A bispecific antibody against CEA5 and NCA-90 and its application

Abstract

This invention relates to the field of biopharmaceuticals, and particularly to a CEA5 and NCA-90 bispecific antibody and its application. The CEA5 and NCA-90 bispecific antibody includes a heavy chain variable region, and the heavy chain variable region of the anti-CD66e antibody includes the following technical features: <1> Including CDR-H1 with an amino acid sequence such as SEQ ID No. 1 or 2; <2> Including the amino acid sequence CDR-H2 as shown in SEQ ID No. 3; <3> The amino acid sequence includes CDR-H3 as shown in SEQ ID No. 4. The CEA5 and NCA-90 bispecific antibody provided by this invention has good affinity. It is used as the antigen-binding domain to construct a chimeric antigen receptor, and CAR-T cells are prepared using this chimeric antigen receptor. These CAR-T cells exhibit killing activity against CEA5 and / or NCA-90 positive tumor cells and can be effectively applied to immunotherapy, which is of great significance for the development of tumor therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceuticals, and in particular to a bispecific antibody against CEA5 and NCA-90 and its applications. Background Technology

[0002] Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), also known as CEA or CD66e, was initially discovered in colon cancer and embryonic tissues. It has since been shown to be widely present in endodermal digestive system tumors such as gastric cancer, liver cancer, pancreatic cancer, and colon cancer, and is also elevated in the serum of breast cancer, lung cancer, and other malignant tumors, making it a broad-spectrum tumor marker. CEA5 belongs to the immunoglobulin (Ig) superfamily and binds to the membrane via glycosylphosphatidylinositol (GPI). It has seven extracellular immunoglobulin-like domains, including a variable IgV-like domain (N domain) and six IgC-like domains. Furthermore, a 34-amino acid signal peptide precedes the Ig domain. The human CEA5 gene is located on chromosome 19q13.2, is approximately 21 kb in length, and consists of nine exons and three non-coding exons. The CEA5 protein consists of 642 amino acids (molecular weight approximately 70 kDa) and has 28 potential N-linked glycosylation sites. A single CEA5 protein molecule contains 24-26 asparagine-linked glycans, with a final molecular weight of approximately 180 kDa.

[0003] Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), also known as CD66c or NCA-90, is a member of the CEA family of immunoglobulin cell adhesion molecules. Its gene is located on chromosome 19, q13.2, and its coding region consists of six exons, encoding a protein containing 344 amino acid residues. NCA-90 is linked to the cell membrane via glycosyl phosphatidylinositol (GPI). Normally, NCA-90 consists of a variable Ig N-terminal domain and six immunoglobulin constant region (IgC) type 2-like domains. These structures are anchored to the cell membrane via GPI. Although they lack intracellular domains, they still influence intracellular signaling events. Their most typical biological function is to mediate cell adhesion by mediated isoform binding with other CEA family members and heteroform binding with integrin receptors (such as NCA-90-CEACAM8, CEA5-NCA-90).

[0004] Studies have found that NCA-90 is highly expressed in many malignant tumors, including tumors of the colon, stomach, pancreas, breast, female reproductive system, and lung (see: Duxbury MS, Matros E, Clancy T, et al. NCA-90 is a novel biomarker in pancreatic adenocarcinoma and PanIN lesions. Ann Surg. 2005; 241(3):491-496.). However, it is expressed at low levels in normal epithelial cells, vascular endothelial cells, granulocytes, T cells, and NK cells (see: Kuijpers TW, Hoogerwerf M, van der Laan LJ, et al. CD66 nonspecific cross-reacting antigens are involved in neutrophil adherence to cytokine-activated endothelial cells. J Cell Biol. 1992; 118(2):457-466. doi:10.1083 / jcb.118.2.457). Abnormally elevated NCA-90 expression is common in malignant tumors of the digestive system, and its expression level is often closely related to the severity of the disease and its prognosis.

[0005] CEA5 and NCA-90 are both important carcinoembryonic antigens. Previous studies have found that CEA5 can be detected in various cancers such as bladder cancer, cholangiocarcinoma, small cell lung cancer, endometrial cancer, and gastric cancer. Currently, CAR-T therapy targeting CEA5 for colorectal cancer has entered the clinical stage and has achieved some efficacy. NCA-90 is also involved in the development of various malignant tumors, such as breast cancer, pancreatic cancer, colon cancer, and non-small cell lung cancer, promoting tumor invasion and metastasis, inhibiting tumor cell anolysm, and promoting angiogenesis at the tumor site. Furthermore, studies have reported that the expression levels of CEA5 and NCA-90 in colon cancer tissues are significantly higher than in adjacent normal tissues. Based on existing research, CEA5 and NCA-90 play important roles in the treatment of colorectal cancer and other cancers, and targeting CEA5 and NCA-90 may be a potential therapeutic approach for treating these cancers. 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. Constructing dual-target CAR-T cells based on CEA5 and NCA-90 may bring new hope to the treatment of cancers such as colorectal cancer.

[0006] In conclusion, CEA5 and NCA-90 can serve as targets for tumor therapy. Providing an antibody with high specificity and affinity against both CEA5 and NCA-90 is of groundbreaking significance for the treatment of malignant tumors. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a CEA5 and NCA-90 bispecific antibody and its application, in order to solve the problems in the prior art.

[0008] To achieve the above and other related objectives, the present invention provides a CEA5 and NCA-90 bispecific antibody, wherein the CEA5 and NCA-90 bispecific antibody includes a heavy chain variable region, and the heavy chain variable region of the anti-CD66e antibody includes the following technical features:

[0009] <1> Including the amino acid sequence CDR-H1 as shown in SEQ ID No. 1 or 2;

[0010] <2> Including the amino acid sequence CDR-H2 as shown in SEQ ID No. 3;

[0011] <3> Including the amino acid sequence CDR-H3 as shown in SEQ ID No.4.

[0012] 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 CEA5 and NCA-90 bispecific antibody.

[0013] The present invention also provides an isolated polynucleotide encoding the CEA5 and NCA-90 bispecific antibody or the polypeptide.

[0014] The present invention also provides a nucleic acid construct containing encoding the isolated polynucleotides described above.

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

[0016] The present invention also provides a lentiviral vector system, which includes the aforementioned nucleic acid construct and helper plasmids or host cells.

[0017] 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.

[0018] As described above, the CEA5 and NCA-90 bispecific antibody of the present invention and its application have the following beneficial effects:

[0019] The dual-targeting nanobody against CEA5 and NCA-90 provided by this invention can efficiently bind to CEA5 and NCA-90 antigens and has good affinity. It can be used as an antigen-binding domain to construct a chimeric antigen receptor, and CAR-T cells can be prepared using this chimeric antigen receptor. The CAR-T cells have killing activity against CEA5 and / or NCA-90 positive tumor cells, and after co-culturing with CEA5 or NCA-90 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

[0020] Figure 1 This is a graph showing the FACS detection results of the anti-CEA5 and NCA-90 nanobodies recognizing the CEA5 antigen in Example 3;

[0021] Figure 2 This is a graph showing the FACS detection results of the anti-CEA5 and NCA-90 nanobodies recognizing the NCA-90 antigen in Example 3;

[0022] Figure 3 This is a graph showing the EC50 detection results of anti-CEA5 and NCA-90 nanobodies binding to CEA5 antigen in Example 4;

[0023] Figure 4 This is a graph showing the EC50 detection results of anti-CEA5 and NCA-90 nanobodies binding to NCA-90 antigen in Example 4;

[0024] Figure 5 This is a map of the chimeric antigen receptor lentiviral vector plasmid targeting CEA5 and NCA-90 in Example 5;

[0025] Figure 6 This is a schematic diagram of the chimeric antigen receptor structure expressing CEA5 and NCA-90 in Example 5;

[0026] Figure 7 This is a flow cytometry result of the chimeric antigen receptor expression rate of T lymphocytes in Example 8;

[0027] Figure 8 The graph shows the killing effect of CAR-T cells on human embryonic kidney cells 293T in Example 9.

[0028] Figure 9 This is a graph showing the killing effect of CAR-T cells on human gastric cancer cells N87-CEA5 in Example 9.

[0029] Figure 10 This is a graph showing the killing effect of CAR-T cells on human orthotopic pancreatic adenocarcinoma cells BxPC3 in Example 9.

[0030] Figure 11 This is a graph showing the killing effect of CAR-T cells on human colon cancer cells SW620 in Example 9;

[0031] Figure 12 This is a graph showing the killing effect of CAR-T cells on human colon cancer cells SW620-NCA-90 in Example 9.

[0032] Figure 13 This is a bar chart showing the secretion of IFN-γ by CAR-T cells killing control cells and CEA5+ target cells in Example 10.

[0033] Figure 14 This is a bar chart showing the secretion of IFN-γ by CAR-T cells killing control cells and NCA-90+ target cells in Example 10. Detailed Implementation

[0034] This invention provides a bispecific antibody for CEA5 and NCA-90, wherein the bispecific antibody for CEA5 and NCA-90 includes a heavy chain variable region, and the heavy chain variable region has one or more of the following technical features;

[0035] <1> Including the amino acid sequence CDR-H1 as shown in SEQ ID No. 1 or 2;

[0036] <2> Including the amino acid sequence CDR-H2 as shown in SEQ ID No. 3;

[0037] <3> Including the amino acid sequence CDR-H3 as shown in SEQ ID No. 4;

[0038] GLTLNYHF (SEQ ID No.1)

[0039] GSIFSINA (SEQ ID No. 2)

[0040] ITNGGST (SEQ ID No. 3)

[0041] AADGGKVTPKYYGMDY (SEQ ID No. 4).

[0042] The numbering and definition scheme is to use the IMGT method to label CDR and FR areas.

[0043] The CDR (complementarity determining region) generally refers to the 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 usually includes three complementarity determining regions: CDR-H1, CDR-H2, and CDR-H3.

[0044] In some embodiments of the present invention, the name of the CEA5 and NCA-90 bispecific antibody is VHH-A039 or A039, and the complementarity-determining region of the heavy chain variable region 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. 3, and the amino acid sequence CDR-H3 as shown in SEQ ID No. 4.

[0045] In some embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody is named VHH-A051 or A051, and the complementarity-determining region of the heavy chain variable region includes the amino acid sequence CDR-H1 as shown in SEQ ID No. 2, the amino acid sequence CDR-H2 as shown in SEQ ID No. 3, and the amino acid sequence CDR-H3 as shown in SEQ ID No. 4.

[0046] The CEA5 and NCA-90 bispecific antibodies are antibody fragments and / or monoclonal antibodies.

[0047] 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.

[0048] In this invention, an unimmunized alpaca was immunized with recombinant CEA5 protein and a cell line overexpressing CEA5 to construct a phage display nanobody library. Antibodies were screened based on this phage display nanobody library to obtain antibodies that can specifically bind to CEA5 and NCA-90 antigens.

[0049] In some embodiments of this invention, the CEA5 and NCA-90 bispecific antibodies are nanobodies (Nb), specifically heavy chain single-domain antibodies (VHH, variable domain of heavy chain of heavy-chain antibody). Nanobodies contain only one heavy chain variable region (VHH) and CH2 and CH3 regions; compared to other antibodies, nanobodies naturally lack the light chain. Nanobody crystals have a diameter of approximately 2.5 nm and a length of approximately 4 nm, representing the smallest naturally occurring fragment capable of binding to antigens. The CEA5 and NCA-90 bispecific antibodies of this invention consist only of the heavy chain variable region, possessing high affinity and specificity, enabling efficient targeting of CEA5 and NCA-90 antigens. They have a simple structure, are easy to prepare, and have significant application value in the field of drug development targeting CEA5 and NCA-90.

[0050] 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.

[0051] 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 framework regions FR1 to FR4 are selected from any of the sequences shown in SEQ ID No. 5 to 11.

[0052] Preferably, the amino acid sequence of FR1 is as shown in SEQ ID No. 5 or 6:

[0053] EVQVVESGGGLVQAGGSLLRLSCAAS(SEQ ID No.5)

[0054] EVRLVESGGGLVQAGGSLLRLSCAAS (SEQ ID No. 6).

[0055] Preferably, the amino acid sequence of FR2 is as shown in SEQ ID No. 7 or 8:

[0056] VAWFRQAPGEQRELVAI(SEQ ID No.7)

[0057] MAWYRQAPGEQRELVAI(SEQ ID No.8)

[0058] Preferably, the amino acid sequence of FR3 is shown in SEQ ID No. 9:

[0059] RYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYC(SEQ ID No.9)

[0060] Preferably, the amino acid sequence of FR4 is as shown in SEQ ID No. 10 or 11:

[0061] WGNGTLVTVSS(SEQ ID No.10)

[0062] WGNGTLGTVSS(SEQ ID No.11)

[0063] In some embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody VHH-A039, wherein the heavy chain variable region of the CEA5 and NCA-90 bispecific antibody includes amino acid sequences as shown in SEQ ID No. 1 (CDR-H1), amino acid sequences as shown in SEQ ID No. 3 (CDR-H2), amino acid sequences as shown in SEQ ID No. 4 (CDR-H3), and amino acid sequences of frame regions FR1 to FR4 as shown in SEQ ID No. 5, 7, 9, and 10, respectively.

[0064] Preferably, the amino acid sequence of the heavy chain variable region of the CEA5 and NCA-90 bispecific antibody VHH-A039 is shown in SEQ ID No. 12.

[0065] EVQVVESGGGLVQAGGSLRLSCAASGLTLNYHFVAWFRQAPGEQRELVAIITNGGST RYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAADGGKVTPKYYGMDYWGN GTLVTVSS(SEQ ID No.12)

[0066] In some embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody VHH-A039 is obtained from a phage antibody library through screening, and its heavy chain variable region nucleotide sequence is shown in SEQ ID No. 13:

[0067] GAGGTGCAAGTGGTCGAGTCCCGGGGGGGGGCTGGTCCAAGCCGGGGGCAGCCTGAGACTCAGCTGCGCCGCTAGCGGCCTGACCCTGAACTACCACTTCGTGGCCTGGTTCAGACAAGCCCCCGGCGAGCAGAGAGAGCTCGTGGCCATCATCACCAACGGCGGGAGCACACGGTACGCCGACAG CGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCATGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGGCGGCAAAGTGACCCCCAAGTACTACGGCATGGACTACTGGGGCAACGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNo.13)

[0068] In some embodiments of the present invention, the heavy chain variable region of the CEA5 and NCA-90 bispecific antibody VHH-A051 includes the amino acid sequences CDR-H1 as shown in SEQ ID No. 2, CDR-H2 as shown in SEQ ID No. 3, CDR-H3 as shown in SEQ ID No. 4, and the amino acid sequences of the frame regions FR1 to FR4 as shown in SEQ ID No. 6, 8, 9, and 11, respectively.

[0069] Preferably, the amino acid sequence of the heavy chain variable region of the CEA5 and NCA-90 bispecific antibody VHH-A051 is shown in SEQ ID No. 14.

[0070] EVRLVESGGGLVQAGGSLRLSCAASGSIFSINAMAWYRQAPGEQRELVAIITNGGSTR YADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAADGGKVTPKYYGMDYWGNG TLGTVSS(SEQ ID No.14)

[0071] In some embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody VHH-A051 is obtained from a phage antibody library through screening, and its heavy chain variable region nucleotide sequence is shown in SEQ ID No. 15:

[0072] GAGGTGCGGCTGGTCGAGTCCCGGCGGGGGGCTGGTGCAAGCCGGGGGCTCCCTGAGACTGAGCTGCGCCGCTAGCGGCAGCATCTTCAGCATCAACGCCATGGCCTGGTACAGACAAGCCCCCGGCGAGCAGAGAGCTGGTGGCCATCATCACCAACGGCGGCAGCACAAGATACGCCGACAG CGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCATGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGGGGGCAAGGTGACCCCCAAGTACTACGGCATGGACTACTGGGGCAACGGCACCCTGGGCACCGTGAGCAGC(SEQ IDNo.15)

[0073] Another aspect of the present invention provides an isolated polynucleotide encoding the CEA5 and NCA-90 bispecific antibody.

[0074] In some embodiments of the present invention, the sequence of the polynucleotide is as shown in SEQ ID No. 13 or 15.

[0075] Another aspect of the present invention provides the use of the CEA5 and NCA-90 bispecific antibodies in the preparation or screening of therapeutic drugs, or in the preparation of diagnostic drugs.

[0076] The therapeutic agent may be an agent that targets the CEA5 or NCA-90 antigen, binds to or acts on the CEA5 or NCA-90 antigen, thereby treating and / or preventing the indication.

[0077] In some embodiments of the present invention, the therapeutic agent may be an antitumor drug. The tumor is a tumor expressing CEA5 or NCA-90. The antitumor drug may be a drug that targets the CEA5 or NCA-90 antigen on the functional surface of tumor cells, binding to or acting on the CEA5 or NCA-90 antigen, thereby treating and / or preventing tumors. The tumor may be a CEA5 or NCA-90 positive tumor, such as colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, ovarian cancer, lung cancer, gastric cancer, gallbladder cancer, etc.

[0078] In some embodiments of the present invention, the therapeutic agent is a chimeric antigen receptor cell.

[0079] 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 CEA5 and NCA-90 bispecific antibody, meaning that the chimeric antigen receptor cells can express the CEA5 and NCA-90 bispecific antibody on their cell surface, thereby guiding the cells to act on the drug that expresses CEA5 or NCA-90 antigens (e.g., tumor cells). The action on cells expressing CEA5 or NCA-90 antigens can include killing cells expressing CEA5 or NCA-90 antigens, etc.

[0080] The diagnostic drug specifically refers to a reagent that targets the CEA5 or NCA-90 antigen and uses the CEA5 or NCA-90 antigen as a biomarker for diagnosis.

[0081] 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 CEA5 and NCA-90 bispecific antibody.

[0082] 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 CEA5 and NCA-90 bispecific antibody, which can efficiently target CEA5 or NCA-90.

[0083] 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.

[0084] For example, the sequence of CD8α can be found in NM_001145873, the sequence of CD28 in NM_006139, and the sequence of DAP10 in NM_014266.

[0085] In some embodiments of the present invention, the intracellular domain may include a signal transduction domain.

[0086] The signal transduction domain includes an immune receptor tyrosine activation motif. This immune receptor tyrosine activation motif may be selected from CD3ζ.

[0087] 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. For further examples, 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 DAP 10 in NM_014266.

[0088] 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.

[0089] In some embodiments of the present invention, the extracellular domain may include a signal peptide, a CEA5 and NCA-90 bispecific antibody, and a hinge region.

[0090] In some embodiments of the present invention, the signal peptide includes the CD8α signal peptide.

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

[0092] In some embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, the CEA5 and NCA-90 bispecific antibody, a transmembrane domain, and an intracellular domain.

[0093] In some specific embodiments of the present invention, the polypeptide comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, a CEA5 and NCA-90 bispecific antibody, 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 comprises, from the N-terminus to the C-terminus, a CD8α signal peptide, a CEA5 and NCA-90 bispecific antibody, 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 described in VHH-A039 is shown in SEQ ID No. 16:

[0096]

[0097] In certain embodiments of the present invention, the polynucleotide sequence encoding the isolated polypeptide of VHH-A051 is shown in SEQ ID No. 17:

[0098] atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggaggtgcggctggtcgagtccggcggggggctggtgcaagccgggggctccctgagactgagctgcgccgctagcggcagcatcttcagcatcaacgccatggcctggtacagacaagcccccggcgagcagagagagctggtggccatcatcaccaacggcggcagcacaagatacgccgacagcgtgaagggcagattcaccatcagcagagacaacgccaagaacaccatgtacctgcagatgaacagcctgaagcccgaggacaccgccgtgtactactgcgccgccgacgggggcaaggtgacccccaagtactacggcatggactactggggcaacggcaccctgggcaccgtgagcagcaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgcagagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgctga(SEQ ID No.17).

[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 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.

[0101] 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.

[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, which is formed by viral packaging of the nucleic acid construct. The lentivirus contains the nucleic acid construct.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[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 that is bound to a membrane.

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

[0110] The immune cells are 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 typically express the polypeptide, which can bind to the CEA5 or NCA-90 antigen. More specifically, it can bind to the CEA5 or NCA-90 antigen via an extracellular domain containing a bispecific antibody against CEA5 and NCA-90. When the polypeptide binds to the CEA5 or NCA-90 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 bispecific antibody against CEA5 and NCA-90 on their surface, thereby guiding the T lymphocytes to act on cells expressing the CEA5 or NCA-90 antigen (e.g., tumor cells). This action can include killing cells expressing the CEA5 or NCA-90 antigen, etc.

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

[0114] The NK cells typically express the polypeptide and can bind to the CEA5 or NCA-90 antigen. More specifically, they can bind to the CEA5 or NCA-90 antigen through an extracellular domain containing a bispecific antibody against CEA5 and NCA-90. 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 bispecific antibody against CEA5 and NCA-90 on their surface, thereby guiding the NK cells to act on cells expressing the CEA5 or NCA-90 antigen (e.g., tumor cells). This action can include killing cells expressing the CEA5 or NCA-90 antigen, etc.

[0115] 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.

[0116] The therapeutic or diagnostic drug may be a drug that targets the CEA5 or NCA-90 antigen, binds to or acts on the CEA5 or NCA-90 antigen, thereby treating and / or preventing the indication.

[0117] In some embodiments of the present invention, the therapeutic drug may be an antitumor drug. The antitumor drug may be a drug that targets CEA5 or NCA-90 antigens functionally expressed on the surface of tumor cells, binding to or acting on CEA5 or NCA-90 antigens to treat and / or prevent tumors. The tumor may be a tumor that is positive for CEA5 or NCA-90 expression, such as gastric cancer, lung cancer, pancreatic cancer, or colorectal cancer.

[0118] The present invention provides a pharmaceutical composition comprising the chimeric antigen receptor immune cells and / or the CEA5 and NCA-90 bispecific antibodies and / or the isolated polypeptides.

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

[0120] "Pharmaceutical acceptable" means that when a drug is properly administered to animals or humans, it will not produce adverse, allergic, or other adverse reactions.

[0121] "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.

[0122] 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.

[0123] 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.

[0124] 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.

[0125] Example 1: Screening of VHH nanobodies

[0126] This embodiment constructs and pans a phage nanobody library, and uses ELISA for preliminary screening. The specific steps are as follows:

[0127] (1) Construction of phage nanobody library

[0128] Alpacas were immunized with recombinant human CEA5 protein and a cell line overexpressing CEA5. 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.

[0129] (2) Screening of phage nanobody libraries

[0130] 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-CEA5 and Colo205-CEA5 / GFP cells.

[0131] 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 subjected to next-generation sequencing. Unexpectedly, two nanobodies that could simultaneously bind to CEA5 and NCA-90 were obtained after screening. The amino acid sequences of the CEA5 and NCA-90 bispecific antibodies are shown in SEQ ID NO.12 and 14, respectively, and are named VHH-A039 and VHH-A051, respectively.

[0132] Example 2: Expression and purification of VHH nanobody.

[0133] To further identify these antibodies, the antibody quality test results are shown in Table 1.

[0134] Table 1

[0135]

[0136] Example 3: Detection of binding between CEA5 and NCA-90 bispecific antibodies and antigens

[0137] This embodiment describes the flow cytometry analysis of the CEA5 and NCA-90 bispecific antibodies obtained in Example 1.

[0138] HGC-27 and HGC-27-CEA5 cells were mixed with purified nanobodies (1 μg / ml), and SW620 and SW620-NCA-90 cells were also mixed with purified nanobodies (1 μg / ml). Each mixture was incubated on ice for 30 min, followed by incubation with APC-labeled anti-human IgG antibody for 30 min. Flow cytometry analysis was performed, and the results are shown below. Figure 1 and 2 As shown, the CEA5 and NCA-90 bispecific antibodies of the present invention can recognize the CEA5 and NCA-90 antigens on the cell surface.

[0139] Example 4: Affinity Detection of CEA5 and NCA-90 Bispecific Antibodies

[0140] In this embodiment, the affinity of the CEA5 and NCA-90 bispecific antibodies from Example 1 was tested, and the EC50 values ​​of their binding to CEA5 and NCA-90 proteins were determined by ELISA.

[0141] 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 CEA5-His (acro, cat#CE5-H5226) and Human NCA-90-His (acro, cat#CE6-H5223) tag antigens with PBS to 1 μg / ml, respectively. Add 100 μl of CEA5-His tag antigen to each well of one plate, and add 100 μl of NCA-90-His tag antigen to each well of the other plate. After sealing the plates, incubate them overnight at 4°C.

[0142] After overnight incubation, remove the coated ELISA plate from the refrigerator, discard the liquid, add 200 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 with 1×NAP to 4 μg / ml, and perform serial 4-fold dilutions for a total of 11-12 concentrations, 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, pat dry on absorbent paper, and repeat the 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. 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, and calculate the EC50 values ​​of antibody binding to CEA5 and NCA-90 proteins. The results are as follows: Figure 3 and 4 As shown, the EC50 of VHH-A039 antibody binding to CEA5 protein was 0.0219 μg / mL, and the EC50 binding to NCA-90 protein was 0.1746 μg / mL, both in the nanomolar range, indicating that it can bind well to both CEA5 and NCA-90 proteins. Similarly, the EC50 of VHH-A051 antibody binding to CEA5 protein was 0.0234 μg / mL, and the EC50 binding to NCA-90 protein was 1.718 μg / mL, both in the nanomolar range, indicating that it can bind well to both CEA5 and NCA-90 proteins.

[0143] Example 5

[0144] This embodiment prepares a lentiviral vector expressing a chimeric antigen receptor (CEA5 / NCA90 CAR) targeting both CEA5 and NCA-90.

[0145] First, the lentiviral vector HD SINO3 anti-CEA5 / NCA90VHH-41BBz carrying the CEA5 / NCA90 CAR chimeric antigen receptor was constructed. The vector map is shown below. Figure 5 As shown in the diagram, a schematic diagram of the chimeric antigen receptor is as follows: Figure 6 As shown, it includes the CD8α signal peptide, CEA5 and NCA-90 bispecific antibody (anti-CEA5 / NCA90 VHH), CD8α hinge region, transmembrane region and immune receptor tyrosine activation motif (CD3ζ).

[0146] The amino acid sequence of the signal peptide is as follows:

[0147] MALPVTALLLPLALLLHAARP (SEQ ID NO. 16).

[0148] The amino acid sequence of anti-CEA5 / NCA90 VHH is shown in SEQ ID NO.12 or 14.

[0149] The amino acid sequences of the CD8α hinge region and transmembrane region are as follows:

[0150] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO. 15).

[0151] The amino acid sequence of the intracellular region of 4-1BB is as follows:

[0152] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO. 16).

[0153] The CD3ζ amino acid sequence is:

[0154] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO. 17).

[0155] The specific preparation method is as follows:

[0156] (1) Prepare the PCR reaction system according to Table 2 (the reagents in the table are from TOYOBO Inc.), amplify each anti-CEA5 nanobody fragment, and perform the PCR reaction according to the PCR procedure shown in Table 3. The primer sequences are as follows:

[0157] CEA5-A039-F (SEQ ID NO.18):

[0158] CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCAAGTGGTCGAGTC

[0159] CEA5-A039-R (SEQ ID NO.19):

[0160] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACCAGG.

[0161] CEA5-A051-F (SEQ ID NO.20):

[0162] CTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGGTGCGGCTGGTCGAGTC.

[0163] CEA5-A051-R (SEQ ID NO.21):

[0164] GCGCTGGCGTCGTGGTGCTGCTCACGGTGCCCAGG

[0165] Table 2

[0166] 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

[0167] Table 3

[0168]

[0169]

[0170] 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.

[0171] (2) Prepare the PCR reaction system according to Table 4, add CD8α signal peptide before the amplification product obtained in step (1), and carry out the reaction according to the PCR procedure shown in Table 3. The primers used are as follows:

[0172] BamH-CD8αsig-F (SEQ ID NO. 22):

[0173] GCTGCAGGTCGACTCTAGAGGATCCCGCCACCATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGC.

[0174] CEA5-A039-R (SEQ ID NO.19):

[0175] GCGCTGGCGTCGTGGTGCTGCTCACGGTCACCAGG.

[0176] CEA5-A051-R (SEQ ID NO.21):

[0177] GCGCTGGCGTCGTGGTGCTGCTCACGGTGCCCAGG

[0178] Table 4

[0179] reagents Volume (μL) 10×PCR Buffer 5 2mM dNTP 5 <![CDATA[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

[0180] 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.

[0181] (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:

[0182] CD8αH-F (SEQ ID NO. 23): ACCACGACGCCAGCGCCGCGAC.

[0183] Vector-R (SEQ ID NO. 24): TCGATAAGCTTGATATCG.

[0184] Table 5

[0185]

[0186]

[0187] After PCR, 1% agarose gel electrophoresis was performed to recover the fragment of about 680 bp, and the fragment was quantified by UV absorption.

[0188] (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.

[0189] 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.

[0190] Table 6

[0191] reagents Usage HD CEA5 / 6CAR skeleton 154.2ng CD8αsingal CEA5 / 6VHH 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

[0192] Single clones were selected from solid culture medium and sequenced for identification; the results were as expected. The sequencing primer sequences are as follows:

[0193] LV-F2 (SEQ ID NO. 25): TCTTGGTTCATTCTCAAGCCTC.

[0194] LV-R (SEQ ID NO. 26): GCAACATAGTTAAGAATACC.

[0195] Example 6

[0196] In this embodiment, the lentiviral vector HD SINO3 anti-CEA5 / NCA90VHH-41BBz prepared in Example 5 was subjected to lentiviral packaging, concentration, and titer detection, including the following steps:

[0197] (1) Lentiviral packaging

[0198] 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 SINO3 anti-CEA5 / NCA90VHH-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.

[0199] 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.

[0200] (2) Lentiviral titer detection

[0201] 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 collected. The cells were resuspended in 100 μL of PBS + 2% FBS. 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody was added to each sample, and the cells were incubated on ice for 30 min. Flow cytometry buffer (containing 2%...) After washing the cells once with PBS buffer, 300 μL of flow cytometry buffer was added to resuspend the cells, and the infection efficiency was detected by flow cytometry. The titer was calculated using the following formula: titer (TU / mL) = number of cells × positive rate / viral volume (mL). The titers of VHH-A039 and VHH-A051 lentiviruses were 8.76E+06 and 4.04E+07 TU / ml, respectively.

[0202] Example 7

[0203] Construction of overexpression cell lines

[0204] CEA5 overexpressing lentivirus was obtained by co-transfection with CEA5-puro plasmid, pMGlg-RRE, pRSV-REV and VSVg plasmid, following the same steps as in Example 5;

[0205] Take 1×10 6 HGC-27 and N87 cells were seeded into 6-well plates, and 1 mL of the CEA5-puro lentivirus obtained above was added to obtain HGC-27-CEA5 and N87-CEA5 cells overexpressing CEA5 protein.

[0206] The overexpressing NCA-90 lentivirus was obtained by co-transfection with NCA-90-puro plasmid, pMGlg-RRE, pRSV-REV and VSVg plasmid, and the steps were the same as in Example 6;

[0207] Take 1×10 6 One SW620 cell was seeded into a 6-well plate, and 1 mL of the NCA-90-puro lentivirus obtained above was added to obtain SW620-NCA-90 cells overexpressing NCA-90 protein.

[0208] Example 8

[0209] This embodiment uses the lentivirus-transduced T lymphocytes prepared in Example 6, and includes the following steps:

[0210] (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;

[0211] (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.

[0212] (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. Each sample was resuspended in 50 μL of 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 of buffer.

[0213] The expression rate of chimeric antigen receptors in T lymphocytes was detected by flow cytometry, and the results are as follows: Figure 7 As shown, the infection efficiencies of CAR-T cells in each group were 69.9% and 38.1%, respectively, indicating that CAR-T cells were successfully constructed.

[0214] Example 9

[0215] This embodiment performs an in vitro toxicity experiment on CAR-T cells, including the following steps:

[0216] (1) Target cell inoculation

[0217] 293T-GPF-luci (CEA5-), N87-GFP-luci-CEA5 (CEA5+), BxPC3-GFP-luci (CEA5+), SW620 (NCA-90-), and SW620-CD66c (NCA-90+) were used as target cells, and the target cell concentration was adjusted to 2 × 10⁻⁶. 5 / mL, take 50μL and inoculate it into a white opaque 96-well plate;

[0218] (2) Effector cell inoculation

[0219] CAR-T cells targeting CEA5 / 6 and control T cells were used as effector cells. The number of cells was added to 96-well plates at effector-to-target ratios of 0.3:1, 1:1 and 3:1.

[0220] (3) Each group has 2 replicates. The experimental group and the control group are as follows:

[0221] Experimental group: each target cell +T / CAR-T; Control group: only target cells were inoculated.

[0222] (5) Detection method:

[0223] 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.

[0224] (5) The formula for calculating the CAR-T killing efficiency is: Killing efficiency % = (1 - experimental group / control group) × 100%.

[0225] The results are as follows Figures 8-12 As shown, the CAR-T cells constructed in this invention have no killing effect on CEA5-negative 293T cells, but have killing activity on CEA5-positive tumor cells. They have weak killing effect on NCA-90-negative SW620 cells, but have good killing activity on NCA-90-positive tumor cells. This indicates that the CAR-T cells constructed in this invention not only have highly efficient tumor killing ability, but also have dual-target specificity.

[0226] Example 10

[0227] In this embodiment, the secretion of CAR-T cytokine IFN-γ was detected using the Human IFN-γ ELISA Kit (Lianke Biotechnology, catalog number: EK180-96).

[0228] 1. Cell culture supernatant

[0229] 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.

[0230] 2. Reagent preparation

[0231] 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.

[0232] 3. Preparation of standard products and samples

[0233] Standards: The stock solution of the standard was diluted 2 times with 5% FBS1640 medium, with a total of 8 dilution gradients, including zero concentration.

[0234] Samples: Dilute the samples using 5% FBS1640 medium as directed.

[0235] 4. Testing Steps

[0236] (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.

[0237] (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;

[0238] (3) Add sample: Add 100 μL of cell culture supernatant to the sample well;

[0239] (4) Add detection antibody: Add 50 μL of diluted detection antibody (1:100 dilution) to each well;

[0240] (5) Incubation: Seal the plate with sealing film, shake at 300 rpm, and incubate at 25°C for 2 hours;

[0241] (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.

[0242] (7) Enzyme incubation: Add 100 μL of diluted horseradish peroxidase-labeled streptavidin (1:100 dilution) to each well;

[0243] (8) Incubation: Seal the plate with a new sealing film, shake at 300 rpm, incubate at 25°C for 45 min, and then wash.

[0244] (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;

[0245] (10) Add stop solution: Add 100 μL of stop solution to each well;

[0246] (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.

[0247] The results of IFN-γ factor secretion were respectively Figure 13 , 14As 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, while co-culture with SW620 cells resulted in the release of only a small amount of IFN-γ; and co-culture with CEA5-positive and NCA-90-positive target cells resulted in CAR-T cells secreting over 1000 pg / mL of IFN-γ. The CAR-T cells constructed in this invention release high levels of cytokines against CEA5- and NCA-90-positive tumor cells, while showing no significant cytokine secretion or only a slight secretion of cytokines against CEA5- and NCA-90-negative cells.

[0248] In summary, the high-affinity CEA5 and NCA-90 bispecific nanobodies screened and prepared in this invention can efficiently bind to CEA5 and NCA-90, and can be used as antigen-binding domains to construct chimeric antigen receptors and CAR-T cells. The resulting CAR-T cells have significant killing activity and dual-target specificity against CEA5 and NCA-90 positive tumor cells, and can secrete tumor-killing cytokines. This indicates that the nanobodies of this invention can be effectively applied to immunotherapy and are of great significance for the development of tumor therapeutic drugs.

[0249] 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. A bispecific antibody against CEA5 and NCA-90, characterized in that, The CEA5 and NCA-90 bispecific antibodies are nanobodies, and the CEA5 and NCA-90 bispecific antibodies include a heavy chain variable region, which is selected from any of the following: (1) 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. 4; (2) 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. 3, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.

4.

2. The CEA5 and NCA-90 bispecific antibody according to claim 1, characterized in that, The heavy chain variable region further includes a framework region, which includes framework regions FR1 to FR4, and the amino acid sequences of framework regions FR1 to FR4 are selected from any of the following: (1) the amino acid sequences of framework regions FR1 to FR4 are shown in SEQ ID No. 5, 7, 9, 10; (2) the amino acid sequences of framework regions FR1 to FR4 are shown in SEQ ID No. 6, 8, 9, 11.

3. The CEA5 and NCA-90 bispecific antibody according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 12 or 14.

4. An isolated polypeptide, characterized in that, The polypeptide is a chimeric antigen receptor, and the polypeptide includes a transmembrane domain, an intracellular domain, and an extracellular domain, wherein the extracellular domain includes the CEA5 and NCA-90 bispecific antibody as described in any one of claims 1 to 3.

5. The polypeptide according to claim 4, characterized in that, It also includes one or more of the following features: The transmembrane domain is selected from any one or more of the CD8α transmembrane region, CD28 transmembrane region, and DAP 10 transmembrane region; The intracellular domain includes a signal transduction domain, which includes an immune receptor tyrosine activation motif and a co-stimulatory molecule. The immune receptor tyrosine activation motif is selected from CD3ζ, and the co-stimulatory molecule is selected from any one or a combination of at least two of 4-1BB, CD28, OX40, ICOS, and DAP 10. The extracellular domain includes a signal peptide, a CEA5 and NCA-90 bispecific antibody, and a hinge region. The signal peptide includes a CD8α signal peptide, and the hinge region is selected from the CD8α hinge region.

6. The polypeptide according to claim 5, characterized in that, The polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, a CEA5 and NCA-90 bispecific antibody, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB domain, and a CD3 zeta signal domain.

7. An isolated polynucleotide, characterized in that, Encoding the CEA5 and NCA-90 bispecific antibody as described in any one of claims 1 to 3 or the polypeptide as described in any one of claims 4 to 6.

8. A nucleic acid construct, characterized in that, It contains the polynucleotide encoding the isolated polynucleotide of claim 7.

9. The nucleic acid construct according to claim 8, characterized in that, The nucleic acid construct is either a retroviral vector or an adeno-associated virus vector, and the retroviral vector includes a lentiviral vector.

10. A lentivirus, characterized in that, The lentivirus is formed by viral packaging of the nucleic acid construct as described in claim 8 or 9.

11. A lentiviral vector system, characterized in that, The lentiviral vector system includes the nucleic acid construct as described in claim 8 or 9, as well as an auxiliary plasmid or host cell.

12. A chimeric antigen receptor immune cell, characterized in that, The chimeric antigen receptor immune cell expresses the isolated polypeptide as described in any one of claims 4 to 6, which is membrane-bound.

13. The chimeric antigen receptor immune cell according to claim 12, characterized in that, The immune cells are selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells, or macrophages.

14. The use of the CEA5 and NCA-90 bispecific antibody according to any one of claims 1 to 3, the isolated polypeptide according to any one of claims 4 to 6, and the chimeric antigen receptor immune cell according to any one of claims 12 to 13 in the preparation of a tumor therapeutic drug, wherein the tumor is selected from colorectal cancer, pancreatic cancer, and gastric cancer.

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