A Nectin-4 monoclonal antibody and its CAR-NK cells

Abstract

This invention provides three monoclonal antibodies against Nectin-4 or their antigen-binding fragments, as well as nucleic acid molecules and expression vectors encoding the above three Nectin-4 monoclonal antibodies. This invention also provides a chimeric antigen receptor polypeptide comprising: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises a monoclonal antibody or antibody fragment that binds to the above-mentioned Nectin-4 protein. This invention also provides a Nectin-4-targeting CAR-NK cell, prepared by co-introducing a Nectin-4-targeting CAR gene expression vector and a CCR7-IRES-IL15 gene expression vector into NK cells to obtain 715N4 CAR-NK cells. 715N4 CAR-NK cells simultaneously possess chemotactic activation and targeting triple functions, exhibiting more comprehensive anti-cancer activity.

Description

Technical Field

[0001] This invention relates to the field of antibody and CAR cell technology, and more specifically, to a Nectin-4 monoclonal antibody and its CAR-NK cells. Background Technology

[0002] Nectin-4 is a type I membrane protein that is overexpressed in various tumor cells and is associated with cancer development. For example, Nectin-4 is highly expressed in urothelial carcinoma, bladder cancer, triple-negative breast cancer, ovarian cancer, pancreatic cancer, esophageal cancer, papillary thyroid carcinoma, and gallbladder cancer. Nectin-4 promotes tumor cell proliferation, differentiation, migration, and invasion by activating the PI3K / Akt pathway. Therefore, Nectin-4 represents a novel target for tumor immunotherapy.

[0003] Nectin-4 can serve not only as an effective prognostic factor for breast cancer but also as an effective therapeutic target for patients with triple-negative breast cancer (TNBC). TNBC patients have a worse prognosis compared to other types of breast cancer. Currently, there is still a lack of effective biological targeted therapies, and systemic chemotherapy remains the primary treatment method. In vitro and in vivo studies have confirmed that Padcev has good efficacy against locally and metastatic TNBC (Ann Oncol. 2017 April 1 online edition). This study analyzed the expression of Nectin-4 / PVRL4 mRNA in 5673 patients with invasive breast cancer and explored its relationship with clinicopathological features, including metastasis-free survival. Immunohistochemistry was used to detect tissue samples from 61 TNBC patients and primary TNBC patients with PDX (pharmacograft). In vitro and in vivo studies examined the efficacy of anti-Nectin-4 in locally and metastatic TNBC PDX. The results showed that high Nectin-4 mRNA expression was associated with poor prognosis, including triple-negative and basal subtypes. Nectin-4 protein is not expressed in healthy adult tissues (including breast tissue). Padcev can act on nectin-4-positive breast cancer cells. In vitro studies show that Padcev has a high affinity for Nectin-4 and binds specifically to it. Padcev's effect on Nectin-4-positive cell lines is dose-dependent; the higher the dose, the greater the cytotoxic effect. In vivo studies have found that Padcev has a rapid, complete, and durable response to Nectin-4-positive PDX TNBC. Therapeutic efficacy depends on the Padcev dose and the expression level of nectin-4 in the tumor.

[0004] In recent years, a novel tumor immunotherapy method—chimeric antigen receptor (CAR) modified T / NK cell therapy (CAR-T / NK)—has attracted widespread attention. The structure of a CAR mainly comprises three basic components: an antigen-binding domain, a transmembrane domain, and an intracellular signal activation domain. It is a genetically engineered fusion protein that combines an extracellular antigen-binding domain with one or more intracellular immune cell activation signaling domains. NK cells are important innate immune cells, playing a crucial role in anti-tumor and anti-infection efforts. Therefore, the development of CAR-T technology naturally spurred the emergence of CAR-NK. NK cells modified with CAR structures can theoretically also efficiently recognize tumor cells and kill them through various means, such as releasing killing mediators and inducing target cell apoptosis. Compared to CAR-T, due to the inherent characteristics of NK cells, CAR-NK therapy does not produce severe cytokine storms during treatment, thus exhibiting higher safety in clinical use.

[0005] Similar to the CAR-T cell construction process, the construction of CAR-NK cells also utilizes gene transfection technology (such as lentivirus or retroviral vectors) to reprogram NK cells to express CAR. This allows NK cells to be redirected to target specific tumor antigens through a mechanism independent of the major histocompatibility complex (MHC-I), thereby enhancing the NK cell's killing effect on tumor cells. The key to this technology lies in which tumor antigen CAR-NK cells selects as their target recognition site. Summary of the Invention

[0006] The purpose of this invention is to overcome the technical deficiencies of existing technologies, such as the limited types of antibodies and CAR-NK cells targeting Nectin-4 and their poor therapeutic effects, and to provide a monoclonal antibody targeting Nectin-4, as well as CAR-NK cells targeting Nectin-4 with chemotactic and activation functions, and their applications.

[0007] The technical solution adopted in this invention is:

[0008] In a first aspect, the present invention provides a monoclonal antibody against Nectin-4 or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment comprising a heavy chain and a light chain; the heavy chain having three complementary regions H-CDR1, H-CDR2 and H-CDR3 in its heavy chain variable region, and the light chain having three complementary regions L-CDR1, L-CDR2 and L-CDR3 in its light chain variable region, wherein the combination of H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, L-CDR3 is any one of the following (I) to (III):

[0009] (Ⅰ) The amino acid sequence of H-CDR1 is VTVNSNP (SEQ ID NO.13).

[0010] The amino acid sequence of H-CDR2 is IRTGRSA (SEQ ID NO.14).

[0011] The amino acid sequence of H-CDR3 is TRGVYPSSY (SEQ ID NO.15).

[0012] The amino acid sequence of L-CDR1 is KGVITR (SEQ ID NO.16).

[0013] The amino acid sequence of L-CDR2 is PPSYR (SEQ ID NO.17).

[0014] The amino acid sequence of L-CDR3 is QKYHSNPLT (SEQ ID NO.18);

[0015] (II) The amino acid sequence of H-CDR1 is FTFNSYA (SEQ ID NO.19).

[0016] The amino acid sequence of H-CDR2 is ISTGGPT (SEQ ID NO.20).

[0017] The amino acid sequence of H-CDR3 is ARGLYYTDY (SEQ ID NO.21).

[0018] The amino acid sequence of L-CDR1 is QSVFTS (SEQ ID NO.22).

[0019] The amino acid sequence of L-CDR2 is SASYR (SEQ ID NO.23).

[0020] The amino acid sequence of L-CDR3 is QQYDSYPFT (SEQ ID NO.24);

[0021] (Ⅲ) The amino acid sequence of H-CDR1 is FKLTTYG (SEQ ID NO.25).

[0022] The amino acid sequence of H-CDR2 is KWGGDAT (SEQ ID NO.26).

[0023] The amino acid sequence of H-CDR3 is ARKFFWYFV (SEQ ID NO.27).

[0024] The amino acid sequence of L-CDR1 is QAVRTN (SEQ ID NO.28).

[0025] The amino acid sequence of L-CDR2 is SGSYR (SEQ ID NO.29).

[0026] The amino acid sequence of L-CDR3 is QQKNSGPST (SEQ ID NO.30).

[0027] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment is any one of the following (I) to (III):

[0028] (I) The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2;

[0029] (II) The amino acid sequence of the light chain variable region is shown in SEQ ID NO.4;

[0030] (III) The amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.6.

[0031] Furthermore, the amino acid sequence of the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment is any one of the following (I) to (III):

[0032] (I) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.8;

[0033] (II) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.10;

[0034] (III) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.12.

[0035] A second aspect of the invention provides a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0036] Furthermore, the sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.1;

[0037] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.4 is as shown in SEQ ID NO.3;

[0038] The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 6 is preferably as shown in SEQ ID NO. 5;

[0039] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 8 is as shown in SEQ ID NO. 7;

[0040] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 10 is as shown in SEQ ID NO. 9;

[0041] The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.12 is preferably as shown in SEQ ID NO.11.

[0042] A third aspect of the invention provides a light chain variable region encoding the monoclonal antibody or an antigen-binding fragment thereof described in the first aspect of the invention;

[0043] The heavy chain variable region;

[0044] The complementary region of the light chain variable region; or,

[0045] The nucleic acid molecule of the complementary region of the heavy chain variable region.

[0046] Furthermore, the sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.1;

[0047] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.4 is as shown in SEQ ID NO.3;

[0048] The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 6 is preferably as shown in SEQ ID NO. 5;

[0049] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 8 is as shown in SEQ ID NO. 7;

[0050] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 10 is as shown in SEQ ID NO. 9;

[0051] The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.12 is preferably as shown in SEQ ID NO.11.

[0052] In a fourth aspect, the present invention provides an expression vector in which a nucleic acid molecule as described in the second or third aspect of the present invention is inserted.

[0053] A fifth aspect of the present invention provides a cell containing a nucleic acid molecule as described in the second or third aspect of the present invention or an expression vector as described in the fourth aspect of the present invention.

[0054] A sixth aspect of the present invention provides a chimeric antigen receptor polypeptide comprising:

[0055] i. An extracellular antigen-binding domain, said extracellular antigen-binding domain comprising the monoclonal antibody or its antigen-binding fragment as described in the first aspect of the invention.

[0056] ii. Transmembrane domains, and

[0057] ⅲ.Intracellular signaling domain.

[0058] A seventh aspect of the present invention provides a nucleic acid molecule encoding the chimeric antigen receptor polypeptide described in the sixth aspect of the present invention.

[0059] Furthermore, the sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.4 is preferably as shown in SEQ ID NO.3;

[0060] The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 6 is preferably as shown in SEQ ID NO. 5;

[0061] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 8 is as shown in SEQ ID NO. 7;

[0062] The preferred sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO. 10 is as shown in SEQ ID NO. 9;

[0063] The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.12 is preferably as shown in SEQ ID NO.11.

[0064] An eighth aspect of the present invention provides a genetically modified NK cell comprising the chimeric antigen receptor polypeptide described in the sixth aspect of the present invention or the nucleic acid molecule described in the seventh aspect of the present invention.

[0065] Furthermore, the genetically modified NK cells according to the eighth aspect of the present invention are prepared by transfecting NK cells with an expression vector containing the nucleic acid molecule of the seventh aspect of the present invention.

[0066] Preferably, the genetically modified NK cells according to the eighth aspect of the present invention further comprise a CCR7 expression sequence;

[0067] More preferably, according to the eighth aspect of the present invention, the genetically modified NK cells further comprise an IL-15 expression sequence.

[0068] Specifically, in the genetically modified NK cells according to the eighth aspect of the present invention, the expression sequence of CCR7 and the expression sequence of IL-15 are inserted into the same expression vector.

[0069] More specifically, an IRES element is preferably linked between the expression sequence of CCR7 and the expression sequence of IL-15.

[0070] By co-transfecting NK cells with an expression vector containing the nucleic acid molecule described in the seventh aspect of the present invention and an expression vector containing the CCR7 and IL-15 expression sequences, CAR-NK cells with chemotactic and activation functions can be obtained; these CAR-NK cells have better anti-cancer effects than CAR-NK cells containing only the chimeric antigen receptor polypeptide described in the sixth aspect of the present invention.

[0071] A ninth aspect of the present invention provides a formulation comprising at least one of the following I to VII:

[0072] I. The monoclonal antibody or its antigen-binding fragment as described in the first aspect of this invention;

[0073] II. The nucleic acid molecule described in the second or third aspect of this invention;

[0074] III. The expression vector described in the fourth aspect of this invention;

[0075] IV. The cell described in the fifth aspect of the present invention;

[0076] V. The chimeric antigen receptor polypeptide described in the sixth aspect of the present invention;

[0077] VI. The nucleic acid molecule described in the seventh aspect of this invention;

[0078] VII. Genetically modified NK cells as described in the eighth aspect of the present invention.

[0079] Furthermore, the formulation is a biomaterial or a drug.

[0080] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0081] A tenth aspect of the present invention provides the use of at least one of the following I to VII in the preparation of an antitumor drug:

[0082] I. The monoclonal antibody or its antigen-binding fragment as described in the first aspect of this invention;

[0083] II. The nucleic acid molecule described in the second or third aspect of this invention;

[0084] III. The expression vector described in the fourth aspect of this invention;

[0085] IV. The cell described in the fifth aspect of the present invention;

[0086] V. The chimeric antigen receptor polypeptide described in the sixth aspect of the present invention;

[0087] VI. The nucleic acid molecule described in the seventh aspect of this invention;

[0088] VII. Genetically modified NK cells as described in the eighth aspect of the present invention.

[0089] Furthermore, according to the application described in the tenth aspect of the present invention, the tumor includes urothelial carcinoma, bladder cancer, triple-negative breast cancer, ovarian cancer, pancreatic cancer, esophageal cancer, papillary thyroid carcinoma, and gallbladder cancer.

[0090] The beneficial effects of this invention are:

[0091] This invention provides three monoclonal antibodies against Nectin-4. The ELISA results of the culture supernatant of the monoclonal antibodies were as follows: N4C1 OD value 2.099, N4C2 OD value 0.919, N4C3 OD value 1.856, positive control (Anti-Nectin-4 antibody, ab155692) OD value 1.048, and negative control OD value 0.104.

[0092] The present invention also provides the amino acid sequences and nucleic acid sequences encoding the above three Nectin-4 monoclonal antibodies, including the hypervariable region, which can be used to prepare Nectin-4 monoclonal antibodies, perform humanization modification, or further prepare bispecific antibodies targeting Nectin-4 and NK cells, or CAR-NK cells, and have the potential to develop new anti-tumor drugs.

[0093] The present invention provides a chimeric antigen receptor polypeptide comprising: an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular antigen-binding domain comprises a monoclonal antibody or antibody fragment that binds to the aforementioned Nectin-4 protein.

[0094] This invention also provides a Nectin-4-targeting CAR-NK cell. 715N4 CAR-NK cells are prepared by co-introducing a Nectin-4-targeting CAR gene expression vector and a CCR7-IRES-IL15 gene expression vector into NK cells. Compared to N4 CAR-NK cells with only targeting or 715NK cells with only lymphochemotaxis and immune cell activation functions, 715N4 CAR-NK cells possess triple functions of chemotaxis, activation, and targeting, exhibiting more comprehensive anti-triple-negative breast cancer activity. 715N4 CAR-NK cells have the potential to develop novel anti-tumor drugs. Attached Figure Description

[0095] Figure 1 Map of the pMSCV-GFP retroviral expression vector.

[0096] Figure 2ELISA results of culture supernatants of three Nectin-4 monoclonal antibodies. The OD values ​​were: N4C1 2.099, N4C2 0.919, N4C3 1.856, positive control (Anti-Nectin-4 antibody, ab155692) 1.048, and negative control 0.104. Detailed Implementation

[0097] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0098] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0099] Example 1: Preparation of Nectin-4 hybridoma monoclonal antibody

[0100] 1.1. Preparation of Nectin-4 hybridoma monoclonal antibody screening

[0101] ① Immunization of BALB / c mice with Nectin-4 protein as the antigen: Four 8-week-old BALB / c mice were administered subcutaneously at three points each, with each point receiving an oil-in-water mixture containing 20 μg of human Nectin-4 protein and 50 μl of complete adjuvant. Second, third, and fourth booster immunizations were administered every 10 days, with each mouse receiving an intraperitoneal injection of 50 μg of Nectin-4 protein. Four days after the fourth immunization, the spleens of the mice were harvested for B cell preparation.

[0102] ②Myeloma cells: The myeloma cells were SP2 / 0. They were cultured in 1640 medium containing 10% fetal bovine serum one week before cell fusion. They grew well and were ready for fusion and use.

[0103] ③ Feeder Cells: Normal BALB / c mice (unimmunized with antigens) were euthanized by cervical dislocation, and the spleens were aseptically removed. The spleen tissue was ground through a 200-mesh stainless steel sieve, and the spleen cells were collected by washing with 1640 medium. 2 × 10⁶ cells were added to each well of a 96-well plate. 5 One spleen cell.

[0104] ④ Fusion of B cells with myeloma cells: On the day of cell fusion, feeder cell preparation methods were used to obtain spleen cells from BALB / c mice immunized with Nectin-4 antigen. Cells containing 1×10-1 myeloma cells were collected. 7 One spleen cell and 3×10 7 Fusing SP2 / 0 myeloma cells. The fusion agent was 50% PEG1500 solution. After fusion, the cells were resuspended in HAT medium and added to 0.1 ml per well of a 96-well cell culture plate containing feeder cells for further culture.

[0105] ⑤ Screening of positive clones: Positive clones in the 96-well plates after fusion were detected using an indirect ELISA method coated with Nectin-4 protein. Three strongly positive hybridoma wells were screened, and subsequent cloning was performed twice consecutively, yielding three positive monoclonal strains: N4C1, N4C2, and N4C3. The ELISA results of the antibody culture supernatant from these three positive monoclonal strains are shown in the appendix. Figure 2 The OD values ​​of N4C1, N4C2, and N4C3 were 2.099, 0.919, and 1.856, respectively. The OD value of the positive control (Anti-Nectin-4 antibody, ab155692) was 1.048, and the OD value of the negative control was 0.104. This indicates that the three monoclonal antibodies prepared have similar effects.

[0106] 1.2. Cloning and sequencing of the variable region genes of the heavy and light chains of Nectin-4 monoclonal antibody

[0107] The cloning and sequencing process of the heavy and light chain variable region genes of Nectin-4 specific N4C1, N4C2, and N4C3 hybridoma cell lines is as follows:

[0108] ① RNA extraction from hybridoma cells: Following the instructions provided with the Trizol reagent, lyse hybridoma cells C1 and C2 separately using Trizol. Transfer all lysed samples to 1.5 ml centrifuge tubes, add 0.2 ml chloroform to each 1 ml of Trizol reagent, extract, and incubate at room temperature for 10 min; then centrifuge at 12000 rpm for 15 min at 4°C. Carefully aspirate RNA from the aqueous phase above, mix the RNA with an equal volume of isopropanol; centrifuge at 12000 rpm for 15 min at 4°C, and discard the supernatant. Wash twice with 75% ethanol, and after the ethanol has completely evaporated, add 50 μl of sterile, RNase-free water to each tube to dissolve the precipitate. Use 10 μl of RNA for subsequent RT-PCR.

[0109] ② Reverse transcription polymerase chain reaction (RT-PCR) amplification of heavy and light chain variable regions: Using the extracted hybridoma cell RNA as a template, cDNA was synthesized by RT-PCR. A set of universal mouse IgG antibody heavy and light chain variable region amplification primers were used for RT-PCR. The PCR amplification products were analyzed by agarose gel electrophoresis to determine band size, and the bands were subsequently recovered and sequenced.

[0110] Two batches of hybridoma monoclonal antibodies were prepared. The light chain variable region genes of the first three hybridoma monoclonal antibodies identified by sequencing results were N4C1L (SEQ ID NO.1), N4C2L (SEQ ID NO.3), and N4C3L (SEQ ID NO.5), with corresponding amino acid (aa) sequences of N4C1Laa (SEQ ID NO.2), N4C2Laa (SEQ ID NO.4), and N4C3Laa (SEQ ID NO.6), respectively. The heavy chain variable region genes were N4C1H (SEQ ID NO.7), N4C2H (SEQ ID NO.9), and N4C3H (SEQ ID NO.11), with corresponding amino acid sequences of N4C1Haa (SEQ ID NO.8), N4C2Haa (SEQ ID NO.10), and N4C3Haa (SEQ ID NO.12), respectively. The underlined sequences correspond to the hypervariable regions in the variable regions, namely CDR1, CDR2, and CDR3, respectively.

[0111] SEQ ID NO.1:N4C1L

[0112] GACATTTGTGGTGACCGAGTCTCAAAAATTCATGGCCACATCAGGAGGAGACGGGGTCAGCGTCACCTGCAAGGCCAGT AAGGGTGTGATTACTCGT GTAGCCAGGTATCAACAGAAATCAGGTCAATCTCCTAAAACACTGATTTAC CCGCCATCCTACAGG TACAGTGGAGGCCCTGATCGCTTCACACGCCGTGGATCTCGGACAGATTTCACTCTCGCCATCAGCAATGTGGAGTCTGAACACTTGGCAGAGTATTTCTGT CAGAAATATCACAGCAATCCGCTCACG ATCCGACGGGGGACCAAGGTGGAAATAAAACGGGCT.

[0113] SEQ ID NO.2:N4C1Laa

[0114] DIVVTESQKFMATSGGDGVSVTCKAS KGVITR VARYQQKSGQSPKTLIY PPSYR YSGGPDRFTRRGSRTDFTLAISNVESEHLAEYFC QKYHSNPLT IRRGTKVEIKRA。

[0115] SEQ ID NO.3:N4C2L

[0116] GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGT CAGAGTGTGTTTACTAGT GTAGCCTGGTATCAACAGAAATCAGGTCAATCTCCTAAAACACTGATTTAC TCGGCATCCTACCGG TACAGTGGAGTCCCTGATCGCTTCACAGGCCGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGT CAGCAATATGACAGCTATCCGTTCACG TTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCT。

[0117] SEQ ID NO.4:N4C2Laa

[0118] DIVMTQSQKFMSTSVGDRVSVTCKAS QSVFTS VAWYQQKSGQSPKTLIY SASYR YSGVPDRFTGRGSGTDFTLTISNVQSEDLAEYFC QQYDSYPFT FGGGTKLEIKRA。

[0119] SEQ ID NO.5:N4C3L

[0120] GGCATTGTGGCCACCCAGCTGCAAAAATTCATGTCCACATCATCAGGAGACAGGGTCAGCGTCACCTGCAAGCGCCTG CAGGCAGTGCGAACTAAT GGCGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTAC TCGGGCTCCTACCGAACCAGTAGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCAACAACAATGCAGTCTGAAGACTTGGCAGAGTATTTCTGT CAGCAAAAAAATAGTGGCCCTTCTACG GCCGGTGCTGGGACCAAGCTGGAGCTGAAACGGGCT。

[0121] SEQ ID NO.6:N4C3No

[0122] GIVATQLQKFMSTSSGDRVSVTCKRL QAVRTN GAWYQQKPGQSPKALIY SGSYR TSSVPDRFTGSGSGTDFTLTINNVQSEDLAEYFC QQKNSGPST AGAGTKLEKRA。

[0123] SEQ ID NO.7:N4C1H

[0124] CAAGTGACAGTGCTGGAGTCGCGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAAATCTCCTGTGCAGCCTCTGGA GTCACTGTCAATAGCAATCCC ATGGCTTGGGTTGGCCAGACTCCAAAGAAGAGGCTGGAGTGGGTCGCATCC ATTCGTACTGGTCGTTCCGCC TATTATCCAGACAGTATGAAGGGCCGATTCACCATCTCCAGATAGTACCAGGAACATCCTGTATCTGGAAATGAACAGTCTGAGGTCTGAAGACACGGCCATGTATTACTGT ACAAGAGGCGT GTACCCTTCCAGCTAC TGGGGCCAAGGCAGGACTGTCACAGTCTCCTCG。

[0125] SEQ ID NO.8:N4C1Haa

[0126] QVTVLESRGGLVQPGGSLKISCAASG VTVNSNP MAWVGQTPKKRLEWVAS IRTGRSA YYPDSMKGRFTISRDSTRNILELEMNSLRSEDTAMYYC TRGVYPSSY WGQGRTVTVSS。

[0127] SEQ ID NO.9:N4C2H

[0128] GAAGTGACACTGGTGGAGTCGGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAAATCTCCTGTGCAGCCTCTGGA TTCACTTTCAATAGCTATGCC ATGTCTTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATCC ATTAGTACTGGTGGTCCCACC TATTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGATAGTGCCAGGAACATCCTGTATCTGCAAATGAACAGTCTGAGGTCTGAAGACACGGCCATGTATTACTGT GCAAGAGGCCT CTACTATACCGACTAC TGGGGCCAAGGCACCACTCTCACAGTCTCCTCG。

[0129] SEQ ID NO.10:N4C2Haa

[0130] EVTLVESGGGLVKPGGSLKISCAASG FTFNSYA MSWVRQTPEKRLEWVAS ISTGGPT YYPDSVKGRFTISRDSARNILYLQMNSLRSEDTAMYYC ARGLYYTDY WGQGTTLTVSS。

[0131] SEQ ID NO.11:N4C3H

[0132] CAGGTGCAGTCAGAGCAGTCATCACCTAGCCTAGACCAGCCCTTACAGAGCCTGTCCCTAACCTGCACAGTCTCTTTC TTCAAGTTAACCACCTATGGT GTACACTGGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTG AAGTGGGGAGGTGACGCAACA GACTACAATGCAGCTAGCATGTCCAGACTGAGCATCACCAAGGACAACTCCCAGAGCCTATGGTTCTTTAAAATGAACAGTCTGCAAGCTGATGACACTGCCAACTACTACTGT GCCAGAAAATT CTTCTGGTACTTCGTC GTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA。

[0133] SEQ ID NO.12:N4C3Haa

[0134] QVQSEQSSPSLDQPLQSLSLTCTVSF FKLTTYG VHWVRQSPGKGLEWLGV KWGGDAT DYNAASMSRLSITKDNSQSLWFFKMNSLQADDTANYYC ARKFFWYFV VWGAGTTVTVSS.

[0135] Example 2: Preparation of N4CAR expression vector containing Nectin-4 monoclonal antibody sequence

[0136] 2.1. The variable region of the Nectin-4 monoclonal antibody obtained in this invention

[0137] 2.2. Construction of a Nectin-4 CAR (N4CAR) gene retroviral expression vector

[0138] ① Following the gene splicing sequence below, three complete N4CAR genes are formed: Igk leader peptide + light chain variable region + Linker + heavy chain variable region + CD8 hinge region + CD28 transmembrane region + CD28 ITAM region + 41BB ITAM region + CD3ζITAM region.

[0139] ② The three N4CAR double-stranded DNA molecules formed were cloned into the T vector and sequenced to confirm that they were correct.

[0140] ③ The three N4CAR genes were cloned into retroviral expression vectors that simultaneously expressed GFP, resulting in three N4CAR gene expression vectors. The N4CARs constructed using the variable regions of the three monoclonal antibodies prepared in Example 1 were named N4CAR1, N4CAR2, and N4CAR3, respectively.

[0141] The expression vectors that can be used include any eukaryotic expression vector.

[0142] In this embodiment, the pMSCV-GFP retroviral expression vector was used, and its image is shown in the attached figure. Figure 1 As shown.

[0143] Example 3: Preparation of N4CAR-NK cells targeting Nectin-4

[0144] 3.1. Preparation of N4CAR retrovirus

[0145] (1) GP2-293 cells were co-transfected with the outer membrane protein of VSV-G using the N4CAR1, N4CAR2 and N4CAR3 vector plasmids, respectively. The culture supernatant contained retroviruses carrying the N4CAR1, N4CAR2 and N4CAR3 genes, respectively.

[0146] (2) The viral load was determined using digital droplet PCR (ddPCR) absolute quantification method, and the viral load was adjusted to 10. 6 / μL, for later use.

[0147] 3.2. Transduction of NK cells using N4CAR retrovirus

[0148] (1) The NK92 cell line was used as the NK cell line;

[0149] (2) Pack NK92 cells at a density of 7 × 10⁶ cells per well 5 Inoculate into 6-well plates (3 mL of culture medium per well);

[0150] (3) The next day, 10 μl of N4CAR1, N4CAR2, and N4CAR3 retroviruses were added to each of two wells, and the NK cells were cultured for another 48 hours to obtain N4CAR-NK1, N4CAR-NK2, and N4CAR-NK3 cells. Flow cytometry analysis showed that 54%, 59%, and 62% of the three types of N4CAR-NK cells were positive for GFP, respectively.

[0151] Example 4: Preparation of CAR-NK cells targeting Nectin-4 with chemotactic and activation functions

[0152] 4.1. Construction of a retroviral expression vector for the Nectin-4 CAR (N4CAR) gene

[0153] (1) The following gene splicing sequence is used to form three complete Nectin-4 CAR genes: Igk leader peptide + light chain variable region + Linker + heavy chain variable region + CD8 hinge region + CD28 transmembrane region + CD28 ITAM region + 41BB ITAM region + CD3ζITAM region.

[0154] (2) The three Nectin-4 CAR double-stranded DNAs formed were cloned into the T vector and sequenced to confirm that they were correct.

[0155] (3) The three Nectin-4 CAR genes were cloned into retroviral expression vectors that simultaneously expressed GFP, resulting in three Nectin-4 CAR gene expression vectors. The N4CARs constructed using the variable regions of the three monoclonal antibodies were named N4CAR1, N4CAR2 and N4CAR3, respectively.

[0156] 4.2. Preparation of Nectin-4-targeted anti-cancer CAR-NK cells

[0157] (1) Preparation of N4CAR retrovirus. ① GP2-293 cells were co-transfected with N4CAR1, N4CAR2, and N4CAR3 vector plasmids and VSV-G outer membrane protein plasmids, respectively. The culture supernatant contained retroviruses carrying the N4CAR1, N4CAR2, and N4CAR3 genes, respectively. ② The viral load was adjusted to 10⁻⁶ using digital droplet PCR (ddPCR) absolute quantification. 6 / μL, for later use.

[0158] (2) The NK92 cell line was used as the NK cell line;

[0159] (3) Pack NK92 cells at a density of 7 × 10⁶ cells per well 5 Inoculate into 6-well plates (3 mL of culture medium per well);

[0160] (4) The next day, 10 μl of N4CAR1, N4CAR2, and N4CAR3 retroviruses were added to each of two wells, and the NK cells were cultured for another 48 hours to obtain CAR-NK1, CAR-NK2, and CAR-NK3 cells. Flow cytometry analysis showed that 54%, 59%, and 62% of the three types of N4CAR-NK cells were positive for GFP, respectively.

[0161] 4.3. Preparation of 715NK cells with chemotactic and activation functions

[0162] Constructing an NK cell-specific CCR7-IRES-IL15 bicistronic retrovirus (715RT) expression vector; preferably, including the following steps:

[0163] (1) Synthesize the CCR7 gene (SEQ-CCR7) and clone it upstream of the IRES of a retroviral expression vector with a bicistronic element to form the CCR7-IRES retroviral expression vector.

[0164] CCR7 gene (SEQ-CCR7) sequence:

[0165]

[0166] (2) Synthesize the IL-15 gene (SEQ-15) and clone it into the downstream of IRES of the CCR7-IRES retroviral expression vector to become the NK cell-specific CCR7-IRES-IL15 retroviral (715RT) expression vector.

[0167] IL-15 gene (SEQ-15) sequence:

[0168] ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGTGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCAGT TGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA (SEQ ID NO.32).

[0169] (3) Preparation of NK cell-specific 715RT virus

[0170] ① GP2-293 cells were co-transfected with 715RT vector plasmid and VSV-G outer membrane protein plasmid. The culture supernatant contained 715RT virus carrying the CCR7-IRES-IL15 bicistronic gene.

[0171] ② The viral load was determined by absolute quantification using digital droplet PCR (ddPCR) and adjusted to 10⁶ / μL for later use.

[0172] (4) Preparation of 715NK cells

[0173] ①The NK92 cell line was used as the NK cell line;

[0174] ②Prepare NK92 cells at a density of 7 × 10⁶ cells per well. 5 Inoculate into 6-well plates (3 mL of culture medium per well);

[0175] ③ The next day, 4 wells of NK92 cells were taken, and 10 μl of N4CAR virus was added to each well. The NK cells were cultured for another 48 hours to obtain 715NK cells. Flow cytometry analysis showed that approximately 54% of the 715NK cells were GFP-positive.

[0176] 4.4. Preparation of anti-cancer 715N4 CAR-NK cells with chemotactic activation and targeting of three functions

[0177] (1) Preparation of 715N4CAR retrovirus

[0178] ① The 715RT expression vector plasmid was mixed with the N4CAR1, N4CAR2 and N4CAR3 vector plasmids respectively, and then co-transfected with the VSV-G outer membrane protein plasmid into GP2-293 cells. The culture supernatant contained retroviruses carrying the corresponding 715N4CAR1, 715N4CAR2 and 715N4CAR3 genes.

[0179] ② Use digital droplet PCR (ddPCR) absolute quantification method to determine the content of each virus, adjust the content of each virus to 10⁶ / μL, and set aside for later use.

[0180] (2) Using NK cell lines as NK cells;

[0181] (3) NK92 cells were seeded at 7 × 10⁵ cells per well in a 6-well plate (3 mL of culture medium per well);

[0182] (4) The next day, add 10 μl of 715N4CAR1, 715N4CAR2, or 715N4CAR3 retrovirus particles to each well and continue culturing the NK cells for 48 hours to obtain 715N4CAR-NK1, 715N4CAR-NK2, or 715N4CAR-NK3 cells. The percentage of GFP-positive 715N4CAR-NK cells is as high as about 58%.

[0183] Example 5: In vitro killing activity of several NK cells against breast cancer cells

[0184] 5.1 In vitro killing activity of several NK cells against breast cancer cells

[0185] Because the breast cancer cell line SK-BR-3 highly expresses Nectin-4, this breast cancer cell line was selected as the target cell for this experiment to determine the in vitro killing activity of effector cells such as NK92 cells, 715NK cells, N4CAR-NK cells, and 715N4CAR-NK cells. U-shaped 96-well culture plates were used, with replicates in each well for the killing experiment.

[0186] (1) Each effector cell was prepared in duplicate wells, and 1×10⁻⁶ cells were added to each well. 5 10 effector cells (NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, 715N4CAR-NK3);

[0187] (2) Add 2×10 to each pore of effector cells 4 Target cells (SK-BR-3);

[0188] (3) Assume it contains only 1×10 5 A replicate well containing effector cells (NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK1, 715N4CAR-NK3);

[0189] (4) Assume it contains only 2×10 4 A replicate well containing SK-BR-3 target cells;

[0190] (5) Add culture medium to each well to 200 μl.

[0191] (6) After incubating in a CO2 incubator at 37℃ for 6 hours, add 10 μl of CCK-8 to each well and shake to mix.

[0192] (7) Continue incubation in the incubator for 1 hour, then remove the 96-well plate and measure the OD value at 450 nm wavelength using a multi-functional microplate reader.

[0193] (8) Calculate the killing activity of each effector cell against SK-BR-3 target cells; the killing activity is expressed as the killing rate. The killing rate formula is: killing rate (%) = 1 - (AB) / C × 100%, where A is the absorbance value of effector cells co-cultured with SK-BR-3 target cells; B is the absorbance value of effector cells alone, and C is the absorbance value of SK-BR-3 target cells.

[0194] (9) Repeat the above killing experiment four times, and the statistical results are shown in Table 1 below;

[0195] Table 1. Comparison of the killing activity of several NK cells against breast cancer cells

[0196]

[0197]

[0198] The results in Table 1 show that:

[0199] (1) NK92 cells showed a certain killing activity against SK-BR-3 breast cancer cells, with an effect rate of 25% ± 3.4%;

[0200] (2) 715 NK cells enhanced the killing activity of NK cells by 42%; compared with NK92 cells, the difference was very significant (p≤0.01).

[0201] (3) N4CAR-NK cells showed a good killing rate against SK-BR-3 breast cancer cells, ranging from 50% to 71%; compared with NK92 and 715NK, there were very significant differences (p≤0.01), indicating that N4CAR-NK cells have the potential to treat breast cancer.

[0202] (4) 715N4CAR-NK cells had the highest killing rate against SK-BR-3 breast cancer cells, ranging from 55% to 84%; compared with N4CAR-NK cells, there was a significant difference (p≤0.05), indicating that 715N4CAR-NK cells have the greatest potential for treating breast cancer.

[0203] 5.2. Chemotaxis of CCR7 on NK cells

[0204] This embodiment uses the CCL19-expressing 293T engineered cell line (CCL19-293T) as a chemotactic target, and a Transwell assay with a membrane pore size of 5 μM is used for cell migration experiments. Primary NK cells and primary T cells expanded in vitro are used as effector cells to prepare NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, and 715N4CAR-NK3, ​​respectively; their chemotactic activity is verified. The implementation process is as follows:

[0205] ① The concentrations of NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, and 715N4CAR-NK3 cells were all adjusted to 10. 6 / ml, in RPMI-20% FCS-500U IL-2 / ml culture medium.

[0206] ②Prepare 2×10⁻⁶ CCL19-293T cell suspensions 5 2 ml of RPMI-20% FCS-500U IL-2 / ml culture medium was added to each well of a 6-well plate and incubated at 37°C for 2 hours until the cells adhered. Then, transwell inserts were placed in the 6-well plates.

[0207] ③ Add 2 ml of NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, and 715N4CAR-NK3 cells to each transwell inserts compartment.

[0208] ④ After the cells were placed in a 37°C CO2 incubator for 24 hours, the inserts chambers were removed, and CCL19-293T cells and NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, and 715N4CAR-NK3 cells that migrated from the transwell inserts chambers were collected from the 6-well plates; the cells were centrifuged at 1000 rpm and washed three times with PBS.

[0209] ⑤ The NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, and 715N4CAR-NK3 cells that migrated to the 6-well plate were stained with anti-CD56-FITC antibody.

[0210] ⑥ Flow cytometry analysis was used to calculate the number of CD56-positive cells. The numbers of NK92, 715NK, N4CAR-NK1, N4CAR-NK2, N4CAR-NK3, ​​715N4CAR-NK1, 715N4CAR-NK2, and 715N4CAR-NK3 cells that migrated to CCL19-293T cells were compared. The chemotactic effect of CCR7 on NK cells is shown in Table 2 below:

[0211] Table 2. Chemotaxis of CCR7 on NK cells

[0212]

[0213] As can be seen from the results in Table 2, NK cells containing CCR7 all showed very significant (p≤0.01) chemotactic activity, indicating that the addition of CCR7 can significantly enhance the anti-cancer effect of 715N4CAR-NK cells in vivo.

[0214] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against Nectin-4 or an antigen-binding fragment thereof, said monoclonal antibody or antigen-binding fragment comprising a heavy chain and a light chain; wherein the heavy chain variable region has three complementarity-determining regions H-CDR1, H-CDR2, and H-CDR3, and the light chain variable region has three complementarity-determining regions L-CDR1, L-CDR2, and L-CDR3, wherein the combination of H-CDR1, H-CDR2, H-CDR3 and L-CDR1, L-CDR2, L-CDR3 is as follows: the amino acid sequence of H-CDR1 is VTVNSNP (SEQ ID NO. 13), the amino acid sequence of H-CDR2 is IRTGRSA (SEQ ID NO. 14), the amino acid sequence of H-CDR3 is TRGVYPSSY (SEQ ID NO. 15), the amino acid sequence of L-CDR1 is KGVITR (SEQ ID NO. 16), and the amino acid sequence of L-CDR2 is... The amino acid sequence of L-CDR3 is PPSYR (SEQ ID NO.17), and the amino acid sequence of L-CDR3 is QKYHSNPLT (SEQ ID NO.18).

2. The monoclonal antibody or antigen-binding fragment thereof of claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO.

2.

3. The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment is shown in SEQ ID NO.

8.

4. A nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1 to 3.

5. A nucleic acid molecule encoding the light chain variable region and heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1 to 3.

6. The nucleic acid molecule according to claim 4 or 5, characterized in that, The sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 is shown in SEQ ID NO.1; the sequence of the nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.8 is shown in SEQ ID NO.

7.

7. An expression vector wherein the nucleic acid molecule of any one of claims 4 to 6 is inserted.

8. A cell comprising the nucleic acid molecule of any one of claims 4 to 6 or the expression vector of claim 7.

9. A chimeric antigen receptor polypeptide comprising: i. an extracellular antigen-binding domain, said extracellular antigen-binding domain comprising a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 3, ii. a transmembrane domain, and iii. an intracellular signaling domain.

10. A nucleic acid molecule encoding the chimeric antigen receptor polypeptide of claim 9.

11. A genetically modified NK cell comprising the chimeric antigen receptor polypeptide of claim 9 or the nucleic acid molecule of claim 10.

12. The genetically modified NK cells according to claim 11, characterized in that, The genetically modified NK cells are prepared by transfecting NK cells with an expression vector containing the nucleic acid molecule of claim 10.

13. The genetically modified NK cells according to claim 11 or 12, characterized in that, The genetically modified NK cells also contain the CCR7 expression sequence.

14. The genetically modified NK cells according to claim 13, characterized in that, The genetically modified NK cells also contain an IL-15 expression sequence.

15. The genetically modified NK cells according to claim 14, characterized in that, The expression sequence of CCR7 and the expression sequence of IL-15 are inserted into the same expression vector; an IRES element is connected between the expression sequence of CCR7 and the expression sequence of IL-15.

16. A formulation comprising at least one of the following I to VII: I. The monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3; II. The nucleic acid molecule according to any one of claims 4 to 6; III. The expression vector according to claim 7; IV. The cell of claim 8; V. The chimeric antigen receptor polypeptide of claim 9; VI. The nucleic acid molecule of claim 10; VII. The genetically modified NK cell of any one of claims 11 to 15.

17. The use of at least one of the following I to VII in the preparation of drugs that kill SK-BR-3 breast cancer cells: I. The monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3; II. The nucleic acid molecule according to any one of claims 4 to 6; III. The expression vector according to claim 7; IV. The cell of claim 8; V. The chimeric antigen receptor polypeptide of claim 9; VI. The nucleic acid molecule of claim 10; VII. The genetically modified NK cell of any one of claims 11 to 15.

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