Engineered immune cells that specifically target mesothelin and uses thereof

Abstract

Disclosed herein are isolated nucleic acid molecules comprising an antibody that specifically recognizes human mesothelin, a polynucleotide encoding a chimeric antigen receptor (CAR) comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; a polynucleotide encoding IL-7; and a polynucleotide encoding CCL19. Also disclosed herein are vectors, modified immune cells, and pharmaceutical compositions comprising the nucleic acid molecules, and methods of use.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 63 / 227,116, filed July 29, 2021, which is incorporated by reference herein in its entirety.

[0002] The present invention relates to immune cells that express a cell surface molecule that specifically recognizes human mesothelin, interleukin 7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19), pharmaceutical compositions comprising the immune cells, expression vectors comprising polynucleotides encoding a cell surface molecule that specifically recognizes mesothelin, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19, methods of use, and a method for producing immune cells that express a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19 (comprising introducing into immune cells a polynucleotide encoding a cell surface molecule that specifically recognizes human mesothelin, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19). [Background technology]

[0003] Malignant tumors are diseases that affect many people around the world, and are generally widely treated with chemotherapy, radiation therapy, or surgery. However, there have been various problems, such as the occurrence of adverse reactions, loss of some functions, and recurrence or metastasis that cannot be treated. As such, in recent years, immune cell therapy has been developed to maintain a higher quality of life (QOL) for patients. Immune cell therapy includes collecting immune cells from a patient, performing a procedure to enhance the immune function of the collected immune cells, amplifying the cells, and re-administering the cells to the patient. For example, immune cell therapy may include collecting T cells from a patient, introducing a nucleic acid encoding a chimeric antigen receptor (constitutive androstane receptor: hereinafter also referred to as "CAR") into the T cells, and re-administering the T cells to the patient. Although early success in blood cancers has been observed with CAR-T therapy, life-threatening toxicity and a lack of substantial efficacy in the treatment of solid tumors have also been observed. As such, improved CAR-T therapy is needed. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2016 / 056228 [Patent Document 2] WO2019 / 124468 [Patent Document 3] WO2013 / 063419 [Non-patent literature]

[0005] [Non-Patent Document 1] Adachi, et al., “IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor,” Nature Biotech, 36(4):346-353, 2018. Summary of the Invention

[0006] Objective technical problem to be solved by the present invention Many challenges exist with immunotherapy using T cells, such as poor transport to solid tumors, high toxicity to normal tissues, inability to overcome the immunosuppressive tumor microenvironment, and poor activation of endogenous immune responses.In addition, immune cells modified to express CAR that specifically recognizes mesothelin have been shown to show minimal therapeutic efficacy.Therefore, the objective technical challenge to be solved is to provide modified immune cells that are optimized to target mesothelin-expressing cancers.

[0007] Means of solving objective technical problems The inventors have discovered that immune cells engineered to express a CAR that specifically recognizes mesothelin, IL-7, and CCL19 can improve the therapeutic efficacy of immunotherapy and improve survival rates.

[0008] In certain embodiments, the invention includes an isolated nucleic acid molecule comprising an antibody that specifically recognizes human mesothelin, a polynucleotide encoding a chimeric antigen receptor (CAR) comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; a polynucleotide encoding IL-7; and a polynucleotide encoding CCL19. In some embodiments, the IL-7 is human IL-7. In some embodiments, the CCL19 is human CCL19. In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity determining regions (CDRs) comprising SEQ ID NOs: 1-3, and the VL comprises three CDRs comprising SEQ ID NOs: 4-6. In some embodiments, the VH comprises SEQ ID NO: 7, and the VL comprises SEQ ID NO: 8. In some embodiments, the antibody comprises a single chain variable fragment (scFv) format. In some embodiments, the antibody comprises SEQ ID NO: 9. In some embodiments, the 4-1BB intracellular region comprises SEQ ID NO: 13. In some embodiments, the CD3 ζ intracellular region comprises SEQ ID NO: 14. In some embodiments, the 4-1BB intracellular region is upstream of the CD3 ζ intracellular region in the isolated nucleic acid molecule. In some embodiments, the CD8 hinge region comprises SEQ ID NO: 11. In some embodiments, the CD8 transmembrane region comprises SEQ ID NO: 12. In some embodiments, the nucleic acid further comprises a peptide linker of 3 to 10 amino acid residues in length linking the antibody and the CD8 hinge region. In some embodiments, the peptide linker comprises AAA. In some embodiments, the isolated nucleic acid molecule further comprises a signaling peptide. In some embodiments, the signaling peptide is located upstream of the antibody that specifically recognizes human mesothelin in the isolated nucleic acid molecule. In some embodiments, the signaling peptide comprises SEQ ID NO: 15. In some embodiments, the polynucleotide encoding IL-7 and the polynucleotide encoding CCL19 are independently transcribed under a promoter that comprises a polynucleotide encoding a self-cleaving 2A peptide (2A peptide). In some embodiments, the 2A peptide is P2A and optionally comprises ATNFSLLKQAGDVEENPGP.In some embodiments, a peptide linker is further added to the N-terminus of the 2A peptide, and the peptide linker comprises GSG. In some embodiments, the IL-7 comprises SEQ ID NO: 18. In some embodiments, the CCL19 comprises SEQ ID NO: 19. In some embodiments, the polynucleotide encoding the CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 are arranged in the nucleic acid molecule from the 5' end to the 3' end as follows: polynucleotide encoding the CAR-polynucleotide encoding IL-7-polynucleotide encoding CCL19. In some embodiments, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 16. In some embodiments, the isolated nucleic acid molecule comprises SEQ ID NO: 17. In some embodiments, the isolated nucleic acid molecule comprises SEQ ID NO: 25.

[0009] In certain embodiments, the invention includes a vector comprising a nucleic acid molecule described herein. In some embodiments, the vector is a viral vector, optionally an expression vector. In some embodiments, the viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is a gamma retroviral vector. In some embodiments, the viral vector is a pSFG vector, a pMSGV vector, or a pMSCV vector. In some embodiments, the vector is a plasmid.

[0010] In certain embodiments, the invention includes immune cells derived from or isolated from a mammal, which comprise a nucleic acid molecule described herein or a vector described herein. In some embodiments, the invention includes immune cells derived from or isolated from a mammal, which express a) an antibody that specifically recognizes human mesothelin, a chimeric antigen receptor (CAR) comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3 zeta intracellular region, b) IL-7, and c) CCL19. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a B cell, an antigen presenting cell, or a granulocyte, optionally a T cell or an NK cell.

[0011] In certain embodiments, the invention includes pharmaceutical compositions comprising the immune cells described herein and a pharma- ceutically acceptable excipient.

[0012] In certain embodiments, the invention includes a method of treating a mesothelin-expressing cancer, the method comprising administering an immune cell described herein or a pharmaceutical composition described herein to a subject in need thereof. In some embodiments, the mesothelin-expressing cancer is a solid tumor, optionally including mesothelioma, colorectal cancer, pancreatic cancer, thymic cancer, bile duct cancer, lung cancer, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, spleen cancer, tracheal cancer, bronchial cancer, gastric cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, and bone cancer. In some embodiments, the mesothelin-expressing cancer is a hematopoietic cancer. In some embodiments, the mesothelin-expressing cancer is a sarcoma, optionally including chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma. In some embodiments, the mesothelin-expressing cancer is a metastatic cancer. In some embodiments, the mesothelin-expressing cancer is a recurrent or refractory cancer. In some embodiments, the method further comprises administering an additional therapeutic agent or an additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof. In some embodiments, the additional therapeutic regimen comprises a first line therapy. In some embodiments, the additional therapeutic regimen comprises surgery. In some embodiments, the immune cells or pharmaceutical composition and the additional therapeutic agent are administered simultaneously. In some embodiments, the immune cells or pharmaceutical composition and the additional therapeutic agent are administered sequentially. In some embodiments, the immune cells or pharmaceutical composition are administered to the subject prior to administration of the additional therapeutic agent. In some embodiments, the immune cells or pharmaceutical composition are administered to the subject after administration of the additional therapeutic agent. In some embodiments, the subject is a human.

[0013] In certain embodiments, the invention includes a method of reducing tumor cell proliferation comprising contacting a tumor cell with an immune cell described herein, thereby reducing tumor cell proliferation. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

[0014] In certain embodiments, the invention includes methods for generating immune cells that express cell surface molecules that specifically recognize human mesothelin, IL-7, and CCL19, to induce expression of cell surface molecules that specifically recognize human mesothelin, IL-7, and CCL19 by the immune cells, the methods comprising introducing into the immune cells a nucleic acid molecule described herein or a vector described herein. In some embodiments, the immune cells are T cells, natural killer (NK) cells, B cells, antigen-presenting cells, or granulocytes, optionally T cells or NK cells.

[0015] In certain embodiments, the invention includes a kit comprising a nucleic acid molecule described herein; a vector described herein, an immune cell described herein, or a pharmaceutical composition described herein, and instructions for use.

[0016] Effect of the Invention The immune cells of the present invention have cytotoxic activity against cancer cells expressing mesothelin (e.g., human mesothelin) and can suppress the formation of tumors expressing mesothelin (e.g., human mesothelin). The immune cells of the present invention also have the effect of suppressing the recurrence of cancer cells. The immune cells of the present invention also have an excellent safety profile. [Brief description of the drawings]

[0017] [Figure 1A] FIG. 1 shows a diagrammatic representation of an exemplary vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19. [Figure 1B] A diagrammatic representation of engineered immune cells expressing a CAR that specifically recognizes mesothelin, IL-7, and CCL19 is shown. [Diagram 2] 1 shows in vitro killing of MSLN-positive tumor cells by engineered immune cells as described herein. [Diagram 3]Showing in vivo efficacy in the Capan-2 xenograft mouse model using engineered immune cells expressing exemplary second and third generation CAR-T systems. [Figure 4A] 1 shows histopathological examination of Capan-2 xenograft tumor tissue from mice treated with an exemplary second generation CAR-T system compared to non-transduced (UTD) T cells. [Figure 4B] 1 shows the in vivo efficacy of an exemplary second generation CAR-T system using the Capan-2 xenograft mouse model. [Figure 5A] FIG. 1 shows bioluminescence imaging (BLI) of tumor cell location in a SKOV3-luc xenograft mouse model in the presence of modified immune cells as described herein. [Figure 5B] 1 shows the in vivo efficacy of an exemplary second generation CAR-T system using the SKOV3-luc BLI model. [Figure 6A] 1 shows the weight change in a Capan-2 xenograft mouse model administered an exemplary second generation CAR-T system compared to non-transduced (UTD) T cells. [Figure 6B] 1 shows the weight change of non-tumor-bearing mice administered an exemplary second generation CAR-T system compared to untransduced (UTD) T cells. [Figure 7A] Shown are histopathology images of lung tissue from a mouse model administered untransduced (UTD) T cells. UTD cells had minimal putative CAR-T infiltration / inflammation in the lung and spleen. [Figure 7B] Shown are histopathological images of lung tissue from a mouse model administered CAR#364 (second 28-28z_7x19). A higher incidence of putative CAR-T infiltration / inflammation in the lungs, spleen, and liver was observed in animals administered UTD cells compared to animals administered UTD cells. [Figure 7C] Shown are histopathological images of lung tissue from a mouse model administered CAR#365 (second 8-BBz_7x19). Animals were observed to have a low incidence of pulmonary mononuclear infiltrates or mixed cell inflammation, and a high incidence of putative CAR-T cells engrafting in the spleen and bone marrow. [Figure 8A] 1 shows flow cytometry analysis of administered T cells in the blood of Capan-2 xenografted mice administered an exemplary second generation CAR-T system. [Figure 8B] 1 shows flow cytometry analysis of administered T cells in tumors of Capan-2 xenografted mice administered an exemplary second generation CAR-T system. [Figure 8C] 1 shows flow cytometry analysis of administered T cells in the spleens of Capan-2 xenografted mice administered an exemplary second generation CAR-T system. [Figure 9] 1 shows human IFN-γ levels from co-culture supernatants of Capan-2 tumor cells and an exemplary second generation CAR-T system pre-incubated with soluble hMSLN. [Figure 10A] Components of the third 8-28BBz_7x19 CAR-T (CAR#348) and the second 8-BBz_7x19 CAR-T (CAR#365) are shown. [Figure 10B] Shows the in vivo efficacy of the third 8-28BBz_7x19 CAR-T (CAR#348) and the second 8-BBz_7x19 CAR-T (CAR#365) using a HepG2-RedFluc xenograft model. [Figure 10C] Shows weight change in a HepG2-RedFluc xenograft mouse model harboring an 8-28BBz_7x19 CAR-T (CAR#348) and a second 8-BBz_7x19 CAR-T (CAR#365). [Figure 11] Shows bioluminescence imaging (BLI) of tumor cell location in a HepG2-RedFluc xenograft mouse model in the presence of a third 8-28BBz_7x19 CAR-T (CAR#348) and a second 8-BBz_7x19 CAR-T (CAR#365). [Figure 12A] FIG. 1 shows the in vivo efficacy of the PBS control in HepG2-RedFluc xenografted mice Group 1 (G1). [Figure 12B]Shows the in vivo efficacy of equivalent total T cell numbers of control untransduced (UTD) at a dose of 3M in HepG2-RedFluc xenografted mice group 2 (G2). [Figure 12C] Shows the in vivo efficacy of the second 8-BBz_7x19 CAR-T (CAR#365) at a dose of 0.3M in HepG2-RedFluc xenografted mice group 3 (G3). [Figure 12D] Shows the in vivo efficacy of the second 8-BBz_7x19 CAR-T (CAR#365) at a dose of 1M in HepG2-RedFluc xenografted mice group 3 (G4). [Figure 12E] Shows the in vivo efficacy of the second 8-BBz_7x19 CAR-T (CAR#365) at a dose of 3M in HepG2-RedFluc xenografted mice group 3 (G5). [Figure 12F] Shows the in vivo efficacy of the third 8-28BBz_7x19 CAR-T (CAR#348) at a dose of 0.3M in HepG2-RedFluc xenografted mice group 3 (G6). [Figure 12G] Shows the in vivo efficacy of the third 8-28BBz_7x19 CAR-T (CAR#348) at a dose of 1M in HepG2-RedFluc xenografted mice group 3 (G7). [Figure 12H] Shows the in vivo efficacy of the third 8-28BBz_7x19 CAR-T (CAR#348) at a dose of 3M in HepG2-RedFluc xenografted mice group 3 (G8). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Engineered immune cells In certain embodiments, disclosed herein are engineered immune cells that express engineered cell surface molecules that specifically bind mesothelin, interleukin 7 (IL-7), and chemokine (CC motif) ligand 19 (CCL19). In some embodiments, the engineered cell surface molecule comprises a chimeric antigen receptor (CAR) that specifically recognizes mesothelin, or a T cell receptor (TCR) that specifically binds mesothelin.

[0019] In some embodiments, the engineered immune cells comprise an exogenous nucleic acid encoding an engineered cell surface molecule, an exogenous nucleic acid encoding IL-7, and an exogenous nucleic acid encoding CCL19, hi some embodiments, the engineered immune cells express a surface molecule that specifically recognizes mesothelin, IL-7, and CCL19.

[0020] Mesothelin (MSLN) is a glycosylphosphatidylinositol (GPI)-anchored protein bound to the cell surface, and its normal expression is restricted to mesothelial cells derived from, for example, the pleura, pericardium, peritoneum, vagina, ovary, or fallopian tube. However, MSLN has also been shown to be overexpressed in a large number of cancers, such as malignant mesothelioma, ovarian cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), pancreatic cancer, lung cancer, gastric cancer, endometrial cancer, cervical cancer, biliary tract cancer, uterine serous carcinoma, cholangiocarcinoma, and childhood acute myeloid leukemia. Furthermore, increased MSLN expression is associated with poor prognosis in patients with TNBC, ovarian cancer, lung adenocarcinoma, cholangiocarcinoma, and pancreatic adenocarcinoma.

[0021] The physiological and biological functions of MSLN have not been fully elucidated. However, MSLN has been shown to be involved in several mechanisms of cancer development. For example, in epithelial ovarian cancer, patients who showed higher levels of MSLN mRNA expression in surgically removed ovarian cancer tissues showed resistance to platinum and cyclophosphamide chemotherapy compared with chemosensitive patients who showed lower MSLN levels (Tang, et al., “The role of mesothelin in tumor progression and targeted therapy,” Anticancer Agents Med Chem. 13(2):276-280 (2013)). MSLN has also been found to bind with high affinity to the surface mucin MUC16 (or CA125), and this binding has been suggested to mediate adhesion of ovarian cancer cells to mesothelial cells and promote metastasis (Rump, et al., “Binding of ovarian cancer antigen CA125 / MUC16 to mesothelin mediates cell adhesion,” J Biol Chem 279(10):9190-9198, 2004; Gubbels, et al., “Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors,” Mol Cancer 5(1):50, 2006). Furthermore, MSLN has been shown to be involved in tumor progression, cell survival, and proliferation in pancreatic cancer both in vitro and in vivo (Li, et al., “Mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer,” Mol Cancer Ther. 7(2):286-296, 2008).

[0022] Chimeric antigen receptors (CARs) A. Anti-mesothelin antibodies In some embodiments, the engineered cell surface molecule comprises a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes mesothelin, hi some embodiments, the antibody specifically recognizes mammalian mesothelin, e.g., rodent mesothelin, non-human primate mesothelin, or human mesothelin.

[0023] Human mesothelin, a 40 kDa protein, is encoded by the MSLN gene. Sequence information of human mesothelin can be appropriately obtained by searching known literature or databases, such as NCBI (www.ncbi.nlm.nih.gov / guide / ). Examples of amino acid sequence information of human mesothelin include GenBank accession numbers NP_037536.2, AAV87530.1, and their isoforms.

[0024] In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising or consisting of CDRH1 set forth in SEQ ID NO: 1, CDRH2 set forth in SEQ ID NO: 2, and CDRH3 set forth in SEQ ID NO: 3; and a light chain variable region (VL) comprising or consisting of CDRL1 set forth in SEQ ID NO: 4, CDRL2 set forth in SEQ ID NO: 5, and CDRL3 set forth in SEQ ID NO: 6. See Table 1.

[0025] [Table 1]

[0026] In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 80% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 80% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 85% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 85% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 90% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 90% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 95% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 95% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 96% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 96% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 97% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 97% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 98% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 98% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence having about 99% sequence identity to SEQ ID NO:7; and a light chain variable region (VL) comprising a sequence having about 99% sequence identity to SEQ ID NO:8.The anti-mesothelin antibody may comprise a heavy chain variable region (VH) comprising SEQ ID NO: 7 and a light chain variable region (VL) comprising SEQ ID NO: 8. The anti-mesothelin antibody may comprise a heavy chain variable region (VH) consisting of SEQ ID NO: 7 and a light chain variable region (VL) consisting of SEQ ID NO: 8.

[0027] In some embodiments, one or more residues in a framework region are modified in an anti-mesothelin antibody to generate 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with either the VH or VL region. The term "framework region" refers to the region of an antibody excluding the complementarity determining regions (CDRs). In some embodiments, an anti-mesothelin antibody contains one or more modifications in a framework region and has a sequence that includes 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:7. In some embodiments, an anti-mesothelin antibody contains one or more modifications in a framework region and has a sequence that includes 85% sequence identity with SEQ ID NO:7. In some embodiments, an anti-mesothelin antibody contains one or more modifications in a framework region and has a sequence that includes 90% sequence identity with SEQ ID NO:7. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 95% sequence identity to SEQ ID NO:7. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 96% sequence identity to SEQ ID NO:7. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 97% sequence identity to SEQ ID NO:7. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 98% sequence identity to SEQ ID NO:7. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 99% sequence identity to SEQ ID NO:7. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody contains one or more modifications in the framework regions and has a sequence that comprises 85% sequence identity to SEQ ID NO: 8. In some embodiments, the anti-mesothelin antibody contains one or more modifications in the framework regions and has a sequence that comprises 90% sequence identity to SEQ ID NO: 8.In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 95% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 96% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 97% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 98% sequence identity to SEQ ID NO:8. In some embodiments, the anti-mesothelin antibody comprises one or more modifications in the framework regions and has a sequence that comprises 99% sequence identity to SEQ ID NO:8.

[0028] In some embodiments, the anti-mesothelin antibody comprises a single chain variable fragment (scFv) format. In some embodiments, the anti-mesothelin scFv antibody comprises a VH comprising or consisting of a CDRH1 set forth in SEQ ID NO:1; a CDRH2 set forth in SEQ ID NO:2; and a CDRH3 set forth in SEQ ID NO:3; and a VL comprising or consisting of a CDRL1 set forth in SEQ ID NO:4, a CDRL2 set forth in SEQ ID NO:5, and a CDRL3 set forth in SEQ ID NO:6. In some embodiments, the anti-mesothelin scFv antibody comprises a VH comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:7; and a VL comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:8.

[0029] In some embodiments, the VH and VL of the anti-mesothelin scFv antibody are linked via a peptide linker. The peptide linker can contain three or more amino acid residues, for example, about 3 to about 30, about 3 to about 20, 3 to about 10, about 5 to about 30, about 5 to about 20, or about 5 to about 10. The peptide linker can contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid residues.

[0030] The peptide linker may comprise multiple polyalanines, polyglycines, or a mixture of alanine and glycine residues. The peptide linker may comprise a (Gly4Ser)n linker, where n is an integer between 1 and 10, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, more preferably 2, 3, 4, or 5. In some embodiments, the peptide linker comprises GGGGSGGGSGGGGGS (SEQ ID NO: 10). In some examples, the peptide linker comprises SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 20). In some cases, the peptide linker comprises SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 21). In some examples, the peptide linker comprises GSGGGGSGGGGSGGGGGS (SEQ ID NO: 22).

[0031] In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS (SEQ ID NO: 9). In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 9. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 90% sequence identity to SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 95% sequence identity to SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 96% sequence identity to SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 97% sequence identity to SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 98% sequence identity to SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence that comprises about 99% sequence identity to SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody comprises SEQ ID NO:9. In some embodiments, the anti-mesothelin scFv antibody consists of SEQ ID NO:9.

[0032] B. Signaling Peptides In some embodiments, the chimeric antigen receptor (CAR) disclosed herein comprises a signaling peptide (e.g., as a leader sequence). The signaling peptide may localize the CAR to the surface of a cell. The signaling peptide may comprise a polypeptide of an immunoglobulin heavy chain, an immunoglobulin light chain, CD8, a T cell receptor alpha and beta chain, CD3zeta, CD28, CD3E, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or a GITR-derived signal peptide (leader sequence).

[0033] In some embodiments, the signaling peptide comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MDWTWRILFLVAAATGAHS (SEQ ID NO: 15). In some embodiments, the signaling peptide comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises a sequence that comprises about 90% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises a sequence that comprises about 95% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises a sequence that comprises about 96% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises a sequence that comprises about 97% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises a sequence that comprises about 98% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises a sequence that comprises about 99% sequence identity to SEQ ID NO: 15. In some embodiments, the signaling peptide comprises SEQ ID NO: 15. In some embodiments, the signaling peptide consists of SEQ ID NO:15.

[0034] C. Transmembrane region In some embodiments, the anti-mesothelin antibody is linked to one or more transmembrane domains and an intracellular signaling domain. The transmembrane domains may be derived from either natural or synthetic sources. Exemplary transmembrane domains may include transmembrane domain polypeptides derived from CD8, T cell receptor alpha and beta chains, CD3zeta, CD28, CD3E (CD3 epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, and GITR. In some embodiments, the transmembrane domain comprises a CD8 transmembrane domain (e.g., a human CD8 transmembrane domain).

[0035] In some embodiments, the transmembrane region comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12). In some embodiments, the transmembrane region comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises a sequence that comprises about 90% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises a sequence that comprises about 95% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises a sequence that comprises about 96% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises a sequence that comprises about 97% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises a sequence that comprises about 98% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises a sequence that comprises about 99% sequence identity to SEQ ID NO: 12. In some embodiments, the transmembrane region comprises SEQ ID NO: 12. In some embodiments, the transmembrane region consists of SEQ ID NO:12.

[0036] In some embodiments, the transmembrane region comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IYIWAPLAGTCGVLLLSLVITLYCN (SEQ ID NO:28). In some embodiments, the transmembrane region comprises a sequence that comprises about 85% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises a sequence that comprises about 90% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises a sequence that comprises about 95% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises a sequence that comprises about 96% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises a sequence that comprises about 97% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises a sequence that comprises about 98% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises a sequence that comprises about 99% sequence identity to SEQ ID NO:28. In some embodiments, the transmembrane region comprises SEQ ID NO: 28. In some embodiments, the transmembrane region consists of SEQ ID NO:28.

[0037] D. Extracellular hinge region An extracellular hinge region comprising or consisting of any oligopeptide or polypeptide may be located between the cell surface molecule that recognizes mesothelin and the transmembrane region. Examples of lengths of the extracellular hinge region may include 1 to 100 amino acid residues, preferably 10 to 70, 10 to 50, or 10 to 30 amino acid residues. Exemplary extracellular hinge regions may include hinge regions derived from CD8, CD28, and CD4, as well as immunoglobulin hinge regions. In some embodiments, the hinge region comprises the hinge region of human CD8.

[0038] In some embodiments, the extracellular hinge region is a CD8 hinge region comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 11). In some embodiments, the CD8 hinge region comprises a sequence having about 85% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having about 90% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having about 95% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having about 96% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having about 97% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having about 98% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having about 99% sequence identity to SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises SEQ ID NO: 11. In some embodiments, the CD8 hinge region consists of SEQ ID NO: 11. In some embodiments, the CD8 hinge region comprises a sequence having PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 29). In some embodiments, the CD8 hinge region comprises a sequence having about 85% sequence identity to SEQ ID NO: 29. In some embodiments, the CD8 hinge region comprises a sequence having about 90% sequence identity to SEQ ID NO: 29. In some embodiments, the CD8 hinge region comprises a sequence having about 95% sequence identity to SEQ ID NO: 29. In some embodiments, the CD8 hinge region comprises a sequence having about 96% sequence identity to SEQ ID NO: 29. In some embodiments, the CD8 hinge region comprises a sequence having about 97% sequence identity to SEQ ID NO: 29. In some embodiments, the CD8 hinge region comprises a sequence having about 98% sequence identity to SEQ ID NO: 29. In some embodiments, the CD8 hinge region comprises a sequence having about 99% sequence identity to SEQ ID NO: 29.In some embodiments, the CD8 hinge region comprises SEQ ID NO: 29. In some embodiments, the CD8 hinge region consists of SEQ ID NO: 29.

[0039] In some embodiments, the anti-mesothelin scFv antibody is linked to the hinge region via a peptide linker. The peptide linker can contain three or more amino acid residues, for example, about 3 to about 30, about 3 to about 20, 3 to about 10, about 5 to about 30, about 5 to about 20, or about 5 to about 10. The peptide linker can contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid residues.

[0040] The peptide linker may comprise multiple polyalanines, polyglycines, or mixtures of alanine and glycine residues, or mixtures of either alanine or glycine with one or more additional amino acids. In some embodiments, the peptide linker comprises AlaAlaAla ("AAA"). In some embodiments, the peptide linker is a triple alanine linker or AlaAlaAla ("AAA"). In some embodiments, the peptide linker comprises ArgAlaAlaAla ("RAAA") (SEQ ID NO: 30). In some embodiments, the peptide linker is ArgAlaAlaAla ("RAAA") (SEQ ID NO: 30).

[0041] In some embodiments, the anti-mesothelin scFv antibody is attached to the hinge region without a linker.

[0042] E. Immune cell activation signaling region In some embodiments, the CAR comprises one or more intracellular signaling regions. The intracellular signaling region may comprise a region capable of transmitting a signal into the cell when a cell surface molecule recognizes mesothelin. The intracellular signaling region may comprise at least one or more members selected from the intracellular regions of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3zeta, or Fc receptor-associated gamma chain polypeptides. In some embodiments, the intracellular signaling region comprises a CD28 intracellular region polypeptide, a 4-1BB intracellular region polypeptide, a CD3 intracellular region polypeptide, or a combination thereof.

[0043] In some embodiments, the CAR comprises a 4-1BB intracellular region. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 13). In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 90% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 95% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 96% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 97% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 98% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises a sequence that comprises about 99% sequence identity to SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region comprises SEQ ID NO: 13. In some embodiments, the 4-1BB intracellular region consists of SEQ ID NO: 13.

[0044] In some embodiments, the CAR further comprises a CD3 zeta intracellular region. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 14). In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 90% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 95% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 96% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 97% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 98% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises a sequence that comprises about 99% sequence identity to SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region comprises SEQ ID NO: 14. In some embodiments, the CD3 zeta intracellular region consists of SEQ ID NO: 14.

[0045] IL-7 and CCL19 Interleukin 7 (IL-7) is involved in the differentiation of multipotent (pluripotent) hematopoietic stem cells into lymphoid progenitors and in the proliferation of lymphoid cells (such as B cells, T cells, and NK cells). IL-7 is produced in non-hematopoietic cells such as bone marrow, thymus, or stromal cells of lymphoid organs or tissues. In the setting of cancer, administration of IL-7 increases the expression of CD8 + and CD4 + Transient disruption of both T cell homeostasis and CD4 + CD25 + Foxp3 + This shows that the proportion of T regulatory cells is decreased.

[0046] In some embodiments, the immune cells described herein express IL-7. In some embodiments, the IL-7 comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH (SEQ ID NO: 18). In some embodiments, the IL-7 comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 18. In some embodiments, the IL-7 comprises a sequence that comprises about 90% sequence identity to SEQ ID NO: 18. In some embodiments, IL-7 comprises a sequence that comprises about 95% sequence identity to SEQ ID NO: 18. In some embodiments, IL-7 comprises a sequence that comprises about 96% sequence identity to SEQ ID NO: 18. In some embodiments, IL-7 comprises a sequence that comprises about 97% sequence identity to SEQ ID NO: 18. In some embodiments, IL-7 comprises a sequence that comprises about 98% sequence identity to SEQ ID NO: 18. In some embodiments, IL-7 comprises a sequence that comprises about 99% sequence identity to SEQ ID NO: 18. In some embodiments, IL-7 comprises SEQ ID NO: 18. In some embodiments, IL-7 consists of SEQ ID NO: 18.

[0047] Chemokine (CC motif) ligand 19 (CCL19), also known as EB11 ligand chemokine (ELC) and macrophage inflammatory protein 3β (MIP-3β), plays a role in lymphocyte recirculation and homing. CCL19 is expressed by dendritic cells or macrophages in lymph nodes and functions to initiate migration of T cells, B cells, or mature dendritic cells via its receptor CCR7.

[0048] In some embodiments, the immune cells described herein further express CCL19. In some embodiments, CCL19 comprises a sequence that comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (SEQ ID NO: 19). In some embodiments, CCL19 comprises a sequence that comprises about 85% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises a sequence that comprises about 90% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises a sequence that comprises about 95% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises a sequence that comprises about 96% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises a sequence that comprises about 97% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises a sequence that comprises about 98% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises a sequence that comprises about 99% sequence identity to SEQ ID NO: 19. In some embodiments, CCL19 comprises SEQ ID NO: 19. In some embodiments, CCL19 consists of SEQ ID NO: 19.

[0049] Additional immune function regulators The immune cells of the present invention may further express additional immune function regulators, such as IL-15, CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, interferon-γ, MIP-1α, GM-CSF, M-CSF, TGF-β, or TNF-α. In some embodiments, the additional immune function regulator comprises IL-15. In some embodiments, the additional immune function regulator comprises IL-2. In some embodiments, the additional immune function regulator comprises interferon-γ. In some embodiments, the additional immune function regulator comprises GM-CSF. In some embodiments, the additional immune function regulator comprises TGF-β. In some embodiments, the additional immune function regulator comprises TNF-α. In some embodiments, the additional immune function regulator is preferably an immune function regulator other than IL-12.

[0050] Arrangement of each area Disclosed herein in certain embodiments is an isolated nucleic acid molecule comprising one or more polynucleotides encoding an engineered cell surface molecule that specifically binds to mesothelin (e.g., a CAR that specifically binds to mesothelin), IL-7, and CCL19. In some embodiments, the isolated nucleic acid molecule comprises an antibody that specifically recognizes human mesothelin, a polynucleotide encoding a CAR comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; a polynucleotide encoding IL-7; and a polynucleotide encoding CCL19. In some embodiments, the polynucleotides encoding the CAR, IL-7, and CCL19 are located in two or more different polynucleotides in the nucleic acid molecule. In other embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR and IL-7, a polynucleotide encoding a CAR and CCL19, or a polynucleotide encoding a CAR, IL-7, or CCL19.

[0051] In some embodiments, the polynucleotide encoding the CAR comprises a signaling peptide upstream of an antibody that specifically recognizes human mesothelin. In some embodiments, the antibody is linked to the CD8 hinge region with a peptide linker (e.g., AlaAlaAla). In some embodiments, the 4-1BB intracellular region is located upstream of the CD3ζ intracellular region in the polynucleotide.

[0052] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSAAATTTP A CAR may be encoded that comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 16). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 16. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 16. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 16. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 16. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 16. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 16. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 16. The CAR may comprise SEQ ID NO: 16. The CAR may consist of SEQ ID NO: 16.

[0053] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAATTT The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 31). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 31. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 31. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 31. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 31. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 31. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 31. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 31. The CAR may comprise SEQ ID NO: 31. The CAR may consist of SEQ ID NO: 31.

[0054] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSTTTPA The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 32). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 32. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 32. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 32. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 32. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 32. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 32. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 32. The CAR may comprise SEQ ID NO: 32. The CAR may consist of SEQ ID NO: 32.

[0055] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSPTTTPA The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 33). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 33. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 33. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 33. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 33. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 33. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 33. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 33. The CAR may comprise SEQ ID NO: 33. The CAR may consist of SEQ ID NO: 33.

[0056] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSTTTPAP The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to RPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 34). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 34. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 34. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 34. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 34. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 34. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 34. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 34. The CAR may comprise SEQ ID NO: 34. The CAR may consist of SEQ ID NO: 34.

[0057] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSPTTTPA The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 35). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 35. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 35. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 35. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 35. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 35. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 35. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 35. The CAR may comprise SEQ ID NO: 35. The CAR may consist of SEQ ID NO: 35.

[0058] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSAAAPTTTP A CAR may be encoded that comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 36). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 36. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 36. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 36. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 36. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 36. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 36. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 36. The CAR may comprise SEQ ID NO: 36. The CAR may consist of SEQ ID NO: 36.

[0059] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSAAATTTP A CAR may be encoded that comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 37). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 37. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 37. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 37. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 37. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 37. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 37. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 37. The CAR may comprise SEQ ID NO: 37. The CAR may consist of SEQ ID NO: 37.

[0060] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSAAAPTTT The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 38). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 38. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 38. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 38. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 38. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 38. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 38. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 38. The CAR may comprise SEQ ID NO: 38. The CAR may consist of SEQ ID NO: 38.

[0061] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAAPTTT The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 39). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 39. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 39. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 39. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 39. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 39. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 39. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 39. The CAR may comprise SEQ ID NO: 39. The CAR may consist of SEQ ID NO: 39.

[0062] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAATTTP A CAR may be encoded that comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 40). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 40. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 40. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 40. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 40. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 40. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 40. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 40. The CAR may comprise SEQ ID NO: 40. The CAR may consist of SEQ ID NO: 40.

[0063] The polynucleotide is: MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAAPTTT The CAR may encode a CAR comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 41). The CAR may comprise a sequence having about 85% sequence identity to SEQ ID NO: 41. The CAR may comprise a sequence having about 90% sequence identity to SEQ ID NO: 41. The CAR may comprise a sequence having about 95% sequence identity to SEQ ID NO: 41. The CAR may comprise a sequence having about 96% sequence identity to SEQ ID NO: 41. The CAR may comprise a sequence having about 97% sequence identity to SEQ ID NO: 41. The CAR may comprise a sequence having about 98% sequence identity to SEQ ID NO: 41. The CAR may comprise a sequence having about 99% sequence identity to SEQ ID NO: 41. The CAR may comprise SEQ ID NO: 41. The CAR may consist of SEQ ID NO: 41.

[0064]

[0065] In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 are each independently transcribed under a promoter that comprises a polynucleotide encoding a self-cleaving 2A peptide (2A peptide) or an internal ribosome entry site (IRES). In some embodiments, the polynucleotide encoding IL-7 and the polynucleotide encoding CCL19 are each independently transcribed under a promoter that comprises a polynucleotide encoding a 2A peptide or an IRES.

[0066] In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a 2A peptide. There are four members of the 2A peptide family: P2A, E2A, F2A, and T2A. P2A is derived from Porcine Teschovirus-1 2A. E2A is derived from Equine Rhinitis A virus. F2A is derived from Foot and Mouth Disease virus 18. T2A is derived from Asignavirus 2A of them. Exemplary sequences of 2A peptide members include the following: P2A-ATNFSLLKQAGDVEENPGP; E2A-QCTNYALLKLAGDVESNPGP; F2A-VKQTLNFDLLKLAGDVESNPGP; and T2A-EGRGSLLTCGDVEENPGP.

[0067] In some embodiments, a peptide linker is further added to the terminus, e.g., the N-terminus, of the 2A peptide. In some embodiments, the peptide linker comprises GSG.

[0068] In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a P2A peptide. The P2A peptide may comprise ATNFSLLKQAGDVEENPGP. In some embodiments, a peptide linker (e.g., GSG) is further added to the N-terminus of the P2A peptide. In some cases, P2A comprises GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 23).

[0069] In some embodiments, the polynucleotide encoding the CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 are arranged in the nucleic acid molecule from the 5' to the 3' end as follows:

[0070] Polynucleotide encoding CAR-Polynucleotide encoding IL-7-Polynucleotide encoding CCL19;

[0071] Polynucleotide encoding CAR-Polynucleotide encoding CCL19-Polynucleotide encoding IL-7;

[0072] Polynucleotide encoding IL-7-Polynucleotide encoding CAR-Polynucleotide encoding CCL19;

[0073] polynucleotide encoding CCL19-polynucleotide encoding CAR-polynucleotide encoding IL-7;

[0074] a polynucleotide encoding IL-7-a polynucleotide encoding CCL19-a polynucleotide encoding CAR; or

[0075] Polynucleotide encoding CCL19-polynucleotide encoding IL-7-polynucleotide encoding CAR.

[0076] In some embodiments, the polynucleotide encoding the first 2A peptide (located between the first polynucleotide and the second polynucleotide) and the polynucleotide encoding the second 2A peptide (located between the second polynucleotide and the third polynucleotide) are non-identical (codon-optimized) polynucleotides to prevent unintended recombination. In some embodiments, the polynucleotide encoding the first P2A peptide comprises GGAAGCGGGAGCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCCTGGACCC (SEQ ID NO: 26) and the polypeptide encoding the second P2A peptide comprises GGCAGCGGGCGCCACCAACTTCTCTCTGCTGAAGCAAGCCGGCGATGTGGAGGAGAATCCCGGCCCC (SEQ ID NO: 27).

[0077]

[0078] vector In some embodiments, the one or more vectors include a polynucleotide encoding a CAR, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19. In some embodiments, the vector (e.g., an expression vector) comprises a nucleic acid molecule comprising a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes human mesothelin, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; a polynucleotide encoding IL-7; and a polynucleotide encoding CCL19. See FIG. 1A. In some embodiments, the polynucleotide encoding IL-7 and the polynucleotide encoding CCL19 are arranged in the vector (e.g., an expression vector) from the 5' end to the 3' end as follows:

[0079] Polynucleotide encoding CAR-Polynucleotide encoding IL-7-Polynucleotide encoding CCL19;

[0080] Polynucleotide encoding CAR-Polynucleotide encoding CCL19-Polynucleotide encoding IL-7;

[0081] Polynucleotide encoding IL-7-Polynucleotide encoding CAR-Polynucleotide encoding CCL19;

[0082] polynucleotide encoding CCL19-polynucleotide encoding CAR-polynucleotide encoding IL-7;

[0083] a polynucleotide encoding IL-7-a polynucleotide encoding CCL19-a polynucleotide encoding CAR; or

[0084] Polynucleotide encoding CCL19-polynucleotide encoding IL-7-polynucleotide encoding CAR.

[0085] In some embodiments, a first vector (e.g., a first expression vector) comprises a polynucleotide encoding a CAR, and a second vector (e.g., a second expression vector) comprises a polynucleotide encoding IL-7 and a polynucleotide encoding CCL19, where the polynucleotide encoding IL-7 and the polynucleotide encoding CCL19 are optionally arranged in the second vector (e.g., the second expression vector) from the 5' end to the 3' end as follows: polynucleotide encoding IL-7-polynucleotide encoding CCL19 or polynucleotide encoding CCL19-polynucleotide encoding IL-7.

[0086] In further embodiments, the first vector (e.g., the first expression vector) comprises a polynucleotide encoding a CAR and either a polynucleotide encoding IL-7 or a polynucleotide encoding CCL19, and the second vector (e.g., the second expression vector) comprises a polynucleotide encoding IL-7 or a polynucleotide encoding CCL19 that is not included in the first vector. In some embodiments, the first vector (e.g., the first expression vector) comprises a polynucleotide encoding a CAR and a polynucleotide encoding IL-7, and the second vector (e.g., the second expression vector) comprises a polynucleotide encoding CCL19. In other embodiments, the first vector (e.g., the first expression vector) comprises a polynucleotide encoding a CAR and a polynucleotide encoding CCL19, and the second vector (e.g., the second expression vector) comprises a polynucleotide encoding IL-7.

[0087] In further embodiments, a first vector (e.g., a first expression vector) comprises a polynucleotide encoding a CAR, a second vector (e.g., a second expression vector) comprises a polynucleotide encoding IL-7, and a third vector (e.g., a third expression vector) comprises a polynucleotide encoding CCL19.

[0088] The vector of the present invention (e.g., expression vector) may contain one or more naturally occurring nucleic acids or artificially synthesized nucleic acids, and can be appropriately selected according to the type of cell into which the vector of the present invention (e.g., expression vector) is to be introduced. These sequence information can be appropriately obtained by searching known literature or databases such as NCBI (www.ncbi.nlm.nih.gov / guide / ).

[0089] The vector of the present invention may be an expression vector that is introduced into an immune cell or a precursor cell thereof by contacting the vector with the cell so that a predetermined encoded protein (polypeptide) can be expressed in the immune cell to generate the modified immune cell of the present invention. The expression vector of the present invention is not particularly limited by any embodiment. Those skilled in the art can design and produce an expression vector that allows the expression of a desired protein (polypeptide) in an immune cell. Examples of the expression vector of the present invention that includes a polynucleotide encoding a cell surface molecule that specifically recognizes human mesothelin, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19 may include any of the expression vectors for generating the immune cell of the present invention.

[0090] The type of expression vector of the present invention may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector. Such vectors may contain a control sequence such as a promoter or a terminator, or a selection marker sequence such as a drug resistance gene or a reporter gene. The polynucleotide encoding CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 may be operably positioned downstream of the promoter sequence so that each of the polynucleotides can be efficiently transcribed.

[0091] Examples of promoters may include virus-derived promoters, such as retroviral LTR promoter, SV40 early promoter, cytomegalovirus promoter, and herpes simplex virus thymidine kinase promoter; and mammalian-derived promoters, such as phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, and RNA polymerase II promoter. In some embodiments, the promoter may preferably include a retroviral LTR promoter. The retroviral LTR promoter may be CTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCC TTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCA (SEQ ID NO: 24). Alternatively, a tetracycline-responsive promoter induced by tetracycline, an Mx1 promoter induced by interferon, etc. may be used. In the expression vector of the present invention, the use of a promoter induced by a specific substance controls the induction of IL-7 and CCL19 expression depending on the course of cancer treatment, and for example, immune cells containing the vector of the present invention are used as a pharmaceutical composition used for cancer treatment.

[0092] Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, and preferably include retroviral vectors, such as gamma retroviral vectors, more preferably pMSGV vectors (Tamada k et al., Clin Cancer Res 18:6436-6445(2002)), pMSCV vectors (manufactured by Takara Bio Inc.), or pSFG vectors. The use of retroviral vectors allows for long-term stable expression of the transgene, since the transgene is integrated into the genome of the host cell.

[0093] One or more assays can be used to confirm incorporation of the expression vector of the present invention into the immune cells. Exemplary assays can include flow cytometry, Northern blotting, Southern blotting, PCR such as RT-PCR, ELISA, or Western blotting to screen for expression of CAR by the engineered immune cells. In some embodiments, the expression vector further comprises a marker gene (e.g., encoding a fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), or yellow fluorescent protein (YFP)) to detect expression of CAR, IL-7, and / or CCL19 by the immune cells.

[0094] Immune cells and methods of production In certain embodiments, the immune cells described herein are modified to express a cell surface molecule that specifically recognizes mesothelin (e.g., human mesothelin), IL-7, and CCL19 (FIG. 1B). Exemplary immune cells may include lymphoid cells, such as T cells, natural killer cells (NK cells), and B cells; antigen-presenting cells, such as monocytes, macrophages, dendritic cells, or granulocytes, such as neutrophils, eosinophils, basophils, or mast cells. Immune cells may include T cells from a mammal, such as a human, dog, cat, pig, or mouse, preferably T cells from or isolated from a human. Immune cells (e.g., T cells) may be obtained through culture, such as ex vivo culture, or directly harvested from a mammal. Immune cells are not limited, so long as the cells are involved in an immune response, express a cell surface molecule that specifically recognizes mesothelin (e.g., human mesothelin), express IL-7, and may express CCL19. The immune cells can be autologous cells taken from a subject in need thereof for subsequent treatment. The immune cells can be allogeneic or syngeneic cells to the subject in need thereof. The immune cells can also be obtained by culturing stem cells (e.g., induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells)) or progenitor cells under appropriate conditions to induce and differentiate into immune cells.

[0095] In certain embodiments, a population of immune cells engineered to express a CAR that specifically recognizes mesothelin, IL-7, and CCL19 is disclosed herein. In some embodiments, the population of immune cells includes engineered T cells (e.g., expanded ex vivo or harvested from a mammal) that express a CAR that specifically recognizes mesothelin, IL-7, and CCL19. The population of immune cells may include about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more engineered T cells. The population of immune cells may include about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% engineered T cells. The population of immune cells may include a substantially pure population of engineered T cells. Exemplary T cells include α-β T cells, γ-δ T cells, CD8 ... + T cells, CD4 + These may include T cells, tumor infiltrating T cells, memory T cells, naive T cells, and natural killer T (NKT) cells.

[0096] In some embodiments, the population of immune cells engineered to express a CAR that specifically recognizes mesothelin, IL-7, and CCL19 contains about 30%, 25%, 20%, 15%, 10%, 5%, or less of contaminating cells. As used herein, the term "contaminating cells" refers to cells that do not express a CAR that specifically recognizes mesothelin, IL-7, and CCL19. Contaminating cells can include T cells that do not express a CAR that specifically recognizes mesothelin, IL-7, and CCL19, and other types of immune cells that do not express a CAR that specifically recognizes mesothelin, IL-7, and CCL19. Contaminating cells can also refer to non-immune cells from bodily fluids (e.g., blood or bone marrow fluid), non-immune cells from tissues (e.g., spleen tissue, thymus, or lymph nodes), or non-immune cells from cancer tissues (e.g., primary tumor tissue, metastatic tumor tissue, or cancerous ascites).

[0097] The method for producing immune cells of the present invention may include a method for introducing a polynucleotide encoding a cell surface molecule, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19 into immune cells. Examples of the production method include the production methods described in WO2016 / 056228, WO2017 / 159736, WO2013 / 176915, WO2015 / 120096, WO2016 / 019300, or Vormittag P et al, Curr Opin Biotechnol 2018;53:164-81. Another example includes a method of obtaining and purifying immune cells from a transgenic mammal produced by transplanting into a fertilized egg an expression vector for a cell surface molecule that specifically recognizes mesothelin (e.g., human mesothelin), IL-7, and / or CCL19, as well as a production method in which, if necessary, an expression vector for a cell surface molecule that specifically recognizes mesothelin (e.g., human mesothelin), IL-7, and / or CCL19 is introduced into immune cells obtained by purification from a transgenic mammal.

[0098] When introducing a polynucleotide encoding a cell surface molecule, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19, or the above-mentioned vector, the method may be any method for introducing a polynucleotide or vector into an immune cell. Examples include electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (Japanese Patent Application Publication No. 2-227075), lipofection (Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)), and virus infection method. Exemplary virus infection methods include transfecting packaging cells (e.g., GP2-293 cells (Takara Bio Inc.), Plat-GP cells (Cosmo Bio Co., Ltd.), PG13 cells (ATCC CRL-10686), or PA317 cells (ATCC CRL-9078)) with a packaging plasmid that produces the vector to be introduced and the recombinant virus, and then infecting immune cells with the recombinant virus (see, for example, WO2017 / 159736).

[0099] In some embodiments, the method comprises introducing into an immune cell one or more vectors comprising a polynucleotide encoding a CAR, a polynucleotide encoding IL-7, and a polynucleotide encoding CCL19. In some embodiments, the method comprises introducing into an immune cell a vector (e.g., an expression vector) comprising a nucleic acid molecule comprising a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes human mesothelin, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; a polynucleotide encoding IL-7; and a polynucleotide encoding CCL19. In some embodiments, the method comprises introducing into an immune cell a first vector (e.g., a first expression vector) comprising a polynucleotide encoding a CAR and a second vector (e.g., a second expression vector) comprising a polynucleotide encoding IL-7 and a polynucleotide encoding CCL19, either together or stepwise. In some embodiments, the method comprises introducing into an immune cell, together or in a stepwise manner, a first vector (e.g., a first expression vector) comprising a polynucleotide encoding a CAR and either a polynucleotide encoding IL-7 or a polynucleotide encoding CCL19, and a second vector (e.g., a second expression vector) comprising a polynucleotide encoding IL-7 or a polynucleotide encoding CCL19 that is not included in the first vector. In some embodiments, the method comprises introducing into an immune cell, together or in a stepwise manner, a first vector (e.g., a first expression vector) comprising a polynucleotide encoding a CAR and a polynucleotide encoding IL-7, and a second vector (e.g., a second expression vector) comprising a polynucleotide encoding CCL19. In some embodiments, the method comprises introducing into an immune cell, together or in a stepwise manner, a first vector (e.g., a first expression vector) comprising a polynucleotide encoding a CAR and a polynucleotide encoding CCL19, and a second vector (e.g., a second expression vector) comprising a polynucleotide encoding IL-7.In some embodiments, the methods include introducing into an immune cell, together or in a stepwise manner, a first vector (e.g., a first expression vector) comprising a polynucleotide encoding a CAR, a second vector (e.g., a second expression vector) comprising a polynucleotide encoding IL-7, and a third vector (e.g., a third expression vector) comprising a polynucleotide encoding CCL19.

[0100] One or more of the polynucleotides encoding the CAR, the polynucleotides encoding IL-7, and the polynucleotides encoding CCL19 can be integrated into the genome of the immune cell. In some embodiments, the polynucleotides encoding the CAR, the polynucleotides encoding IL-7, and the polynucleotides encoding CCL19 are not integrated into the genome (e.g., episomally).

[0101] How to use In certain embodiments, methods of treating mesothelin-expressing cancer are disclosed herein. In some embodiments, the methods include administering to a subject in need thereof immune cells described herein that have been modified to express engineered cell surface molecules that specifically bind to mesothelin, interleukin 7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19). In some embodiments, the immune cells are modified to express engineered cell surface molecules that include a chimeric antigen receptor (CAR) that specifically recognizes mesothelin, or a T cell receptor (TCR) that specifically binds to mesothelin. In some embodiments, the immune cells are modified to express an antibody that specifically recognizes human mesothelin, a CAR that includes a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3 zeta intracellular region; IL-7; and CCL19.

[0102] In some embodiments, the mesothelin-expressing cancer is a solid tumor. In some embodiments, the solid tumor comprises mesothelioma, colorectal cancer, pancreatic cancer, thymic cancer, bile duct cancer, lung cancer, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, stomach cancer, spleen cancer, tracheal cancer, bronchial cancer, gastric cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, or bone cancer. In some embodiments, the mesothelin-expressing cancer is ovarian cancer. In some embodiments, the mesothelin-expressing cancer is mesothelioma. In some embodiments, the mesothelin-expressing cancer is gastric cancer. In some embodiments, the mesothelin-expressing cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung carcinoid tumor, lung adenosquamous carcinoma, large cell neuroendocrine carcinoma, or salivary gland type lung cancer). In some embodiments, the mesothelin-expressing cancer is NSCLC (eg, adenocarcinoma, squamous cell carcinoma, large cell undifferentiated carcinoma, sarcomatoid carcinoma, or adenosquamous carcinoma of the lung).

[0103] Mesothelin-expressing cancer can be hematopoietic cancer.Hematopoietic cancer can be B-cell hematopoietic cancer, T-cell hematopoietic cancer, Hodgkin's lymphoma, or non-Hodgkin's lymphoma.Hematopoietic cancer can be acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal lymphoma, Burkitt's lymphoma, or Waldenstrom's macroglobulinemia.

[0104] The hematopoietic cancer may be a sarcoma. The sarcoma may include chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, or soft tissue sarcoma.

[0105] The mesothelin-expressing cancer can be a metastatic cancer, such as a metastatic solid tumor or a metastatic hematopoietic cancer. The metastatic mesothelin-expressing cancer can be metastatic ovarian cancer, metastatic mesothelioma, metastatic gastric cancer, or metastatic lung cancer (e.g., metastatic NSCLC).

[0106] The mesothelin-expressing cancer can be a recurrent or refractory cancer, such as a recurrent or refractory solid tumor or a recurrent or refractory hematopoietic cancer. The recurrent or refractory mesothelin-expressing cancer can be a recurrent or refractory ovarian cancer, a recurrent or refractory mesothelioma, a recurrent or refractory gastric cancer, or a recurrent or refractory lung cancer (e.g., recurrent or refractory NSCLC).

[0107] In some embodiments, the method further comprises administering an additional therapeutic agent or additional treatment regimen to the subject. The additional therapeutic agent may comprise a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, a radiation therapy, or a combination thereof. Examples of additional therapeutic agents include alkylating agents, such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxalaplatin, temozolomide, or thiotepa; antimetabolites, such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, or pemetrexed; anthracyclines, such as daunorubicin. , doxorubicin, epirubicin, or idarubicin; topoisomerase I inhibitors, such as topotecan or irinotecan (CPT-11); topoisomerase II inhibitors, such as etoposide (VP-16), teniposide, or mitoxantrone; mitotic inhibitors, such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine, or vinorelbine; or corticosteroids, such as prednisone, methylprednisolone, or dexamethasone.

[0108] In some embodiments, the additional therapeutic agent comprises a first-line therapy. As used herein, "first-line therapy" includes a primary treatment for a subject with cancer. In some embodiments, the cancer is a primary cancer. In other embodiments, the cancer is a metastatic or recurrent cancer. In some embodiments, the first-line therapy comprises chemotherapy. In other embodiments, the first-line therapy comprises radiation therapy. Those skilled in the art will readily appreciate that different first-line therapies may be applicable to different types of cancer.

[0109] In some embodiments, the additional therapeutic agent comprises an inhibitor of the enzyme poly ADP ribose polymerase (PARP). Exemplary PARP inhibitors include, but are not limited to, olaparib (AZD-2281, Lynparza®, manufactured by Astra Zeneca), rucaparib (PF-01367338, Rubraca®, manufactured by Clovis Oncology), niraparib (MK-4827, Zejula®, manufactured by Tesaro), talazoparib (BMN-673, manufactured by BioMarin Pharmaceutical Inc.), veliparib (ABT-888, manufactured by AbbVie), CK-102 (formerly CEP 9722, manufactured by Teva Pharmaceutical Industries Ltd.), E7016 (manufactured by Eisai), iniparib (BSI 201, manufactured by Sanofi), and pamiparib (BGB-290, manufactured by BeiGene).

[0110] In some embodiments, the additional therapeutic agent comprises an immune checkpoint inhibitor. In some embodiments, the checkpoint inhibitor comprises pembrolizumab, nivolumab, tremelimumab, or ipilimumab. In some embodiments, the checkpoint inhibitor comprises an inhibitor of PD-L1, PD-L2, PD-1, CTLA-4, LAG3, B7-H3, KIR, CD137, PS, TFM3, CD52, CD30, CD20, CD33, CD27, OX40, GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM. The inhibitor may be an antibody or a fragment thereof (e.g., a monoclonal antibody, a human antibody, a humanized antibody, or a chimeric antibody), an RNAi molecule, or a small molecule.

[0111] In some embodiments, the additional therapeutic agent comprises an antibody, e.g., alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab.

[0112] In some embodiments, the additional therapeutic agent comprises a cytokine. Exemplary cytokines include, but are not limited to, IL-1β, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, or TNFα.

[0113] In some embodiments, the additional therapeutic agent comprises a receptor agonist. In some embodiments, the receptor agonist comprises a Toll-like receptor (TLR) ligand. In some embodiments, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some embodiments, the TLR ligand comprises a synthetic ligand, e.g., Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, poly I:C, poly A:U, AGP, MPL A, RC-529, MDF2p, CFA, or flagellin.

[0114] In some embodiments, the additional therapeutic agent comprises fludarabine and cyclophosphamide.

[0115] In some embodiments, the additional therapeutic agent comprises tisagenlecleucel (KYMRIAH®), axicabtagene ciloleucel (YESCARTA®), or brexcabtagene autoleucel (TECARTUS®).

[0116] In some embodiments, the additional therapeutic regimen comprises surgery.

[0117] In some embodiments, the immune cells described herein or the pharmaceutical composition described herein and the additional therapeutic agent are administered simultaneously.

[0118] In some embodiments, the immune cells described herein or pharmaceutical compositions described herein and the additional therapeutic agent are administered sequentially. In some embodiments, the immune cells described herein or pharmaceutical compositions described herein are administered to the subject prior to administration of the additional therapeutic agent. In other embodiments, the immune cells described herein or pharmaceutical compositions described herein are administered to the subject after administration of the additional therapeutic agent.

[0119] In some embodiments, the subject is a human.

[0120] Also described herein, in some embodiments, is a method of generating immune cells that express cell surface molecules that specifically recognize mesothelin (e.g., human mesothelin), IL-7, and CCL19. The method includes introducing a nucleic acid molecule or a vector containing a nucleic acid molecule described herein into an immune cell to induce expression by the immune cell of cell surface molecules that specifically recognize human mesothelin, IL-7, and CCL19. In some embodiments, the immune cell is a T cell, a natural killer (NK) cell, a B cell, an antigen-presenting cell, or a granulocyte, optionally a T cell or an NK cell.

[0121] Pharmaceutical Compositions In certain embodiments, the immune cells are formulated as a pharmaceutical composition. In some embodiments, the pharmaceutical composition is administered to a subject by multiple routes of administration, including but not limited to parenteral, oral, sublingual, or transdermal routes of administration. In some embodiments, parenteral administration includes intravenous, subcutaneous, intramuscular, intranasal, intraarterial, intraarticular, intradermal, intraosseous injection, intraperitoneal, intrathecal, intracranial, intrasynovial, intratumoral, intradermal, intramedullary, intracardiac, or intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for local administration. In other embodiments, the pharmaceutical composition is formulated for systemic administration.

[0122] In some embodiments, the pharmaceutical composition comprises a pharma- ceutically acceptable additive. Examples of additives may include saline, buffered saline, cell culture medium, glucose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, isotonicity agents, bulking agents, lubricants, or combinations thereof.

[0123] In some embodiments, the pharmaceutical composition further comprises a pH adjusting or buffering agent (including acids, such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid; bases, such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and trishydroxymethylaminomethane; and buffers, such as citrate / dextrose, sodium bicarbonate, and ammonium chloride). Such acids, bases, and buffers are included in amounts necessary to maintain the pH of the composition in an acceptable range.

[0124] In some embodiments, the pharmaceutical composition includes one or more salts in an amount necessary to render the osmolality of the composition tolerable. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, with suitable salts including sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0125] In exemplary methods, the pharmaceutical compositions of the invention can be administered independently, either once or in several divided doses, such as four times a day, three times a day, two times a day, or once a day, at intervals of one, two, three, four, or five days, once a week, seven, eight, or nine days, twice a week, once a month, twice a month, three or more times a month.

[0126] In the event that the patient's condition improves, and at the discretion of the physician, administration of the composition may be continued, or the dose of the composition administered may be temporarily reduced or temporarily stopped for a specified period of time (i.e., a "drug holiday"). In some embodiments, the length of the drug holiday may vary from 2 days to 1 year, including, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. Dose reductions during drug holidays can be from 10% to 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0127] In some embodiments, the amount of a given engineered T cell that corresponds to such an amount will vary depending on factors such as the severity of the disease, the personality (e.g., weight) of the subject or host requiring treatment, but is nevertheless routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, for example, the particular agent being administered, the route of administration, and the subject or host being treated. In some embodiments, the desired dose is conveniently presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., as two, three, four or more divided doses per day.

[0128] The above ranges are merely suggestions, since the number of variables for any particular treatment regimen is large and significant deviations from these recommendations are not uncommon. Such dosages will vary depending on many variables, including, but not limited to, the activity of the compound being used, the disease or condition being treated, the method of administration, the requirements of the particular subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0129] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens, including but not limited to, determination of LD50 (the dose lethal to 50% of the population) and ED50 (the dose therapeutically effective in 50% of the population), are determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio of LD50 and ED50. Compounds that exhibit high therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to determine a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with minimal toxicity. Dosages vary within this range depending on the dosage form employed and the route of administration utilized.

[0130] Kits / Products In certain embodiments, a kit comprising the above-mentioned nucleic acid molecule, a vector comprising the above-mentioned nucleic acid molecule, a CAR that specifically recognizes mesothelin (e.g., human mesothelin), an immune cell expressing IL-7 and CCL19, or a pharmaceutical composition is disclosed herein. In some embodiments, the kit may contain one or more packaging materials, such as a package insert, a label, a package, etc., that describe the method of use for use in the treatment of cancer. Since the immune cells in the pharmaceutical composition of the present invention have an inhibitory effect on tumor recurrence, the pharmaceutical composition of the present invention may function as a pharmaceutical composition used for suppressing tumor recurrence. Such a pharmaceutical composition used for suppressing tumor recurrence may contain one or more packaging materials, such as a package insert, a label, a package, etc., that describe the method of use for use in suppressing tumor recurrence.

[0131] The term "packaging material" refers to a physical structure that houses the components of the kit. The material can maintain the components sterile and can be made of materials commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

[0132] The kit of the present invention may include a label or insert. The label or insert includes "printed matter", such as paper or cardboard, or separate from or attached to the component, kit, or packaging material (e.g., box), or affixed to the ampoule, tube, or vial containing the kit component. The label or insert may further include a computer readable medium, such as a disk (e.g., floppy diskette, ZIP disk), optical disk, such as CD-ROM / RAM or DVD-ROM / RAM, DVD, MP3, magnetic tape, or electrical storage medium, such as RAM and ROM, or hybrids thereof, such as magnetic / optical storage medium, FLASH media, or memory type card.

[0133] The label or insert may include identification of one or more components therein (e.g., binding agent or pharmaceutical composition), dosage, clinical pharmacology, pharmacokinetics and pharmacodynamics of the active agent(s) including mechanism of action. The label or insert may include information identifying manufacturer information, lot number, and location and date of manufacture.

[0134] The label or insert may include information regarding the disease for which the kit components can be used.The label or insert may include instructions for the clinician or subject to use one or more of the kit components in a method or treatment protocol or treatment regimen.The instructions may include dosage, frequency or duration, and instructions for carrying out any of the methods, treatment protocols, or treatment regimens described herein.

[0135] The label or insert may include information regarding any benefits, e.g., therapeutic benefits, that the component may provide. The label or insert may include information regarding potential adverse side effects, e.g., warnings to the subject or clinician regarding situations in which it is not appropriate to use a particular composition (e.g., modified immune cells described herein). For example, adverse side effects are generally more likely to occur with high doses, frequency, or duration of the active agent, and thus the instructions may include recommendations for high doses, frequency, or duration. Adverse side effects may also occur if the subject is taking, planning to take, or currently taking one or more other drugs that may be incompatible with the composition, or if the subject is taking, planning to take, or currently taking another treatment protocol or therapeutic regimen that is incompatible with the composition, and thus the instructions may include information regarding such incompatibilities.

[0136] definition As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0137] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, is intended to mean excluding other elements of any essential importance to the combination for the intended use. For example, a composition consisting essentially of the elements defined herein would not exclude trace contaminants from the isolation and purification methods and pharma- ceutically acceptable carriers, e.g., phosphate buffered saline, preservatives, and the like. "Consisting of" is intended to mean excluding more than trace elements of other components and substantial method steps for administering the compositions disclosed herein. The embodiments defined by each of these transition terms are within the scope of the present disclosure.

[0138] As used herein, the term "about" is used to indicate that a value includes the standard deviation of error of the device or method being used to determine the value. When used before specifying a numerical value (e.g., temperature, time, amount, and concentration) that includes a range, the term "about" indicates an approximation that may vary by (+) or (-) 15%, 10%, 5%, 3%, 2%, or 1%.

[0139] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0140] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and is meant to include embodiments in which the event or circumstance occurs and embodiments in which it does not occur.

[0141] As used herein, the term "antibody" refers to a heavy chain variable domain and a light chain variable domain, respectively, V H and V LThe term "variable region" or "variable domain" refers to a protein that binds to other molecules (antigens, e.g., mesothelin) via a VH or VL domain. The term "variable region" or "variable domain" refers to the domain of an antibody that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally share a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a particular antigen may be isolated using the VH or VL domain from an antibody that binds the antigen, and a library of complementary VL or VH domains, respectively, may be screened. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0142] The antibodies of the present disclosure include monoclonal antibodies. The term "monoclonal" when used in reference to an antibody refers to an antibody that is based on, obtained from, or derived from a single clone, including any eukaryotic, prokaryotic, or phage clone. Thus, a "monoclonal" antibody is defined herein structurally, not the method by which it is produced.

[0143] Monoclonal antibodies are produced by methods known in the art (Kohler et al., Nature, 256:495 (1975); and Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1999). Briefly, monoclonal antibodies can be obtained by injecting a mouse with an antigen. The polypeptide or peptide used to immunize the animal can be derived from translated DNA or chemically synthesized and conjugated to a carrier protein. Commonly used carriers that are chemically coupled to the immunizing peptide include, for example, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. Antibody production is verified by analyzing serum samples, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies against the antigen, and isolating the antibodies from the hybridoma cultures. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of established techniques, including, for example, affinity chromatography using protein A sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan et al., Current Protocols in Immunology sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; and Barnes et al., "Methods in Molecular Biology," 10:79-104, Humana Press (1992)).

[0144] The antibodies of the present disclosure may belong to any antibody class, IgM, IgG, IgE, IgA, IgD, or subclass. Exemplary subclasses of IgG are IgG1, IgG2, IgG3, and IgG4.

[0145] The antibody of the present disclosure may be a humanized antibody. The term "humanized" refers to an antibody sequence having non-human amino acid residues in one or more complementarity determining regions (CDRs) that specifically bind to an antigen in an acceptor human immunoglobulin molecule, and one or more human amino acid residues in the framework regions (FRs) adjacent to the CDRs. Any mouse, rat, guinea pig, goat, non-human primate (e.g., ape, chimpanzee, macaque, orangutan, etc.) or other animal antibody may be used as a CDR donor to generate a humanized antibody. Human framework region residues can be replaced with corresponding non-human residues (e.g., from the donor variable region). Thus, human framework region residues can be replaced with corresponding residues in the non-human CDR donor antibody. A humanized antibody may include residues that are not found in human antibodies or in the donor CDR or framework sequences. The use of antibody components derived from humanized monoclonal antibodies reduces problems associated with the immunogenicity of non-human regions. Methods for producing humanized antibodies are known in the art (e.g., U.S. Pat. Nos. 5,225,539, 5,530,101, 5,565,332, and 5,585,089; Riechmann et al., (1988) Nature 332:323; EP 239,400; W091 / 09967; EP 592,106; EP 519,596; Padlan Molecular Immunol. (1991) 28:489; Studnicka et al., Protein Engineering (1994) 7:805; Singer et al., J. Immunol. (1993) 150:2844; and Roguska et al., Proc. Nat'l. Acad. Sci. USA (1994) 91:969).

[0146] The antibody of the present disclosure may be a chimeric antibody. The term "chimeric antibody" refers to an antibody in which different portions are derived from different animal species, such as an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region, such as a humanized antibody. In some embodiments, techniques developed for the production of "chimeric antibodies" by splicing genes from a mouse antibody molecule of appropriate antigen specificity and genes from a human antibody molecule of appropriate biological activity (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used.

[0147] The antibodies of the present disclosure include binding fragments thereof. Exemplary antibody fragments include Fab, Fab', F(ab')2, Fv, Fd, single chain Fv (scFv), disulfide-linked Fv (sdFv), light chain variable region V, L , heavy chain variable region V H , trispecific (Fab3), bispecific (Fab2), diabody ((V L -V H )2 or (V H -V L )2), triabody (trivalent), tetrabody (tetravalent), minibody ((scF V -C H ))2), bispecific single chain Fv (Bis-scFv), IgGδCH2, scFv-Fc, (scFv)2-Fc, and IgG4PE. Such fragments may have the binding affinity of a full-length antibody, the binding specificity of a full-length antibody, or one or more activities or functions of a full-length antibody, such as a function or activity of a mesothelin-binding antibody.

[0148] Antibody fragments can be combined. For example, V L or V H The subsequences are joined by a linker sequence, thereby forming V L -V HA combination of single chain Fv (scFv) sequences may be linked with a linker sequence, thereby forming a scFv-scFv chimera. Antibody fragments include single chain antibodies or variable region(s) alone or in combination with all or part of other sequences.

[0149] Antibody fragments can be produced in a variety of ways, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment that does not occur in nature (such as having two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers) and / or contains an arrangement that may not be produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is an scFv.

[0150] Antibody fragments can also be prepared by proteolytic hydrolysis of antibodies, for example, by pepsin or papain digestion of whole antibodies. Antibody fragments produced by enzymatic cleavage with pepsin provide a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage with pepsin produces two monovalent Fab' fragments and an Fc fragment directly (see, for example, U.S. Pat. Nos. 4,036,945 and 4,331,647; and Edelman et al., Methods Enymol. 1:422 (1967)). Other methods of cleaving antibodies can also be used, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic or chemical methods.

[0151] In some embodiments, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

[0152] As used herein, "identical", "sequence identity", or percent "identity", when used in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences that are the same or have a certain percentage of nucleotide or amino acid residues over a specified region (e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity). Alignment and sequence identity can be determined using software programs known in the art, such as those described in: Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for the alignment. A preferred alignment program is BLAST using default parameters. Particularly preferred programs are BLASTN and BLASTP using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expectation=10; matrix=BLOSUM62; description=50 sequences; sort=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address ncbi.nlm.nih.gov / cgi-bin / BLAST. The terms "identical", "sequence identity", or percent "identity" can also refer to or apply to the complement of a test sequence. The terms also include sequences having deletions and / or additions, as well as sequences having substitutions. As described herein, preferred algorithms may take into account gaps and the like. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or, more preferably, over a region that is at least 50-100 amino acids or nucleotides in length.An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences disclosed herein.

[0153] The terms "protein", "peptide" and "polypeptide" are used interchangeably and in the broadest sense refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, and the like. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that may comprise a protein or peptide sequence. Polypeptides include full-length naturally occurring polypeptides and "modified" forms, such as subsequences, variant sequences, fusion / chimeric sequences, and dominant negative sequences. As used herein, the term "amino acid" refers to any of the natural and / or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs, and peptidomimetics.

[0154] Peptides include L and D isomers, as well as combinations thereof. Peptides may include modifications typically associated with post-translational processing of proteins, such as cyclization (e.g., disulfide or amide bonds), phosphorylation, glycosylation, carboxylation, ubiquitination, myristylation, or lipidation. Modified peptides may have one or more amino acid residues replaced with another residue, added to the sequence, or deleted from the sequence. Particular examples include one or more amino acid substitutions, additions, or deletions (e.g., 1-3, 3-5, 5-10, 10-20, or more).

[0155] As used herein, the terms "modification" and "modified" refer to a mutation, substitution, addition, or deletion of one or more amino acid residues of an antibody, protein, or polypeptide compared to a reference antibody, protein, or polypeptide, which is equivalent to the antibody, protein, or polypeptide without the modification. In some embodiments, a modification comprises a conservative substitution.

[0156] A "conservative substitution" is the replacement of one amino acid with a biologically, chemically, or structurally similar residue. Biologically similar means that the substitution is compatible with the activity or function of the unsubstituted sequence. Structurally similar means that the amino acids have side chains of similar length, or similar size, such as alanine, glycine, and serine. Chemically similar means that the residues have the same charge, or are both hydrophilic or hydrophobic. Particular examples include the replacement of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, or the replacement of one polar residue for another (e.g., arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, serine for threonine, etc.).

[0157] As used herein, the term "nucleic acid" refers to DNA or RNA that includes natural, synthetic, or artificial nucleotide analogs or bases. In some embodiments, the nucleotide analogs or artificial nucleotide bases include nucleic acids that have modifications at the 2' hydroxyl group of the ribose moiety. In some embodiments, the modifications include H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some embodiments, the alkyl moiety further includes a modification. In some embodiments, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate group or a cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some embodiments, the alkyl moiety further comprises heterosubstitution. In some embodiments, a carbon of a heterocyclic group is replaced with nitrogen, oxygen, or sulfur. In some embodiments, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

[0158] In some embodiments, the nucleotide analogs include modified bases, such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N-dimethyladenine, 2-propyladenine, 2 propylguanine, 2-aminoadenine, 1-methylinosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (e.g., 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (e.g., 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queosine, alkeosine, naphthyl and substituted naphthyl groups, any O-alkylated and N-alkylated purines and pyrimidines (e.g., N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that function as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety and nucleotides having sugars or analogs thereof that are not ribosyl. For example, in some embodiments, the sugar moiety is or is based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose, and other sugars, heterocycles, or carbocycles. The term nucleotide also includes what are known in the art as universal bases.By way of example, universal bases include, but are not limited to, 3-nitropyrrole, 5-nitroindole, or nebularine.

[0159] The nucleic acid molecules of the present invention can be produced by known methods, such as chemical synthesis or PCR amplification, based on the base sequence information of each nucleic acid. The codons selected to code for amino acids can be manipulated to optimize nucleic acid expression in the host cell of interest.

[0160] As used herein, the term "substantially" when describing a population of T cells refers to a population that contains about 30%, 25%, 20%, 15%, 10%, 5%, or less of contaminating cells. In some embodiments, the contaminating cells are less than about 20% of the T cell population. In some embodiments, the contaminating cells are less than about 15% of the T cell population. In some embodiments, the contaminating cells are less than about 10% of the T cell population.

[0161] As used herein, the terms "treat", "treatment" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be therapeutic in terms of amelioration of symptoms of a disease, or partial or complete curing of a disease and / or adverse reactions resulting from a disease. In one aspect, the term "treatment" does not include prevention.

[0162] As used herein, "treating" further includes the general amelioration of symptoms associated with the pathology and / or delaying the onset of symptoms. The clinical and potential evidence of "treatment" varies with the condition, the individual, and the procedure. In one aspect, treatment does not include prevention.

[0163] The term "ameliorate" refers to a detectable improvement in a subject's condition. Detectable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limitation, or control in the occurrence, frequency, severity, progression, or duration of a symptom (e.g., one or more adverse symptoms) caused by or associated with a disease, disorder, illness, condition, disease, or complication caused by or associated with a disease, or an improvement in the underlying cause or effects of a disease or reversal of a disease.

[0164] Thus, treatment may result in the reduction, reduction, inhibition, suppression, restriction, control, or prevention of a disease, or associated symptoms or results, or underlying causes; the reduction, reduction, inhibition, suppression, restriction, control, or aggravation of the progression or worsening of a disease, condition, symptom, or effect, or underlying cause; or the further worsening or occurrence of one or more additional symptoms or symptoms of a condition. Thus, a successful treatment outcome provides a "therapeutic effect" or "benefit" that reduces, reduces, inhibits, suppresses, restricts, controls, or prevents the occurrence, frequency, severity, progression, or duration of one or more symptoms or underlying causes or effects of a condition, disease, or symptom in a subject, such as one or more adverse symptoms, disorders, illnesses, pathologies, diseases, or complications caused by or associated with a disease or condition. Thus, a treatment that affects one or more underlying causes of a condition, disease, or symptom is considered to be beneficial. Stabilizing a disorder or condition is also a good treatment outcome.

[0165] Thus, a therapeutic benefit or improvement does not necessarily require the complete elimination of any one, most, or all of the symptoms, complications, effects, or underlying causes associated with a condition or disease. Thus, a satisfactory endpoint is achieved when there is a gradual improvement in the condition of the subject; or a partial reduction, reduction, inhibition, suppression, restriction, control, or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal of one or more associated adverse symptoms or complications or effects or underlying causes; a deterioration or progression (e.g., stabilization of one or more symptoms or complications of a condition, disorder, or disease) of one or more physiological, biochemical, or cellular signs or characteristics of a disorder or disease (e.g., one or more adverse symptoms, disorders, illnesses, lesions, diseases, or complications caused by or associated with a disease or condition) over a short or long period (hours, days, weeks, months, etc.).

[0166] The terms "acceptable," "effective," or "sufficient," when used to describe the selection of any components, ranges, dosage forms, etc. disclosed herein, are intended to mean that the components, ranges, dosage forms, etc. are suitable for the purposes of the disclosure.

[0167] The terms "subject," "host," "individual," and "patient" are used interchangeably herein to refer to an animal, typically a mammal. Any suitable mammal can be treated with the methods, cells, or compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), domestic animals (e.g., dogs and cats), livestock (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (e.g., an adult, teenager, child, infant, or mammal in utero). The mammal can be male or female. The mammal can be a pregnant female. In some embodiments, the subject is a human. EXAMPLES

[0168] These examples are provided for illustrative purposes only and are not intended to limit the scope of the claims provided herein.

[0169] Example 1 Preparation of CAR-T cells For transduction of MSGV γ-retroviral vectors, the plasmids were transfected into GP2-293 packaging cell line using Lipofectamine 3000 (Thermo Fisher Scientific, MA, USA) together with pAmpho vector to generate viral supernatants. 48 h after transfection, the supernatants were collected and centrifuged on plates coated with Retronectin (Takarabio, Shiga, Japan) to prepare virus-binding plates. Peripheral blood mononuclear cells (PBMCs) were activated with solidified anti-human CD3 Ab (OKT3, 5 μg / mL) and cultured for 3 days in medium containing recombinant human IL-2 (400 IU / mL). PBMCs were added to the virus-binding plate and cultured for 24 h for the first round of infection. The cultured PBMCs were then transferred to another virus-binding plate for the second round of infection, and after 4 h of incubation, the infected cells were grown for 3 days in fresh medium containing 400 IU / mL of recombinant human IL-2. For transduction of SFGγ-retroviral vectors, the plasmids were transfected into PhoenixAmpho packaging cell line using FuGENE® HD transfection reagent (Promega Corp., WI, USA) together with gag / pol genes (Cell Biolabs Inc., CA, US) and VSV-G vector (Takarabio, Shiga, Japan). T cells were isolated from PBMCs using EasySep™ human T cell isolation kit (Stem Cell Technologies, BC, Canada) and cultured with T cell TransAct (Miltenyi Biotech, Bergisch Gladbach, Germany) and 10 ng / mL recombinant human IL-2 (Miltenyi, Bergisch Gladbach, Germany) for 2 days. T cells were cultured in medium containing 10ng / mL recombinant human IL-2, and 3 days after isolation, T cells were transduced with viral supernatant for 5 hours on plates coated with 20ug / mL retronectin. Transduced T cells were expanded for 4 days using G-Rex (WilsonWolf, MN, USA).Transduction efficiency was measured by flow cytometry. Either X-VIVO™ 15 Medium (Lonza, Basel, Switzerland) or CTS™ OpTmizer™ T Cell Expansion SFM (Thermo Fisher Scientific, MA, USA) was used as the culture medium.

[0170] Evaluation of in vitro tumor cytotoxicity of CAR-T cells The in vitro target tumor cell killing activity of CAR-T cells was evaluated against human MSLN-expressing cells. Untransduced (UTD) T cells were evaluated in parallel as a control substance. The target cells used in this study were Capan-2 cells and MSTO-211H-Luc cells, which endogenously express human MSLN. Target cells were seeded and then diluted CAR-T cells or UTD T cells were added to obtain a six-point series of effector (CAR positive): target cell (E:T) ratios: 3:1, 1:1, 0.3:1, 0.1:1, 0.03:1, and 0.01:1. After 48 hours of co-culture, the viability of the target cell lines was measured after washing out the T cells using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega Corp., WI, USA). Relative killing activity was calculated by comparing the cell viability of target cells in T cell co-culture conditions with that in non-T cell co-culture conditions.

[0171] The in vitro target cell killing activity of CAR-T cells and UTD T cells against Capan-2 and MSTO-211H-Luc cells is shown in Figure 2. All CAR-T cells tested showed a dose-dependent increase in target cell killing activity against Capan-2 and MSTO-211H-Luc cells. In contrast, UTD T cells show limited ability to kill Capan-2 and MSTO-211H-Luc cells, even at the highest E:T ratio. The second 8-28z_7x19 CAR-T (CAR#305), second 8-BBz_7x19 CAR-T (CAR#309), and second 28-28z_7x19 CAR-T (CAR#311) cells showed higher target cell killing activity and similar activity compared to the third 8-28BBz_7x19 CAR-T cells (CAR#301).

[0172] A description of the anti-MSLN CAR-IL-7-CCL19 constructs used in the experiments is provided below.

[0173] CAR#301 (third 8-28BBz_7x19 containing F2A, pMSGV) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human CD8, CD28, 4-1BB, and CD3z

[0174] CAR#305 (second 8-28z_7x19 containing F2A, pMSGV) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human CD28 and CD3z

[0175] CAR#309 (second 8-BBz_7x19 containing F2A, pMSGV) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human 4-1BB and CD3z

[0176] CAR#311 (28-28z_7x19, pMSGV, containing F2A) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD28, intracellular signaling domains of human CD28 and CD3z

[0177] CAR#334 (third 8-28BBz_7x19 containing F2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human CD8, CD28, 4-1BB, and CD3z

[0178] CAR#314 (second 8-28z_7x19 containing F2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human CD28 and CD3z

[0179] CAR#318 (second 8-BBz_7x19 containing F2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human 4-1BB and CD3z

[0180] CAR#323 (second 28-28z_7x19 containing F2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD28, intracellular signaling domains of human CD28 and CD3z

[0181] CAR#345 (second 28-28z_7x19 containing P2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD28, intracellular signaling domains of human CD28 and CD3z

[0182] CAR#347 (second 8-28z_7x19 containing P2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human CD28 and CD3z

[0183] CAR#348 (third 8-28BBz_7x19 containing P2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human CD8, CD28, 4-1BB, and CD3z

[0184] CAR#349 (second 8-BBz_7x19 containing P2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human 4-1BB and CD3z

[0185] CAR#357 (second 8-BBz_7x19 containing F2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human 4-1BB and CD3z

[0186] CAR#358 (second 8-BBz_7x19 containing T2A, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human 4-1BB and CD3z

[0187] CAR#364 (second 28-28z_7x19, pSFG, containing a non-identical nucleotide P2A sequence) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD28, intracellular signaling domains of human CD28 and CD3z

[0188] CAR#365 (a second 8-BBz_7x19 with a non-identical nucleotide P2A sequence, pSFG) Anti-MSLN CAR DNA fragment encoding anti-MSLN scFv, hinge and transmembrane regions of human CD8, intracellular signaling domains of human 4-1BB and CD3z

[0189] In the constructs used in the above experiments, GSG (peptide linker) was added to the N-terminus of each of the P2A, F2A, and T2A sequences.

[0190] Evaluation of the in vivo antitumor activity of CAR-T cells Female NSG mice were subcutaneously inoculated with 2 million (M) MSLN-positive Capan-2 tumor cells. Seven days after inoculation, CAR-T cells were administered intravenously once at several doses (0.8M, 2M, and 5M for the third 8-28BBz_7x19 CAR-T (CAR#334), 3.2M for the second 8-28z_7x19 (CAR#314) and the second 8-BBz_7x19 CAR-T (CAR#318), and 5M for the second 8-28z_7x19 CAR-T cells (CAR#323) as CAR-positive cell counts). Vehicle control phosphate buffered saline (PBS) or control UTD T cells were administered as control groups. The tumor volume (TV) of each mouse was measured twice weekly.

[0191] Tumor volumes (TV) of mice in each treatment group (5 mice per group) are presented in Figure 3. In the third 8-28BBz_7x19 CAR-T (CAR#334) group at 5M and the second 8-28z_7x19 CAR-T (CAR#314) group at 3.2M, TV tended to gradually increase during the first 3 weeks after administration, and a trend toward a mild decrease in TV was observed. Meanwhile, in the second 28-28z_7x19 CAR-T (CAR#323) group at 5M and the second 8-BBz_7x19 CAR-T (CAR#318) group at 3.2M, tumor shrinkage was observed within 2 weeks after CAR-T treatment, and disappearance of tumor tissue was demonstrated in 3 out of 5 mice in each group, suggesting a complete response (CR). These results demonstrated that second-generation CAR-Ts enhanced with IL-7 and CCL19 had higher antitumor efficacy compared to the third-generation 8-28BBz_7x19 CAR-T construct.

[0192] Assessment of the antitumor activity of CAR-T cells using histopathological evaluation of xenografted tumor tissue Female NSG mice were subcutaneously inoculated with 2 million (M) mesothelin-positive Capan-2 tumor cells. Seven days after inoculation, a single dose of 5M CAR-positive cells was administered intravenously. The constructs tested were the second 8-BBz_7x19 CAR-T (CAR#349) and the second 28-28z_7x19 CAR-T cells (CAR#345). As control groups, the vehicle control phosphate buffered saline (PBS) or control UTD T cells with an equivalent total T cell number were administered. The tumor volume (TV) of each mouse was measured twice weekly. At the endpoint of each group (day 18 for the second 28-28z_7x19 CAR-T (CAR#345) group and day 32 for the UTD or second 8-BBz_7x19 CAR-T (CAR#349) group), tumor xenografts were collected and microscopic examination of tissue slides was performed. See Figure 4A.

[0193] In Figure 4B, the mean tumor volume (TV) of each group was plotted. The second 28-28z_7x19 CAR-T (CAR#345) treatment group showed a rapid increase in mean TV from day 8 after CAR-T administration, and all mice died on day 18 due to GvHD-like symptoms, which were evaluated as a humane endpoint. Meanwhile, in the second 8-BBz_7x19 CAR-T (CAR#349) treatment group, an increase in mean TV was observed from day 15 to day 22, and these enlarged tumor xenografts rapidly decreased by day 32. At day 32, 3 out of 5 mice showed tumor disappearance, suggesting a complete response of the second 8-BBz_7x19 CAR-T cells (CAR#349). Tumor xenografts treated with UTD T cells retained a glandular pattern of tumor cells, and mild infiltration of human CD3-positive T cells was observed. A significant infiltration of human CD3 positive T cells was observed in the tumor xenografts of the second 28-28z_7x19 CAR-T (CAR#345) treatment group. Tumor xenografts taken from the second 8-BBz_7x19 CAR-T (CAR#349) treated mice showed tumor shrinkage at the endpoint, but the remaining xenografts showed moderate infiltration of human CD3 positive T cells. In these xenograft tissues of CAR-T treated mice, the glandular growth pattern of tumor cells was lost and the tissue was filled with infiltrating human T cells, which was quite different from that of UTD T cell treated mice. These results indicated that the increase in tumor burden observed in CAR-T treated mice was the result of accumulation of administered human T cells in the xenografted tumor microenvironment, which can be considered as pseudoprogression of the tumor. This suggests a MOA-driven indication of efficacy resulting from IL-7-dependent T cell proliferation and CCL19-dependent accumulation of T cells.

[0194] Assessment of antitumor activity of CAR-T cells using bioluminescence imaging (BLI) of tumor cells Although mean tumor volume is a common means of assessing tumor burden, as would be expected with the use of CAR-T or other similar test substances, the effect measured by tumor volume (TV) may be complicated if the test substance stimulates the accumulation of immune cells. Despite the absence of observed changes or increases in tumor volume due to inflammation, in vivo assessment of tumor volume using luciferase-expressing tumor cell lines may be a more specific measure of antitumor efficacy in these models, as tumor cell reduction can be specifically measured. Female NSG mice were inoculated intraperitoneally with 5 million (M) MSLN-positive SKOV3-luc tumor cells. Four days after inoculation, CAR-T cells were administered intravenously in a single dose at several doses (0.1M, 0.3M, and 1M for the second 8-BBz_7x19 CAR-T (CAR#365) and the second 28-28z_7x19 CAR-T cells (CAR#364) as CAR-positive cell counts). As a control group, vehicle control phosphate buffered saline (PBS) or control UTD T cells with equivalent total T cell numbers were administered to the 0.3M and 1M UTD groups. Mice were monitored for 28 days. Total flux (TF), a measure of SKOV3-luc cell luminescence, which is proportional to the number of SKOV3-luc tumor cells present in the animal and can be used to evaluate antitumor efficacy, was measured once a week. See Figure 5A.

[0195] The duration of this study was 28 days, but all of the second 28-28z_7x19 CAR-T (CAR#364) treatment groups at doses of 0.1M, 0.3M, and 1M were sacrificed on day 14 after CAR-T injection due to a humane endpoint. Both CAR-T treatment groups (CAR#364 and CAR#365) at a dose of 1M CAR-T cells showed a reduction in mean total flux on day 14, which was first observed at the first measurement on day 7 after CAR-T injection. The reduction in mean TF was sustained throughout the study in the second 8-BBz_7x19 CAR-T (CAR#365) treatment groups at doses of 0.3M and 1M. In the second 8-BBz_7x19 (CAR#365) CAR-T 0.1M dose group, tumor cells remained present throughout the study. TFs in both vehicle control and UTD T cell dose groups increased continuously from day 7 to endpoint. These results show evidence of dose-dependent antitumor activity (measured by a significant reduction in mean TFs, a measure of luminescence) of both the second 8-BBz_7x19 CAR-T (CAR#365) and second 28-28z_7x19 CAR-T (CAR#364) cells in NSG mice bearing SKOV3-luc xenografts. See Figure 5B.

[0196] To evaluate the antigen-independent in vivo cell proliferation ability of CAR-T cells, we investigated the body weight (BW) change of mice after CAR-T administration with or without antigen-expressing tumors. BW loss is usually observed as a symptom of graft-versus-host disease (GvHD) after human T cell injection into mice.

[0197] Female NSG mice were subcutaneously inoculated with 2 million (M) MSLN-positive Capan-2 tumor cells. Seven days after inoculation, a single dose of 3M CAR-positive cells CAR-T cells was administered intravenously. The CAR-T constructs tested were the second 8-28z_7x19 (CAR#347), the second 8-BBz_7x19 CAR-T (CAR#349), and the second 28-28z_7x19 CAR-T (CAR#345). As a control group, a comparable total cell number of UTD T cells (4.4M cells) was administered. These T cells were administered to non-tumor-bearing mice on the same day. The body weight (BW) of each mouse was measured twice weekly.

[0198] The mean percent body weight change for each mouse group of 5 animals is plotted in Figures 6A and 6B.

[0199] In the Capan-2 tumor xenograft model, acute BW loss was observed 11 days after administration of the second 8-28z_7x19 CAR-T cells (CAR#347). BW loss was also observed 14 days after administration of the second 28-28z_7x19 CAR-T cells (CAR#345), but no significant BW loss was observed in either the UTD T cell treatment group or the second 8-BBz_7x19 CAR-T (CAR#349) treatment group. In the non-tumor-bearing state, a significant decrease in mean BW was observed only in the second 8-28z_7x19 CAR-T (CAR#347) treatment group at day 11, but not in the other CAR-T treatment groups or the UTD group. This suggests a high in vivo proliferation capacity of the second 8-28z_7x19 CAR-T (CAR#347) without antigen stimulation, which may be considered a risk of off-target toxicity.

[0200] In vivo safety evaluation of CAR-T cells In tumor-bearing female NSG mice, 7.5 × 10 6The safety of constructs CAR#365 (second 8-BBz_7x19), CAR#364 (second 28-28z_7x19), and non-transduced (UTD) cells was evaluated after a single dose of total cells (equivalent to 3M CAR+ cells). In this study, animals were humanely euthanized 17 days after CAR-T administration and evaluated for changes in serum chemistry, organ weights, and gross and microscopic pathology.

[0201] There were no significant changes in serum chemistries or organ weights among the three groups, and all animals survived until terminal necropsy. Macroscopic findings were present in animals receiving CAR#364 and consisted of lung discoloration and spleen enlargement, both of which were associated with minimal increases in organ weights and microscopically associated with CAR-T cells in these tissues, i.e., the presence of mixed cellular inflammation (lungs) and increased cellularity in the white pulp (spleen).

[0202] UTD cells had minimal putative CAR-T infiltration / inflammation in the lungs and spleen. As the pulmonary pattern was typical, it is believed that this finding may be related to graft-versus-host disease (GvHD). Animals receiving CAR#365 were similar to those receiving UTD cells, with a lower incidence of pulmonary mononuclear infiltrates or mixed cell inflammation, and a higher incidence of putative CAR-T cells engrafting in the spleen and even bone marrow. An additional finding was present in the liver of one animal each (minimal putative CAR-T infiltration, likely consistent with GvHD).

[0203] Animals receiving CAR#364 had more severe disease than those receiving UTD cells, and also had liver findings, suggesting persistent signaling leading to exacerbation of the effects of GvHD and / or other mechanisms of CAR-T activation related to cytokine protection.

[0204] Overall, in non-tumor-bearing female NSG mice, construct CAR#365 was well tolerated, with minimal findings similar to those seen in UTD cells, indicating that findings may be related to GvHD. In normal tissues, construct CAR#365 also showed a superior safety profile compared to construct CAR#364, which showed signs of uncontrolled cell proliferation. See Figures 7A-7C.

[0205] Flow cytometric analysis of T cells administered to Capan-2 xenografted NSG mice Female NOG MHC class I / II KO mice were subcutaneously inoculated with 2 million (M) mesothelin-positive Capan-2 tumor cells. Seven days after tumor inoculation, CAR-T cells were administered intravenously in a single dose of 5M CAR-positive cells. The CAR-T constructs tested were the second 8-BBz_7x19 CAR-T (CAR#364) and the second 28-28z_7x19 CAR-T cells (CAR#365). As a control group, UTD T cells (12.5M cells) with an equivalent total cell number were administered. Blood, spleen, and xenograft tumor tissues were collected on days 13, 27, and 41 after CAR-T injection for the second 8-BBz_7x19 CAR-T (CAR#365) administration group. Similarly, blood and tissues were collected in the second 28-28z_7x19 CAR-T (CAR#364) cell dose group on day 13 after CAR-T injection. All animals in this group were killed on day 13 after CAR-T injection for humane endpoints. For tumor samples, cells were collected using a tumor tissue dissociation reagent kit (TTDR, BD Biosciences). Spleen samples were dissociated and filtered to obtain cell suspensions. Cells were incubated with his-tagged mesothelin in FACS buffer (500 mL DPBS- / 5 mL NaN3 / 10 mL FBS) for 30 minutes at room temperature in the dark. Cells were washed, centrifuged, and subsequently incubated with Zombie NIR in PBS for 15 minutes at room temperature in the dark. Cells were incubated with antibody mixtures (CD3 / FITC, CD8 / BV510, CTLA4 / PE-Cy7, LAG-3 / Alexa Fluor 647, PD-1 / BV421, TIM-3 / PerCP-Cy5.5, anti-hisAb / PE). Washed cells were incubated with BD FACS lysis buffer (BD Biosciences) in DW. After a washing step, cells were resuspended in FACS buffer (500 mL DPBS- / 5 ml NaN3 / 10 mL FBS) and analyzed on a BD FACSLyric™ flow cytometer. Data analysis was performed using FlowJo software (BD Biosciences).

[0206] Expression of exhaustion markers (CTLA4, PD-1, LAG-3, and TIM3) in blood, tumor, and spleen CD3+ cells is shown in Figures 8A-8C. In the periphery (blood and spleen), CAR#365 showed less expression of exhaustion markers than CAR#364. In the tumor, both #364 and #365 had low expression of LAG-3 and TIM-3. For CTLA-4 and PD-1, CAR#365 showed slower expression than CAR#364.

[0207] Evaluation of soluble human mesothelin for CAR-T activation To evaluate the blocking activity of soluble MSLN on CAR-T function, the second 8-BBz_7x19 CAR-T (CAR#349) and the second 28-28z_7x19 CAR-T cells (CAR#345) were pre-incubated with soluble MSLN (sMSLN) at several doses (0.01, 0.03, 0.1, 0.3, and 1 μg / mL) for 24 hours, respectively, and the washed CAR-T cells were incubated with MSLN-positive Capan-2 tumor cells. After 48 hours of co-culture of tumor and CAR-T cells, the co-culture supernatants were collected and human IFNγ secretion levels were measured by ELISA.

[0208] As shown in Figure 9, about 10,000 pg / mL of human IFNγ was secreted from the co-culture supernatant of Capan-2 cells and secondary 28-28z_7x19 CAR-T (CAR#345) cells pre-incubated with vehicle PBS. IFNγ secretion from secondary 8-BBz_7x19 CAR-T cells (CAR#349) was about 7,000 pg / mL under the same conditions. IFNγ secretion from antigen-stimulated secondary 28-28z_7x19 CAR-T cells (CAR#345) was dose-dependently decreased by pre-incubation with sMSLN, but dose-dependent inhibition in IFNγ secretion from antigen-stimulated secondary 8-BBz_7x19 CAR-T cells (CAR#349) was not induced by sMSLN.

[0209] Comparison of IL-7 and CCL19 secretion levels from CAR-T cells The secretion levels of IL-7 and CCL19 were assessed by ELISA using culture supernatants collected from cultured second 8-BBz_7x19 CAR-T (containing P2A, CAR#349), second 8-BBz_7x19 CAR-T (containing F2A, CAR#357), and second 8-BBz_7x19 CAR-T (containing T2A, CAR#358) cells. Quantification of IL-7-CCL19 fusion protein secreted from each CAR-T cell was performed using the MSD system.

[0210] MSD GOLD 96-well streptavidin SECTOR plates (MSD Cat. No.: L15SA-5) were incubated with 250 μL / well of 1× PBST containing 3% BSA for >30 min. The plates were washed 3 times with 1× PBST and blotted to remove excess buffer (hereafter referred to as wash step). Biotin anti-human MIP-3β antibody from the U-PLEX Human MIP-3β Antibody Set (MSD, Cat. No.: B21VA-3) was used as the capture reagent. 25 μL / well of 1× capture antibody was added and incubated for 1 h at room temperature. After the wash step, samples were diluted with an equal volume of assay buffer (1× PBST containing 3% BSA). 50 μL / well of 2× diluted samples were added to the plate and incubated for 1.5 h at room temperature with gentle shaking on a shaker. Following a wash step, a 1x detection antibody solution was prepared by diluting detection antibody from the U-PLEX Human IL-7 Antibody Set (MSD Catalog Number: B21UP-3) 100-fold in assay buffer (1x PBST containing 3% BSA). 50 μL / well of 1x detection antibody solution was added and incubated for 1.5 hours at room temperature with gentle shaking on a shaker. After a wash step, 150 μL of 2x MSD reading buffer was added and the plate was immediately read on the MSD plate reader.

[0211] The secretion levels of IL-7, CCL19, and IL-7 / CCL19 fusion protein from the culture supernatant of each CAR-T cell were shown in Table 2. The second 8-BBz_7x19 CAR-T (containing P2A, CAR#349) cells showed the highest expression levels of IL-7 and CCL19 from the same amount of culture supernatant. The second 8-BBz_7x19 CAR-T (P2A, #349) showed the highest cleaved IL-7 and CCL19 concentrations. In the case of IL-7 / CCL19 fusion protein, the ratio of cleaved / uncleaved fusion protein of the P2A construct was comparable to that of the second 8-BBz_7x19 CAR-T (containing T2A, #358) and smaller than that of the second 8-BBz_7x19 CAR-T (containing F2A, #357).

[0212] [Table 2]

[0213] Assessment of the in vivo antitumor activity of second- and third-generation CAR-T cells using bioluminescence imaging of tumor cells To fairly compare the efficacy of the third-generation 7x19 CAR-T and the second-generation 7x19 CAR-T, the third-generation 8-28BBz_7x19 CAR-T (CAR#348) and the second-generation 8-BBz_7x19 CAR-T (CAR#365) cells were generated using the same retroviral vector components containing the P2A peptide sequence, as shown in FIG. 10A. Female NSG mice were subcutaneously inoculated with 5 million (M) mesothelin-positive HepG2-RedFluc cells. Seven days after inoculation, the third-generation 8-28BBz_7x19 CAR-T (CAR#348) and the second-generation 8-BBz_7x19 CAR-T (CAR#365) cells were administered intravenously once at several doses (0.3M, 1M, and 3M as the number of CAR-positive cells). As control groups, the UTD 3M group was administered either the vehicle control phosphate-buffered saline (PBS) or control untransduced (UTD) T cells with an equivalent total T cell number relative to the CAR-positive cells in 3M.To evaluate the antitumor efficacy of CAR-T cells, the total flux (TF), a measure of the luminescence of HepG2-RedFluc cells proportional to the number of tumor cells present in the animal, was measured once a week.

[0214] Both the second 8-BBz_7x19 CAR-T (CAR#365) and the third 8-28-BBz_7x19 CAR-T (CAR#348) showed a dose-dependent reduction in mean total flux. In the 3M CAR-T group, both groups showed comparable efficacy, but 3 of 5 mice in the third 8-28-BBz_7x19 CAR-T (CAR#348) group died by the end of the study due to a humane endpoint. Significant reductions in TF were also observed in both 1M CAR-T groups by the endpoint, but 1 of 5 mice in the third 8-28-BBz_7x19 CAR-T (CAR#348) had tumors regrow after day 14. In the 0.3M CAR-T group, comparable levels of TF reduction were observed in both CAR-T groups at the endpoint, but the second 8-BBz_7x19 CAR-T (CAR#365) showed good tumor control in all mice tested, whereas one of five tested mice in the third 8-28-BBz_7x19 CAR-T (CAR#348) group did not show tumor regression throughout the study period. These results indicate that the second 8-BBz_7x19 CART (CAR#365) has higher and longer-lasting antitumor activity compared to the third 8-28BBz_7x19 CART (CAR#348). See Figures 10A-B, 11, and 12A-H.

[0215] Example 2 An Open-Label, Dose-Escalation, Phase 1, First-In-Human Study of Engineered Immune Cells Expressing the CAR, IL-7, and CCL19 Described Herein in Adult Patients with Mesothelin-Expressing Advanced or Metastatic Solid Tumors Research design: This is an open-label, non-randomized, Phase 1, first-in-human study evaluating the safety and tolerability of engineered immune cells expressing CAR, IL-7, and CCL19 described herein administered intravenously (IV) to patients with mesothelin-expressing advanced or metastatic solid tumors for which there is no standard alternative treatment with established clinical benefit. Ovarian cancer, mesothelioma, gastric cancer, and non-small lung cell carcinoma (NSCLC) are high-priority target populations for engineered immune cells expressing CAR, IL-7, and CCL19 described herein. Patients with other cancer types will be enrolled based on discussion between the investigator(s) and sponsor. Up to 21 DLT-evaluable patients will be treated with engineered immune cells expressing CAR, IL-7, and CCL19 described herein in one of five proposed dose cohorts. Dose escalation will be guided with a Bayesian optimal interval (BOIN) design based on DLT rates observed at each dose level. Dose escalation / de-escalation decisions will be made within the BOIN recommended dose range, taking into account safety, efficacy, and cell kinetics (CK) other than DLT, and will be made jointly by the sponsor and investigator at meetings, including end-of-cohort meetings. When feasible, additional new translational data (e.g., soluble mesothelin, cancer antigen 125 [CA125]) may also be used to support the decision to dose additional patients at a given dose level. The recommended phase 2 dose (RP2D) will be determined based on the combined observations of safety, efficacy, CK, and biomarker assessments at each dose level. In any dose cohort, the infusion of the modified immune cells expressing CAR, IL-7, and CCL19 described herein to the first and second patients will be separated by 14 days (or more). Subsequent patients may be co-administered. Dose cohorts will be separated by at least 28 days (from the last infusion of the previous cohort to the first infusion of the subsequent cohort). Toxicity will be assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) version 5.0. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) will be assessed according to the American Society for Transplantation and Cellular Therapy (ASTCT) consensus.The CARTOX (CAR-T-cell Therapy Associated Toxicity) recommendations will be used to monitor, grade, and manage toxicities associated with immune effector cell therapy.

[0216] The study consists of the following phases: pre-screening, screening, pre-treatment, treatment and primary follow-up, and secondary follow-up. The pre-screening phase may begin on the date that the patient signs an Institutional Review Board / Independent Ethics Committee (IRB / IEC) approved informed consent form (ICF) for the evaluation of mesothelin expression in tumor cells. The screening phase begins on the date that any one of the procedures of the screening phase is performed. Patients will be provided with a study-specific patient population on the date of the pre-screening ICF or the date of the main ICF, whichever comes first. The screening phase includes confirmation of eligibility and enrollment in the study and ends with the start of the leukapheresis transfusion procedure. The pre-treatment phase is defined as the period from the start of the leukapheresis transfusion procedure through conditioning chemotherapy to the start of the infusion. During the pre-treatment phase, leukapheresis, bridging therapy, and conditioning chemotherapy will be performed. The treatment and primary follow-up phase begins with the administration of the modified immune cells expressing CAR, IL-7, and CCL19 described herein and continues until the 13th month. The secondary follow-up phase begins at the end of the treatment and primary follow-up periods and continues until Month 37. During the pre-treatment, treatment, and primary and secondary follow-up phases, patients may cease participation in the study due to death, withdrawal of consent, or other pre-specified circumstances.

[0217] Patients will be followed according to a visit schedule to ensure that appropriate data are collected on appropriate assessment of the primary and secondary objectives of the study. Patients may voluntarily withdraw or be excluded from the treatment and primary follow-up phase at any time at the investigator's discretion. It is anticipated that patients may leave the primary follow-up and transition to the secondary follow-up for reasons including: -Disease progression - Voluntary withdrawal of the patient from primary follow-up

[0218] Patients who discontinued treatment and the primary follow-up phase before 13 months will continue to be followed in the secondary follow-up phase to collect data required by health authorities (e.g., protocol-defined AEs) up to 3 years after infusion of the engineered immune cells expressing the CAR, IL-7, and CCL19 described herein.

[0219] The first visit in the secondary follow-up phase will be determined according to the time the patient discontinues treatment and the primary follow-up phase. For example, if the patient discontinues treatment and the primary follow-up phase at month 7, the first visit in the secondary follow-up phase will be at month 10.

[0220] Main purpose: -To evaluate the safety and tolerability of the engineered immune cells expressing CAR, IL-7, and CCL19 described herein. - Determining the RP2D of engineered immune cells expressing the CAR, IL-7, and CCL19 described herein.

[0221] Secondary Objectives: -To evaluate the anti-tumor activity of engineered immune cells expressing CAR, IL-7, and CCL19 as described herein. - Characterizing the CK of engineered immune cells expressing the CAR, IL-7, and CCL19 described herein. - Determining the number and percentage of embodiments with a replication-competent retrovirus (RCR) positive test result.

[0222] Target population: Patients with advanced or metastatic solid tumors that express mesothelin. The study will include up to 21 DLT-evaluable patients.

[0223] Dose Levels: The following cohorts of escalating dose levels should be studied: Cohort(-1): 0.3×10 7 chimeric antigen receptor (CAR)(+) cells / individual (if cohort 1 is not tolerated) Cohort 1: 1×10 7CAR(+) cells / individual [starting dose]. Cohort 2: 1×10 8 CAR(+) cells / individual. Cohort 3: 5×10 8 CAR(+) cells / individual. Cohort 4: 1×10 9 CAR(+) cells / individual.

[0224] Route of administration: Intravenous

[0225] Treatment duration: A single IV infusion. Conditioning chemotherapy precedes treatment. (A cohort that does not receive conditioning chemotherapy can be studied.)

[0226] Main criteria for acceptance: 1. Male or female patients aged 20 years or older at the time of signing informed consent. 2. Histologically or cytologically confirmed advanced or metastatic solid tumors for which no standard treatment options with proven clinical benefit are available or are intolerable. 3. Mesothelin expression (positive in ≥50% of viable tumor cells with intensity 2+ and / or 3+) must be determined locally in the tumor by immunohistochemistry using a sponsor-confirmed validated assay, scoring, and staining. A fresh biopsy specimen must be used for eligibility assessment unless an archived biopsy specimen obtained within 6 months prior to the leukapheresis procedure is available. 4. Life expectancy of 12 weeks or more. 5. Eastern Cooperative Oncology Group performance status of 0 or 1. 6. Adequate organ function as determined by laboratory values ​​as specified below: a) Total bilirubin ≤ 1.5 x upper limit of normal (ULN) (except for patients with Gilbert's syndrome). Patients with Gilbert's syndrome may be enrolled with a direct bilirubin ≤ 3 x ULN for direct bilirubin. Elevated indirect bilirubin due to post-transfusion hemolysis is permitted. b) Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) must be less than 3xULN. AST and ALT may be elevated up to 5xULN if the elevation can be reasonably attributed to the presence of metastatic disease of the liver. c) Calculated creatinine clearance >50 mL / min (Cockcroft-Gault formula). d) Hemoglobin must be greater than or equal to 9 g / dL. e) Neutrophil count is 1000 / mm 3 It has to be super. f) Absolute lymphocyte count is 500 / mm 3 It has to be super. g) The platelet count is 75,000 / mm 3 It has to be super. 7. Patients must have radiographically measurable disease as defined by Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST1.1). 8.Female patients who: a) Postmenopausal for at least 1 year (spontaneous amenorrhea) prior to the screening visit, or b) be surgically sterile, or c) if of childbearing potential, agree to simultaneously practice one highly effective method of non-hormonal contraception and one additional effective (barrier) method of contraception from the time of signing the informed consent and through at least 12 months after infusion of the engineered immune cells expressing CAR, IL-7, and CCL19 as described herein, or d) Agree to practice true abstinence when consistent with the subject's preferences and usual lifestyle. Note: Periodic abstinence (e.g., calendar, ovulation, symptomatic, postovulatory methods), withdrawal, spermicide only, and lactational amenorrhea are not acceptable methods of contraception. 9. Male patients (even if surgically sterilized (i.e. after a vasectomy)): a) agree to practice effective barrier contraception from the time of signing the informed consent through at least 12 months following infusion of the engineered immune cells expressing CAR, IL-7, and CCL19 as described herein, or b) Agree to practice true abstinence when consistent with the subject's preferences and usual lifestyle. Note: Periodic abstinence (e.g., calendar, ovulation, symptomatic, postovulatory methods), withdrawal, spermicide only, and lactational amenorrhea are not acceptable methods of contraception. 10. Voluntary written consent must be given prior to carrying out any study-related procedures that are not part of standard medical care, provided that consent may be withdrawn by the patient at any time without affecting future medical care. 11. Willingness and ability to comply with scheduled clinic visits and study procedures.

[0227] Main criteria for exclusion: 1. Active systemic infection. 2. Known hepatitis B surface antigen (HBsAg) positivity or known or suspected active hepatitis C virus (HCV) infection. Patients with hepatitis B core antibody (HBcAb) or hepatitis B surface antibody (HBsAb) positivity may also be enrolled but must have an undetectable hepatitis B virus (HBV) viral load. Patients with hepatitis C virus antibody (HCVAb) positivity must have an undetectable HCV viral load. 3. Coagulation disorders or other major medical illnesses, e.g., respiratory or immune system, and obstructive / restrictive pulmonary disease. 4. Patients with known cardiovascular and cardiopulmonary disease defined as unstable angina, clinically significant arrhythmias, myocardial infarction, congestive heart failure, left ventricular ejection fraction (LVEF) < 45%, or starting oxygen saturation on room air < ​​93%. Well-controlled atrial fibrillation is not excluded, but uncontrolled atrial fibrillation will be excluded. 5. Any serious medical or psychiatric illness that, in the opinion of the investigator, may prevent the completion of treatment according to this protocol. 6. History of malignancy other than non-melanoma skin cancer or carcinoma in situ (e.g., cervix, bladder, breast) unless disease-free for ≥3 years. 7. Disease requiring systemic steroid treatment. 8. Any prior use of cell and gene therapy. 9. Treatment with any investigational product (excluding cell or gene therapy) within 14 days prior to a leukapheresis procedure or 28 days prior to conditioning chemotherapy or treatment with engineered immune cells expressing CAR, IL-7, and CCL19 as described herein. 10. Treatment with leukapheresis transfusion procedures or conditioning chemotherapy or systemic anti-cancer therapy (including immuno-oncology therapy) and radiation therapy within 14 days prior to treatment with modified immune cells expressing CAR, IL-7, and CCL19 as described herein. 11. Treatment with leukapheresis transfusion procedures or conditioning chemotherapy or major surgery within 28 days prior to treatment with modified immune cells expressing CAR, IL-7, and CCL19 as described herein (minor surgical procedures such as catheter placement are not an exclusion criterion). 12. Prior treatment with any mesothelin-targeted therapy. 13. Any non-resolving toxicity of grade 3 or greater from prior anticancer therapy. 14. Patients at risk for bleeding as determined by the investigator. 15. Presence of central nervous system metastases or other significant neurological conditions (patients with central nervous system metastases that have been effectively treated and are stable may be enrolled, if appropriate). 16. Patients who are human immunodeficiency virus (HIV) seropositive and / or human T-cell lymphotropic virus (HTLV) seropositive. 17. Patients with a history of organ transplantation or awaiting organ transplantation. 18. Patients with severe immediate hypersensitivity to any of the agents containing cyclophosphamide, fludarabine, or streptomycin. 19. Admission or evidence of illegal drug use, drug abuse, or alcohol abuse. 20. Live vaccines within 6 weeks prior to initiation of the conditioning regimen. 21. Lactating and breastfeeding female patients or those with a positive serum pregnancy test (a urine pregnancy test is permitted prior to conditioning chemotherapy and treatment with engineered immune cells expressing CAR, IL-7, and CCL19 as described herein).

[0228] NOTE: Lactating female patients will be eligible if they discontinue breastfeeding prior to treatment with engineered immune cells expressing CAR, IL-7, and CCL19 as described herein.

[0229] Main criteria for evaluation and analysis: Primary Endpoint: Incidence of dose-limiting toxicities (DLTs). Incidence of treatment-emergent adverse events (TEAEs). Incidence of clinically significant adverse events (AEs), including severe ICANS, CRS, hemophagocytic lymphohistiocytosis, macrophage activation syndrome, and tumor lysis syndrome.

[0230] Secondary Endpoints: Overall response rate (ORR), disease control rate (DCR), duration of response (DOR), time to progression (TTP), and progression-free survival (PFS) assessed by investigator according to RECIST 1.1 and iRECIST.

[0231] Overall survival (OS).

[0232] CK-related parameters assessed by CAR vector copy number (C max [Maximum (peak) observed peripheral blood drug concentration after a single dose], t max [Time when maximum observed peripheral blood concentration first occurred], C last [Last observed quantifiable concentration in peripheral blood], t last [persistence: time of last observed quantifiable concentration in peripheral blood (days)], AUC [area under the blood concentration-time curve]).

[0233] The number and percentage of embodiments with a positive RCR test result.

[0234] Statistical considerations: Incidence and rates of DLTs and TEAEs will be summarized by International Conference on Harmonisation (ICH) International Medical Dictionary (MedDRA) organ system class and preferred term, and by grade. In addition, the following events will be summarized in the same manner: - Treatment-related TEAEs. - Grade ≥ 3 TEAEs. - Grade ≥ 3 treatment-related TEAEs. - Serious adverse events (regardless of related and unrelated).

[0235] Clinical AEs and RCRs will be summarized by number and percentage.

[0236] For secondary efficacy endpoints, point estimates and two-sided 95% exact binomial confidence intervals will be calculated for binary endpoints, and time-to-event endpoints will be analyzed descriptively using the Kaplan-Meier method.

[0237] CK parameters will be summarized using descriptive statistics. Individual concentration-time data and individual CK parameters will be presented in lists and tabulated with summary statistics by dose cohort. Individual mean concentration-time profiles will be plotted by dose cohort.

[0238] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0239] The technology illustratively described herein can be suitably implemented in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, terms such as "comprise", "include", "contain", etc., should be interpreted expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the illustrated and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the technology claimed.

[0240] Thus, the materials, methods, and examples provided herein are of preferred embodiments and are exemplary, and should not be understood as limiting the scope of the technology.

[0241] The present technology has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the scope of the generic disclosure also form part of the present technology. This includes conditionally negative limitations that remove any subject matter from the general description of the technology, or from the genus, regardless of whether the excised material is specifically described herein.

[0242] Additionally, when features or aspects of the technology are described in terms of a Markush group, one of skill in the art will recognize that the technology is also described in terms of any individual member or subgroup of members of the Markush group.

[0243] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each was individually incorporated by reference. In the case of conflict, the present specification, including definitions, will control.

[0244] Other aspects are within the scope of the following claims.

Claims

**Claim 1** An isolated nucleic acid molecule comprising: a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes human mesothelin, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; a polynucleotide encoding IL-7; and a polynucleotide encoding CCL19 said isolated nucleic acid molecule. **Claim 2** The isolated nucleic acid molecule according to claim 1, wherein said IL-7 is human IL-7. **Claim 3** The isolated nucleic acid molecule according to claim 1, wherein said CCL19 is human CCL19. **Claim 4** The isolated nucleic acid molecule according to claim 1, wherein said antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), said VH comprises three complementarity-determining regions (CDRs) comprising SEQ ID NOs: 1-3, said VL comprises three CDRs comprising SEQ ID NOs: 4-6, optionally, said VH comprises SEQ ID NO: 7, and said VL comprises SEQ ID NO:

8. **Claim 5** The isolated nucleic acid molecule according to claim 1, wherein said antibody comprises a single-chain variable fragment (scFv) format, optionally, said antibody comprises SEQ ID NO:

9. **Claim 6** The isolated nucleic acid molecule according to claim 1, wherein said 4-1BB intracellular region comprises SEQ ID NO: 13, optionally, said CD3ζ intracellular region comprises SEQ ID NO: 14, optionally, said CD8 hinge region comprises SEQ ID NO: 11, optionally, said CD8 transmembrane region comprises SEQ ID NO: 12, and optionally, said 4-1BB intracellular region is upstream of said CD3ζ intracellular region in said isolated nucleic acid molecule. **Claim 7** The isolated nucleic acid molecule according to claim 1, wherein said nucleic acid further comprises a peptide linker 3-10 amino acid residues in length that links said antibody and said CD8 hinge region, optionally, said peptide linker comprises AAA. **Claim 8** The isolated nucleic acid molecule according to claim 1, wherein said isolated nucleic acid molecule further comprises a signal transduction peptide, optionally, said signal transduction peptide is located upstream of said antibody that specifically recognizes human mesothelin in said isolated nucleic acid molecule, and optionally, said signal transduction peptide comprises SEQ ID NO:

15. **Claim 9** The polynucleotide encoding IL-7 and the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a self-cleaving 2A peptide (2A peptide), optionally, the 2A peptide is P2A, optionally, the 2A peptide comprises ATNFSLLKQAGDVEENPGP (SEQ ID NO: 42), and optionally, a peptide linker is further added to the N-terminus of the 2A peptide, and the peptide linker comprises GSG, the isolated nucleic acid molecule according to claim 1.

10. The isolated nucleic acid molecule according to claim 1, wherein the IL-7 comprises SEQ ID NO:

18.

11. The isolated nucleic acid molecule according to claim 1, wherein the CCL19 comprises SEQ ID NO:

19.

12. The polynucleotide encoding the CAR, the polynucleotide encoding IL-7, and the polynucleotide encoding CCL19 are arranged in the nucleic acid molecule in the order of the polynucleotide encoding the CAR - the polynucleotide encoding IL-7 - the polynucleotide encoding CCL19 from the 5'-end to the 3'-end, the isolated nucleic acid molecule according to claim 1.

13. The isolated nucleic acid molecule according to claim 1, wherein the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 16, and optionally, the isolated nucleic acid molecule comprises SEQ ID NO:

17.

14. A vector comprising the nucleic acid molecule according to any one of claims 1 to 13.

15. The vector according to claim 14, wherein the vector is a viral vector, optionally, the vector is an expression vector, and optionally, the vector is a plasmid.

16. The viral vector is selected from a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated virus (AAV) vector, and optionally, the viral vector is a pSFG vector, a pMSG vector, or a pMSCV vector, the vector according to claim 15.

17. An immune cell derived from or isolated from a mammal and comprising the isolated nucleic acid molecule according to any one of claims 1 to 13 or the vector according to any one of claims 14 to 16.

18. Derived from or isolated from a mammal, an immune cell expressing: a) an antibody that specifically recognizes human mesothelin, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region, a chimeric antigen receptor (CAR); b) IL-7; and c) CCL19.

19. The immune cell according to claim 17 or 18, wherein the immune cell is a T cell, a natural killer (NK) cell, a B cell, an antigen-presenting cell, or a granulocyte, optionally a T cell or an NK cell.

20. A pharmaceutical composition comprising the immune cell according to any one of claims 17 to 19 and a pharmaceutically acceptable additive.

21. A method for treating mesothelin-expressing cancer, comprising administering to a subject in need thereof the immune cell according to any one of claims 17 to 19 or the pharmaceutical composition according to claim 20.

22. The method according to claim 21, wherein the mesothelin-expressing cancer is a solid tumor, optionally selected from mesothelioma, colorectal cancer, pancreatic cancer, thymic cancer, cholangiocarcinoma, lung cancer, skin cancer, breast cancer, prostate cancer, bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, spleen cancer, tracheal cancer, bronchial cancer, gastric cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, and bone cancer; optionally, the mesothelin-expressing cancer is a hematopoietic cancer; optionally, the mesothelin-expressing cancer is a sarcoma, optionally selected from chondrosarcoma, Ewing sarcoma, malignant angioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma; optionally, the mesothelin-expressing cancer is a metastatic cancer; optionally, the mesothelin-expressing cancer is a recurrent or refractory cancer.

23. The method according to claim 21, further comprising administering to the subject an additional therapeutic agent or an additional treatment regimen.

24. The method according to claim 23, wherein the additional therapeutic agent comprises a chemotherapeutic agent, an immunotherapeutic agent, a targeted therapy, radiation therapy, or a combination thereof; optionally, the additional treatment regimen comprises a first-line therapy; optionally, the additional treatment regimen comprises surgery.

25. The method according to claim 24, wherein the immune cell according to any one of claims 17 to 19 or the pharmaceutical composition according to claim 20 and the additional therapeutic agent are administered simultaneously or sequentially.

26. The method according to claim 25, wherein the immune cell according to any one of claims 17 to 19 or the pharmaceutical composition according to claim 20 is administered to the subject before or after administration of the additional therapeutic agent.

27. A method for reducing the growth of tumor cells, the method comprising contacting the tumor cells with an immune cell according to any one of claims 17 to 19, thereby reducing the growth of the tumor cells.

28. The use according to claim 27, wherein the method is an in vitro method.

29. A method for producing an immune cell that expresses a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19, the method comprising: introducing the nucleic acid molecule according to any one of claims 1 to 13 or the vector according to any one of claims 14 to 16 into an immune cell to induce the expression of a cell surface molecule that specifically recognizes human mesothelin, IL-7, and CCL19 by the immune cell comprising the above.

30. The method according to claim 29, wherein the immune cell is a T cell, a natural killer (NK) cell, a B cell, an antigen-presenting cell, or a granulocyte, optionally a T cell or an NK cell.

31. A kit comprising the nucleic acid molecule according to any one of claims 1 to 13; the vector according to any one of claims 14 to 16; the immune cell according to any one of claims 17 to 19; or the pharmaceutical composition according to claim 20, and an instruction manual.

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