A fully human antibody or antibody fragment targeting MSLN and its chimeric antigen receptor and applications

Abstract

This invention discloses a fully human antibody or antibody fragment targeting MSLN, its chimeric antigen receptor, and its applications. The chimeric antigen receptor comprises, in sequence, a signal peptide sequence, an MSLN-binding domain, a detection tag, a hinge region, a transmembrane domain, and a functional signal transduction domain. This invention utilizes lentiviral gene transduction to induce NK cells to express a CAR fusion protein targeting MSLN, resulting in CAR-NK cells. The technical solution of this invention has a positive effect on the treatment of solid tumors with MSLN protein overexpression and can be used for the independent or combined treatment of tumors with MSLN protein overexpression, such as ovarian cancer and pancreatic cancer, with broad clinical application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fully human antibody or antibody fragment targeting MSLN, its chimeric antigen receptor, and its applications. Background Technology

[0002] Natural killer (NK) cells are a key component of the innate immune system. Their cytotoxicity is regulated by a complex of various activating and inhibitory receptors. Activating receptors include natural cytotoxic receptors (NCRs), NKG2D, CD16 (FcγRIIIa), FasL, TRAIL, and other co-stimulatory receptors such as LFA-1, CD244 (2B4), and CD137 (4-1BB). Inhibitory receptors include KIRs (cytotoxic cell immunoglobulin-like receptors) and NKG2A. These receptors recognize changes on the cell surface, thus determining whether NK cells are activated. NK cells can rapidly recognize and destroy infected and tumor cells without prior sensitization. Therefore, genetically modifying allogeneic NK cells using chimeric antigen receptor (CAR) constructs can enhance their killing power and persistence against tumor cells expressing specific antigens. Furthermore, CAR-NK therapy has a higher safety profile compared to CAR-T therapy.

[0003] Mesothelin (MSLN) is a glycosylphosphatidylinositol-linked membrane glycoprotein present at relatively low levels in the mesothelial cells of the pleura, peritoneum, and pericardium of healthy individuals. However, MSLN is overexpressed in many different cancers, including mesothelioma, gastric cancer, prostate cancer, pancreatic cancer, lung cancer, cholangiocarcinoma, breast cancer, and ovarian cancer. The high expression of MSLN in various cancers and its limited expression in normal tissues make it an attractive target for CAR-NK immunotherapy.

[0004] Ovarian cancer is the second leading cause of cancer death in gynecology and the leading cause of death. Although combination chemotherapy is effective for patients with advanced cancer, the overall treatment outcome is poor. Most patients experience severe chemotherapy side effects, and approximately 60% of patients relapse after receiving first-line chemotherapy. Currently, there are no other effective drug treatment options. To address the poor prognosis and high recurrence rate of current ovarian cancer treatments, it is urgent to explore new clinical treatment options to improve treatment outcomes. Summary of the Invention

[0005] The purpose of this invention is to provide a fully human antibody or antibody fragment targeting MSLN, its chimeric antigen receptor, and its application. The fully human antibody or antibody fragment targeting MSLN can bind to human MSLN protein with high specificity. Experiments have shown that CAR-NK cells prepared based on the chimeric antigen receptor provided by this invention can target MSLN-overexpressing tumor cells with high specificity and achieve significant killing effect.

[0006] To achieve the above objectives, the present invention provides a fully human antibody or antibody fragment targeting MSLN, wherein the antibody or antibody fragment comprises: a heavy chain variable region and a light chain variable region;

[0007] The heavy chain variable region includes three complementarity-determining regions: HCDR1, HCDR2, and HCDR3. The amino acid sequence of HCDR1 includes the sequence shown in SEQ ID NO: 3; the amino acid sequence of HCDR2 includes the sequence shown in SEQ ID NO: 4; and the amino acid sequence of HCDR3 includes the sequence shown in SEQ ID NO: 5.

[0008] The light chain variable region includes three complementarity-determining regions: LCDR1, LCDR2, and LCDR3. The amino acid sequence of LCDR1 includes the sequence shown in SEQ ID NO: 6; the amino acid sequence of LCDR2 includes the sequence shown in SEQ ID NO: 7; and the amino acid sequence of LCDR3 includes the sequence shown in SEQ ID NO: 8.

[0009] Preferably, the MSLN binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is the sequence shown in SEQ ID NO: 1.

[0010] Preferably, the MSLN binding domain of the antibody or antibody fragment is scFv, and the nucleotide sequence of the scFv is the sequence shown in SEQ ID NO: 2.

[0011] In another aspect, the present invention provides a chimeric antigen receptor comprising a fully human antibody or antibody fragment targeting MSLN as described above.

[0012] Preferably, the chimeric antigen receptor comprises, in sequence: a signal peptide sequence, an MSLN binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signal transduction domain;

[0013] The signal peptide sequence comprises CSF2RA or 2B4', wherein the amino acid sequence of CSF2RA is the sequence shown in SEQ ID NO: 9, and the amino acid sequence of 2B4' is the sequence shown in SEQ ID NO: 10;

[0014] The detection tag contains eGFP, and the amino acid sequence of the eGFP is the sequence shown in SEQ ID NO: 11;

[0015] The MSLN binding domain includes scFv, and the scFv contains the fully human antibody or antibody fragment targeting MSLN.

[0016] The hinge region includes CD8', and the amino acid sequence of CD8' is the sequence shown in SEQ ID NO: 12;

[0017] The transmembrane domain comprises any one of CD8”, 2B4”, or NKG2D, wherein the amino acid sequence of CD8” is the sequence shown in SEQ ID NO: 13, the amino acid sequence of 2B4” is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of NKG2D is the sequence shown in SEQ ID NO: 15.

[0018] The functional signal transduction domain comprises at least one of 2B4”', 4-1BB, DAP12, DAP10, and CD3 Zeta, wherein the amino acid sequence of 2B4”' is the sequence shown in SEQ ID NO: 16, the amino acid sequence of 4-1BB is the sequence shown in SEQ ID NO: 17, the amino acid sequence of DAP12 is the sequence shown in SEQ ID NO: 18, the amino acid sequence of DAP10 is the sequence shown in SEQ ID NO: 19, and the amino acid sequence of CD3 Zeta is the sequence shown in SEQ ID NO: 20.

[0019] In another aspect, the present invention provides a host cell comprising the aforementioned chimeric antigen receptor.

[0020] Preferably, the host cell is an NK cell.

[0021] In another aspect, the present invention provides a lentiviral plasmid comprising a nucleotide sequence capable of encoding the aforementioned fully human antibody or antibody fragment targeting MSLN.

[0022] The aforementioned fully human antibodies or antibody fragments targeting MSLN, or the aforementioned chimeric antigen receptors, or the aforementioned host cells, or the aforementioned lentiviral plasmids, are used in the preparation of antitumor drugs.

[0023] Preferably, the tumor is an MSLN antigen overexpression-related tumor.

[0024] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0025] (1) The fully human antibody or antibody fragment targeting MSLN provided by this invention can bind to human MSLN protein with high specificity. The EC50 value of the protein level of the lead antibody 2MH6 is 0.15 nM. Furthermore, by using chimeric antigen receptor technology (CAR) to engineer and express the human antibody fragment that binds MSLN integrated into the CAR in NK cells, the resulting CAR-NK cells can be used to treat hematologic cancers related to MSLN expression.

[0026] (2) The chimeric antigen receptor of the present invention has an intracellular signal transduction domain that is different from that of CAR-T technology, including NK cell endogenous gene proteins such as 2B4”', DAP10, and DAP12. The chimeric antigen receptor of the present invention also has different structures and combinations of different signal transduction domains, which can better activate the targeted killing activity of NK cells while preventing excessive activation of CAR-NK cells, prolonging the half-life, and achieving a better tumor cell killing effect. Attached Figure Description

[0027] Figure 1 Output results for three rounds of screening of single-chain scFv antibodies targeting MSLN.

[0028] Figure 2 To detect the binding of the three-round phage library to the MSLN protein using ELISA.

[0029] Figure 3 ELISA assay to detect the binding activity of candidate fully human anti-MSLN scFv antibodies to MSLN antigen protein.

[0030] Figure 4 The results are obtained by flow cytometry analysis of the binding activity of candidate fully human anti-MSLN scFv antibodies to MSLN antigen proteins on the cell surface.

[0031] Figure 5 To detect the expression rate of CAR on the surface of NK cells containing different anti-MSLN-CAR by flow cytometry; where:

[0032] a represents the results of the FCARB group; b represents the results of the SCARA, SCARC, SCARD, SCARE, TCARF, TCARG, TCARH, and TCARI groups.

[0033] Figure 6 To detect the cytotoxic activity of NK cells containing different anti-MSLN-CARs against SK-OV-3 tumor cells; among which:

[0034] a represents the results of the FCARB group; b represents the results of the SCARA, SCARC, SCARD, SCARE, TCARF, TCARG, TCARH, and TCARI groups. Detailed Implementation

[0035] Terminology Definition

[0036] An "antibody" is a glycoprotein, or its antigen-binding portion, that comprises at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds. Antibodies include single-chain antibodies.

[0037] The “heavy chain” consists of the heavy chain variable region (VH) and the heavy chain constant region.

[0038] A “light chain” consists of a light chain variable region (VL) and a light chain constant region.

[0039] “scFv”: Single-chain antibody fragment, which is composed of the variable regions of the antibody heavy chain and light chain linked by a short peptide (linker) of 15 to 20 amino acids.

[0040] The "heavy chain variable region" and "light chain variable region" can be further divided into hypervariable regions, called "complementarity-determining regions" (CDRs), which are scattered in more conserved regions called "framework regions" (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that can interact with the antigen. In this invention, the CDR1, CDR2, and CDR3 of the heavy chain variable region are represented as HCDR1, HCDR2, and HCDR3, respectively; and the CDR1, CDR2, and CDR3 of the light chain variable region are represented as LCDR1, LCDR2, and LCDR3, respectively.

[0041] "Constant regions" can mediate the binding of immunoglobulins to host tissues or factors.

[0042] An "antigen-binding domain" refers to one or more segments of an antibody that retain the ability to specifically bind to an antigen (such as an MSLN).

[0043] "Monoclonal antibody" refers to an antibody molecule composed of a single molecule. Monoclonal antibody compositions exhibit specific binding and affinity for a specific epitope (the antigenic portion that is specifically recognized by the antigen receptor).

[0044] The "chimeric antigen receptor" consists of an extracellular antigen-binding region (composed of light and heavy chains derived from monoclonal antibodies, connected by a tough hinge region to form a single-chain antibody), a transmembrane region, and an intracellular signal transduction region.

[0045] "95-99% identity" refers to a homology of more than 95% (ideally more than 98%).

[0046] "Modification" is a variation of an amino acid or nucleotide, including the deletion, insertion, and / or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids or nucleotides, as well as the addition of one or more (usually up to 20, preferably up to 10, more preferably up to 5) amino acids or nucleotides at the C-terminus and / or N-terminus, without altering the function of the protein or nucleic acid.

[0047] In this application, "anti-MSLN" and "targeted MSLN" can be used interchangeably.

[0048] This invention provides a fully human antibody or antibody fragment targeting MSLN, wherein the MSLN binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is shown in Table 1.

[0049] Table 1. Amino acid sequence of scFv

[0050]

[0051] The nucleotide sequence encoding scFv provided by this invention includes the sequence shown in SEQ ID NO: 2. After translation into an amino acid sequence based on the codons corresponding to the bases in each sequence, the corresponding amino acid sequence of scFv is the sequence shown in SEQ ID NO: 1. The sequences shown in SEQ ID NO: 3 to SEQ ID NO: 8 are the variable region sequences of SEQ ID NO: 1.

[0052] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0053] The sources of some materials are explained below:

[0054] MSLN antigen protein: Human MSLN, purchased from Kaika, catalog number MSL-HM480B-100μg.

[0055] Phage antibody library of human single-chain antibodies: constructed by Changzhou Felos Pharmaceutical Technology Co., Ltd.

[0056] Streptavidin magnetic beads: purchased from Invitrogen.

[0057] Enzyme-linked immunosorbent assay (ELISA) microplate: 96-well low-permeability flat-bottom microplate, purchased from Corning.

[0058] Anti-M13 HRP antibody: purchased from Thermo Fisher.

[0059] VCSM13 helper phage: purchased from Invitrogen.

[0060] SOC culture medium: purchased from Shanghai Sangon Biotech.

[0061] pCGMT3 phage vector: purchased from Addgene.

[0062] XL1-blue bacteria: purchased from Agilent Technologies, product number 200228.

[0063] LB solid culture medium plates: Dissolve 5g yeast extract, 10g peptone, 10g sodium chloride and 10g agar powder in 1L of double-distilled water, autoclave at 121℃, and then pour onto plates.

[0064] Developer ABTS solution: purchased from Thermo Fisher, product number 002024.

[0065] Gelred nucleic acid dye: purchased from Thermo Fisher.

[0066] pFUSE expression vector: purchased from Invitrogen.

[0067] Plasmid extraction kit: purchased from QIAGEN.

[0068] Restriction endonucleases: purchased from Takara.

[0069] Recombinase: purchased from Novoprotein.

[0070] lipo3000 reagent: purchased from Thermo Fisher Scientific.

[0071] Polybrene reagent: purchased from Thermo Fisher Scientific.

[0072] RMPI 1640 medium: purchased from Gibco.

[0073] DMEM medium: purchased from Gibco.

[0074] Myc-Tag(9B11)Mouse mAb(PE Conjugate): Purchased from Cell Signaling.

[0075] 293Fectin transfection reagent: purchased from Invitrogen, catalog number 12347500.

[0076] 293 Freestyle suspension cells: purchased from Thermo Fisher.

[0077] Immunoglobulin IgG1 constant region Fc segment: purchased from Nanjing Genscript Biotech Co., Ltd.

[0078] BCA protein quantification kit: purchased from Pierce, catalog number 23252.

[0079] CBS antigen fixation solution: Dissolve 1.59g Na2CO3 and 2.93g NaHCO3 in 1L of water and adjust the pH to 9.6.

[0080] Anti-Human Fc HRP secondary antibody: purchased from Thermo Fisher.

[0081] 96-hole bottom plate: purchased from Corning.

[0082] Biacore Instrument: Purchased from GE, T200.

[0083] Protein A chip: purchased from GE.

[0084] Example 1: Screening of single-chain antibodies targeting human Mesothelin (MSLN)

[0085] (1) Establish a phage antibody library of fully human single-chain antibodies

[0086] Primers were designed to amplify the heavy and light chain variable regions of the fully human single-chain antibody. The heavy and light chain variable regions were ligated together using (GGGGS)3-Linker via overlap extension PCR to obtain the full-length PCR product. The PCR product and pCGMT3 phage vector were digested with SfiI enzyme, and after reaction with T4 ligase at 16°C, the ligation transformation product was electroporated into XL1-blue competent cells. Then, 3 mL of SOC medium was added to the competent cells, and the cells were cultured at 30°C for 1 h. Finally, ampicillin and tetracycline were added to a final concentration of 50 μg / mL, and the cells were cultured at 37°C with shaking for 2 h.

[0087] Then add a concentration of 10. 1350 μL of VCSM13 helper phage was added and incubated at room temperature for 1 h, with gentle shaking every 10 min. After 2 h of shaking culture at 37 °C, kanamycin was added to a final concentration of 70 μg / mL and cultured overnight at 30 °C. The supernatant was collected by centrifugation, and 10% PEG-8000 / sodium chloride solution (PEG is polyethylene glycol) was added. The mixture was placed on ice for 1 h, centrifuged at 8000 rpm and 4 °C, and the supernatant was discarded. The precipitate was completely dissolved in 2 mL of PBS solution (phosphate buffer) containing 1% BSA (bovine serum albumin). The supernatant was collected by centrifugation, which is the phage-displayed fully human single-chain antibody library.

[0088] (2) Screening of a fully human antibody library targeting the MSLN antigen protein

[0089] Take 200 μL (containing 1 × 10⁶ bacteriophages) 13 A phage antibody library expressing fully human single-chain antibodies ( / mL) was mixed with 5 μg of His-tagged MSLN antigen protein and incubated at room temperature for 2 hours. Then, 50 μL of streptavidin magnetic beads were added. Phages that bound the antigen were captured by the streptavidin magnetic beads. Unbound phages were removed by rinsing with 0.5% Tween-20 PBS solution (phosphate buffer). Phages that were stably bound to the magnetic beads were eluted with glycine hydrochloride solution (pH 2.2) for later use.

[0090] Inoculate 20 mL of XL1-Blue Escherichia coli and shake until the OD 600 (absorbance at 600 nm) reaches 0.6. Then add the eluted bacteriophage and incubate with XL1-Blue Escherichia coli at 37°C for 30 min. Spread the bacterial suspension on ampicillin-resistant plates. The next day, wash and collect the bacterial cells from the ampicillin-resistant plates. Infect 1×10⁶ cells / mL of the bacterial suspension. 12 After amplifying VCSM13 helper phages at pfu / mL (pfu, plaque forming unit), the next round of screening was performed, for a total of 2-3 rounds. The XL1-Blue bacterial suspensions in the input and output phage libraries were thoroughly diluted, and then plated onto LB agar plates resistant to ampicillin. Single clones on each plate were counted, and the enrichment level of the screened phages was determined based on the counts from the output and input.

[0091] The results are as follows Figure 1As shown, after three rounds of panning using MSLN antigen proteins targeting the His tag, the output in the third round increased by more than 100 times compared to the first round, while the output in the second round increased by more than 10 times. Monoclonal antibodies were picked from the phage library (output) plates after screening, and the phage libraries from each round of panning were validated using phage enzyme-linked immunosorbent assay (ELISA).

[0092] (3) Enzyme-linked immunosorbent assay (ELISA) of phage library

[0093] Each batch of phage library input was diluted 10-fold, 50-fold, and 100-fold, respectively. ELISA microplates were coated with MSLN antigen protein and incubated overnight at 4°C. After elution with PBST (phosphate-buffered saline containing Tween-20), the plates were blocked. The diluted phage library input was then added, and the plates were incubated at room temperature for 2 hours. After washing the plates 8 times with PBST, Anti-M13 HRP antibody was added and incubated for 30 minutes. The plates were then washed 8 times with PBST, and 50 μL of ABTS developing solution was added.

[0094] like Figure 2 As shown, according to OD 450 After three rounds of screening, the phage pools at different dilutions showed a significant increase in binding to MSLN antigen, indicating that the screening of human MSLN antigen yielded a significantly enriched antibody library.

[0095] (4) Monoclonal phage enzyme-linked immunosorbent assay

[0096] XL1-Blue monoclonal bacteria infected with phages were selected from the phage library obtained after the third round of screening and inoculated into 2 mL 96-well bacterial culture plates (purchased from Corning). 500 μL of SB medium containing tetracycline resistance was added, and the plates were incubated at 37°C for 4-6 hours at 200 rpm. Once the OD600 value approached 0.6, 1 μL of helper phage was added, and the plates were incubated overnight at 30°C. The next day, the plates were centrifuged at 3000g for 15 minutes, and the supernatant was collected for later use. Microplates containing MSLN antigen protein were coated and incubated overnight at 4°C. After elution with PBST (phosphate-buffered saline containing Tween-20), the plates were blocked. The prepared phage supernatant was then added, and the plates were incubated at room temperature for 2 hours. After washing 8 times with PBST, Anti-M13 HRP antibody was added and incubated for 30 minutes. After washing 8 times with PBST, 50 μL of ABTS developing solution was added. According to the rule that positive clones are identified by an OD450nm value > 1, positive clones are selected for sequencing to obtain the DNA sequence of the positive antibody. The obtained sequences are then compared and analyzed. The more duplicate clones there are, the higher the enrichment of the antibody sequence, thus determining the effectively enriched nucleotide coding sequence.

[0097] Example 2: Preparation of anti-MSLN single-chain antibody

[0098] (1) Expression and purification

[0099] Plasmids were extracted from the positive monoclonal strains screened in Experiment Example 1, and after digestion with restriction endonuclease SfiⅠ, the fragments were ligated into the pFuse expression vector (the pFuse expression vector is ligated by restriction endonuclease SfiⅠ at the restriction site) using T4 ligase, thereby obtaining the pFuse expression vector containing anti-MSLN single-chain antibody.

[0100] The 293Fectin transfection reagent was mixed with the eukaryotic antibody expression vector obtained above at a volume-to-weight ratio of 30 μL:30 μg. 30 mL of 293Freestyle suspension cells were added, and the mixture was cultured in a shaker at 37°C at 125 rpm for 48-72 hours. After centrifugation, the supernatant was collected, and the antibody protein was purified using Protein A HP columns on a protein purification instrument to obtain purified anti-MSLN antibody test samples 2MG10, MD3, 2MC9, 2MD12, 2MC11, 2MH6, 2MD6, 2MB1, and MA8. Finally, the antibody concentration was detected according to the instructions of the BCA method protein quantification kit.

[0101] Example 3: Detection of the lead antibody

[0102] (1) Enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibody to human MSLN protein.

[0103] The binding affinity of the purified anti-MSLN single-chain antibody obtained in Example 2 to human MSLN protein (purchased from Kaika) was verified. The human MSLN protein was diluted to a final concentration of 1.0 μg / mL with CBS antigen fixation solution, and then 50 μL was added to each well of a 96-well ELISA plate. The plate was sealed with plastic film and incubated at 4°C overnight. The next day, the plate was washed twice with washing buffer [PBS containing 0.01% (v / v) Tween 20], and then blocked at room temperature for 1 hour with blocking buffer [PBS containing 0.01% (v / v) Tween 20 and 1% (v / v) BSA]. Discard the blocking buffer. The purified human MSLN protein antibodies 2MG10, MD3, 2MC9, 2MD12, 2MC11, 2MH6, 2MD6, 2MB1, MA8, and the negative control human Fc protein (hFc) prepared in Example 2 were serially diluted 3-fold, starting at 300 nM, for a total of 10 gradients. 50 μL of each solution was added to each well of the ELISA plate. After incubation at 37°C for 1 hour, the plate was washed 8 times with wash buffer [PBS containing 0.01% (v / v) Tween 20]. HRP (horseradish peroxidase)-labeled rabbit anti-human IgG Fc secondary antibody was added, and after incubation at 37°C for 1 hour, the plate was washed 8 times with wash buffer [PBS containing 0.01% (v / v) Tween 20]. 100 μL of TMB substrate was added to each well, and after incubation at room temperature for 30 minutes, 100 μL of stop solution (1.0N HCl) was added to each well. The A450nm value was read using a multi-functional microplate reader (SpectraMax iD5), and the EC50 value was calculated by fitting a curve.

[0104] The results are as follows Figure 3 As shown, the purified anti-MSLN single-chain antibody exhibited specific binding ability to human MSLN recombinant protein at the ELISA level. Lead antibodies 2MC9, 2MD12, 2MC11, 2MH6, 2MD6, 2MB1, and MA8 were selected for cellular-level binding experiments. Among them, lead antibody 2MH6 had an EC50 of 0.15 nM and showed good protein-level binding activity, indicating potential for further development.

[0105] (2) Flow cytometry (FACS) assay to detect the binding of antibody to human MSLN-overexpressing cells

[0106] A nucleotide sequence encoding the full-length human MSLN protein was cloned into the pLenti plasmid via homologous recombination. This plasmid was then transfected into the CHOK1 cell line to obtain a stable CHOK1 cell line overexpressing human MSLN (referred to here as the CHOK1-hMesothelin stable cell line). The CHOK1-h4-1BB stable cell line was cultured in 10 cm cell culture dishes until 90% confluence. The culture medium was aspirated, and the cells were washed twice with FACS buffer. After cell counting, the cells were diluted to 2 × 10⁻⁶ cells with FACS buffer.6 Cells / ml were collected and 1% goat serum blocking buffer was added (the percentage is by weight). The cells were incubated on ice for 30 minutes, then washed twice by centrifugation with FACS buffer. The collected cells were resuspended in FACS buffer (PBS containing 1% BSA, the percentage is by weight) to a concentration of 2 × 10⁻⁶ cells / ml. 6 Cells / ml were added at a rate of 100 μL per well to a 96-well FACS plate. The purified MSLN antibody samples (2MC9, 2MD12, 2MC11, 2MH6, 2MB1, MA8) prepared in Example 2, along with the negative control human Fc protein (hFC) and the positive control MSLN Tab1 protein, were serially diluted 5-fold, starting at 200 nM, for a total of 7 gradients. 100 μL of the diluted antibody was added to each well, and the plates were incubated on ice for 1 hour. Cells were washed twice with FACS buffer by centrifugation. 100 μL of fluorescent (Alexa 488) labeled goat anti-human IgG Fc secondary antibody (from Abcam) was added to each well, and the plates were incubated on ice for 1 hour. Cells were washed three times with FACS buffer by centrifugation. Cells were resuspended in 100 μL of FACS buffer, and the results were detected and analyzed using FACS (FACS Calibur, from BD). The EC50 value was calculated from the fitted curve.

[0107] The results are as follows Figure 4 As shown, the purified anti-MSLN single-chain antibodies prepared in Example 2 can all specifically recognize and bind to cells overexpressing MSLN protein on their cell surface. Among them, the lead antibody 2MH6 has an EC50 of 0.79 nM and exhibits high activity in specifically recognizing MSLN protein on the cell surface, showing potential for further development. The negative control is human Fc protein, and the data in the table are the average fluorescence intensity values ​​of the cell population measured by MFI.

[0108] Example 4: Preparation of CAR-NK cells targeting MSLN

[0109] (1) Constructing different CAR structures containing human cortisol single-chain antibody

[0110] The DNA fragment of the lead antibody 2MH6 obtained in Experiment 3 was inserted into the pCDH lentiviral vector to obtain the recombinant lentiviral vector pLenti-anti-MSLN-CAR. The anti-MSLN-CAR construct of this invention comprises, in sequence: an EF-1α promoter, a signal peptide sequence, an MSLN binding domain, a detection tag, a hinge region, and a transmembrane domain, as well as a functional signal transduction domain, represented as: promoter-SP-scFv-hinge-TM-CD / s-SD-eGFP. Wherein:

[0111] The signal peptide sequence (SP) includes CSF2RA (SP1) or 2B4' (SP2), wherein the amino acid sequence of CSF2RA is the sequence shown in SEQ ID NO: 9, and the amino acid sequence of 2B4' is the sequence shown in SEQ ID NO: 10.

[0112] The detection tag contains eGFP, and the amino acid sequence of the eGFP is the sequence shown in SEQ ID NO: 11.

[0113] The MSLN binding domain includes scFv, which contains the fully human antibody or antibody fragment targeting MSLN.

[0114] The hinge region contains CD8', and the amino acid sequence of CD8' is the sequence shown in SEQ ID NO: 12.

[0115] The transmembrane domain (TM) comprises any one of CD8”, 2B4”, or NKG2D, wherein the amino acid sequence of CD8” is the sequence shown in SEQ ID NO: 13, the amino acid sequence of 2B4” is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of NKG2D is the sequence shown in SEQ ID NO: 15.

[0116] The functional signal transduction domain (CD / s-SD) comprises at least one of 2B4”', 4-1BB, DAP12, DAP10, and CD3Zeta (CD3ζ), wherein the amino acid sequence of 2B4”' is the sequence shown in SEQ ID NO: 16, the amino acid sequence of 4-1BB is the sequence shown in SEQ ID NO: 17, the amino acid sequence of DAP12 is the sequence shown in SEQ ID NO: 18, the amino acid sequence of DAP10 is the sequence shown in SEQ ID NO: 19, and the amino acid sequence of CD3Zeta (CD3ζ) is the sequence shown in SEQ ID NO: 20.

[0117] Through design and selection, this embodiment yielded nine different anti-MSLN-CAR constructs, as shown in the table below.

[0118] Table 2 shows the nine different anti-MSLN-CAR constructs in Example 4.

[0119] CARs Target protein Component (SP-scFv-hinge-TM-CD / s-SD) Label SCARA Mesothelin SP1-2MH6-CD8'-CD8”-4-1BB-CD3ζ eGFP FCARB Mesothelin SP2-2MH6-2B4”-2B4”' / SCARC Mesothelin SP1-2MH6-CD8'-CD8”-2B4”’-CD3ζ eGFP SCARD Mesothelin SP1-2MH6-CD8'-NKG2D-4-1BB-CD3ζ eGFP SCARE Mesothelin SP1-2MH6-CD8'-NKG2D-2B4”’-CD3ζ eGFP TCARF Mesothelin SP1-2MH6-CD8'-NKG2D-2B4″'-DAP10-CD3ζ eGFP TCARG Mesothelin SP1-2MH6-CD8'-NKG2D-2B4”’-DAP12-CD3ζ eGFP TCARH Mesothelin SP1-2MH6-CD8'-NKG2D-4-1BB-2B4”’-CD3ζ eGFP TCARI Mesothelin SP1-2MH6-CD8'-NKG2D-2B4”'-4-1BB-CD3ζ eGFP

[0120] (2) Packaging Lentiviral

[0121] Take 5 μg, 3.2 μg, and 1.8 μg of the different recombinant lentiviral vectors pLenti-anti-Mesothelin-CAR, PspaX2 plasmid, and PMD2.0G plasmid (PspaX2 plasmid and PMD2.0G plasmid are lentiviral packaging plasmids) prepared in step (1), respectively, mix them, and transfect them into 293T cells (approximately 3 × 10⁻⁶ cells). 6 Cells were transfected into 10 cm plates in 10 mL of DMEM medium containing 10% (v / v) FBS. After 72 hours, the viral stock solution was collected, filtered through a 0.45 μm filter membrane, and different groups of CAR-NK lentiviruses were obtained. The titers were detected using a kit.

[0122] (3) Recombinant lentivirus infection of NK cells

[0123] Resuscitate NK cells (obtained from peripheral blood culture), culture overnight, centrifuge at 300g for 10 min, collect cells, and resuspend in NK cell culture medium. Take a 12-well plate and add 5 × 10⁵ cells to each well. 5 100 μL of CAR-NK lentivirus, polybrene, and IL2 were mixed to obtain a culture system. In this culture system, the concentration of polybrene was 8 μg / mL and the concentration of IL2 was 500 U / mL. The system was incubated at 37°C in a 5% CO2 incubator to obtain CAR-NK cells targeting mesothelin protein.

[0124] (4) Detection of CAR expression levels

[0125] Take 2×10 5 NK cells were washed twice with FACS buffer, incubated with fluorescently labeled MSLN to detect proteins, washed twice with FACS buffer, and then the CAR expression rate was detected by flow cytometry.

[0126] The results are as follows Figure 5 As shown in Figures a and b, CAR-NK cells containing different anti-MSLN-CARs were prepared by lentiviral transfection, with transfection efficiencies ranging from 18% to 60%.

[0127] Example 5: Detection of the killing effect of CAR-NK cells targeting mesothelin on ovarian cancer tumor cells.

[0128] The CAR-NK cells or human NK cells (obtained from peripheral blood isolation and culture) prepared in Example 4 were used as effector cells, and SKOV3 ovarian cancer tumor cells (ATCC product) that highly express human MSLN protein were used as target cells to detect cytotoxic activity. 1×10⁻⁶ cells were used. 4Ovarian cancer SKOV3 tumor cells were incubated with CAR-NK or human NK effector cells at different effector-to-target ratios. The number of effector cells in each incubation system was 5 × 10⁻⁶. 4 1, 2.5 × 10 4 1×10 4 One or 0.4×10 4 Individual cells were incubated at 37°C and 5% CO2 for 4 hours. The killing efficiency of effector cells against ovarian cancer tumor cells was detected using a DELFA kit (killing efficiency = number of viable ovarian cancer tumor cells after incubation / 10). 4 The ratio of effector cells to ovarian cancer tumor cells was used to plot a kill curve with the kill efficiency as the x-axis and the kill efficiency as the y-axis.

[0129] The results are as follows Figure 6 As shown, compared with human NK cells, the CAR-NK cells containing different anti-MSLN-CARs prepared in Example 4 all showed better killing effects on SKOV3 ovarian cancer tumor cells.

[0130] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A fully human antibody or antibody fragment targeting MSLN, characterized in that, The antibody or antibody fragment comprises: a heavy chain variable region and a light chain variable region; The heavy chain variable region includes three complementarity-determining regions: HCDR1, HCDR2, and HCDR3, wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 3; the amino acid sequence of HCDR2 is shown in SEQ ID NO: 4; and the amino acid sequence of HCDR3 is shown in SEQ ID NO:

5. The light chain variable region includes three complementary determinant regions: LCDR1, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR1 is shown in SEQ ID NO: 6; the amino acid sequence of LCDR2 is shown in SEQ ID NO: 7; and the amino acid sequence of LCDR3 is shown in SEQ ID NO:

8.

2. The fully human antibody or antibody fragment targeting MSLN according to claim 1, characterized in that, The MSLN binding domain of the antibody or antibody fragment is scFv, and the amino acid sequence of the scFv is the sequence shown in SEQ ID NO:

1.

3. The fully human antibody or antibody fragment targeting MSLN according to claim 1, characterized in that, The MSLN binding domain of the antibody or antibody fragment is scFv, and the nucleotide sequence of the scFv is the sequence shown in SEQ ID NO:

2.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor contains a fully human antibody or antibody fragment targeting MSLN as described in any one of claims 1-3, wherein the fully human antibody is a single-chain antibody.

5. The chimeric antigen receptor according to claim 4, characterized in that, The chimeric antigen receptor comprises, in sequence: a signal peptide sequence, an MSLN binding domain, a detection tag, a hinge region and a transmembrane domain, and a functional signal transduction domain. The signal peptide sequence comprises CSF2RA or 2B4', wherein the amino acid sequence of CSF2RA is the sequence shown in SEQ ID NO: 9, and the amino acid sequence of 2B4' is the sequence shown in SEQ ID NO: 10; The detection tag contains eGFP, and the amino acid sequence of the eGFP is the sequence shown in SEQ ID NO: 11; The MSLN binding domain includes scFv, and the scFv contains the fully human antibody or antibody fragment targeting MSLN. The hinge region includes: CD8', the amino acid sequence of which is the sequence shown in SEQ ID NO: 12; The transmembrane domain comprises any one of CD8'', 2B4'', or NKG2D, wherein the amino acid sequence of CD8'' is the sequence shown in SEQ ID NO: 13, the amino acid sequence of 2B4'' is the sequence shown in SEQ ID NO: 14, and the amino acid sequence of NKG2D is the sequence shown in SEQ ID NO:

15. The functional signal transduction domain comprises at least one of 2B4''', 4-1BB, DAP12, DAP10, and CD3 Zeta, wherein the amino acid sequence of 2B4''' is the sequence shown in SEQ ID NO: 16, the amino acid sequence of 4-1BB is the sequence shown in SEQ ID NO: 17, the amino acid sequence of DAP12 is the sequence shown in SEQ ID NO: 18, the amino acid sequence of DAP10 is the sequence shown in SEQ ID NO: 19, and the amino acid sequence of CD3 Zeta is the sequence shown in SEQ ID NO:

20.

6. A host cell, characterized in that, The host cell comprises the chimeric antigen receptor as described in claim 4 or 5, and the host cell is an NK cell.

7. A lentiviral plasmid, characterized in that, The lentiviral plasmid contains a nucleotide sequence capable of encoding a fully human antibody or antibody fragment targeting MSLN as described in any one of claims 1-3.

8. The use of the fully human antibody or antibody fragment targeting MSLN according to any one of claims 1-3, or the chimeric antigen receptor according to claim 4 or 5, or the host cell according to claim 6, or the lentiviral plasmid according to claim 7 in the preparation of an antitumor drug, characterized in that, The tumor is ovarian cancer.

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