An antibody against ceacam5 and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to an anti-CEACAM5 antibody and application thereof, the anti-CEACAM5 antibody has the following technical features: the heavy chain variable region includes CDR-H1 with the amino acid sequence shown in SEQ ID No.1, CDR-H2 with the amino acid sequence shown in SEQ ID No.2 and CDR-H3 with the amino acid sequence shown in SEQ ID No.3.The anti-CEACAM5 antibody has high 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 on tumor cells positive for CEACAM5, and after co-culturing with CEACAM5-positive cells, the CAR-T cell can efficiently secrete the cytokine IFN-gamma, the nanobody can be effectively applied to immunotherapy, and has important significance for developing tumor treatment drugs.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an anti-CEACAM5 antibody and its uses. Background Technology

[0002] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CEA or CD66e, is the first member of the CEACAM family. CEACAM5 was initially discovered in colon cancer and embryonic tissues (see: Gold P, Freedman SO. Specific carcinoembryonic antigens of the human digestive system. J Exp Med. 1965 Sep 1; 122(3):467-81.), and was later found to be widely present in digestive system tumors of endodermal origin such as gastric cancer, liver cancer, pancreatic cancer, and colon cancer. It is also elevated in the serum of breast cancer, lung cancer, and other malignant tumors, making it a broad-spectrum tumor marker (see: Han ZW, Lyv ZW, Cui B, et al. The old CEACAMs find their new role in tumor immunotherapy. Invest New Drugs. 2020 Dec; 38(6):1888-1898.).

[0003] CEACAM5 belongs to the immunoglobulin (Ig) superfamily and binds to the membrane via glycosylphosphatidylinositol (GPI). It has seven extracellular immunoglobulin-like domains, including one variable IgV-like domain (N domain) and six IgC-like domains. Additionally, a 34-amino acid signal peptide precedes the Ig domain. The human CEACAM5 gene is located on chromosome 19q13.2, is approximately 21 kb in length, and consists of nine exons and three non-coding exons. The CEACAM5 protein is composed of 642 amino acids (approximately 70 kDa) and has 28 potential N-linked glycosylation sites. A single CEACAM5 protein molecule contains 24-26 asparagine-linked sugar chains, with a final molecular weight of approximately 180 kDa (see: Beauchemin N, Arabzadeh A. Carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) in cancer progression and metastasis. Cancer Metastasis Rev. 2013 Dec; 32(3-4):643-71.).

[0004] CEACAM5 expression begins in the early stages of embryonic and fetal development (9-14 weeks) and persists in certain cells. In normal adult tissues, CEACAM5 is primarily found in the columnar epithelium and goblet cells of the colon, especially in the upper third of the crypts and on the surface of the free lumen. It is also present in the stomach, tongue, esophagus, cervix, sweat glands, and prostate (see: S. The carcinoembryonic antigen (CEA) family: structures, suggested functions and expression in normal and malignant tissues. Semin Cancer Biol. 1999 Apr; 9(2):67-81.). Interestingly, CEACAM5 expression in normal lung and gastrointestinal epithelial cells is limited to the apical surface of the epithelial cell membrane facing the lumen, making it unrecognizable by immune cells. When tumors develop, CEACAM5 in luminal epithelial cells loses its apical polarity, thereby entering capillaries and increasing serum soluble CEACAM5 levels (see: Han ZW, Lyv ZW, Cui B, et al. The old CEACAMs find their new role in tumor immunotherapy. Invest New Drugs. 2020 Dec; 38(6):1888-1898.). Therefore, CEACAM5's unique localization pattern makes it a potential target for related antigens in cancer immunotherapy.

[0005] Adoptive immunotherapy is considered a promising anti-tumor therapy. CAR-T cells express CAR transgenes and bind to tumor antigens through antigen-antibody recognition, thereby eliminating cancer cells. These CAR-T cells can directly recognize tumor cells, unaffected by antigen processing and MHC limitations. Currently, CAR-T therapy has shown promising results in hematologic malignancies, and researchers are conducting further studies in solid tumors by identifying various tumor-associated antigens.

[0006] Due to its high expression and unique expression pattern in various tumor cells, CEACAM5 has become a popular target. A Phase I clinical trial report on CEACAM5+ colorectal cancer (NCT02349724) indicated that intravenous infusion of CEACAM5 CAR-T into patients with metastatic, recurrent, and refractory colorectal cancer was well tolerated by patients, even at high doses, and researchers observed certain therapeutic effects in most patients (see: Zhang C, Wang Z, Yang Z, et al. Phase I Escalating-Dose Trial of CAR-T Therapy Targeting CEA+Metastatic Colorectal Cancers. Mol Ther. 2017 May 3; 25(5):1248-1258.). Furthermore, Steven C. Katz et al. treated six patients with CEACAM5+ liver metastases (NCT01373047) via hepatic artery infusion of CEACAM5 CAR-T. The results showed that these patients tolerated the treatment well and did not experience severe cytokine release syndrome or neurotoxicity (see: Katz SC, Hardaway J, Prince E, et al. HITM-SIR: phase Ib trial of intraarterial chimeric antigen receptor T-cell therapy and selective internal radiation therapy for CEA+ liver metastases. Cancer Gene Ther. 2020 May; 27(5):341-355.). These results support the view that CEACAM5, as an autoantigen with a strictly luminal expression pattern, can be safely targeted by CAR-T cell therapy, thereby producing a certain therapeutic effect on solid tumors.

[0007] In conclusion, CEACAM5 can serve as a target for tumor therapy. Providing an anti-CEACAM5 antibody with high specificity and affinity is of great significance for the treatment of malignant tumors. Summary of the Invention

[0008] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide an antibody against CEACAM5 and its uses, in order to solve the problems in the prior art.

[0009] To achieve the above and other related objectives, the present invention provides an anti-CEACAM5 antibody, characterized in that the anti-CEACAM5 antibody includes a heavy chain variable region, and the anti-CEACAM5 antibody has one or more of the following technical features;

[0010] <1> The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1;

[0011] <2> The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2;

[0012] <3> The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3.

[0013] The present invention also provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises the aforementioned anti-CEACAM5 antibody.

[0014] The present invention also provides an isolated polynucleotide encoding the anti-CEACAM5 antibody or the polypeptide.

[0015] The present invention also provides a nucleic acid construct containing encoding the isolated polynucleotides described above.

[0016] The present invention also provides a lentivirus, which is formed by viral packaging of the aforementioned nucleic acid construct.

[0017] The present invention also provides a lentiviral vector system, which includes the aforementioned nucleic acid construct and helper plasmids or host cells.

[0018] The present invention also provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the isolated polypeptide that is bound to the membrane.

[0019] As described above, the anti-CEACAM5 antibody and its use according to the present invention have the following beneficial effects:

[0020] (1) In this invention, a phage display nanobody library was constructed by immunizing unimmunized alpacas with recombinant CEACAM5 protein and cells overexpressing CEACAM5. Antibodies against CEACAM5 were screened based on the phage display nanobody library. The obtained nanobodies can specifically bind to CEACAM5 antigen and have good affinity.

[0021] (2) The anti-CEACAM5 nanobody provided by the present invention has high affinity. It is used as an antigen-binding domain to construct a chimeric antigen receptor, and CAR-T cells are prepared using the chimeric antigen receptor. The CAR-T cells have killing activity against CEACAM5 positive tumor cells, and after co-culturing with CEACAM5 positive cells, they efficiently secrete the cytokine IFN-γ. The nanobody can be effectively applied to immunotherapy and is of great significance for the development of tumor therapeutic drugs. Attached Figure Description

[0022] Figure 1 This is a graph showing the FACS detection results of the anti-CEACAM5 nanobody recognizing the CEACAM5 antigen in Example 3;

[0023] Figure 2 This is a graph showing the EC50 detection results of the anti-CEACAM5 nanobody binding to the CEACAM5 antigen in Example 4;

[0024] Figure 3 This is a map of the chimeric antigen receptor lentiviral vector plasmid targeting CEACAM5 in Example 5;

[0025] Figure 4 This is a schematic diagram of the chimeric antigen receptor expressing CEACAM5 in Example 5;

[0026] Figure 5 This is a flow cytometry result of the chimeric antigen receptor expression rate of T lymphocytes in Example 8;

[0027] Figure 6 This is a graph showing the killing effect of CAR-T cells on CEACAM5-negative human embryonic kidney cells 293T in Example 9;

[0028] Figure 7 The graph shows the killing effect of CAR-T cells on CEACAM5-positive human gastric cancer cells N87-CEACAM5 in Example 9.

[0029] Figure 8 The graph shows the killing effect of CAR-T cells on CEACAM5-positive human orthotopic pancreatic adenocarcinoma cells BxPC3 in Example 9.

[0030] Figure 9 This is a bar chart showing the secretion of IFN-γ by CAR-T cells in Example 10. Detailed Implementation

[0031] This invention provides an anti-CEACAM5 antibody A031, the anti-CEACAM5 antibody comprising a heavy chain variable region, and the anti-CEACAM5 antibody having one or more of the following technical features;

[0032] <1> The heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1;

[0033] <2> The heavy chain variable region includes the amino acid sequence CDR-H2 as shown in SEQ ID No. 2;

[0034] <3> The heavy chain variable region includes the amino acid sequence CDR-H3 as shown in SEQ ID No. 3;

[0035] GRTFSNYAYA (SEQ ID No. 1)

[0036] ISRSGDST (SEQ ID No. 2)

[0037] AAVLTDYERAYYRPTEYKY (SEQ ID No. 3).

[0038] The numbering and definition scheme is to use the IMGT method to label CDR and FR areas.

[0039] A CDR (complementarity determining region) generally refers to a region in an antibody that is spatially complementary to the antigenic determinant. The variability in an antibody is usually not uniformly distributed throughout its variable region. The heavy chain variable region of a monoclonal antibody typically has three hypervariable regions (HVRs). These regions are often spatially complementary to the antigenic determinant, hence the term complementarity determining region (CDR). In other words, the heavy chain variable region typically includes three CDRs: HCDR1, HCDR2, and HCDR3.

[0040] In some embodiments of the present invention, the complementarity-determining region of the heavy chain variable region of the anti-CEACAM5 antibody includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 1, the amino acid sequence CDR-H2 as shown in SEQ ID No. 2, and the amino acid sequence CDR-H3 as shown in SEQ ID No. 3.

[0041] The anti-CEACAM5 antibody is an antibody fragment and / or a monoclonal antibody.

[0042] An "antibody fragment" comprises a portion of a complete antibody, preferably including 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.

[0043] In this invention, an unimmunized alpaca was immunized with recombinant CEACAM5 protein and a cell line overexpressing CEACAM5 to construct a phage display nanobody library. Based on this phage display nanobody library, CEACAM5 antibodies were screened to obtain monoclonal antibodies that can specifically bind to the CEACAM5 antigen.

[0044] In some embodiments of this invention, the anti-CEACAM5 antibody is a nanobody (Nb), specifically a heavy-chain single-domain antibody (VHH, variable domain of heavy chain). A nanobody contains only one heavy-chain variable region (VHH) and CH2 and CH3 regions; compared to other antibodies, the light chain is naturally absent in nanobodies. The nanobody crystal has a diameter of approximately 2.5 nm and a length of approximately 4 nm, representing the smallest naturally occurring fragment capable of binding to the antigen. The anti-CEACAM5 antibody of this invention comprises only the heavy-chain variable region, possessing high affinity and specificity, enabling efficient targeting of the CEACAM5 antigen. It has a simple structure, is easy to prepare, and has significant application value in the field of drug development targeting CEACAM5.

[0045] In some embodiments of the present invention, the heavy chain variable region may further include a framework region, which may be located between complementarity-determining regions or at both ends of the complementarity-determining regions. In some specific embodiments of the present invention, the framework region sequence is a human monoclonal antibody variable region or a mouse monoclonal antibody variable region framework region sequence obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0046] In some embodiments of the present invention, the heavy chain variable region further includes a framework region. The framework region includes framework regions FR1 to FR4. The amino acid sequences of the framework regions FR1 to FR4 are selected from any of the sequences shown in SEQ ID No. 4 to 7.

[0047] Preferably, the amino acid sequence of FR1 is as shown in SEQ ID No. 4: EVQVQESGGGLVQAGDSLRLSCAAS (SEQ ID No. 4).

[0048] Preferably, the amino acid sequence of FR2 is as shown in SEQ ID No. 5: MGWFRQAPGKEREFVGA (SEQ ID No. 5)

[0049] Preferably, the amino acid sequence of FR3 is as shown in SEQ ID No. 6: YFADFVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYC (SEQ ID No. 6)

[0050] Preferably, the amino acid sequence of FR4 is as shown in SEQ ID No. 7: WGQGTQVTVSS (SEQ ID No. 7)

[0051] In some embodiments of the present invention, the anti-CEACAM5 antibody is designated as VHH-A031 or anti-CEACAM5-A031, and the variable region of the heavy chain of the anti-CEACAM5 antibody includes amino acid sequences as shown in SEQ ID No. 1 (CDR-H1), amino acid sequences as shown in SEQ ID No. 2 (CDR-H2), amino acid sequences as shown in SEQ ID No. 3 (CDR-H3), and amino acid sequences of the frame regions FR1 to FR4 as shown in SEQ ID No. 4 to 7.

[0052] Preferably, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 8.

[0053] EVQVQESGGGLVQAGDSLRLSCAASGRTFSNYAYAMGWFRQAPGKEREFVGAISRSG DSTYFADFVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCAAVLTDYERAYYRPTEYKY WGQGTQVTVSS(SEQ ID No.8)

[0054] In some embodiments of the present invention, the anti-CEACAM5 antibody is obtained from a phage antibody library through screening, and its heavy chain variable region nucleotide sequence is shown in SEQ ID No. 9:

[0055] GAGGTCCAAGTGCAAGAGAGCGGGGGGGCCTGGTCCAAGCTGGCGATAGCCTGAGACTGAGCTGCGCCGCTAGCGGCAGAACCTTCAGCAACTACGCCTACGCCATGGGCTGGTTCAGACAAGCCCCCGGCAAGGAGAGAGTTCGTGGGCGCCATCAGCAGAAGCGGCGACAGCACCTACTTCGCCGACTT CGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAGCACCGTGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGTGCTGACCGACTACGAGAGAGCCTACTACAGACCCACCGAGTACAAGTACTGGGGCCAAGGCACCCAAGTGACCGTGAGCAGC(SEQ ID No.9)

[0056] In another aspect, the present invention provides an isolated polynucleotide encoding the anti-CEACAM5 antibody.

[0057] In some embodiments of the present invention, the sequence of the polynucleotide is shown in SEQ ID No. 9.

[0058] Another aspect of the present invention provides the use of the anti-CEACAM5 antibody in the preparation or screening of therapeutic drugs, or in the preparation of diagnostic drugs.

[0059] The therapeutic agent may be an agent that targets the CEACAM5 antigen, binds to or acts on the CEACAM5 antigen, thereby treating and / or preventing the indication.

[0060] In some embodiments of the present invention, the therapeutic drug may be an antitumor drug. The tumor is a tumor expressing CEACAM5. The antitumor drug may be a drug that targets the CEACAM5 antigen on the functional surface of tumor cells, binding to or acting on the CEACAM5 antigen to treat and / or prevent tumors. The tumor may be a CEACAM5-positive tumor such as colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, ovarian cancer, lung cancer, gastric cancer, or gallbladder cancer.

[0061] In some embodiments of the present invention, the therapeutic agent is a chimeric antigen receptor cell.

[0062] The chimeric antigen receptor cell therapy typically includes chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, etc. The chimeric antigen receptor T cells typically include T lymphocytes, which also include chimeric antigen receptors. The chimeric antigen receptor NK cells typically include NK cells, which also include chimeric antigen receptors. The chimeric antigen receptor includes a transmembrane domain, an intracellular domain, and an extracellular domain. In some embodiments of the present invention, the extracellular domain includes the anti-CEACAM5 antibody, meaning that the chimeric antigen receptor cells can express the anti-CEACAM5 antibody on their cell surface, thereby guiding the cells to act on cells expressing CEACAM5 antigen (e.g., tumor cells). The action on cells expressing CEACAM5 antigen can include killing cells expressing CEACAM5 antigen, etc.

[0063] The diagnostic drug specifically refers to a reagent that targets the CEACAM5 antigen and uses the CEACAM5 antigen as a biomarker for diagnosis.

[0064] Another aspect of the present invention provides an isolated polypeptide comprising a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain comprises the anti-CEACAM5 antibody.

[0065] In some embodiments of the present invention, the polypeptide is a chimeric antigen receptor. In this invention, a chimeric antigen receptor is constructed using the anti-CEACAM5 antibody, and this chimeric antigen receptor can efficiently target CEACAM5.

[0066] In some embodiments of the present invention, the transmembrane domain may be selected from any one or more transmembrane structural domains such as CD8α transmembrane region, CD28 transmembrane region, and DAP 10 transmembrane region.

[0067] For example, the sequence of CD8α can be found in NM_001145873, the sequence of CD28 can be found in NM_006139, and the sequence of DAP10 can be found in NM_014266.

[0068] In some embodiments of the present invention, the intracellular domain may include a signal transduction domain.

[0069] The signal transduction domain includes an immune receptor tyrosine activation motif. This immune receptor tyrosine activation motif may be selected from CD3ζ.

[0070] Preferably, the signal transduction domain further includes a co-stimulatory molecule. For example, the co-stimulatory molecule may be selected from any one or a combination of at least two protein molecules such as 4-1BB, CD28, OX40, ICOS, and DAP 10. As another example, the amino acid sequence of 4-1BB may include the following:

[0071] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID No.12)

[0072] For example, the sequence of 4-1BB can be found in NM_001561, the sequence of CD28 in NM_006139, the sequence of OX40 in NM_003327, the sequence of ICOS in NM_012092, the sequence of CD3 zeta in NM_198053, and the sequence of DAP10 in NM_014266.

[0073] In one specific embodiment of the present invention, the intracellular domain includes 4-1BB and CD3 zeta sequentially from the N-terminus to the C-terminus.

[0074] In some embodiments of the present invention, the extracellular domain may include a signal peptide, an anti-CEACAM5 antibody, or a hinge region.

[0075] In some embodiments of the present invention, the signal peptide includes the CD8α signal peptide.

[0076] In some embodiments of the present invention, the hinge region is selected from the CD8α hinge region.

[0077] In some embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the anti-CEACAM5 antibody, a transmembrane domain, and an intracellular domain.

[0078] In some specific embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an anti-CEACAM5 nanobody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule, and CD3 zeta.

[0079] In some specific embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, an anti-CEACAM5 nanobody, a CD8α hinge region, a CD8α transmembrane region, 4-1BB, and CD3 zeta.

[0080] In certain embodiments of the present invention, the polynucleotide sequence encoding the isolated polypeptide is shown in SEQ ID No. 21:

[0081]

[0082] The present invention also provides a nucleic acid construct containing a polynucleotide encoding the isolated polypeptide.

[0083] The nucleic acid construct can be a lentiviral vector, a retroviral vector, or an adeno-associated virus vector. Taking a lentiviral vector as an example, a lentiviral vector includes a vector backbone, 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.

[0084] The terms "nucleic acid construct" or "vector" refer 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. A vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expression of a target protein in an organism. The DNA can be produced in vitro by PCR or one or more suitable techniques known to those skilled in the art.

[0085] The term "expression frame" refers to a sequence that has the potential to encode a protein.

[0086] The present invention also provides a lentivirus, which is formed by viral packaging of the nucleic acid construct. The lentivirus contains the nucleic acid construct.

[0087] The present invention also provides a lentiviral vector system, characterized in that the lentiviral vector system includes the aforementioned nucleic acid construct and auxiliary plasmid.

[0088] Furthermore, the helper plasmid encodes one or more nucleotide sequences of the gag, pol, Rev, and VSVg proteins, as well as nucleotide sequences of other essential viral packaging components. These helper plasmids are commercially available, for example, Addgene catalog numbers 14887 and 8454.

[0089] Furthermore, the lentiviral vector system also includes a host cell, which can be a cell that produces lentiviruses, such as a mammalian cell, specifically a 293T cell.

[0090] The lentivirus can be obtained by transfecting host cells using the nucleic acid construct and helper plasmid in the lentiviral vector system. The host cell can be a mammalian cell.

[0091] Another aspect of the present invention provides a chimeric antigen receptor immune cell, wherein the chimeric antigen receptor immune cell expresses the isolated polypeptide that is bound to a membrane.

[0092] Preferably, the chimeric antigen receptor immune cell comprises the nucleic acid construct and / or the lentivirus.

[0093] The immune cells are selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells, or macrophages.

[0094] In another specific embodiment of the present invention, the chimeric antigen receptor immune cell is a T lymphocyte.

[0095] The T lymphocytes typically express the polypeptide, which can bind to the CEACAM5 antigen. More specifically, it can bind to the CEACAM5 antigen through an extracellular domain containing the anti-CEACAM5 antibody. When the polypeptide binds to the CEACAM5 antigen, the T lymphocytes can typically be activated and / or stimulated to proliferate. In some embodiments of the present invention, the T lymphocytes, i.e., chimeric antigen receptor T cells, can express the anti-CEACAM5 antibody on their surface, thereby guiding the T lymphocytes to act on cells expressing the CEACAM5 antigen (e.g., tumor cells). This action can include killing cells expressing the CEACAM5 antigen, etc.

[0096] In another specific embodiment of the present invention, the chimeric antigen receptor immune cell is an NK cell.

[0097] The NK cells typically express the polypeptide and can bind to the CEACAM5 antigen, more specifically, through an extracellular domain containing the anti-CEACAM5 antibody. When the polypeptide binds to the antigen, the NK cells are typically activated and / or stimulated to proliferate. In some embodiments of the invention, the NK cells, i.e., chimeric antigen receptor NK cells, can express the anti-CEACAM5 antibody on their surface, thereby guiding the NK cells to act on cells expressing the CEACAM5 antigen (e.g., tumor cells), such action as killing cells expressing the CEACAM5 antigen.

[0098] Another aspect of the present invention provides the use of the isolated polypeptides, isolated polynucleotides, nucleic acid constructs, lentiviruses, and chimeric antigen receptor immune cells in the preparation or screening of therapeutic drugs, or in the preparation of diagnostic drugs.

[0099] The therapeutic or diagnostic drug may be a drug that targets the CEACAM5 antigen, binds to or acts on the CEACAM5 antigen, and thereby treats and / or prevents indications.

[0100] In some embodiments of the present invention, the therapeutic drug may be an antitumor drug. The antitumor drug may be a drug that targets the CEACAM5 antigen functionally expressed on the surface of tumor cells, binding to or acting on the CEACAM5 antigen to treat and / or prevent tumors. The tumor may be a CEACAM5-positive tumor such as gastric cancer, lung cancer, pancreatic cancer, or colorectal cancer.

[0101] The present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cells and / or the anti-CEACAM5 antibody and / or the isolated polypeptide.

[0102] Preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier or excipient.

[0103] "Pharmaceutical acceptable" means that when a drug is properly administered to animals or humans, it will not produce adverse, allergic, or other adverse reactions.

[0104] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, meaning they can be miscible with it without significantly reducing the efficacy of the drug under normal circumstances. Pharmaceutically acceptable carriers or excipients are selected from any one or a combination of at least two of the following: surfactants, disintegrants, coating materials, excipients, solubilizers, diluents, pH adjusters, binders, wetting agents, colorants, emulsifiers, antibacterial agents, cosolvents, osmotic pressure regulators, fillers, antioxidants, or buffers. 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 methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum 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, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or aroma when taken orally.

[0105] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0106] Before further describing 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 terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0107] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0108] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0109] Example 1

[0110] This embodiment constructs and pans a phage nanobody library, and uses ELISA for preliminary screening. The specific steps are as follows:

[0111] (1) Construction of phage nanobody library

[0112] Alpacas were immunized with recombinant human CEACAM5 protein and a cell line overexpressing CEACAM5. After serum titers were detected by ELISA, peripheral blood was collected. Lymphocytes were isolated, total RNA was extracted, and then reverse transcribed into cDNA. The VHH gene was then amplified by nested PCR. The VHH gene was inserted into the PhD-dAb-1 vector, electroporated into SS320 competent cells, and amplified. Phages were isolated and purified to obtain an antibody library. The concentration was adjusted, aliquoted, and stored at -80°C for later use.

[0113] (2) Screening of phage nanobody libraries

[0114] First, the phage antibody library was screened in two rounds, one in liquid phase and one in solid phase. Then, the screened antibody library was negatively screened by co-incubating with AsPC-1, Colo205 and SNU5 cells. After that, it was positively screened by incubating with AsPC-1-CEACAM5 and Colo205-CEACAM5 / GFP cells.

[0115] The phage library was isolated and purified for the next round of ELISA screening. After enrichment, the VHH region was amplified using the obtained phage as a template, and next-generation sequencing was performed to obtain the anti-CEACAM5 nanobody. The amino acid sequence is shown in SE1 ID NO.21 and it was named VHH-A031.

[0116] Example 2

[0117] To further identify the antibody, this embodiment describes the expression, purification, and antibody affinity determination of the VHH Fc nanobody.

[0118] The antibody quality test results are shown in Table 1.

[0119] Table 1

[0120]

[0121] Example 3

[0122] This embodiment describes the flow cytometry analysis of the anti-CEACAM5 nanobody from Example 1.

[0123] HGC-27 and HGC-27-CEACAM5 cells (prepared in Example 7) were mixed with purified anti-CEACAM5 nanobodies and incubated on ice for 30 min. Then, the cells were incubated with APC-labeled anti-human IgG antibody for 30 min. Flow cytometry analysis was performed, and the results are shown below. Figure 1 As shown, the anti-CEACAM5 nanobody of the present invention can recognize the CEACAM5 antigen on the cell surface.

[0124] Example 4

[0125] In this embodiment, the anti-CEACAM5 nanobody from Example 1 was detected by ELISA to determine its EC50 value of binding to the CEACAM5 protein.

[0126] First, take two clean ELISA empty plates, wash them once with PBS, discard the PBS, and pat them dry on absorbent paper. Dilute the Human CEACAM5-His (acro, cat#CE5-H5226) tag antigen with PBS to 1 μg / mL, add 100 μL of CEACAM5-His tag antigen to each well of a plate, seal the plate, and incubate overnight at 4°C.

[0127] After overnight incubation, remove the coated ELISA plate from the refrigerator, discard the liquid, add 300 μL of PBS to each well, discard the supernatant, and pat dry on absorbent paper. Add 300 μL of 1×NAP blocking buffer to each well, incubate at room temperature for 1 hour, discard the supernatant, and pat dry on absorbent paper. Dilute the primary antibody to 4 μg / mL with 1×NAP, and perform 11 sequential 4-fold dilutions, 50 μL per well. Add an equal volume of 1×NAP to the blank wells as a control. Seal the plate with sealing film, incubate at room temperature for 1 hour, discard the supernatant, and pat dry on absorbent paper. Add 300 μL of TBST to each well for washing, soak for 30 seconds, discard the liquid, and pat dry on absorbent paper. Repeat washing 4–5 times. Dilute the secondary antibody (0.4 μg / mL, Goat Anti-Human IgG HRP H&L, Cat#ab6858) with 1×NAP and add 50 μL to each reaction well. Incubate at room temperature for 1 hour. Discard the supernatant, add 300 μL of TBST to each well for washing, soak for 30 seconds, then discard the liquid, pat dry on absorbent paper, and repeat the washing 4–5 times. Add 50 μL of HRP substrate to each well and incubate at room temperature in the dark for 5–30 minutes. Add 50 μL of 1N HCl to each well to terminate the substrate reaction. Measure the absorbance at 450 nm using a microplate reader.

[0128] The results are as follows Figure 2 As shown, the EC50 of the CEACAM5 nanobody VHH-A031 binding to the CEACAM5 protein is approximately 0.0150 μg / mL, which is in the nanomolar range, indicating that VHH-A031 can bind well to the CEACAM5 protein.

[0129] Example 5

[0130] This embodiment prepares a lentiviral vector expressing a chimeric antigen receptor (CEACAM5 CAR) targeting CEACAM5.

[0131] First, the lentiviral vector HD SIN03 anti-CEACAM5VHH-41BBz carrying the CEACAM5 CAR chimeric antigen receptor was constructed. The vector map is shown below. Figure 3 As shown in the diagram, a schematic diagram of the chimeric antigen receptor is as follows: Figure 4 As shown, it includes CD8α signal peptide, anti-CEACAM5 nanobody (anti-CEACAM5 VHH), CD8α hinge region, transmembrane region, co-stimulatory molecule and immune receptor tyrosine activation motif (CD3ζ).

[0132] The amino acid sequence of the signal peptide (SEQ ID NO.10) is as follows:

[0133] MALPVTALLLPLALLLHAARP.

[0134] The amino acid sequence of anti-CEACAM5 VHH is shown in SEQ ID NO.8.

[0135] The amino acid sequences (SEQ ID NO.11) of the CD8α hinge region and transmembrane region are as follows:

[0136] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC.

[0137] The amino acid sequence of the 4-1BB co-stimulatory molecule (SEQ ID NO.12) is as follows:

[0138] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0139] The CD3ζ amino acid sequence (SEQ ID NO.13) is as follows:

[0140] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0141] The specific preparation method is as follows:

[0142] (1) Prepare the PCR reaction system according to Table 2 (the reagents in the table are from TOYOBO Inc.), amplify each anti-CEACAM5 nanobody fragment, and perform the reaction according to the PCR procedure shown in Table 3. The primer sequences are as follows:

[0143] CEACAM5-A031-F(SEQ ID NO.14):

[0144] CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTCCAAGTGCAAGAGAG.

[0145] CEACAM5-A031-R(SEQ ID NO.15):

[0146] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACTTGG.

[0147] Table 2

[0148] reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 <![CDATA[25mM MgSO4]]> 3 10μM upstream primer 2 10μM downstream primer 2 Template DNA (cDNA clone) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0149] Table 3

[0150]

[0151] After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis, and the fragment of about 400 bp was recovered and quantified by ultraviolet absorption method.

[0152] (2) Prepare the PCR reaction system according to Table 4. Add the CD8α signal peptide before the amplification product obtained in step (1), and perform the PCR reaction according to the PCR procedure shown in Table 3. The primers are:

[0153] BamH-CD8αsig-F (SEQ ID NO.16):

[0154] GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC.

[0155] CEACAM5-A031-R(SEQ ID NO.15):

[0156] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACTTGG.

[0157] Table 4

[0158] reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 25mM MgSO4 3 10μM upstream primer 2 10μM 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

[0159] 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 ultraviolet absorption method.

[0160] (3) Prepare the PCR reaction system according to Table 5. After preparation, perform the PCR reaction as shown in Table 3 to amplify the CD8αhinge-TM-41BB-CD3Z fragment. The primers used are as follows:

[0161] CD8αH-F (SEQ ID NO. 17): ACCACGACGCCAGCGCCGCGAC.

[0162] Vector-R (SEQ ID NO. 18): TCGATAAGCTTGATATCG.

[0163] Table 5

[0164] reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 25mM MgSO4 3 10 μM upstream primer CD8αH-F 2 10μM downstream primer Vector-R 2 Template DNA (HD CEACAM5 CAR) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0165] After PCR, 1% agarose gel electrophoresis was performed to recover the fragment of about 680 bp, and the fragment was quantified by UV absorption.

[0166] (4) 25 μg of the laboratory-constructed HD SIN03 GUCY2C A3-41BBz plasmid was digested with BamHI and EcoRI, reacted in a water bath at 37℃ for 1 h, and then the vector was recovered.

[0167] The two recovered fragments and the vector backbone were ligated with recombinase. The recombinant reaction system is shown in Table 6. After preparation, the reaction was carried out in a water bath at 37°C for 0.5 h and then transformed into Escherichia coli Stbl3 competent cells according to conventional methods.

[0168] Table 6

[0169] reagents Usage HD CEACAM5 CAR skeleton 154.2ng CD8αsingal CEACAM5 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 12μL

[0170] Single clones were selected from solid culture medium and sequenced for identification; the results were as expected. The sequencing primer sequences are as follows:

[0171] LV-F2 (SEQ ID NO. 19): TCTTGGTTCATTCTCAAGCCTC.

[0172] LV-R (SEQ ID NO. 20): GCAACATAGTTAAGAATACC.

[0173] Example 6

[0174] In this embodiment, the lentiviral vector HD SIN03 anti-CEACAM5 VHH-41BBz prepared in Example 5 was subjected to lentiviral packaging, concentration, and titer detection, including the following steps:

[0175] (1) Lentiviral packaging

[0176] 293T cells at an appropriate concentration were inoculated into 15cm culture dishes and cultured overnight at 37°C with 5% CO2 to prepare for virus packaging. The culture medium was DMEM containing 10% fetal bovine serum. 14.5μg of lentiviral vector HD SIN03 anti-CEACAM5VHH-41BBz, 16.7μg of helper plasmid pMDLg-RRE (Addgene, 12251), 16.7μg of helper plasmid pRSV-REV (Addgene, 12253), and 6.5μg of envelope plasmid VSVg (Addgene, 8454) were dissolved in 2mL of serum-free DMEM medium and mixed well.

[0177] 163.2 μg PEI (1 μg / μL) was dissolved in 2 mL of serum-free DMEM medium, vortexed at 1000 rpm for 5 s, and incubated at 25 °C for 5 min. The PEI mixture was then added to the DNA mixture, gently mixed, and incubated at 25 °C for 20 min to form a transfection complex. 4 mL of the transfection complex was then added dropwise to 18 mL of DMEM medium containing 293T cells. After 6 h, the medium was replaced with fresh medium. After 48 h, the viral supernatant was collected.

[0178] (2) Lentiviral titer detection

[0179] 500 μL Jurkat (1×10 5 Cells were seeded into 24-well plates, and the collected viral supernatant was added to the cell suspension at volumes of 1 μL, 5 μL, and 25 μL, respectively. Polybrene was added to a final concentration of 8 μg / mL. After incubation at 37°C and 5% CO2 for 72 h, the cells were centrifuged at 500g for 5 min, the supernatant was discarded, and the cells were resuspended in 100 μL of PBS + 2% FBS. 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 flow cytometry buffer (PBS containing 2% FBS), the cells were resuspended in 300 μL of flow cytometry buffer, and the infection efficiency was detected by flow cytometry. The titer was calculated using the following formula: Titer (TU / mL) = Cell count × Positive rate / Virus volume (mL). The viral titer calculated according to the titer formula was 1.08E+0.7TU / mL.

[0180] Example 7 Construction of overexpression cell lines

[0181] Lentiviral viruses overexpressing CEACAM5 were obtained by co-transfection with CEACAM5-puro plasmid, pMGlg-RRE, pRSV-REV and VSVg plasmid, following the same steps as in Example 6;

[0182] Take 1×10 6 HGC-27 and N87 cells were seeded into 6-well plates, and 1 mL of the CEACAM5-puro lentivirus obtained above was added to obtain HGC-27-CEACAM5 and N87-CEACAM5 overexpressing CEACAM5 protein.

[0183] Example 8

[0184] This embodiment uses the lentivirus-transduced T lymphocytes prepared in Example 6, and includes the following steps:

[0185] (1) Human PBMCs were adjusted to a density of 1×10⁻⁶ cells / mL using T cell culture medium (X-VIVO + 10% FBS + 300 U / mL IL-2). 6 / mL, add 1 / 100 volume of T Cell TransAct (commercially available magnetic beads coupled with CD3 and CD28) to activate for 24h;

[0186] (2) Collect activated T cells and adjust the cell density to 1×10⁻⁶. 6 Lentiviral virus was added at a concentration of 8 μg / mL according to a multiplicity of infection (MOI) of 10, and polybrene was added to a final concentration of 8 μg / mL. The culture was incubated overnight at 37°C and 5% CO2, and then replaced with fresh medium. The culture was passaged every 3 days.

[0187] (3) Eight days after T cell infection, 3×10⁻⁶ cells were collected. 5 T cells were centrifuged at 500g for 5 min at 4℃, the supernatant was discarded, and the cells were washed once with flow cytometry buffer. The cells were resuspended in 50 μL buffer, and 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody was added. The cells were incubated on ice for 30 min. After washing once with buffer, the cells were resuspended in 300 μL buffer.

[0188] The expression rate of chimeric antigen receptors in T lymphocytes was detected by flow cytometry, and the results are as follows: Figure 5 As shown, the infection efficiencies of the CAR-T cells in each group were 71.1%, 78.8%, and 67.8%, respectively, indicating that CAR-T cells were successfully constructed.

[0189] Example 9

[0190] This embodiment includes an in vitro toxicity experiment of CAR-T cells, comprising the following steps:

[0191] (1) Target cell inoculation

[0192] 293T-GPF-luci(CEACAM5) - ), N87-GFP-luci-CEACAM5(CEACAM5 + ), and BxPC3-GFP-luci (CEACAM5) + As target cells, the target cell concentration was adjusted to 2 × 10⁻⁶. 5 / mL, take 50μL and inoculate it into a white opaque 96-well plate;

[0193] (2) Effector cell inoculation

[0194] CAR-T cells targeting CEACAM5 and control T cells were used as effector cells. CAR-T cells and control T cells were added to 96-well plates at effector-to-target ratios of 0.3:1, 1:1 and 3:1.

[0195] (3) Each group has 2 replicates. The experimental group and the control group are as follows:

[0196] Experimental group: each target cell +T / CAR-T; Control group: only target cells were inoculated.

[0197] (5) Detection method:

[0198] After co-culturing effector cells and target cells for 18 hours, 70 μL of Steady-Lumi was added to each well. TM Firefly luciferase reporter gene assay reagent (Beyotime, catalog number: RG058M), reaction time 5 min, bioluminescent signal detected using a multi-functional microplate reader.

[0199] (5) The formula for calculating the CAR-T killing efficiency is: Killing efficiency % = (1 - experimental group / control group) × 100%.

[0200] The results are as follows Figures 6 to 8 As shown, the CAR-T cells constructed in this invention have no killing effect on CEACAM5-negative 293T cells, but have killing activity on CEACAM5-positive tumor cells, indicating that the CAR-T cells constructed in this invention not only have highly efficient tumor killing ability, but also have high specificity.

[0201] Example 10

[0202] In this embodiment, the secretion of CAR-T cytokine IFN-γ was detected using the HumanIFN-γ ELISA Kit (Lianke Biotechnology, catalog number: EK180-96).

[0203] 1. Cell culture supernatant

[0204] Centrifuge 400g of cell culture with an effect-to-target ratio of 1:1 for 10 minutes to remove the precipitate, and store the supernatant at -80℃ for later testing.

[0205] 2. Reagent preparation

[0206] Before testing, restore all reagents and samples to 25°C. If concentrated reagents crystallize, incubate at 37°C until all crystals dissolve. Prepare 1× washing solution and 1× test buffer according to the instructions.

[0207] 3. Preparation of standard products and samples

[0208] Standards: The stock solution of the standard was diluted 2 times using 5% FBS1640 medium, with a total of 8 dilution gradients, including zero concentration.

[0209] Samples: Dilute the samples using 5% FBS1640 medium as directed.

[0210] 4. Testing Steps

[0211] (1) Soaking the microplate: Add 300 μL of 1× washing solution and let it stand for 30 seconds. Discard the washing solution and pat the microplate dry on absorbent paper.

[0212] (2) Add standard: Add 100 μL of 2-fold serially diluted standard to the standard well and add 100 μL of standard diluent to the blank well;

[0213] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0214] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0215] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0216] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well and wash the plate 6 times. After each wash, pat the plate dry on absorbent paper.

[0217] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0218] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, incubate at 25°C for 45 min, and then wash.

[0219] (9) Adding substrate for color development: Add 100 μL of TMB substrate to each well, incubate in the dark at 25°C for 20 min;

[0220] (10) Add stop solution: Add 100 μL of stop solution to each well;

[0221] (11) Detection reading: Within 30 minutes, use an ELISA reader to perform dual-wavelength detection and measure the OD value at the maximum absorption wavelength of 450nm and the reference wavelength; the calibrated OD value is the measured value at 450nm minus the measured value at the reference wavelength.

[0222] The results of IFN-γ factor secretion were respectively Figure 9As shown, the spontaneous MOCK group consisted of CAR-T cells alone, with virtually no detectable cytokine release; the CAR-T cell co-culture group with 293T cells also showed virtually no detectable cytokines; and after co-culture with CEACAM5-positive target cells, the CAR-T cells secreted IFN-γ exceeding 500 pg / mL. The CAR-T cells constructed in this invention release cytokines against CEACAM5-positive tumor cells, but show no significant cytokine secretion against CEACAM5-negative cells.

[0223] In summary, the anti-CEACAM5 nanobody screened and prepared in this invention possesses high affinity and can efficiently and specifically bind to CEACAM5. Using it as an antigen-binding domain, chimeric antigen receptors and CAR-T cells can be constructed. The resulting CAR-T cells exhibit significant killing activity and specificity against CEACAM5-positive tumor cells and can secrete tumor-killing cytokines. This demonstrates that the nanobody of this invention can be effectively applied to immunotherapy and is of great significance for the development of tumor therapeutic drugs.

[0224] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the 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 this invention.

Claims

1. An anti-CEACAM5 antibody, characterized in that, The anti-CEACAM5 antibody is a nanobody, the anti-CEACAM5 antibody comprises heavy chain variable region CDR-H1-3, the heavy chain variable region CDR-H1 amino acid sequence is shown as SEQ ID No. 1; the heavy chain variable region CDR-H2 amino acid sequence is shown as SEQ ID No. 2; the heavy chain variable region CDR-H3 amino acid sequence is shown as SEQ ID No.

3.

2. The anti-CEACAM5 antibody according to claim 1, characterized in that, 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 shown as SEQ ID No. 4-7.

3. The anti-CEACAM5 antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown as SEQ ID No.

8.

4. Use of the anti-CEACAM5 antibody according to any one of claims 1-3 in the preparation of a medicament for treating a tumor, the tumor being selected from the group consisting of pancreatic cancer, gastric cancer.

5. Use according to claim 4, characterized in that, The tumor treatment drug is a chimeric antigen receptor T cell.

6. An isolated polypeptide, comprising, The polypeptide is a chimeric antigen receptor, the polypeptide comprises a transmembrane domain, an intracellular domain and an extracellular domain, and the extracellular domain is selected from the anti-CEACAM5 antibody according to any one of claims 1-3.

7. The polypeptide of claim 6, wherein, Further comprising the following features: The transmembrane domain is selected from any one or more of CD8a transmembrane region, CD28 transmembrane region, DAP 10 transmembrane region; The intracellular domain comprises a signal transduction domain, the signal transduction domain comprises an immune receptor tyrosine activation motif and a costimulatory molecule; The extracellular domain comprises a signal peptide, an anti-CEACAM5 antibody, and a hinge region, the signal peptide comprises a CD8a signal peptide, and the hinge region is selected from a CD8a hinge region; The polypeptide comprises, in order from N-terminus to C-terminus, a CD8a signal peptide, an anti-CEACAM5 antibody, a CD8a hinge region, a CD8a transmembrane region, a costimulatory molecule, and a CD3 zeta signal domain.

8. The polypeptide of claim 7, wherein, The immune receptor tyrosine activation motif is selected from CD3 zeta; and / or, the costimulatory molecule is selected from any one or a combination of at least two of 4-1BB, CD28, OX40, ICOS, and DAP 10.

9. An isolated polynucleotide, comprising: The anti-CEACAM5 antibody according to any one of claims 1-4 or the polypeptide according to any one of claims 6-8.

10. A nucleic acid construct, characterized in that, The nucleic acid construct is any one of a retroviral vector or an adeno-associated viral vector.

11. The nucleic acid construct of claim 10, wherein, The nucleic acid construct is a lentiviral vector.

12. The nucleic acid construct of claim 10, wherein, The lentivirus is packaged from the nucleic acid construct according to any one of claims 10-12.

13. A lentivirus, characterized in that, The lentiviral vector system comprises the nucleic acid construct according to any one of claims 10-12 and a helper plasmid or a host cell.

14. A lentiviral vector system, characterized in that, The chimeric antigen receptor immune cell expresses a membrane-bound isolated polypeptide according to any one of claims 6-8.

15. A chimeric antigen receptor immune cell, comprising: The immune cell is selected from a T lymphocyte.

16. The chimeric antigen receptor immune cell of claim 15, wherein, 17. Use of the isolated polypeptide according to any one of claims 6-8 or the chimeric antigen receptor immune cell according to any one of claims 15-16 in the preparation of a medicament for treating a tumor, the tumor being selected from the group consisting of gastric cancer and pancreatic cancer. ​ 18. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the chimeric antigen receptor immune cell according to any one of claims 15-16 and a pharmaceutically acceptable carrier.

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