Nectin-4-targeting nano antibody, chimeric antigen receptor CAR and application

Abstract

The invention belongs to the technical field of biology, and provides a nanometer antibody targeting Nectin-4, and a nucleic acid molecule and an expression vector for coding the nanometer antibody targeting Nectin-4. The invention also provides a chimeric antigen receptor which comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular domain, and the extracellular antigen binding domain is a Nectin-4 nano antibody; the invention also provides a chimeric antigen receptor CAR which contains the nano antibody as scFv and co-expresses CXCR1, a CAR-T cell constructed by the chimeric antigen receptor CAR, and an enhanced CAR-T cell obtained by modifying the CAR-T cell with microbial short-chain fatty acid, so as to improve the infiltration and anti-tumor activity of the CAR-T cell targeting Nectin-4.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a nano antibody targeting nectin-4, a chimeric antigen receptor CAR and applications thereof. Background Art

[0002] Chimeric antigen receptor T cell (CAR-T) therapy has shown significant therapeutic effects in hematological malignancies. However, the application of CAR-T cells in the treatment of solid tumors remains a challenge due to the complex inhibitory tumor microenvironment, target defects, lack of specific antigens, poor delivery, and continuous stimulation of antigens leading to T cell exhaustion.

[0003] Most candidate target antigens are usually co-expressed on non-malignant tissues and lack highly tumor-specific cell surface targets, which poses a risk of low off-tumor / on-target toxicity. 2+ Nectin-4 is an immunoglobulin-like protein that is mainly involved in cell-cell adhesion. It is mostly expressed in placenta and embryonic tissues, and rarely expressed in healthy adult tissues and serum. However, it is highly expressed in some tumor cells, such as urothelial carcinoma, breast cancer, skin cancer, gastric cancer, esophageal cancer, cervical cancer, prostate cancer and many other cancers. Given that Nectin-4 is limitedly expressed in normal human tissues and overexpressed in some solid tumors, Nectin-4 has attracted much attention as a new target for the treatment of solid tumors.

[0004] Nanobody (Nb) is an artificially designed antibody molecule, also known as single-domain antibodies (sdAbs), VHH antibodies or camelid antibodies. In 1993, Belgian scientists discovered a heavy chain antibody with a natural light chain deletion in camel serum, with a molecular weight of about 95kDa, including two constant regions (CH2 and CH3), a hinge region and a variable heavy chain domain (VHH). Then they cloned a single domain antibody containing only one heavy chain variable region, namely VHH antibody. The crystal structure of VHH antibody is an oval of 4nm×2.5nm×3nm, and the molecular weight is only 1 / 10 of that of ordinary antibodies, about 12-14kDa. It is the smallest complete antigen-binding fragment, so it is also called nanobody. Nanobody (VHH) has the same structural domains as ordinary antibody VH, namely 4 conserved framework regions (FR1 / 2 / 3 / 4) and 3 complementary determining regions (CDR1 / 2 / 3).

[0005] The molecular weight of nanobodies is only 10% of that of traditional antibodies, and they retain the complete antigen binding ability of HCAbs, with strong specificity, good affinity, and high stability. They are widely used in biochemical mechanism research, structural biology, and diagnosis and treatment of diseases such as tumors. Most of the targeted monoclonal antibodies developed in the prior art are single-chain antibodies, which contain heavy chains and light chains. Although single-chain antibodies have the characteristics of small molecular weight, strong penetration, and weak antigenicity compared to traditional antibodies, nanobodies have excellent chemical and physical properties such as higher solubility, stability, smaller size, and lower production costs compared to single-chain antibodies.

[0006] The migration and persistence of CAR-T cells are essential factors for durable clinical responses in cancer patients. The active transport of T cells to tumor masses depends in part on the compatibility between chemokines in the tumor and chemokine receptors presented on T cells, so mismatched tumor-induced chemokines / chemokine receptors are considered to be one of the key factors limiting T cell infiltration. IL-8 is a chemokine that participates in the formation of the immunosuppressive environment of the tumor microenvironment, promotes tumor cell mesenchymalization and angiogenesis, and enhances tumor metastasis. It is a major obstacle to anti-tumor therapy. CXCR1 and CXCR2 are IL8 receptors, and IL-8 produced by tumors is used to guide IL-8 receptor-modified CAR-T cells to migrate into tumors and induce enhanced anti-tumor responses in solid tumors.

[0007] In addition, the intestinal microbiota has been shown to directly affect the efficacy of specific cancer immunotherapy. Acetate, propionate, and butyrate are the main intestinal microbial metabolites and belong to the short-chain fatty acids (SCFAs). Studies have shown that short-chain fatty acids can promote cell proliferation and improve the function of effector T cells. However, there is no clear report on whether short-chain fatty acids can directly affect the therapeutic effect of CAR-T cells on solid tumors.

[0008] Therefore, combining microbial short-chain fatty acids with CXCR1-modified CAR-T cells, innovating and developing new CAR-T cell drugs, and improving the infiltration and anti-tumor activity of CAR-T cells targeting Nectin-4 are of great significance and have broad application value for improving clinical efficacy. It provides treatment options for patients with advanced tumors, especially triple-negative breast cancer patients, promotes the commercialization of CAR-T cells, and benefits more patients. Summary of the invention

[0009] The purpose of the present invention is to overcome the technical defect of poor efficacy of CAR-T cells in solid tumors, provide a microbial short-chain fatty acid-treated and CXCR1-modified CAR-T cell targeting Nectin-4, and prove its therapeutic effect. Although the patent of this invention takes the killing of breast cancer cells as an example, the drug is also applicable to other tumor types expressing Nectin-4, such as urothelial carcinoma, breast cancer, skin cancer, gastric cancer, esophageal cancer, cervical cancer, prostate cancer, etc.

[0010] The first object of the present invention is to provide a nanobody targeting nectin-4, wherein the nanobody targeting nectin-4 comprises a complementary determining region, wherein the complementary determining region consists of CDR1, CDR2 and CDR3; the amino acid sequence of the CDR1 is shown in SEQ ID NO.2; the amino acid sequence of the CDR2 is shown in SEQ ID NO.3; and the amino acid sequence of the CDR3 is shown in SEQ ID NO.4.

[0011] SEQ ID NO.2: PTFNIYA;

[0012] SEQ ID NO.3: LVLVVP;

[0013] SEQ ID NO.4: VRGIYYE;

[0014] Furthermore, the nanobody targeting nectin-4 also includes framework regions FR1, FR2, FR3 and FR4; the amino acid sequence of the FR1 is shown in SEQ ID NO.5; the amino acid sequence of the FR2 is shown in SEQ ID NO.6; the amino acid sequence of the FR3 is shown in SEQ ID NO.7; and the amino acid sequence of the FR4 is shown in SEQ ID NO.8.

[0015] SEQ ID NO.5 sequence is: GVTLVESGGALVKPGGSLKISCAASG;

[0016] SEQ ID NO.6 sequence: MSWVRQTPEKRLEWVAS;

[0017] SEQ ID NO.7 sequence is:

[0018] LSRQCEDRFTISRDSARNILYLQMNSLRSEDTAMYYC;

[0019] The sequence of SEQ ID NO.8 is: YWGQGTTLTVSS.

[0020] Furthermore, the full-length amino acid sequence of the antibody is shown in SEQ ID NO.1.

[0021] SEQ ID NO.1 sequence is:

[0022] GVTLVESGGALVKPGGSLKISCAASGPTFNIYAMSWVRQTPEKRLEWVAS LVLVVPLSRQCEDRFTISRDSARNILYLQMNSLRSEDTAMYYCVRGIYYEYW GQGTTLTVSS.

[0023] The second object of the present invention is to provide an isolated nucleic acid, which comprises a nucleic acid sequence encoding the above-mentioned nanoantibody targeting nectin-4 or its complementary sequence. Its nucleic acid sequence is SEQ ID NO.9: GGAGTGACACTGGTGGAGTCGGGGGGAGCCTTAGTGAAGCCTGGAGGGTCCCTGAAGATCTCTTGTGCAGCCTCTGGACCCACTTTCAATATCTATGCCATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCCATTGGTACTTGTGGTCCCACCTATTATCCAGACAGTGTGAAGATCGATTCACCATCTCCAGAGATAGTGCCAGGAACATCCTGTATCTGCAAATGAACAGTCTGAGGTCTGAAGACACGGCCATGTATTACTGTGTAAGAGGCATCTACTATGAGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCG.

[0024] The third object of the present invention is to provide an expression vector or host cell comprising the isolated nucleic acid. The expression vector contains the nucleic acid. The host cell contains the expression vector.

[0025] The fourth object of the present invention is to provide a chimeric antigen receptor CAR comprising the above-mentioned nanobody, that is, the above-mentioned nanobody is used as the antigen recognition domain (scFv) of the chimeric antigen receptor CAR, and constitutes an extracellular domain with a hinge domain (Hinge), and the chimeric antigen receptor CAR also includes a transmembrane domain, an intracellular domain and a co-expressed CXCR1 chemokine receptor; the intracellular domain includes a co-stimulatory domain and a signal transduction domain;

[0026] The transmembrane domain is selected from one or more transmembrane domains of CD3ζ, CD4, CD5, CD8, CD8α, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, ICOS, CD134, CD137, CD154, NKG2D, 2B4 and DNAM1. Preferably, the transmembrane domain is NKG2D, and accordingly, its nucleotide base sequence is preferably the sequence shown in SEQ ID NO.10: CCATTTTTTTTCTGCTGCTTCATCGCTGTAGCCATGGGAATCCGTTTCATTA TTATGGTAGCA.

[0027] The costimulatory domain is selected from: CD27, CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4, CD5, CD28, CD30, CD40, CD134, CD137, ICOS, CD154, 4-1BB, OX40, CD7, LIGHT, NKG2C, B7-H3, DAP10, DAP12, NKp44, NKG2D, NKp46, NKp30, TNFR family or SLAM-related receptor family such as 2B4 or one or more combinations. Among them, TNFR family refers to any one of 4-1BB, OX40 and CD27. And preferably, the costimulatory domain uses 4-1BB, and accordingly, the nucleotide sequence of the expression gene of the intracellular costimulatory domain is shown in SEQ ID NO.11:

[0028] AGGTTCAGTGTCGTGAAGAGAGGCCGGAAGAAGCTGCTGTACATCTT CAAGCAGCCTTTCATGAGGCCCGTGCAGACTACCCAGGAGGAAGATGGAT GCAGCTGTAGATTCCCTGAAGAGGAGGAAGGAGGCTGTGAGCTGAGA.

[0029] Preferably, the signal transduction domain is CD3ζ, and correspondingly, the nucleotide sequence of the expression gene of the signal transduction domain is shown in SEQ ID NO.12:

[0030] GTGAAGTTCTCCCGAAGCGCAGATGCCCCAGCCTATCAGCAGGGACAGAA

[0031] TCAGCTGTACAACGAGCTGAACCTGGGAAGACGGGAGGAATACGATGTG

[0032] CTGGACAAAAGGCGGGGCAGAGATCCTGAGATGGGCGGCAAACCAAGAC

[0033] GGAAGAACCCCCAGGAAGGTCTGTATAATGAGCTGCAGAAAGACAAGAT

[0034] GGCTGAGGCCTACTCAGAAATCGGGATGAAGGGCGAAAGAAGGAGAGGA

[0035] AAAGGCCACGACGGACTGTACCAGGGGCTGAGTACAGCAACAAAAGACACCTATGACGCTCTGCACATGCAGGCTCTGCCACCAAGA。

[0036] The nucleotide sequence of the co-expressed CXCR1 chemokine receptor expression gene is shown in SEQ ID NO.13:

[0037] ATGTCAAATA TTACAGATCC ACAGATGTGG GATTTTGATG ATCTAAAT

[0038] TT CACTGGCATGCCACCTGCAG ATGAAGATTA CAGCCCCTGT ATGCTA

[0039] GAAA CTGAGACACT CAACAAGTATGTTGTGATCA TCGCCTATGC CCT

[0040] AGTGTTC CTGCTGAGCC TGCTGGGAAA CTCCCTGGTGATGCTGGTCA T

[0041] CTTATACAG CAGGGTCGGC CGCTCCGTCA CTGATGTCTA CCTGCTGAA

[0042] CCTGGCCTTGG CCGACCTACT CTTTGCCCTG ACCTTGCCCA TCTGGGC

[0043] CGC CTCCAAGGTGAATGGCTGGA TTTTTGGCAC ATTCCTGTGC AAGGT

[0044] GGTCT CACTCCTGAA GGAAGTCAACTTCTACAGTG GCATCCTGCT GTT

[0045] GGCCTGC ATCAGTGTGG ACCGTTACCT GGCCATTGTCCATGCCACAC G

[0046] CACACTGAC CCAGAAGCGT CACTTGGTCA AGTTTGTTTG TCTTGGCTG

[0047] CTGGGGACTGT CTATGAATCT GTCCCTGCCC TTCTTCCTTT TCCGCCAG

[0048] GC TTACCATCCAAACAATTCCA GTCCAGTTTG CTATGAGGTC CTGGGA

[0049] AATGACACAGCAAAATGGCGGATGGTGTTGCGGA TCCTGCCTCA CACCT

[0050] TTGGC TTCATCGTGC CGCTGTTTGT CATGCTGTTCTGCTATGGAT TCAC

[0051] CCTGCG TACACTGTTT AAGGCCCACA TGGGGCAGAA GCACCGAGCCA

[0052] TGAGGGTCA TCTTTGCTGT CGTCCTCATC TTCCTGCTTT GCTGGCTGCC

[0053] CTACAACCTGGTCCTGCTGG CAGACACCCT CATGAGGACC CAGGTGA

[0054] TCC AGGAGAGCTG TGAGCGCCGC AACAACATCG GCCGGGCCCT GGA

[0055] TGCCACT GAGATTCTGG GATTTCTCCA TAGCTGCCTCAACCCCATCA T

[0056] CTACGCCTT CATCGGCCAA AATTTTCGCC ATGGATTCCTCAAGATCCTG

[0057] GCTATGCATGGCCTGGTCAGCAAGGAGTTCTTGGCACGTCATCGTGTTAC

[0058] CTCCTACACTTCTTCGTCTG TCAATGTCTC TTCCAACCTC

[0059] The fifth object of the present invention is to provide a CAR-T cell expressing the above-mentioned chimeric antigen receptor, or to provide an enhanced CAR-T cell expressing the above-mentioned chimeric antigen receptor and treated with short-chain fatty acids.

[0060] The sixth object of the present invention is to provide a method for preparing the above-mentioned CAR-T cells and enhanced CAR-T cells, which specifically comprises the following steps:

[0061] Step S1, peripheral blood of healthy donors is obtained, PBMCs are separated, and activated by CD3 / CD28 stimulation.

[0062] Step S2, 24 hours later, infecting the activated T cells with a lentivirus carrying the chimeric antigen receptor gene;

[0063] Step S3, the infected T cells are replaced with fresh culture medium after 24 hours. If only cytokines are added and cultured for a further 72 hours, CAR-T cells expressing chimeric antigen receptors are obtained; if cytokines and a certain concentration of short-chain fatty acids are added and cultured for a further 72 hours, the enhanced CAR-T cells are obtained.

[0064] Wherein, the cytokine is a mixture of IL7, IL15 and IL21. Preferably, the cytokine is a mixture of IL7 with a concentration of 10 ng / ml, IL15 with a concentration of 15 ng / ml, and IL21 with a concentration of 15 ng / ml.

[0065] The short-chain fatty acid is acetic acid, propionic acid, butyric acid or valeric acid. Preferably, the concentration of the short-chain fatty acid is 0.25-2.5 mM, and more preferably, the concentration of the short-chain fatty acid is 0.5-1.0 mM.

[0066] The seventh purpose of the present invention is to provide the use of the above-mentioned nanoantibodies, chimeric antigen receptors CAR, or CAR-T cells in the preparation of drugs or products against Nectin-4 cell malignant tumors or Nectin-4 cell proliferative diseases.

[0067] Compared with the prior art, the beneficial technical effects achieved by the present invention are as follows:

[0068] (1) The nanoantibodies or antigen-binding fragments thereof targeting nectin-4 provided in the present application have good binding ability with nectin-4 or tumor cells expressing nectin-4, and have high specificity and affinity.

[0069] (2) The CAR-T formed based on the nanoantibodies or antigen-binding fragments thereof that specifically bind to nectin-4 provided in the present application can migrate to tumor cells, solving the problem of lack of T cell infiltration into the tumor site;

[0070] (3) Nectin-4-targeting CAR-T cells treated with microbial short-chain fatty acids and co-expressing CXCR1 can significantly inhibit tumor cell proliferation, reduce tumor volume, and improve survival rate, thereby more effectively treating diseases related to abnormal Nectin-4 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 Indicates the recognition ability of NCT antibody and commercial Nectin-4-abcam antibody against tumor cell lines (ABC-1, MCF-1, HCI-H441) that endogenously express Nectin-4;

[0072] Figure 2A Schematic diagram of the structure of NCT-CXCR-CAR-T;

[0073] Figure 2B It is a lentiviral DNA vector of CAR against Nectin-4;

[0074] Figure 3 The expression efficiency of four different groups of cells: A, B, C, and D;

[0075] Figure 4 The cell proliferation of four different groups of cells, A, B, C, and D, within 12 days;

[0076] Figure 5 The ABC-1 cleavage after the four groups of cells were co-incubated with ABC-1 endogenously expressing nectin-4 at different effector-target ratios;

[0077] Figure 6 The lysis of MCF-1 after the four groups of cells were co-incubated with MCF-1 endogenously expressing nectin-4 at different effector-target ratios;

[0078] Figure 7 The expression of cytokine TNF-α in the supernatant of ABC-1 in each group and each effector-target ratio;

[0079] Figure 8 The expression of cytokine TNF-α in the supernatant of MCF-1 in each group and each effector-target ratio;

[0080] Fig. 9 The expression of cytokine IFN-γ in the supernatant of ABC-1 in each group and each effector-target ratio;

[0081] Fig.10 The expression of cytokine IFN-γ in the supernatant of MCF-1 in each group and each effector-target ratio. DETAILED DESCRIPTION

[0082] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with specific embodiments and accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are not intended to limit the protection scope of the present invention.

[0083] Example 1 Screening and purification of human nanobodies against nectin-4

[0084] The human nanobody against nectin-4 provided in the present application is based on the heavy chain variable region amino acid sequence of the existing monoclonal antibody against nectin-4, such as the sequence shown in SEQ ID NO.14, to construct a mutant library, and then use phage display technology to screen the optimal mutant antibody sequence from the mutant antibody library. The sequence shown in SEQ ID NO.14 is specifically:

[0085] EVTLVESGGGLVKPGGSLKISCAASGFTFNSYAMSWVRQTPEKRLEWVASISTGGPTYYPDSVKGRFTISRDSARNILYLQMNSLRSEDTAMYYCARGLYYTDYWGQGTTLTVSS

[0086] The amino acid sequence of the screened anti-nectin-4 human nanobody is shown in SEQ ID NO.1:

[0087] GVTLVESGGALVKPGGSLKISCAASGPTFNIYAMSWVRQTPEKRLEWVAS LVLVVPLSRQCEDRFTISRDSARNILYLQMNSLRSEDTAMYYCVRGIYYEYW GQGTTLTVSS;

[0088] The specific screening process of human nanoantibodies against Nectin-4 is as follows:

[0089] (1) Using error-prone PCR technology, a target gene fragment based on the sequence shown in SEQ ID NO. 14 was amplified, and then agarose gel electrophoresis was used to recover the amplified target fragment.

[0090] (2) Connect the target gene fragment to the pCANTAB5E vector after restriction digestion. According to the requirements of the PCR reaction system, add the target gene fragment and vector at a molar ratio of 4:1, T4 ligase, 10x buffer and sterile water to the reaction system, and place it on the PCR instrument overnight to connect the target gene to the vector after restriction digestion. Use the DNA purification recovery kit to recover the ligation product according to the instructions and dissolve it in ultrapure water.

[0091] (3) Transformation of TG1: Pre-cool the sterile electroporation cup on ice, add 200ng of the recovered ligation product to the thawed 100μl TG1 competent medium, transfer to the pre-cooled electroporation cup, and electroporate at 2.5KV for 6ms. Quickly add 1ml SOC medium and incubate at 37℃ for 2 hours; dilute the suspension after gradient culture, spread on SOBAG plates for overnight growth, and calculate the library capacity.

[0092] (4) The antibody library obtained above was inoculated into 2×YT medium, and cultured at 37°C, 220rpm with shaking until OD600 reached 0.5. After adding helper phage at 10 times the volume, continue to culture for 1 hour. After centrifugation at 4000rpm and 4°C for 15 minutes, the supernatant was discarded, and 200ml of culture medium containing ampicillin and kanamycin was added to resuspend the bacterial liquid. After the resuspended liquid was cultured at 37°C for 2 hours, it was centrifuged at 13000rpm for 10 minutes, the supernatant was transferred to a new centrifuge tube and 1 / 4 volume of pre-cooled 5*PEG8000 / NaCl was added, and incubated on ice overnight. Centrifuged at 13000rpm and 4°C for 10 minutes, the supernatant was discarded, and the precipitate was resuspended with 1ml of culture medium. After adding 250 μl 5X PEG8000 / NaCl again, incubate on ice for 10 min, centrifuge at 16000 g for 15 min at 4°C, discard the supernatant, dissolve the precipitate in 1 ml PBS to obtain the phage library, and store it at -20°C for future use.

[0093] (5) The obtained phages were graded diluted, infected with TG1 bacteria, coated on SOBAG plates, and the phage library titer was calculated by colony counting.

[0094] (6) Through "adsorption-elution-amplification", antibodies were selected from the phage-displayed antibody library, and finally a nano-antibody with high killing function targeting Nectin-4 was screened out, which was named NCT-1, i.e., the human nano-antibody against Nectin-4 with the sequence shown in SEQ ID NO.1.

[0095] Example 2 NCT-1 affinity verification

[0096] The purified NCT-1 antibody corresponding to the nucleic acid sequence of the gene fragment shown in SEQ ID NO.9 and the commercial Abcam Nectin-4 antibody gene fragment were respectively combined with breast cancer cells expressing Nectin-4, such as ABC-1, MCF-1, and HCI-H441, and the binding activity of NCT-1 was verified by flow cytometry. The test results are as follows Figure 1 As shown in the figure, the NCT-1 antibody has similar and better affinity to the commercial Nectin-4 antibody, which proves that the screened antibody NCT-1 can well recognize the Nectin-4 target.

[0097] Example 3 Construction of Lentivirus CAR Plasmid

[0098] The nano antibody NCT-1 gene fragment obtained above was combined with the NKG2D transmembrane domain, 4-1BB, CD3ζ co-stimulatory domain, and CXCR1 receptor to construct a CAR structure NCT-CXCR-CAR-T targeting Nectin-4, with the structure as shown Figure 2A As shown. The CAR structure is connected to the plasmid vector, and the vector structure Figure 2B The plasmid was commissioned to Anhui General Biotechnology Co., Ltd. for construction. After competent transformation and identification, the plasmid was extracted using the ZymoPURE endotoxin-free plasmid extraction kit for subsequent stable cell line construction.

[0099] Example 4 Lentivirus-mediated cell transfection

[0100] The constructed BSTN#1 plasmid was used as the target plasmid and the virus was packaged using the four-plasmid lentiviral packaging system of our laboratory. 24 hours before transfection, the density of 293F suspension cells that had been passed for more than 3 generations was adjusted to 4.0-5.0×10 6 After overnight growth, the cells were diluted to 4.7 × 10 cells / ml with fresh medium. 6 The prepared DNA / transfection reagent complex was slowly transferred to a shaking flask under 8% CO 2 Cultivate under shaking conditions for 5-6 hours, add 4% transfection enhancer, and continue culturing. Collect the supernatant 48-55 hours after transfection, centrifuge at 5000rpm and 4℃ for 10 minutes. Discard the precipitate, filter the supernatant with a 0.45um filter membrane, add it to an ultra-high-speed centrifuge tube containing sucrose solution, and centrifuge it at 4℃ and 25000rpm for 2 hours. Discard the supernatant, resuspend the precipitate with PBS without calcium and magnesium to obtain the virus suspension, and freeze it at -80℃; use QPCR to detect the virus titer, and the virus titer can reach 10 8 TU / ml.

[0101] Example 5 Preparation of CXCR1-modified nectin-4-targeted CAR-T cells treated with microbial short-chain fatty acids

[0102] The experiment was divided into four groups:

[0103] A: T cell group; B: Nectin-4 single chain antibody group; C: Nectin-4 nano antibody group; D: Nectin-4 nano antibody + valeric acid culture group;

[0104] It should be noted that the scFv of the CAR-T cells in group B was the Nectin-4 single-chain antibody; the scFv of the CAR-T cells in groups C and D were both the Nectin-4 nanoantibodies screened in the present invention; the only difference between group D and group C was that valeric acid with a final concentration of 1.0 mM was added to the culture system of group D for continued culture;

[0105] The experimental steps are as follows:

[0106] (1) Peripheral blood was obtained from healthy volunteers and PBMCs were isolated using lymphocyte separation technique;

[0107] (2) After adding CD3 / CD28 (200 ng / ml) to stimulate each group A, B, C, and D for 24 hours, group A was left untreated, while groups B, C, and D were transduced with the corresponding viruses;

[0108] (3) Serum must be removed during transduction. 24 hours after transduction, fresh X-VIVO15 medium containing IL7 (10 ng / ml), IL15 (20 ng / ml), IL21 (20 ng / ml), and 5% serum was used for continued culture;

[0109] (4) After 72 hours, valeric acid was added to the culture system of group D at a final concentration of 1.0 mM, and the culture was continued for 72 hours.

[0110] (5) Use flow cytometry to detect the infection efficiency and proliferation capacity of CAR-T cells.

[0111] The infection efficiency of CAR-T cells is as follows Figure 3 As shown in Figure 2, the expansion capacity of CAR-T cells is as follows Figure 4 As shown in the figure, Donor1 and Donor2 represent the experimental results of two repeated samples. Among them, the infection efficiency of groups B, C, and D were 51.93%, 63.43%, and 66.73%, respectively. There was no significant difference in the expansion ability of CAR-T cells among the three groups.

[0112] Example 6 Killing of breast cancer cells by CXCR1-modified nectin-4-targeted CAR-T cells treated with microbial short-chain fatty acids

[0113] Breast cancer cell lines (ABC-1 and MCF-1) in the logarithmic growth phase and highly expressing nectin-4 were co-incubated with four groups of cells, A, B, C, and D, at different effector-target ratios including 1:5, 1:2, 1:1, 2:1, 5:1, and 10:1. The killing efficacy of CAR-T was detected by cell lysis experiments and the release of cytokines IFN-γ and TNF-α in the supernatant. The results are shown in Figures 5 to 10 As shown, Figure 5 As shown, the ABC-1 cleavage of four groups of cells after co-incubation with ABC-1 endogenously expressing nectin-4 at different effector-target ratios; Figure 6 The lysis of MCF-1 cells after co-incubation with MCF-1 cells endogenously expressing nectin-4 at different effector-target ratios shown in the figure; Figure 5 and Figure 6 From the results shown: the cytotoxicity of groups B, C, and D was significantly higher than that of the control T cell group, i.e., group A; and further, compared with group B, group C showed that the nectin-4 nanoantibody screened in the present application had a better cancer cell killing effect than the single-chain antibody targeting nectin-4 developed in the prior art; compared with group C, group D showed that the enhanced CAR-T cells treated with short-chain fatty acids showed a stronger killing effect than the CAR-T cells not treated with microbial short-chain fatty acids.

[0114] like Figure 7 Shown is the expression of cytokine TNF-α in the supernatant of ABC-1 in each group and at each effector-target ratio; Figure 8 Shown is the expression of cytokine TNF-α in the supernatant of MCF-1 in each group and at each effector-target ratio; Fig. 9 The expression of cytokine IFN-γ in the supernatant of ABC-1 in each group and each effector-target ratio is shown; Fig.10 The expression of cytokine IFN-γ in the supernatant of MCF-1 cells in each group and at each effector-target ratio is shown. Figure 7-10 The results of cytokine release further verified the above conclusions, indicating that the CAR-T cells targeting Nectin-4 and co-expressing CXCL1 treated with microbial short-chain fatty acids in this application have stronger cytotoxic effects. It can also be proved that the damage to cancer cells is indeed related to the enhancement of the killing ability of CAR-T cells, which is a direct result of the increase in the release of cytokines IFN-γ and TNF-α in the supernatant.

Claims

1. A nanobody targeting nectin-4, characterized in that: The nanobody includes a heavy chain variable region, which includes three complementary determining regions consisting of CDR1, CDR2 and CDR3, the amino acid sequence of CDR1 is shown in SEQ ID NO.2, the amino acid sequence of CDR2 is shown in SEQ ID NO.3, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

4.

2. The nanobody targeting Nectin-4 according to claim 1, characterized in that: The nanobody also includes framework regions FR1, FR2, FR3 and FR4; the amino acid sequence of the FR1 is shown in SEQ ID NO.5; the amino acid sequence of the FR2 is shown in SEQ ID NO.6; and the amino acid sequence of the FR3 is shown in SEQ ID NO.

7.

3. The nanobody targeting nectin-4 according to claim 1, characterized in that: The full-length amino acid sequence of the nanobody is shown in SEQ ID NO.

1.

4. An isolated nucleic acid, characterized in that The nucleic acid comprises a nucleic acid sequence encoding the Nanobody according to any one of claims 1 to 3 or a complementary sequence thereof.

5. The nucleic acid according to claim 4, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

8.

6. An expression vector or host cell comprising the isolated nucleic acid of claim 5.

7. A chimeric antigen receptor CAR comprising the Nanobody according to any one of claims 1 to 3, characterized in that: The nanobody serves as the antigen recognition domain of the chimeric antigen receptor CAR and constitutes an extracellular domain with the hinge domain. The chimeric antigen receptor CAR also includes a transmembrane domain, an intracellular domain and a co-expressed CXCR1 chemokine receptor; the intracellular domain includes a co-stimulatory domain and a signal transduction domain.

8. The chimeric antigen receptor CAR according to claim 7, characterized in that: The transmembrane domain is selected from one or more transmembrane domains of CD3ζ, CD4, CD5, CD8, CD8α, CD9, CD16, CD19, CD22, CD28, CD33, CD37, CD45, ICOS, CD134, CD137, CD154, NKG2D, 2B4 and DNAM1; the co-stimulatory domain is selected from CD27, CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD2, CD4 , CD5, CD28, CD30, CD40, CD134, CD137, ICOS, CD154, 4-1BB, OX40, CD7, LIGHT, NKG2C, B7-H3, DAP10, DAP12, NKp44, NKG2D, NKp46, NKp30, one or more combinations of TNFR family or SLAM-related receptor family such as 2B4, wherein TNFR family refers to any one of 4-1BB, OX40 and CD27; the signal transduction domain is CD3ζ.

9. A CAR-T cell expressing the chimeric antigen receptor CAR of claim 7, or an enhanced CAR-T cell expressing the chimeric antigen receptor of claim 7 and treated with short-chain fatty acids.

10. Use of the nanobody according to any one of claims 1 to 3, the chimeric antigen receptor CAR according to claim 7, the CAR-T cell or enhanced CAR-T cell according to claim 9 in an active drug or product against Nectin-4(+) cancer cells.

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