CEA5 and NCA-90 bispecific antibody and application thereof

Abstract

The invention relates to the field of biological medicines, in particular to a CEA5 and NCA-90 bispecific antibody and application thereof, the CEA5 and NCA-90 bispecific antibody comprises a heavy chain variable region, and the heavy chain variable region of the anti-CD66e antibody comprises the following technical characteristics: 1t; 1gt; comprising CDR-H1 of which the amino acid sequence is shown as SEQ ID No.1 or 2; lt; 2gt; comprising CDR-H2 with an amino acid sequence as shown in SEQ ID No.3; lt; 3gt; comprising CDR-H3 with an amino acid sequence as shown in SEQ ID No.4. The CEA5 and NCA-90 bispecific antibody provided by the invention has relatively good affinity, the CEA5 and NCA-90 bispecific antibody is used as an antigen binding structural domain to construct a chimeric antigen receptor, the chimeric antigen receptor is used for preparing a CAR-T cell, and the CAR-T cell has killing activity on CEA5 and / or NCA-90 positive tumor cells, can be effectively applied to immunotherapy, and has a good application prospect. The important significance is realized on the development of tumor treatment medicines.

Description

Technical Field

[0001] The present invention relates to the field of biological medicines, and in particular to a CEA5 and NCA-90 bispecific antibody and application thereof. Background Art

[0002] Carcinoembryonic antigen related cell adhesion molecule 5 (CEACAM5) is also called CEA or CD66e. CEA5 was first discovered in colon cancer and embryonic tissues, and was later confirmed 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. It is a broad-spectrum tumor marker. CEA5 is a member of the immunoglobulin (Ig) superfamily. It binds to the membrane through glycosylphosphatidylinositol (GPI). There are 7 immunoglobulin-like domains in the extracellular space, including a variable lgV-like domain (N domain) and six lgC-like domains. In addition, there is a 34-amino acid signal peptide before the lg domain. The human CEA5 gene is located on chromosome 19q13.2. The gene length is about 21kb, consisting of 9 exons and 3 non-coding exons. The CEA5 protein consists of 642 amino acids (molecular weight of about 70 kDa) and has 28 potential N-linked glycosylation sites. One CEA5 protein molecule contains 24-26 asparagine-linked sugar chains, and its final molecular weight is about 180 kDa.

[0003] Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), also known as CD66c and NCA-90, is a member of the immunoglobulin cell adhesion molecule CEA family. Its gene is located on chromosome 19q13.2, and the coding region consists of 6 exons. The protein it encodes contains 344 amino acid residues. NCA-90 is connected to the cell membrane through glycosylphosphatidylinositol (GPI). Generally, NCA-90 consists of an Ig variable N-terminal domain and 6 immunoglobulin constant region (IgC) type 2-like domains. These structures are anchored to the cell membrane through GPI. Although they lack intracellular domains, they still affect intracellular signaling events. Their most typical biological function is to mediate intercellular adhesion by mediating homotypic binding with other CEA family members and heterotypic 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 in the colon, stomach, pancreas, breast, female reproductive system, lung, etc. (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 lowly expressed 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 expression of NCA-90 is common in digestive system malignancies, and its expression level is often closely related to the severity of the disease and its prognosis.

[0005] Both CEA5 and NCA-90 are important carcinoembryonic antigens. Previous studies have found that CEA5 can be detected in a variety of cancers such as bladder cancer, bile duct cancer, small cell lung cancer, endometrial cancer, and gastric cancer. At present, CAR-T research targeting CEA5 for the treatment of colorectal cancer has entered the clinical stage and has achieved certain therapeutic effects. NCA-90 is also involved in the occurrence and development of a variety of malignant tumors, such as breast cancer, pancreatic cancer, colon cancer, and non-small cell lung cancer. It has the effects of promoting tumor invasion and metastasis, inhibiting tumor cell anoikis, and promoting angiogenesis at the tumor site. In addition, some studies have reported that the expression levels of CEA5 and NCA-90 in colon cancer tissues are significantly higher than those in adjacent normal tissues. Comprehensive existing studies have shown that CEA5 and NCA-90 play an important role in the treatment of colorectal cancer and other cancers. Using CEA5 and NCA-90 as therapeutic targets will be a potential treatment for colorectal cancer and other cancers. Adoptive immune cell therapy is considered a promising anti-tumor therapy. T cells expressing CAR transgenes can bind to tumor antigens through antigen-antibody recognition, thereby eliminating cancer cells. These CAR-T cells can directly recognize tumor cells without being restricted by antigen processing and MHC. The construction of dual-target CAR-T cells with CEA5 and NCA-90 may bring new hope for the treatment of cancers such as colorectal cancer.

[0006] In summary, CEA5 and NCA-90 can be used as targets for tumor treatment. Providing an antibody with high specificity and affinity against CEA5 and NCA-90 dual targets is of breakthrough significance for the treatment of malignant tumors. Summary of the invention

[0007] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a CEA5 and NCA-90 bispecific antibody and application thereof, so as to solve the problems in the prior art.

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

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

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

[0011] <3> It includes CDR-H3 with an amino acid sequence as shown in SEQ ID No.4.

[0012] The present invention also provides a separated polypeptide, which comprises a transmembrane domain, an intracellular domain and an extracellular domain, wherein the extracellular domain comprises the 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 comprising a polynucleotide encoding the isolated protein.

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

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

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

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

[0019] The anti-CEA5 and NCA-90 dual-targeted nanoantibodies provided by the present invention can efficiently bind to CEA5 and NCA-90 antigens with good affinity. They are used as antigen binding domains to construct chimeric antigen receptors, and the chimeric antigen receptors are used to prepare CAR-T cells. The CAR-T cells have killing activity against CEA5 and / or NCA-90 positive tumor cells, and after co-culture with CEA5 or NCA-90 positive cells, they efficiently secrete the cytokine IFN-γ. The nanoantibodies can be effectively used in immunotherapy, which is of great significance for the development of tumor treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram 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 diagram 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 test results of the anti-CEA5 and NCA-90 nanoantibodies binding to the CEA5 antigen in Example 4;

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

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

[0025] Figure 6 Schematic diagram of the chimeric antigen receptor structure expressing and targeting CEA5 and NCA-90 in Example 5;

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

[0027] Figure 8 This is a graph showing the killing effect of CAR-T cells on human embryonic kidney cells 293T in Example 9;

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

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

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

[0031] Fig.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] Fig.13 This is a bar graph showing the secretion of IFN-γ by CAR-T cells killing control cells and CEA5+ target cells in Example 10.

[0033] Fig.14 This is a bar graph showing the secretion of IFN-γ by CAR-T cells killing control cells and NCA-90+ target cells in Example 10. DETAILED DESCRIPTION

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

[0035] <1> comprising a CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1 or 2;

[0036] <2> comprising a CDR-H2 having an amino acid sequence as shown in SEQ ID No. 3;

[0037] <3> comprising a CDR-H3 having an amino acid sequence 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 uses the IMGT method to annotate CDR and FR regions.

[0043] CDR (complementarity determining region) usually refers to the region in an antibody that can form complementarity with an antigenic determinant in terms of spatial structure. The variability in an antibody is usually not evenly distributed throughout the variable region of the antibody. The heavy chain variable region of a monoclonal antibody usually has three hypervariable regions (HVRs), which can usually form complementarity with an antigenic determinant in terms of spatial structure, so the hypervariable region is also called the complementarity determining region (CDR), that is, the heavy chain variable region usually includes three complementarity determining regions, namely CDR-H1, CDR-H2 and CDR-H3.

[0044] In certain embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody is named VHH-A039 or A039, and the complementarity determining region of the heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID No.1, CDR-H2 with an amino acid sequence as shown in SEQ ID No.3, and CDR-H3 with an amino acid sequence as shown in SEQ ID No.4.

[0045] In certain 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 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.

[0046] The CEA5 and NCA-90 bispecific antibody is an antibody fragment and / or a monoclonal antibody.

[0047] "Antibody fragments" include a portion of an intact antibody, preferably its antigen binding region or variable region. For example, antibody fragments include nanobodies (VHH), single-chain antibodies (scFv), Fab, Fab', F(ab') or F(ab')2.

[0048] In the present invention, an unimmunized alpaca is immunized with CEA5 recombinant protein and a cell line overexpressing CEA5, a phage display nanoantibody library is constructed, and antibodies are screened based on the phage display nanoantibody library to obtain antibodies that can specifically bind to CEA5 and NCA-90 antigens.

[0049] In certain embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody is a nanobody (Nb), i.e., a heavy chain single domain antibody VHH (variable domain of heavy chain of heavy-chain antibody). Nanobodies only contain a heavy chain variable region (VHH) and CH2, CH3 regions. Compared with other antibodies, nanobody light chains are naturally absent. The nanobody crystal has a diameter of about 2.5nm and a length of about 4nm. It is the smallest naturally occurring fragment that can bind to an antigen. The CEA5 and NCA-90 bispecific antibody of the present invention only includes a heavy chain variable region, has high affinity and specificity, can efficiently target CEA5 and NCA-90 antigens, has a simple structure, is easy to prepare, and has important application value in the preparation of drugs targeting CEA5 and NCA-90.

[0050] In certain embodiments of the present invention, the heavy chain variable region may further include a framework region, which may be located between the complementary determining regions or at both ends of the complementary determining regions. In certain specific embodiments of the present invention, the sequence of the framework region is a human monoclonal antibody variable region, or a framework region sequence of a mouse monoclonal antibody variable region obtained by replacing, deleting or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence and the framework region sequence of the human monoclonal antibody variable region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology.

[0051] In certain embodiments of the present invention, the heavy chain variable region further comprises a framework region. The framework region comprises framework regions FR1 to FR4. The amino acid sequence of the framework regions FR1 to FR4 is selected from any sequence shown in SEQ ID No. 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 certain embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody VHH-A039, the CEA5 and NCA-90 bispecific antibody heavy chain variable region includes a CDR-H1 with an amino acid sequence as shown in SEQ ID No.1, a CDR-H2 with an amino acid sequence as shown in SEQ ID No.3, a CDR-H3 with an amino acid sequence as shown in SEQ ID No.4, and the amino acid sequences of the framework regions FR1 to FR4 are shown in SEQ ID Nos.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 as shown in SEQ ID No.12.

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

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

[0067] GAGGTGCAAGTGGTCGAGTCCCGGGGGGGGGCTGGTCCAAGCCGGGGGCAGCCTGAGACTCAGCTGCGCCGCTAGCGGCCTGACCCTGAACTACCACTTCGTGGCCTGGTTCAGACAAGCCCCCGGCGAGCAGAGAGAGCTCGTGGCCATCATCACCAACGGCGGGAGCACACGGTACGCCGACAG CGTGAAGGGCAGATTCACCATCAGCAGAGACAACGCCAAGAACACCATGTACCTGCAGATGAACAGCCTGAAGCCCGAGGACACCGCCGTGTACTACTGCGCCGCCGACGGCGGCAAAGTGACCCCCAAGTACTACGGCATGGACTACTGGGGCAACGGCACCCTGGTGACCGTGAGCAGC(SEQ IDNo.13)

[0068] In certain embodiments of the present invention, the heavy chain variable region of the CEA5 and NCA-90 bispecific antibody VHH-A051 includes a CDR-H1 with an amino acid sequence as shown in SEQ ID No. 2, a CDR-H2 with an amino acid sequence as shown in SEQ ID No. 3, a CDR-H3 with an amino acid sequence as shown in SEQ ID No. 4, and the amino acid sequences of the framework regions FR1 to FR4 are shown in SEQ ID Nos. 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 as shown in SEQ ID No.14.

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

[0071] In certain embodiments of the present invention, the CEA5 and NCA-90 bispecific antibody VHH-A051 is obtained by screening from a phage antibody library, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No. 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 certain embodiments of the present invention, the sequence of the polynucleotide is shown as SEQ ID No.13 or 15.

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

[0076] The therapeutic drug may be a drug that uses CEA5 or NCA-90 antigen as a target, binds to or acts on the CEA5 or NCA-90 antigen, thereby treating and / or preventing the indication.

[0077] In certain embodiments of the present invention, the therapeutic drug may be a tumor therapeutic drug. The tumor is a tumor expressing CEA5 or NCA-90. The tumor therapeutic drug may be a drug that targets the CEA5 or NCA-90 antigen on the functional surface of the tumor cell surface, binds to or acts on the CEA5 or NCA-90 antigen, thereby treating and / or preventing tumors. The tumor may be a tumor that is positive for CEA5 or NCA-90 expression, such as colorectal cancer, pancreatic cancer, breast cancer, bladder cancer, ovarian cancer, lung cancer, gastric cancer, gallbladder cancer, lung cancer, etc.

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

[0079] The chimeric antigen receptor cell therapy drug generally includes a chimeric antigen receptor cell, and the chimeric antigen receptor cell can be a chimeric antigen receptor T cell, a chimeric antigen receptor NK cell, etc. The chimeric antigen receptor T cell generally includes a T lymphocyte, which also includes a chimeric antigen receptor. The chimeric antigen receptor NK cell generally includes an NK cell, which also includes a chimeric antigen receptor. The chimeric antigen receptor includes a transmembrane domain, an intracellular domain, and an extracellular domain. In certain embodiments of the present invention, the extracellular domain includes the CEA5 and NCA-90 bispecific antibodies, that is, the chimeric antigen receptor cell can express the CEA5 and NCA-90 bispecific antibodies on the cell surface, so as to guide the cell to act on cells (such as tumor cells) expressing CEA5 or NCA-90 antigens. The cell expressing CEA5 or NCA-90 antigens can be killed by killing cells expressing CEA5 or NCA-90 antigens, etc.

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

[0081] Another aspect of the present invention provides an isolated polypeptide, which comprises a transmembrane domain, an intracellular domain and an extracellular domain, wherein the extracellular domain comprises the CEA5 and NCA-90 bispecific antibody.

[0082] In certain embodiments of the present invention, the polypeptide is a chimeric antigen receptor. In the present invention, the CEA5 and NCA-90 bispecific antibodies are used to construct a chimeric antigen receptor, and the chimeric antigen receptor can efficiently target CEA5 or NCA-90.

[0083] In certain embodiments of the present invention, the transmembrane domain can be selected from any one or more transmembrane domains of CD8α transmembrane region, CD28 transmembrane region, DAP 10 transmembrane region, etc.

[0084] For another example, the sequence of CD8α can be referenced to NM_001145873, the sequence of CD28 can be referenced to NM_006139, and the sequence of DAP10 can be referenced to NM_014266.

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

[0086] The signal transduction domain includes an immunoreceptor tyrosine-based activation motif. The immunoreceptor tyrosine-based activation motif can be selected from CD3ζ.

[0087] Preferably, the signal transduction domain also includes a costimulatory molecule. For example, the costimulatory molecule can be selected from any one of 4-1BB, CD28, OX40, ICOS, DAP 10, or a combination of at least two protein molecules. For another example, the sequence of 4-1BB can refer to NM_001561, the sequence of CD28 can refer to NM_006139, the sequence of OX40 can refer to NM_003327, the sequence of ICOS can refer to NM_012092, the sequence of CD3 zeta can refer to NM_198053, and the sequence of DAP 10 can refer to NM_014266.

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

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

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

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

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

[0093] In some specific embodiments of the present invention, the polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, 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 includes, 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, 4-1BB, and CD3 zeta.

[0095] In certain embodiments of the present invention, the polynucleotide sequence encoding the isolated polypeptide of 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 viral vector. Taking a lentiviral vector as an example, the lentiviral vector includes a vector skeleton, i.e., an empty vector and an expression framework. That is, the nucleic acid construct is a vector containing the coding gene of the chimeric antigen receptor.

[0101] The term "nucleic acid construct" or "vector" refers to a nucleic acid fragment or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into a target cell or tissue. Typically, a vector is used to introduce exogenous DNA into another cell or tissue. The vector may contain bacterial resistance genes for growth in bacteria and promoters for expressing a target protein in an organism. DNA may be produced in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.

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

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

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

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

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

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

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

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

[0110] The immune cell is selected from any one of T lymphocytes, B lymphocytes, NK cells, mast cells or macrophages.

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

[0112] The T lymphocytes can usually express the polypeptide, which can usually bind to CEA5 or NCA-90 antigens, and more specifically can bind to CEA5 or NCA-90 antigens through the extracellular domain of the CEA5 and NCA-90 bispecific antibody. When the polypeptide binds to the CEA5 or NCA-90 antigen, the T lymphocytes can usually be activated and / or stimulated to proliferate. In certain embodiments of the present invention, the T lymphocytes, i.e., chimeric antigen receptor T cells, can express the CEA5 and NCA-90 bispecific antibodies on the surface of the T lymphocytes, thereby guiding the T lymphocytes to act on cells (e.g., tumor cells) expressing the CEA5 or NCA-90 antigens, and the action can be to kill cells expressing the CEA5 or NCA-90 antigens, etc.

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

[0114] The NK cell can usually express the polypeptide and can usually bind to the CEA5 or NCA-90 antigen, more specifically, it can bind to the CEA5 or NCA-90 antigen through the extracellular domain of the CEA5 and NCA-90 bispecific antibody. When the polypeptide binds to the antigen, the NK cell can usually be activated and / or stimulated to proliferate. In certain embodiments of the present invention, the NK cell, i.e., the chimeric antigen receptor NK cell, can express the CEA5 and NCA-90 bispecific antibody on the surface of the NK cell, thereby guiding the NK cell to act on cells (e.g., tumor cells) expressing the CEA5 or NCA-90 antigen, and the action can be to kill cells expressing the CEA5 or NCA-90 antigen, etc.

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

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

[0117] In certain embodiments of the present invention, the therapeutic drug may be a tumor therapeutic drug. The tumor therapeutic drug may be a drug that targets CEA5 or NCA-90 antigens functionally expressed on the surface of tumor cells, binds to or acts on CEA5 or NCA-90 antigens, and thereby treats and / or prevents tumors. The tumor may be a tumor that is positive for CEA5 or NCA-90 expression, such as gastric cancer, lung cancer, pancreatic cancer, intestinal cancer, etc.

[0118] The present invention provides a pharmaceutical composition, which comprises the chimeric antigen receptor immune cell and / or the CEA5 and NCA-90 bispecific antibody and / or the separated polypeptide.

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

[0120] "Pharmaceutically acceptable" means that the drugs do not produce adverse, allergic or other untoward reactions when they are properly administered to animals or humans.

[0121] "Pharmaceutically acceptable carriers or excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the effect of the drug under normal circumstances. Pharmaceutically acceptable carriers or excipients are selected from any one or a combination of at least two of surfactants, disintegrants, coating materials, excipients, solubilizers, diluents, pH regulators, binders, wetting agents, colorants, emulsifiers, antibacterial agents, cosolvents, osmotic pressure regulators, fillers, antioxidants or buffers. Specific examples may be sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, etc. These substances are used as needed to help the stability of the formulation or to help improve the activity or its bioavailability or to produce an acceptable taste or smell in the case of oral administration.

[0122] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

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

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

[0125] Example 1 VHH Nanobody Screening

[0126] In this example, a phage nanobody library was constructed and panned, and ELISA was used for preliminary screening. The specific steps are as follows:

[0127] (1) Construction of phage nanoantibody library

[0128] Alpacas were immunized with human CEA5 recombinant protein and cell lines overexpressing CEA5. After the serum titer was detected by ELISA, peripheral blood was drawn. Lymphocytes were separated, total RNA was extracted, and then reverse transcribed into cDNA. Then, VHH genes were amplified by nested PCR. VHH genes were inserted into PhD-dAb-1 vectors, electroporated into SS320 competent cells, and after amplification, phages were separated and purified to obtain antibody libraries. The concentration was adjusted and the cells were aliquoted and frozen in a -80℃ refrigerator for later use.

[0129] (2) Screening of phage nanoantibody library

[0130] First, the phage antibody library was screened in two rounds, liquid phase and solid phase, respectively. Then, the screened antibody library was incubated with AsPC-1, Colo205 and SNU5 cells for negative screening, and then incubated with AsPC-1-CEA5 and Colo205-CEA5 / GFP cells for positive screening.

[0131] The phage library was separated and purified for the next round of ELISA screening. After enrichment, the obtained phage was used as a template to amplify the VHH region and perform second-generation sequencing. After screening, two nanoantibodies were unexpectedly obtained that could simultaneously bind to CEA5 and NCA-90. The amino acid sequences of the CEA5 and NCA-90 bispecific antibodies are shown in SEQ ID NOs. 12 and 14, respectively, and were named VHH-A039 and VHH-A051, respectively.

[0132] Example 2 VHH nanobody expression and purification.

[0133] In order to further identify these antibodies, the antibody quality test results obtained 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] In this example, the CEA5 and NCA-90 bispecific antibodies obtained in Example 1 were subjected to flow cytometry.

[0138] HGC-27 and HGC-27-CEA5 cells were mixed with purified nanoantibodies (1 μg / ml), and SW620 and SW620-NCA-90 cells were mixed with purified nanoantibodies (1 μg / ml), and each was incubated on ice for 30 min, and then incubated with APC-labeled anti-human IgG antibody for 30 min. The results were detected by flow cytometry. Figure 1 and 2 As shown, it is demonstrated that the CEA5 and NCA-90 bispecific antibody of the present invention can recognize CEA5 and NCA-90 antigens on the cell surface.

[0139] Example 4 Affinity detection of CEA5 and NCA-90 bispecific antibodies

[0140] In this example, the affinity of the bispecific antibody of CEA5 and NCA-90 of Example 1 was tested, and the EC50 value of its binding to CEA5 and NCA-90 proteins was determined by ELISA.

[0141] First, take two clean ELISA blank plates, wash them with PBS, discard the PBS, and pat dry on absorbent paper. Dilute Human CEA5-His (acro, cat#CE5-H5226) and Human NCA-90-His (acro, cat#CE6-H5223) tag antigens to 1 μg / ml with PBS, 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, seal the plates, and place them in a 4°C refrigerator overnight for coating.

[0142] After overnight, take out the coated ELISA plate from the refrigerator, discard the liquid, add 200μL PBS to each well, pour out the supernatant, and pat dry on absorbent paper. Add 300μL 1×NAP blocking solution to each well, incubate at room temperature for 1 hour, pour out the supernatant, and pat dry on absorbent paper. Dilute the primary antibody to 4ug / ml with 1×NAP, and dilute it 4 times in sequence, a total of 11-12 concentrations, 50μL per well. Add an equal volume of 1×NAP to the blank well as a control, seal the plate with a sealing film, incubate at room temperature for 1 hour, pour out the supernatant, and pat dry on absorbent paper. Add 300μL TBST to each well for washing, soak for 30s, pour out the liquid, pat dry on absorbent paper, and repeat washing 4 to 5 times. Dilute the secondary antibody (0.4ug / ml, Goat Anti-Human IgG HRP H&L, Cat#ab6858) with 1×NAP and add it to the reaction well, 50μL per well. Incubate at room temperature for 1 hour. Add 300 μL TBST to each well for washing. After soaking for 30 seconds, pour out the liquid, pat dry on absorbent paper, and repeat washing 4 to 5 times. Add 50 μL HRP substrate to each well and incubate at room temperature in the dark for 5 to 30 minutes. Add 50 μL 1N HCL to each well to terminate the substrate reaction. Use an enzyme reader to measure the absorbance at 450 nm and calculate the EC50 value of the 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 is 0.0219μg / mL, and the EC50 of binding to NCA-90 protein is 0.1746μg / mL, both of which are nanomolar, indicating that it can bind to CEA5 and NCA-90 proteins well. The EC50 of VHH-A051 antibody binding to CEA5 protein is 0.0234μg / mL, and the EC50 of binding to NCA-90 protein is 1.718μg / mL, both of which are nanomolar, indicating that it can bind to CEA5 and NCA-90 proteins well.

[0143] Example 5

[0144] In this example, a lentiviral vector was prepared that expresses a chimeric antigen receptor (CEA5 / NCA90 CAR) targeting both CEA5 and NCA-90.

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

[0146] Among them, the amino acid sequence of the signal peptide is:

[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 hinge and transmembrane regions of CD8α are:

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

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

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

[0153] The amino acid sequence of CD3ζ 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 react according to the PCR program shown in Table 3. The primer sequences are:

[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 10 μM upstream primer 2 10 μM downstream primer 2 Template DNA (cDNA clone) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[0167] Table 3

[0168]

[0169]

[0170] After the reaction, the PCR products were subjected to 1% agarose gel electrophoresis, and fragments of about 400 bp were recovered and quantified by UV absorption;

[0171] (2) Prepare a PCR reaction system according to Table 4, add CD8α signal peptide before the amplified product obtained in step (1), and perform the reaction according to the PCR program shown in Table 3. The primers 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 10 μM upstream primer 2 10 μM downstream primer 2 Template DNA (VHH fragment PCR reaction solution) 2 Sterile deionized water (PCR grade water) 30 KOD-Plus-Neo High-Fidelity PCR Enzyme 1

[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 UV absorption method;

[0181] (3) Prepare a PCR reaction system according to Table 5. After the preparation, perform a PCR reaction according to Table 3 to amplify the CD8αhinge-TM-41BB-CD3Z fragment. The primers used are as follows:

[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 quantify it by UV absorption method;

[0188] (4) 25 μg of the HD SIN03 GUCY2C A3-41BBz plasmid constructed in the laboratory was double-digested with BamHI and EcoRI, reacted in a 37°C water bath for 1 h, and then the vector was recovered;

[0189] The two fragments recovered above and the vector backbone were connected with the recombinase. The recombination reaction system was shown in Table 6. After the preparation, the reaction was carried out in a water bath at 37° C. for 0.5 h and transformed into Escherichia coli Stbl3 competent cells according to the conventional method.

[0190] Table 6

[0191] Reagents Usage HD CEA5 / 6CAR backbone 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 12 μL

[0192] Single clones were selected from the solid culture medium for sequencing and identification, and the results were consistent with expectations. The sequencing primer sequences are as follows:

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

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

[0195] Example 6

[0196] In this example, the lentiviral vector HD SINO3 anti-CEA5 / NCA90VHH-41BBz prepared in Example 5 was packaged, concentrated and titered, including the following steps:

[0197] (1) Lentiviral packaging

[0198] Inoculate 293T cells of appropriate concentration in a 15 cm culture dish and culture overnight at 37°C and 5% CO2 to prepare virus packaging. The culture medium is DMEM containing 10% fetal bovine serum. Dissolve 14.5 μg HD SINO3 anti-CEA5 / NCA90VHH-41BBz, 16.7 μg auxiliary plasmid pMDLg-RRE (Addgene, 12251), 16.7 μg auxiliary plasmid pRSV-REV (Addgene, 12253) and 6.5 μg envelope plasmid VSVg (Addgene, 8454) in 2 mL serum-free DMEM culture medium and mix well.

[0199] Dissolve 163.2 μg PEI (1 μg / μL) in 2 mL serum-free DMEM culture medium, vortex at 1000 rpm for 5 seconds, and incubate at 25°C for 5 minutes; add the PEI mixture to the DNA mixture, mix gently, and incubate at 25°C for 20 minutes to form a transfection complex; then add 4 mL of the transfection complex dropwise to 18 mL of DMEM culture medium containing 293T cells, replace with fresh culture medium after 6 hours; collect the supernatant of the virus solution after 48 hours;

[0200] (2) Lentiviral titer detection

[0201] 500 μL Jurkat (1×10 5 ) cells were inoculated into a 24-well culture plate, and the collected virus supernatant was added to the cell suspension in a volume of 1 μL, 5 μL and 25 μL, respectively, and polybrene was added to a final concentration of 8 μg / mL. After culturing at 37°C and 5% CO2 for 72 hours, the cells were centrifuged at 500 g for 5 minutes, the supernatant was discarded, the cells were collected, and 100 μL PBS+2% FBS was added to resuspend the cells. 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody was added to each sample, and incubated on ice for 30 minutes; flow buffer (containing 2% After washing once with FBS (PBS) buffer, 300 μL flow buffer was added to resuspend the cells, and the infection efficiency was detected by flow cytometry; the titer was calculated, and the titer calculation formula was as follows: titer (TU / mL) = number of cells × positive rate / virus volume (mL), and the titers of VHH-A039 and VHH-A051 lentiviruses were 8.76E+06 and 4.04E+07TU / ml, respectively.

[0202] Example 7

[0203] Construction of overexpression cell lines

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

[0205] Take 1×10 6 HGC-27 cells 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 overexpressing CEA5 protein.

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

[0207] Take 1×10 6 SW620 cells were inoculated into 6-well plates, 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 example uses the lentivirus prepared in Example 6 to transduce T lymphocytes, comprising the following steps:

[0210] (1) Human PBMCs were adjusted to a density of 1×10 using T cell culture medium (X-VIVO + 10% FBS + 300 U / mL IL-2). 6 / mL, add 1 / 100 volume of T Cell TransAct (commercial magnetic beads coupled to CD3 and CD28) and activate for 24h;

[0211] (2) Collect activated T cells and adjust the cell density to 1×10 6 / mL, add lentivirus at a multiplicity of infection (MOI) of 10, add polybrene to a final concentration of 8 μg / mL; culture overnight at 37°C, 5% CO2, replace with fresh medium, and passage every 3 days;

[0212] (3) 8 days after T cell infection, 3×10 5 T cells were centrifuged at 4°C and 500g for 5 min, the supernatant was discarded, and the cells were washed once with flow cytometry buffer; 50 μL of buffer was added to each sample to resuspend the cells, 0.5 μg of Rabbit Anti-Camelid VHH (iFluor488) antibody was added, and the cells were incubated on ice for 30 min; after washing once with buffer, 300 μL of buffer was added to resuspend the cells;

[0213] Flow cytometry was used to detect the expression rate of chimeric antigen receptors on T lymphocytes. Figure 7 As shown, the infection efficiency of CAR-T cells in each group was 69.9% and 38.1%, respectively, indicating that CAR-T cells were successfully constructed.

[0214] Example 9

[0215] In this example, the CAR-T cell in vitro toxicity experiment was performed, 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 5 / mL, take 50 μL and inoculate 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, and the cell numbers were added to 96-well plates at effector-target ratios of 0.3:1, 1:1, and 3:1;

[0220] (3) Each group has two replicate wells, and each experimental group and each 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 the effector cells and target cells were co-cultured for 18 h, 70 μL of Steady-Lumi TM Firefly luciferase reporter gene detection reagent (Biyuntian, catalog number: RG058M) was used for reaction for 5 min, and the bioluminescent signal was detected using a multifunctional microplate reader.

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

[0225] The results are as follows Figure 8-Figure 12 As shown, the CAR-T cells constructed by the present invention have no killing effect on CEA5-negative 293T cells, have killing activity on CEA5-positive tumor cells, have weak killing effect on NCA-90-negative SW620 cells, and have good killing activity on NCA-90-positive tumor cells, indicating that the CAR-T cells constructed by the present invention not only have efficient tumor killing ability, but also have dual-targeting specificity.

[0226] Example 10

[0227] In this example, the secretion of CAR-T cell factor IFN-γ was detected, and the kit used was Human IFN-γ ELISA Kit (Lianke Bio, catalog number: EK180-96).

[0228] 1. Cell culture supernatant

[0229] The cell culture with an effector-target ratio of 1:1 was centrifuged at 400 g for 10 min to remove the precipitate, and the supernatant was stored at -80°C for testing.

[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× detection buffer according to the instructions.

[0232] 3. Preparation of standards and samples

[0233] Standards: Use 5% FBS1640 medium to dilute the standard stock solution 2-fold, with a total of 8 dilution gradients, including zero concentration.

[0234] Sample: Dilute the sample using 5% FBS1640 medium.

[0235] 4. Detection steps

[0236] (1) Soaking the microplate: Add 300 μL of 1× washing solution and let it soak for 30 seconds. Discard the washing solution and pat the microplate dry on absorbent paper.

[0237] (2) Adding standard: Add 100 μL of 2-fold diluted standard to the standard wells, and add 100 μL of standard dilution to the blank wells;

[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 a sealing film, shake at 300 rpm, and incubate at 25°C for 2 h;

[0241] (6) Washing: Discard the liquid, add 300 μL of washing solution to each well, wash 6 times, and pat dry on absorbent paper after each wash;

[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 wash;

[0244] (9) Add substrate for color development: Add 100 μL of color development substrate TMB to each well, protect from light, and incubate at 25°C for 20 min;

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

[0246] (11) Detection reading: Within 30 min, dual-wavelength detection was performed using an ELISA reader to measure the OD values ​​at the maximum absorption wavelength of 450 nm and the reference wavelength. The calibrated OD value was the measured value at 450 nm minus the measured value at the reference wavelength.

[0247] The results of IFN-γ secretion were Fig.13 , 14As shown, the spontaneous MOCK group is a separate CAR-T cell group, and cytokine release is basically undetectable; the CAR-T cell and 293T cell co-culture group, basically no cytokines are detected, and when co-cultured with SW620 cells, only a small amount of cytokine IFN-γ is released; after co-culture with CEA5-positive and NCA-90-positive target cells, the IFN-γ secreted by CAR-T cells exceeds 1000pg / mL. The CAR-T cells constructed by the present invention release high levels of cytokines to CEA5 and NCA-90-positive tumor cells, but have no obvious cytokine secretion or only slightly secrete a small amount of cytokines to CEA5 and NCA-90-negative cells.

[0248] In summary, the high-affinity CEA5 and NCA-90 bispecific nanoantibodies screened and prepared by the present invention can efficiently bind to CEA5 and NCA-90, and use them as antigen-binding domains to construct chimeric antigen receptors and CAR-T cells. The resulting CAR-T cells have obvious killing activity and dual-targeting specificity for CEA5 and NCA-90-positive tumor cells, and can secrete tumor-killing cytokines, indicating that the nanoantibodies of the present invention can be effectively used in immunotherapy, which is of great significance for the development of tumor treatment drugs.

[0249] The above examples are for the purpose of illustrating the embodiments disclosed by the present invention and are not to be construed as limiting the present invention. In addition, the various modifications listed herein and the variations of the methods in the invention are obvious to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been specifically described in conjunction with various specific preferred embodiments of the present invention, it should be understood that the present invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of the present invention.

Claims

1. A bispecific antibody of CEA5 and NCA-90, characterized in that: The CEA5 and NCA-90 bispecific antibody includes a heavy chain variable region, and the heavy chain variable region includes the following technical features: <1> comprising a CDR-H1 having an amino acid sequence as shown in SEQ ID No. 1 or 2; <2> comprising a CDR-H2 having an amino acid sequence as shown in SEQ ID No. 3; <3> It includes 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 also includes a framework region, and the framework region includes framework regions FR1 to FR4. The amino acid sequences of the framework regions FR1 to FR4 are shown in any of SEQ ID No. 5 to 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. The CEA5 and NCA-90 bispecific antibody according to claim 1, characterized in that: The CEA5 and NCA-90 bispecific antibody is a nanobody.

5. An isolated polypeptide, characterized in that The polypeptide comprises a transmembrane domain, an intracellular domain and an extracellular domain, and the extracellular domain comprises the CEA5 and NCA-90 bispecific antibody according to any one of claims 1 to 4.

6. The polypeptide according to claim 5, characterized in that Also includes any one or more of the following features: a) the polypeptide is a chimeric antigen receptor; b) the transmembrane domain is selected from any one or more of the CD8α transmembrane region, the CD28 transmembrane region, and the DAP 10 transmembrane region; c) the intracellular domain includes a signal transduction domain; preferably, the signal transduction domain includes an immunoreceptor tyrosine-based activation motif and a co-stimulatory molecule; more preferably, the immunoreceptor tyrosine-based activation motif is selected from CD3ζ; preferably, the co-stimulatory molecule is selected from any one of 4-1BB, CD28, OX40, ICOS, and DAP 10, or a combination of at least two thereof; d) the extracellular domain comprises a signal peptide, a CEA5 and NCA-90 bispecific antibody, and a hinge region; preferably, the signal peptide comprises a CD8α signal peptide; preferably, the hinge region is selected from a CD8α hinge region; e) The polypeptide includes, from N-terminus to C-terminus, a CD8α signal peptide, a CEA5 and NCA-90 bispecific antibody, a CD8α hinge region, a CD8α transmembrane region, a co-stimulatory molecule, and a CD3 zeta signal domain.

7. An isolated polynucleotide, characterized in that Encoding the CEA5 and NCA-90 bispecific antibody according to any one of claims 1 to 4 or the polypeptide according to any one of claims 5 to 6.

8. A nucleic acid construct, characterized in that Contains a polynucleotide encoding the isolated protein according to claim 7.

9. The nucleic acid construct according to claim 8, characterized in that The nucleic acid construct is any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector.

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

11. A lentiviral vector system, characterized in that: The lentiviral vector system comprises the nucleic acid construct of claim 8 or 9 and a helper plasmid or a host cell.

12. A chimeric antigen receptor immune cell, characterized in that: The chimeric antigen receptor immune cell expresses the membrane-bound isolated polypeptide according to any one of claims 5 to 6.

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

14. Use of the CEA5 and NCA-90 bispecific antibody according to any one of claims 1 to 4, the isolated polypeptide according to any one of claims 5 to 6, or the chimeric antigen receptor immune cell according to any one of claims 12 to 13 in the preparation or screening of tumor therapeutic drugs.

15. The use according to claim 14, characterized in that The tumor is a tumor expressing CEA5 and / or NCA-90; preferably, the tumor is selected from liver cancer, colorectal cancer, melanoma, pancreatic cancer, thyroid cancer, breast cancer, bladder cancer, ovarian cancer, lung cancer, gastric cancer or gallbladder cancer.

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