Anti-mesothelin nanobody chimeric antigen receptor and its applications

Abstract

The present invention relates to an anti-mesothelin nanobody chimeric antigen receptor and its application, specifically, a chimeric antigen receptor comprising a mesothelin-binding molecule whose antigen-binding domain comprises an anti-mesothelin single domain antibody. Immune cells comprising the chimeric antigen receptor described herein have a higher cell killing effect.

Description

[Technical field]

[0001] The present invention relates to the field of chimeric antigen receptor therapy, specifically to anti-mesothelin nanobody-chimeric antigen receptors and their applications. [Background technology]

[0002] The mesothelin (MSLN) gene is located on chromosome 1p13.3 and has a total length of 8 kD. The gene contains an open reading frame of 1884 bp, 17 exons, and encodes 628 amino acids. The precursor protein of MSLN is a glycoprotein with a length of 69 kD, anchored to the cell membrane with glycosylphosphopeptide inositol, and can be hydrolyzed by proteolytic enzymes into two parts, the N-terminus of which is a soluble protein of 31 kD with megakaryocyte stimulating activity, called megakaryocyte potentiating factor (MPF), and the C-terminus of which is a membrane-bound protein of about 40 kD with cell adhesive properties, called MSLN. The structure of MSLN in the membrane protein can be divided into three different regions, of which the I region is the binding site for the ligand CA125.

[0003] Mesothelin is highly expressed in various malignant tumors as a differentiation antigen and is deeply involved in the development and progression of tumors. This target is one of the most promising antitumor targets, and many major pharmaceutical companies have been developing drugs against this target, including CAR-T, monoclonal antibodies, and ADCs. Over the past 20 years, various anti-mesothelin monoclonal antibodies have been developed, including mainly SS1P immunotoxin and MORAB-009. SS1P is a recombinant immunotoxin composed of a fusion of scFv and a truncated Pseudomonas aeruginosa exotoxin, which mainly mediates cell killing. Meanwhile, MORAB-009 is an IgG1 antibody engineered with the SS1P sequence, which mainly induces antibody-dependent cell-mediated cytotoxicity (ADCC).

[0004] The SS1P antibody binds to the mesothelin I region and can block the binding of CA125 to mesothelin. This antibody is also used to treat CAR-T, but all of them have insufficient therapeutic effects (Jiang, H. et al., Protein Cell 8, 926-931, 2017; Adusumilli, PS et al., Sci. Transl. Med. 6, 261ra151, 2014; Lanitis, E. et al., Mol. Ther. 20, 633-643, 2012). Nanobodies have the natural advantages of high stability, high osmotic pressure, and broad binding epitopes (Muyldermans S. Annu Rev Biochem. 2013;82:775-97.). There have been no reports in the field of using MSLN nanobodies for the treatment of CAR-T.

[0005] Currently, CAR-T therapy has achieved great success in the treatment of blood tumors, but the response rate against solid tumors is low. CAR-T therapy combined with PD-1 inhibitors is an emerging therapy for the treatment of solid tumors. Researchers at the Memorial Sloan Kettering Cancer Center in the United States have constructed CAR-T cells that can secrete anti-PD-1 single-chain antibodies (scFv) (Sarwish Rafiq et al., Nat Biotechnol 2018 Oct; 36(9): 847-856). However, research has shown that traditional scfv antibodies have low expression levels in T cells, making it difficult to fully exert the effects of PD-1 inhibitors, which is mainly due to the properties of the scfv antibodies themselves, such as their tendency to aggregate, their tendency to mismatch due to folding, and their low thermal stability (Martin N et al., Macromol Biosci. 2017 Feb;17(2)). On the other hand, nanobodies have natural advantages such as easy expression, high stability, and high affinity. Due to the advantages of the nanobody itself and the biological mechanisms of MSLN and PD-1, the development of MSLN nanobody-based CAR-T and its simultaneous combination with autocrine PD-1 nanobody has become an urgent task to be solved. Summary of the Invention

[0006] One aspect of the present invention is a method for producing a mesothelin single domain antibody comprising any signal peptide sequence, The present invention provides a chimeric antigen receptor comprising a mesothelin-binding molecule comprising a hinge region, a transmembrane region, and an intracellular region.

[0007] In one or more embodiments, the intracellular region comprises an intracellular costimulatory domain and / or an intracellular signaling domain.

[0008] In one or more embodiments, the anti-mesothelin single domain antibody is a single domain antibody specific for the mesothelin III domain (membrane proximal end).

[0009] In one or more embodiments, the anti-mesothelin single domain antibody comprises CDR1, CDR2 and CDR3, wherein CDR1 comprises the sequence set forth in SEQ ID NO:1, CDR2 comprises the sequence set forth in SEQ ID NO:2, and CDR3 comprises the sequence set forth in SEQ ID NO:3.

[0010] In one or more embodiments, the CDR1 of the anti-mesothelin single domain antibody comprises a sequence set forth in any one of SEQ ID NOs:4-27, 73. Preferably, the CDR1 comprises a sequence set forth in any one of SEQ ID NOs:5, 11, 14, 15, 17, 21, 27, 73.

[0011] In one or more embodiments, the CDR2 of the anti-mesothelin single domain antibody comprises a sequence set forth in any one of SEQ ID NOs: 28-47, 74. Preferably, the CDR2 comprises a sequence set forth in any one of SEQ ID NOs: 29, 35, 37, 38, 39, 43, 40, 74.

[0012] In one or more embodiments, the CDR3 of the anti-mesothelin single domain antibody comprises a sequence set forth in any one of SEQ ID NOs: 48-72, 75. Preferably, the CDR3 comprises a sequence set forth in any one of SEQ ID NOs: 49, 55, 58, 59, 61, 65, 72, 75.

[0013] In one or more embodiments, the FR region of the anti-mesothelin single domain antibody is a FR region of any one of VHH selected from SEQ ID NOs:76-120.

[0014] In one or more embodiments, the anti-mesothelin single domain antibody VHH is set forth in any one of SEQ ID NOs:76-120. Preferably, the anti-mesothelin single domain antibody is set forth in SEQ ID It is shown as one of NO:77, 83, 85, 86, 87, 89, 93, 100, or 103.

[0015] In one or more embodiments, the mesothelin binding molecule is a monovalent or multivalent single domain antibody comprising one, two or more of said single domain antibodies, a multispecific single domain antibody, a heavy chain antibody or antigen-binding fragment thereof, an antibody or antigen-binding fragment thereof.

[0016] In one or more embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a signal peptide, a mesothelin binding molecule comprising an anti-mesothelin single domain antibody, a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain.

[0017] In one or more embodiments, the signal peptide is a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, or a light chain signal peptide, more preferably a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is set forth in amino acid residues 1 to 22 of SEQ ID NO:121.

[0018] In one or more embodiments, the hinge region is selected from a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, and an IgG4 Fc CH2CH3 hinge region; preferably, the hinge region is a CD8 hinge region; more preferably, the amino acid sequence of the CD8 hinge region is set forth in amino acid residues 149 to 203 of SEQ ID NO:121.

[0019] In one or more embodiments, the transmembrane domain is one of the CD28 transmembrane domain, the CD8 transmembrane domain, the CD3ζ transmembrane domain, the CD134 transmembrane domain, the CD137 transmembrane domain, the ICOS transmembrane domain, and the DAP10 transmembrane domain; preferably the CD28 transmembrane domain, more preferably the amino acid sequence of which is set forth in amino acid residues 204 to 231 of SEQ ID NO:121.

[0020] In one or more embodiments, the intracellular costimulatory domain comprises an intracellular domain of a costimulatory signal molecule, including the intracellular domains of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T-cell costimulatory factor (ICOS), and DNAX-activation protein 10; preferably, the intracellular costimulatory domain is the intracellular domain of CD28; preferably, the amino acid sequence of the CD28 intracellular domain is set forth in amino acid residues 231 to 272 of SEQ ID NO:121.

[0021] In one or more embodiments, the intracellular signaling domain is a CD3ζ intracellular signaling domain or an FcεRIγ intracellular signaling domain; preferably a CD3ζ intracellular signaling domain, and preferably, the amino acid sequence of the CD3ζ intracellular signaling domain is set forth in amino acid residues 273 to 384 of SEQ ID NO:121.

[0022] In one or more embodiments, the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a CD8 signal peptide, an anti-MSLN nanobody, a CD8 hinge region, a CD28 transmembrane region, a CD28 costimulatory domain, and a CD3 zeta intracellular signaling domain.

[0023] In one or more embodiments, the amino acid sequence of the chimeric antigen receptor is set forth in SEQ ID NO:121, 122 or 123.

[0024] The present invention also provides a chimeric antigen receptor, the antigen binding domain of which comprises an anti-mesothelin single domain antibody.

[0025] In one or more embodiments, the anti-mesothelin single domain antibody may be as described in any of the embodiments above.

[0026] The present invention also provides a nucleic acid molecule comprising a sequence selected from: (1) a coding sequence for a chimeric antigen receptor described in any embodiment herein; (2) The complementary sequence of (1); (3) A 5-50 bp fragment of either the sequence of (1) or (2).

[0027] In one or more embodiments, the fragment is a primer.

[0028] In one or more embodiments, the coding sequence is DNA or RNA.

[0029] A second aspect of the invention also provides a nucleic acid construct comprising a nucleic acid molecule according to any embodiment of the invention.

[0030] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for a PD-1 binding molecule.

[0031] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for a transposase and / or a coding sequence for a PD-1 binding molecule, where the transposase is used to transpose the coding sequence for the chimeric antigen receptor and / or the PD-1 binding molecule, e.g., to integrate the coding sequence or expression cassette for the chimeric antigen receptor and / or the PD-1 binding molecule into the genome.

[0032] In one or more embodiments, the coding sequence for the transposase is DNA or RNA.

[0033] In one or more embodiments, the coding sequence for the PD-1 binding molecule is DNA or RNA.

[0034] In one or more embodiments, the PD-1 binding molecule comprises a PD-1 antibody or antigen-binding fragment thereof, a PD-1 heavy chain antibody or antigen-binding fragment thereof, a monovalent or multivalent single domain antibody including one, two or more PD-1 single domain antibodies, a multispecific single domain antibody.

[0035] In one or more embodiments, the PD-1 binding molecule (e.g., a PD-1 single domain antibody) also comprises an Fc region. Preferably, the Fc region is an IgG4 Fc region, the sequence of which is shown in SEQ ID NO:133.

[0036] In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector or an integrating vector.

[0037] In one or more embodiments, the expression vector is a constitutive expression vector. Preferably, the constitutive expression vector is a transfer vector.

[0038] In one or more embodiments, the coding sequence for the chimeric antigen receptor is located between the recognition sequences for the transposase.

[0039] In one or more embodiments, the coding sequence for the PD-1 binding molecule is located between the recognition sequences for the transposase.

[0040] In one or more embodiments, the transposase is PiggyBac transposase (PBase).

[0041] In one or more embodiments, the transposase is as described in CN105154473A.

[0042] In one or more embodiments, the nucleic acid construct comprises an expression cassette for a CAR. In one or more embodiments, the nucleic acid construct further comprises an expression cassette for a transposase and / or an expression cassette for a PD-1 binding molecule. The one, two or three expression cassettes are contained in one or more vectors.

[0043] In one or more embodiments, the nucleic acid construct comprises an expression cassette for the chimeric antigen receptor and an expression cassette for the transposase, or the nucleic acid construct is an expression cassette, and the coding sequence for the chimeric antigen receptor and the coding sequence for the transposase are within the expression cassette.

[0044] In one or more embodiments, the nucleic acid construct comprises an expression cassette for the chimeric antigen receptor and an expression cassette for the PD-1-binding molecule, or the nucleic acid construct is an expression cassette, and the coding sequence for the chimeric antigen receptor and the coding sequence for the PD-1-binding molecule are within the expression cassette.

[0045] In one or more embodiments, the nucleic acid construct comprises an expression cassette for the chimeric antigen receptor, an expression cassette for the transposase, and an expression cassette for the PD-1 binding molecule; or the nucleic acid construct comprises two expression cassettes, one of which expresses the chimeric antigen receptor. one of the coding sequence for the chimeric antigen receptor, the coding sequence for the transposase, and the coding sequence for the PD-1 binding molecule, and the other of the coding sequences for the remaining ones; or the nucleic acid construct is an expression cassette, and the coding sequence for the chimeric antigen receptor, the coding sequence for the transposase, and the coding sequence for the PD-1 binding molecule are in the expression cassette.

[0046] In one or more embodiments, the nucleic acid construct is one or more vectors, each vector comprising one or two or three expression cassettes as described in any embodiment herein.

[0047] In one or more embodiments, the nucleic acid construct comprises: One vector containing (a) an expression cassette for a CAR, and, optionally, (b) an expression cassette for a transposase and / or an expression cassette for a PD-1 binding molecule; two vectors, (a) each containing an expression cassette for a CAR and an expression cassette for a PD-1 binding molecule, optionally one of which also contains an expression cassette for a transposase, or (b) two vectors, one of which contains an expression cassette for a transposase and the other of which contains an expression cassette for a CAR and an expression cassette for a PD-1 binding molecule; Three vectors each containing an expression cassette for a CAR, an expression cassette for a PD-1 binding molecule, and an expression cassette for a transposase Includes.

[0048] Another aspect of the present invention also provides a combination of coding sequences comprising a coding sequence for a chimeric antigen receptor according to any one of claims 1 to 4 and a coding sequence for a transposase and / or a coding sequence for a PD-1 binding molecule.

[0049] In one or more embodiments, any of the coding sequences in the combination of coding sequences can be freely selected to be DNA or RNA, for example, all three coding sequences are DNA, or all three coding sequences are RNA, or any one or two of the three are DNA.

[0050] In one or more embodiments, the combination of coding sequences includes a coding sequence for a chimeric antigen receptor, a coding sequence for a transposase, and a coding sequence for a PD-1 binding molecule, and all three coding sequences are DNA.

[0051] In one or more embodiments, the combination of coding sequences includes a coding sequence for a chimeric antigen receptor, a coding sequence for a transposase, and a coding sequence for a PD-1 binding molecule, and the coding sequence for the chimeric antigen receptor and the coding sequence for the PD-1 binding molecule are DNA and the coding sequence for the transposase is RNA, more preferably mRNA.

[0052] In one or more embodiments, in the combination of coding sequences, the coding sequences may be located on the same nucleic acid molecule or on different nucleic acid molecules, for example, the coding sequence for the chimeric antigen receptor, the coding sequence for the transposase, and the coding sequence for the PD-1 binding molecule are located on the same nucleic acid molecule, or the coding sequence for the chimeric antigen receptor and the coding sequence for the transposase are located on the same nucleic acid molecule and the coding sequence for the PD-1 binding molecule is located on another nucleic acid molecule, or the coding sequence for the chimeric antigen receptor, the coding sequence for the transposase, and the coding sequence for the PD-1 binding molecule are each located on three nucleic acid molecules.

[0053] Another aspect of the present invention provides a host cell, said host cell comprising (1) comprises and / or expresses a chimeric antigen receptor as described in any embodiment herein, and / or optionally comprises, expresses and / or secretes a PD-1 binding molecule, and / or (2) comprising a nucleic acid molecule and / or a nucleic acid construct described herein; and / or (3) Contains a combination of the coding sequences described herein.

[0054] The transposase and the PD-1 binding molecule are as described in the second aspect herein.

[0055] In one or more embodiments, the cell is an immune cell, preferably a T cell.

[0056] In one or more embodiments, the cell (a) comprises a coding sequence for a chimeric antigen receptor described in any embodiment herein; and optionally (b) comprises a coding sequence for a PD-1 binding molecule described in any embodiment herein.

[0057] In one or more embodiments, the cell comprises a coding sequence for a chimeric antigen receptor described in any embodiment herein and a coding sequence for a PD-1 binding molecule described in any embodiment herein.

[0058] Another aspect of the present invention is a composition comprising a pharmaceutical composition comprising: (1) a chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally including (i) a transposase and / or its coding sequence, and / or (ii) a PD-1 binding molecule and / or its coding sequence, or (2) A nucleic acid molecule, a nucleic acid construct, a combination of coding sequences, or a cell according to any embodiment of the present specification. The present invention provides a pharmaceutical composition comprising:

[0059] The transposase and the PD-1 binding molecule are as described in the second aspect herein.

[0060] In one or more embodiments, the pharmaceutical composition is utilized to treat cancer.

[0061] In one or more embodiments, the cancer comprises a mesothelin-mediated cancer. Preferably, the cancer also comprises a PD-1-mediated cancer.

[0062] In one or more embodiments, the cancer comprises one or more selected from mesothelioma, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, and non-Hodgkin's lymphoma.

[0063] Another aspect of the present invention provides a use of a reagent in the preparation of activated immune cells (e.g., T cells), the reagent comprising: (1) a chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally including (i) a transposase and / or its coding sequence, and / or (ii) a PD-1 binding molecule and / or its coding sequence, or (2) A nucleic acid molecule, a nucleic acid construct, a combination of coding sequences, or a cell according to any embodiment of the present specification. Includes.

[0064] The transposase and the PD-1 binding molecule are as described in the second aspect herein.

[0065] Another aspect of the present invention provides a use of the reagent in preparing a medicament for treating cancer, the reagent comprising: (1) a chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally including (i) a transposase and / or its coding sequence, and / or (ii) a PD-1 binding molecule and / or its coding sequence, or (2) A nucleic acid molecule, a nucleic acid construct, a combination of coding sequences, or a cell according to any embodiment of the present specification. Includes.

[0066] The transposase and the PD-1 binding molecule are as described in the second aspect herein.

[0067] In one or more embodiments, the cancer comprises a mesothelin-mediated cancer. Preferably, the cancer also comprises a PD-1-mediated cancer.

[0068] In one or more embodiments, the cancer comprises one or more selected from mesothelioma, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, and non-Hodgkin's lymphoma.

[0069] The present invention also provides a method for treating or preventing cancer, said method comprising administering to a patient in need thereof a therapeutically effective amount of a cell or pharmaceutical composition according to any of the embodiments of the present invention.

[0070] In one or more embodiments, the cancer comprises a mesothelin-mediated cancer. Preferably, the cancer also comprises a PD-1-mediated cancer.

[0071] In one or more embodiments, the cancer is mesothelioma, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, or non-Hodgkin's lymphoma.

[0072] In one or more embodiments, the therapeutically effective amount is 1-100×10 6 cells / mouse, preferably 5 x 10 6 Cells / mouse.

[0073] In one or more embodiments, the cells or pharmaceutical compositions are administered by intravenous injection. [Brief description of the drawings]

[0074] [Figure 1]Growth curves and proliferation folds of MSLN CAR-T cells at D5-D13. Annotations are shown in Figure 1D. AC: D5-D13 proliferation curves of CAR-T cells prepared from peripheral blood of three volunteers (AC-201908B, AC-201909A, AC-201910A), D: average proliferation curves of CAR-T cells prepared from peripheral blood of the above three volunteers at D5-D13, E: average proliferation fold curves of CAR-T cells prepared from peripheral blood of the above three volunteers at D5-D13. [Diagram 2] Cell viability curve of MSLN CAR-T culture D5-D13. [Diagram 3] Changes in the number of MSLN CAR-positive T cells and the CAR positivity rate at D13. A: Number of MSLN CAR-positive T cells, B: CAR positivity rate. [Figure 4] Percentage of CD4+ and CD8+ T cells among CD3+ cells detected on D13 [Diagram 5] Percentage of memory T cells in the cell population on day 13 [Figure 6] Statistical results of in vitro killing of various mesothelin-negative (A: H520) and -positive (B: H226, C: ASPC-1) tumor cells by cryo-resuscitated MSLN CAR-T in a preliminary experiment. [Figure 7] Killing rates of mock-T and MSLN CAR-T against two mesothelin-positive tumor cells (E:T=1:1) at 72 h. A: SKOV3, B: H226. [Figure 8] Cytokine release after 72 h of co-incubation of mock-T and various MSLN CAR-T with mesothelin-positive tumor cells. A: IFN-gamma, B: IL-2, C: TNF-alpha. [Figure 9] D5-13 growth curves and proliferation folds of cells. AC: D5-D13 proliferation curves of CAR-T prepared from peripheral blood of three volunteers (AC201906A, AC201908A, AC201909A), D: average proliferation curves of CAR-T prepared from peripheral blood of the above three volunteers, E: average proliferation fold curves of CAR-T prepared from peripheral blood of the above three volunteers. Annotations are shown in Figure 9D. [Figure 10] Cell viability of cell culture D5-13. [Figure 11] Changes in the number of MSLN CAR-positive T cells and the CAR positivity rate on Day 13. A: Number of MSLN CAR-positive T cells, B: CAR positivity rate. [Figure 12] Percentage of CD4+ T cells and CD8+ T cells among CD3+ cells detected on D13. [Figure 13] Percentage of memory T cells in the cell population on day 13. [Figure 14] Changes in the proportion of two types of memory T cells before and after antigen stimulation. [Figure 15] Comparison of antibody concentrations secreted from cells before and after antigen stimulation. [Figure 16] Killing rates of mock-T and CAR-T against SKOV3-PDL1 at different ET ratios at 48 h. [Figure 17] Killing rates of mock-T and CAR-T against H226 at 48 h with different ET ratios. [Figure 18] Killing curves of mock-T and MSLN CAR-T against tumor cells. A: Killing rate statistics of CAR-T cells against H226 cells and SKOV-PDL1 cells (E:T=1:4, killing time 72h), B: Representative killing curve against H226 cells, C: Representative killing curve against SKOV3-PDL1 cells. [Figure 19] Cytokine release after 48 h of co-incubation of mock-T and MSLN CAR-T with mesothelin & PDL1 positive tumor cells. A: IL-2, B: IFN-gamma, C: TNF-alpha. [Figure 20] Changes in NCI-H226-PDL1 tumor volume at high, medium, and low doses. [Figure 21] Changes in Skov3-PDL1 tumor volume at high, medium, and low doses. [Figure 22] Body weight changes in NCI-H226-PDL1 tumor-bearing mice at high, medium, and low doses. [Diagram 23] Body weight changes in Skov3-PDL1 tumor-bearing mice at high, medium, and low doses. [Figure 24] Survival curves of NCI-H226-PDL1 tumor-bearing mice at high, medium, and low doses. [Diagram 25] Survival curves of Skov3-PDL1 tumor-bearing mice at high, medium, and low doses. [Figure 26] Changes in blood levels of PD1 antibody in NCI-H226-PDL1 tumor-bearing mice at high, medium, and low doses. A, B: low dose, C, D: medium dose, E, F: high dose. [Figure 27] Changes in blood levels of PD1 antibody in Skov3-PDL1 tumor-bearing mice at high, medium, and low doses. A, B: low dose, C, D: medium dose, E, F: high dose. [Figure 28] NCI-H226-PDL1 tumor volume change at high doses. [Figure 29] Body weight change in NCI-H226-PDL1 tumor-bearing mice at high doses. [Diagram 30] Survival curves of NCI-H226-PDL1 tumor-bearing mice at high doses. [Diagram 31] Changes in blood concentrations of PD1 antibodies in NCI-H226-PDL1 tumor-bearing mice at high doses (A: mass concentration, B: molar concentration). [Diagram 32] Changes in blood concentrations of CAR-T per microliter of blood (A: CD3+ T cells, B: CD8+ T cells, C: CAR+ T cells, D: percentage of CAR+ T cells among CD3+ T cells) in NCI-H226-PDL1 tumor-bearing mice at high doses. [Diagram 33] Changes in peripheral blood cytokine concentrations in NCI-H226-PDL1 tumor-bearing mice at high doses. A: IL-2, B: IL-4, C: TNF-alpha, D: IL-6, E: IFN-gamma, F: IL-10. [Diagram 34] Changes in tumor volume and body weight at common doses (A and C: changes in tumor volume and body weight for NCI-H226-PDL1, B and D: changes in tumor volume and body weight for Skov3-PDL1). [Diagram 35] CAR-T cell preparation data. A: proliferation rate, B: cell viability, C: CAR positive rate, D: Tcm and Tem, E: CD4 / 8, F: antibody secretion. [Diagram 36] Amount of PD-1 antibody secreted and cytokine secreted from CAR-T cells. A: Amount of PD-1 antibody secreted from CAR-T cells 6 and 48 hours after antigen stimulation. B: Amount of cytokine secreted from CAR-T cells 6 and 48 hours after antigen stimulation without and with antigen stimulation. [Figure 37] Killing ability of CAR-T cells. AB: Killing rate of Mock-T and CAR-T against H226-PDL1 tumor cells prepared from PBMCs from two different volunteers (09B and 09A01) at 80 h. CD: Killing curves of Mock-T and CAR-T against H226-PDL1 tumor cells prepared from PBMCs from two different volunteers (09B and 09A01). [Figure 38] Killing ability of CAR-T cells. AB: Killing rate of Mock-T and CAR-T against SKOV3-PDL1 tumor cells prepared from PBMCs from three different volunteers (09B, 09A01, 12A) at 100 h. CD: Killing curves of Mock-T and CAR-T against SKOV3-PDL1 tumor cells prepared from PBMCs from two different volunteers (09B and 09A01). [Figure 39] Schematic diagram of the in vivo experiment procedure using mice. [Diagram 40] The inhibitory effect of different doses of CAR-T on tumors. AD shows the results of doses of 0.2E6, 1E6, 5E6, and 20E6, respectively. [Diagram 41] Body weight and survival rate of mice at different doses. AD represents the body weight of mice at doses of 0.2E6, 1E6, 5E6, and 20E6, respectively, and E: survival rate of mice at dose of 20E6. [Diagram 42] Peripheral blood CD3+ T cell counts at different doses. [Diagram 43] Percentage of CAR-positive cells. A: Percentage of peripheral blood CAR+ T cells among CD3+ T cells at different doses after CAR-T infusion over 14 days. B: Percentage of CAR+ cells in each group before infusion. [Diagram 44] Diagram of the structure of the pNB338B-1444-CAR vector. [Diagram 45]Changes in blood concentrations of PD1 antibodies at common dosages (A: NCI-H226-PDL1, B: Skov3-PDL1). [Diagram 46] Changes in blood concentrations of CAR-T at common dosages (AD: NCI-H226-PDL1, EH: Skov3-PDL1). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0075] The present invention provides a chimeric antigen receptor (CAR) that targets mesothelin, which comprises an optional signal peptide sequence, a mesothelin-binding molecule including an anti-mesothelin single domain antibody sequence, a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain.

[0076] As used herein, a "binding molecule" of an antigen is a protein that specifically binds to an antigen, including, but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target-binding domains of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, single-chain molecules, and antibody fragments (particularly antigen-binding fragments, e.g., Fab, F(ab')2, and Fv). As used herein, the terms "immunoglobulin" (Ig) and "antibody" are interchangeable.

[0077] A "heavy chain antibody" as described herein is an antibody derived from a camelid or elasmobranch organism. Compared to the above four-chain antibodies, a heavy chain antibody lacks a light chain and a heavy chain constant region 1 (CH1), and comprises only two heavy chains consisting of a variable region (VHH) and another constant region, the variable region being linked to the constant region by a structure similar to a hinge region structure. Each heavy chain of a camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of an elasmobranch heavy chain antibody comprises one variable region and five constant regions (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. A heavy chain antibody can have the CH2 and CH3 of human IgG Fc by fusion with the constant region of human IgG Fc. Exemplarily, the heavy chain constant region comprises the sequence shown in SEQ ID NO:132.

[0078] As used herein, the terms "single domain antibody", "heavy chain variable region domain of a heavy chain antibody", "VHH", "nanobody" and "single variable region" are used interchangeably and all refer to a single domain polypeptide or protein that specifically recognizes and binds to an antigen. A single domain antibody is the variable region of a heavy chain antibody. Typically, a single domain antibody contains three CDRs and four FRs. Single domain antibodies are the smallest functional antigen-binding fragments. Generally, Typically, an antibody that naturally lacks a light chain and heavy chain constant region 1 (CH1) is obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0079] A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more different specificity single domain antibodies is a multispecific single domain antibody. A multivalent single domain antibody or a multispecific single domain antibody is linked to the multiple single domain antibodies by a linker. The linker usually consists of 1-15 amino acids selected from G and S.

[0080] In this specification, heavy chain antibody and antibody are intended to distinguish different combinations of antibodies. Due to structural similarities, the structural description of antibodies below applies not only to light chains but also to heavy chain antibodies.

[0081] The "variable region" or "variable domain" of an antibody is the amino-terminal domain of either the heavy or light chain of an antibody. The heavy and light chain variable domains are designated "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0082] The term "variable" means that certain segments in the variable domains differ extensively in sequence among antibodies. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, the variability is not uniformly distributed over the entire amino acid span of the variable domain. Instead, it is concentrated in three segments (present in both light and heavy chain variable domains) called hypervariable regions (HVRs): HCDR1, HCDR2, HCDR3 (which may be abbreviated as CDR1, CDR2, CDR3 in heavy chain antibodies) in the heavy chain variable region and LCDR1, LCDR2, LCDR3 in the light chain variable region. The more highly conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions (FR1, FR2, FR3, and FR4), which usually adopt a β-pleated sheet structure, but are connected by three HVRs that form a cyclic link and sometimes form part of the β-pleated sheet structure. The HVRs in each chain are held in close proximity by the FR regions and, together with the HVRs in the other chain, promote the formation of the antigen-binding site of the antibody. Usually, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity.

[0083] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain, which mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined, for example, as in Kabat, as the sequence segment from an amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, with its numbering according to the EU index. Each heavy chain of a camelid heavy chain antibody comprises one variable domain (VHH) and two constant domains (CH2 and CH3). The heavy chain antibody may have the CH2 and CH3 of human IgG Fc by fusion with the constant domain of human IgG Fc.

[0084] An "antibody fragment" refers to a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. The antibody fragments include the Fab, Fab', F(ab')2 and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, scFv-Fc fragments, multispecific antibodies formed by antibody fragments, and any fragment whose half-life can be increased by chemical modification or incorporation into liposomes. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, and one remaining "Fc" fragment, the name reflecting its ability to crystallize easily. The Fab fragments are composed of complete light and heavy chain variable domains (VH) and one heavy chain first constant domain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces one large F(ab')2 fragment, which roughly corresponds to two Fab fragments linked by disulfide bonds, with different antigen-binding activities, and still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by the addition of several other residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments are naturally produced as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are already known. Fc fragments contain the carboxy terminal portions of two heavy chains held together by disulfide bonds. The effector functions of antibodies are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcR) found on several types of cells. Antigen-binding fragments of heavy chain antibodies include VHHs and single heavy chain antibodies.

[0085] As used herein, antibodies include "chimeric" antibodies, which are antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, so long as the desired biological activity is exhibited, as well as fragments of such antibodies.

[0086] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Thus, a "humanized antibody" is typically a non-human antibody in which the variable domain framework regions have been replaced with sequences found in a human antibody. Typically, in a humanized antibody, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). The CDRs (either partially or completely encoded by nucleic acid derived from a non-human organism) are grafted onto the β-pleated sheet framework of a human antibody variable region to produce an antibody whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art and include, for example, the generation of mice with genetically engineered immune systems.

[0087] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced from a human and / or produced using any technique disclosed herein for producing human antibodies. This definition of a human antibody expressly excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies may be produced using a variety of techniques known in the art, including phage display libraries. The binding molecules according to any embodiment of the invention are chimeric or fully human antibodies, preferably fully human antibodies.

[0088] The term "immune cell" refers to a cell involved in or associated with an immune response, examples of which include, but are not limited to, lymphocytes, dendritic cells, monocytes / macrophages, granulocytes, mast cells, etc. Preferably, the immune cells are, for example, T cells, NK cells, NKT cells, tumor-infiltrating lymphocytes, and macrophages. In some embodiments, the immune cells of the present invention include T cells, such as CD4+ T cells and / or CD8+ T cells.

[0089] The "mesothelin binding molecules" described herein include anti-mesothelin single domain antibodies, the complementarity determining regions (CDRs) of said single domain antibodies comprising CDR1, CDR2 and CDR3, wherein CDR1 is selected from the group consisting of SEQ ID NO: CDR1 comprises the sequence shown in SEQ ID NO:1, CDR2 comprises the sequence shown in SEQ ID NO:2, and CDR3 comprises the sequence shown in SEQ ID NO:3.

[0090] In one or more embodiments, SEQ ID NO:1 is X1X2X3X4X5X6X7X8X9X 10 X 11 where X1 is G, E, T or A, X2 is S, N, P, A, H, D, K, I or F, X3 is I, S, V, T, D, L, A, M or H, X4 is F, S, I, A, L or G, X5 is N, S, G, A, D, T, E, R or H, X6 is I, L, F, Y, E or N, X7 is N, A, G, D, K, Y, L or S, X8 is A, Y, V, D or N, X9 is E or none, and X 10 is F or nothing, and X 11is A or none. In one or more embodiments, SEQ ID NO:1 is X1X2X3X4X5X6X7X8, where X1 is G, E, T or A, X2 is S, N, P, A, H, D, K, I or F, X3 is I, S, V, T, D, L, A, M or H, X4 is F, S, I, A, L or G, X5 is N, S, G, A, D, T, E or H, X6 is I, L, F, Y or N, X7 is N, A, G, D, K, Y or S, and X8 is A, Y, V or N. In one or more embodiments, SEQ ID NO:1 is GX2X3X4X5X6X7A, where X2 is S, N, A, D, I or F, X3 is I, V, T, D, A, L or S, X4 is F, S, I, A or L, X5 is N, S, A, D, E, T or H, X6 is I, F, N or Y, and X7 is N, G, D or Y. Preferably, SEQ ID NO:1 is X1X2X3X4X5X6X7X8, where X1 is G, E, T or A, X2 is S, N, P, H or I, X3 is I, S, T, D, L or A, X4 is F, I or L, X5 is S, T or E, X6 is I, F or Y, X7 is N, A, D or Y, and X8 is A or Y.

[0091] In one or more embodiments, the CDR1 of the anti-mesothelin single domain antibody comprises the sequence set forth in any one of SEQ ID NOs:4-27, 73. Preferably, the CDR1 comprises the sequence set forth in any one of SEQ ID NOs:5, 11, 14, 15, 17, 21, 27, 73.

[0092] In one or more embodiments, SEQ ID NO:2 is X1X2X3X4X5X6X7X8X9X 10 X 11 X 12where X1 is I, M, A, T or L, X2 is S, G, N, D, T or V, X3 is S, N, A, R, G or T, X4 is S, T, G, D or N, X5 is N, G, T or I, X6 is S, R, N, D, K, T or G, X7 is D, T, S, I or K, X8 is N, T, G, D or none, X9 is T, K, G or none, and X 10 is G, R or none, and X 11 is V, T or none, X 12 is T or none. In one or more embodiments, X3 is S, N, A, R or T. In one or more embodiments, SEQ ID NO:2 is IX2X3X4X5X6X7X8X9, where X2 is S, G, N or D, X3 is S, N, A or T, X4 is S, T, G, D or N, X5 is N or G, X6 is S, R, N, D or K, X7 is D, T, S or K, X8 is N, T or none, and X9 is T, K or none. Preferably, SEQ ID NO:2 is X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 where X1 is I, A or T, X2 is S, G, N or T, X3 is S or R, X4 is T, G or D, X5 is N, G or T, X6 is S, R, N, K or T, X7 is D, T, S, I or K, X8 is N, G or none, X9 is T, G or none, and X 10 is G or nothing, X 11 is V or nothing, X 12 is T or nothing.

[0093] In one or more embodiments, the CDR2 of the anti-mesothelin single domain antibody comprises a sequence set forth in any one of SEQ ID NOs:28-47, 74. Preferably, the CDR2 comprises a sequence set forth in any one of SEQ ID NOs:29, 35, 37, 38, 39, 43, 40, 74.

[0094] In one or more embodiments, SEQ ID NO:3 is X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 with the proviso that X1 is N, H, A or Q, X2 is L, A, G or V, X3 is S, R, E, G, D or T, X4 is N, A, R, G, D, K, V, T, I, E or Q, X5 is Y, F, K, S, C, I, D, G, H or R, X6 is D, A, V, G, R, N, P, T, C, Q or S, X7 is R, Y, T, A, D, K, L, E, S or G, and X8 is K , S, F, T, D, Q, E, G, P, R, Y or N, and X9 is D, G, I, H, S, Y, Q, K, T, R or V, and X 10 is R, Y, H, N, T, D, P, V, K, C, L or S, and X 11 is Y, D, E, P, F, S, L, I or none, and X 12 is P, V, Q, A, Y, F, N, D, R or None, and X 13 is D, A, Y, V, Q, P or none, and X 14 is Y, L, P, A, E or none, and X 15 is C, S, M, P, T or none, X 15 is C, S, M, P or none, X 16 is V, D, S, Y or none, and X 17 is L, F, V, D or none, and X 18 is R, G, M, S or None, and X 19 is D, N, S or none, X 20 is Y, L or nothing, and X 21 is Y or nothing, and X 22 is A or nothing, X 23is D or nothing. In one or more embodiments, X1 is N, H, A or Q, X2 is L, A, G or V, X3 is S, R, E, G, D or T, X4 is N, A, R, G, D, K, V, T, I or Q, X5 is Y, F, K, S, C, I, D, G, H or R, X6 is D, A, V, G, R, N, P, T, C, Q or S, X7 is R, Y, T, A, D, K, L, E, S or G, X8 is K, S, F, T, D, Q, E, G, P, R, Y or N, X9 is D, G, I, H, S, Y, Q, K, T or V, and X 10 is R, Y, H, N, T, D, P, V, K, C or S, and X 11 is Y, D, E, P, F, S, L or none, X 12 is P, V, Q, A, Y, F, N, D or None, and X 13 is D, A, Y, V, Q or none, and X 14 is Y, L, P, A, E or none, and X 15 is C, S, M, P or none, X 16 is V, D, S, Y or none, and X 17 is L, F, V, D or none, and X 18 is R, G, M, S or None, and X 19 is D, N, S or none, X 20 is Y, L or nothing, and X 21 is Y or nothing, and X 22 is A or nothing, X 23 is D or absent. In one or more embodiments, SEQ ID NO:3 is AX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19with the proviso that X2 is A, G or V, X3 is S, E, G, D or T, X4 is N, R, D, K, T or I, X5 is Y, F, K, I, D, G, H or R, X6 is D, A, G, N, P, T, C, Q or S, X7 is R, Y, T, A, D, K, L or G, X8 is K, S, F, D, Q, G, P, Y or N, X9 is D, G, I, S, Y, Q, K or V, and X 10 is R, Y, H, T, D, P, V, K or C, and X 11 is Y, D, E, F, S, L or none, X 12 is P, V, A, Y, F, N, D or None, and X 13 is D, A, Y, V or none, and X 14 is Y, P, E or none, and X 15 is S, P or none, X 16 is D, Y or None, X 17 is F, D or none, X 18 is G, S or none, X 19 is N or absent. Preferably, SEQ ID NO:3 is AX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 with the proviso that X2 is A or V, X3 is S, E, G or D, X4 is N, R, D or I, X5 is Y, F, K, D or H, X6 is D, G, P, C or S, X7 is R, Y, D, K or L, X8 is K, Q, G or N, X9 is D, S, Y or Q, and X 10 is R, Y, D, V or S, and X 11 is Y, D, F, S, L or none, and X 12 is P, F, or none, and X 13 is D, V or none, X 14 is P or nothing, and X 15 is S or nothing, X 16 is D or nothing, X17 is F or nothing, and X 18 is G or nothing, X 19 is N or None.

[0095] In one or more embodiments, the CDR3 of the anti-mesothelin single domain antibody comprises a sequence set forth in any one of SEQ ID NOs: 48-72, 75. Preferably, the CDR3 comprises a sequence set forth in any one of SEQ ID NOs: 49, 55, 58, 59, 61, 65, 72, 75.

[0096] In one or more embodiments, the CDR1 of the anti-mesothelin single domain antibody comprises a sequence set forth in any one of SEQ ID NOs: 4-27, 73, the CDR2 comprises a sequence set forth in any one of SEQ ID NOs: 28-47, 74, and the CDR3 comprises a sequence set forth in any one of SEQ ID NOs: 48-72, 75. Preferably, the anti-mesothelin single domain antibody comprises CDR1, CDR2 and CDR3 set forth in any one of Groups a1 to a26 below:

[0097] [Table 1]

[0098] In one or more embodiments, FR1 of the anti-mesothelin single domain antibody is selected from FR1 of the VHH of each antibody number in Table A, FR2 of the VHH is selected from FR2 of the VHH of each antibody number in Table 1, FR3 of the VHH is selected from FR3 of the VHH of each antibody number in Table 1, and FR4 of the VHH is selected from FR4 of the VHH of each antibody number in Table 1. In one or more embodiments, the FR region of the anti-mesothelin single domain antibody is the FR region of any one of the VHHs selected from SEQ ID NOs:76-120.

[0099] In one or more embodiments, the anti-mesothelin single domain antibody is set forth in any one of SEQ ID NOs:76-120. Preferably, the single domain antibody is set forth in any one of SEQ ID NOs:77, 83, 85, 86, 87, 89, 93, 100, 103.

[0100] The mesothelin binding molecules described herein are monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen-binding fragments thereof, or antibodies or antigen-binding fragments thereof, including one, two or more of the anti-mesothelin single domain antibodies described herein. The multivalent single domain antibodies or multispecific single domain antibodies are linked to the multiple single domain antibodies by a linker. The linker consists of 1-15 amino acids selected from G and S. The heavy chain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody. The antigen-binding fragment of the heavy chain antibody is a single-chain heavy chain antibody. The heavy chain antibody further comprises a heavy chain constant region. In one or more embodiments, the heavy chain constant region is a camelid heavy chain antibody constant region and comprises CH2 and CH3. In one or more embodiments, the CH2 and CH3 are human I The CH2 and CH3 of IgG Fc, for example, the CH2 and CH3 of IgG4.

[0101] The mesothelin binding molecules and anti-mesothelin single domain antibodies referred to in this specification include all mesothelin binding molecules and anti-mesothelin single domain antibodies described in CN202011584014 or CN202011582948, the entire text of the aforementioned patent applications being incorporated herein by reference in their entirety, each embodiment of which is specifically and individually incorporated herein by reference.

[0102] Any signal peptide on the CAR can be selected as needed. For example, CD8 signal peptide, CD28 signal peptide, CD4 signal peptide, or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used for CARs in the art. In some embodiments, the amino acid sequence of the human CD8 signal peptide can be as shown in amino acids 1-22 of SEQ ID NO:121.

[0103] The hinge region of the CAR is selected from the CD8α hinge region, the IgD hinge region, the IgG1 Fc CH2CH3 hinge region, or the IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of one of ordinary skill in the art. The CD8 hinge region suitable for the present invention may be any of the various human CD8 hinge region sequences commonly used for CARs in the art. In some embodiments, the amino acid sequence of the human CD8 hinge region may be as shown in amino acids 149 to 203 of SEQ ID NO:121.

[0104] The transmembrane region of the CAR is selected from one of the following: CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region, and DAP10 transmembrane region; preferably CD28 transmembrane region, the sequence of which is within the knowledge of one of ordinary skill in the art. The human CD28 transmembrane region suitable for the present invention may be any of various human CD28 transmembrane region sequences commonly used for CARs in the art. In some embodiments, the amino acid sequence of the human CD28 transmembrane region is as shown in amino acids 204 to 231 of SEQ ID NO:121.

[0105] If necessary, an appropriate intracellular costimulatory domain can be selected, such as an intracellular domain having a costimulatory signal molecule, such as CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T-cell costimulator (ICOS), and intracellular domain of DNAX-activating protein 10. The human CD28 costimulatory domain suitable for the present invention can be various human CD28 costimulatory domain sequences commonly used for CARs in the art. In some embodiments, the amino acid sequence of the CD28 costimulatory domain is as shown in the amino acid sequence at positions 231 to 272 of SEQ ID NO:121.

[0106] Similarly, the intracellular signal domain of the CAR can be selected as needed, including, but not limited to, the CD3ζ intracellular signal domain or the FcεRIγ intracellular signal domain. The CD3ζ intracellular signal domain suitable for the present invention can be any of the various CD3ζ intracellular signal domains used in CARs known in the art. In some embodiments, the amino acid sequence of the CD3ζ intracellular signal domain is as shown in the amino acid sequence at positions 273 to 384 of SEQ ID NO:121.

[0107] The above moieties forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, anti-MSLN nanobody, CD8 hinge region, CD28 transmembrane region, CD28 costimulatory domain, and CD3ζ intracellular signal domain, can be directly linked to each other or linked by a linker sequence. The linker sequence can be a linker sequence suitable for antibodies known in the art, such as a linker sequence containing G and S. Typically, the linker contains one or more repeat motifs. For example, the motifs are GGGS, GGGGS, SSSSG, GSGSA, GGSGG. Preferably, the motifs are adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence can contain 1, 2, 3, 4, or 5 repeat motifs. Linker The length of the linker sequence may be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, but is usually 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also include other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). As an example, the linker may be composed of any of the amino acid sequences of SEQ ID NOs:7-18. In some embodiments, the linker sequence is a (GGGGS)n linkage, where n is an integer from 1 to 5.

[0108] In exemplary embodiments, the CAR comprises, in order from N-terminus to C-terminus, a CD8 signal peptide, an anti-MSLN nanobody, a CD8 hinge region, a CD28 transmembrane region, a CD28 costimulatory domain, and a CD3 zeta intracellular signal domain. In specific examples, an exemplary CAR having the above structure is set forth in any one of SEQ ID NOs:121-123. In some embodiments, the amino acid sequence of the CAR of the invention is set forth in amino acids 23-384 of SEQ ID NO:121, or amino acids 23-377 of SEQ ID NO:122, or amino acids 23-378 of SEQ ID NO:123.

[0109] It should be understood that gene cloning always requires the design of an appropriate enzyme cleavage site, so that one or more extraneous residues are inevitably introduced at the end of the expressed amino acid sequence, but do not affect the activity of the target sequence. To construct a fusion protein, promote recombinant protein expression, obtain a recombinant protein that is automatically secreted outside the host cell, or aid in recombinant protein purification, it is always necessary to add specific amino acids to the N-terminus, C-terminus, or other suitable region of the protein. Other suitable regions of the protein include, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, for example. Thus, the amino or carboxyl terminus of the CAR of the present invention may also include one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0110] The present invention also includes mutants of the CAR described in any embodiment. These mutants comprise an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retains the biological activity of the CAR (e.g., T cell activation). The sequence identity between two sequences to be compared can be calculated, for example, using BLASTp from NCBI.

[0111] Mutants also include amino acid sequences having one or more mutations (insertion, deletion, or substitution) in the amino acid sequence of the CAR described in any embodiment, while retaining the biological activity of the CAR. The number of mutations is usually 1 to 10, for example, 1 to 8, 1 to 5, or 1 to 3. The substitution is preferably a conservative substitution. For example, conservative substitution with an amino acid having close or similar properties in the art usually does not change the function of the protein or polypeptide. "Amino acids with close or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., threonine, valine, isoleucine). (e.g., tyrosine, phenylalanine, tryptophan, histidine) and therefore, substitution of one or more sites in a polypeptide of the invention with another amino acid residue from the same side chain class does not substantially affect its activity.

[0112] The present invention includes polynucleotide sequences encoding the fusion proteins of the present invention. The polynucleotide sequences of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The present invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e. nucleotide sequences that encode the same amino acid sequence but have different nucleotide sequences.

[0113] The polynucleotide sequences described herein can be obtained by PCR amplification. Specifically, primers are designed according to the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the relevant sequences are obtained by amplifying a commercially available cDNA library or a cDNA library prepared by a conventional method well known to those skilled in the art as a template. For long sequences, PCR amplification is usually performed twice or multiple times, and then the amplified fragments are joined in the correct order.

[0114] The present invention also relates to a nucleic acid construct, where the term "nucleic acid construct" or "polynucleotide construct" refers to one or more single-stranded or double-stranded nucleic acid molecules isolated from a naturally occurring gene or modified to contain a nucleic acid fragment in a non-naturally occurring manner. The term "nucleic acid molecule" refers primarily to the physical nucleic acid molecule, and the term "nucleic acid sequence" refers primarily to the sequence of nucleotides on a nucleic acid molecule, but the two terms can be used interchangeably, especially with respect to a nucleic acid molecule or a nucleic acid sequence that can code for a protein or protein domain. The nucleic acid construct comprises a polynucleotide sequence as described herein and one or more regulatory sequences operably linked to these sequences. The polynucleotide sequence of the present invention may be manipulated in various ways to ensure expression of the CAR. The nucleic acid construct may be manipulated according to differences or requirements of the expression vector before it is inserted into a vector. Techniques for modifying polynucleotide sequences by recombinant DNA methods are known in the art.

[0115] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence from which the protein is to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence may be a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in the selected host cell may be used in the present invention. The regulatory sequence may be a suitable leader sequence, which is a non-translated region of the mRNA that is important for host cell translation. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that functions in the selected host cell may be used in the present invention.

[0116] In some embodiments of the invention, the nucleic acid construct further comprises a coding sequence for a PD-1 binding molecule, including, but not limited to, a PD-1 antibody or antigen-binding fragment thereof, a PD-1 heavy chain antibody or antigen-binding fragment thereof, a monovalent or polyvalent single domain antibody, including one, two or more PD-1 single domain antibodies, and a multispecific single domain antibody. The PD-1 binding molecules and anti-PD-1 single domain antibodies referred to herein include all PD-1 binding molecules and all anti-PD-1 single domain antibodies, respectively, described in CN202011582908, the entirety of which is incorporated herein by reference in its entirety for each embodiment. As described in that patent application, the PD-1 binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof, comprising one, two or more of the above-mentioned PD-1 single domain antibodies.

[0117] Thus, the complementarity determining regions (CDRs) of the anti-PD-1 single domain antibodies described herein comprise CDR1, CDR2, and CDR3, where CDR1, CDR2, and CDR3 comprise the sequences set forth in SEQ ID NOs: 1, 2, and 3 of CN202011582908, respectively. Illustratively, in the anti-PD-1 single domain antibodies described herein, CDR1 comprises the sequence set forth in any one of SEQ ID NOs: 4-39, 320 of CN202011582908, CDR2 comprises the sequence set forth in any one of SEQ ID NOs: 40-75 of CN202011582908, and CDR3 comprises the sequence set forth in any one of SEQ ID NOs: 76-183 of CN202011582908. In one or more embodiments, the anti-PD-1 single domain antibodies described herein comprise CDR1, CDR2, and CDR3 as set forth in any one of Group a1 to Group a136 of CN202011582908. The anti-PD-1 single domain antibody VHH described herein is set forth in any one of SEQ ID NOs:184-319 of CN202011582908. Preferably, the anti-PD-1 single domain antibodies described herein are set forth in any one of SEQ ID NOs:197, 200, 223-309 of CN202011582908. Additionally, the CDRs or full length of each of the anti-PD-1 single domain antibodies described herein may be as set forth in any embodiment of CN202011582908.

[0118] Exemplarily, the CDR1 of the anti-PD-1 single domain antibody is shown in SEQ ID NO:124 or 128, the CDR2 is shown in SEQ ID NO:125 or 129, and the CDR3 is shown in SEQ ID NO:126 or 130; the sequence of the anti-PD-1 single domain antibody VHH is shown in SEQ ID NO:127 or 131.

[0119] The PD-1 binding molecules described herein can be monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen-binding fragments thereof, or antibodies or antigen-binding fragments thereof, including one, two or more of the anti-PD-1 single domain antibodies described herein. The multivalent single domain antibodies or multispecific single domain antibodies are linked to the multiple single domain antibodies by a linker. The linker consists of 1-15 amino acids selected from G and S. As used herein, the heavy chain antibody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody. The antigen-binding fragment of the heavy chain antibody is a single chain heavy chain antibody. The heavy chain antibody further comprises a heavy chain constant region. In one or more embodiments, the heavy chain constant region is a camelid heavy chain antibody constant region and comprises a CH2 and a CH3. In one or more embodiments, the CH2 and CH3 are the CH2 and CH3 of human IgG Fc, e.g., the CH2 and CH3 of IgG4.

[0120] Thus, in one or more embodiments, the nucleic acid construct comprises coding sequences for a CAR and a PD-1 binding molecule. In one or more embodiments, the nucleic acid construct comprises an expression cassette for the chimeric antigen receptor and an expression cassette for the PD-1 binding molecule. The two expression cassettes are contained in one or two vectors. Alternatively, the nucleic acid construct is a single expression cassette, and the coding sequence for the chimeric antigen receptor and the coding sequence for the PD-1 binding molecule are in the expression cassette.

[0121] In some embodiments, the nucleic acid construct is a vector. The term "vector" is capable of introducing a gene sequence into a target cell. In general, "vector construct", "expression vector" and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and introducing a gene sequence into a target cell. This can be achieved by genomic integration of the whole or part of the vector, or by transient or heritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning vectors, expression vectors and integrating vectors. In general, expression of the polynucleotide sequence of the invention is achieved by operably linking the polynucleotide sequence of the invention to a promoter and introducing the construct into an expression vector. The vector is capable of inducing replication in eukaryotic cells. A nucleic acid construct is suitable for preparation and integration. A typical cloning vector contains transcription and translation terminators, initiation sequences and a promoter that are utilized to regulate the expression of the desired nucleic acid sequence. The nucleic acid construct may be one or more vectors, each vector containing one, two or three expression cassettes as described in any embodiment herein.

[0122] The polynucleotide sequence of the present invention may be cloned into various types of vectors. For example, it may be cloned into a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Furthermore, the vector is an expression vector. The expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks in virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0123] Suitable vectors usually contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0124] The nucleic acid construct may be a cloning vector or an expression vector. The expression vector is preferably a constitutive expression vector, such as a transfer vector (or "transposon vector"). In some embodiments, the transposon vector is a eukaryotic expression vector comprising a transposon element selected from piggybac, sleeping beauty, frog prince, Tn5, or Ty. Such transposon vectors comprise the 5' inverted terminal repeat (5'ITR) of a corresponding transposon and the 3' inverted terminal repeat (3'ITR) of a corresponding transposon. Thus, in some embodiments of the invention, the nucleic acid construct further comprises a coding sequence for a transposase, which is used to transfer the coding sequence of the chimeric antigen receptor and / or PD-1 binding molecule, e.g., to integrate the coding sequence or expression cassette of the chimeric antigen receptor and / or PD-1 binding molecule into the genome. The transposase may be any transposase known in the art suitable for human cells, such as a transposase from piggybac, sleeping beauty, frog prince, Tn5 or Ty transposon system. If a transposase from a different transposon system is used, the sequences of the 5'ITR and 3'ITR in the vector are appropriately changed to sequences compatible with the transposon system, which can be easily determined by a person skilled in the art. Between the 5'ITR and 3'ITR, an expression cassette of the CAR or antibody of the present invention is included, including the corresponding promoter sequence, the coding sequence of the CAR or antibody, and a polyA tailing signal sequence. An exemplary transposase is PBase. In some embodiments, the transposon 5' inverted terminal repeat sequence is as shown in SEQ ID NO:1 of CN105154473A (the contents of which are incorporated herein by reference). In some embodiments, the transposon 3' inverted terminal repeat sequence is as shown in SEQ ID NO:4 of CN105154473A. In some embodiments, the piggybac transposase is a transposase that includes a coding sequence for the c-myc nuclear localization signal.In some embodiments, the coding sequence of the piggybac transposase is as shown in SEQ ID NO:5 of CN 201510638974.7. Exemplary vector backbones are pNB338B and pS338B, described in CN111206043A and CN114729361A.

[0125] Thus, in one or more embodiments, the nucleic acid construct comprises coding sequences for a CAR and a transposase. In one or more embodiments, the nucleic acid construct comprises an expression cassette for the chimeric antigen receptor and an expression cassette for the transposase. The two expression cassettes are contained in one or two vectors. Alternatively, the nucleic acid construct is wherein the chimeric antigen receptor coding sequence and the transposase coding sequence are within the expression cassette.

[0126] In some embodiments, the nucleic acid construct described herein comprises coding sequences for CAR, transposase and PD-1 binding molecule. The nucleic acid construct can comprise three expression cassettes, namely, the expression cassette for the chimeric antigen receptor, the expression cassette for the transposase and the expression cassette for the PD-1 binding molecule; the three expression cassettes may be comprised in one, two or three vectors. Alternatively, the nucleic acid construct comprises two expression cassettes, one of which comprises the coding sequence for the chimeric antigen receptor, the coding sequence for the transposase and the coding sequence for the PD-1 binding molecule, and the other of which comprises the remaining coding sequences; the two expression cassettes may be comprised in one or two vectors; or, the nucleic acid construct comprises one expression cassette, in which the coding sequence for the chimeric antigen receptor, the expression cassette for the transposase and the coding sequence for the PD-1 binding molecule are in the expression cassette.

[0127] In one or more embodiments, the nucleic acid construct comprises: (a) one vector comprising an expression cassette for a CAR and, optionally, (b) an expression cassette for a transposase and / or an expression cassette for a PD-1 binding molecule; two vectors, each comprising (a) an expression cassette for a CAR and an expression cassette for a PD-1 binding molecule, optionally either of which also comprises an expression cassette for a transposase, or (b) two vectors, one of which comprises an expression cassette for a transposase and the other of which comprises an expression cassette for a CAR and an expression cassette for a PD-1 binding molecule; or three vectors, each of which comprises an expression cassette for a CAR, an expression cassette for a PD-1 binding molecule, and an expression cassette for a transposase.

[0128] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α). Another example of a suitable promoter is the IFNγ-driven promoter. However, other constitutive promoter sequences can also be used, including but not limited to the Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, EB virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, including but not limited to, for example, actin promoter, myosin promoter, protoheme promoter, and creatine kinase promoter. In addition, the use of inducible promoters is also contemplated. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0129] To assess the expression of the CAR polypeptide or a portion thereof, the expression vector introduced into the cells may contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the cell population that is sought to be transfected or infected by the viral vector. In other embodiments, the selectable marker may be carried on a single DNA and utilized in a co-transfection step. Both the selectable marker and the reporter gene may have appropriate regulatory sequences to allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0130] Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences. After the DNA is introduced into the recipient cells, expression of the reporter gene is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are well known and may be prepared using known techniques or obtained commercially.

[0131] Methods for introducing genes into cells and expressing genes in cells are known in the art.Vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art.For example, expression vectors can be introduced into host cells by physical, chemical or biological means.

[0132] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing a polynucleotide of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macrocomplexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0133] T cells suitable for the present invention can be various types of T cells from various sources. For example, T cells can be derived from PBMCs of patients with B cell malignancies. In some embodiments, after obtaining T cells, they can be stimulated and activated with an appropriate amount (e.g., 30 to 80 ng / ml, e.g., 50 ng / ml) of CD3 antibody, and then cultured in an IL2 medium containing an appropriate amount (e.g., 30 to 80 IU / ml, e.g., 50 IU / ml).

[0134] Thus, in some embodiments, the invention provides genetically modified T cells that comprise a polynucleotide sequence described herein, or that comprise a nucleic acid construct described herein, or that are prepared using the methods described herein, or that stably express a CAR and any anti-PD-1 binding molecule described herein. The cells (e.g., T cells) described herein can comprise or express a transposase or a coding sequence thereof.

[0135] The CAR-T cells of the invention can undergo robust in vivo T cell expansion, persist at high levels in the blood and bone marrow for extended periods of time, and form specific memory T cells. Without wishing to be bound by any particular theory, the CAR-T cells of the invention can differentiate in vivo into a central memory-like state after encountering and subsequently eliminating target cells expressing alternative antigens.

[0136] The present invention further includes a type of cell therapy in which T cells are genetically modified to express the CAR and any anti-PD-1 binding molecule described herein, and the CAR-T cells are infused into a recipient in need thereof. The infused cells can kill tumor cells in the recipient. Unlike antibody therapy, CAR-T cells can replicate in vivo, resulting in long-term persistence leading to sustained tumor control.

[0137] The anti-tumor immune response elicited by CAR-T cells can be an active or passive immune response. The CAR-mediated immune response can also be part of an adoptive immunotherapy process in which CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.

[0138] Thus, the disease treatable with the CAR, its coding sequence, nucleic acid construct, expression vector, and CAR-T cell of the present invention is preferably a mesothelin-mediated cancer. Preferably, said disease also benefits from anti-PD-1 antibody therapy and is a PD-1-mediated cancer.

[0139] Specifically, as used herein, "mesothelin-mediated cancer" includes, among others, various types of ovarian cancer, pleural mesothelioma (e.g., epithelial mesothelioma), pancreatic cancer, and squamous cell carcinoma of the cervix, endometrium, head, neck, vagina, lung, and esophagus. In some embodiments, the mesothelin-mediated disease is malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, and lung adenocarcinoma. Diseases that would "benefit from anti-PD-1 antibody therapy" or "PD-1-mediated cancer" include gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, and the like.

[0140] The CAR-modified T cells of the present invention can be administered as a pharmaceutical composition alone or in combination with other components such as diluents and / or associated cytokines and cell populations. Briefly, the pharmaceutical compositions of the present invention can include a combination of the CAR-T cells described herein with one or more pharma- ceutical or physiologically acceptable additives (e.g., vectors, diluents or excipients). Such compositions can include buffers such as neutral buffered saline, sulfate buffered saline, carbohydrates such as glucose, mannose, sucrose or dextran, amino acids such as proteins, polypeptides or glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide) and preservatives.

[0141] The pharmaceutical compositions of the present invention may be administered in a manner suitable for treating (or preventing) a disease. The amount and frequency of administration will be determined by factors such as the medical condition of the patient, and the type and severity of the patient's disease.

[0142] When an "immunologically effective amount", "antitumor effective amount", "tumor suppression effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the age, weight, tumor size, degree of infection or metastasis, and condition of the patient (subject). Generally, the pharmaceutical compositions comprising the T cells described herein are administered in an amount of 10 4 ~10 9 Cells / kg body weight, preferably 10 5 ~10 6 It is possible to administer the T cell composition at a dose of 1000 cells / kg body weight. The T cell composition can also be administered multiple times at these doses. The cells can be administered using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one of ordinary skill in the medical arts by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0143] The subject compositions may be administered by any convenient method, including aerosolization, injection, swallowing, infusion, implantation or transplantation. The compositions herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous or intraperitoneal injection. In one embodiment, the T cell compositions of the present invention may be administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions may be injected directly into a tumor, lymph node or infection.

[0144] In some embodiments of the present invention, the CAR-T cells of the present invention or compositions thereof may be combined with other therapies known in the art, including but not limited to chemotherapy, radiation therapy, and immunosuppressants. For example, the CAR-T cells may be combined with radiation therapy or chemotherapy preparations for treating mesothelin-mediated diseases known in the art.

[0145] As used herein, the term "antitumor effect" refers to a biological effect such as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms related to cancer. do.

[0146] "Patient," "subject," "individual," and the like, may be used interchangeably herein and refer to a living organism, such as a mammal, in which an immune response can be elicited. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0147] The present invention is described in more detail with reference to the following experimental examples. These examples are presented for illustrative purposes and are not limiting unless otherwise specified. Therefore, the present invention is not limited to the following examples, and includes any and all variations made clear by the teachings provided by this specification. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified. EXAMPLES

[0148] Testing Method: Isolation and freezing of human peripheral blood mononuclear cells (PBMCs) First, human apheresis blood was diluted 1-2 times with DPBS, then the diluted blood was gently added to the upper layer of ficoll so as not to mix the two solutions (blood:ficoll=3:4); centrifuged at 800g for 20 min (centrifuge acceleration 1, deceleration 0), after centrifugation, the white membrane layer was sucked into a new centrifuge tube with a pipette; after adding 3 times the amount of DPBS and mixing thoroughly, it was placed in a centrifuge and centrifuged at 1500 rpm, after 10 min the supernatant was removed, and this operation was repeated 2-3 times; after counting, AIM-V medium was supplemented at a volume of 5 x 10^6 cells / ml and cultured overnight in a 5% CO2 incubator. After culture, the suspended cells were collected, counted and frozen.

[0149] CAR-T preparation procedure Human mononuclear cells (PBMCs) were isolated from peripheral blood of healthy human donors using Ficoll density gradient centrifugation (GE). The isolated PBMCs were 1 × 10 6 The cells were cultured overnight in AIM-V medium at a concentration of 1 × 10 / mL and then cryopreserved in serum-free cryopreservation medium (NCM Biotech). 7 Fresh or frozen resuscitated PBMCs were electroporated using a Lonza 4D electroporator, and the electroporation process was optimized according to the operating procedures in the electroporator manual. 6PBMCs were resuspended in 100ul of electroporation solution, and 6ug of pNB338B-1444-CAR plasmid, or 4ug of pNB338B-1444-CAR + 4ug of pS338B-1182 plasmid were added to the cell suspension, and electroporation was performed using the EO-115 program. The electroporated PBMCs were added to a 12-well plate containing 2ml of AIM-V (+2%FBS) culture medium pre-warmed to 37℃, and after 4-6h of culture, they were transferred to an antigen-coated 6-well plate (5ug / ml CD28 antibody + 5ug / ml MNSL antigen), supplemented with 4ml of culture medium, and added with IL-2 at a final concentration of 500IU / ml. The negative control group was electroporated with 6ug Mock-T plasmid, and the cell culture plate was coated with 5ug / ml CD3 antibody + 5ug / ml CD28 antibody, and other operations were the same as the above experimental groups and performed simultaneously. The electroporated PBMCs were cultured in the above culture medium for 5 days, after which the first passage was performed, and then passaged every 2-3 days, completing the preparation of CAR-T cells on day 13. The PBMCs during passage were cultured in AIM-V culture medium containing 2% FBS and 100 IU / ml IL-2, and the passage cell density was 5 × 10 5 The cell count and cell viability were detected and recorded using a CountStar.

[0150] During and after CAR-T cell preparation, CAR positivity and other surface markers on the cell surface were detected by flow cytometry using the following antibodies: anti-mesothelin, anti-human CD3 PE-Cy5 / CD4 PE / CD8 FITC Cocktail (Biolegend), anti-CD45RO (Biolegend), anti-CD62L (Biolegend), anti-CCR7 (Biolegend), anti-CD107a, anti-PD1, anti-TIM3, anti-LAG3, and anti-PDL1.

[0151] RTCA cell killing The killing effect of CAR-T cells on tumor cells was detected using an xCELLigence RTCA instrument (Roche Applied Science, Canada), and the detection process was carried out according to the operating instructions of the instrument. 3 -2×10 4 ) of target cells (tumor cells) were resuspended in 50ul of culture medium and inoculated into a microplate (16-well or 96-well) attached to the RTCA device. The tumor cells were cultured in the RTCA device for about 24h, and when the cell index (CI, indicating cell proliferation) reached about 1.5, different numbers of effector cells (CAR-T cells) with different ET ratios (effector cells:target cells) were resuspended in 50ul of culture medium and mixed with the tumor cells, and continued to be cultured in the RTCA device for 3-5 days. The cell killing curves were recorded by the RTCA device, and the data were analyzed with RTCA Pro 2.3.0 software.

[0152] Detection of antibody secretion The amount of PD1 nanobody secretion during and after the preparation of MSLN-targeting CAR-T cells that autosecrete PD1 nanobody was detected by enzyme-linked immunosorbent assay (ELISA). 5 The cells were subcultured at a density of 5 × 10 / mL, and the supernatants on days 5-7 and 11-13 were collected to detect the amount of PD1 antibody secreted. 5 CAR-T cells at 100μg / mL were inoculated onto cell culture plates coated with 5ug / mL MSLN antigen, and after 48h of culture, the supernatant was collected to detect the amount of PD1 antibody secretion. The following antibody was used for ELISA detection of PD1 nanobody secretion: anti-1182-VHH (Singhwan Nano Co., Ltd., amino acid sequence shown in SEQ ID NO:134).

[0153] Example 1, CAR and CAR-T Cells Containing Mesothelin Nanobodies Plasmids were constructed using nanobody-derived MSLN CARs with different sequences and benchmarker MSLN CARs, and non-viral vector-based MSLN CAR-T cells were constructed by electroporating PBMCs derived from healthy subjects, and the performance of CAR-T cells in vitro was verified, including indicators such as proliferation ability, CAR positive rate, in vitro target cell killing ability, and cytokine secretion levels.

[0154] 1.1. Construction of MSLN CAR-T cell plasmid Laboratory equipment: conventional PCR equipment (Jena, TRIO), electrophoresis equipment (Bio-Rad, 1645050), electrophoresis tank (Bio-Rad, 1704483), tabletop microcentrifuge (Thermo, Pico17), metal bath heating module (Thermo, DCD), electric thermostatic water bath (Shanghai Yiheng, DK-8D), automated gel imaging device (Tenno, Tanon, 2500)

[0155] [Table 2]

[0156] Experimental Method Based on the sequence of MSLN nanobody, the full-length nucleic acid sequence and primers were synthesized, a number of target fragments were obtained by PCR, and the pNB338B vector (the sequence and structure of which can be found in "pNB338B-E" in CN109988759A) was subjected to double enzyme cleavage (EcoR1 and Sal1) to obtain vector fragments, and the target fragments and the vector fragments were ligated by DNA ligase to obtain a plasmid. The sequence of the constructed vector was determined, and if the sequence was correct, the plasmid was extracted in large quantities. Among them, the vector structure of pNB338B-1444-CAR is shown in Figure 44. The amino acid sequence of MSLN3

[1444] nanobody is shown in SEQ ID NO: 83 (the CAR containing it is shown in SEQ ID NO: 122), and the amino acid sequence of MSLN3

[1450] nanobody is shown in SEQ ID NO: 85 (the CAR containing it is shown in SEQ ID NO: 123). The amino acid sequence of MSLN3[Scfv] is shown in SEQ ID NO:3 of CN109306014A.

[0157] 1.2 Preparation of mesothelin-targeted CAR-T cells Mock-T and MSLN CAR-T were prepared by electroporation. Laboratory equipment: electroporator (Lonza 4D), automated cell counter (Invitrogen Countess IIFL), microscope (Jiangnan XD-202), biological safety cabinet (Thermo Fisher Scientific), refrigerator (Qingdao Haier), carbon dioxide incubator (Thermo), tabletop low-speed centrifuge (Xiangyi), Pico17 tabletop microcentrifuge (Thermo).

[0158] [Table 3]

[0159] Experimental Method Isolation and freezing of human peripheral blood mononuclear cells (PBMCs) Please see Experimental Methods for further details.

[0160] Resuscitation of PBMC cells and preparation of MSLN CAR-T The densities of three donor PBMCs (AC-201909A, AC-201908B, AC-201910A) ranged from 6.08E+06 to 9.67E+06 cells / mL, and the viabilities were all above 90%. For single plasmid electroporation, the plasmid concentration was 6 µg per sample. For more details, please see Experimental Methods.

[0161] [Table 4]

[0162] 1.3. Detection of MSLN CAR-T cell proliferation and positivity rate in vitro Since the culture cycle after the preparation of non-viral vector CAR-T is usually 13 days, and the final yield of CAR-T cells is ultimately obtained by the proliferation of total cells and the final percentage of CAR-positive cells, the purpose of this experiment was to evaluate the quality of the prepared mock-T cells and MSLN CAR-T cells in terms of proliferation status and positivity rate.

[0163] Laboratory equipment: automated cell counter (Invitrogen Countess IIFL), microscope (Jiangnan XD-202), biological safety cabinet (Thermo Fisher Scientific), refrigerator (Qingdao Haier), carbon dioxide incubator (Thermo), tabletop low-speed centrifuge (Xiangyi), flow cytometry (Navios, AZ46353), Pico17 tabletop microcentrifuge (Thermo).

[0164] [Table 5]

[0165] Experimental Method 1) Cultivation and passaging of CAR-T cells By cell counting and isodensity passage, the proliferation fold of cells in each group was calculated and cell proliferation curves were plotted. The passage density was 5 × 10 5The cell concentration was 100 U / mL.

[0166] 2) Detection of the proportion of MSLN CAR-T positive cells The change in CAR positivity rate in cell proliferation after antigen enrichment was detected by flow cytometry and compared with different CAR sequences. For details, please see Experimental Methods.

[0167] Experimental Results The results are shown in Figure 1-3 and Table 1-3. The results showed that there were certain differences in the proliferation ability of CAR-T cells prepared from PBMCs from three donors; two of them proliferated well with a proliferation fold of about 20-38 times, and one donor proliferated poorly with only 1.3-3.7 times; overall, MSLN3

[1444] CAR-T cells could be successfully proliferated, and their proliferation ability was comparable to that of Mock-T cells and MSLN3[Scfv]CAR-T cells (Figure 1). Furthermore, similar to Mock-T cells and MSLN3[Scfv]CAR-T cells, MSLN3

[1444] CAR-T cells showed a high survival rate (over 90%) (Figure 2). The CAR positive rate of MSLN3

[1444] CAR-T cells reached about 60%, which was comparable to that of MSLN3[Scfv]CAR-T (Figure 3).

[0168] [Table 6]

[0169] [Table 7]

[0170] [Table 8]

[0171] 1.4. Detection of MSLN CAR-T cell phenotype The phenotype of the prepared CART cells was evaluated by flow cytometry to detect CD3 / CD4 / CD8, TCM / TEM of MSLN CART. Laboratory equipment: automated cell counter (Invitrogen Countess IIFL), microscope (Kangnam XD-202), Pico17 tabletop microcentrifuge (Thermo), flow cytometry (Navios, AZ46353)

[0172] [Table 9]

[0173] Experimental Method 1) Detection of MSLN CART CD3 / CD4 / CD8 Anti-human CD3 PE-Cy5 / CD4 PE / CD8 FITC Cocktail antibody for flow cytometry. For details of the experimental process, please refer to the Experimental Methods.

[0174] 2) Detection of MSLN CART TCM / TEM 2 μL PE / Cy5 anti-human CD45RO, 2 μL FITC anti-human CD197, and 2 μL PE anti-human CD62L flow cytometry antibodies. For details of the experimental process, please refer to the Experimental Methods.

[0175] Experimental Results The results are shown in Table 4-5 and Figure 4-5. According to the results, the CD3 positive rate of CAR-T cells was high, about 85%-97%, in both mock-T and nanobody mesothelin groups (1444 and 1450); the CD3 positive rate of MSLN3[Scfv]CAR-T cells was slightly lower, about 75%-84%. The overall trends of CD4 and CD8 rates of CD3 positive cells were consistent in all groups, with the CD4 rate being higher than the CD8 rate. The overall trends of Tcm and Tem rates were consistent in all groups, with the Tcm rate being higher than the Tem rate. Compared with mock-T cells, the CD4 / CD8 and Tcm / Tem of MSLN3[Scfv]CAR-T cells and MSLN3

[1444] CAR-T cells were both decreased to some extent (Figure 4, Figure 5).

[0176] [Table 10]

[0177] [Table 11]

[0178] 1.5. Detection of MSLN CAR-T cytotoxicity in vitro In this example, we investigated the in vitro pharmacodynamic properties of MSLN CAR-T with different sequences, detected cytokine secretion after co-culture with mesothelin-positive expressing tumor cells, and detected the specific killing ability of target cells. In vitro killing experiments were performed using mesothelin-negative and mesothelin-positive tumor cells. This was carried out and demonstrated that the candidate sequences had specific killing activity against the mesothelin antigen.

[0179] Experimental sample Sample information for AC-201908B, AC-201909A, AC-201910A shown in Table 2, and YP-201903A04, YP-201903A23, and YP-201903A24 obtained in a similar manner is shown in the table below.

[0180] [Table 12]

[0181] Target cell information is shown in the table below: [Table 13]

[0182] Laboratory equipment: Automated cell counter (Invitrogen Countess IIFL YSB195), microscope (Jiangnan XD-202 YSB126), biological safety cabinet, refrigerator (TCL), carbon dioxide incubator (Thermo), real-time cell analyzer (ACEA XCELLIgence RTCA TP, SB0475, included consumables: E-Plate 16, E-Plate 96), benchtop centrifuge (small) (Thermo Fisher), benchtop centrifuge (large) (Xiangyi)

[0183] [Table 14]

[0184] Experimental Method 1) Cell killing (1) YP-201903A04, YP-201903A23, YP-201903A24: Target cells were H520, AsPC-1, and H226, and the ET ratio (E:T) was 1:1. (2) AC-201908B, AC-201909A, AC-201910A: target cells were SKOV3 and H226, and the ET ratio (E:T) was 1:1, 1:4, and 1:16

number

[0185] 2) Detection of cytokine release After co-incubation with target cells for 72 h, the 96-well plates were removed and cytokine release in the supernatant was detected by ELISA.

[0186] Experimental Results The results are shown in Figures 6-8 and Table 6. The CAR-T cells we prepared using the transposase plasmid system were shown to have the ability to specifically kill mesothelin-positive tumor cells (Figures 6 and 7). Co-culture with mesothelin-positive tumor cells could effectively stimulate MSLN3[Scfv]CAR-T cells and MSLN3

[1444] CAR-T cells to secrete IFN-γ, IL-2, and TNF-α (Figure 8).

[0187] [Table 15]

[0188] According to the above results, nanobody-derived MSLN CAR sequences have the same or higher proliferation ability, MSLN CAR positivity rate, CD4+T / CD8+T subpopulation ratio, and memory T cell percentage compared to single-chain antibody sequences in in vitro screening, and are capable of specifically killing mesothelin-positive tumor cells.

[0189] Example 2. In vitro experiment of MSLN CAR-T cells autocreting PD1 nanobody A plasmid was constructed using the PD1 antibody sequence derived from the nanobody and the sequence of the benchmarker Keytruda, and the plasmid was transfected into PBMCs derived from healthy subjects using a double plasmid electroporation system. We constructed non-viral vector aPD1 MSLN CAR-T cells by transducing the vector, and verified the function of CAR-T cell autocrine antibodies by comparing the performance of CAR-T cells in vitro, including proliferation capacity, CAR positive rate, antibody secretion level, and the blocking effect of secreted antibodies against immune checkpoint inhibitors.

[0190] 2.1. Construction of plasmid for autocrine secretion of PD1 nanobody An exemplary PD1 nanobody (aPD1

[1182] , SEQ ID NO: 127) was used as the target fragment, and the pS338B (see CN111206043A) vector was used as the backbone vector to perform double enzyme cleavage (EcoR1 and BamH1), and other experimental equipment, experimental reagents and experimental methods were the same as those in 1.1 of Example 1.

[0191] 2.2 Preparation of MSLN CAR-T cells autocreting PD1 nanobodies Mock-T, aPD1-MSLN CAR-T were prepared by electroporation, and cell and cell supernatant samples were obtained for detection and analysis.

[0192] The experimental equipment, reagents and methods were the same as in 1.2 of Example 1. aPD1-MSLN CAR-T is a CAR-T cell electroporated with double plasmids (plasmids constructed in 1.1 of Example 1 and 2.1 of Example 2) expressing nanobody-based MSLN CAR and PD1 nanobody. For single plasmid electroporation, the plasmid concentration was 6 μg per sample; for double plasmid electroporation, the concentration of each plasmid was 4 μg per sample.

[0193] [Table 16]

[0194] 2.3. Detection of aPD1-MSLN CAR-T proliferation and positivity rate in vitro When the double plasmid system allows MSLN CAR-T cells to self-secrete PD1 (aPD1-MSLN CAR-T), it was verified whether normal cell proliferation could be achieved and a certain MSLN CAR positive rate could be ensured, and samples of cells and cell supernatant were obtained to detect the situation in which cells secrete PD1 antibody. The experimental equipment, reagents and methods were the same as those in 1.3 of Example 1.

[0195] Experimental Results The results are shown in Table 7-9 and Figures 9-11. [Table 17] [Table 18] [Table 19]

[0196] 2.4. Detection of cell phenotype Purpose of the experiment The phenotype of the prepared CART cells was evaluated by flow cytometry to detect CD3 / CD4 / CD8, TCM / TEM of autocrine PD1 antibody MSLN CART. The experimental equipment, reagents and methods were the same as those in 1.4 of Example 1. Experimental Results The results are shown in Table 10-11 and Figure 12-13 (AC-201908A).

[0197] [Table 20] [Table 21]

[0198] 2.5 Detection of autocrine antibodies in cells During the cell preparation, ELISA was used to detect whether the cells had the ability to autocrete antibody before the end of the preparation and the range of antibody concentration secreted. Experimental equipment: Multi-function microplate reader (TECAN Spark)

[0199] [Table 22]

[0200] Experimental Method 5 x 10 cells cultured for 2 days 5A sample of the cell supernatant was collected, and the antibody secretion concentration in the supernatant was detected by ELISA, and the amount and concentration of the substance secreted by the cells in the supernatant were calculated. For details, please refer to the Experimental Methods. Calculation formula: mass concentration of substance = mass concentration / molecular weight in kDa / 1000 The cell supernatant samples in this experiment were diluted 4-fold, 40-fold, and 400-fold.

[0201] Experimental Results The results, shown in Table 12, indicate that mesothelin CAR-T cells that autocrete PD1 nanobodies can successfully secrete PD1 nanobodies during preparation, and that their ability to secrete PD1 nanobodies tends to decrease over time.

[0202] [Table 23]

[0203] 2.6 Detection of cell functions The prepared CAR-T cells were stimulated to a certain extent with the antigen targeted by the CAR-T cells, and changes in the cell phenotype and antibody secretion were detected to evaluate the response ability of the CAR-T cells after stimulation with a specific antigen.

[0204] The experimental equipment, reagents and methods were the same as those in Example 1, 1.4. Experimental method: 12-well cell culture plates were coated with mesothelin antigen at a concentration of 5 μg / mL, 5×10^5 cell samples were added, and cultured in 1 mL culture system for 48 h. Cells and cell supernatants were collected and subjected to flow cytometry and ELISA detection, respectively. Then, the proportion of Tcm (CD45RO+CCR7+CD62L+) and Tem (CD45RO+CCR7-CD62L-) and the amount of PD1 nanobody secretion were analyzed according to the analytical methods in 2.4 and 2.5. Experimental Results The results are shown in Tables 13-14 and Figures 14-15. The results showed that after CAR-T cells were stimulated with antigen, the proportion of Tcm decreased and the proportion of Tem increased compared to before stimulation; after CAR-T cells were stimulated with antigen, the secretion amount of PD1 nanobody was significantly increased compared to before stimulation.

[0205] [Table 24] [Table 25]

[0206] 2.7. Detection of aPD1-MSLN CAR-T cytotoxicity in vitro We verified the in vitro pharmacodynamic properties of MSLN CAR-T cells expressing PD1 antibody autocrinely, detected cytokine secretion after co-culture with mesothelin-positive and PDL1-high expressing tumor cells, and evaluated the specific killing ability of CAR-T cells against target cells.

[0207] [Table 26] [Table 27]

[0208] The experimental equipment, reagents and methods were the same as those in 1.5 of Example 1. The target cells were SKOV3-PDL1 and H226, and the ET ratio (E:T) was 1:1, 1:4, and 1:16.

[0209] For detection of cytokine release, all experimental samples of IL-2 were diluted 5-fold and 10-fold for detection; all experimental samples of TNF-α were diluted 3-fold for detection; all experimental samples of IFN-γ were diluted 10-fold and 100-fold for detection.

[0210] Experimental Results The results are shown in Figures 16-18 and Table 15. The autocrine PD1 antibody significantly improved the killing efficiency of mesothelin CAR-T against H226 cells (Figure 16), but had no significant effect on the killing efficiency against SKOV3-PDL1 cells (Figure 17). This indicates that the mesothelin-targeting CAR-T cells that autocrine PD1 nanobody we prepared can effectively kill mesothelin-positive tumor cells, and that the mesothelin-targeting CAR-T cells against a certain type of mesothelin and PDL1 double-positive tumor cells can effectively kill mesothelin-positive tumor cells. These results suggest that co-culture with H226 cells and Skov3-PDL1 tumor cells can improve the killing effect of CAR-T. Co-culture with H226 cells and Skov3-PDL1 tumor cells can improve the ability of CAR-T cells to secrete cytokines IFN-γ and TNF-α; moreover, co-culture of mesothelin CAR-T cells that autocrete PD1 antibodies with tumor cells significantly increased the secretion of IFN-γ and TNF-α, compared with mesothelin cells that do not secrete PD1 antibodies (Figure 19).

[0211] [Table 28]

[0212] Example 3: In vivo efficacy study of MSLN CAR-T cells secreting PD1 nanobody 3.1. Experimental animals and cells 6-8 weeks NPSG severe immune combined deficiency prkdc mice scid Il2rg null (Beijing Phenotek Biotechnology Co., Ltd.) Tumor cells Skov3-PDL1 (ATCC) expresses high levels of PDL1 NCI-H226-PDL1 (ATCC) expresses high levels of PDL1 Construction process of CAR-T cells autocrine Fc-containing PD1 antibody: aPD1

[1182] nanobody sequence was constructed in pCDNA3.4-IgG4 vector to obtain pCDNA3.4-1182-Fc vector, and expressed by ExpiCHOTM (Thermo Fisher) expression system for 1 week, and then the supernatant was collected for Protein A (GE) purification. Then, the protein mass was detected by Nanodrop, and the protein purity was detected by HPLC. The 1182-Fc coding sequence was obtained from pCDNA3.4-1182-FC vector by double enzyme cleavage and introduced into pS338B backbone vector, and other experimental instruments, experimental reagents, and experimental methods were the same as those in Example 2.

[0213] 3.2. Vaccination and administration schedule Tumor cells were inoculated subcutaneously into NPSG severe immunodeficient mice. [Table 29]

[0214] Tumor volume 100-150 mm 3 Dosing was initiated when the dose reached 100 mg / kg. Dosing was scheduled according to Table 17. [Table 30]

[0215] Among the drugs in Table 17, CD19-1182-VHH refers to the CAR-T targeting CD19, which autocrine 1182-VHH, and the amino acid sequence and coding sequence of CAR are as shown in SEQ ID NO:9 and SEQ ID NO:10 of CN109971712A, respectively, 1444 refers to MSLN3

[1444] CAR-T, 1444-1182-VHH refers to aPD1

[1182] -MSLN3

[1444] CAR-T, and 1444-1182-FC refers to aPD1[1182-Fc]-MSLN3

[1444] CAR-T.

[0216] 3.3 Changes in tumor volume at high, medium, and low doses The results of H226 are shown in Figure 20. In the autocrine PD1 antibody administration group, significant differences in efficacy were observed at multiple doses: 0.05x10^6 dose group (1444 VS 1444-1182-VHH: P=0.0283, 1444 VS 1444-1182-FC: P=0.0010), 0.5x10^6 dose group (1444 VS 1444-1182-VHH: P=0.1722, 1444 VS 1444-1182-FC: P=0.0104), and 5x10^6 dose group (1444 VS 1444-1182-VHH: P=0.0051, 1444 VS 1444-1182-FC: P=0.0006).

[0217] The results of Skov3 are shown in Figure 21. In the autocrine PD1 antibody administration group, significant differences in efficacy were observed at multiple doses: 0.05x10^6 dose group (1444 VS 1444-1182-VHH: P=0.7479, 1444 VS 1444-1182-FC: P=0.0757), 0.5x10^6 dose group (1444 VS 1444-1182-VHH: P=0.0002, 1444 VS 1444-1182-FC: P<0.0001).

[0218] 3.4. Changes in animal weight at high, medium, and low doses The results of H226 are shown in Figure 22. The autocrine PD1 antibody-treated groups showed less change in body weight compared to the other treatment groups, and maintained a mild or elevated state except for the high-dose treatment group, which was relatively stable over the long term. Overall, weight loss in the autocrine PD1 antibody-treated groups did not exceed 20%. The results of Skov3 are shown in Figure 23. The autocrine PD1 antibody-administered group showed less change in body weight compared to the other administration groups, and maintained a mild or elevated state except for the low-dose administration group, which was relatively stable over the long term. Overall, the weight loss in the autocrine PD1 antibody-administered group did not exceed 20%.

[0219] 3.5 Survival rates at high, medium and low doses The results of H226 are shown in Figure 24. The survival time of the autocrine PD1 antibody-treated group showed a clear advantage over the other treatment groups, remaining relatively stable over the long term, with the mortality rate increasing slightly as the dose increased.

[0220] The results of Skov3 are shown in Figure 25. The survival time of the autocrine PD1 antibody treatment group had a clear advantage over the other treatment groups, remaining relatively stable over the long term, with the mortality rate increasing slightly as the dose increased.

[0221] 3.6. Changes in blood levels of PD1 antibodies at high, medium, and low doses The results of H226 are shown in Figure 26. In the autocrine PD1 antibody administration group, the blood concentration of PD1 antibody showed a positive correlation with the dose, and F C The blood concentration of PD1 antibody with segment F C Significantly higher than non-segmented PD1 antibodies and F C It was shown that the segmented PD1 antibody has a long half-life in vivo. The results of Skov3 are shown in Figure 27. In the autocrine PD1 antibody administration group, the blood concentration of PD1 antibody showed a positive correlation with the dose, and F C The blood concentration of PD1 antibody with segment F C Significantly higher than non-segmented PD1 antibodies and F C It has been shown that PD1 antibodies containing segments have a long half-life in the body.

[0222] Example 4. High-dose drug safety of MSLN CAR-T cells autocreting PD1 nanobodies 4.1 Test materials Experimental animals and cells: 6-8 week MHC-I KO B-NDG severe immune combined deficiency mice prkdc scid Il2rg null (Biocytogen Biotechnology Co., Ltd.). MHC-I knockdown can minimize graft-versus-host disease (GVHD) that occurs after CAR-T infusion, making it easier to monitor the safety of the drug over the long term. Tumor cells: NCI-H226-PDL1 (ATCC) that highly express PDL1 Experimental process: same as Example 3

[0223] 4.2. Inoculation and administration schedule [Table 31] [Table 32]

[0224] Among the drugs in Table 19, CD19-CAR-T refers to CAR-T targeting CD19, and the amino acid sequence and coding sequence of the CAR are as shown in SEQ ID NO:9 and SEQ ID NO:10 of CN109971712A, respectively. BMK-CAR-T refers to CAR-T whose CAR antigen-binding domain is YP218scFv. The difference with MSLN3

[1444] CAR-T is that YP218 is an scfv-type antibody and 1444 is a nanobody, both of which bind to the mesothelin III region, and YP218scFv is as shown in YP218scFv of CN105026429A.

[0225] 4.3. Changes in tumor volume As shown in Figure 28, all autocrine PD1 antibody groups were able to completely regress tumors at this dose, and a significant difference in efficacy was observed compared to the positive control drug BMK (BMK VS 1444-1182-VHH: P<0.0001, BMK VS 1444-1182-FC: P<0.0001).

[0226] 4.4. Weight change From Figure 29, F C In the group administered the autocrine segmented PD1 antibody, the decrease in body weight change was smaller than in the other groups, and the body weight was relatively stable over the long term. In the long-term observation up to 100 days, most of the body weight did not decrease by 20%.

[0227] 4.5. Survival time From FIG. 30, the autocrine PD1 antibody administration group had a clear advantage in terms of survival time compared with the other administration groups, and the change trend was relatively stable over the long term. C It can be seen that the survival time of mice administered the autocrine segmented PD1 antibody exceeded 100 days.

[0228] 4.6. Changes in blood levels of PD-1 antibodies As shown in FIG. 31, in the autocrine PD1 antibody administration group, F C The blood concentration of PD1 antibody with segment F C Significantly higher than non-segmented PD1 antibodies and F C The segmented PD1 antibody has a long half-life in vivo, and as the tumor shrinks after administration, antibody secretion gradually decreases.

[0229] 4.7. Changes in peripheral blood CAR-T cell counts Figure 32 shows that CAR-T cells showed a peak proliferation after administration, and then tended to decrease as the tumor regressed. Among them, the change trends of CD3+, CD8+, and CAR+ cells were almost the same. The CAR positive rate decreased for a while after administration, and then remained basically stable. The lower the number of blood cells per microliter, the larger the detection error was accordingly.

[0230] 4.8. Changes in peripheral blood cytokine concentrations The results in Figure 33 show that interferon-γ and interleukin-6 were significantly secreted after administration, and interleukin-6 secretion was concentrated in the negative control group and the BMK positive control group, whereas it was hardly secreted in the autocrine PD1 antibody administration group. Interferon-γ was significantly secreted in each group, which correlated with the killing function of T cells. The remaining cytokines, interleukin-2, interleukin-4, interleukin-10, and TNF No significant secretion was detected, the lower limit of detection was approximately 10 pg / ml, and it is thought that concentrations close to this range would not be detected.

[0231] 4.9. Clinical Observations of Test Animals [Table 33]

[0232] As a result of clinical observation, the negative control group, the BMK positive control group, and the 1444-1182-VHH (antibody F C The survival status of mice with the 1444-1182-F C The results were slightly worse than those in the control group. C All treated animals survived for over 100 days.

[0233] Example 5: Verification study of efficacy, pharmacokinetics and safety at common doses 5.1 Test materials Experimental animals and cells Test mice: same as in Example 4 Tumor cells: Skov3-PDL1 (ATCC) expresses high levels of PDL1 NCI-H226-PDL1 (ATCC) expresses high levels of PDL1 Experimental process: same as Example 3 The preparation process of BMK-1182 CAR-T cells refers to "CAR-T Preparation Procedure". The process is briefly described as follows: fresh or frozen resuscitated PBMCs were electroporated using a Lonza 4D electroporator, the electroporation system was 100ul, the plasmid dose was 4ug pNB338B-1444-CAR+4ug pS338B-1182 plasmid, the electroporation program was EO-115, and the passaging process, detection time, and detection index were all consistent with other CAR-T cells.

[0234] 5.2. Inoculation and administration schedule [Table 34] [Table 35]

[0235] Of the drugs in Table 22, CD19-1182 is the same as CD19-1182-VHH described above, Meso3CAR(BMK) is the same as BMK-CAR-T described above, and the difference between Meso3CAR(BMK)-1182-FC and Meso3CAR(BMK) is that 1182-FC is also autocrine.

[0236] [Table 36]

[0237] 5.3. Changes in tumor volume and body weight FIG. 34 shows that both the positive control and test groups were able to achieve complete regression of the tumors at this dose.

[0238] 5.4. Changes in blood levels of PD-1 antibodies The results are shown in Figure 45. As a result, in the autocrine PD1 antibody administration group, the secreted F C The in vivo drug metabolism profiles of the segmented PD1 antibodies were relatively similar, and both showed a tendency to initially increase and then decrease with tumor clearance and regression, as verified in two different tumor-bearing mouse models. Furthermore, the PD1 antibodies had the lowest blood concentrations, the lowest risk of systemic toxic side effects, and the highest safety without affecting efficacy.

[0239] 5.5. Changes in peripheral blood CAR-T cell counts The results are shown in Figure 46, which shows that CAR-T cells showed a peak proliferation after administration, and then tended to decrease as the tumor regressed. Among them, the change trends of CD3+, CD8+, and CAR+ cells were almost the same. The CAR positive rate decreased for a while after administration, and then remained basically stable. Compared with CD19 CAR-T targeting blood tumors, CAR-T targeting mesothelin had a significantly lower peripheral blood concentration, suggesting that these CAR-Ts are mainly present in the tumor local area rather than in the peripheral blood, thus achieving the purpose of precision targeted therapy.

[0240] Example 6. In vivo analysis of other MSLN CAR-T cells and MSLN CAR-T cells that autocrine PD1 nanobodies In vitro experiments In this example, the expression sequence expressing PBase on the CAR plasmid used in CAR-T cells was removed, and PBase was added in the form of mRNA to the electroporation system, which was then electroporated together with the plasmid into T cells to be expressed; changes to the PD1 antibody secretion plasmid: the promoter was changed from a CMV promoter to an IFNγ-driven promoter.

[0241] A brief description of the CAR-T preparation method: the electroporation system, electroporation program and passaging process are all the same as the previous preparation of CAR-T, and the addition of plasmids and other components in the electroporation system is single plasmid 6ug + 20ug PBase mRNA + 200IU RNase inhibitor, or double plasmid 4ug CAR plasmid + 4ug secretion plasmid + 20ug PBase mRNA + 200IU RNase inhibitor.

[0242] The inventors have constructed the following: CAR-T cells comprising the mesothelin nanobody MSLN3

[1444] (abbreviated as 1444), CAR-T cells comprising the mesothelin nanobody MSLN3

[1444] (abbreviated as "1444+1194nla" or "1444-1194") that autocrete the PD1 nanobody 1194nla (SEQ ID NO:131), the CAR sequence of which is shown in SEQ ID NO:122; and CAR-T cells containing the mesothelin nanobody MSLN3

[2339] (SEQ ID NO:103) (abbreviated as 2339); CAR-T cells containing the mesothelin nanobody MSLN3

[2339] (abbreviated as "2339+1194nla" or "2339-1194") that autocrete the PD1 nanobody 1194nla; the CAR sequence of the above cells is SEQ ID NO As shown at :121.

[0243] The results are shown in Figure 35-38: Figure 35 shows that all CAR-T cells could proliferate effectively, and all had high survival rates (over 90%) and high CAR positivity rates; the CD3 / 4 / 8 ratios of the remaining donors were slightly different, about 1:1, except for Donor 04A, which was low; and the difference between Tcm and Tem donors was small. Figure 36 shows that during preparation, the secretion level of anti-PD1 (1194nla) was low at 0-150ng / ml; as time passed, the secretion amount of 1194nla gradually decreased; there was no significant difference in the secretion amount between the 1444+1194nla group and the 2339+1194nla group. 6h after stimulation, the secretion amount of 1194nla was low at 100ng / ml or less; 48h after stimulation, the secretion amount of 1194nla increased obviously from 6h; 48h after stimulation, the secretion amount of the 1444+1194nla group was slightly higher than that of the 2339+1194nla group. 48h after antigen stimulation, the cytokine production ability of CAR-T cells in each group was significantly increased compared to before stimulation. The difference in the IL-2, IFNγ, and TNFα production ability of CAR-T between the 1444 group and the 2339 group was small, and all were higher than BMK (meso3CAR). FIG. 37 shows that both 1444 and 2339 groups could effectively kill H226-PDL1 cells; the killing ability of 2339 group against H226-PDL1 was stronger than that of 1444 group; and autocrine 1194nla improved the killing ability of MLSN CAR-T to some extent. FIG. 38 shows that both the 1444 group and the 2339 group can effectively kill SKOV3-PDL1 cells; the 1444 group has stronger killing ability against SKOV3-PDL1 than the 2339 group; and the autocrine 1194nla can improve the killing ability of MLSN CAR-T to a certain extent.

[0244] Example 7. In vivo analysis of other MSLN CAR-T cells and MSLN CAR-T cells that autocrine PD1 nanobodies In vivo dose-effect experiments Experimental process Tumor cells 1E7 (NCI-H226-PDL1) were subcutaneously inoculated into severely immunodeficient NPSG mice (Phenotek) for approximately 21 days, and tumors were grown to 100-120 mm 3 After the mice were grown to 1000 mg / kg, they were divided into groups and intravenously injected with CAR-T. The injection dose of each group was divided into four dose gradients (0.2E6, 1E6, 5E6, and 20E6, respectively), and then the efficacy, body weight, and survival rate were observed. Peripheral blood was collected once a week to detect the percentage and quantity of CD3 in the peripheral blood. The experimental design is shown in the table below and Figure 39.

[0245] [Table 37]

[0246] The experimental results are shown in Figures 40-43. Figure 40 shows that 2339-1194 and 1444-1194 could inhibit tumor growth at different doses (1E6, 5E6, 20E6) with an inhibition rate of more than 70%. 2339-1194 and 1444-1194 showed comparable tumor inhibition effects. Figure 41 shows that the body weight change of mice was within 20% at various doses, and the safety was good. There was no significant difference in body weight change and survival rate between 2339-1194 and 1444-1194. FIG. 42 shows that peripheral blood CD3+ T cells peaked on day 14 and fell to a nadir on day 21, at which time the tumors essentially regressed. Figure 43 shows that the percentage of CAR+ in peripheral blood at day 14 after CAR-T infusion was relatively stable (60%-70%) at different doses.

[0247] conclusion MSLN CAR-T cells express a chimeric antigen receptor that targets the mesothelin antigen and can secrete PD1 antibodies. They have the ability to autocrete immune checkpoint antibodies and the specific killing ability to target specific antigens, and have stronger cytokine secretion ability and a higher killing rate than conventional CAR-T cells.

Claims

1. 1. A chimeric antigen receptor comprising a mesothelin-binding molecule, the antigen-binding domain of which comprises an anti-mesothelin single domain antibody, Preferably, the anti-mesothelin single domain antibody comprises CDR1, CDR2 and CDR3, wherein the CDR1 comprises the sequence shown in SEQ ID NO:1, the CDR2 comprises the sequence shown in SEQ ID NO:2 and the CDR3 comprises the sequence shown in SEQ ID NO:3, among which: SEQ ID NO: 1 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 where X 1 is G, E, T or A, and X 2 is S, N, P, A, H, D, K, I or F, and X 3 is I, S, V, T, D, L, A, M or H, and X 4 is F, S, I, A, L or G, and X 5 is N, S, G, A, D, T, E, R or H, and X 6 is I, L, F, Y, E or N, and X 7 is N, A, G, D, K, Y, L or S, and X 8 is A, Y, V, D or N, and X 9 is E or nothing, X 10 is F or nothing, and X 11 is A or nothing, SEQ ID NO:2 is X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 where X 1 is I, M, A, T or L, and X 2 is S, G, N, D, T or V, and X 3 is S, N, A, R, G or T, and X 4 is S, T, G, D or N, and X 5 is N, G, T or I, and X 6 is S, R, N, D, K, T or G, and X 7 is D, T, S, I or K, and X 8 is N, T, G, D or none, X 9 is T, K, G or none, X 10 is G, R or none, and X 11 is V, T or none, X 12 is T or nothing, SEQ ID NO: 3 X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21 X 22 X 23 where X 1 is N, H, A or Q, and X 2 is L, A, G or V, and X 3 is S, R, E, G, D or T, and X 4 is N, A, R, G, D, K, V, T, I, E or Q, and X 5 is Y, F, K, S, C, I, D, G, H or R, and X 6 is D, A, V, G, R, N, P, T, C, Q or S, and X 7 is R, Y, T, A, D, K, L, E, S or G, and X 8 is K, S, F, T, D, Q, E, G, P, R, Y or N, and X 9 is D, G, I, H, S, Y, Q, K, T, R or V, and X 10 is R, Y, H, N, T, D, P, V, K, C, L or S, and X 11 is Y, D, E, P, F, S, L, I or none, and X 12 is P, V, Q, A, Y, F, N, D, R or None, and X 13 is D, A, Y, V, Q, P or none, and X 14 is Y, L, P, A, E or none, and X 15 is C, S, M, P, T or none, X 16 is V, D, S, Y or none, and X 17 is L, F, V, D or none, and X 18 is R, G, M, S or None, and X 19 is D, N, S or none, X 20 is Y, L or nothing, and X 21 is Y or nothing, and X 22 is A or nothing, X 23 is D or none, Preferably, the chimeric antigen receptor comprises a mesothelin-binding molecule comprising an anti-mesothelin single domain antibody, a hinge region, a transmembrane region, and an intracellular region.

2. The chimeric antigen receptor of claim 1, wherein the mesothelin-binding molecule is a monovalent or polyvalent single domain antibody comprising one, two or more of the single domain antibodies, a multispecific single domain antibody, a heavy chain antibody or antigen-binding fragment thereof, or an antibody or antigen-binding fragment thereof.

3. the CDR1 of the anti-mesothelin single domain antibody comprises a sequence as set forth in any one of SEQ ID NOs: 4-27, 73; and / or CDR2 of the anti-mesothelin single domain antibody comprises a sequence as set forth in any one of SEQ ID NOs: 28-47, 74; and / or The chimeric antigen receptor of claim 2, wherein the CDR3 of the anti-mesothelin single domain antibody comprises any one of the sequences shown in SEQ ID NOs: 48-72 and 75.

4. The chimeric antigen receptor also has the following characteristics: the signal peptide comprises a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide, or a light chain signal peptide; the hinge region comprises a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, or an IgG4 Fc CH2CH3 hinge region; the transmembrane domain comprises a CD28 transmembrane domain, a CD8 transmembrane domain, a CD3ζ transmembrane domain, a CD134 transmembrane domain, a CD137 transmembrane domain, an ICOS transmembrane domain, or a DAP10 transmembrane domain; the intracellular region comprises an intracellular costimulatory domain and / or an intracellular signaling domain; the intracellular costimulatory domain comprises the intracellular domain of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell costimulatory factor (ICOS), or DNAX-activation protein 10; The chimeric antigen receptor according to claim 2, characterized in that the intracellular signal domain comprises one or more selected from the group consisting of a CD3ζ intracellular signal domain and an FcεRIγ intracellular signal domain.

5. (1) A coding sequence for the chimeric antigen receptor according to any one of claims 1 to 4, (2) a complementary sequence of (1), and (3) A nucleic acid molecule comprising a sequence selected from a 5-50 bp fragment of any one of the sequences (1) or (2), Preferably, the fragment is a primer, and / or The coding sequence is a DNA or RNA nucleic acid molecule.

6. A nucleic acid construct comprising the nucleic acid molecule of claim 5, Preferably, said nucleic acid construct also has the following characteristics: the nucleic acid construct further comprises a coding sequence for a PD-1 binding molecule; the nucleic acid construct further comprises a coding sequence for a transposase used to transpose the coding sequence for the chimeric antigen receptor and / or the PD-1 binding molecule; the coding sequence for the transposase is DNA or RNA; the coding sequence for the PD-1 binding molecule is DNA or RNA; the nucleic acid construct is a cloning vector, an expression vector or an integration vector; The transposase is a PiggyBac transposase.

7. 5. A combination of coding sequences comprising a coding sequence for a chimeric antigen receptor according to any one of claims 1 to 4 and a coding sequence for a transposase and / or a coding sequence for a PD-1 binding molecule, preferably wherein said coding sequence is DNA or RNA and said transposase is a PiggyBac transposase.

8. A cell, the cell comprising: (1) (a) comprising and / or expressing a chimeric antigen receptor according to any one of claims 1 to 4, and optionally (b) comprising, expressing and / or secreting a PD-1 binding molecule, and / or (2) comprising a nucleic acid molecule according to claim 5 and / or a nucleic acid construct according to claim 6; and / or (3) A combination of the coding sequences according to claim 7, Preferably, the cell is a T cell.

9. A pharmaceutical composition comprising a pharma- ceutically acceptable excipient; (1) (i) a chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally (ii) a transposase and / or its coding sequence, and / or (iii) a PD-1 binding molecule and / or its coding sequence, or (2) A pharmaceutical composition comprising a nucleic acid molecule according to claim 5, a nucleic acid construct according to claim 6, a combination of coding sequences according to claim 7, or a cell according to claim 8.

10. The use of a reagent in the preparation of activated immune cells, said reagent comprising (1) (i) a chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally (ii) a transposase and / or its coding sequence, and / or (iii) a PD-1 binding molecule and / or its coding sequence, or (2) An application comprising a combination of the nucleic acid molecule of claim 5, the nucleic acid construct of claim 6 or the coding sequence of claim 7.

11. The application of a reagent in the preparation of a drug for treating cancer, comprising: (1) (i) a chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally (ii) a transposase and / or its coding sequence, and / or (iii) a PD-1 binding molecule and / or its coding sequence, or (2) A nucleic acid molecule according to claim 5, a nucleic acid construct according to claim 6, a combination of coding sequences according to claim 7, or a cell according to claim 8, Preferably, the cancer comprises a mesothelin-mediated cancer, and optionally further comprises a PD-1-mediated cancer; More preferably, the cancer is selected from mesothelioma, pancreatic cancer, ovarian cancer, cervical cancer, endometrial cancer, rectal cancer, esophageal cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, and non-Hodgkin's lymphoma.

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