. The calibrated OD value was the measured value at 450 nm minus the measured value at the reference wavelength.

[0239] The results of IFN-γ secretion were Fig. 9As shown, the spontaneous MOCK group is a single CAR-T cell group, and cytokine release is basically undetectable; the CAR-T cell and 293T cell co-culture group also basically cannot detect cytokines; after co-culture with CD66e-positive target cells, the IFN-γ secreted by CAR-T cells exceeds 500pg / mL. The CAR-T cells constructed by the present invention release cytokines to CD66e-positive tumor cells, but have no obvious cytokine secretion to CD66e-negative cells.

[0240] In summary, the anti-CD66e nanoantibodies with high affinity screened and prepared by the present invention can efficiently and specifically bind to CD66e, and are used as antigen binding domains to construct chimeric antigen receptors and CAR-T cells. The resulting CAR-T cells have obvious killing activity and specificity for CD66e-positive tumor cells, and can secrete tumor-killing cytokines, indicating that the nanoantibodies of the present invention can be effectively used in immunotherapy, which is of great significance for the development of tumor treatment drugs.

[0241] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. An anti-CD66e antibody, characterized in that The anti-CD66e antibody comprises a heavy chain variable region, and the heavy chain variable region of the anti-CD66e antibody has the following technical features: <1> comprising a CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1 or 2; <2> comprising a CDR-H2 having an amino acid sequence as shown in SEQ ID No. 3 or 4; <3> It includes CDR-H3 with an amino acid sequence as shown in SEQ ID No. 5 or 6.

2. The anti-CD66e antibody according to claim 1, characterized in that The heavy chain variable region of the anti-CEACAM5CD66e antibody includes CDR-H1 as shown in SEQ ID No.1, CDR-H2 as shown in SEQ ID No.23, and CDR-H3 as shown in SEQ ID No.35; or, the heavy chain variable region of the anti-CD66e antibody includes CDR-H1 as shown in SEQ ID No.2, CDR-H2 as shown in SEQ ID No.4, and CDR-H3 as shown in SEQ ID No.

6.

3. The anti-CD66e antibody according to claim 1, characterized in that The heavy chain variable region also includes a framework region, and the framework region includes FR1 to FR4. The amino acid sequence of FR1 to FR4 is any sequence shown in SEQ ID No. 7 to 13.

4. 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 or 16.

5. The anti-CD66e antibody according to claim 1, characterized in that The anti-CD66e antibody is a nanobody.

6. An isolated polypeptide, characterized in that The polypeptide comprises a transmembrane domain, an intracellular domain and an extracellular domain, and the extracellular domain comprises the anti-CD66e antibody according to any one of claims 1 to 5.

7. The polypeptide according to claim 6, characterized in that Also includes any one or more of the following features: a) the polypeptide is a chimeric antigen receptor; b) the transmembrane domain is selected from any one or more of the CD8α transmembrane region, the CD28 transmembrane region, and the DAP 10 transmembrane region; c) the intracellular domain includes a signal transduction domain; preferably, the signal transduction domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory molecule; more preferably, the immunoreceptor tyrosine-based activation motif is selected from CD3ζ; preferably, the co-stimulatory molecule is selected from any one of 4-1BB, CD28, OX40, ICOS, and DAP 10, or a combination of at least two thereof; d) the extracellular domain comprises a signal peptide, an anti-CD66e antibody, and a hinge region; preferably, the signal peptide comprises a CD8α signal peptide; preferably, the hinge region is selected from a CD8α hinge region; e) The polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, an anti-CD66e antibody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule, and a CD3 zeta signal domain.

8. An isolated polynucleotide, characterized in that Encoding the anti-CD66e antibody according to any one of claims 1 to 5 or the polypeptide according to any one of claims 6 to 7.

9. A nucleic acid construct, characterized in that Contains a polynucleotide encoding the isolated protein of claim 8.

10. The nucleic acid construct according to claim 9, characterized in that 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 formed by viral packaging of the nucleic acid construct according to 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, characterized in that: The chimeric antigen receptor immune cell expresses the membrane-bound isolated polypeptide according to any one of claims 6 to 7.

14. The chimeric antigen receptor immune cell according to claim 13, characterized in that The immune cell is selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells or macrophages.

15. Use of the anti-CD66e antibody according to any one of claims 1 to 5, the isolated polypeptide according to any one of claims 6 to 7, or the chimeric antigen receptor immune cell according to any one of claims 13 to 14 in the preparation or screening of tumor therapeutic drugs.

16. The use according to claim 15, characterized in that The tumor is a tumor expressing CD66e; preferably, the tumor is selected from liver cancer, colorectal cancer, pancreatic cancer, melanoma, breast cancer, bladder cancer, ovarian cancer, lung cancer, thyroid cancer, gastric cancer or gallbladder cancer.

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