Anti-mesothelin antibodies and uses thereof

Abstract

The application belongs to the technical field of tumor immunotherapy, and particularly relates to an anti-mesothelin antibody and application thereof, and the application finds three mouse-derived antibodies with high affinity, and the antibodies are subjected to humanization treatment; the affinity is significantly higher than that of SS1 antibody. On this basis, a second-generation CAR-T cell based on the above antibody is constructed, and the in-vitro killing effect is better than that of a conventional SS1 CAR-T, and the CAR-T cell in a humanized form has tumor cell killing capacity equivalent to that of a mouse-derived CAR-T cell, and the cell exhaustion level is significantly reduced within a long time of 0-14 days after being stimulated by an antigen. The chimeric antigen receptor T cell has strong killing activity on mesothelin-positive tumor cells, uses a fully human antibody as an scfv, has no immunogenicity, can be repeatedly administered, and enhances the treatment effect of the CAR-T cell.

Description

Technical Field

[0001] This invention belongs to the field of tumor immunotherapy technology, specifically relating to an anti-mesothelin antibody and its application. Background Technology

[0002] Mesothelin, or MSLN for short, is highly expressed in solid tumors such as ovarian cancer, pancreatic cancer, and mesothelioma, making it an ideal therapeutic target. Existing anti-MSLN antibodies, such as SS1 antibodies, have low affinity and strong tetanic signals, which limits CAR-T cell activity. Existing anti-MSLN CAR-T cells, such as SS1 scfv, are overly activated upon contact with free antigens in the peripheral circulation system, resulting in high CAR-T cell depletion upon reaching the tumor site and weakened tumor-killing ability. Therefore, there is an urgent need to develop a novel mesothelin antibody. Summary of the Invention

[0003] The purpose of this invention is to provide a fully human anti-mesothelin antibody, which solves the problems existing in the prior art.

[0004] The technical solution adopted in this invention is:

[0005] This invention provides an anti-mesothelin antibody, wherein the anti-mesothelin antibody comprises HCDR1~HCDR3 and LCDR1~LCDR3;

[0006] The amino acid sequence of HCDR1 is selected from any one of cases 1) to 12):

[0007] 1) The amino acid sequence as shown in SEQ ID NO.24;

[0008] 2) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.24;

[0009] 3) The amino acid sequence as shown in SEQ ID NO.32;

[0010] 4) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.32;

[0011] 5) The amino acid sequence as shown in SEQ ID NO.40;

[0012] 6) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 40;

[0013] 7) The amino acid sequence as shown in SEQ ID NO.30;

[0014] 8) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 30;

[0015] 9) The amino acid sequence as shown in SEQ ID NO.38;

[0016] 10) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 38;

[0017] 11) The amino acid sequence as shown in SEQ ID NO.46;

[0018] 12) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 46;

[0019] The amino acid sequence of HCDR2 is selected from any one of cases 13) to 24):

[0020] 13) The amino acid sequence as shown in SEQ ID NO.25;

[0021] 14) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 25;

[0022] 15) The amino acid sequence as shown in SEQ ID NO.33;

[0023] 16) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 33;

[0024] 17) The amino acid sequence as shown in SEQ ID NO.41;

[0025] 18) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.41;

[0026] 19) The amino acid sequence shown in SEQ ID NO.31;

[0027] 20) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.31;

[0028] 21) The amino acid sequence as shown in SEQ ID NO.39;

[0029] 22) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.39;

[0030] 23) The amino acid sequence as shown in SEQ ID NO.47;

[0031] 24) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.47;

[0032] The amino acid sequence of HCDR3 is selected from any one of the following: (25) to (30).

[0033] 25) The amino acid sequence as shown in SEQ ID NO.26;

[0034] 26) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 26;

[0035] 27) The amino acid sequence as shown in SEQ ID NO.34;

[0036] 28) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.34;

[0037] 29) The amino acid sequence shown in SEQ ID NO.42;

[0038] 30) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 42;

[0039] The amino acid sequence of LCDR1 is selected from any one of cases 31) to 36):

[0040] 31) The amino acid sequence as shown in SEQ ID NO.27;

[0041] 32) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.27;

[0042] 33) The amino acid sequence as shown in SEQ ID NO.35;

[0043] 34) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 35;

[0044] 35) The amino acid sequence shown in SEQ ID NO.43;

[0045] 36) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 43;

[0046] The amino acid sequence of LCDR2 is selected from any one of the following: (37) to (42).

[0047] 37) The amino acid sequence as shown in SEQ ID NO. 28;

[0048] 38) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 28;

[0049] 39) The amino acid sequence shown in SEQ ID NO.36;

[0050] 40) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.36;

[0051] 41) The amino acid sequence as shown in SEQ ID NO.44;

[0052] 42) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 44;

[0053] The amino acid sequence of LCDR3 is selected from any one of cases 43) to 48):

[0054] 43) The amino acid sequence as shown in SEQ ID NO. 29;

[0055] 44) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO. 29;

[0056] 45) The amino acid sequence as shown in SEQ ID NO.37;

[0057] 46) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.37;

[0058] 47) The amino acid sequence as shown in SEQ ID NO.45;

[0059] 48) An amino acid sequence that has at least 80% identity with the amino acid sequence shown in SEQ ID NO.45.

[0060] Preferably, the amino acid sequence of the heavy chain variable region of the anti-mesothelin antibody is any one of the sequences shown in SEQ ID NO.1, SEQ ID NO.7, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.3, SEQ ID NO.9, SEQ ID NO.21, SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.17, and SEQ ID NO.18.

[0061] The amino acid sequence of the light chain variable region is any one of the sequences shown in SEQ ID NO.2, SEQ ID NO.8, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.4, SEQ ID NO.10, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.6, SEQ ID NO.12, SEQ ID NO.19, and SEQ ID NO.20.

[0062] Preferably, the anti-mesothelin antibody further comprises a heavy chain constant region and / or a light chain constant region.

[0063] A second aspect of the present invention provides a chimeric antigen receptor comprising a nucleotide sequence encoding the heavy chain variable region and / or the light chain variable region.

[0064] A third aspect of the present invention provides a method for preparing the chimeric antigen receptor, the method being as follows:

[0065] The chimeric antigen receptor is obtained by linking the heavy chain variable region, the light chain variable region, and the second-generation CAR sequence through homologous recombination.

[0066] The second-generation CAR sequence includes the intracellular signaling domain CD3ζ, CD8a signal peptide, CD8a hinge region, CD8a transmembrane region, and 4-1BB co-stimulatory signaling region.

[0067] The amino acid sequence of the intracellular signaling domain CD3ζ is shown in SEQ ID NO.48;

[0068] The amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO.49;

[0069] The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.50;

[0070] The amino acid sequence of the transmembrane region of CD8a is shown in SEQ ID NO.51;

[0071] The amino acid sequence of the 4-1BB co-stimulatory signal region is shown in SEQ ID NO.52.

[0072] A fourth aspect of the present invention provides a recombinant expression vector comprising the chimeric antigen receptor described above.

[0073] A fifth aspect of the present invention provides a CAR-T cell comprising the recombinant expression vector described above.

[0074] The fifth aspect of the present invention provides an application of the aforementioned anti-mesothelin antibody, the aforementioned chimeric antigen receptor, the aforementioned recombinant expression vector, and the aforementioned CAR-T cells, wherein the application refers to the preparation of drugs for treating cancer.

[0075] Preferably, the cancer includes any one of breast cancer, pancreatic cancer, ovarian cancer, and stomach cancer.

[0076] Compared with the prior art, the beneficial effects of the present invention are:

[0077] This invention provides an anti-mesothelin antibody comprising HCDR1-HCDR3 and LCDR1-LCDR3. This invention also discovered three high-affinity murine antibodies, 2B6, 2E5, and M10A6, and humanized them; among them, clones 2B6 and M10A6 showed significantly higher affinity than the SS1 antibody. Based on these antibodies, second-generation CAR-T cells were constructed, exhibiting superior in vitro killing effects compared to traditional SS1 CAR-T cells. Furthermore, the humanized CAR-T cells possess tumor cell killing capabilities comparable to those of the murine counterparts, while showing significantly reduced cell exhaustion levels over a prolonged period of 0-14 days after antigen stimulation. The chimeric antigen receptor T cells of this invention exhibit strong killing activity against mesothelin-positive tumor cells. Using a fully human antibody as the scfv, it is non-immunogenic, allowing for repeated administration and enhancing the therapeutic effect of CAR-T cells. Attached Figure Description

[0078] Figure 1 For comparison of the affinity of different antibodies, A: mouse 2E5; B: mouse 2B5; C: mouse 10A6.

[0079] Figure 2 The concentration gradient curves of different antibodies and mesothelin-stable cell lines HEK293T-mesothelin binding and the EC50 values ​​of antibody-cell binding are shown. A: SS1 antibody; B: mouse 2B6; C: mouse M10A6; D: mouse 2E5.

[0080] Figure 3 This is a schematic diagram of the chimeric antigen receptor in this invention.

[0081] Figure 4 Figure showing the comparison of the lysis and killing ability of mesothelin-CAR-T cells against target cells HCC1806.

[0082] Figure 5 Figure showing the comparison of the lytic and killing abilities of mesothelin-CAR-T cells against target cells SKOV3.

[0083] Figure 6Figure showing the comparison of the lysis and killing ability of mesothelin-CAR-T cells against KATO3 target cells.

[0084] Figure 7 Figure 1 shows the comparison of the ability of mesothelin-CAR-T cells to secrete IFN-γ and TNF-α. A: IFN-γ content in the supernatant co-cultured with 293T cells; B: IFN-γ content in the supernatant co-cultured with KATO3 cells; C: IFN-γ content in the supernatant co-cultured with HCC1806 cells; D: IFN-γ content in the supernatant co-cultured with SKOV3 cells; E: TNF-α content in the supernatant co-cultured with 293T cells; F: TNF-α content in the supernatant co-cultured with KATO3 cells; G: TNF-α content in the supernatant co-cultured with HCC1806 cells; H: TNF-α content in the supernatant co-cultured with SKOV3 cells.

[0085] Figure 8 A comparison of mesothelin-CAR-T cell exhaustion levels.

[0086] Figure 9 The therapeutic effects of different combinations of VH and VL sequences of M10A6 in different tumor cells are shown in the following: A: HCC1806; B: SKOV3; C: KATO3; D: 293T.

[0087] Figure 10 The therapeutic effects of different VH and VL sequence combinations of 2B6 and 2E5 in different tumor cells are shown in the following: A: HCC1806; B: SKOV3; C: KATO3; D: 293T. Detailed Implementation

[0088] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0089] The inventive concept of this invention is as follows:

[0090] The second-generation CAR structure includes: CD8 Hinge for enhanced spatial flexibility, CD8 transmembrane region for stable membrane localization, 41BB co-stimulatory domain for enhanced T cell persistence, and CD3ζ activation signaling domain.

[0091] The vector construction of this invention involves cloning the CAR sequence into a lentiviral vector and achieving stable expression via T-cell transduction. This invention also provides a vector containing the aforementioned nucleic acid encoding a chimeric antigen receptor protein expressed on the surface of immune effector cells. In one specific embodiment, the vector used in this invention is a lentiviral plasmid vector pwslv04. This plasmid belongs to a third-generation self-inactivated lentiviral vector system, which contains three plasmids: a packaging plasmid encoding the Gag / Pol protein, a packaging plasmid psPAX2 encoding the Rev protein, an envelope plasmid PMD2.G encoding the VSV-G protein, and an empty vector pwslv04, which can be used for recombination to introduce the target nucleic acid sequence, i.e., the nucleic acid sequence encoding CAR. The expression of enhanced green fluorescent protein in the empty vector pwslv04 is regulated by the SFFV promoter. The recombinant expression vector pwslv04-CAR-T2A-eGFP containing the target nucleic acid sequence encoding CAR achieves co-expression of eGFP and CAR through a ribosomal skipping sequence 2A from the Thosea asigna virus. It should be understood that other expression vectors are also available. This invention also includes viruses comprising the vectors described above. The viruses of this invention include packaged infectious viruses, as well as unpackaged viruses containing the components necessary for packaging into infectious viruses. Other viruses and their corresponding plasmid vectors known in the art for transferring exogenous genes into immune effector cells can also be used in this invention.

[0092] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0093] The list of abbreviations for this invention is shown in Table 1.

[0094]

[0095] Example 1

[0096] An anti-mesothelin antibody, as detailed below:

[0097] 1. This invention screened three mouse-derived antibodies through mouse hybridomas: mouse anti-mesothelin M10A6, mouse anti-mesothelin 2E5, and mouse anti-mesothelin 2B6.

[0098] The heavy chain amino acid sequence of the mouse anti-mesothelin M10A6 scFv is shown in SEQ ID NO.1, and the light chain amino acid sequence is shown in SEQ ID NO.2. The heavy chain amino acid sequence of the mouse anti-mesothelin 2E5 scFv is shown in SEQ ID NO.3, and the light chain amino acid sequence is shown in SEQ ID NO.4. The heavy chain amino acid sequence of the mouse anti-mesothelin 2B6 scFv is shown in SEQ ID NO.5, and the light chain amino acid sequence is shown in SEQ ID NO.6.

[0099] 2. Human-centered design.

[0100] The CDR regions of three antibodies—mouse anti-mesothelin M10A6, mouse anti-mesothelin 2E5, and mouse anti-mesothelin 2B6—were transplanted into the human IgG framework, specifically the FR region of human IgG. Site-directed mutagenesis was used to reduce immunogenicity, resulting in three fully human anti-mesothelin antibodies: fully human anti-mesothelin M10A6, fully human anti-mesothelin 2E5, and fully human anti-mesothelin 2B6. The CDR region sequences of the mouse antibodies were optimized for humanization to retain high specificity.

[0101] The heavy chain amino acid sequence of the fully human anti-mesothelin H10A6 scFv is shown in SEQ ID NO. 7, and the light chain amino acid sequence is shown in SEQ ID NO. 8. The heavy chain amino acid sequence of the fully human anti-mesothelin H2E5 scFv is shown in SEQ ID NO. 9, and the light chain amino acid sequence is shown in SEQ ID NO. 10. The heavy chain amino acid sequence of the fully human anti-mesothelin 2B6 HscFv is shown in SEQ ID NO. 11, and the light chain amino acid sequence is shown in SEQ ID NO. 12.

[0102] 3. Reverse mutation of humanized antibodies.

[0103] The heavy chain amino acid sequence of the reversion mutant humanized anti-mesothelin M10A6 scFv is shown in SEQ ID NO. 13, and the light chain amino acid sequence is shown in SEQ ID NO. 15. The heavy chain amino acid sequence of the reversion mutant humanized anti-mesothelin 2B6 scFv is shown in SEQ ID NO. 17, and the light chain amino acid sequence is shown in SEQ ID NO. 19. The heavy chain amino acid sequence of the reversion mutant humanized anti-mesothelin 2E5 scFv is shown in SEQ ID NO. 21, and the light chain amino acid sequence is shown in SEQ ID NO. 22.

[0104] 4. Further reverse mutations based on the reverse mutation.

[0105] The heavy chain amino acid sequence of the further reverse mutation-humanized anti-mesothelin M10A6 scFv is shown in SEQ ID NO. 14, and the light chain amino acid sequence is shown in SEQ ID NO. 16. The heavy chain amino acid sequence of the further reverse mutation-humanized anti-mesothelin 2B6 scFv is shown in SEQ ID NO. 18, and the light chain amino acid sequence is shown in SEQ ID NO. 20. The light chain amino acid sequence of the further reverse mutation-humanized anti-mesothelin 2E5 scFv is shown in SEQ ID NO. 23.

[0106] The amino acid sequences of M10A6-related antibodies are shown in Table 2; the amino acid sequences of 2E5-related antibodies are shown in Table 3; and the amino acid sequences of 2B6-related antibodies are shown in Table 4.

[0107] Table 2. Amino acid sequences of M10A6-related antibodies

[0108]

[0109] Table 3. Amino acid sequences of 2E5-related antibodies

[0110]

[0111] Table 4. Amino acid sequences of 2B6-related antibodies

[0112]

[0113] 5. Predict the CDR region of the anti-mesothelin antibody using the Kabat algorithm and the Chothia algorithm.

[0114] The amino acid sequences of the M10A6 CDR region are shown in Table 5; the amino acid sequences of the 2E5 CDR region are shown in Table 6; and the amino acid sequences of the 2B6 CDR region are shown in Table 7.

[0115] Table 5. Amino acid sequence of the M10A6 CDR region

[0116]

[0117] Table 6. Amino acid sequence of the 2E5 CDR region

[0118]

[0119] Table 7. Amino acid sequence of the 2B6 CDR region

[0120]

[0121] 6. Verify the protein affinity of the anti-mesothelin antibody described in this embodiment.

[0122] According to the literature "Chowdhury PS, Viner JL, Beers R, Pastan I. Isolation of a high-affinity stable single-chain Fv specific for mesothelin from DNA-immunized mice by phage display and construction of a recombinant immunotoxin with anti-tumor activity. Proc Natl Acad Sci US A. 1998 Jan 20;95(2):669-74. doi: 10.1073 / pnas.95.2.669. PMID: 9435250; PMCID: PMC18478," the KD of the existing anti-MSLN antibody SS1 is 11 nM. Therefore, the surface plasmon resonance (SPR) assay was performed on the above three mouse antibodies. The modified anti-mesothelin M10A6 scFv and anti-mesothelin 2B6 scFv antibodies showed a KD value of less than 5 nM against MSLN, which was significantly better than that of the SS1 antibody. See results Figure 1 .

[0123] 7. Verify the cell affinity of the anti-mesothelin antibody described in this embodiment.

[0124] Using the known antibody SS1 as a control, the three murine antibodies were subjected to concentration gradient incubation and flow cytometry analysis. The EC50 of antibody-cell binding was calculated using the MFI value of the antibody binding to the secondary antibody fluorescein. Flow cytometry analysis showed that the EC50 of M10A6 scFv and M2B6 scFv antibodies against mesothelin-stable 293T cells were 109.6 mg / ml and 78.04 mg / ml, respectively, significantly better than the SS1 antibody (158.5 ng / ml). The results are shown in [Figure number missing]. Figure 2 .

[0125] Example 2

[0126] The application of an anti-mesothelin antibody is as follows:

[0127] This embodiment constructs CAR-T cells targeting MSLN based on the anti-mesothelin antibody described in Example 1. First, a chimeric antigen receptor was constructed, the structural diagram of which is shown below. Figure 3 The amino acid sequences of each element in the second-generation CAR sequence are shown in SEQ ID NO.48~SEQ ID NO.52.

[0128] (1) The amino acid sequence of the intracellular signaling domain CD3ζ is shown in SEQ ID NO.48.

[0129] SEQ ID NO.48:

[0130] RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0131] (2) The extracellular signal peptide is the CD8a signal peptide, and the amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO.49.

[0132] SEQ ID NO49: MALPVTALLLPLALLLHAARP.

[0133] (3) The hinge region is the CD8a hinge region, and the amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.50.

[0134] SEQ ID NO.50:

[0135] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD.

[0136] (4) The transmembrane region is the CD8a transmembrane region, and the amino acid sequence of the CD8a transmembrane region is shown in SEQ ID NO.51.

[0137] SEQ ID NO. 51: IYIWAPLAGTCGVLLLSLVITLYC.

[0138] (5) The intracellular co-stimulatory signaling domain contains the 4-1BB co-stimulatory signaling region, and the amino acid sequence of the 4-1BB co-stimulatory signaling region is shown in SEQ ID NO.52.

[0139] SEQ ID NO.52:

[0140] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0141] 1. The CAR-T cell construction process is as follows:

[0142] 1.1 Carrier construction.

[0143] In the empty vector pwslv04-eGFP, the built-in SFFV promoter regulates the expression of enhanced green fluorescent protein eGFP. After inserting the scFv sequence from Example 1 and the aforementioned second-generation CAR sequence into the empty vector, multiple fragments are ligated using Gibson homologous recombination to form a recombinant expression vector encoding the target gene CAR. Co-expression of eGFP and the target gene CAR is achieved via T2A. In immunotherapy based on chimeric antigen receptor gene-modified T lymphocytes, the 2A sequence is often used to achieve co-expression of the target gene with GFP or eGFP. CAR expression can be indirectly detected by detecting GFP or eGFP. Once the recombinant expression vector is successfully constructed and its sequence is confirmed to be correct, it can be prepared for lentiviral packaging. CAR-T2A-eGFP is transcribed into one mRNA, but ultimately translated into two peptide chains: eGFP and an anti-MSLN chimeric antigen receptor. Guided by the CD8a signal peptide, the anti-MSLN chimeric antigen receptor will localize on the cell membrane.

[0144] The vectors containing CARs for each purpose were obtained as follows:

[0145] Pwslv04-SS1-CAR-T2A-eGFP, Pwslv04-2B6-CAR-T2A-eGFP, Pwslv04-2E5-CAR-T2A-eGFP, and Pwslv04-M10A6-CAR-T2A-eGFP.

[0146] 1.2 Lentiviral preparation.

[0147] When the cell density of 293 cells is close to 90%, passage is performed. Preheated trypsin at 37°C is added for digestion. After digestion and centrifugation, the cells are seeded into 10cm culture dishes. The cells are incubated in a 37°C incubator containing 5% CO2 for 24 hours. When the cells adhere to the culture dish and occupy more than 50% of the total area, lentiviral plasmids are transfected.

[0148] Take a sterile 1.5ml EP tube, add 800µL serum-free DMEM, 2.5µg pMD2G, 7.5µg psPAX2, 10µg recombinant expression vector, and 24µl PEI; vortex for 15s, and incubate at room temperature for 20min. Add the mixture to a cell culture dish, gently shake to mix, and then incubate at 37℃ with 5% CO2. After 16h, aspirate the medium, add an appropriate amount of preheated 20% FBS complete medium at 37℃, and continue incubation. Collect the supernatant containing lentivirus at approximately 48h and 72h post-transfection. Centrifuge at 1500rpm for 5min to remove cell debris. Filter the supernatant through a 0.45μm filter membrane and collect the viral solution. Spread the experimental cells to be transfected on 35mm culture dishes, change the medium after 24h, with an optimal density of 50%. Add the viral solution collected 24h post-transfection for the first time. Re-infection with viral fluid can be performed as needed. After the cells have reached confluence, they can be passaged or their expression can be detected.

[0149] 1.3 Isolation of primary T cells.

[0150] The PBMC cell density was adjusted to 1×10⁻⁶. 8 Cells / mL, total volume not exceeding 2.5 mL, resuspended in a 5 mL round-bottom tube. Remove the magnetic beads and mix thoroughly by pipetting up and down at least 5 times. Add 100 μL of magnetic beads / mL to the above sample, mix thoroughly, and incubate at room temperature for 30 min. Add complete culture medium to a total volume of 2.5 mL, open the tube and insert it into the magnetic pole, incubate at room temperature for 5 min. After incubation, leave the tube in the magnetic pole, gently invert, and pour out the cells. Repeat the washing once. Resuspend the cells in X-vivo15 medium and add 100 U / mL IL-2, 5 ng / mL IL-15, and 10 ng / mL IL-7.

[0151] 1.4. Preparation of CAR-T cells via T cell lentiviral infection: The specific experimental steps are as follows:

[0152] Calculate the required viral load based on an MOI of 20. After removing the virus from the -80°C freezer, thaw it rapidly in a 37°C water bath. Add the calculated viral load to a 24-well plate, along with DEAE and IX, mix thoroughly, seal the plate with sealing film, and centrifuge at 800g for 1 hour. After centrifugation, remove the sealing film and incubate the 24-well plate at 37°C with 5% CO2 for another 24 hours. Centrifuge at 250g for 10 minutes, remove the virus-containing supernatant, resuspend the cell pellet in fresh culture medium, transfer the cells to a new 24-well plate, and continue culturing for 6 days.

[0153] 2. Verification of the killing effect of CAR-T cells. The specific experimental steps are as follows:

[0154] Adjust the target cells to the logarithmic growth phase and passage them twice before the experiment. Digest the adherent target cells with trypsin and resuspend them in complete culture medium. Take a new 96-well plate and seed the target cells at a rate of 100 μL / well. Add 100 μL of sterile water to each of the unused wells around the perimeter of the 96-well plate to prevent water evaporation from the central wells. Place the plate in a 5% CO2 incubator at 37°C and incubate overnight.

[0155] The CAR-T cells prepared above were collected by centrifugation and resuspended in serum-free X-VIVO15 medium. The 96-well plate containing the target cells was removed from the incubator, the medium was completely aspirated from the wells, and the cells were gently washed once with sterile PBS. Then, CAR-T cells were added at an effector-to-target ratio of E / T = 1:1, and the final volume was brought to 100 μL / well. The 96-well plate was placed in a 5% CO2 incubator at 37°C and incubated for 72 h. After incubation, the plate was removed from the incubator, and images were taken to detect the killing effect on the target cells. The results are as follows: Figures 4-6 As shown, chimeric antigen receptor T cells targeting mesothelin can efficiently mediate the killing of tumor cells by T cells at an effector-to-target ratio of 1:1. The combined use of CAR-T cells from 2B6, 2E5, and M10A6 to verify the killing effect yielded similar results to their individual effects, and will not be presented separately here.

[0156] CAR-T cells constructed using humanized recombinant expression vectors showed that they could still efficiently mediate the killing of tumor cells by T cells. Results are shown in [Figure number missing]. Figure 9 and Figure 10 Therefore, it can be concluded that the humanization of antibody sequences does not affect the killing of target cells by CAR-T cells.

[0157] Figure 9This study illustrates the therapeutic effects of different VH and VL sequence combinations of M10A6 in different tumor cells. M10A6: VH and VL are both mouse-derived; HG-LG: VH and VL are both human-derived; HG-L5A: VH is human-derived, VL is a reversion mutation; HG-LIN: VH is human-derived, VL is a further reversion mutation; H5A-LG: VH is a reversion mutation, VL is human-derived; H5A-L5A: VH is a reversion mutation, VL is a reversion mutation; H5A-LIN: VH is a reversion mutation, VL is a further reversion mutation; HIN-LG: VH is a further reversion mutation, VL is human-derived; HIN-L5A: VH is a further reversion mutation, VL is a reversion mutation; HIN-LIN: VH is a further reversion mutation, VL is a further reversion mutation; NC is the control group; HBEST-LBEST represents the results of the best-performing VH and VL pairing.

[0158] Figure 10 The therapeutic effects of different VH and VL sequence combinations for 2B6 and 2E5 in different tumor cells are shown. The first three rows are the results for 2B6, and the last three rows are the results for 2E5. 2B6: VH and VL are both mouse-derived; 2E5: VH and VL are both mouse-derived; HGL5A: VH is human-derived, VL is a reversion mutation; H5ALG: VH is a reversion mutation, VL is human-derived; HINLG: VH is a further reversion mutation, VL is human-derived; HGLIN: VH is human-derived, VL is a further reversion mutation; HGLG: VH and VL are both human-derived.

[0159] 3. Detection of CAR-T cytokine secretion levels.

[0160] After incubation, the plate was removed from the incubator and centrifuged at 400g at room temperature for 5 min. The plate was then gently removed, and 150 μL of culture supernatant was transferred from each well. The expression of IFN-γ and TNF-α was detected using an ELISA kit, and OD values ​​were read using a microplate reader. The obtained data were plotted using GraphPad 9.0. The results are shown below. Figure 7 As shown, chimeric antigen receptor T cells targeting mesothelin can effectively activate primary T cells and increase the expression of cytokines after binding to targeted tumor cells.

[0161] 0. Detection of CAR-T cell exhaustion level.

[0162] Three days after T cell activation, the cells were transfected with SS1, 2E5, and 2B6 viruses. On days 13, 17, and 24 after activation, the expression of PD1 and Tim3 in the infected T cells was detected by flow cytometry. Results are as follows: Figure 8As shown, 2B6-CAR-T cells had the lowest exhaustion level, followed by 2E5-CAR-T cells, while the exhaustion level of SS1-CAR T cells was much higher than that of 2E5-CAR-T and 2B6-CAR-T cells from day 13 onwards.

[0163] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0164] 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 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. An anti-mesothelin antibody, characterized in that, The anti-mesothelin antibody comprises HCDR1~HCDR3 and LCDR1~LCDR3; When using the Kabat algorithm, the sequences of HCDR1 to HCDR3 are shown in SEQ ID NO.40, SEQ ID NO.41, and SEQ ID NO.42, respectively; the sequences of LCDR1 to LCDR3 are shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, respectively. When using the Chothia algorithm, the sequences of HCDR1 to HCDR3 are shown in SEQ ID NO.46, SEQ ID NO.47, and SEQ ID NO.42, respectively, and the sequences of LCDR1 to LCDR3 are shown in SEQ ID NO.43, SEQ ID NO.44, and SEQ ID NO.45, respectively.

2. The anti-mesothelin antibody of claim 1, wherein The amino acid sequence of the heavy chain variable region of the anti-mesothelin antibody is any one of the sequences shown in SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.17 and SEQ ID NO.18; The amino acid sequence of the light chain variable region is any one of those shown in SEQ ID NO.6, SEQ ID NO.12, SEQ ID NO.19 and SEQ ID NO.

20.

3. The anti-mesothelin antibody of claim 1, wherein The anti-mesothelin antibody also includes a heavy chain constant region and / or a light chain constant region.

4. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises a nucleotide sequence encoding the heavy chain variable region and / or the light chain variable region as described in claim 2.

5. The method for preparing the chimeric antigen receptor as described in claim 4, characterized in that, The method is as follows: The chimeric antigen receptor is obtained by linking the heavy chain variable region, the light chain variable region, and the second-generation CAR sequence through homologous recombination. The second-generation CAR sequence includes the intracellular signaling domain CD3ζ, CD8a signal peptide, CD8a hinge region, CD8a transmembrane region, and 4-1BB co-stimulatory signaling region. The amino acid sequence of the intracellular signaling domain CD3ζ is shown in SEQ ID NO.48; The amino acid sequence of the CD8a signal peptide is shown in SEQ ID NO.49; The amino acid sequence of the CD8a hinge region is shown in SEQ ID NO.50; The amino acid sequence of the transmembrane region of CD8a is shown in SEQ ID NO.51; The amino acid sequence of the 4-1BB co-stimulatory signal region is shown in SEQ ID NO.

52.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the chimeric antigen receptor as described in claim 4.

7. A CAR-T cell, characterized in that, The CAR-T cells comprise the recombinant expression vector of claim 6.

8. The use of the anti-mesothelin antibody according to claim 1, the chimeric antigen receptor according to claim 4, the recombinant expression vector according to claim 6 and the CAR-T cell according to claim 7, characterized in that, The application refers to its use in the preparation of drugs for treating cancer; The cancer type is any one of breast cancer, ovarian cancer, and stomach cancer.

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