Cells expressing anti-mesothelin CAR

Abstract

Some aspects of the present disclosure relate to engineered innate lymphoid cells that contain a heterologous targeting construct specific for mesothelin and uses thereof. Some aspects of the present disclosure relate to polynucleotides that encode a chimeric antigen receptor (CAR) that specifically binds human mesothelin, host cells that contain the polynucleotides, and methods of using the same to treat a disease or condition in a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This PCT application claims the benefit of priority to UK Application No. GB2204927.4, filed April 4, 2022, and U.S. Provisional Application No. 63 / 482,752, filed February 1, 2023, each of which is incorporated by reference in its entirety.

[0002] Referencing sequence listings submitted electronically via EFS-Web The contents of the Sequence Listing submitted electronically with this application in an ASCII text file (Name: 3817_165PC01_Seqlisting_ST26, Size: 38,145 bytes, and Creation Date: April 3, 2023) are hereby incorporated by reference in their entirety.

[0003] Field of Disclosure The present invention relates to engineered innate lymphoid cells comprising a heterologous targeting construct specific for mesothelin. The present invention also relates to pharmaceutical compositions comprising said engineered innate lymphoid cells, methods of treatment using said engineered innate lymphoid cells or pharmaceutical compositions, and said engineered innate lymphoid cells or pharmaceutical compositions for use in therapy. The present invention also includes methods of making engineered innate lymphoid cells, and isolated cell populations comprising a plurality of said engineered innate lymphoid cells. [Background technology]

[0004] Mesothelin, encoded by the MSLN gene located on human chromosome 16p.13.3, is a 40 kDa glycophosphatidylinositol (GPI)-linked cell surface glycoprotein expressed by mesothelial cells. Mesothelin is present on the cell surface of normal mesothelial cells, but is also overexpressed in several human tumors, including ovarian, cervical, uterine, gastric, pancreatic, and lung adenocarcinomas. Thus, mesothelin is an attractive candidate for the development of targeted cancer therapies.

[0005] Thus, there is a need for therapies that can target mesothelin-expressing cells. Summary of the Invention

[0006] According to a first aspect of the present invention, there is provided an engineered innate lymphoid cell comprising a heterologous targeting construct specific for mesothelin.

[0007] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising the engineered innate lymphoid cells as described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients.

[0008] According to a further aspect of the present invention there is provided a method of treating a patient in need of treatment using the engineered innate lymphoid cells or pharmaceutical compositions described herein.

[0009] According to a further aspect of the invention there is provided a use of the engineered innate lymphoid cells or pharmaceutical composition described herein for treating a condition in a subject.

[0010] According to a further aspect of the invention there is provided an engineered innate lymphoid cell or a pharmaceutical composition as described herein for use in therapy.

[0011] According to a further aspect of the present invention there is provided a method of making the engineered innate lymphoid cells described herein.

[0012] According to a further aspect of the present invention there is provided an isolated cell population comprising a plurality of engineered innate lymphoid cells as described herein.

[0013] Some embodiments of the present disclosure relate to a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a variable heavy (VH) domain and a variable light (VL) domain, the VH comprising a VH-complementarity determining region 3 (CDR3) comprising the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the CAR further comprises a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the CAR further comprises a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the CAR further comprises a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the CAR further comprises a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the CAR further comprises a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.

[0014] In some embodiments, the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1; a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4; a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.

[0015] In some embodiments, the VH comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:7.

[0016] In some embodiments, the VL comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO:8.

[0017] In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:7 and the VL comprises the amino acid sequence set forth in SEQ ID NO:8.

[0018] In some embodiments, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof. In some embodiments, the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, and (iv) an intracellular signaling domain.

[0019] In some embodiments, the hinge region comprises a hinge region from CD8, CD28, or an immunoglobulin. In some embodiments, the immunoglobulin is selected from the group consisting of IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof. In some embodiments, the hinge region is derived from a CD8 hinge region. In some embodiments, the hinge region comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO:9.

[0020] In some embodiments, the transmembrane domain is selected from the group consisting of CD8, KIRDS2, OX40, CD2, CD4, CD28, CD45, PD1, CD152, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA- 1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, CD19, or any combination thereof. In some embodiments, the transmembrane domain is derived from the CD8 transmembrane domain. In some embodiments, the transmembrane domain comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10. In some embodiments, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0021] In some embodiments, the costimulatory domain comprises a costimulatory domain derived from 4-1BB / CD137, interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, CD28 lacking Lck binding (ICA), BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds CD83, or any combination thereof. In some embodiments, the costimulatory domain is derived from the 4-1BB costimulatory domain. In some embodiments, the costimulatory domain comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO:11.

[0022] In some embodiments, the intracellular signaling domain comprises a CD3 zeta activation domain, a CD3 delta activation domain, a CD3 epsilon activation domain, a CD3 eta activation domain, a CD79A activation domain, a DAP12 activation domain, a FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof. In some embodiments, the intracellular signaling domain comprises a CD3 zeta activation domain. In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0023] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: (a) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (b)(i) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9; (b)(ii) a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: 10, (b)(iii) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11, (b)(iv) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12, or (b)(v) any combination of (b)(i) through (b)(iv).

[0024] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: (i) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; and (ii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0025] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: (i) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; and (ii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0026] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising: (i) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0027] In some embodiments, the polynucleotide further comprises a second nucleotide sequence encoding an armor protein. In some embodiments, the armor protein comprises an IL-15Rβ polypeptide, an IL-15Rα polypeptide, an IL-2Rβ polypeptide, an IL-15 polypeptide, or any combination thereof.

[0028] In some embodiments, the Armor protein comprises an IL-2Rβ polypeptide. In some embodiments, the Armor protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the Armor protein comprises the amino acid sequence set forth in SEQ ID NO: 15.

[0029] In some embodiments, the armor protein comprises an IL-2Rβ polypeptide and the armor protein does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0030] In some embodiments, the armor protein comprises (i) an IL-15Rα polypeptide or (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the armor protein comprises an IL-15Rα polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the armor protein comprises an IL-15Rα polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17.

[0031] In some embodiments, the Armor protein comprises an IL-15 polypeptide. In some embodiments, the Armor protein comprises an IL-15 polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the Armor protein comprises an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16.

[0032] In some embodiments, the armor protein comprises an IL-15Rα polypeptide connected to an IL-15 polypeptide by a linker. In some embodiments, the linker comprises a peptide bond. In some embodiments, the linker comprises a Gly-Ser linker. In some embodiments, the linker comprises an amino acid sequence selected from SEQ ID NOs: 20-25, or any combination thereof.

[0033] In some embodiments, the armor protein comprises (i) an IL-2Rβ polypeptide, (ii) a cleavable linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the cleavable linker comprises a P2A sequence. In some embodiments, the cleavable linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, the cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0034] In some embodiments, the polynucleotide is (a) a chimeric antigen receptor (CAR), comprising: (i) an antigen-binding domain that specifically binds human mesothelin, the antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12; and (b) an armor protein comprising an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15.

[0035] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding (a) a chimeric antigen receptor (CAR), the CAR comprising: (i) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12; and (b) an armor protein, the CAR comprising an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15.

[0036] In some embodiments, the polynucleotide does not comprise a nucleotide sequence encoding an IL-15Rα polypeptide or an IL-15 polypeptide.

[0037] In some embodiments, the polynucleotide comprises: (a) a chimeric antigen receptor (CAR), comprising: (i) an antigen binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 12. and (b) (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18;and (iii) an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein (a) the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and (b) the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;

[0038] In some embodiments, the polynucleotide is (a) a chimeric antigen receptor (CAR), comprising: (i) an antigen-binding domain that specifically binds human mesothelin, the antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; and (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10. (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12; and (b) an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0039] Some aspects of the present disclosure relate to a vector or a set of vectors comprising the polynucleotide disclosed herein.In some embodiments, the vector comprises one or more promoters.In some embodiments, the vector is a retrovirus vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV), a lentivirus vector, or any combination thereof.

[0040] Some aspects of the present disclosure pertain to polypeptides encoded by the polynucleotides disclosed herein.

[0041] Some aspects of the disclosure pertain to host cells comprising a polynucleotide disclosed herein, a vector or set of vectors disclosed herein, or a polypeptide disclosed herein.

[0042] In some embodiments, the host cell is an immune cell. In some embodiments, the immune cell is an innate lymphoid cell. In some embodiments, the immune cell is selected from a T cell, a NK cell, a B cell, or any combination thereof. In some embodiments, the T cell is selected from a γδ T cell, an αβ T cell, or any combination thereof. In some embodiments, the T cell is selected from a V51 T cell and a V52 T cell.

[0043] In some embodiments, the host cell is obtained from a human subject. In some embodiments, the host cell is obtained from a blood sample obtained from a human subject, a skin sample obtained from a human subject, a tumor sample obtained from a human subject, an intestinal tissue sample obtained from a human subject, or any combination thereof. In some embodiments, the host cell is derived from an induced pluripotent stem cell (iPSC).

[0044] Some embodiments of the present disclosure relate to a population of cells comprising the host cells disclosed herein. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the total number of cells in the population of cells are composed of host cells.

[0045] Some aspects of the present disclosure relate to a pharmaceutical composition comprising a polynucleotide disclosed herein, a vector or set of vectors disclosed herein, a polypeptide disclosed herein, a host cell disclosed herein, or a population of cells disclosed herein, and a pharma- ceutically acceptable excipient.

[0046] Some aspects of the present disclosure relate to a method of treating a disease or condition in a subject in need of treatment, comprising administering to the subject a polynucleotide disclosed herein, a vector or set of vectors disclosed herein, a polypeptide disclosed herein, a host cell disclosed herein, a population of cells disclosed herein, or a pharmaceutical composition disclosed herein.

[0047] In some embodiments, the disease or condition comprises cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or acute leukemia, acute myeloid leukemia, chronic myelogenous ... The cancer includes leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, childhood solid tumor, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasm, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including asbestos-induced), or any combination thereof. In some embodiments, the cancer includes small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, mesothelioma, or any combination thereof.

[0048] In some embodiments, the cancer is locally advanced, in some embodiments, the cancer is metastatic, in some embodiments, the cancer is refractory or recurrent.

[0049] In some embodiments, the method further comprises administering an additional anti-cancer agent. In some embodiments, the additional anti-cancer therapy comprises chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof. In some embodiments, the additional anti-cancer therapy comprises chemotherapy.

[0050] In some embodiments, the additional anti-cancer therapy comprises an immune checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy comprises a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR antagonist, or any combination thereof. In some embodiments, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-1. In some embodiments, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-L1. [Brief description of the drawings]

[0051] [Figure 1]Proof of concept for functionality of CAR engineered cells against Meso+ve targets. (A) Blood-derived γδ T cells transduced with a YP218 anti-mesothelin CAR construct and unengineered blood-derived Vδ1 cells ("GDX012") were examined for functionality against solid tumor-targeted OVCAR3 cells. Cryopreserved CAR-modified blood Vδ1 T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1:1, 1:1:2.5 and 1:5 for 18-24 hours in media without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. These data show that engineering GDX012 to express mesothelin-CAR significantly enhanced the intrinsic killing capacity of Vδ1 T cells against OVCAR3 cells. (B) Blood-derived γδ T cells (n=1) transduced with the YP218 anti-mesothelin CAR construct were cultured with HeLa cells at an E:T ratio of 2:1 in media alone or in media supplemented with IL-15 (10 ng / ml). On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. These data indicate that engineering GDX012 to express the mesothelin-CAR significantly improved the intrinsic killing capacity of Vδ1 T cells against HeLa tumor targets and that in addition to CAR signaling, IL-15 is required for the survival and proliferation of mesothelin CAR-modified blood Vδ1 T cells. [Diagram 2] As a proof of concept for targeting solid tumors, GDX012 was engineered with the YP218 anti-mesothelin CAR construct. Mesothelin was selected as a proof of concept solid tumor target because it is overexpressed in a variety of cancer indications and is an established target in a variety of solid tumor models and clinical trials. Expression of mesothelin by healthy tissues is very low, making it an ideal solid tumor target. The anti-MSLN Vδ1 product was functionally tested to understand the phenotype, cytotoxicity, proliferation and in vivo activity to demonstrate solid tumor targeting and establish a framework for future engineered products. [Diagram 3] Unlike skin-resident γδ T cells, GDX01 shows a moderate intrinsic ability to target solid tumor targets, which can be improved by genetic engineering. (A) GDX012 (n=3) and skin-resident γδ T cells (n=3) were examined for functionality against HeLa cells, a mesothelin+ target cell line. Cryopreserved GDX012 and skin-resident γδ T cells were thawed and immediately cultured with target cells without added cytokines in medium at E:T ratios of 5:1, 2.5:1, and 1.25:1 for 18-24 h. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. These data indicate that skin-resident γδ T cells have an intrinsic killing ability against solid tumor targets, lysing more than 80% of tumor targets at an E:T ratio of 5:1, more than 60% at an E:T ratio of 2.5, and more than 50% at an E:T ratio of 1.25. Target cell lysis by GDX012 did not exceed >20% lysis at all E:T ratios profiled against HeLa cells. (B) Blood-derived γδ T cells transduced with YP218 anti-mesothelin CAR construct and GDX012 were examined for functionality against OVCAR3 cells. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1 for 18-24 h in medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. These data demonstrate that engineering GDX012 to express mesothelin-CAR significantly enhanced the intrinsic killing capacity of Vδ1 T cells against OVCAR3 tumor targets, lysing >90% of tumor targets at an E:T ratio of 5:1, >75% at an E:T ratio of 2.5:1, >50% at an E:T ratio of 1.1, and >20% at an E:T ratio of 1:2.5, whereas target cell lysis by GDX012 did not exceed 30% at all E:T ratios profiled against OVCAR3 cells. [Figure 4]Mesothelin CAR-modified blood Vδ1 T cells demonstrated robust cytotoxicity against HeLa (A) and OVCAR3 (B) cells, and this cytotoxicity was comparable to CAR-modified αβ T cells. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were examined for functionality against HeLa and OVCAR3 tumor cells. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18-24 h in medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. [Diagram 5] Mesothelin CAR-modified blood Vδ1 T cells demonstrated robust cytotoxicity against HT29 (A) and A549 (B) cells engineered to overexpress mesothelin, and this cytotoxicity was comparable to CAR-modified αβ T cells. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=2) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were examined for functionality against HT29.MSLN. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were tested for functionality against A549.MSLN cells. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18–24 h in culture medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. [Figure 6]Experimental design for repetitive antigen stimulation (RAS) assay against solid tumor targets. Effector cells are thawed and co-cultured with solid tumor target cancer cell lines at a 2:1 E:T ratio in medium supplemented with IL-15 (10 ng / ml). Day +2: Cultures are replenished with cytokines with a 50% medium change. Day +4: Targets and cytokines are replenished with a 50% medium change. Day +7: Cultures are harvested. Effectors are counted and the same number of effectors as plated on day 0 are re-plated at a 2:1 E:T ratio in medium supplemented with IL-15 (10 mg / ml) with target cells. Additional effectors are used for phenotypic analysis by flow cytometry or plated in a 24-hour killing assay with target cells. Overall, effector cells are subjected to 21 days of culture with a final 24-hour killing assay proving 6 intra-assay target cell killing and a 7th consecutive killing. [Figure 7] Mesothelin CAR modified blood Vδ1 T cells are expanded (A) and enriched for CAR expression (B) after six tumor challenges with Hela cells. Blood-derived γδ T cells (n=2) transduced with YP218 anti-mesothelin CAR construct were cultured with Hela cells at an E:T ratio of 2:1 in medium supplemented with IL-15 (10 ng / ml). On days 7, 14 and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The fold expansion of effector cells at each time point was calculated by dividing the cell number of harvested effector cells by the number of cells plated 7 days prior. The total or cumulative fold expansion was calculated at each time point by multiplying the fold expansion by the previous fold expansion. Cells expanded 60-fold over 21 days. CAR expression was measured on Vδ1 T cells by flow cytometry on days 0, 7, 14 and 21. CAR-expressing V51 T cells were enriched over 21 days, increasing from >20% expression to >90% expression at the end of the assay. Data are representative of two independent experiments. [Figure 8]Mesothelin CAR modified blood Vδ1 T cells are expanded (A) and enriched in response to sequential killing of OVCAR3 and A549.MSLN cells in a RAS assay (B). Blood-derived γδ T cells (n=3) transduced with the YP218 anti-mesothelin CAR construct were cultured with OVCAR3 and A549.MSLN cells at an E:T ratio of 2:1 in medium supplemented with IL-15 (10 ng / ml). On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The fold expansion of effector cells at each time point was calculated by dividing the cell number of harvested effector cells by the number of cells plated 7 days prior. The total (cumulative) fold expansion was calculated at each time point by multiplying the fold expansion by the previous fold expansion. Cells cultured with OVCAR3 cells expanded 20-fold over the 21 days. Cells cultured with A549.MSLN cells expanded 400-fold over 21 days. CAR expression was measured on V51 T cells by flow cytometry at days 0, 7, 14 and 21. CAR-expressing V51 T cells cultured with OVCAR3 and A549.MSLN cells were enriched over 21 days, increasing to >95% expression at day 21. C, V51 frequency was retained over the course of the analysis. [Figure 9]Mesothelin CAR-modified blood Vδ1 T cells remain cytotoxic even after 21 days of RAS against HeLa tumor cells. (A) Blood-derived γδ T cells (n=2) transduced with the YP218 anti-mesothelin CAR construct were cultured with HeLa cells at a 2:1 ratio in medium supplemented with IL-15 (10 ng / ml) for 21 days. On days 7, 14 and 21, fresh target cells were harvested and viable cell counts were obtained using an NC250 cell counter. Target cells were plated at 2×105 cells / well and allowed to attach for 4 h. Effectors were harvested, counted and cultured with adherent target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 for 18–24 h in medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay and data are representative of 7 consecutive kills. Error bars indicate the mean ± SD of triplicate wells for each condition. (B) Flow cytometry analysis of live CAR+ γδ T cells after 7, 14, and 21 days of RAS assay on HeLa cells. Phenotypic analysis of Vδ1 T cells showed that NKG2D, CD56, NKp30, NKG2C, and PD1 expression were upregulated from day 0 to day 21, while CD27, indicative of effector status, was downregulated from day 0 to day 21 by Vδ1 T cells. Data are representative of two independent experiments. [Figure 10]Mesothelin CAR-modified blood Vδ1 T cells remain cytotoxic even after 21 days of RAS against OVCAR3 and A549.MSLN cells. Blood-derived γδ T cells (n=3) transduced with the YP218 anti-mesothelin CAR construct were cultured with OVCAR3 (A-C) and A549.MSLN (D-F) cells at a 2:1 ratio in medium supplemented with IL-15 (10 ng / ml). On days 7 (A and D), 14 (B and E), and 21 (C and F), fresh target cells were harvested and viable cell counts were obtained using an NC250 cell counter. Target cells were plated at 2×105 cells / well and allowed to attach for 4 hours. Effectors were harvested, counted, and cultured with adherent target cells without added cytokines in culture medium for 18-24 h at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. [Figure 11] Mesothelin CAR-modified blood Vδ1 T cells have higher cytotoxicity against MSLN-expressing A549(A) tumor cells than unmodified A549(A) tumor cells. Blood-derived γδ T cells transduced with YP218 anti-mesothelin CAR construct (n=4) and blood-derived αβ T cells transduced with YP218 anti-mesothelin construct (n=1) were tested for functionality against A549 tumor cells and A549 tumor cells modified to overexpress MSLN. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18-24 h in medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. [Figure 12]Skin-derived Vd1+γδ T cells can be efficiently transduced with Meso-CAR. Skin-resident cells were thawed and immediately processed by MACS selection to remove αβ T cells. The resulting negatively selected γδ T cells were then transduced with vectors encoding either the P4 or YP218 anti-mesothelin CAR constructs. The transduced cells were then expanded in the presence of IL-15 (80 ng / ml) and IL21 (11.25 ng / ml) for 14 days before being harvested and cryopreserved. The fold expansion of γδ T cells was recorded on day 14 of the expansion culture (A). The percentage of cells positive for each mesothelin-specific CAR was measured by flow cytometry on day 14 (B). [Figure 13] Skin-derived γδ T cells are cytotoxic against the mesothelin-positive target cell, HeLa cell line. Skin-resident γδ T cells were transduced and expanded as shown in Figures 12A-12B. Cryopreserved cells were then thawed and immediately tested for functionality against the mesothelin+ HeLa cell line. Cells were cultured for 17-20 hours at various effector-to-target ratios without added cytokines. Specific cell lysis for P4 (A) and YP218 (B) binders was detected using the CellTitreGLO cell counting assay. Open circles indicate untransduced skin-resident γδ T cells. Grey dots indicate mesothelin-specific CAR-transduced skin-resident γδ T cells. [Figure 14] Skin-derived γδ T cells are cytotoxic against A459, a mesothelin-positive target cell. Skin-resident γδ T cells transduced with a YP218 anti-mesothelin CAR construct were tested for functionality against the mesothelin+ A549 target cell line. Cryopreserved YP218+ expanded skin-resident γδ T cells were thawed and immediately cultured with target cells for 17–20 h without the addition of cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. [Figure 15]Mesothelin CAR-modified cutaneous Vδ1 T cells are cytotoxic against HT29.MSLN and A549.MSLN tumor cells. Skin-resident γδ and αβ T cells (n=1) transduced with the YP218 anti-mesothelin CAR construct were tested for functionality against the mesothelin+ A549 target cell line. Cryopreserved YP218+ expanded skin-resident γδ T cells were thawed and immediately cultured with target HT29.MSLN (A) and A549.MSLN (B) cells for 18–24 h without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay. Error bars indicate the mean ± SD of triplicate wells for each condition. [Figure 16A] 1 is a graphical representation showing that disarmed P4 and YP218 CAR γδ T cells are effective in a disseminated A549 tumor model in NSG mice. The CARs contained either "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domains (as indicated), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. Data are provided showing that γδ T cells transduced with either binder exhibit effective tumor suppression ("Binder Y"=YP218, "Binder P"=P4). [Figure 16B] 1 is a graphical representation showing that disarmed P4 and YP218 CAR γδ T cells are effective in a disseminated A549 tumor model in NSG mice. The CARs contained either the "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domain (as shown), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. Data are provided showing that anti-MSLN CAR-expressing γδ T cells were able to suppress tumor growth when administered as little as 1 million total cells ("Binder Y"=YP218, "Binder P"=P4). [Figure 16C]1 is a graphical representation showing that disarmed P4 and YP218 CAR γδ T cells are efficacious in a disseminated A549 tumor model in NSG mice. The CARs contained either the "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domain (as indicated), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. Data are provided showing that all γδ T cells expressing either anti-MSLN binder persisted to comparable levels in the bone marrow of the niches evaluated ("Binder Y"=YP218, "Binder P"=P4). [Figure 16D] 1 is a graphical representation showing that disarmed P4 and YP218 CAR γδ T cells are effective in a disseminated A549 tumor model in NSG mice. The CARs contained either the "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domain (as indicated), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. Data are provided showing that all γδ T cells expressing either anti-MSLN binder persisted to comparable levels in the spleen of the niches evaluated ("Binder Y"=YP218, "Binder P"=P4). [Figure 16E] 1 is a graphical representation showing that disarmed P4 and YP218 CAR γδ T cells are effective in a disseminated A549 tumor model in NSG mice. The CARs contained either the "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domain (as indicated), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. Data are provided showing that all γδ T cells expressing either anti-MSLN binder persisted to comparable levels in the lung of the assessed niches ("Binder Y"=YP218, "Binder P"=P4). [Figure 16F]1 is a graphical representation showing that disarmed P4 and YP218 CAR γδ T cells are effective in a disseminated A549 tumor model in NSG mice. The CARs contained either the "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domain (as indicated), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ intracellular signaling domain. Data are provided showing that all γδ T cells expressing either anti-MSLN binder persisted to comparable levels in the blood of the assessed niches ("Binder Y"=YP218, "Binder P"=P4). [Figure 17A] FIG. 1 is a schematic diagram of the two novel armed constructs that were generated. [Figure 17B] Schematic diagram of the corresponding polycistronic gammaretroviral vector in which the chimeric antigen receptor (CAR) coding sequence is fused in-frame to individual IL-15 receptor chain components encoding wild-type or IL-15 fusion (tethered) chain variants. [Figure 18A] 13 is a bar graph showing the percentage of V51 CAR+armed+ cells based on the codon optimization algorithm for formulations transduced with the P4 α.15.β codon sequence. [Figure 18B] 1 is a bar graph showing the armed maximum fluorescence intensity (MFI) based on the codon optimization algorithm for formulations transduced with the P4 α.15.β codon sequence. [Figure 18C] 1 is a bar graph showing the percentage of V51 CAR+armed+ cells based on the codon optimization algorithm for formulations transduced with the YP218 α.15.β codon sequence. [Figure 18D] 1 is a bar graph showing the armed maximum fluorescence intensity (MFI) based on the codon optimization algorithm for formulations transduced with the YP218 α.15.β codon sequence. [Figure 18E] 1 shows the cytotoxicity of mesothelin-targeted CAR engineered cells that were unarmed (Meso-CAR) or armed with mbIL-15 and IL-15Rβ (Meso-CAR.α.15.β). [Figure 19A] 1 is a graphical representation of the results of a sequential killing assay for cells transduced with a disarmed binder P construct or a P4 β construct. [Figure 19B] 1 is a graphical representation of fold proliferation for cells transduced with disarmed binder P or P4 β constructs. [Figure 19C] 1 is a graphical representation of the percentage of cells expressing CAR for cells transduced with disarmed binder P construct or P4 β construct. [Figure 20A] Schematic and results of evaluating the in vivo performance of disarmed and armed MesoCAR modified V51 T cells with mbIL-15 and IL-15Rβ (P4 CAR and P4 CAR.α.15.β, respectively) in a disseminated A549 tumor model. The A549 cell line is engineered to express mesothelin (A549-Meso). A schematic of the model used is shown. [Figure 20B] Schematic and results of evaluating the in vivo performance of disarmed and armed MesoCAR modified V51 T cells with mbIL-15 and IL-15Rβ (P4 CAR and P4 CAR.α.15.β, respectively) in a disseminated A549 tumor model. The A549 cell line is engineered to express mesothelin (A549-Meso). The in vivo efficacy of the tested products is shown. [Figure 21A] FIG. 11 is a graph assessing the effect of in vitro survival and repeated antigen exposure on two different mesothelin-targeting CAR (YP218(1) or P4(2)) modified V51 T that are unarmed, armed with mbIL-15 and IL-15Rβ (CAR.α.15.β) or armed with IL-15Rβ (CAR.β) after cryopreservation. Survival of unarmed and α.15.β armed CAR T cells is shown. [Figure 21B]FIG. 11 is a graph assessing the effect of in vitro survival and repeated antigen exposure on two different mesothelin-targeting CAR (YP218(1) or P4(2)) modified V51 T that are unarmed, armed with mbIL-15 and IL-15Rβ (CAR.α.15.β) or armed with IL-15Rβ (CAR.β) after cryopreservation. Survival of unarmed and α.15.β armed CAR T cells is shown. [Figure 21C] Graph assessing the effect of in vitro survival and repeated antigen exposure on two different mesothelin-targeting CAR (YP218(1) or P4(2)) modified V51 T that were unarmed after cryopreservation, armed with mbIL-15 and IL-15Rβ (CAR.α.15.β) or armed with IL-15Rβ (CAR.β). Tumor confluence measured by following GFP fluorescence over time after treatment with different Meso-CART cell products is shown. [Figure 21D] FIG. 11 is a graph assessing the effect of in vitro survival and repeated antigen exposure on two different mesothelin-targeting CAR (YP218(1) or P4(2)) modified V51 T that are unarmed after cryopreservation, armed with mbIL-15 and IL-15Rβ (CAR.α.15.β) or armed with IL-15Rβ (CAR.β). Fold expansion of CAR T cells following repeated antigen stimulation assays is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Some aspects of the disclosure relate to antigen-binding moieties that specifically bind to human mesothelin. Some aspects of the disclosure relate to a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain that binds to human mesothelin. Some aspects of the disclosure relate to a host cell, e.g., an immune cell, e.g., an innate lymphoid cell, comprising a polynucleotide disclosed herein. Further aspects of the disclosure relate to a method of treating a subject in need of treatment, comprising administering to the subject a polynucleotide and / or a cell.

[0053] I. Terminology In order that this specification may be more readily understood, certain terms are first defined. Further definitions are set forth throughout the detailed description.

[0054] It should be noted that the term "a" element or "an" element refers to one or more of that element; for example, "a nucleotide sequence" is understood to represent one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0055] Furthermore, as used herein, "and / or" shall be construed as specifically disclosing the two specified features or components with or without the other. Thus, the term "and / or" when used in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" when used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0056] Whenever an embodiment is described herein using the term "comprising", it is understood that otherwise similar embodiments described with the terms "consisting of" and / or "consisting essentially of" are also provided. As used herein, the terms "comprise" and "include" and variations thereof (e.g., "comprises", "comprising", "includes" and "including") are understood to indicate the inclusion of a specified component, feature, element, or step or group of components, features, elements, or steps, but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms "comprising", "consisting essentially of" and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide those of ordinary skill in the art with a general dictionary for many of the terms used in this disclosure.

[0058] Units, prefixes, and symbols are indicated in the form recognized by the International System of Units (SI). Numeric ranges are inclusive of the numbers that define the range. When a range of values ​​is listed, it is understood that each intervening integer value between the upper and lower limits of the range listed, and each fraction thereof, is also specifically disclosed, along with each subrange between such values. The upper and lower limits of any range may be independently included or excluded from the range, and each range in which either, neither, or both limits are included is also encompassed by the present disclosure. Thus, ranges listed herein are understood to be shorthand for all values ​​within the range, including the recited endpoints. For example, a range of 1 to 10 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.

[0059] When a value is explicitly recited, it is understood that values ​​of approximately the same quantity or amount as the recited value are also included in the scope of the disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and included in the scope of the disclosure. Conversely, when different elements or groups of elements are individually disclosed, the combination is also disclosed. When any element of a disclosure is disclosed with multiple options, examples of the disclosure in which each option is excluded alone or in any combination with other options are also disclosed herein. Multiple elements of a disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0060] Nucleotides are represented by their widely accepted single-letter symbols. Unless otherwise noted, nucleotide sequences are written left to right in a 5' to 3' orientation. Nucleotides are represented herein by their widely accepted single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Thus, "a" represents adenine, "c" represents cytosine, "g" represents guanine, "t" represents thymine, and "u" represents uracil.

[0061] Amino acid sequences are written left to right in amino to carboxy direction. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0062] The term "about" refers to a value or composition that is within an acceptable error range for a particular value or composition, as determined by one of ordinary skill in the art, which will depend in part on the method of measuring or determining the value or composition, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation, as is customary in the art. Alternatively, "about" may mean a range of up to 10%. Furthermore, particularly with respect to biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When a particular value or composition is given in the present application and claims, unless otherwise specified, the meaning of "about" should be considered to be within an acceptable error range for that particular value or composition.

[0063] The terms "administration," "administering," and grammatical variations thereof, refer to the introduction of a composition of the disclosure (e.g., a polynucleotide encoding a CAR or cells expressing a CAR) into a subject by a pharma- ceutically acceptable route. Introduction of a composition of the disclosure (e.g., a polynucleotide encoding a CAR or cells expressing a CAR) into a subject is by any suitable route, including intratumoral, oral, intrapulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intrathecal, periocular, or topical.

[0064] Administration includes self-administration and administration by another person. A suitable route of administration allows the composition or agent to perform its intended function. For example, if a suitable route is intravenous, the composition is administered by introducing the composition or agent into the subject's vein.

[0065] The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immunocompetent cells, or both. Those skilled in the art will readily appreciate that any macromolecule, including virtually any protein or peptide, can function as an antigen. Additionally, antigens may be derived from recombinant or genomic DNA.

[0066] As used herein, the term "epitope" refers to a portion of an antigen that specifically interacts with an antigen-binding portion, e.g., a CAR or antibody molecule. Such portions, referred to herein as epitopic determinants, typically include or are part of elements such as amino acid side chains or sugar side chains. Epitopes can be defined, for example, by methods known in the art, e.g., by crystallography or hydrogen-deuterium exchange. At least one or some of the portions on an antibody molecule that specifically interact with an epitopic determinant are generally located in the CDR(s). Generally, epitopes have certain three-dimensional structural characteristics. Generally, epitopes have certain charge characteristics. Some epitopes are linear epitopes, while others are conformational epitopes.

[0067] The term "autologous" refers to any material derived from the same individual that is later reintroduced.

[0068] The term "chimeric antigen receptor" or "CAR" as used herein refers to a recombinant polypeptide construct comprising an extracellular antigen-binding domain, a transmembrane domain, and, optionally, an intracellular domain that propagates an activation signal and / or a costimulatory signal to activate the cell. In some embodiments, the CAR comprises an optional leader sequence at the N-terminus of the CAR fusion protein. In some embodiments, the CAR lacks an intracellular (e.g., signaling) domain. Expression of a CAR on the surface of a cell, e.g., an immune cell, allows the cell to target and bind to a specific antigen. In some embodiments, the CAR is expressed by an immune cell, e.g., an αβ T cell, a γδ T cell, or an NK cell. In some embodiments, the antigen-binding domain comprises a Fab, Fab', F(ab')2, Fd, Fv, a single chain variable region fragment (scFv), a single chain antibody, a VHH, a vNAR, a nanobody (single domain antibody), or any combination thereof. In some embodiments, the transmembrane domain comprises a transmembrane domain selected from the transmembrane domains of CD8, CD4, or CD28. In some embodiments, the intracellular domain comprises a costimulatory domain or a portion thereof. In some embodiments, the intracellular domain comprises a costimulatory domain selected from the group consisting of a 4-1BB costimulatory domain, a CD3z, a CD28 costimulatory domain, a CD27 costimulatory domain, and any combination thereof. The CAR may further comprise a "hinge" or "spacer" domain. Non-limiting examples of hinge / spacer domains include a CD8 hinge or an immunoglobulin hinge / spacer domain, such as an IgG1 hinge domain, and an IgG2 hinge domain, an IgG3 hinge domain, or an IgG4 hinge domain.

[0069] The term "intracellular signaling domain" as used herein refers to the intracellular portion of a molecule. The intracellular signaling domain can generate a signal that promotes immune effector function of a cell comprising a CAR, such as an anti-ROR1 CAR T cell described herein. For example, non-limiting examples of immune effector function of a CAR T cell include helper activity, including cytolytic activity and secretion of cytokines. In some embodiments, the intracellular signaling domain is the portion of a protein that transmits effector function signals and prompts a cell to perform a specialized function.

[0070] The term "complementarity determining region" or "CDR" as used herein refers to a sequence of amino acids in an antigen-binding variable region that confers antigen specificity and binding affinity. For example, there are generally three CDRs in each heavy chain variable region (e.g., VH-CDR1, VH-CDR2, and VH-CDR3) and three CDRs in each light chain variable region (VL-CDR1, VL-CDR2, and VL-CDR3). The exact amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those set forth by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme), or a combination thereof. Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (VH-CDR1), 50-65 (VH-CDR2), and 95-102 (VH-CDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (VL-CDR1), 50-56 (VL-CDR2), and 89-97 (VL-CDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids within VH are numbered 26-32 (VH-CDR1), 52-56 (VH-CDR2), and 95-102 (VH-CDR3), and the CDR amino acid residues within VL are numbered 26-32 (VL-CDR1), 50-52 (VL-CDR2), and 91-96 (VL-CDR3). Under the combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both.For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (VH-CDR1), 50-65 (VH-CDR2), and 95-102 (VH-CDR3) in a VH, e.g., a mammalian VH, e.g., a human VH, and amino acid residues 24-34 (VL-CDR1), 50-56 (VL-CDR2), and 89-97 (VL-CDR3) in a VL, e.g., a mammalian VL, e.g., a human VL.

[0071] In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule, wherein the antigen binding domain and the transmembrane domain are linked by a CAR hinge / spacer. In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain by a CAR hinge / spacer and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain by a CAR hinge / spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an antigen binding domain linked to a transmembrane domain by a CAR hinge / spacer and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises an optional leader sequence at the amino terminus (N-terminus) of the CAR. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the antigen binding domain, which is optionally cleaved from the antigen binding domain (e.g., scFv) during cellular processing and localization of the CAR to the cell membrane.

[0072] Although the present application often uses CAR to describe various aspects of the subject matter of the present disclosure, it will be apparent to one of skill in the art that the relevant disclosures provided herein are equally applicable to other chimeric binding proteins. As used herein, the term "chimeric binding protein" refers to a protein that can bind to one or more antigens (e.g., including antigen-binding moieties) and is created by the linkage of two or more heterologous polynucleotides that originally encode separate proteins or fragments of proteins or multiple fragments of the same protein linked in a non-naturally occurring orientation. Non-limiting examples of other chimeric binding proteins include T cell receptors (TCRs) (e.g., engineered TCRs), chimeric antibody-T cell receptors (caTCRs), chimeric signaling receptors (CSRs), T cell receptor mimetics (TCR mimetics), and combinations thereof. Thus, unless otherwise indicated, the term CAR may in some embodiments encompass other types of chimeric binding proteins known in the art, such as those described herein.

[0073] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (e.g., epitope) with its cognate binding domain (e.g., paratope). As used herein, the term "avidity" refers to the overall stability of the complex between a population of epitopes and paratopes (i.e., antigens and antigen-binding domains).

[0074] The term "binds to the same epitope" in reference to two or more antigen-binding moieties means that the antigen-binding moieties bind to the same segment of amino acid residues. An antigen-binding moiety that "competes with another antibody for binding to a target" refers to an antigen-binding moiety that inhibits (partially or fully) binding of the other antibody to the target.

[0075] As used herein, the terms "specific binding," "selective binding," "selectively binds," and "specifically binds" refer to an antigen-binding moiety (e.g., a CAR) that binds to an epitope on a given antigen. Generally, an antigen-binding moiety (e.g., a CAR) exhibits (i) a binding affinity of approximately 10, as measured by surface plasmon resonance (SPR) technology, e.g., in a BIACORE® 2000 instrument, using a given antigen, e.g., human mesothelin, as the target polypeptide and CAR as the ligand. -7 Less than M, e.g., about 10 -8 M, 10 -9 M or 10 -10 and (ii) binds to a given antigen with an affinity that is at least two-fold higher than the affinity of binding to a nonspecific antigen other than the given antigen or a closely related antigen. Thus, an antigen-binding moiety (e.g., a CAR) that "specifically binds to human mesothelin" is one that is at least 10 -7 M or less, for example, about 10 -8 Less than M, 10 -9 Less than M or 10 -10 It refers to an antigen-binding portion (e.g., CAR) that binds to human mesothelin with a KD of less than M.

[0076] As used herein, the term "engineered γδ T cell" refers to a γδ T cell that expresses a transgene (i.e., a gene transduced into the engineered γδ T cell or a parent cell thereof).

[0077] As used herein, the term "stimulated γδ T cells" refers to a starting population (e.g., an endogenous population of γδ T cells) that has been affected by the culture conditions. In some examples, stimulated γδ T cells have a different functional viral entry receptor profile compared to their unstimulated counterparts prior to experiencing the culture conditions. In some embodiments, the stimulated population of γδ T cells is an expanded population of γδ T cells.

[0078] As used herein, an "expanded population of γδ cells" refers to a population of hematopoietic cells comprising γδ T cells that have been cultured under conditions and for a period of time that cause the expansion of γδ cells, i.e., that increases the number of γδ cells. Similarly, an "expanded population of V51 T cells" as used herein refers to a population of hematopoietic cells comprising V51 T cells that have been cultured under conditions and for a period of time that cause the expansion of V51 T cells, i.e., that increases the number of V51 cells. Similarly, an "expanded population of V52 T cells" as used herein refers to a population of hematopoietic cells comprising V52 T cells that have been cultured under conditions and for a period of time that cause the expansion of V52 T cells, i.e., that increases the number of V52 cells.

[0079] As used herein, a "population" of γδ T cells refers to three or more γδ T cells (e.g., at least 10, at least 10 2 , at least 10 3 , at least 10 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , at least 10 12 , or at least 10 13 ) refers to a population of γδ T cells (e.g., engineered γδ T cells). A population of a particular cell type (e.g., a population of endogenous γδ T cells, a population of stimulated γδ T cells, or a population of engineered γδ T cells) refers to that type of cell, and not to different types of cells within the broader population. For example, 8 If 10% of the cells in a starting population of T cells are γδ T cells, then the starting population of γδ T cells is 10 7 It is.

[0080] As used herein, "Armor protein" refers to a protein encoded by a transgene that, when expressed by an immune cell, e.g., a NK cell or a γδ T cell (e.g., a γδ T cell expressing a CAR), enhances the persistence and / or proliferation of the immune cell, e.g., a NK cell or a γδ T cell, and / or the cytotoxicity of the immune cell, e.g., a NK cell or a γδ T cell against a target cell, e.g., by signaling (e.g., cytokine signaling), thereby improving, e.g., cell persistence, cell survival, activation, and other desirable properties. Armor proteins may be membrane-bound or soluble proteins. For example, armor proteins include membrane-bound proteins, e.g., membrane-bound receptors (e.g., IL-15R (e.g., IL-15Rβ and IL-15Rα or fragments thereof) and / or IL-2R (e.g., IL-2Rβ or fragments thereof), αβ, IL-15Rα, IL-2Rβ, IL-2 ... Armor proteins include TCRs, natural cytotoxicity receptors (e.g., NKp30, NKp44, or NKp46), cytokine receptors (e.g., IL-12 receptor), and / or chemokine receptors (e.g., CCR2 receptor), and / or membrane-bound ligands or cytokines (e.g., membrane-bound IL-15, membrane-bound IL-7, membrane-bound CD40L, membrane-bound 4-1BB, membrane-bound 4-1BBL, membrane-bound CCL19). Additionally or alternatively, the armor protein may be a soluble protein, e.g., a soluble ligand or cytokine (e.g., soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, and / or soluble CCL19). In some embodiments, the armor protein is not antigen-specific. In some embodiments, the armor protein is IL-2Rβ. In some embodiments, the IL-2Rβ polypeptide comprises a polypeptide or a fragment thereof. In some embodiments, the IL-2Rβ polypeptide is a recombinant polypeptide. In some embodiments, the IL-2Rβ polypeptide is expressed without IL-2Rα and tethered to IL-15. In some embodiments, the IL-2Rβ polypeptide is expressed without IL-2Rα and tethered to IL-15.

[0081] In some embodiments, the Armor protein comprises two polypeptides. In some embodiments, the Armor protein comprises two polypeptides that are fused, i.e., as contiguous polypeptides. In some embodiments, the Armor protein comprises two polypeptides that combine to form a dimer. In some embodiments, the Armor protein comprises (i) a fusion protein comprising IL-15Rα and IL-15, and (ii) a dimer comprising IL-2Rβ.

[0082] As used herein, "IL-15" refers to native or recombinant IL-15 or variants thereof (e.g., muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity to an isometric portion that acts as an agonist for one or more IL-15 receptor (IL-15R) subunits. IL-15 is a known T cell growth factor that, like IL-2, can support the proliferation of the IL-2-dependent cell line, CTLL-2. IL-15 was first reported as a 114 amino acid mature protein by Grabstein et al. (Science 264.5161:965-969,1994). The term "IL-15" as used herein refers to native or recombinant IL-15 and its muteins, analogs, subunits, or complexes thereof (e.g., receptor complexes described in PCT Publication No. WO2007 / 046006, e.g., sushi peptides), each of which can stimulate proliferation of CTLL-2 cells. In a CTLL-2 proliferation assay, supernatants of cells transfected with recombinantly expressed precursor and in-frame fusions of the mature form of IL-15 can induce CTLL-2 cell proliferation. As used herein, the term "mbIL-15" refers to membrane-bound IL-15Rα tethered to IL-15 (see, e.g., FIG. 17A).

[0083] Human IL-15 can be obtained according to the procedure described by Grabstein et al. (Science 264.5161:965-969, 1994) or by conventional procedures such as polymerase chain reaction (PCR). Human IL-15 cDNA was deposited with the ATCC on February 19, 1993 and assigned accession number 69245.

[0084] The amino acid sequence of human IL-15 (Gene ID 3600) is found in Genbank under accession numbers NP000576.1 GI: 10835153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2). The mouse (Mus musculus) IL-15 amino acid sequence (Gene ID 16168) is found in Genbank under accession number NP_001241676.1 GI: 363000984.

[0085] IL-15 may also refer to IL-15 from various mammalian species, including, for example, human, ape, cow, pig, horse, and mouse. An IL-15 "mutant protein," as referred to herein, is a polypeptide having an amino acid sequence that differs from a native mammalian IL-15 polypeptide due to one or more amino acid deletions, insertions, and / or substitutions. A variant may include a conservative substitution sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions are well known, such as the substitution of whole regions with similar hydrophobic properties. Naturally occurring IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15 protein, where IL-15 binding properties are retained. Alternative splicing of the mRNA can result in truncated but biologically active IL-15 proteins. Mutations resulting from proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-15 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang et al. Cancer 76:687-694, 1995). In some embodiments, the termini or interiors of the protein can be modified with additional amino acids (Clark-Lewis et al. PNAS 90:3574-3577, 1993).

[0086] As used herein, "IL-2 receptor β subunit," "IL-2Rβ," "IL-15 receptor β subunit," and "IL-15Rβ" refer to native or recombinant IL-15Rβ or variants thereof (e.g., muteins, analogs, fragments, isoforms, and peptidomimetics thereof) having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity to an isometric portion that acts as a receptor for one or more IL-2 or IL-15. IL-15Rβ includes full-length and fragments thereof. IL-2Rβ, also known as IL-15Rβ, is a known T cell growth factor receptor that can support the proliferation of the IL-2-dependent cell line, CTLL-2. IL-2Rβ is a mature protein of 551 amino acids. The term "IL-15Rβ" as used herein means native or recombinant IL-2Rβ and its muteins, analogs, subunits, or complexes thereof.

[0087] The amino acid sequence of human IL-2Rβ (gene ID 3560) is found in Genbank under accession numbers NP_000869, NP_001333151, and NP_001333152. The mouse (Mus musculus) IL-2Rβ amino acid sequence (gene ID 16185) is found in Genbank under accession number NP_032394.

[0088] IL-15Rβ may also refer to IL-15Rβ from various mammalian species, including, for example, human, ape, cow, pig, horse, and mouse. IL-15Rβ "mutant protein", as referred to herein, is a polypeptide having an amino acid sequence that differs from a native mammalian IL-15Rβ polypeptide due to one or more amino acid deletions, insertions, and / or substitutions. Variants may include conservative substitution sequences, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, e.g., Lys for Arg, Glu for Asp, or Gln for Asn. Other such conservative substitutions are well known, e.g., substitutions of whole regions with similar hydrophobic properties. Naturally occurring IL-15Rβ variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15Rβ protein, where IL-15Rβ binding properties are retained. Alternative splicing of the mRNA can result in truncated but biologically active IL-15Rβ proteins. Mutations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-15Rβ protein.

[0089] As used herein, "IL-15 receptor alpha subunit" and "IL-15Rα" refer to natural or recombinant IL-15Rα or variants thereof (e.g., muteins, analogs, fragments, isoforms, and peptidomimetics thereof) that act as receptors for IL-15. IL-15Rα includes full-length and fragments thereof. IL-15Rα is a known T cell growth factor receptor. IL-15Rα is a mature protein of 267 amino acids. The term "IL-15Rα" as used herein refers to natural or recombinant IL-15Rα and its muteins, analogs, subunits, or complexes thereof. Fragments of IL-15Rα include soluble fragments, such as sushi peptides described in PCT Publication No. WO2007 / 046006. Soluble fragments (e.g., sushi peptides, e.g., fragments comprising residues 31-95 of IL-15Rα or variants thereof having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity to an isometric portion) also include muteins, analogs, fragments, isoforms, and peptidomimetics thereof. In some embodiments, the soluble fragment is linked to the Fc domain, e.g., Fc-sushi or sushi-Fc.

[0090] The amino acid sequence of human IL-15Rα (gene ID 3601) is found in Genbank under accession numbers NP_001230468, NP_001243694, NP_002180, NP_751950, and NP_001338024. The mouse (Mus musculus) IL-15Rα amino acid sequence (gene ID 16169) is found in Genbank under accession number NP_001258426.

[0091] IL-15Rα may also refer to IL-15Rα from various mammalian species, including, for example, human, ape, cow, pig, horse, and mouse. An IL-15Rα "mutant protein," as referred to herein, is a polypeptide having an amino acid sequence that differs from a native mammalian IL-15Rα polypeptide due to one or more amino acid deletions, insertions, and / or substitutions. A variant may include a conservatively substituted sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, e.g., Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, e.g., between Lys and Arg, Glu and Asp, or Gln and Asn. Other such conservative substitutions, e.g., substitutions of whole regions with similar hydrophobic properties, are well known. Naturally occurring IL-15Rα variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15Rα protein, where IL-15Rα binding properties are retained. Alternative splicing of the mRNA can result in truncated but biologically active IL-15Rα proteins. Mutations resulting from proteolysis include, for example, differences in the N-terminus or C-terminus upon expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (typically 1-10 amino acids) from the IL-15Rα protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang et al. Cancer 76:687-694, 1995). In some embodiments, the termini or interiors of the protein can be modified with additional amino acids (Clark-Lewis et al. PNAS 90:3574-3577, 1993).

[0092] A "polypeptide" refers to a chain comprising at least two consecutively linked amino acid residues, with no upper limit on the length of the chain. One or more amino acid residues of a protein may contain modifications, such as, but not limited to, glycosylation, phosphorylation, or disulfide bond formation. A "protein" may include one or more polypeptides.

[0093] The term "mesothelin" or "CAK1 antigen" as used herein refers to a glycosylphosphatidylinositol-anchored membrane polypeptide involved in cell adhesion (see UNIPROT Reference No. Q13421). Overexpression of mesothelin has been observed in various types of cancer, making it a promising target for CAR-T therapy.

[0094] The term "nucleic acid molecule" as used herein is intended to include DNA molecules and RNA molecules. A nucleic acid molecule may be single-stranded or double-stranded and may be cDNA.

[0095] "Conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, a predicted non-essential amino acid residue in a mesothelin binding moiety (e.g., anti-mesothelin CAR) is replaced with another amino acid residue from the same side chain family.

[0096] The term "tether" or "tethered," as used herein, refers to a polypeptide that, when present, links two or more polypeptides (e.g., IL-15Rα or a fragment thereof (e.g., the sushi domain) and IL-15 or a variant thereof). Generally, a tether can be, for example, about 1-100 (e.g., 5-50, 5-30, 5-20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100) amino acids in length. In some embodiments, the tether is highly flexible and can be rich in glycine (G) and / or serine (S) residues, and can be in the form of GS repeats. In some embodiments, the tether can link the C-terminus of a first protein to the N-terminus of a second protein, while in other embodiments, the tether can attach the C-terminus of a second protein to the N-terminus of a first protein.

[0097] As used herein, the term "percent identity" refers to the percentage of amino acid residues in a candidate sequence, e.g., a VH, VL, or IL-15Rβ variant, that are identical to the amino acid residues of a reference sequence, e.g., a wild-type IL-15Rβ polypeptide, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps may be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison). Alignment to determine percent identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percent amino acid sequence identity of a given candidate sequence to, with, or compared to a given reference sequence (alternatively, it may be expressed as a given candidate sequence having or containing a particular percent amino acid sequence identity to, with, or compared to a given reference sequence) is calculated as follows: 100 x (A / B ratio) where A is the number of amino acid residues recorded as identical in an alignment of the candidate and reference sequences, and B is the total number of amino acid residues in the reference sequence. In some embodiments where the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid sequence identity of the candidate sequence to the reference sequence will not be equal to the percent amino acid sequence identity of the reference sequence to the candidate sequence.

[0098] As used herein, "2A peptide" refers to a class of 18-22 amino acids long peptides that share a core sequence motif of DxExNPGP, where X is any amino acid. The 2A peptide may be the foot and mouth disease virus 18 2A (F2A) peptide, the equine rhinitis A virus 2A (E2A) peptide, the porcine teschovirus-1 2A (P2A) peptide, or the thosea asigna virus 2A (T2A) peptide.

[0099] "Suicide gene" refers to a gene that causes a cell to kill itself. In some embodiments, the suicide gene causes a cell to kill itself by apoptosis. Non-limiting examples of suicide genes include viral thymidine kinase, cytosine deaminase, intracellular antibodies against antioxidant enzymes (AOEs), bacterial nitroreductase, caspase, and DNase.

[0100] In one embodiment, the suicide gene is a viral thymidine kinase (TK). Thymidine kinase is an ATP-thymidine 5'-phosphotransferase that converts deoxythymidine to deoxythymidine 5'-monophosphate, which is further phosphorylated by viral thymidine kinase and nucleoside diphosphate kinase to deoxythymidine diphosphate and then to deoxythymidine triphosphate, respectively. Deoxythymidine triphosphate is incorporated into synthetic DNA molecules by DNA polymerase. Some dNTP analogs, such as ganciclovir (GCV), a synthetic analog of 2'-deoxy-guanosine, have the ability to terminate DNA synthesis upon incorporation into synthetic DNA. Termination of synthesis triggers an apoptotic signal cascade. GCV is not recognized by human thymidine kinase, but is recognized as a substrate for some viral thymidine kinases, such as herpes simplex virus-1 thymidine kinase (HSV-TK). As a result, human cells expressing HSV-TK convert GCV to GCV phosphate, which is further phosphorylated and incorporated into synthetic DNA, leading to termination of synthesis and apoptosis. Variants of HSV-TK include, but are not limited to, HSV-TK, such as TK007 (see Preuss et al., Hum Gene Ther. 2010 Aug;21(8):929-41).

[0101] In some embodiments, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyzes cytosine to uracil and releases ammonia. Under physiological conditions, the modified site is recognized by endonuclease, then the phosphorylation bond in DNA is broken and repair is initiated by the incorporation of new cytosine. However, cytosine deaminase can also convert 5-fluorocytosine to 5-fluorouracil (5-FU). Thus, when providing the non-toxic prodrug 5-FC, cytosine deaminase converts it to the highly toxic 5-FU (suicide inhibitor of thymidylate synthase), resulting in the inhibition of cell proliferation and apoptosis.

[0102] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be ligated. Another type of vector is a viral vector into which additional DNA fragments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of inducing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors that are useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions are also included.

[0103] The term "recombinant host cell" (or unit "host cell"), as used herein, is intended to refer to a cell that contains a nucleic acid that does not naturally occur in the cell, which may be a cell into which a recombinant expression vector has been introduced. Of course, such a term is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

[0104] "Immune response," as understood in the art, generally refers to a biological response in a vertebrate to foreign agents or abnormalities (e.g., cancerous cells) that protects the organism from such agents and the diseases they cause. The immune response is mediated by the action of one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules produced by these cells or the liver, including antibodies, cytokines, and complement, which results in the selective targeting, binding, damage, destruction, and / or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues. An immune response can include, for example, the activation or inhibition of T cells (e.g., effector T cells, Th cells, CD4+ cells, CD8+ T cells, or Treg cells), or the activation or inhibition of any other cell of the immune system (e.g., NK cells).

[0105] "Immunotherapy" refers to treating a subject suffering from a disease or at risk of relapsing or suffering from a disease by methods involving inducing, enhancing, suppressing, or otherwise modifying the immune system or immune response.

[0106] As used herein, the terms "treat", "treatment", or "treatment of" when used in connection with treating a disease or condition, e.g., cancer, in a subject, refer to reducing disease pathology, reducing or eliminating disease symptoms, promoting increased survival, and / or reducing discomfort. For example, treating can refer to the ability of a therapy to reduce one or more disease symptoms, signs, or causes when administered to a subject. Treating can also refer to the alleviation or reduction of one or more clinical symptoms and / or inhibiting or delaying the progression of a condition and / or preventing or delaying the onset of a disease or condition.

[0107] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division can form malignant tumors or cells that can invade adjacent tissues and metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0108] As used herein, the term "effective amount" or "therapeutically effective amount" of an administered therapeutic agent, e.g., immune cells comprising a polynucleotide encoding a CAR, is an amount sufficient to achieve a specifically stated or intended purpose, e.g., to treat or effect cancer treatment. An "effective amount" can be determined empirically in a routine manner for the stated purpose.

[0109] As used herein, the terms "subject," "individual," or "patient" refer to any subject for which diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, etc.

[0110] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM", respectively.

[0111] Various aspects described herein are described in further detail in the following subsections.

[0112] II. Compositions of the Disclosure Some aspects of the disclosure relate to antigen-binding moieties that specifically bind to human mesothelin. Some aspects of the disclosure relate to a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), the CAR comprising an antigen-binding domain that binds to human mesothelin. Some aspects of the disclosure relate to a host cell, e.g., an immune cell, e.g., an innate lymphoid cell, comprising a polynucleotide disclosed herein. Further aspects of the disclosure relate to a method of treating a subject in need of treatment, comprising administering to the subject a polynucleotide and / or a cell.

[0113] According to a first aspect of the present invention, there is provided an engineered innate lymphoid cell comprising a heterologous targeting construct specific for mesothelin.

[0114] Mesothelin, encoded by the MSLN gene located on human chromosome 16p.13.3, is a 40 kDa glycophosphatidylinositol (GPI) linked cell surface glycoprotein expressed by mesothelial cells. The MSLN gene encodes a 71 KD precursor that is cleaved at arginine 295 (Arg295) into two products, a 31 KD soluble N-terminal protein called megakaryocyte potentiating factor (MPF) and mesothelin.

[0115] In one embodiment, the innate lymphoid cells are natural killer (NK) cells. In another embodiment, the innate lymphoid cells are gamma delta (γδ) T cells. In another embodiment, the γδ T cells are Vd1+ γδ cells.

[0116] Reference herein to a "heterologous targeting construct" relates to any targeting construct that is not normally expressed by a cell.

[0117] In one embodiment, the heterologous targeting construct is a chimeric antigen receptor (CAR). CAR is composed of a single chain variable fragment (scFv) consisting of an antibody variable heavy (VH) chain and a variable light (VL) chain fused by a peptide linker. This antigen-binding scFv of CAR is linked to an intracellular CD3ζ signaling domain. First generation CARs contain only CD3ζ, while second generation chimeric antigen receptors contain additional signaling domains such as CD28 or 4-1BB. Third generation CARs containing multiple costimulatory signaling modules have also been reported.

[0118] In another embodiment, the CAR is a full-length CAR or a non-signaling CAR. Reference herein to "full-length chimeric antigen receptor" or "full-length CAR" or "FL CAR" refers to a CAR that includes an intracellular signaling domain. Reference herein to "non-signaling chimeric antigen receptor", "non-signaling CAR", "naked chimeric antigen receptor", "naked CAR" or "NKD CAR" refers to a CAR that lacks the intracellular CD3ζ signaling domain that is typically present in CARs.

[0119] In another embodiment, the CAR is an armed CAR. Reference herein to "armed chimeric antigen receptor" or "armed CAR" refers to CAR cells engineered to secrete cytokines or express ligands known to enhance or interact with endogenous immune cells, such as dendritic cells (DCs), macrophages, or regulatory T cells (Treg cells). The "armor" can enhance the cytotoxicity and specificity of the CAR, avoid immune suppression, avoid host rejection, and extend the therapeutic half-life of the CAR.

[0120] II.A. Chimeric Antigen Receptors Some embodiments of the present disclosure provide innate lymphoid cells (e.g., blood- or skin-derived Vd1 cells) that contain a heterologous nucleic acid encoding a CAR. +In some embodiments, the innate lymphoid cells are selected from the group consisting of blood-derived Vd1 cells, which contain a heterologous nucleic acid molecule encoding a CAR, and in some embodiments, the innate lymphoid cells are selected from the group consisting of blood-derived Vd1 cells, which contain a heterologous nucleic acid molecule encoding a CAR. + In some embodiments, the cells are γδ cells, and the CAR comprises an antigen-binding domain that specifically binds to human mesothelin (e.g., the antigen-binding domain comprises any of the sequences of SEQ ID NOs: 1-8, 41-48). In some embodiments, the cells further comprise a 4-1BB and a CD3 zeta domain. In yet other embodiments, the cells are a population of γδ T cells (e.g., blood- or skin-derived Vd1 ) comprising a γδ T cell that comprises a heterologous nucleic acid encoding a CAR. + In yet another embodiment, the cells are a population of γδ T cells (e.g., a blood- or skin-derived Vd1 CAR or a γδ T cell population) comprising γδ T cells comprising a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to human mesothelin (e.g., the antigen-binding domain comprises any of the sequences of SEQ ID NOs: 1-8, 41-48), and the γδ T cells further comprise a CD8 transmembrane domain. +In another embodiment, the γδ T cells described herein are directed to a γδ T cell (a population of γδ cells) comprising a CAR, the CAR comprises an antigen binding domain that specifically binds human mesothelin (e.g., the antigen binding domain comprises a sequence of any of SEQ ID NOs: 1-8, 41-48), and the γδ T cells further comprise (a) IL-15R-beta or (b)(i) IL-15R-alpha or a variant thereof tethered to IL-15 and (ii) IL-15R-beta. In another embodiment, the γδ T cells described herein comprise a heterologous nucleic acid encoding a CAR, the CAR comprises an antigen binding domain that specifically binds human mesothelin (e.g., the antigen binding domain comprises a sequence of any of SEQ ID NOs: 1-8, 41-48), and may further comprise 4-1BB and a CD3 zeta domain and / or a CD8 transmembrane domain, and (a) IL-15R-beta or (b)(i) IL-15R-alpha or a variant thereof tethered to IL-15 and (ii) IL-15R-beta. In some embodiments, the IL-2Rβ polypeptide is expressed without IL-2Rα and tethered to IL-15. In some embodiments, the IL-15Rβ polypeptide is expressed without IL-2Rα and tethered to IL-15.

[0121] In some embodiments, an antigen binding domain that specifically binds human mesothelin comprises a variable heavy (VH) complementarity determining region-1 (CDR1), a VH-CDR2, a VH-CDR3, a variable light chain (VL) CDR-1, a VL-CDR2, and a VL-CDR3. In some embodiments, the VH-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:3. In some embodiments, the VH-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:2. In some embodiments, the VH-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the VL-CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, the VL-CDR2 comprises the amino acid sequence set forth in SEQ ID NO:5. In some embodiments, the VL-CDR3 comprises the amino acid sequence set forth in SEQ ID NO:6.

[0122] In some embodiments, the innate lymphoid cells are blood-derived Vd1 cells that contain a heterologous nucleic acid molecule encoding a CAR.+ The CAR is a gamma-delta cell, and the CAR comprises an antigen-binding domain that specifically binds to human mesothelin, and the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6.

[0123] In some embodiments, the innate lymphoid cells are blood-derived Vd1 cells comprising a heterologous nucleic acid molecule encoding a CAR. + and a γδ cell, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, (ii) a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8, or (iii) both (i) and (ii).

[0124] In some embodiments, the innate lymphoid cells are blood-derived Vd1 cells comprising a heterologous nucleic acid molecule encoding a CAR. + and a γδ cell, wherein the CAR comprises an antigen-binding domain that specifically binds to human mesothelin, and the CAR comprises (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 7, (ii) a VL comprising the amino acid sequence set forth in SEQ ID NO: 8, or (iii) both (i) and (ii).

[0125] In some embodiments, the antigen-binding domain that specifically binds to human mesothelin comprises the P4 antigen-binding domain (Table 1).

[0126] [Table 1]

[0127] In some embodiments, the innate lymphoid cells are blood-derived Vd1 cells comprising a heterologous nucleic acid molecule encoding a CAR. + The CAR is a gamma-delta cell, and the CAR comprises an antigen-binding domain that specifically binds to human mesothelin, and the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41; a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46.

[0128] In some embodiments, the innate lymphoid cells are blood-derived Vd1 cells comprising a heterologous nucleic acid molecule encoding a CAR. + and a γδ cell, wherein the CAR comprises an antigen-binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47; (ii) a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48; or (iii) both (i) and (ii).

[0129] In some embodiments, the innate lymphoid cells are blood-derived Vd1 cells comprising a heterologous nucleic acid molecule encoding a CAR. +and a γδ cell, wherein the CAR comprises an antigen-binding domain that specifically binds to human mesothelin, and the CAR comprises (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 47, (ii) a VL comprising the amino acid sequence set forth in SEQ ID NO: 48, or (iii) both (i) and (ii).

[0130] II.A.1. Hinge Region In some embodiments, the CAR further comprises a hinge region between the antigen-binding domain that specifically binds to mesothelin and the transmembrane domain. In some embodiments, the hinge is derived from an immunoglobulin (e.g., from a hinge region or a loop region). In certain embodiments, these hinges comprise, for example, an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge region, a fragment thereof (alone or capped with additional sequences, e.g., sequences of the CH1 or CH2 regions), or a combination of fragments of the IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM hinge region. In some embodiments, the hinge comprises, for example, an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM constant domain loop region, a fragment thereof (either alone or capped, for example, with additional sequence from an adjacent beta strand), or a combination of fragments of an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM loop region. In some embodiments, the hinges of the present disclosure comprise sequences from a hinge region, sequences from a loop region, or a combination thereof.

[0131] In some embodiments, the hinge region is selected from a CD8 hinge, a CD28 hinge, and an immunoglobulin hinge. In some embodiments, the hinge comprises a CD8 hinge. In some embodiments, the CAR comprises a hinge comprising the amino acid sequence set forth in SEQ ID NO: 9 (Table 2).

[0132] [Table 2]

[0133] Thus, in some embodiments, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region that comprises the hinge region of CD8, (iii) a transmembrane domain, and (iv) an intracellular domain. In some embodiments, the disclosure provides a CAR (or a polynucleotide encoding a CAR), comprising (i) an antigen binding domain that specifically binds mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region that comprises the amino acid sequence set forth in SEQ ID NO:9, (iii) a transmembrane domain, and (iv) an intracellular domain.

[0134] II.A.2. Transmembrane Domains In some embodiments, the CAR further comprises a transmembrane domain. The transmembrane domain may be derived from either natural or recombinant sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In some embodiments, the transmembrane domain can transmit a signal to the intracellular domain(s) whenever the CAR of the present disclosure binds to a target.

[0135] In some embodiments, the transmembrane domain is selected from the group consisting of, e.g., CD8, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R alpha, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, C and may include at least the transmembrane region(s) of D11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, or CD19.

[0136] In some embodiments, the TM domain is from CD8, CD2, CD4, CD28, CD45, PD1, CD152, or any combination thereof. In some embodiments, the TM domain is from CD8.

[0137] In some embodiments, the TM domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10. In certain embodiments, the TM domain comprises the amino acid sequence set forth in SEQ ID NO:10.

[0138] II.A.3. Costimulatory Domains In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the costimulatory domain comprises a costimulatory domain of 4-1BB / CD137, interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, CD28 lacking Lck binding (ICA), BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor epsilon chain, a ligand that specifically binds CD83, or any combination thereof. In some embodiments, the CAR comprises a 4-1BB costimulatory domain.

[0139] In some embodiments, the costimulatory domain comprises an amino acid sequence having at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11. In certain embodiments, the TM domain comprises the amino acid sequence set forth in SEQ ID NO:11.

[0140] II.A.4. Intracellular Signaling Domains In some embodiments, the CAR further comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a CD3zeta activation domain, a CD3delta activation domain, a CD3epsilon activation domain, a CD3etta activation domain, a CD79A activation domain, a DAP12 activation domain, a FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof. In some embodiments, the intracellular signaling domain comprises a CD3zeta activation domain.

[0141] In some embodiments, the intracellular signaling domain comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 12. In certain embodiments, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO:12.

[0142] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the CAR further comprises a hinge region.

[0143] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain; and (iii) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:10; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the CAR further comprises a hinge region.

[0144] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain; (iii) a costimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR further comprises a hinge region.

[0145] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises: (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0146] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0147] In some embodiments, the CAR comprises: (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9; and (iii) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1. (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0148] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0149] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0150] In some embodiments, the CAR comprises an antigen-binding domain that specifically binds human mesothelin, comprising: (i) a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, and a VL comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; and (ii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0151] In some embodiments, the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, wherein the CAR comprises a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12.

[0152] In some embodiments, the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0153] In some embodiments, the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the CAR further comprises a hinge region.

[0154] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR further comprises a costimulatory domain. In some embodiments, the CAR further comprises a hinge region.

[0155] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a costimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain.In some embodiments, the CAR further comprises a hinge region.

[0156] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises: (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0157] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0158] In some embodiments, the CAR comprises: (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 9; and (iii) a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 47. (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0159] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0160] In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0161] In some embodiments, the CAR comprises an antigen-binding domain that specifically binds human mesothelin, comprising: (i) a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:47, and a VL comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48; and (ii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0162] In some embodiments, the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, wherein the CAR comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 48; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0163] In some embodiments, the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 48; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0164] II.A.4. Weaponization In some embodiments, an engineered immune cell disclosed herein (e.g., an NK cell or a γδ T cell expressing a CAR of the present disclosure) further expresses an Armor protein. In some embodiments, the Armor protein enhances the persistence of the engineered immune cell compared to a similarly engineered immune cell that does not express the Armor protein. In some embodiments, the Armor protein increases the proliferation of the engineered immune cell compared to a similarly engineered immune cell that does not express the Armor protein. In some embodiments, the Armor protein enhances the cytotoxicity of the engineered immune cell compared to a similarly engineered immune cell that does not express the Armor protein.

[0165] In some embodiments, the armor protein comprises a membrane bound polypeptide. In some embodiments, the armor protein comprises a membrane bound receptor. In some embodiments, the membrane bound receptor comprises IL-15R (e.g., IL-15Rβ and IL-15Rα). In some embodiments, the membrane bound receptor comprises IL-2R (e.g., IL-2Rβ). In some embodiments, the membrane bound receptor comprises an αβ TCR. In some embodiments, the membrane bound receptor comprises a natural cytotoxicity receptor (e.g., NKp30, NKp44, or NKp46). In some embodiments, the membrane bound receptor comprises a cytokine receptor (e.g., an IL-12 receptor). In some embodiments, the membrane bound receptor comprises a chemokine receptor (e.g., a CCR2 receptor). In some embodiments, the armor protein comprises a membrane bound ligand or cytokine. In some embodiments, the membrane-bound ligand or cytokine comprises membrane-bound IL-15, membrane-bound IL-7, membrane-bound CD40L, membrane-bound 4-1BB, membrane-bound 4-1BBL, membrane-bound CCL19, or any combination thereof.

[0166] In some embodiments, the armor protein comprises a soluble polypeptide. In some embodiments, the soluble protein comprises a soluble ligand. In some embodiments, the soluble protein comprises a cytokine. In some embodiments, the armor protein comprises soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, soluble CCL19, or any combination thereof.

[0167] In some embodiments, the armor proteins include membrane-bound and soluble polypeptides.

[0168] In some embodiments, the armor protein comprises an IL-2Rβ polypeptide. In some embodiments, the armor protein comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15 (Table 3). In some embodiments, the armor protein comprises the amino acid sequence set forth in SEQ ID NO: 15. In some embodiments, the armor protein further comprises a signal peptide, which is cleaved post-translationally. Any signal peptide may be used. In some embodiments, the armor protein comprises an IL-2Rβ polypeptide, and the armor protein does not comprise an IL-15Rα polypeptide or a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0169] [Table 3]

[0170] In some embodiments, the armor protein comprises an IL-15Rα polypeptide. In some embodiments, the armor protein comprises a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the armor protein comprises an (i) IL-2Rβ polypeptide and (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the armor protein comprises a construct comprising an IL-15Rα polypeptide tethered to an (i) IL-2Rβ polypeptide and (ii) an IL-15 polypeptide.

[0171] In some embodiments, the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the IL-15Rα polypeptide comprises a signal peptide sequence.

[0172] In some embodiments, the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the IL-15 polypeptide comprises a signal peptide sequence.

[0173] In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker. In some embodiments, the linker comprises one or more peptide bonds. In some embodiments, the linker comprises a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the linker is a rigid linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises one or more repeats of the sequence GGGS, GGGS (SEQ ID NO: 19), or a combination thereof. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 20. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 21. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 22. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 23. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 24. In some embodiments, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide by a linker comprising the amino acid sequence set forth in SEQ ID NO: 25.

[0174] In some embodiments, a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide is arranged in the following order from N-terminus to C-terminus: (i) the N-terminus, (ii) the IL-15 polypeptide, (iii) a peptide linker disclosed herein (e.g., a polypeptide comprising GGGS, GGGS (SEQ ID NO: 19), or a combination thereof), (iv) the IL-15Rα polypeptide, (v) the C-terminus. In some embodiments, a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide is arranged in the following order from N-terminus to C-terminus: (i) the N-terminus, (ii) an IL-15 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 16, (iii) a peptide linker disclosed herein (e.g., a polypeptide comprising GGGS, GGGS (SEQ ID NO: 19), or a combination thereof), (iv) an IL-15Rα polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 17, (v) the C-terminus.

[0175] In some embodiments, the Armor protein is expressed as a single polypeptide. In some embodiments, the Armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide, (ii) a linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the Armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide, (ii) a cleavable linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the cleavable linker comprises a P2A sequence. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18. In some embodiments, the armor protein is expressed as a single polypeptide comprising (i) an IL-2Rβ polypeptide, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:18, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0176] In some embodiments, the armor protein comprises: (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18; and (iii) an IL-15 polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18. and a IL-15Rα polypeptide having an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and an IL-15 polypeptide having an amino acid sequence that has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.

[0177] In some embodiments, the armor protein is expressed as a single polypeptide comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:18; and (iii) an IL-15Rα polypeptide linked to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0178] II.B. Nucleic acid molecules Some aspects of the disclosure relate to a polynucleotide or set of polynucleotides encoding a CAR disclosed herein, i.e., a CAR that specifically binds to mesothelin. In some aspects, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to human mesothelin, and the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1; a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2; a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4; a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5; and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6.

[0179] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 7.

[0180] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 8.

[0181] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, wherein the CAR comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7; and (ii) a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8.

[0182] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and (ii) a VL comprising the amino acid sequence set forth in SEQ ID NO:8.

[0183] Some aspects of the disclosure relate to a polynucleotide or set of polynucleotides encoding a CAR disclosed herein, i.e., a CAR that specifically binds to mesothelin. In some aspects, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen-binding domain that specifically binds to human mesothelin, and the CAR comprises a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41; a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42; a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43; a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44; a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45; and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46.

[0184] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 47. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VH comprising the amino acid sequence set forth in SEQ ID NO: 47.

[0185] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 48. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and wherein the CAR comprises a VL comprising the amino acid sequence set forth in SEQ ID NO: 48.

[0186] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, wherein the CAR comprises a VH comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:47; and (ii) a VL comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48.

[0187] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds human mesothelin, and the CAR comprises (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and (ii) a VL comprising the amino acid sequence set forth in SEQ ID NO: 48.

[0188] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, the CAR comprising (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein, (ii) a transmembrane domain as disclosed herein, and (iii) an intracellular signaling domain as disclosed herein. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, the CAR comprising (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein, (ii) a CD8 transmembrane domain, and (iii) an intracellular signaling domain as disclosed herein. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, the CAR comprising (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein, (ii) a transmembrane domain as disclosed herein, and (iii) a CD3 zeta intracellular signaling domain. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, the CAR comprising (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein, (ii) a CD8 transmembrane domain, and (iii) a CD3 zeta intracellular signaling domain.

[0189] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen binding domain disclosed herein, (ii) a transmembrane domain comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 10, and (iii) an intracellular signaling domain disclosed herein. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen binding domain disclosed herein, (ii) a transmembrane domain disclosed herein, and (iii) an intracellular signaling domain comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen binding domain as disclosed herein, (ii) a transmembrane domain comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:10, and (iii) an intracellular signaling domain comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:12.

[0190] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, and the CAR further comprises a hinge region as disclosed herein. In some embodiments, the hinge region comprises a CD8 hinge region. In some embodiments, the hinge region comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:9. In some embodiments, the hinge region comprises the amino acid sequence set forth in SEQ ID NO:9.

[0191] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, and the CAR further comprises a costimulatory region as disclosed herein. In some embodiments, the costimulatory region comprises a 4-1BB costimulatory region. In some embodiments, the costimulatory region comprises an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the amino acid sequence set forth in SEQ ID NO:11. In some embodiments, the costimulatory region comprises the amino acid sequence set forth in SEQ ID NO:11.

[0192] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain; (iii) a costimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a transmembrane domain; (iii) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (iv) an intracellular signaling domain comprising a CD3ζ activation domain.In some embodiments, the CAR further comprises a hinge region.

[0193] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises: (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO:9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0194] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0195] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, the CAR comprising: (i) an antigen-binding domain that specifically binds human mesothelin, the antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; and (ii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0196] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, the antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:10, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12.

[0197] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO:4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO:5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO:6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0198] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, and a VL comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; and (ii) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0199] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, the CAR comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12.

[0200] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0201] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a costimulatory domain; and (iv) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a costimulatory domain comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain.In some embodiments, the CAR comprises (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some embodiments, the CAR further comprises a hinge region.

[0202] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain. In some embodiments, the CAR comprises: (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.In some embodiments, the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0203] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0204] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, the CAR comprising: (i) an antigen-binding domain that specifically binds human mesothelin, the antigen-binding domain comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; and (ii) an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 9. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0205] In some embodiments, the polynucleotide or set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10, (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0206] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0207] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:47, and a VL comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48; and (ii) an antigen binding domain that specifically binds human mesothelin, the antigen binding domain comprising a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48. (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12.

[0208] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, the CAR comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 48; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12.

[0209] In some embodiments, a polynucleotide of the set of polynucleotides encodes a CAR, wherein the CAR comprises: (i) an antigen binding domain that specifically binds human mesothelin, comprising a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 48; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB costimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0210] Some aspects of the disclosure relate to a polynucleotide or set of polynucleotides that encode an Armor protein, i.e., an Armor protein as disclosed herein. In some embodiments, the polynucleotide or set of polynucleotides encodes an Armor protein, and the Armor protein comprises an IL-2Rβ polypeptide (e.g., a recombinant IL-2Rβ polypeptide) (e.g., the Armor comprises a recombinant IL-2Rβ polypeptide, and the polynucleotide or set of polynucleotides does not express an IL-15Rα polypeptide linked to an IL-15 polypeptide). In some embodiments, the polynucleotide or set of polynucleotides encodes an Armor protein, which comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the polynucleotide or set of polynucleotides encodes an Armor protein comprising the amino acid sequence set forth in SEQ ID NO:15. In some embodiments, the polynucleotide or set of polynucleotides encodes an Armor protein, the Armor protein comprising (i) a signal peptide and (ii) an IL-2Rβ polypeptide, the signal peptide being post-translationally cleaved. In some embodiments, the Armor protein comprises an IL-2Rβ polypeptide, the Armor protein does not comprise an IL-15Rα polypeptide or a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0211] In some embodiments, the polynucleotide or set of polynucleotides (i) encodes an IL-2Rβ polypeptide disclosed herein and (ii) does not encode an IL-15Rα polypeptide or a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0212] In some embodiments, the polynucleotide or set of polynucleotides encodes a construct comprising: (i) an IL-2Rβ polypeptide disclosed herein, and (ii) an IL-15Rα polypeptide disclosed herein or an IL-15Rα polypeptide tethered to an IL-15 polypeptide disclosed herein.

[0213] In some embodiments, the polynucleotide or set of polynucleotides encodes an armor protein comprising an IL-15Rα polypeptide. In some embodiments, the polynucleotide or set of polynucleotides encodes an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the polynucleotide or set of polynucleotides encodes an armor protein comprising (i) an IL-2Rβ polypeptide and (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0214] In some embodiments, the polynucleotide or set of polynucleotides encodes an armor protein comprising an IL-15Rα polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In some embodiments, the polynucleotide or set of polynucleotides encodes a signal peptide immediately upstream of the sequence encoding the IL-15Rα polypeptide.

[0215] In some embodiments, the polynucleotide or set of polynucleotides encodes an armor protein comprising an IL-15 polypeptide, wherein the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the polynucleotide or set of polynucleotides encodes a signal peptide immediately upstream of the sequence encoding the IL-15 polypeptide.

[0216] In some embodiments, a single polynucleotide of the disclosure encodes an IL-15Rα polypeptide linked to an IL-15 polypeptide by a linker. In some embodiments, the linker comprises one or more peptide bonds. In some embodiments, the linker is a Gly-Ser linker. In some embodiments, the linker comprises one or more repeats of the sequence GGGS, GGGS (SEQ ID NO: 19), or a combination thereof. In some embodiments, a single polynucleotide of the disclosure encodes an IL-15Rα polypeptide linked to an IL-15 polypeptide by a linker, the linker comprising an amino acid sequence selected from SEQ ID NOs: 20-25.

[0217] In some embodiments, a single polynucleotide of the disclosure encodes (i) an IL-2Rβ polypeptide, (ii) a linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, a single polynucleotide encodes (i) an IL-2Rβ polypeptide, (ii) a cleavable linker, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some embodiments, the cleavable linker comprises a P2A sequence. In some embodiments, the linker comprises an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 18. In some embodiments, a single polynucleotide of the disclosure encodes (i) an IL-2Rβ polypeptide, (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO: 18, and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0218] In some embodiments, the disclosed single polynucleotide comprises: (i) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18; (iii) an IL- and an IL-15Rα polypeptide linked to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16.

[0219] In some embodiments, a single polynucleotide of the disclosure encodes (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:18; and (iii) an IL-15Rα polypeptide linked to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17, and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0220] In some embodiments, (i) the CAR and (ii) the Armor protein are both encoded by a single polynucleotide. In some embodiments, the nucleic acid sequence of the single polynucleotide encoding (i) the CAR and the nucleic acid sequence of the single polynucleotide encoding (ii) the Armor protein are separated by an IRES. In some embodiments, the nucleic acid sequence of the single polynucleotide encoding (i) the CAR and the nucleic acid sequence of the single polynucleotide encoding (ii) the Armor protein are expressed under the control of the same promoter sequence. In some embodiments, the nucleic acid sequence of the single polynucleotide encoding (i) the CAR is expressed under the control of a first promoter, and the nucleic acid sequence of the single polynucleotide encoding (ii) the Armor protein is expressed under the control of a second promoter. In some embodiments, the first promoter and the second promoter are the same. In some embodiments, the first promoter and the second promoter are different.

[0221] Some embodiments of the disclosure include: (A) an antigen-binding domain that specifically binds to human mesothelin, comprising: (i) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a VL-CDR1 having at least about 90% identity with the sequence set forth in SEQ ID NO: 9, at least about (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO: 12; (ii) an IL-2Rβ polypeptide comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;

[0222] Some embodiments of the disclosure include: (A) (i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:7, and a VL comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12. (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;

[0223] Some embodiments of the present disclosure include: (A) an antigen-binding domain that specifically binds to human mesothelin, comprising: (i) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12; and (B) a polynucleotide encoding an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO: 18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0224] Some embodiments of the disclosure relate to a polynucleotide encoding (A) a CAR comprising: (i) an antigen binding domain that specifically binds human mesothelin, the CAR comprising: (i) a VH comprising the amino acid sequence set forth in SEQ ID NO:7 and a VL comprising the amino acid sequence set forth in SEQ ID NO:8; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12; and (B) an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0225] Some embodiments of the disclosure include: (A) an antigen-binding domain that specifically binds to human mesothelin, comprising: (i) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12. (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;

[0226] Some embodiments of the disclosure include: (A)(i) an antigen-binding domain that specifically binds human mesothelin, comprising a VH comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:47, and a VL comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:48; (ii) a hinge region comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:9; (iii) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:10; (iv) a transmembrane domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:11; (B) a CAR comprising (i) an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:15; and (v) an intracellular signaling domain comprising an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 98%, at least about 98%, or at least about 99% sequence identity to the sequence set forth in SEQ ID NO:12. (ii) a linker comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:18;and (iii) a polynucleotide encoding an armor protein comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:17, and the IL-15 polypeptide comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:16;

[0227] Some embodiments of the disclosure include: (A) an antigen-binding domain that specifically binds to human mesothelin, comprising: (i) a VH-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 46; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a CDR comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO:12; and (B) a polynucleotide encoding an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO:15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO:18; and (iii) an IL-15Rα polypeptide linked to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0228] Some embodiments of the disclosure relate to a polynucleotide encoding (A) a CAR comprising: (i) an antigen-binding domain that specifically binds human mesothelin, the CAR comprising: (i) a VH comprising the amino acid sequence set forth in SEQ ID NO: 47 and a VL comprising the amino acid sequence set forth in SEQ ID NO: 48; (ii) a hinge region comprising the amino acid sequence set forth in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence set forth in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence set forth in SEQ ID NO: 12; and (B) an armor protein comprising: (i) an IL-2Rβ polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 15; (ii) a linker comprising the amino acid sequence set forth in SEQ ID NO: 18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide, wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17 and the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0229] In some embodiments, the polynucleotide or set of polynucleotides further comprises a suicide gene as disclosed herein, in some embodiments, the suicide gene encodes a viral thymidine kinase, a cytosine deaminase, an intracellular antibody against an antioxidant enzyme (AOE), a bacterial nitroreductase, a caspase, and a DNase.

[0230] Some aspects of the disclosure relate to vectors comprising a polynucleotide or set of polynucleotides disclosed herein. In some embodiments, the disclosure relates to vectors or set of vectors comprising a polynucleotide encoding a CAR described herein. In some embodiments, the set of vectors comprises a first vector and a second vector, the first vector comprising a nucleic acid sequence encoding a CAR disclosed herein, and the second vector comprising a nucleic acid sequence encoding an armor protein disclosed herein. In some embodiments, the disclosure relates to vectors comprising (i) a nucleic acid sequence encoding a CAR disclosed herein and (ii) a nucleic acid sequence encoding an armor protein disclosed herein.

[0231] Any vector may be suitable for the present disclosure.In some embodiments, vector is a virus vector.In some embodiments, vector is a retrovirus vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenovirus vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adeno-associated virus (AAV), a lentivirus vector, or any combination thereof.

[0232] II.C. Engineered Cells Some aspects of the disclosure relate to engineered cells comprising (i) a polynucleotide encoding a CAR disclosed herein, (ii) a CAR disclosed herein, (iii) a polynucleotide encoding an Armor protein disclosed herein, (iv) an Armor protein disclosed herein, or (v) any combination of (i)-(iv). In some embodiments, the engineered cell is an immune cell. In some embodiments, the engineered cell is an innate lymphoid cell. In some embodiments, the engineered cell is selected from a T cell, a NK cell, a B cell, or any combination thereof. In some embodiments, the T cell is a γδ T cell. In some embodiments, the engineered cell is a Vδ1 T cell. In some embodiments, the engineered cell is a Vδ2 T cell. In some embodiments, the T cell is an αβ T cell. In some embodiments, the engineered cell is a tumor infiltrating lymphocyte (TIL).

[0233] The engineered cells can be obtained from any source prior to engineering the cells. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from a human subject. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from a healthy human subject. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from a human subject suffering from a disease or condition. In some embodiments, the disease or condition involved activation of an immune response in the subject. In some embodiments, the disease or condition comprises cancer. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from a tumor sample of a human subject. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from peripheral blood of a human subject. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from a skin sample obtained from a human subject. In some embodiments, the pre-engineered cells, e.g., innate lymphoid cells, are obtained from an intestinal tissue sample obtained from a subject.

[0234] In some embodiments, the engineered cell comprises a T cell, wherein the T cell comprises a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR. In some embodiments, the engineered cell comprises a T cell, wherein the T cell comprises (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and (ii) a polynucleotide encoding an armor protein disclosed herein.

[0235] In some embodiments, the engineered cell comprises a γδ T cell, wherein the γδ T cell comprises a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR. In some embodiments, the engineered cell comprises a γδ T cell, wherein the γδ T cell comprises (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and (ii) a polynucleotide encoding an armor protein disclosed herein.

[0236] In some embodiments, the engineered cell comprises a V51 T cell, wherein the V51 T cell comprises a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR. In some embodiments, the engineered cell comprises a V51 T cell, wherein the V51 T cell comprises (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and (ii) a polynucleotide encoding an armor protein disclosed herein.

[0237] In some embodiments, the engineered cell comprises a V52 T cell, wherein the V52 T cell comprises a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR. In some embodiments, the engineered cell comprises a V52 T cell, wherein the V52 T cell comprises (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and (ii) a polynucleotide encoding an armor protein disclosed herein.

[0238] In some embodiments, the engineered cell comprises a NK cell, and the NK cell comprises a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR. In some embodiments, the engineered cell comprises a NK cell, and the NK cell comprises (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and (ii) a polynucleotide encoding an armor protein disclosed herein.

[0239] In some embodiments, the engineered cells are derived from induced pluripotent stem cells (iPSCs). In some embodiments, the iPSCs are differentiated into cells expressing one or more markers of innate lymphoid cells, e.g., T cells (e.g., γδ T cells) or NK cells. In some embodiments, the iPSCs are transfected with (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and / or (ii) a polynucleotide encoding an armor protein disclosed herein, prior to differentiation into cells expressing one or more markers of innate lymphoid cells, e.g., T cells (e.g., γδ T cells) or NK cells. In some embodiments, the iPSCs are transfected with (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and / or (ii) a polynucleotide encoding an armor protein disclosed herein, following differentiation into cells expressing one or more markers of innate lymphoid cells, e.g., T cells (e.g., γδ T cells) or NK cells. In some embodiments, iPSCs are transfected with (i) a polynucleotide encoding a CAR disclosed herein, e.g., an anti-mesothelin CAR, and / or (ii) a polynucleotide encoding an Armor protein disclosed herein during differentiation into cells expressing one or more markers of innate lymphoid cells, e.g., T cells (e.g., γδ T cells) or NK cells.

[0240] According to a further aspect of the invention, there is provided an isolated cell population comprising a plurality of engineered innate lymphoid cells as described herein. Some aspects of the present disclosure relate to a population of cells comprising a plurality of engineered innate lymphoid cells as described herein.

[0241] In some embodiments, the engineered innate lymphoid cells may represent more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the total number of cells in the isolated cell population. In some embodiments, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the cells in the population of cells are engineered innate lymphoid cells as disclosed herein.

[0242] II.D. Pharmaceutical Compositions According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising the engineered innate lymphoid cells described herein in combination with one or more pharma- ceutical or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants, such as aluminum hydroxide; and preservatives. Cryopreservation solutions that may be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The compositions may be formulated, for example, for intravenous administration.

[0243] III. Methods of the Disclosure According to further aspects of the present invention, there are provided methods of treating a patient in need of treatment using the engineered innate lymphoid cells or pharmaceutical compositions described herein. Some aspects of the present disclosure relate to methods of treating a disease or condition in a subject in need of treatment, comprising administering to the subject the engineered innate lymphoid cells disclosed herein.

[0244] According to a further aspect of the invention there is provided a use of the engineered innate lymphoid cells or pharmaceutical composition described herein for treating a condition in a subject.

[0245] According to a further aspect of the invention there is provided an engineered innate lymphoid cell or a pharmaceutical composition as described herein for use in therapy.

[0246] The engineered innate lymphoid cells or pharmaceutical compositions may be useful for the treatment of cancer. "Cancer" as used herein refers to the abnormal growth or division of cells. Generally, the growth and / or life span of cancer cells exceeds and is not coordinated with the growth and / or life span of the surrounding normal cells and tissues. Cancer may be benign, pre-malignant, or malignant. Cancers arise in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, reproductive system, (e.g., uterus, ovaries, prostate, testes, etc.), urinary system (e.g., bladder, kidneys, ureters, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0247] In some embodiments, the disease or condition comprises cancer, e.g., the subject is afflicted with cancer. In some embodiments, the cancer comprises a solid tumor type of cancer. In some embodiments, the cancer comprises lung cancer (e.g., small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC)), lung adenocarcinoma, mesothelioma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, chronic or The cancer includes acute leukemia, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, neoplasms of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer (including asbestos-induced), or any combination thereof. In some embodiments, the cancer is locally advanced. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is refractory. In some embodiments, the cancer is recurrent. In some embodiments, the cancer is refractory or recurrent after one or more prior anti-cancer therapies. In some embodiments, the one or more prior anti-cancer therapies include standard of care.

[0248] In some embodiments, the compositions disclosed herein are administered in combination with an additional anti-cancer therapy. In some embodiments, the additional anti-cancer therapy comprises chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof. In some embodiments, the additional anti-cancer therapy comprises chemotherapy. In some embodiments, the additional anti-cancer therapy comprises an immune checkpoint inhibitor. In some embodiments, the additional anti-cancer therapy comprises a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, a LAG-3 antagonist, a GITR antagonist, or any combination thereof. In some embodiments, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-1. In some embodiments, the anti-cancer therapy comprises an antibody or antigen-binding portion thereof that specifically binds to and inhibits PD-L1.

[0249] In some embodiments, the method further comprises pretreating the subject prior to administering the population of immune cells. In some embodiments, the subject is administered chemotherapy prior to administering the population of immune cells. In some embodiments, the subject is administered immunoablative chemotherapy prior to administering the population of immune cells. In some embodiments, the immunoablative chemotherapy comprises cyclophosphamide, fludarabine, or both.

[0250] In some embodiments, the method comprises administering to the subject (i) an engineered innate lymphoid cell disclosed herein and (ii) a cytokine. In some embodiments, the cytokine comprises IL-2, an analog thereof, a variant thereof, or a fragment thereof.

[0251] In some embodiments, the cells of the present disclosure are at least about 1 x 10 6 Cells, at least about 2 x 10 6 Cells, at least about 3 x 10 6 Cells, at least about 4 x 10 6 Cells, at least about 5 x 10 6 cells, 1 x 10 7 Cells, at least about 2 x 107 Cells, at least about 3 x 10 7 Cells, at least about 4 x 10 7 Cells, at least about 5 x 10 7 cells, 1 x 10 8 Cells, at least about 2 x 10 8 Cells, at least about 3 x 10 8 Cells, at least about 4 x 10 8 Cells, at least about 5 x 10 8 cells, 1 x 10 9 Cells, at least about 2 x 10 9 Cells, at least about 3 x 10 9 Cells, at least about 4 x 10 9 cells, or at least about 5×10 9 A dose of cells is administered to the subject.

[0252] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of those in the art and are fully explained in the literature. For example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); DNGlover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. Synthesis; Mullis et al. U.S. Patent No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press)(1986);Perbal(1984)A Practical Guide To Molecular Cloning;the treatise,Methods In Enzymology(Academic Press,Inc.,NY);Miller and Calos eds.(1987)Gene Transfer Vectors For Mammalian Cells,(Cold Spring Harbor Laboratory);Wu et al.,eds.,Methods In Enzymology,Vols.154 and 155;Mayer and Walker,eds.(1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-IV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1986)); Crooke, Antisense drug Technology: Principles, Strategies and Applications,2. nd Ed. CRC Press (2007); and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0253] All references cited above, and all references cited herein, are hereby incorporated by reference in their entirety.

[0254] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0255] Materials and Methods cell culture Retroviral transduction Retroviral vectors were transduced into γδ T cells grown in Retronectin-coated (20 mg / mL) cell expansion bags (PermaLife bags, OriGen Biomedical) at a defined multiplicity of infection (MOI). MOI refers to the number of infectious particles added per cell during transduction (measured by flow cytometry). Viral vectors were diluted in CTS OpTmizer medium. Transduction efficiency was determined at regular intervals 3 days after transduction using flow cytometry.

[0256] Flow cytometry Immunophenotyping was performed using a BD FACSLyric™ flow cytometry instrument. Dead cells were excluded using the LIVE / DEAD™ Fixable Aqua Dead Cell Stain kit (Invitrogen). Flow data is gated on live single Pan γδ+, Vδ1+, CAR+ events.

[0257] Transduction with gammaretroviral vectors encoding anti-MSLN chimeric antigen receptors Cells were grown in the presence of RetroNectin (20 μg / mL) and transduced with a gamma-retroviral vector encoding a mesothelin (MSLN)-targeted chimeric antigen receptor construct. The viral vector was mixed with immune cells diluted in a CTS OpTmizer overnight at 37°C. Transduction efficiency was determined by flow cytometry 4 to 9 days after transduction.

[0258] Thawing of cryopreserved cell products Frozen cryovials were thawed in a 37°C water bath and added to pre-warmed OpTmizer + 2.5% allogeneic plasma. Cells were spun down at 300g for 7 minutes, counted, assessed for viability, and then resuspended at 2x106 cells per mL for phenotyping and downstream assays.

[0259] Viability assay Cells were cultured in 96-well plates for 13 days in the absence of IL-15, and duplicate wells from each condition were harvested on days 3, 7, 10, and 13. Viable Pan- γδ+, CAR+ cells were quantified by flow cytometry using a BD FACS Lyric.

[0260] Repeated antigen stimulation assay Formulations were thawed and co-cultured with A549-MLSN-fluc-gfp cells in 24-well plates (300,000 effectors to 150,000 targets) at a 2:1 E:T ratio (in the absence or presence of 1 ng / mL IL-15) in an Incucyte to follow GFP fluorescence over time as a surrogate for tumor cell lysis. On days 2 and 9 of culture, cells were fed with fresh medium plus or minus 1 ng / mL IL-15 and 50% of the well volume was replaced by careful aspiration after centrifugation. On days 4 and 11 of culture, cells were fed with fresh medium containing targets (150,000 per well) plus or minus 1 ng / mL IL-15 and 50% of the well volume was replaced by careful aspiration after centrifugation. On day 7, all wells were harvested, counted, and replated at 2:1 E:T as on day 0.

[0261] In vivo testing Study 1 (Figure 16): 6-8 week old NSG mice were implanted with 1x106 A549-MSLN-fluc-GFP cells via intravenous (IV) injection. 19 days after implantation, mice were injected with vehicle (n=7, Cryostor CS5, group 1), YP218 γδ CAR T cells totaling 10, 3, or 1 million cells per mouse (n=7, groups 2-4), or P4 γδ CAR T cells totaling 10 million cells per mouse. Starting on day 19, mice were further administered 1ug of human IL-15 daily via IP injection. Mice were imaged twice weekly using an IVIS camera following intraperitoneal (IP) injection of 150mg / kg d-luciferin.

[0262] Study 2 (Figure 20): 6-8 week old NSG mice were implanted with 1x106 A549-MSLN-fluc-GFP cells via intravenous (IV) injection. 19 days after implantation, mice were injected with vehicle (n=7, Cryostor CS5, group 1), P4 disarmed γδ CAR T cells totaling 10 million cells per mouse (n=7, group 2), P4 α.15.β γδ CAR T cells totaling 10 million cells per mouse (n=7, group 3), or P4 β γδ CAR T cells totaling 10 million cells per mouse (n=7, group 4). Mice were imaged twice weekly using an IVIS camera following intraperitoneal (IP) injection of 150 mg / kg d-luciferin. Group 4 mice were administered 1 μg IL-15 daily via IP injection.

[0263] Example 1 Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct and unengineered blood-derived Vδ1 cells ("GDX012") were examined for functionality against solid tumor-targeted OVCAR3 cells. Cryopreserved CAR-modified blood Vδ1 T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1:1, 1:1:2.5 and 1:5 for 18-24 h in culture medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay (Figure 1A). These data indicate that engineering GDX012 to express the mesothelin-CAR significantly enhanced the intrinsic killing capacity of Vδ1 T cells against OVCAR3.

[0264] Blood-derived γδ T cells (n=1) transduced with the YP218 anti-mesothelin CAR construct were cultured with HeLa cells at an E:T ratio of 2:1 in media alone or in media supplemented with IL-15 (10 ng / ml). On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter (Figure 1B). These data indicate that engineering GDX012 to express the mesothelin-CAR significantly improved the intrinsic killing capacity of Vδ1 T cells against HeLa tumor targets and that in addition to CAR signaling, IL-15 is required for the survival and proliferation of mesothelin CAR-modified blood Vδ1 T cells.

[0265] As a proof of concept for targeting solid tumors, GDX012 was engineered with the YP218 anti-mesothelin CAR construct (Figure 2). Mesothelin was selected as a proof of concept solid tumor target because it is overexpressed in a variety of cancer indications and is an established target in a variety of solid tumor models and clinical trials. Expression of mesothelin by healthy tissues is very low, making it an ideal solid tumor target. The anti-MSLN Vδ1 product was functionally tested to understand the phenotype, cytotoxicity, proliferation and in vivo activity to demonstrate solid tumor targeting and establish a framework for future engineered products.

[0266] Unlike skin-resident γδ T cells, GDX01 shows a modest intrinsic ability to target solid tumor targets, which can be improved by genetic engineering. GDX012 (n=3) and skin-resident γδ T cells (n=3) were treated with mesothelin +Functionality against the target cell line, Hela cells, was examined. Cryopreserved GDX012 and skin-resident γδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, and 1.25:1 for 18–24 h in medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay (Figure 3A). These data indicate that skin-resident γδ T cells have an intrinsic killing capacity against solid tumor targets, with >80% of tumor targets lysed at an E:T ratio of 5:1, >60% at an E:T ratio of 2.5, and >50% at an E:T ratio of 1.25. Target cell lysis by GDX012 did not exceed >20% lysis at all E:T ratios profiled against Hela cells. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct and GDX012 were examined for functionality against OVCAR3 cells. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells without added cytokines in culture medium for 18–24 h at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125:1. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay (Figure 3B). These data indicate that engineering GDX012 to express mesothelin-CAR significantly enhanced the intrinsic killing capacity of Vδ1 T cells against OVCAR3 tumor targets, lysing >90% of tumor targets at an E:T ratio of 5:1, >75% at an E:T ratio of 2.5:1, >50% at an E:T ratio of 1.1, and >20% at an E:T ratio of 1:2.5, whereas target cell lysis by GDX012 did not exceed 30% at all E:T ratios profiled against OVCAR3 cells.

[0267] Mesothelin CAR-modified blood Vδ1 T cells demonstrated robust cytotoxicity against HeLa and OVCAR3 cells, and this cytotoxicity was comparable to that of CAR-modified αβ T cells. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were examined for functionality against HeLa tumor cells (Figure 4A) and OVCAR3 tumor cells (Figure 4B). Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18–24 h in culture medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay.

[0268] Mesothelin CAR-modified blood Vδ1 T cells demonstrated robust cytotoxicity against HT29 (FIG. 5A) and A549 (FIG. 5B) cells engineered to overexpress mesothelin, and this cytotoxicity was comparable to CAR-modified αβ T cells. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=2) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were examined for functionality against HT29.MSLN. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were tested for functionality against A549.MSLN cells. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18–24 h in culture medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay.

[0269] Repeated antigen stimulation (RAS) assay for solid tumor targets Effector cells are thawed and co-cultured with solid tumor target cancer cell lines at a 2:1 E:T ratio in medium supplemented with IL-15 (10 ng / ml) (Figure 6). Day +2: Cultures are replenished with cytokines by 50% medium change. Day +4: Target and cytokines are replenished by 50% medium change. Day +7: Cultures are harvested. Effectors are counted and the same number of effectors as plated on day 0 are re-plated at a 2:1 E:T ratio in medium supplemented with IL-15 (10 mg / ml) with target cells. Additional effectors are used for phenotypic analysis by flow cytometry or plated in a 24-hour killing assay with target cells. Overall, effector cells are subjected to 21 days of culture with a final 24-hour killing assay proving 6 intra-assay target cell killing and a 7th consecutive killing.

[0270] Mesothelin CAR modified blood Vδ1 T cells are expanded and enriched for CAR expression after six tumor challenges with Hela cells. Blood-derived γδ T cells (n=2) transduced with YP218 anti-mesothelin CAR construct were cultured with Hela cells at an E:T ratio of 2:1 in medium supplemented with IL-15 (10 ng / ml). On days 7, 14 and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The fold expansion of effector cells at each time point was calculated by dividing the cell number of harvested effector cells by the number of cells plated 7 days prior. The total or cumulative fold expansion was calculated at each time point by multiplying the fold expansion by the previous fold expansion. Cells expanded 60-fold over 21 days (Figure 7A). CAR expression was measured on Vδ1 T cells by flow cytometry on days 0, 7, 14 and 21. CAR-expressing V51 T cells were enriched over 21 days, increasing from >20% expression to >90% expression at the end of the assay (Figure 7B).

[0271] Mesothelin CAR modified blood Vδ1 T cells are expanded and enriched in response to sequential killing of OVCAR3 and A549.MSLN cells in a RAS assay. Blood-derived γδ T cells (n=3) transduced with the YP218 anti-mesothelin CAR construct were cultured with OVCAR3 and A549.MSLN cells at an E:T ratio of 2:1 in medium supplemented with IL-15 (10 ng / ml). On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The fold expansion of effector cells at each time point was calculated by dividing the cell number of harvested effector cells by the number of cells plated 7 days prior. The total (cumulative) fold expansion was calculated at each time point by multiplying the fold expansion by the previous fold expansion. Cells cultured with OVCAR3 cells expanded 20-fold over 21 days, and cells cultured with A549.MSLN cells expanded 400-fold over 21 days (Figure 8A). CAR expression was measured on V51 T cells by flow cytometry on days 0, 7, 14 and 21. CAR-expressing V51 T cells cultured with OVCAR3 and A549.MSLN cells were enriched over 21 days, increasing to >95% expression at day 21 (Figure 8B).

[0272] Blood-derived γδ T cells (n=2) transduced with the YP218 anti-mesothelin CAR construct were cultured with HeLa cells at a 2:1 ratio in medium supplemented with IL-15 (10 ng / ml) for 21 days. Fresh target cells were harvested on days 7, 14, and 21, and viable cell counts were obtained using an NC250 cell counter. Target cells were cultured at 2×10 5 Effectors were plated at 1000 cells / well and allowed to adhere for 4 h. Effectors were harvested, counted, and cultured with adherent target cells without added cytokines in medium for 18–24 h at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay, and data are representative of seven consecutive killings (Figure 9A). Live CAR cells after 7, 14, and 21 days of RAS assay on HeLa cells. +Flow cytometric analysis of γδ T cells was performed. Phenotypic analysis of Vδ1 T cells showed that NKG2D, CD56, NKp30, NKG2C and PD1 expression was upregulated from day 0 to day 21, while CD27, indicative of effector status, was downregulated by Vδ1 T cells from day 0 to day 21 (Figure 9B).

[0273] Mesothelin CAR-modified blood Vδ1 T cells remain cytotoxic after 21 days of RAS against OVCAR3 and A549.MSLN cells. Blood-derived γδ T cells (n=3) transduced with the YP218 anti-mesothelin CAR construct were cultured with OVCAR3 and A549.MSLN cells at a 2:1 ratio in medium supplemented with IL-15 (10 ng / ml) for 21 days. On days 7, 14, and 21, fresh target cells were harvested and viable cell counts were obtained using an NC250 cell counter. Target cells were cultured at 2×10 5 Cells / well were plated and allowed to attach for 4 h. Effectors were harvested, counted, and cultured with adherent target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18-24 h in medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay (Figures 10A-10F).

[0274] Mesothelin CAR-modified blood Vδ1 T cells have higher cytotoxicity against MSLN-expressing A549 (Figure 11B) tumor cells than unmodified A549 (Figure 11A) tumor cells. Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=4) and blood-derived αβ T cells transduced with the YP218 anti-mesothelin construct (n=1) were tested for functionality against A549 tumor cells and A549 tumor cells modified to overexpress MSLN. Cryopreserved CAR-modified blood Vδ T cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 for 18-24 h in culture medium without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay.

[0275] Skin-derived Vd1+ γδ T cells can be efficiently transduced with Meso-CAR. Skin-resident cells were thawed and immediately processed by MACS selection to remove αβ T cells. The resulting negatively selected γδ T cells were then transduced with vectors encoding either the P4 or YP218 anti-mesothelin CAR constructs. The transduced cells were then expanded in the presence of IL-15 (80 ng / ml) and IL21 (11.25 ng / ml) for 14 days before being harvested and cryopreserved. The fold expansion of γδ T cells was recorded on day 14 of the expansion culture (Figure 12A). The percentage of cells positive for each mesothelin-specific CAR was measured by flow cytometry on day 14 (Figure 12B).

[0276] Skin-derived γδ T cells are cytotoxic to the mesothelin-positive target cell, the HeLa cell line (FIGS. 13A-13B). Skin-resident γδ T cells were transduced and expanded as shown in FIGS. 12A-12B. Cryopreserved cells were then thawed and mesothelin-positive cells were then transduced and expanded. + Functionality against the HeLa cell line was immediately tested. Cells were cultured for 17-20 h at various effector-to-target ratios without added cytokines. Specific cell lysis was detected using the CellTitreGLO cell counting assay. Open circles represent untransduced skin-resident γδ T cells, grey dots represent mesothelin-specific CAR-transduced skin-resident γδ T cells.

[0277] Skin-derived γδ T cells are cytotoxic to mesothelin-positive target cells, A459 (FIG. 14). Skin-resident γδ T cells transduced with the YP218 anti-mesothelin CAR construct were cytotoxic to mesothelin-positive target cells, A459 (FIG. 15). + Functionality was tested against the A549 target cell line. Cryopreserved YP218+ expanded skin-resident γδ T cells were thawed and immediately cultured with target cells for 17–20 h without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay.

[0278] Mesothelin CAR-modified cutaneous V51 T cells are cytotoxic to HT29.MSLN (Figure 15A) and A549.MSLN (Figure 15B) tumor cells. Skin-resident γδ and αβ T cells (n=1) transduced with the YP218 anti-mesothelin CAR construct were treated with mesothelin + Functionality against the A549 target cell line was tested. Cryopreserved YP218+ expanded skin-resident γδ T cells were thawed and immediately cultured with target HT29.MSLN and A549.MSLN cells for 18–24 h without added cytokines. Specific cell lysis was detected by a luciferase-based fluorescent cell counting assay.

[0279] Example 2 Blood-derived Vd1+ γδ are transduced with a transgene encoding an anti-mesothelin CAR comprising a P4 antigen-binding domain (e.g., the antigen-binding domain comprises any of the sequences of SEQ ID NOs: 1-8). Optionally, the cells may also be transduced to express 4-1BB and CD3 zeta domains. Optionally, the cells may also be transduced to express a CD8 transmembrane domain and hinge region. The cells are further engineered to express (a) IL-15R-beta or (b) (i) IL-15R-alpha or a variant thereof tethered to IL-15 and (ii) IL-15R-beta. The CAR-expressing cells are assayed for target cell binding and lysis.

[0280] Example 3 Blood-derived γδ T cells transduced with either the YP218 or P4 anti-mesothelin CAR constructs (containing an anti-mesothelin binder, CD8 hinge region, CD8 transmembrane domain, 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain) were evaluated for functionality in a disseminated A549 tumor model. Figure 16A shows that γδ T cells transduced with either binder exhibited effective tumor suppression. Figure 16B shows that anti-MSLN CAR-expressing γδ T cells were able to suppress tumor growth when administered as little as 1 million total cells. 18 days after formulation administration, all mice were treated and bone marrow aspirates from both femurs, spleens, lungs, and peripheral blood (collected into lithium heparin tubes) were harvested. Figures 16C-16F show that γδ T cells expressing either anti-MSLN binder persisted to comparable levels in the niches evaluated.

[0281] Example 4 To improve survival of V51 γδ CAR T cells either in the presence or absence of low IL-15 concentrations, a series of novel arming constructs were designed and evaluated utilizing both YP218 and P4 binders. Polycistronic gammaretroviral vectors were generated in which the chimeric antigen receptor (CAR) coding sequence was fused in frame with the IL-15 receptor chain components. Figure 17A shows a schematic of the arming method used to co-express the CAR and arming moiety(s). Figure 17B shows the domain structure of a conventional armed CAR encoding gammaretroviral vector. Cells transduced with IL-15Rβ alone are referred to as "β" and cells transduced with IL-15Rβ + IL-15Rα tethered to IL-15 are referred to as "α.15.β".

[0282] Example 5 For each of the P4 and YP218 binders armed with α.15.β, two different codon-optimized sequences generated using various optimization algorithms were examined to determine whether this could affect the frequency and relative protein expression of each component of the arming cassette. Preparations were generated from two leukapheresis donors, donor 1 (square) and donor 2 (circle), using each of four different vectors (P4 α.15.β codon sequence 1, P4 α.15.β codon sequence 2, YP218 α.15.β codon sequence 1, YP218 α.15.β codon sequence 2). Preparations transduced with vectors encoding P4 α.15.β codon sequences 1 and 2 both expressed approximately equal amounts of each arming component in terms of percentage expression (Figure 18A) and relative levels of protein (Figure 18B). In contrast, preparations transduced with YP218 α.15.β codon sequence 2 showed a higher percentage of cells expressing each armoury component than YP218 α.15.β codon sequence 1 (Figure 18C). The relative levels of protein expression were also increased for both IL15Rβ and IL15Rα carrying codon sequence 2 compared to codon sequence 1 (Figure 18D).

[0283] Significantly enhanced cytotoxicity by tethered IL-15 and IL-15Rβ co-expressing cells was readily observed when targeting antigen-expressing tumor targets (FIG. 18E).

[0284] Example 6 The response of β-armed binder P to repeated antigen exposure in the presence of 1 ng / mL IL-15 compared to its disarmed counterpart was examined. Tumor confluence was examined by measuring GFP fluorescence over time. The β-armed binder P4 construct showed superior tumor suppression over the 14-day repeated exposure assay. These results indicate that the enhanced sensitivity to exogenous IL-15 provided by upregulation of IL-2Rβ results in a γδ CAR T formulation with superior functionality (Figure 19A). This enhanced functionality significantly correlated with a higher proliferative capacity in response to low levels of exogenous IL-15 (1 ng / mL) compared to the disarmed counterpart, reflecting enhanced sensitivity to soluble exogenous IL-15 (Figure 19B). The enrichment of CAR-modified cells increased throughout the assay, indicating that CAR-transduced cells are the major population controlling the tumor (Figure 19C).

[0285] P4 α.15.β and P4 β transduced blood-derived γδ T cells were evaluated for functionality in a disseminated A549 tumor model (Figure 20A). Data in Figure 20B show that α.15.β armed γδ CAR T cells confer greater tumor suppression than their disarmed counterparts in the absence of IL-15 support, with P4 α.15.β treated mice having on average 10-fold less BLI signal than mice administered disarmed cells at the end of the study (19 days post-treatment). Consistent with the in vitro experiments, in the presence of IL-15, β armed γδ CAR T cells show faster initial tumor clearance kinetics but overall tumor suppression comparable to the α.15.β armed (without IL-15 support) γδ CAR T treated group.

[0286] Example 7 Mesothelin-targeted blood-derived CAR γδ T cells co-expressing IL-15Rα and IL-15Rβ tethered to IL-15 survived up to 14 days after cryopreservation, whereas disarmed constructs decayed rapidly. This is the case when examined between the two different Meso-CAR binders used, YP218 CAR (Figure 21A) and P4 CAR (Figure 21B). Figure 21C shows that IL-15-tethered IL-15Rα and IL-15Rβ chain-armed Meso-CAR T cells are able to dominantly suppress tumors over the course of repeated antigen stimulation assays in the absence of exogenous IL-15. Again, this functionality is observed regardless of the CAR binder used (YP218 or P4). Furthermore, the enhanced tumor suppression exhibited by IL-15-tethered IL-15Rα and IL-15R β chain armed CAR T cells does not necessarily correlate with proliferation potential. Instead, proliferation potential appears to be driven by the CAR molecule and is a functionality specific to the binder used. Proliferation was observed when using the YP218 binder but not the P4 binder (Figure 21D).

Claims

1. An engineered γδ T cell comprising a chimeric antigen receptor (CAR), The CAR comprises an antigen-binding domain that specifically binds to mesothelin. The antigen-binding domain comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain. The aforementioned VH includes VH-complementarity determination region 1 (VH-CDR1) containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 3, and The VL comprises a manipulated γδ T cell containing VL-CDR1 having the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 having the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 having the amino acid sequence described in SEQ ID NO:

6.

2. The engineered γδ T cell according to claim 1, further comprising an exogenous gene encoding an armor protein containing IL-15Rβ polypeptide.

3. The γδ T cell according to claim 2, wherein the armor protein further comprises an IL-15Rα polypeptide.

4. The engineered γδ T cell according to claim 2 or 3, wherein the armor protein further comprises an IL-15 polypeptide.

5. The engineered γδ T cell according to claim 4, wherein the armor protein further comprises an IL-15Rα polypeptide linked to the IL-15 polypeptide.

6. The manipulated γδ T cell according to any one of claims 1 to 3, which is a Vd1+ γδ cell.

7. (i) The VH comprises an amino acid sequence having at least about 75% sequence identity with the amino acid sequence described in Sequence ID No. 7, (ii) The VL contains an amino acid sequence having at least about 75% sequence identity with the amino acid sequence described in Sequence ID No. 8, or (iii) Both (i) and (ii) The manipulated γδ T cell according to any one of claims 1 to 3.

8. (i) The VH comprises the amino acid sequence described in Sequence ID No. 7, (ii) The VL contains the amino acid sequence described in Sequence ID No. 8, or (iii) Both (i) and (ii) The manipulated γδ T cell according to any one of claims 1 to 3.

9. The engineered γδ T cell according to any one of claims 1 to 3, wherein the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a costimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof.

10. (i) The hinge region includes a hinge region derived from CD8, CD28, or immunoglobulin, (ii) The transmembrane domains are CD8, KIRDS2, OX40, CD2, CD4, CD28, CD45, PD1, CD152, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (K LRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R Beta, IL2R Gamma, IL7R α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, I TGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226) , SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), Includes transmembrane domains derived from PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C, CD19, or any combination thereof. (iii) The aforementioned co-stimulatory domains are 4-1BB / CD137, interleukin-2 receptor (IL-2R), interleukin-12 receptor (IL-12R), IL-7, IL-21, IL-23, IL-15, CD2, CD3, CD4, CD7, CD8, CD27, CD28, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, OX40, DAP10, B7-H3, CD28 (ICA) lacking Lck binding, BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor ε chain, costimulatory domains derived from ligands that specifically bind to CD83, or any combination thereof. (iv) The intracellular signaling domain includes a CD3ζ activation domain, a CD3δ activation domain, a CD3ε activation domain, a CD3η activation domain, a CD79A activation domain, a DAP12 activation domain, an FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof, (v) Any combination of (i) to (iv) The manipulated γδ T cell according to claim 9.

11. (i) The hinge region contains an immunoglobulin selected from the group consisting of IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof, or the hinge region is derived from the CD8 hinge region. (ii) The transmembrane domain is derived from the CD8 transmembrane domain. (iii) The aforementioned co-stimulatory domain is derived from the 4-1BB co-stimulatory domain. (iv) The intracellular signaling domain includes a CD3ζ activation domain, or (v) Any combination of (i) to (iv) The manipulated γδ T cell according to claim 9.

12. The CAR is, (a) an antigen-binding domain that specifically binds to human mesothelin, comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 3, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6, (b) (i) A hinge region comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence described in Sequence ID No. 9, (b) (ii) A transmembrane domain comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence described in Sequence ID No. 10, (b) (iii) A co-stimulatory domain comprising an amino acid sequence having at least about 90% sequence identity with the amino acid sequence described in Sequence ID No. 11, (b) (iv) comprising an intracellular signaling domain having at least about 90% sequence identity with the amino acid sequence described in Sequence ID No. 12, The manipulated γδ T cell according to any one of claims 1 to 3.

13. The CAR is, (i) an antigen-binding domain that specifically binds to human mesothelin, comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 3, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6, (ii) A hinge region containing the amino acid sequence described in Sequence ID No. 9, (iii) A transmembrane domain comprising the amino acid sequence described in Sequence ID No. 10, (iv) A co-stimulatory domain comprising the amino acid sequence described in Sequence ID No. 11, (v) comprising an intracellular signaling domain containing the amino acid sequence described in Sequence ID No. 12, The manipulated γδ T cell according to any one of claims 1 to 3.

14. The armor protein is (i) an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in Sequence ID No. 15, (ii) An IL-15 polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in Sequence ID No. 16, or (iii) A polypeptide comprising an amino acid sequence having at least 70% sequence identity with the amino acid sequence described in Sequence ID No. 17, The manipulated γδ T cell according to any one of claims 1 to 3.

15. The engineered γδ T cell according to any one of claims 1 to 3, wherein the armor protein comprises an IL-15Rα polypeptide linked to an IL-15 polypeptide by a linker.

16. (a) A chimeric antigen receptor (CAR), (i) an antigen-binding domain that specifically binds to human mesothelin, comprising VH-CDR1 containing the amino acid sequence described in SEQ ID NO: 1, VH-CDR2 containing the amino acid sequence described in SEQ ID NO: 2, VH-CDR3 containing the amino acid sequence described in SEQ ID NO: 3, VL-CDR1 containing the amino acid sequence described in SEQ ID NO: 4, VL-CDR2 containing the amino acid sequence described in SEQ ID NO: 5, and VL-CDR3 containing the amino acid sequence described in SEQ ID NO: 6; (ii) Hinge region containing the amino acid sequence described in Sequence ID No. 9; (iii) A transmembrane domain comprising the amino acid sequence described in Sequence ID No. 10; (iv) A co-stimulatory domain comprising the amino acid sequence described in Sequence ID No. 11; and (v) The CAR comprising an intracellular signaling domain containing the amino acid sequence described in Sequence ID No. 12, (b) Armor proteins, (i) IL-2Rβ polypeptide comprising the amino acid sequence described in Sequence ID No. 15; (ii) A linker comprising the amino acid sequence described in Sequence ID No. 18; and (iii) An IL-15Rα polypeptide linked to an IL-15 polypeptide, wherein the IL-15Rα polypeptide contains the amino acid sequence described in SEQ ID NO: 17, and the IL-15 polypeptide contains the amino acid sequence described in SEQ ID NO: 16, wherein the IL-15Rα polypeptide linked to an IL-15 polypeptide The armor protein includes, The manipulated γδ T cell according to any one of claims 1 to 3.

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