Anti-mesothelin nano antibody chimeric antigen receptor and application thereof

Abstract

The invention relates to an anti-mesothelin nano-antibody chimeric antigen receptor and application thereof, and particularly provides a chimeric antigen receptor, and an antigen binding domain of the chimeric antigen receptor comprises a mesothelin binding molecule containing an anti-mesothelin single-domain antibody. The immune cell containing the chimeric antigen receptor disclosed by the invention has a higher cell killing effect.

Description

Technical Field

[0001] The present invention relates to the field of chimeric antigen receptor therapy, and in particular to an anti-mesothelin nanoantibody chimeric antigen receptor and applications thereof. Background Art

[0002] The mesothelin (MSLN) gene is located on chromosome 1p13.3, with a total length of 8kD. The gene contains an open reading frame of 1884bp, encoding 17 exons and 628 amino acids. The precursor protein of MSLN is a glycoprotein of about 69kD anchored to the cell membrane by glycosylphosphatidylinositol, which can be hydrolyzed by proteolytic enzymes into two parts, of which the N-terminal is a 31kD soluble protein with megakaryocyte stimulating activity, known as megakaryocyte potentiating factor (MPF); and the C-terminal is a membrane-bound protein of about 40kD with cell adhesion, known as MSLN. The structure of the membrane protein of MSLN can be divided into three different segments, of which zone I is the binding site of the ligand CA125.

[0003] Mesothelin, as a differentiation antigen, is highly expressed in a variety of malignant tumors and is closely related to the occurrence and development of tumors. This target is one of the most promising anti-tumor targets. Many large pharmaceutical companies have targeted this target for development. Currently, the drug forms entering clinical trials include CAR-T, monoclonal antibodies, ADC, etc. In the past nearly 20 years, a variety of anti-mesothelin monoclonal antibodies have been developed, mainly including SS1P immunotoxin and MORAB-009. SS1P is a recombinant immunotoxin composed of a fusion of scFv and a truncated pseudoexotoxin, which mainly mediates cell killing; MORAB-009 is an IgG1 antibody recombined with the SS1P sequence, which mainly causes antibody-dependent cell-mediated cytotoxicity (ADCC).

[0004] SS1P antibody binds to the mesothelin I region and can block the binding of CA125 and mesothelin. This antibody has also been used in the treatment of CAR-T, but the efficacy is not ideal (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, strong penetration, and wide binding epitopes (Muyldermans S. Annu Rev Biochem. 2013; 82: 775-97.). There is no report on the use of MSLN nanobodies for the treatment of CAR-T in this field.

[0005] At present, although CAR-T therapy has achieved great results in the treatment of hematological tumors, its response rate for solid tumors is relatively 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 constructed a CAR-T cell that can secrete anti-PD-1 single-chain antibody (scFv) (Sarwish Rafiq et al., Nat Biotechnol 2018 Oct; 36 (9): 847-856). However, studies have found that traditional scFv antibodies have low expression levels in T cells and are difficult to fully exert the effect of PD-1 inhibitors. This is mainly due to the properties of scFv antibodies themselves, such as easy aggregation, easy mismatch in folding, and low thermal stability (Martin N et al., Macromol Biosci. 2017 Feb; 17 (2)). Nanobodies have natural advantages such as easy expression, high stability, and high affinity. Based on the advantages of nanoantibodies themselves, the biological mechanisms of MSLN and PD-1, developing a CAR-T based on MSLN nanoantibodies and a therapy that combines autocrine PD-1 nanoantibodies has become an urgent problem to be solved. Summary of the invention

[0006] In one aspect, the present invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a mesothelin binding molecule comprising an anti-mesothelin single domain antibody, a hinge region, a transmembrane region, and an intracellular region.

[0007] In one or more embodiments, the intracellular region includes an intracellular co-stimulatory 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 region (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 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.

[0010] In one or more embodiments, the CDR1 of the anti-mesothelin single domain antibody comprises a sequence as shown in any one of SEQ ID NOs: 4-27, 73. Preferably, the CDR1 comprises a sequence as shown 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 as shown in any one of SEQ ID NOs: 28-47, 74. Preferably, the CDR2 comprises a sequence as shown 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 as shown in any one of SEQ ID NOs: 48-72, 75. Preferably, the CDR3 comprises a sequence as shown 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 the FR region of any one VHH selected from SEQ ID NOs: 76-120.

[0014] In one or more embodiments, the anti-mesothelin single domain antibody VHH is shown in any one of SEQ ID NOs: 73 to 120. Preferably, the anti-mesothelin single domain antibody is shown in any one of SEQ ID NOs: 77, 83, 85, 86, 87, 89, 93, 100, 103.

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

[0016] In one or more embodiments, the chimeric antigen receptor contains, 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 co-stimulatory 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, it is a CD8 signal peptide; preferably, the amino acid sequence of the CD8 signal peptide is as shown in amino acid residues 1-22 of SEQ ID NO:121.

[0018] In one or more embodiments, the hinge region is selected from the group consisting of a CD8 hinge region, an IgD hinge region, an IgG1 FcCH2CH3 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 as shown in amino acid residues 149-203 of SEQ ID NO:121.

[0019] In one or more embodiments, the transmembrane region is one of the CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region; preferably it is the CD28 transmembrane region, and more preferably its amino acid sequence is shown in amino acid residues 204-231 of SEQ ID NO:121.

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

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

[0022] In one or more embodiments, the chimeric antigen receptor contains, from N-terminus to C-terminus, a CD8 signal peptide, an anti-MSLN nanobody, a CD8 hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal domain.

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

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

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

[0026] The present invention also provides a nucleic acid molecule comprising a sequence selected from the group consisting of:

[0027] (1) a coding sequence of a chimeric antigen receptor as described in any embodiment of the present invention;

[0028] (2) the complementary sequence of (1);

[0029] (3) A 5-50 bp fragment of any of the sequences in (1) or (2).

[0030] In one or more embodiments, the fragment is a primer.

[0031] In one or more embodiments, the coding sequence is DNA or RNA.

[0032] The second aspect of the present invention also provides a nucleic acid construct, which comprises the nucleic acid molecule described in any embodiment of the present invention.

[0033] In one or more embodiments, the nucleic acid construct further comprises a coding sequence for a PD-1 binding molecule.

[0034] In one or more embodiments, the nucleic acid construct further comprises a coding sequence of a transposase and / or a coding sequence of a PD-1 binding molecule, wherein the transposase is used to transpose the coding sequence of the chimeric antigen receptor and / or the PD-1 binding molecule, for example, to integrate the coding sequence or expression cassette of the transposed chimeric antigen receptor and / or the PD-1 binding molecule into the genome.

[0035] In one or more embodiments, the coding sequence for the transposase is DNA or RNA.

[0036] In one or more embodiments, the coding sequence of the PD-1 binding molecule is DNA or RNA.

[0037] In one or more embodiments, the PD-1 binding molecules include: PD-1 antibodies or antigen-binding fragments thereof, PD-1 heavy chain antibodies or antigen-binding fragments thereof, monovalent or multivalent single-domain antibodies, and multispecific single-domain antibodies comprising one, two or more PD-1 single-domain antibodies.

[0038] In one or more embodiments, the PD-1 binding molecule (eg, PD-1 single domain antibody) further comprises an Fc region. Preferably, the Fc region is the Fc region of IgG4, the sequence of which is shown in SEQ ID NO: 133.

[0039] In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, or an integrating vector.

[0040] In one or more embodiments, the expression vector is a constitutive expression vector. Preferably, the constitutive expression vector is a transposable vector.

[0041] In one or more embodiments, the coding sequence of the chimeric antigen receptor is located between the recognition sequences of the transposase.

[0042] In one or more embodiments, the coding sequence of the PD-1 binding molecule is located between the recognition sequences of the transposase.

[0043] In one or more embodiments, the transposase is PiggyBac transposase (PBase).

[0044] In one or more embodiments, the transposase is as described in CN105154473A.

[0045] In one or more embodiments, the nucleic acid construct comprises an expression cassette for 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.

[0046] In one or more embodiments, the nucleic acid construct contains an expression cassette of the chimeric antigen receptor and an expression cassette of the transposase; or the nucleic acid construct is an expression cassette, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are within the expression cassette.

[0047] In one or more embodiments, the nucleic acid construct contains an expression frame of the chimeric antigen receptor and an expression frame of the PD-1 binding molecule; or the nucleic acid construct is an expression frame, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the PD-1 binding molecule are in the expression frame.

[0048] In one or more embodiments, the nucleic acid construct contains an expression cassette of the chimeric antigen receptor, an expression cassette of the transposase, and an expression cassette of the PD-1 binding molecule; or the nucleic acid construct is two expression cassettes, one of which contains any one of the coding sequence of the chimeric antigen receptor, the coding sequence of the transposase, and the coding sequence of the PD-1 binding molecule, and the other contains the remaining coding sequences; or the nucleic acid construct is an expression cassette, wherein the coding sequence of the chimeric antigen receptor, the coding sequence of the transposase, and the coding sequence of the PD-1 binding molecule are in the expression cassette.

[0049] In one or more embodiments, the nucleic acid construct is one or more vectors, each vector comprising one, two or three expression cassettes as described in any of the embodiments herein.

[0050] In one or more embodiments, the nucleic acid construct comprises:

[0051] A vector comprising (a) an expression cassette for CAR and optionally (b) an expression cassette for a transposase and / or an expression cassette for a PD-1 binding molecule;

[0052] Two vectors, wherein the two vectors (a) respectively comprise an expression cassette for CAR and an expression cassette for PD-1 binding molecule, and optionally, either of the two vectors further comprises an expression cassette for transposase, or (b) one comprises an expression cassette for transposase, and the other comprises an expression cassette for CAR and an expression cassette for PD-1 binding molecule;

[0053] Three vectors, which respectively contain the expression frame of CAR, the expression frame of PD-1 binding molecule and the expression frame of transposase.

[0054] Another aspect of the present invention provides a host cell, wherein:

[0055] (1) comprising and / or expressing a chimeric antigen receptor as described in any of the embodiments herein, and / or optionally comprising, expressing and / or secreting a PD-1 binding molecule, and / or

[0056] (2) comprising the nucleic acid molecules and / or nucleic acid constructs described herein.

[0057] The transposase and PD-1 binding molecule are as described in the second aspect of this invention.

[0058] In one or more embodiments, the cell is an immune cell, preferably a T cell.

[0059] In one or more embodiments, the cell contains: (a) the coding sequence of the chimeric antigen receptor described in any embodiment herein; and optionally contains (b) the coding sequence of the PD-1 binding molecule described in any embodiment herein.

[0060] In one or more embodiments, the cell contains: a coding sequence for a chimeric antigen receptor as described in any embodiment herein and a coding sequence for a PD-1 binding molecule as described in any embodiment herein.

[0061] Another aspect of the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and:

[0062] (1) the chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally further comprising (i) a transposase and / or its coding sequence, and / or (ii) a PD-1 binding molecule and / or its coding sequence, or

[0063] (2) The nucleic acid molecule, nucleic acid construct or cell according to any one of the embodiments herein.

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

[0065] In one or more embodiments, the pharmaceutical composition is used to treat cancer.

[0066] In one or more embodiments, the cancer comprises a mesothelin-mediated cancer. Preferably, the cancer further comprises a PD-1-mediated cancer.

[0067] In one or more embodiments, the cancer comprises one or more selected from mesothelioma, pancreatic cancer, ovarian cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma.

[0068] Another aspect of the present invention provides the use of a reagent in preparing activated immune cells (such as T cells), wherein the reagent comprises:

[0069] (1) the chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally further comprising (i) a transposase and / or its coding sequence, and / or (ii) a PD-1 binding molecule and / or its coding sequence, or

[0070] (2) The nucleic acid molecule, nucleic acid construct or cell according to any one of the embodiments herein.

[0071] The transposase and PD-1 binding molecule are as described in the second aspect of this invention.

[0072] Another aspect of the present invention provides the use of a reagent in the preparation of a drug for treating cancer, wherein the reagent comprises:

[0073] (1) the chimeric antigen receptor and / or its coding sequence as described in any embodiment herein, optionally further comprising (i) a transposase and / or its coding sequence, and / or (ii) a PD-1 binding molecule and / or its coding sequence, or

[0074] (2) The nucleic acid molecule, nucleic acid construct or cell according to any one of the embodiments herein.

[0075] The transposase and PD-1 binding molecule are as described in the second aspect of this invention.

[0076] In one or more embodiments, the cancer comprises a mesothelin-mediated cancer. Preferably, the cancer further comprises a PD-1-mediated cancer.

[0077] In one or more embodiments, the cancer comprises one or more selected from mesothelioma, pancreatic cancer, ovarian cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma.

[0078] The present invention also provides a method for treating or preventing cancer, comprising administering a therapeutically effective amount of the cell or pharmaceutical composition according to any embodiment of the present invention to a patient in need thereof.

[0079] In one or more embodiments, the cancer comprises a mesothelin-mediated cancer. Preferably, the cancer further comprises a PD-1-mediated cancer.

[0080] In one or more embodiments, the cancer is mesothelioma, pancreatic cancer, ovarian cancer, lung adenocarcinoma, gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, or the like.

[0081] In one or more embodiments, the therapeutically effective amount is 1-100*10 6 cells / mouse, preferably 5*10 6 cells / mouse.

[0082] In one or more embodiments, the cells or pharmaceutical compositions are administered by intravenous injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 :D5-D13 proliferation curve and proliferation multiple of MSLN CAR-T cells. Figure 1 , as shown in D. AC: D5-D13 proliferation curve of CAR-T prepared from peripheral blood of 3 volunteers (AC-201908B, AC-201909A, AC-201910A); D: D5-D13 average proliferation curve of CAR-T prepared from peripheral blood of the above 3 volunteers; E: D5-D13 average proliferation fold curve of CAR-T prepared from peripheral blood of the above 3 volunteers.

[0084] Figure 2 : Cell viability curve of MSLN CAR-T culture D5-D13.

[0085] Figure 3 :Changes in the number of CAR-positive T cells in MSLN and the CAR-positive rate at D13. A: Number of CAR-positive T cells in MSLN; B: CAR-positive rate.

[0086] Figure 4 :The ratio of CD4+T cells and CD8+T cells in CD3+ cells detected on D13

[0087] Figure 5 :The proportion of memory T cells in the cell population on Day13

[0088] Figure 6 : Statistical results of in vitro killing of different tumor cells, mesothelin-negative (A: H520) and mesothelin-positive (B: H226; C: ASPC-1), by MSLN CAR-T cells frozen and thawed in preliminary experiments.

[0089] Figure 7:72h killing rate of mock-T and MSLN CAR-T on two mesothelin-positive tumor cells (E:T=1:1). A: SKOV3; B: H226.

[0090] Figure 8 :Cytokine release of mock-T and different MSLN CAR-T cells after 72 hours of co-incubation with mesothelin-positive tumor cells. A: IFN-gamma; B: IL-2; C: TNF-alpha.

[0091] Fig. 9 : Cell D5-13 proliferation curve and proliferation fold. AC: D5-D13 proliferation curve of CAR-T prepared from peripheral blood of 3 volunteers (AC201906A, AC201908A, AC201909A); ​​D: Average D5-D13 proliferation curve of CAR-T prepared from peripheral blood of the above 3 volunteers; E: Average D5-D13 proliferation fold curve of CAR-T prepared from peripheral blood of the above 3 volunteers. Label as Fig. 9 , as shown in D.

[0092] Fig.10 : Cell viability of cell culture D5-13.

[0093] Fig.11 :Changes in the number of CAR-positive T cells in MSLN and the CAR-positive rate on Day 13. A: Number of CAR-positive T cells in MSLN; B: CAR-positive rate.

[0094] Fig.12 :The ratio of CD4+T cells and CD8+T cells in CD3+ cells detected on D13.

[0095] Fig.13 : The proportion of memory T cells in the cell population on Day13.

[0096] Fig.14 :Changes in the ratio of two types of memory T cells before and after antigen stimulation.

[0097] Fig.15 : Comparison of antibody concentrations secreted by cells before and after antigen stimulation.

[0098] Fig.16 :The killing rate of mock-T and CAR-T against SKOV3-PDL1 at different effector-target ratios at 48h.

[0099] Fig.17 :48h killing rate of mock-T and CAR-T against H226 at different effector-target ratios.

[0100] Fig.18 : Killing curves of mock-T and MSLN CAR-T cells against tumor cells. A: Statistics of killing rates of CAR-T cells against H226 and SKOV-PDL1 cells (E:T=1:4, killing time 72h); B: Representative killing curves against H226 cells; C: Representative killing curves against SKOV3-PDL1 cells.

[0101] Fig.19 :Cytokine release of mock-T and MSLN CAR-T after 48h of co-incubation with mesothelin&PDL1-positive tumor cells. A: IL-2; B: IFN-gamma; C: TNF-alpha.

[0102] Fig. 20 : Changes in NCI-H226-PDL1 tumor volume at high, medium, and low doses.

[0103] Fig.21 : Changes in Skov3-PDL1 tumor volume at high, medium and low dosages.

[0104] Fig. 22 : Body weight changes of NCI-H226-PDL1 tumor-bearing mice at high, medium and low dosages.

[0105] Fig.23 : Changes in body weight of Skov3-PDL1 tumor-bearing mice at high, medium and low doses.

[0106] Fig.24 : Survival curves of NCI-H226-PDL1 tumor-bearing mice at high, medium and low dosages.

[0107] Fig.25 : Survival curves of Skov3-PDL1 tumor-bearing mice at high, medium and low dosages.

[0108] Fig.26 : Changes in blood concentration 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.

[0109] Fig. 27 : Changes in the blood concentration 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.

[0110] Fig.28 : Changes in NCI-H226-PDL1 tumor volume at high dosages.

[0111] Fig.29 : Body weight changes of NCI-H226-PDL1 tumor-bearing mice under high dosage.

[0112] Fig.30 : Survival curve of NCI-H226-PDL1 tumor-bearing mice under high dosage.

[0113] Fig.31 : Changes in blood concentration of PD1 antibody in NCI-H226-PDL1 tumor-bearing mice under high dosage (A: mass concentration; B: molar concentration).

[0114] Fig.32 : Changes in blood drug concentration of CAR-T (A: CD3+T cells, B: CD8+T cells, C: CAR+T cells, D: ratio of CAR+T cells in CD3+T cells) per microliter of blood in NCI-H226-PDL1 tumor-bearing mice at high dosage.

[0115] Fig.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.

[0116] Fig.34 : Changes in tumor volume and body weight at commonly used dosages (A and C: changes in tumor volume and body weight of NCI-H226-PDL1; B and D: changes in tumor volume and body weight of Skov3-PDL1).

[0117] Fig.35 : CAR-T cell preparation data. A: proliferation times; B: cell viability; C: CAR positive rate; D: Tcm and Tem; E: CD4 / 8; F: antibody secretion.

[0118] Fig.36 : PD-1 antibody secretion and cytokine secretion of CAR-T cells. A: PD-1 antibody secretion of CAR-T cells 6h and 48h after antigen stimulation; B: cytokine secretion of CAR-T cells without antigen stimulation and 6h and 48h after antigen stimulation.

[0119] Fig.37 : Killing performance of CAR-T cells. AB: Killing rate of Mock-T and CAR-T prepared from PBMCs from two different volunteers (09B and 09A01) on H226-PDL1 tumor cells at 80 hours; CD: Killing curve of Mock-T and CAR-T prepared from PBMCs from two different volunteers (09B and 09A01) on H226-PDL1 tumor cells.

[0120] Fig.38 : Killing performance of CAR-T cells. AB: Killing rate of Mock-T and CAR-T prepared from PBMCs from three different volunteers (09B, 09A01, 12A) against SKOV3-PDL1 tumor cells at 100 hours; CD: Killing curve of Mock-T and CAR-T prepared from PBMCs from two different volunteers (09B and 09A01) against SKOV3-PDL1 tumor cells.

[0121] Fig.39 : Schematic diagram of the in vivo experimental process in mice.

[0122] Fig.40 :The inhibitory effect of different doses of CAR-T on tumors. AD represents the results of 0.2E6, 1E6, 5E6, and 20E6 doses, respectively.

[0123] Fig.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; E: survival rate of mice at a dose of 20E6.

[0124] Fig.42 : The number of peripheral blood CD3+T cells under different doses.

[0125] Fig.43 :The proportion of CAR-positive cells. A:The proportion of peripheral blood CAR+T cells in CD3+T cells at different doses 14 days after CAR-T transfusion. B:The proportion of CAR+ cells in each group before transfusion.

[0126] Fig.44 :PNB338B-1444-CAR vector structure diagram.

[0127] Fig.45 : Changes in blood concentrations of PD1 antibodies at commonly used dosages (A: NCI-H226-PDL1, B: Skov3-PDL1).

[0128] Fig.46 :Changes in CAR-T blood drug concentrations at commonly used dosages (AD: NCI-H226-PDL1, EH: Skov3-PDL1). DETAILED DESCRIPTION

[0129] The present invention provides a chimeric antigen receptor (CAR) targeting mesothelin. The CAR contains an optional signal peptide sequence, a mesothelin binding molecule containing an anti-mesothelin single domain antibody sequence, a hinge region, a transmembrane region, an intracellular costimulatory domain and an intracellular signaling domain.

[0130] Herein, the "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 regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines. Herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies, which have an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), double antibodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, e.g., Fab, F(ab')2 and Fv). Herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably.

[0131] The "heavy chain antibody" described herein is an antibody derived from a camelid or cartilaginous fish. Compared with the above-mentioned 4-chain antibody, the heavy chain antibody lacks the light chain and the heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of variable regions (VHH) and other constant regions, and the variable regions are connected to the constant regions by a structure similar to the hinge region. Each heavy chain of the camelid heavy chain antibody contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of the cartilaginous fish heavy chain antibody contains 1 variable region and 5 constant regions (CH1-CH5). The antigen-binding fragment of the heavy chain antibody includes VHH and a single-chain heavy chain antibody. By fusion with the constant region of human IgG Fc, the heavy chain antibody can have CH2 and CH3 of human IgG Fc. Exemplarily, the heavy chain constant region comprises the sequence shown in SEQ ID NO:132.

[0132] As used herein, the terms "single domain antibody", "heavy chain variable region domain of heavy chain antibody", "VHH", "nanobody", and "single variable domain" are used interchangeably and refer to a single domain polypeptide or protein that specifically recognizes and binds to an antigen. A single domain antibody is a variable region of a heavy chain antibody. Typically, a single domain antibody contains three CDRs and four FRs. A single domain antibody is the smallest functional antigen-binding fragment. Usually, an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1) is first 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.

[0133] A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody; a binding molecule comprising two or more single domain antibodies of different specificities is a multispecific single domain antibody. A multivalent single domain antibody or a multispecific single domain antibody connects multiple single domain antibodies via a linker. The linker is generally composed of 1-15 amino acids selected from G and S.

[0134] In this article, heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies. Due to the similarity between the two structures, the following structural description of antibodies is also applicable to heavy chain antibodies in addition to light chains.

[0135] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy or light chain of an antibody. The variable domains of the heavy and light chains may be referred to as "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.

[0136] The term "variable" refers to the situation that certain segments in the variable domain vary widely in the antibody sequence. The variable domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, the variability is not evenly distributed over all the amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (in both the light chain and heavy chain variable domains), namely, HCDR1, HCDR2, HCDR3 (heavy chain antibodies can be referred to as CDR1, CDR2, CDR3) of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region. The more highly conserved parts of the variable domain are called framework regions (FRs). The variable domains of natural heavy and light chains each contain four FR regions (FR1, FR2, FR3 and FR4), most of which adopt a β-folded conformation, connected by three HVRs that form a loop connection and in some cases form a part of the β-folded structure. The HVR in each chain is held together in close proximity by the FR region and contributes to the formation of the antigen binding site of the antibody together with the HVR of the other chain. Typically, 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 domain is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity.

[0137] "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that 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 binding 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, the CH2 domain and the CH3 domain from 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 can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is according to the EU index, as in Kabat. Each heavy chain of a camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3). By fusing with the constant region of human IgG Fc, the heavy chain antibody can have the CH2 and CH3 of human IgG Fc.

[0138] "Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding and / or variable regions of an intact antibody. Antibody fragments are preferably antigen binding fragments of antibodies. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragments that should be able to increase half-life by chemical modification or by incorporation into liposomes. Digestion of antibodies with papain produces two identical antigen binding fragments called "Fab" fragments, and a residual "Fc" fragment, the name of which reflects its ability to crystallize easily. Fab fragments consist of an intact light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH1). Each Fab fragment is monovalent in terms of antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produces a larger F(ab')2 fragment, which is roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still able to cross-link antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F(ab')2 antibody fragments were originally generated as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known. The Fc fragment contains the carboxyl terminal portions of the 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 certain types of cells. Antigen binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies.

[0139] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. The fragment consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each in the heavy chain and light chain) protrude from the folding of these two domains, contributing the amino acid residues for antigen binding and giving the antibody antigen binding specificity. However, even a single variable domain (or half an Fv containing only three HVRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of a complete binding site. "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising an antibody VH and VL domains connected into a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen binding structure. The Fv of a heavy chain antibody is VHH.

[0140] Antibodies herein also include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0141] A "humanized" form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework region is exchanged with sequences found in human antibodies. 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 (some or all of which are encoded by nucleic acids derived from non-human organisms) are transplanted into the β-folded framework of the variable region of a human antibody to produce an antibody whose specificity is determined by the transplanted CDRs. Methods for producing such antibodies are well known in the art, for example, using mice with a genetically engineered immune system.

[0142] "Human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and / or produced using any of the techniques disclosed herein for producing human antibodies. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries. The binding molecules described in any embodiment of the present invention are chimeric antibodies or fully human antibodies; preferably fully human antibodies.

[0143] The "mesothelin binding molecule" described herein comprises an anti-mesothelin single domain antibody, the complementarity determining region CDR of the single domain antibody comprises CDR1, CDR2 and CDR3, wherein 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.

[0144] In one or more embodiments, SEQ ID NO: 1 is X1X2X3X4X5X6X7X8X9X 10 X 11 , wherein 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, X10 is N, A, G, D, K, Y, L or S, X11 is N, A, G, D, K, Y, L or S, X12 is N, A, G, D, K, Y, L or S, X13 is N, A, G, D, K, Y, L or S, X14 is N, A, G, D, K, Y, L or S, X15 is N, A, G, D, K, Y, L or S, X16 is N, A, G, D, K, Y, L or S, X17 is N, A, G, D, K, Y, L or S, X18 is N, A, Y, V, D or S, X19 is E or none, X20 is N, A, G, D, L or S, X21 is N, A, G, D, L or S, X22 is N, A, Y, V, D or S, X23 is N, A, Y, V, D or S, X24 is N, A, Y, V, D or S, X25 is N, A, S, V, D or S, X26 is N, A, G, D, L or S, X27 is N, A, G, D, L or S, X28 is N, A, Y, V, D or S, X29 is E or none, X30 is N, A, S, V, T, D, L or S, X31 is N, A, S 10 F or None, X 11 In one or more embodiments, SEQ ID NO: 1 is X1X2X3X4X5X6X7X8, wherein 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 GX2XXXX4XXXX7A, wherein 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 X1XXXX4XXXX6XXXX8, wherein 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.

[0145] In one or more embodiments, the CDR1 of the anti-mesothelin single domain antibody comprises the sequence shown in any one of SEQ ID NOs: 4-27, 73. Preferably, CDR1 comprises the sequence shown in any one of SEQ ID NOs: 5, 11, 14, 15, 17, 21, 27, 73.

[0146] In one or more embodiments, SEQ ID NO: 2 is X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 , wherein 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, X 10 G, R or None, X 11 V, T or None, X 12 In one or more embodiments, X3 is S, N, A, R or T. In one or more embodiments, SEQ ID NO: 2 is IX2XXXX4XXXX6XXXX9, wherein 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 X1X2XXXX4XXXX6XXXX7XXXX9X 10 X 11 X 12 , wherein 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, X 10 G or None, X 11 V or None, X 12 T or None.

[0147] In one or more embodiments, the CDR2 of the anti-mesothelin single domain antibody comprises the sequence shown in any one of SEQ ID NOs: 28-47, 74. Preferably, CDR2 comprises the sequence shown in any one of SEQ ID NOs: 29, 35, 37, 38, 39, 43, 40, 74.

[0148] In one or more embodiments, SEQ ID NO:3 is X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X16X 17 X 18 X 19 X 20 X21 X 22 X 23 , wherein 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, 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, R or V, X 10 R, Y, H, N, T, D, P, V, K, C, L or S, X 11 Y, D, E, P, F, S, L, I or none, X 12 P, V, Q, A, Y, F, N, D, R or None, X 13 D, A, Y, V, Q, P or none, X 14 Y, L, P, A, E or None, X 15 C, S, M, P, T or none, X 15 C, S, M, P or None, X 16 V, D, S, Y or none, X 17 L, F, V, D or none, X 18 R, G, M, S or none, X 19 D, N, S or None, X 20 Y, L or None, X 21 is Y or None, X 22 A or None, X 23 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, X 10 is R, Y, H, N, T, D, P, V, K, C or S, X 11 Y, D, E, P, F, S, L or none, X 12 P, V, Q, A, Y, F, N, D or None, X 13 D, A, Y, V, Q or none, X 14 Y, L, P, A, E or None, X 15 C, S, M, P or None, X16 V, D, S, Y or none, X 17 L, F, V, D or none, X 18 R, G, M, S or none, X 19 D, N, S or None, X 20 Y, L or None, X 21 is Y or None, X 22 A or None, X 23 In one or more embodiments, SEQ ID NO: 3 is AX2X3X4X5X6X7X8X9X 10 X 11 X12X 13 X 14 X 15 X 16 X 17 X 18 X 19 , wherein 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, X 10 R, Y, H, T, D, P, V, K or C, X 11 Y, D, E, F, S, L or none, X 12 P, V, A, Y, F, N, D or None, X 13 D, A, Y, V or none, X 14 is Y, P, E or none, X 15 S, P or None, X 16 D, Y or None, X 17 F, D or None, X 18 G, S or None, X 19 Preferably, SEQ ID NO: 3 is AX2X3X4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X17X 18 X 19 , wherein 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, X 10R, Y, D, V or S, X 11 Y, D, F, S, L or none, X 12 P, F, or None, X 13 D, V or None, X 14 P or None, X 15 S or None, X 16 D or None, X 17 F or None, X 18 G or None, X 19 N or None.

[0149] In one or more embodiments, the CDR3 of the anti-mesothelin single domain antibody comprises a sequence as shown in any one of SEQ ID NOs: 48-72, 75. Preferably, the CDR3 comprises a sequence as shown in any one of SEQ ID NOs: 49, 55, 58, 59, 61, 65, 72, 75.

[0150] In one or more embodiments, the CDR1 of the anti-mesothelin single domain antibody comprises a sequence as shown in any one of SEQ ID NOs: 4-27 and 73, the CDR2 comprises a sequence as shown in any one of SEQ ID NOs: 28-47 and 74, and the CDR3 comprises a sequence as shown in any one of SEQ ID NOs: 48-72 and 75. Preferably, the anti-mesothelin single domain antibody comprises CDR1, CDR2 and CDR3 as shown in any one of the following groups a1 to a26:

[0151] Group CDR1 CDR2 CDR3 a1 4 28 48 a2 5 29 49 a3 6 30 50 a4 7 31 51 a5 8 32 52 a6 9 33 53 a7 10 34 54 a8 11 35 55 a9 12 36 56 a10 13 32 57 a11 14 37 58 a12 15 38 59 a13 16 31 60 a14 17 39 61 a15 18 40 62 a16 19 41 63 a17 20 42 64 a18 21 43 65 a19 22 32 66 a20 13 32 67 a21 23 44 68 a22 24 45 69 a23 25 46 70 a24 26 47 71 a25 27 40 72 a26 73 74 75

[0152] In one or more embodiments, FR1 of the anti-mesothelin single domain antibody can be selected from FR1 of the VHH of each antibody number in Table A, FR2 of the VHH can be selected from FR2 of the VHH of each antibody number in Table 1, FR3 of the VHH can be selected from FR3 of the VHH of each antibody number in Table 1, and FR4 of the VHH can be 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 VHH selected from SEQ ID NOs: 76-120.

[0153] In one or more embodiments, the anti-mesothelin single domain antibody is as shown in any one of SEQ ID NOs: 76 to 120. Preferably, the single domain antibody is as shown in any one of SEQ ID NOs: 77, 83, 85, 86, 87, 89, 93, 100, 103.

[0154] The mesothelin binding molecule described herein is a monovalent or multivalent single domain antibody, a multispecific single domain antibody, a heavy chain antibody or an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof comprising one, two or more anti-mesothelin single domain antibodies described herein. The multivalent single domain antibody or multispecific single domain antibody connects multiple single domain antibodies via a linker. The linker consists of 1-15 amino acids selected from G and S. The heavy chain antibody is a camel 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 constant region of a camel heavy chain antibody, comprising CH2 and CH3. In one or more embodiments, the CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG4.

[0155] The mesothelin binding molecules and anti-mesothelin single domain antibodies mentioned herein include all mesothelin binding molecules and anti-mesothelin single domain antibodies described in CN202011584014 or CN202011582948, and the entire text of the above patent application is incorporated herein by reference to each embodiment thereof specifically and individually.

[0156] The optional signal peptide on CAR can be selected as needed. For example, a CD8 signal peptide, a CD28 signal peptide, a CD4 signal peptide or a light chain signal peptide, the sequence of which is 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 in CAR in the art. In certain embodiments, the amino acid sequence of the human CD8 signal peptide can be as shown in SEQ ID NO: 121 amino acids 1-22.

[0157] The hinge region of CAR is selected from CD8α hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 Fc CH2CH3 hinge region, and its sequence is within the knowledge of those skilled in the art. The CD8 hinge region suitable for the present invention can be various human CD8 hinge region sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the human CD8 hinge region can be as shown in SEQ ID NO: 121 amino acids 149-203.

[0158] The transmembrane region of CAR is selected from one of the CD28 transmembrane region, CD8 transmembrane region, CD3ζ transmembrane region, CD134 transmembrane region, CD137 transmembrane region, ICOS transmembrane region and DAP10 transmembrane region; preferably the CD28 transmembrane region, whose sequence is within the knowledge of those skilled in the art. The human CD28 transmembrane region suitable for the present invention can be various human CD28 transmembrane region sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the human CD28 transmembrane region is shown in SEQ ID NO: 121 amino acids 204-231.

[0159] Suitable intracellular co-stimulatory domains can be selected as needed, including intracellular domains with co-stimulatory signal molecules, such as CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulatory factor (ICOS) and DNAX activation protein 10. The human CD28 co-stimulatory domain suitable for the present invention can be various human CD28 co-stimulatory domain sequences commonly used in CAR in the art. In certain embodiments, the amino acid sequence of the CD28 co-stimulatory domain is shown in the amino acid sequence of positions 231-272 of SEQ ID NO:121.

[0160] Similarly, the intracellular signaling domain of CAR can be selected as needed, including but not limited to CD3ζ intracellular signaling domain or FcεRIγ intracellular signaling domain. The CD3ζ intracellular signaling domain suitable for the present invention can be various CD3ζ intracellular signaling domains for CAR known in the art. In certain embodiments, the amino acid sequence of the CD3ζ intracellular signaling domain is shown in the amino acid sequence of positions 273-384 of SEQ ID NO:121.

[0161] The above-mentioned parts forming the chimeric antigen receptor of the present invention, such as CD8 signal peptide, anti-MSLN nanobody, CD8 hinge region, CD28 transmembrane region, CD28 co-stimulatory domain, CD3ζ intracellular signal domain, etc., can be directly connected to each other, or can be connected through 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. Generally, the linker contains one or more motifs repeated before and after. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motif is adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence can be composed of 1, 2, 3, 4 or 5 repeated motifs. The length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycine in the linker sequence is not particularly limited, and is generally 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), etc. As an example, the linker may be composed of any amino acid sequence in SEQ ID NO: 7-18. In certain embodiments, the linker sequence is (GGGGS)n connection, where n is an integer of 1 to 5.

[0162] In an exemplary embodiment, CAR contains a CD8 signal peptide, an anti-MSLN nanobody, a CD8 hinge region, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ intracellular signal domain from the N-terminus to the C-terminus. In a specific embodiment, an exemplary CAR having the above structure is shown in any of SEQ ID NOs: 121-123. In certain embodiments, the amino acid sequence of the CAR of the present invention is shown in SEQ ID NO: 121 amino acids 23-384 or in SEQ ID NO: 122 amino acids 23-377 or in SEQ ID NO: 123 amino acids 23-378.

[0163] It should be understood that in gene cloning operations, it is often necessary to design suitable restriction sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, which does not affect the activity of the target sequence. In order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus or other suitable regions within the protein of the recombinant protein, for example, including but not limited to suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino or carboxyl terminus of the CAR of the present invention may also contain 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 to purify proteins.

[0164] The present invention also includes mutants of CAR described in any embodiment. These mutants include: an amino acid sequence having 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 retaining the biological activity (such as activated T cells) of the CAR. The sequence identity between the two aligned sequences can be calculated using, for example, NCBI's BLASTp.

[0165] Mutants also include: an amino acid sequence having one or more mutations (insertions, deletions or substitutions) in the amino acid sequence of the CAR described in any embodiment, while still retaining the biological activity of the CAR. The several mutations generally refer to within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, in the art, when conservative substitutions are made with amino acids with similar or similar properties, the function of the protein or polypeptide is generally not changed. "Amino acids with similar 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 non-polar 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., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or several sites in the polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0166] The present invention includes polynucleotide sequences encoding the fusion proteins of the present invention. The polynucleotide sequences of the present invention may be in the form of DNA or RNA. The DNA forms include cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand. The present invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0167] The polynucleotide sequences described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA library prepared by conventional methods known to those skilled in the art can be used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each amplification together in the correct order.

[0168] The present invention also relates to a nucleic acid construct containing a polynucleotide sequence as described herein, and one or more regulatory sequences operably connected to these sequences. The polynucleotide sequence of the present invention can be manipulated in a variety of ways to ensure the expression of the CAR. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the different or required expression vectors. The technology of using recombinant DNA methods to change polynucleotide sequences is known in the art.

[0169] The regulatory sequence can be a suitable promoter sequence. The promoter sequence is usually operably connected to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably connected to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, a non-translated region of an mRNA that is important for host cell translation. The leader sequence is operably connected to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention.

[0170] In some embodiments of the present invention, the nucleic acid construct further comprises a coding sequence for a PD-1 binding molecule. PD-1 binding molecules include, but are not limited to: PD-1 antibodies or antigen-binding fragments thereof, PD-1 heavy chain antibodies or antigen-binding fragments thereof, monovalent or multivalent single-domain antibodies, and multispecific single-domain antibodies comprising one, two or more PD-1 single-domain antibodies. The PD-1 binding molecules and anti-PD-1 single-domain antibodies mentioned herein respectively include all PD-1 binding molecules and all anti-PD-1 single-domain antibodies described in CN202011582908, and the full text of the patent application is incorporated herein by reference to each embodiment thereof specifically and individually. As described in the 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 an antigen-binding fragment thereof, an antibody or an antigen-binding fragment thereof comprising one, two or more of the PD-1 single-domain antibodies.

[0171] Therefore, the complementary determining region CDR of the anti-PD-1 single domain antibody described herein comprises CDR1, CDR2 and CDR3, wherein CDR1, CDR2 and CDR3 respectively comprise the sequences shown in SEQ ID NO: 1, 2 and 3 in CN202011582908. Exemplarily, in the anti-PD-1 single domain antibody described herein, CDR1 comprises the sequence shown in any one of SEQ ID NO: 4-39, 320 in CN202011582908; CDR2 comprises the sequence shown in any one of SEQ ID NO: 40-75 in CN202011582908; CDR3 comprises the sequence shown in any one of SEQ ID NO: 76-183 in CN202011582908. In one or more embodiments, the anti-PD-1 single domain antibody described herein comprises CDR1, CDR2 and CDR3 shown in any one of group a1 to group a136 in CN202011582908. The anti-PD-1 single domain antibody VHH described herein may be as shown in any one of SEQ ID NOs: 184-319 in CN202011582908. Preferably, the anti-PD-1 single domain antibody described herein is as shown in any one of SEQ ID NOs: 197, 200, 223-309 in CN202011582908. In addition, each CDR or full length of the anti-PD-1 single domain antibody described herein may be as described in any embodiment of CN202011582908.

[0172] Exemplarily, CDR1 of the anti-PD-1 single domain antibody is shown as SEQ ID NO: 124 or 128, CDR2 is shown as SEQ ID NO: 125 or 129, and CDR3 is shown as SEQ ID NO: 126 or 130; the sequence of the anti-PD-1 single domain antibody VHH is shown as SEQ ID NO: 127 or 131.

[0173] The PD-1 binding molecules described herein may be monovalent or multivalent single domain antibodies, multispecific single domain antibodies, heavy chain antibodies or antigen-binding fragments thereof, antibodies or antigen-binding fragments thereof comprising one, two or more anti-PD-1 single domain antibodies described herein. The multivalent single domain antibody or multispecific single domain antibody connects multiple single domain antibodies via a linker. The linker consists of 1-15 amino acids selected from G and S. Herein, the heavy chain antibody is a camel 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 constant region of a camel heavy chain antibody, comprising CH2 and CH3. In one or more embodiments, the CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG4.

[0174] Therefore, in one or more embodiments, the nucleic acid construct comprises the coding sequence of CAR and PD-1 binding molecule. In one or more embodiments, the nucleic acid construct contains the expression frame of the chimeric antigen receptor and the expression frame of the PD-1 binding molecule. The two expression frames are contained in one or two vectors. Alternatively, the nucleic acid construct is an expression frame, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the PD-1 binding molecule are in the expression frame.

[0175] In certain embodiments, the nucleic acid construct is a vector. Usually, the expression of the polynucleotide sequence of the present invention is achieved by operably connecting the polynucleotide sequence of the present invention to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. Typical cloning vectors include transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. The nucleic acid construct may be one or more vectors, each of which includes one or two or three expression cassettes described in any embodiment of this article.

[0176] The polynucleotide sequence of the present invention can be cloned into many types of vectors. For example, it can be cloned into plasmids, phagemids, phage derivatives, animal viruses and cosmids. Further, the vector is an expression vector. The expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses.

[0177] Typically, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0178] 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 transposition vector (or "transposon vector"). In certain embodiments, the transposon vector is a eukaryotic expression vector containing a transposition element selected from piggybac, sleeping beauty, frog prince, Tn5 or Ty. Such transposon vectors contain the 5' inverted terminal repeat (5'ITR) of the corresponding transposon and the 3' inverted terminal repeat (3'ITR) of the corresponding transposon. Therefore, in some embodiments of the present invention, the nucleic acid construct further comprises a coding sequence for a transposase, which is used to transpose the coding sequence of the chimeric antigen receptor and / or PD-1 binding molecule, for example, integrating 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 the piggybac, sleeping beauty, frog prince, Tn5 or Ty transposition system. When using transposases from different transposition systems, the sequences of 5'ITR and 3'ITR in the vector are also changed accordingly to sequences compatible with the transposition system, which can be easily determined by those skilled in the art. The expression frame of the CAR or antibody of the present invention is contained between 5'ITR and 3'ITR, including the corresponding promoter sequence, the coding sequence of CAR or antibody, and the polyA tailing signal sequence. An exemplary transposase is a PB enzyme. In certain embodiments, the 5' reverse terminal repeat sequence of the transposon is shown in SEQ ID NO: 1 of CN105154473A (the contents of which are incorporated herein by reference). In certain embodiments, the 3' reverse terminal repeat sequence of the transposon is shown in SEQ ID NO: 4 of CN105154473A. In certain embodiments, the piggybac transposase is a transposase containing a c-myc nuclear localization signal coding sequence. In certain embodiments, the coding sequence of the piggybac transposase is shown in SEQ ID NO: 5 of CN 201510638974.7. Exemplary vector backbones are pNB338B and pS338B described in CN105154473A and CN111206043A.

[0179] Therefore, in one or more embodiments, the nucleic acid construct comprises the coding sequence of CAR and transposase. In one or more embodiments, the nucleic acid construct contains the expression cassette of the chimeric antigen receptor and the expression cassette of the transposase. The two expression cassettes are contained in one or two vectors. Alternatively, the nucleic acid construct is an expression cassette, wherein the coding sequence of the chimeric antigen receptor and the coding sequence of the transposase are in the expression cassette.

[0180] In some cases, the nucleic acid construct described herein comprises the coding sequences of CAR, transposase and PD-1 binding molecules. The nucleic acid construct may contain three expression frames, namely the expression frame of the chimeric antigen receptor, the expression frame of the transposase and the expression frame of the PD-1 binding molecule; the three expression frames may be contained in one, two or three vectors. Alternatively, the nucleic acid construct contains two expression frames, one of which contains the coding sequence of the chimeric antigen receptor, the coding sequence of the transposase and the coding sequence of the PD-1 binding molecule, and the other remaining coding sequence; the two expression frames may be contained in one or two vectors; or, the nucleic acid construct contains an expression frame, wherein the coding sequence of the chimeric antigen receptor, the expression frame of the transposase and the coding sequence of the PD-1 binding molecule are in the expression frame.

[0181] In one or more embodiments, the nucleic acid construct comprises: a vector comprising (a) an expression cassette of CAR and optionally (b) an expression cassette of a transposase and / or an expression cassette of a PD-1 binding molecule; two vectors, wherein the two vectors (a) respectively comprise an expression cassette of CAR and an expression cassette of a PD-1 binding molecule, and optionally, either of the two vectors further comprises an expression cassette of a transposase, or (b) one comprises an expression cassette of a transposase and the other comprises an expression cassette of a CAR and an expression cassette of a PD-1 binding molecule; or three vectors comprising an expression cassette of a CAR, an expression cassette of a PD-1 binding molecule, and an expression cassette of a transposase, respectively.

[0182] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive any polynucleotide sequence operably connected thereto to express at a high level. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoter, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Further, it is also possible to consider the use of an inducible promoter. The use of an inducible promoter provides a molecular switch that can open the expression of a polynucleotide sequence operably connected to an inducible promoter when the deadline is expressed, and close the expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter.

[0183] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector introduced into the cell may also include any one or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a cell population seeking to be transfected or infected by a viral vector. In other aspects, selectable markers may be carried on a single DNA segment and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences so that they can be expressed in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo and the like.

[0184] Reporter genes are used to identify cells of potential transfection and to evaluate the functionality of regulatory sequences. After DNA has been introduced into recipient cells, the expression of reporter genes is measured at the appropriate time. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein genes. Suitable expression systems are known and can utilize known techniques to prepare or commercially obtain.

[0185] Methods for introducing genes into cells and expressing genes into cells are known in the art. Vectors can be easily introduced into host cells, for example, mammalian, bacterial, yeast or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical or biological means.

[0186] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing polynucleotides 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 macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0187] 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 certain embodiments, after obtaining T cells, they can be first stimulated and activated with an appropriate amount (e.g., 30 to 80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in a medium containing an appropriate amount (e.g., 30 to 80 IU / ml, such as 50 IU / ml) of IL2 for standby use.

[0188] Therefore, in certain embodiments, the present invention provides a genetically modified T cell, the genetically modified T cell containing a polynucleotide sequence as described herein, or containing a nucleic acid construct as described herein, or prepared using the method described herein, or stably expressing CAR as described herein and an optional anti-PD-1 binding molecule. The cell described herein (eg, T cell) may also contain or express a transposase or its coding sequence.

[0189] The CAR-T cells of the present invention can undergo robust in vivo T cell expansion and persist at high levels in the blood and bone marrow for an extended amount of time, and form specific memory T cells. Without wishing to be bound by any particular theory, after encountering and subsequently eliminating target cells expressing alternative antigens, the CAR-T cells of the present invention can differentiate into a central memory-like state in vivo.

[0190] The present invention also includes a class of cell therapies in which T cells are genetically modified to express the CAR described herein and optionally the anti-PD-1 binding molecule, and the CAR-T cells are injected into a recipient in need thereof. The injected cells are capable of killing the recipient's tumor cells. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0191] The anti-tumor immune response caused by CAR-T cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy procedure, in which the CAR-T cells induce an immune response specific to the antigen-binding portion in the CAR.

[0192] Therefore, the disease that can be treated with the CAR, its coding sequence, nucleic acid construct, expression vector and CAR-T cell of the present invention is preferably mesothelin-mediated cancer. Preferably, the disease also benefits from anti-PD-1 antibody therapy, which is PD-1-mediated cancer.

[0193] Specifically, herein, "mesothelin-mediated cancers" include, among others, various types of ovarian cancer, pleural mesothelioma (such as epithelial mesothelioma), pancreatic cancer, and squamous cell carcinoma of the cervix, head, neck, vagina, lung, and esophagus. In certain embodiments, the mesothelin-mediated disease is malignant pleural mesothelioma, pancreatic cancer, ovarian cancer, and lung cancer. Diseases that "benefit from anti-PD-1 antibody therapy" or "PD-1-mediated cancers" include gastric cancer, melanoma, lung cancer, head and neck cancer, renal cell carcinoma, urothelial carcinoma, non-Hodgkin's lymphoma, and the like.

[0194] The CAR-modified T cells of the present invention may be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as related cytokines or cell groups. Simply put, the pharmaceutical composition of the present invention may include CAR-T cells as described herein, combined with one or more pharmaceutically or physiologically acceptable excipients (e.g., carriers, diluents, or excipients). Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0195] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by such factors as the patient's condition, and the type and severity of the patient's disease.

[0196] When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive 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, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can be generally stated that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 The dosage is preferably 10 cells / kg body weight. 5 Up to 10 6The T cell composition can also be administered multiple times at these doses. The cells can be administered using well-known infusion techniques in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dose and treatment regimen for a particular patient can be easily determined by a medical technician by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0197] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The composition of the T cell can be directly injected into a tumor, lymph node or infection site.

[0198] In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention may be combined with other therapies known in the art. The therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, the treatment may be combined with radiotherapy or chemotherapy agents known in the art for treating mesothelin-mediated diseases.

[0199] Herein, "anti-tumor effect" refers to a biological effect which can be represented by 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 associated with cancer.

[0200] "Patient", "subject", "individual" and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0201] The present invention is further described in detail by reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should never be construed as being limited to the following examples, but should be construed as including any and all variations that become apparent due to the teachings provided herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art.

[0202] Example

[0203] Experimental operation method:

[0204] Isolation and cryopreservation of human peripheral blood mononuclear cells (PBMCs)

[0205] First, dilute the human single blood with DPBS 1-2 times, then gently add the diluted blood to the upper layer of ficoll to avoid mixing the two solutions (blood:Ficoll=3:4); centrifuge at 800g for 20min (centrifuge speed is 1, speed reduction is 0), and after centrifugation, use a pipette to draw the white film layer into a new centrifuge tube; add 3 times the volume of DPBS and mix thoroughly, then centrifuge at 1500rpm for 10min and remove the supernatant, repeat this step 2-3 times; after counting, add AIM-V medium at a volume of 5*10^6 cells / ml and incubate in a 5% CO2 incubator overnight. After culture, collect the suspended cells and count them and freeze them.

[0206] CAR-T preparation steps

[0207] Human mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy human donors using Ficoll density gradient centrifugation (GE). The isolated PBMCs were expressed as 1×10 6 After overnight culture in AIM-V medium, the cells were cryopreserved in serum-free cryopreservation medium (New Cyme) and stored in the cell bank of Shanghai Cell Therapy Group at a cryopreservation density of 1×10 7 / mL. Fresh or frozen PBMCs were electroporated using Lonza 4D electroporator. The electroporation process was optimized according to the electroporator manual. 100ul electroporation solution was resuspended in 5-7×10 6 PBMCs, 6ug pNB338B-1444-CAR plasmid, or 4ug pNB338B-1444-CAR + 4ug pS338B-1182 plasmid was added to the cell suspension and electroporated using the EO-115 program. The electroporated PBMCs were added to a 12-well plate containing 2ml of 37°C preheated AIM-V (+2% FBS) culture medium and cultured for 4-6h, then transferred to an antigen-coated six-well plate (5ug / ml CD28 antibody + 5ug / ml MNSL antigen), the culture medium was supplemented to 4ml, and IL-2 was added 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. The rest of the operation process was the same as the above experimental group and was performed simultaneously. After 5 days of culture in the above culture medium, the electroporated PBMCs were subcultured for the first time, and then subcultured every 2-3 days. On the 13th day, CAR-T cells were prepared. During the subculture process, PBMCs were cultured in AIM-V culture medium containing 2% FBS and 100 IU / ml IL-2, and the cell density was 5×10 5 / mL. The cell number and cell viability were detected and recorded using CountStar.

[0208] CAR-T cell preparation process and preparation completion, flow cytometry was used to detect the cell surface CAR positive rate and other surface markers. The following antibodies were used for flow cytometry detection: 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.

[0209] RTCA cell killing

[0210] The killing effect of CAR-T cells on tumor cells was detected using xCELLigence RTCA equipment (Roche Applied Science, Canada), and the detection process was carried out according to the equipment operating instructions. 3 -2×10 4 ) Target cells (tumor cells) were resuspended in 50ul culture medium and inoculated into the microplate (16-well or 96-well) of the RTCA device. Tumor cells were cultured on the RTCA device for about 24 hours. When the cell index (CI, indicating cell growth) reached about 1.5, different numbers of effector cells (CAR-T cells) were resuspended in 50ul culture medium according to different effector-target ratios (effector cells: target cells), mixed with tumor cells, and continued to be cultured on the RTCA device for 3-5 days. The cell killing curve was recorded by the RTCA device, and the data was analyzed using RTCA Pro 2.3.0 software.

[0211] Antibody secretion detection

[0212] The secretion of PD1 nanoantibodies during and after the preparation of CAR-T cells targeting MSLN was detected by ELISA (Enzyme-linked immunosorbent assay). 5 / mL density, and the supernatant was collected on days 5-7 and 11-13 to detect the secretion of PD1 antibody. 5 / mL CAR-T cells were inoculated into cell culture plates coated with 5ug / ml MSLN antigen. After 48h of culture, the supernatant was collected to detect PD1 antibody secretion. The following antibodies were used for ELISA to detect the secretion of PD1 nanoantibodies: anti-1182-VHH (Chenghuang Nano, amino acid sequence as shown in SEQ ID NO: 134).

[0213] Example 1, CAR and CAR-T cells containing mesothelin nanobodies

[0214] By using MSLN CAR derived from nanoantibodies of different sequences and MSLN CAR in the benchmark to construct plasmids and transferring the genes into PBMCs from healthy subjects by electroporation, we constructed non-viral vector MSLN CAR-T cells and verified the in vitro performance of CAR-T cells, including proliferation ability, CAR positivity rate, in vitro killing ability of target cells, cytokine secretion level and other indicators.

[0215] 1.1. Construction of MSLN CAR-T cell plasmid

[0216] Experimental instruments: ordinary PCR instrument (Jena, TRIO), electrophoresis instrument (Bio-Rad, 1645050), electrophoresis tank (Bio-Rad, 1704483), desktop microcentrifuge (Thermo, Pico17), metal bath heating module (Thermo, DCD), electric constant temperature water bath (Shanghai Yiheng, DK-8D), automatic gel imager (Tianneng Tanon, 2500)

[0217] Experimental reagents

[0218]

[0219] Experimental methods

[0220] According to the sequence of the MSLN nanobody, the full-length nucleic acid sequence and primers were synthesized, and a large number of target fragments were obtained by PCR. The pNB338B vector (its sequence and structure are shown in "pNB338B-E" in CN109988759A) was double-digested (EcoR1 and Sal1) to obtain the vector fragment, and the target fragment and the vector fragment were connected by DNA ligase to obtain a plasmid. The constructed vector was sequenced, and the plasmid was extracted in large quantities after the sequence was correct. Among them, the vector structure of pNB338B-1444-CAR is as follows Fig.44The amino acid sequence of the MSLN3

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

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

[0221] 1.2. Preparation of mesothelin-targeted CAR-T cells

[0222] Mock-T and MSLN CAR-T were prepared by electroporation.

[0223] Experimental instruments: electroporator (Lonza 4D), automated cell counter (Invitrogen CountessⅡFL), microscope (Jiangnan XD-202), biological safety cabinet (Thermo Fisher Scientific), refrigerator (Qingdao Haier), carbon dioxide incubator (Thermo), desktop low-speed centrifuge (Xiangyi), Pico17 desktop microcentrifuge (Thermo),

[0224] Experimental reagents

[0225]

[0226] Experimental methods

[0227] Isolation and cryopreservation of human peripheral blood mononuclear cells (PBMCs)

[0228] See experimental procedure for details.

[0229] PBMC cell recovery and MSLN CAR-T preparation

[0230] The density of three donor PBMCs (AC-201909A, AC-201908B, AC-201910A) was 6.08E+06~9.67E+06 / mL, and the viability was over 90%. When single plasmid was electroporated, the plasmid concentration was 6μg per sample. See the experimental operation method for details.

[0231] Experimental Results

[0232]

[0233] 1.3. Detection of in vitro proliferation and positive rate of MSLN CAR-T cells

[0234] The culture cycle after preparation of non-viral vector CAR-T is generally 13 days. The final yield of CAR-T cells is finally obtained by the proliferation of total cells and the final CAR-positive cell ratio. Therefore, the purpose of this experiment is to evaluate the quality of the prepared mock-T cells and MSLN CAR-T cells by proliferation and positive rate.

[0235] Experimental instruments: automated cell counter (Invitrogen CountessⅡFL), microscope (Jiangnan XD-202), biological safety cabinet (Thermo Fisher Scientific), refrigerator (Qingdao Haier), carbon dioxide incubator (Thermo), desktop low-speed centrifuge (Xiangyi), flow cytometer (Navios, AZ46353), Pico17 desktop microcentrifuge (Thermo)

[0236] Experimental reagents

[0237] Reagent name Manufacturer AIM-V medium GIBCO FBS GIBCO DPBS Hyclone Recombinant human interleukin-2 for injection Shandong Quangang Pharmaceutical Co., Ltd. Serum-free cell freezing medium New Saimei 0.4% Trypan blue dye Gibco Biotinylated human mesothelin antigen Kaixia Biology PE Streptavidin BD

[0238] Experimental methods

[0239] 1) CAR-T cell culture and passage

[0240] By counting cells and passing them at equal density, the proliferation times of each group of cells were calculated and the cell proliferation curve was drawn. The passaging density was 5×10 5 / mL, passaged and counted on Day 5, 8, and 11; IL-2 concentration was 100U / mL. See the experimental procedure for details.

[0241] 2) Detection of the proportion of MSLN CAR-T positive cells

[0242] The flow cytometry method was used to detect the changes in CAR positive rate during the proliferation of cells after antigen enrichment to compare different CAR sequences. See the experimental operation method for details.

[0243] Experimental Results

[0244] The results are as follows Figure 1-3 As shown in Tables 1-3. The results showed that the proliferation ability of CAR-T cells prepared from PBMCs from three donors was different. Two of the donors had good proliferation, with amplification times of about 20-38 times, and one donor had poor proliferation, only 1.3-3.7 times. Overall, MSLN3

[1444] CAR-T cells could be successfully expanded, and their proliferation ability was comparable to that of Mock-T cells and MSLN3[Scfv]CAR-T cells ( Figure 1In addition, MSLN3

[1444] CAR-T cells, like Mock-T cells and MSLN3[Scfv]CAR-T cells, had a high viability (>90%) ( Figure 2 ). The CAR positivity rate of MSLN3

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

[0245] Table 1. MSLN CAR-T cell proliferation data from D5 to D13

[0246]

[0247] Table 2. Cell viability of MSLN CAR-T cells at D5-D13

[0248]

[0249] Table 3. Changes in the positive rate during MSLN CAR-T culture and the number of CAR-positive cells at D13

[0250]

[0251] 1.4. Phenotypic detection of MSLN CAR-T cells

[0252] The phenotype of the CART cells we prepared was evaluated by detecting the CD3 / CD4 / CD8 and TCM / TEM of MSLN CART using flow cytometry.

[0253] Experimental instruments: automated cell counter (Invitrogen CountessⅡFL), microscope (Jiangnan XD-202), Pico17 desktop microcentrifuge (Thermo), flow cytometer (Navios, AZ46353)

[0254] Experimental reagents

[0255]

[0256] Experimental methods

[0257] 1) MSLN CART CD3 / CD4 / CD8 detection

[0258] Anti-human CD3 PE-Cy5 / CD4 PE / CD8 FITC Cocktail flow cytometry antibody. For details of the experimental process, please refer to the experimental operation method.

[0259] 2) MSLN CART TCM / TEM inspection

[0260] 2μL PE / Cy5 anti-human CD45RO, 2μL FITC anti-human CD197 and 2μL PE anti-human CD62L flow cytometry antibodies. For details of the experimental process, please refer to the experimental operation method.

[0261] Experimental Results

[0262] The results are shown in Tables 4-5 and Figure 4-5 As shown. The results showed that the CD3 positive ratio of CAR-T cells in the mock-T group and the nano-antibody mesothelin group (1444 and 1450) was high, about 85%-97%; the CD3 positive ratio of MSLN3[Scfv]CAR-T cells was slightly lower, about 75%-84%. The overall trend of the CD4 to CD8 ratio in CD3-positive cells in all groups was consistent, and the CD4 ratio was higher than the CD8 ratio. The overall trend of the Tcm and Tem ratio in all groups was consistent, and the Tcm ratio was higher than the Tem ratio. Compared with Mock-T cells, the CD4 / CD8 and Tcm / Tem of MSLN3[Scfv]CAR-T cells and MSLN3

[1444] CAR-T cells decreased to a certain extent ( Figure 4 , Figure 5 ).

[0263] Table 4. T cell populations of MSLN CAR-T detected on Day 13

[0264]

[0265]

[0266] Table 5. Clustering of memory T cells in Day 13 detection cells

[0267]

[0268] 1.5. Detection of MSLN CAR-T cytotoxicity in vitro

[0269] This example studies the in vitro pharmacodynamic properties of MSLN CAR-T with different sequences, and detects the secretion of cytokines after co-culture with mesothelin-positive tumor cells and their specific killing ability on target cells. In vitro killing experiments were performed using mesothelin-negative tumor cells and mesothelin-positive tumor cells to prove that the candidate sequence has the ability to specifically kill the mesothelin antigen.

[0270] Experimental samples

[0271] The sample information of AC-201908B, AC-201909A and AC-201910A shown in Table 2 and YP-201903A04, YP-201903A23 and YP-201903A24 obtained using the same method are shown in the following table.

[0272]

[0273]

[0274] The target cell information is shown in the following table:

[0275] Cell Type Surface MSLN+ Live rate H520 10.3％ 91％ AsPC-1 78.1％ 96％ H226 99.1％ 98％ SKOV3 75.4％ 96％

[0276] Experimental instruments: automated cell counter (Invitrogen CountessⅡFL YSB195), microscope (Jiangnan XD-202YSB126), biological safety cabinet, refrigerator (TCL), carbon dioxide incubator (Thermo), real-time cell analyzer (ACEA XCELLIgence RTCA TP, SB0475, supporting consumables: E-Plate 16, E-Plate 96), desktop centrifuge (small) (Thermo Fisher), desktop centrifuge (large) (Xiangyi)

[0277] Experimental reagents

[0278] Reagent name Manufacturer Specification Part Number AIM-V medium Gibco 1000mL A3021002 Fetal bovine serum (FBS) Gibco 500mL 10099141 IL-2 Shandong Quangang Pharmaceutical Co., Ltd. 2 million U / unit National Medicine Approval No. 20020004 Trypan Blue Stain (0.4%) Invitrogen 100mL 15250-061 DMEM cell culture medium CORNING 500mL / bottle 10-013-CER 0.25% trypsin digestion solution New Saimei 125mL C125C1 Human IL-2ValukineTM ELISA KIT NOVUS 96-well VAL110 Human TNF-αValukineTM ELISA KIT NOVUS 96-well VAL105 Human IFN-γValukineTM ELISA KIT NOVUS 96-well VAL104

[0279] Experimental methods

[0280] 1) Cell killing

[0281] (1) YP-201903A04, YP-201903A23 and YP-201903A24: target cells are H520, AsPC-1 and H226, and the effector-target ratio (E:T) is 1:1

[0282] (2) AC-201908B, AC-201909A and AC-201910A: Target cells: SKOV3, H226, effector-target ratio (E:T) = 1:1, 1:4, 1:16

[0283]

[0284] Cell index: Cell index reflects the growth status and cell number of tumor cells.

[0285] For details of the experimental process, please refer to the experimental operation method.

[0286] 2) Cytokine release detection

[0287] After incubation with target cells for 72 h, the 96-well plate was taken out and the cytokine release in the supernatant was detected by ELISA.

[0288] Experimental Results

[0289] The results are as follows Figure 6-8 As shown in Table 6, the results show that the CAR-T cells prepared by our transposase plasmid system have the function of specifically killing mesothelin-positive tumor cells ( Figure 6 , Figure 7 ). Co-culture with mesothelin-positive tumor cells can effectively stimulate MSLN3[Scfv]CAR-T cells and MSLN3

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

[0290] Table 6. Cytokine release after 72 h of co-incubation of mock-T and MSLN CAR-T with mesothelin-positive tumor cells

[0291]

[0292] The above results show that the MSLN CAR sequence derived from the nanoantibody has similar or better proliferation ability, MSLN CAR positivity rate, CD4+T / CD8+T clustering ratio, and memory T cell ratio in in vitro screening compared with the single-chain antibody sequence; it has the ability to specifically kill mesothelin-positive tumor cells.

[0293] Example 2, in vitro experiment of MSLN CAR-T cells with autocrine PD1 nanoantibodies

[0294] By constructing plasmids using the PD1 antibody sequence derived from nanoantibodies and the sequence of benchmark Keytruda, and transferring the genes into PBMCs from healthy subjects through a dual-plasmid electroporation system, we constructed non-viral vector aPD1MSLN CAR-T cells. By comparing the in vitro performance of these CAR-T cells, including proliferation ability, CAR positivity rate, antibody secretion level, and the blocking effect of secreted antibodies on immune checkpoint inhibitors, we verified the function of CAR-T cell autocrine antibodies.

[0295] 2.1. Plasmid construction of autocrine PD1 nanobody

[0296] 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 for double enzyme digestion (EcoR1 and BamH1). The remaining experimental instruments, experimental reagents, and experimental methods were the same as those in 1.1 of Example 1.

[0297] 2.2 Preparation of MSLN CAR-T cells with autocrine PD1 nanoantibodies

[0298] Mock-T and aPD1-MSLN CAR-T were prepared by electroporation, and cells and cell supernatant samples were obtained for detection and analysis.

[0299] The experimental instruments, reagents and methods are the same as those in 1.2 of Example 1. Among them, 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), which expresses MSLN CAR and PD1 nanoantibodies based on nanoantibodies. When single plasmid electroporation is performed, the plasmid concentration is 6 μg per sample; when double plasmid electroporation is performed, the concentration of each plasmid is 4 μg per sample.

[0300] Experimental Results

[0301]

[0302] 2.3. Detection of in vitro proliferation and positive rate of aPD1-MSLN CAR-T

[0303] Verify whether normal cell proliferation and a certain MSLN CAR positive rate can be achieved when MSLN CAR-T cells are able to auto-secrete PD1 (aPD1-MSLN CAR-T) under the dual plasmid system, and obtain cells and cell supernatant samples for detecting the secretion of PD1 antibodies by cells. The experimental instruments, reagents and methods are the same as those in 1.3 of Example 1.

[0304] Experimental Results

[0305] The results are shown in Tables 7-9 and Figure 9-11 shown.

[0306] Table 7, total number and proliferation times of cells D5-D13

[0307]

[0308] Table 8. Cell viability of cell culture D5-13

[0309]

[0310]

[0311] Table 9. Changes in CAR positive rate of 13MSLN cells at D8 of cell culture and the amount of CAR+ cells obtained

[0312]

[0313] 2.4 Cell phenotype detection

[0314] Purpose

[0315] The phenotype of the CART cells prepared by us was evaluated by detecting CD3 / CD4 / CD8 and TCM / TEM of the autocrine PD1 antibody MSLN CART by flow cytometry. The experimental instruments, reagents and methods were the same as those in 1.4 of Example 1.

[0316] Experimental Results

[0317] The results are shown in Tables 10-11 and Figure 12-13 (AC-201908A).

[0318] Table 10. T cell populations of MSLN CAR-T detected on Day 13

[0319]

[0320] Table 11. Clustering of memory T cells on day 13 in MSLN CAR-T

[0321]

[0322]

[0323] 2.5 Cell autocrine antibody detection

[0324] During the cell preparation process, the ELISA method is used to detect whether the cells have the ability to secrete antibodies before the end of preparation and the range of secreted antibody concentration.

[0325] Experimental instrument: Multifunctional microplate reader (TECAN Spark)

[0326] Experimental reagents:

[0327]

[0328] Experimental methods

[0329] Take 5×10 5 The cell supernatant samples of cells cultured for two days were tested by ELISA to detect the concentration of antibody secretion in the supernatant, and the concentration of the substance secreted by the cells in the supernatant was calculated. See the experimental operation method for details.

[0330] The calculation formula is: substance concentration = mass concentration / molecular weight kDa / 1000

[0331] In this experiment, the cell supernatant samples were set to 4-fold, 40-fold, and 400-fold dilutions.

[0332] Experimental Results

[0333] The results are shown in Table 12, which indicate that the mesothelin CAR-T cells that secrete the PD1 nanoantibody can successfully secrete the PD1 nanoantibody during the preparation process, and the secretion capacity of the PD1 nanoantibody tends to decrease over time.

[0334] Table 12. D11-13 antibody secretion concentration after cell preparation

[0335]

[0336] 2.6 Cell function detection

[0337] By using the antigen targeted by the CAR-T cells to give the prepared CAR-T cells a certain stimulation, detecting changes in cell phenotype and changes in antibody secretion, the responsiveness of CAR-T cells after being stimulated by specific antigens can be evaluated.

[0338] The experimental instruments, reagents and methods are the same as those in 1.4 of Example 1.

[0339] Experimental methods

[0340] After coating a 12-well cell culture plate with 5 μg / mL mesothelin antigen, 5×10^5 cells were added and cultured in 1 mL of culture system for 48 hours. The cells and cell supernatant were collected for flow cytometry and ELISA detection respectively. The ratio of Tcm (CD45RO+CCR7+CD62L+) and Tem (CD45RO+CCR7-CD62L-) and the secretion of PD1 nanoantibodies were analyzed according to the analysis methods of 2.4 and 2.5.

[0341] Experimental Results

[0342] The results are shown in Tables 13-14 and Figure 14-15 The results showed that after CAR-T cells were stimulated by antigens, the proportion of Tcm decreased and the proportion of Tem increased compared with before stimulation; after CAR-T cells were stimulated by antigens, the secretion of PD1 nanoantibodies increased significantly compared with before stimulation.

[0343] Table 13. Memory T cell population ratio in cells 48 hours after mesothelin antigen stimulation

[0344]

[0345] Table 14. Antibody concentration in cell supernatant after 48h of mesothelin antigen stimulation

[0346]

[0347]

[0348] 2.7. aPD1-MSLN CAR-T in vitro cytotoxicity assay

[0349] To verify the in vitro pharmacodynamic properties of MSLN CAR-T cells with autocrine PD1 antibodies, detect the secretion of cytokines after co-culture with mesothelin-positive and PDL1-high expressing tumor cells and their specific killing ability against target cells, and then evaluate CAR-T cells.

[0350] Effector cells

[0351]

[0352] Target cells

[0353] Cell Type Surface MSLN+ Surface PDL1 Live rate SKOV3-PDL1 72.3％ 99.9％ 96％ H226 98.3％ 37.7％ 96％

[0354] The experimental instruments, reagents and methods were the same as those in 1.5 of Example 1. The target cells selected were SKOV3-PDL1 and H226, and the effector-target ratio (E:T) was 1:1, 1:4, and 1:16.

[0355] In the cytokine release test, all experimental samples of IL-2 were uniformly diluted 5 times and 10 times before testing; all experimental samples of TNF-α were uniformly diluted 3 times before testing; all experimental samples of IFN-γ were uniformly diluted 10 times and 100 times before testing.

[0356] Experimental Results

[0357] The results are as follows Figure 16-18 As shown in Table 15, the results showed that autocrine PD1 antibody can significantly improve the killing efficiency of mesothelin CAR-T on H226 cells ( Fig.16 ), but had no significant effect on the killing efficiency of SKOV3-PDL1 cells ( Fig.17 ). This shows that the mesothelin-targeting CAR-T cells with autocrine PD1 nanoantibodies we prepared can effectively kill mesothelin-positive tumor cells, and can improve the killing effect of mesothelin CAR-T on certain types of mesothelin and PDL1 double-positive tumor cells. 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, compared with mesothelin cells that do not secrete PD1 antibodies, the secretion of IFN-γ and TNF-α by mesothelin CAR-T cells with autocrine PD1 antibodies was significantly increased after co-culture with tumor cells ( Fig.19 ).

[0358] Table 15. Cytokine release after 48h co-incubation of mock-T and MSLN CAR-T with mesothelin & PDL1-positive tumor cells

[0359]

[0360] Example 3, in vivo efficacy experiment of MSLN CAR-T cells secreting D1 nanoantibody

[0361] 3.1 Experimental animals and cells

[0362] 6-8 weeks NPSG severe combined immunodeficiency mice prkdc scid Iq null (Beijing Finok Biotechnology Co., Ltd.)

[0363] Tumor cells

[0364] Skov3-PDL1 (ATCC) that highly expresses PDL1

[0365] NCI-H226-PDL1 (ATCC) with high expression of PDL1

[0366] The process of constructing CAR-T cells with autocrine Fc-containing PD1 antibodies: The aPD1

[1182] nanoantibody sequence was constructed into the pCDNA3.4-IgG4 vector to obtain the pCDNA3.4-1182-Fc vector, which was then expressed using the ExpiCHOTM (Thermo Fisher) expression system. After one week of expression, the supernatant was collected for Protein A (GE) purification. The protein quality was then detected using Nanodrop, and the protein purity was detected using HPLC. The 1182-Fc coding sequence was obtained by double enzyme digestion from the pCDNA3.4-1182-FC vector and introduced into the pS338B backbone vector. The remaining experimental instruments, experimental reagents, and experimental methods were the same as in Example 2.

[0367] 3.2. Vaccination and Dosage Schedule

[0368] NPSG severe combined immunodeficient mice were subcutaneously inoculated with tumor cells.

[0369] Table 16, Vaccination schedule and grouping

[0370]

[0371] When the tumor volume reaches 100-150mm 3 The dosing schedule was as per Table 17.

[0372] Table 17, NPSG mouse dosing regimen and grouping

[0373]

[0374] In the drugs in Table 17, CD19-1182-VHH refers to a CD19-targeting CAR-T that secretes 1182-VHH. The amino acid sequence and coding sequence of CAR are shown in SEQ ID NO:9 and SEQ ID NO:10 in 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.

[0375] 3.3 Changes in tumor volume at high, medium, and low doses

[0376] H226 results are as follows Fig. 20 As shown, the autocrine PD1 antibody administration groups showed significant differences in efficacy at multiple administration doses. 0.05*10^6 dose group (1444VS1444-1182-VHH: P=0.0283, 1444VS 1444-1182-FC: P=0.0010), 0.5*10^6 dose group (1444VS1444-1182-VHH: P=0.1722, 1444VS1444-1182-FC: P=0.0104), 5*10^6 dose group (1444VS1444-1182-VHH: P=0.0051, 1444VS1444-1182-FC: P=0.0006).

[0377] Skov3 results are as follows Fig.21 As shown, the autocrine PD1 antibody administration groups showed significant differences in efficacy at multiple administration doses, including 0.05*10^6 dose group (1444VS1444-1182-VHH: P=0.7479, 1444VS1444-1182-FC: P=0.0757), 0.5*10^6 dose group (1444VS1444-1182-VHH: P=0.0002, 1444VS1444-1182-FC: P<0.0001).

[0378] 3.4. Changes in animal body weight at high, medium and low doses

[0379] H226 results are as follows Fig. 22 As shown, the weight change in the group receiving the autocrine PD1 antibody was smaller than that in the other groups, and was relatively stable in the long run. Except for the high-dose group, all groups remained stable or increased. Overall, the weight loss in the group receiving the autocrine PD1 antibody did not exceed 20%.

[0380] Skov3 results are as follows Fig.23 As shown, the weight change in the group receiving the autocrine PD1 antibody was smaller than that in the other groups, and was relatively stable in the long run. Except for the low-dose group, all groups remained stable or increased. Overall, the weight loss in the group receiving the autocrine PD1 antibody did not exceed 20%.

[0381] 3.5 Survival rate at high, medium and low doses

[0382] H226 results are as follows Fig.24 As shown, the survival period of the group treated with autocrine PD1 antibodies was significantly better than that of other groups and was relatively stable in the long run. The mortality rate increased slightly with the increase in the dosage.

[0383] Skov3 results are as follows Fig.25 As shown, the survival period of the group treated with autocrine PD1 antibodies was significantly better than that of other groups and was relatively stable in the long run. The mortality rate increased slightly with the increase in the dosage.

[0384] 3.6 Changes in PD1 antibody blood concentrations at high, medium, and low doses

[0385] H226 results are as follows Fig.26 As shown, in the group receiving autocrine PD1 antibody, the PD1 antibody blood concentration was positively correlated with the dosage, and the F C The blood concentration of PD1 antibody in the F segment was significantly higher than that in the non-F C PD1 antibody with F C The half-life of PD-1 antibodies in the body is longer.

[0386] Skov3 results are as follows Fig. 27 As shown, in the group receiving autocrine PD1 antibody, the PD1 antibody blood concentration was positively correlated with the dosage, and the F C The blood concentration of PD1 antibody in the F segment was significantly higher than that in the non-F C PD1 antibody with F C The half-life of PD-1 antibodies in the body is longer.

[0387] Example 4. High-dose drug safety of MSLN CAR-T cells with autocrine PD1 nanoantibodies

[0388] 4.1 Test materials

[0389] Experimental animals and cells: 6-8 week MHC-I KO B-NDG severe combined immunodeficiency mice prkdc scid Iq null(Biocytogen Biotech Co., Ltd.). MHC-I knockout can minimize the graft-versus-host disease (GVHD) caused by CAR-T transfusion, which is conducive to long-term observation of the safety of the drug.

[0390] Tumor cells: NCI-H226-PDL1 (ATCC) that highly expresses PDL1

[0391] Experimental process: Same as Example 3

[0392] 4.2. Vaccination and Dosage Schedule

[0393] Table 18. Vaccination schedule

[0394]

[0395] Table 19. Dosage regimen and grouping

[0396]

[0397] Among the drugs in Table 19, CD19-CAR-T refers to CAR-T targeting CD19, and the amino acid sequence and coding sequence of CAR are shown in SEQ ID NO:9 and SEQ ID NO:10 in CN109971712A, respectively. BMK-CAR-T refers to CAR-T whose antigen binding domain is YP218scFv, which differs from MSLN3

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

[0398] 4.3 Changes in Tumor Volume

[0399] Fig.28 The results showed that the autocrine PD1 antibody-treated group was able to completely regress the tumor at this dosage, and the efficacy was significantly different from that of the positive control drug BMK (BMK VS1444-1182-VHH: P < 0.0001, BMK VS1444-1182-FC: P < 0.0001).

[0400] 4.4 Weight Change

[0401] Fig.29 Autocrine band F C The weight change of the PD1 antibody-treated group was smaller than that of the other treatment groups and was relatively stable in the long run. After long-term observation for 100 days, most of the weight did not drop by 20%.

[0402] 4.5. Survival

[0403] Fig.30 The results showed that the survival time of the group treated with autocrine PD1 antibody was significantly better than that of other groups, and the trend of change was relatively stable in the long term. C The survival time of mice in the group treated with PD-1 antibodies exceeded 100 days.

[0404] 4.6 Changes in PD-1 Antibody Blood Concentration

[0405] Fig.31 The group of patients receiving autocrine PD1 antibody showed that the F C The blood concentration of PD1 antibody in the F segment was significantly higher than that in the non-F C PD1 antibody with F C The half-life of the PD-1 antibody in vivo is longer. After administration, as the tumor regresses, the antibody secretion gradually decreases.

[0406] 4.7 Changes in the number of CAR-T cells in peripheral blood

[0407] Fig.32 It shows that CAR-T cells reach a peak of proliferation after administration, and then there is a downward trend as the tumor disappears. The change trends of CD3+, CD8+ and CAR+ cells are almost the same. The CAR positive rate drops briefly after administration, and then remains basically stable. The fewer the number of cells per microliter of blood, the greater the detection error.

[0408] 4.8 Changes in peripheral blood cytokine concentrations

[0409] Fig.33 The results showed that after administration, interferon-γ and interleukin-6 were significantly secreted, among which interleukin-6 secretion was mainly concentrated in the negative control group and the BMK positive control group, while there was almost no secretion in the group receiving the autocrine PD1 antibody. Interferon-γ was significantly secreted in all groups, which was related to the killing function of T cells. No significant secretion was detected for the remaining cytokines, interleukin-2, interleukin-4, interleukin-10, and TNF, and the detection limit was about 10pg / ml. It can be considered undetectable close to this concentration.

[0410] 4.9 Clinical Observation of Experimental Animals

[0411] Table 20: Clinical manifestations of NCI-H226-PDL1 tumor-bearing mice at high doses

[0412]

[0413]

[0414] Clinical observation results showed that the negative control group, BMK positive control group and 1444-1182-VHH (antibody without F C The survival of mice in the three groups was slightly worse than that in 1444-1182-F C Administration group. 1444-1182-F C The survival time of animals in the drug-treated group was more than 100 days.

[0415] Example 5, efficacy, pharmacokinetics and safety verification test at commonly used dosages

[0416] 5.1 Test materials

[0417] Experimental animals and cells

[0418] Test mice: Same as Example 4

[0419] Tumor cells:

[0420] Skov3-PDL1 (ATCC) that highly expresses PDL1

[0421] NCI-H226-PDL1 (ATCC) with high expression of PDL1

[0422] Experimental process: Same as Example 3

[0423] The preparation process of BMK-1182CAR-T cells refers to the "CAR-T preparation operation steps". The process is briefly described as follows: fresh or frozen PBMCs are revived and electroporated using a Lonza 4D electroporator. The electroporation system is 100ul, the plasmid dosage is 4ugpNB338B-1444-CAR+4ug pS338B-1182 plasmid, and the electroporation program is EO-115. The passage process, detection time and detection indicators are consistent with other CAR-T cells.

[0424] 5.2. Vaccination and Dosage Schedule

[0425] Table 21. Vaccination schedule and grouping

[0426]

[0427] Table 22. Dosage regimen and grouping (NCI-H226-PDL1)

[0428]

[0429]

[0430] Among the drugs in Table 22, CD19-1182 is the same as the above-mentioned CD19-1182-VHH, Meso3CAR (BMK) is the same as the above-mentioned BMK-CAR-T, and the difference between Meso3CAR (BMK)-1182-FC and Meso3CAR (BMK) is that it also autosecretes 1182-FC.

[0431] Table 23. Dosage regimen and grouping (Skov3-PDL1)

[0432]

[0433] 5.3 Changes in tumor volume and body weight

[0434] Fig.34 It showed that both the positive control group and the experimental group could achieve complete tumor regression at this dosage.

[0435] 5.4 Changes in PD-1 Antibody Blood Concentration

[0436] The results are as follows Fig.45 The results showed that the autocrine PD1 antibody administration group secreted F C The drug metabolism characteristics of the PD1 antibodies of the two segments in the body are relatively similar. With the clearance and regression of the tumor, they all show a trend of first rising and then falling, and this has been verified in two different tumor-bearing mouse models. Moreover, without affecting the efficacy, the PD1 antibody has the lowest blood drug concentration, the lowest risk of systemic toxic side effects, and the highest safety.

[0437] 5.5 Changes in the number of CAR-T cells in peripheral blood

[0438] The results are as follows Fig.46 As shown in the figure, after administration, CAR-T cells showed a peak of proliferation, and then there was a downward trend as the tumor subsided. The change trends of CD3+, CD8+ and CAR+ cells were almost the same. The CAR positive rate dropped briefly after administration, and then remained basically stable. Compared with CD19 CAR-T targeting hematological tumors, the concentration of CAR-T targeting mesothelin in peripheral blood was significantly reduced, indicating that these CAR-Ts were mainly located in the tumor rather than in the peripheral blood, thereby achieving the purpose of precise targeted therapy.

[0439] Example 6, in vitro experiments of other MSLN CAR-T cells and MSLN CAR-T cells expressing autocrine PD1 nanoantibodies

[0440] In this embodiment, the expression sequence for expressing PB enzyme on the CAR plasmid used for CAR-T cells is removed, and the PB enzyme is added to the electroporation system in the form of mRNA and electroporated into T cells together with the plasmid for expression; the PD1 antibody secretory plasmid is changed: the promoter is replaced from the CMV promoter to the IFNγ-driven promoter.

[0441] Brief description of CAR-T preparation method: The electroporation system, electroporation procedure and subculture process are the same as the previous CAR-T preparation. The addition of plasmids and other components in the electroporation system: single plasmid 6ug + 20ug PBase mRNA + 200IU RNase inhibitor or dual plasmid 4ug CAR plasmid + 4ug secretory plasmid + 20ug PBase mRNA + 200IU RNase inhibitor.

[0442] The inventors constructed:

[0443] CAR-T cells containing mesothelin nanobody MSLN3

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

[1444] with autocrine PD1 nanobody 1194nla (SEQ ID NO: 131) (abbreviated as "1444+1194nla" or "1444-1194"), the CAR sequence of the above cells is shown in SEQ ID NO: 122; and

[0444] CAR-T cells containing mesothelin nanoantibody MSLN3

[2339] (SEQ ID NO: 103) (abbreviated as 2339), CAR-T cells containing mesothelin nanoantibody MSLN3

[2339] with autocrine PD1 nanoantibody 1194nla (abbreviated as "2339+1194nla" or "2339-1194"), the CAR sequence of the above cells is shown in SEQ ID NO: 121.

[0445] The results are as follows Figure 35-38 As shown:

[0446] Fig.35 The results showed that all CAR-T cells could proliferate effectively and had high viability (more than 90%) and high CAR positivity rate. The CD3 / 4 / 8 ratio was relatively low for Donor 04A, while the other donors had little difference, about 1:1. The differences among Tcm and Tem donors were relatively small.

[0447] Fig.36The results showed that the secretion level of anti-PD1 (1194nla) was low during the preparation process, between 0-150ng / ml; the secretion of 1194nla gradually decreased over time; there was no significant difference between the secretion of the 1444+1194nla group and the 2339+1194nla group. After 6h of stimulation, the secretion of 1194nla was low, below 100ng / ml; after 48h of stimulation, the secretion of 1194nla was significantly higher than that of 6h; after 48h of stimulation, the secretion of the 1444+1194nla group was slightly higher than that of the 2339+1194nla group. After 48h of antigen stimulation, the ability of CAR-T cells in each group to produce cytokines was significantly increased compared with that before stimulation. The ability of CAR-T in the 1444 group and the 2339 group to produce IL-2, IFNγ and TNFα was slightly different, all of which were higher than BMK (meso3CAR).

[0448] Fig.37 The results showed that both group 1444 and group 2339 could effectively kill H226-PDL1 cells; group 2339 had stronger killing ability against H226-PDL1 than group 1444; autocrine 1194nla had a certain improvement on the killing ability of MLSN CAR-T.

[0449] Fig.38 The results showed that both group 1444 and group 2339 could effectively kill SKOV3-PDL1 cells; group 1444 had stronger killing ability against SKOV3-PDL1 than group 2339; autocrine 1194nla had a certain improvement on the killing ability of MLSN CAR-T.

[0450] Example 7, in vivo dose-effect experiment of other MSLN CAR-T cells and MSLN CAR-T cells with autocrine PD1 nanoantibodies

[0451] Experimental Procedure

[0452] Severely immunodeficient NPSG mice (Phenom) were subcutaneously inoculated with 1E7 NCI-H226-PDL1 tumor cells. About 21 days after inoculation, the tumors grew to 100-120 mm 3 , grouping and intravenous CAR-T transfusion, the transfusion volume of each group is divided into 4 dose gradients (0.2E6, 1E6, 5E6, 20E6), and the efficacy, body weight and survival are observed. Peripheral blood is collected once a week to detect the proportion and number of CD3 in peripheral blood. The experimental scheme is as follows and Fig.39 shown.

[0453] Group Number of mice drug Dosage (10^6) 1 6 Vehicle -- 2 6 MOCK-T 0.2 3 6 2339-1194 0.2 4 6 1444-1194 0.2 5 6 MOCK-T 1 6 6 2339-1194 1 7 6 1444-1194 1 8 6 MOCK-T 5 9 6 2339-1194 5 10 6 1444-1194 5 11 6 MOCK-T 20 12 6 2339-1194 20 13 6 1444-1194 20

[0454] The experimental results are as follows Figure 40-43 shown.

[0455] Fig.40 The results showed that 2339-1194 and 1444-1194 could inhibit tumor growth at different doses (1E6, 5E6 and 20E6), with the inhibition rate exceeding 70%. 2339-1194 and 1444-1194 had comparable tumor inhibition effects.

[0456] Fig.41 The results showed that the body weight of mice was within 20% at different dosages, and the safety was good. There was no significant difference in body weight change and survival rate between 2339-1194 and 1444-1194.

[0457] Fig.42 The results showed that the number of peripheral blood CD3+T cells reached a peak at 14 days and dropped to a low point at 21 days, at which time the tumor had basically subsided.

[0458] Fig.43 The results showed that 14 days after CAR-T infusion, the proportion of CAR+ in peripheral blood was relatively stable (between 60% and 70%) at different dosages.

[0459] in conclusion

[0460] MSLN CAR-T cells that secrete PD1 antibodies can secrete PD1 antibodies and express chimeric antigen receptors that target mesothelin antigens. It has both the ability to secrete immune checkpoint antibodies and the specific killing ability to target specific antigens. It has a stronger ability to secrete cytokines than ordinary CAR-T cells and a higher killing rate. It can be used as a candidate drug to enter in vivo experiments for in vivo efficacy evaluation experiments.

Claims

1. A chimeric antigen receptor, wherein the antigen binding domain comprises a mesothelin binding molecule comprising an anti-mesothelin single domain antibody, wherein the anti-mesothelin single domain antibody comprises CDR1, CDR2 and CDR3, wherein: The CDR1 comprises the sequence shown in any one of SEQ ID NOs: 4-27, 73, and / or the CDR2 comprises the sequence shown in any one of SEQ ID NOs: 28-47, 74, and / or the CDR3 comprises the sequence shown in any one of SEQ ID NOs: 48-72, 75.

2. The chimeric antigen receptor according to claim 1, wherein The chimeric antigen receptor comprises a mesothelin binding molecule containing an anti-mesothelin single domain antibody, a hinge region, a transmembrane region, and an intracellular region.

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

4. The chimeric antigen receptor according to claim 2, wherein The chimeric antigen receptor also has one or more characteristics selected from the following: The chimeric antigen receptor further comprises a signal peptide, wherein 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 includes a CD8 hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region or an IgG4 Fc CH2CH3 hinge region, The transmembrane region includes a CD28 transmembrane region, a CD8 transmembrane region, a CD3ζ transmembrane region, a CD134 transmembrane region, a CD137 transmembrane region, an ICOS transmembrane region or a DAP10 transmembrane region, The intracellular region includes an intracellular co-stimulatory domain and / or an intracellular signaling domain. The intracellular co-stimulatory domain includes the intracellular domain of CD28, CD134 / OX40, CD137 / 4-1BB, lymphocyte-specific protein tyrosine kinase, inducible T cell co-stimulator (ICOS) or DNAX activation protein 10, The intracellular signaling domain includes a CD3ζ intracellular signaling domain or a FcεRIγ intracellular signaling domain.

5. A nucleic acid molecule comprising a sequence selected from the group consisting of: (1) a coding sequence of the chimeric antigen receptor according to any one of claims 1 to 4, and (2) The complementary sequence of (1), Preferably, the coding sequence is DNA or RNA.

6. A nucleic acid construct comprising the nucleic acid molecule according to claim 5, Preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

7. The nucleic acid construct according to claim 6, wherein The nucleic acid construct further comprises a coding sequence for a PD-1 binding molecule, Preferably, the coding sequence of the PD-1 binding molecule is DNA or RNA.

8. The nucleic acid construct according to claim 6, wherein The nucleic acid construct further comprises a coding sequence of a transposase, wherein the transposase is used to transpose the coding sequence of the chimeric antigen receptor and / or the PD-1 binding molecule. Preferably, the coding sequence of the transposase is DNA or RNA, Preferably, the transposase is PiggyBac transposase.

9. A cell, wherein: (1) (a) comprises and / or expresses a chimeric antigen receptor according to any one of claims 1 to 4, and optionally (b) comprises, expresses and / or secretes a PD-1 binding molecule, and / or (2) comprising the nucleic acid molecule according to claim 5 and / or the nucleic acid construct according to any one of claims 6 to 8, Preferably, the cell is a T cell.

10. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and: (1) (i) the chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally further comprising (ii) a transposase and / or its coding sequence, and / or (iii) a PD-1 binding molecule and / or its coding sequence, or (2) The nucleic acid molecule according to claim 5, the nucleic acid construct according to any one of claims 6 to 8, or the cell according to claim 9.

11. Use of a reagent in preparing activated immune cells, the reagent comprising: (1) (i) the chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally further comprising (ii) a transposase and / or its coding sequence, and / or (iii) a PD-1 binding molecule and / or its coding sequence, or (2) The nucleic acid molecule according to claim 5 or the nucleic acid construct according to any one of claims 6 to 8.

12. Use of a reagent in the preparation of a drug for treating cancer, the reagent comprising: (1) (i) the chimeric antigen receptor and / or its coding sequence according to any one of claims 1 to 4, optionally further comprising (ii) a transposase and / or its coding sequence, and / or (iii) a PD-1 binding molecule and / or its coding sequence, or (2) the nucleic acid molecule according to claim 5, the nucleic acid construct according to any one of claims 6 to 8, or the cell according to claim 9, in, The cancer is selected from the group consisting of mesothelioma, pancreatic cancer, ovarian cancer, lung cancer and gastric cancer, Preferably, the cancer is lung adenocarcinoma.

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