Cell expressing anti-mesothelin CAR

Abstract

Some aspects of the present disclosure relate to engineered congenital lymphoid cells comprising a heterologous targeting construct that is specific for mesothelin and uses thereof. Some aspects of the present disclosure relate to polynucleotides encoding chimeric antigen receptors (CARs) that specifically bind to human mesothelin, host cells comprising the polynucleotides, and methods of using the same to treat a disease or condition in a subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This PCT application claims the benefit of priority to UK Application No. GB ​​2204927.4, filed on April 4, 2022, and U.S. Provisional Application No. 63 / 482,752, filed on February 1, 2023, each of which is incorporated herein by reference in its entirety.

[0003] References to sequence listings submitted electronically via EFS-WEB

[0004] The contents of the electronically submitted sequence listing in the ASCII text file (Name: 3817_165PC01_Seqlisting_ST26; Size: 38,145 bytes; Creation Date: April 3, 2023) submitted with this application are incorporated herein by reference in their entirety. Technical Field

[0005] The present invention relates to an engineered innate lymphoid cell comprising a heterologous targeting construct specific for mesothelin. The present invention also relates to a pharmaceutical composition comprising the engineered innate lymphoid cell, a method of treatment using the engineered innate lymphoid cell or the pharmaceutical composition, and the engineered innate lymphoid cell or the pharmaceutical composition for use in therapy. The present invention also includes a method for preparing an engineered innate lymphoid cell and an isolated cell population comprising a plurality of the engineered innate lymphoid cells. Background Art

[0006] 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. Although mesothelin is present on the cell surface of normal mesothelial cells, it is also overexpressed in several human tumors, including ovarian cancer, cervical cancer, uterine cancer, gastric cancer, pancreatic cancer, and lung adenocarcinoma. Therefore, mesothelin is an attractive candidate for the development of targeted cancer therapies.

[0007] There is therefore a need for therapies that can target mesothelin-expressing cells. Summary of the invention

[0008] 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.

[0009] According to another 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 pharmaceutically or physiologically acceptable carriers, diluents or excipients.

[0010] According to another aspect of the present invention, there is provided a method of treating a patient in need thereof using the engineered innate lymphoid cells or the pharmaceutical composition as described herein.

[0011] According to another aspect of the present invention, there is provided use of an engineered innate lymphoid cell or a pharmaceutical composition as described herein for treating a condition in a subject.

[0012] According to another aspect of the present invention, there is provided an engineered innate lymphoid cell or a pharmaceutical composition as described herein for use in therapy.

[0013] According to another aspect of the present invention, there is provided a method of preparing an engineered innate lymphoid cell as described herein.

[0014] According to another aspect of the present invention, there is provided an isolated cell population comprising a plurality of engineered innate lymphoid cells as described herein.

[0015] Some aspects of the present disclosure relate to a polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH comprises a VH-complementarity determining region 3 (CDR3), and the VH-complementarity determining region 3 comprises the amino acid sequence shown in SEQ ID NO: 3. In some aspects, the CAR also comprises a VH-CDR2, and the VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 2. In some aspects, the CAR also comprises a VH-CDR3, and the VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO: 3. In some aspects, the CAR also comprises a VL-CDR1, and the VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO: 4. In some aspects, the CAR also comprises a VL-CDR2, and the VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO: 5. In some aspects, the CAR further comprises a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:6.

[0016] In some aspects, the CAR comprises: a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

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

[0018] In some aspects, 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 aspects, the VL comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0019] In some aspects, VH comprises the amino acid sequence set forth in SEQ ID NO:7, and wherein VL comprises the amino acid sequence set forth in SEQ ID NO:8.

[0020] In some aspects, CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a co-stimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof. In some aspects, CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a co-stimulatory domain, and (iv) an intracellular signaling domain.

[0021] In some aspects, the hinge region includes a hinge region derived from CD8, CD28 or an immunoglobulin. In some aspects, the immunoglobulin is selected from the group consisting of: IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM and any combination thereof. In some aspects, the hinge region is derived from the CD8 hinge region. In some aspects, 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 shown in SEQ ID NO:9. In some aspects, the hinge region comprises an amino acid sequence shown in SEQ ID NO:9.

[0022] In some aspects, the transmembrane domain comprises a transmembrane domain derived from 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, IL2Rβ, IL2Rγ, IL7Rα, 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 aspects, the transmembrane domain is derived from a CD8 transmembrane domain. In some aspects, 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 aspects, the transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0023] In some aspects, the costimulatory domain includes a costimulatory domain derived from the 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 deleted due to 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 to CD83, or any combination thereof. In some aspects, the costimulatory domain is derived from a 4-1BB costimulatory domain. In some aspects, 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 shown in SEQ ID NO: 11. In some aspects, the costimulatory domain comprises an amino acid sequence shown in SEQ ID NO: 11.

[0024] In some aspects, the intracellular signaling domain comprises a CD3 ζ activation domain, a CD3 δ activation domain, a CD3 ε activation domain, a CD3 η activation domain, a CD79A activation domain, a DAP 12 activation domain, a FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof. In some aspects, the intracellular signaling domain comprises a CD3 ζ activation domain. In some aspects, 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 with the amino acid sequence shown in SEQ ID NO: 12. In some aspects, the costimulatory domain comprises an amino acid sequence shown in SEQ ID NO: 12.

[0025] In some aspects, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; and (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 shown in SEQ ID NO: 9; (b)(ii) 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 shown in SEQ ID NO: 10. NO:10 has 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; (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 shown 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 shown in SEQ ID NO:12; or any combination of (b)(v)(b)(i) to (b)(iv).

[0026] In some aspects, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 10; NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0027] In some aspects, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 10; NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0028] In some aspects, the polynucleotide comprises a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12.

[0029] In some aspects, the polynucleotide further comprises a second nucleotide sequence encoding an armorin. In some aspects, the armorin comprises an IL-15Rβ polypeptide, an IL-15Rα polypeptide, an IL-2Rβ polypeptide, an IL-15 polypeptide, or any combination thereof.

[0030] In some aspects, the armorin comprises an IL-2Rβ polypeptide. In some aspects, the armorin 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 with the amino acid sequence shown in SEQ ID NO: 15. In some aspects, the armorin comprises an amino acid sequence shown in SEQ ID NO: 15.

[0031] In some aspects, the armorin comprises an IL-2Rβ polypeptide, and wherein the armorin does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

[0032] In some aspects, the armorin comprises (i) an IL-15Rα polypeptide or (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some aspects, the armorin 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 shown in SEQ ID NO: 17. In some aspects, the armorin comprises an IL-15Rα polypeptide comprising an amino acid sequence shown in SEQ ID NO: 17.

[0033] In some aspects, the armorin comprises an IL-15 polypeptide. In some aspects, the armorin 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 with the amino acid sequence shown in SEQ ID NO: 16. In some aspects, the armorin comprises an IL-15 polypeptide comprising an amino acid sequence shown in SEQ ID NO: 16.

[0034] In some aspects, the armorin comprises an IL-15Rα polypeptide tethered to an IL-15 polypeptide via a linker. In some aspects, the linker comprises a peptide bond. In some aspects, the linker comprises a Gly-Ser linker. In some aspects, the linker comprises an amino acid sequence selected from SEQ ID NO: 20-25 or any combination thereof.

[0035] In some aspects, the armorin comprises (i) an IL-2Rβ polypeptide; (ii) a cleavable linker; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some aspects, the cleavable linker comprises a P2A sequence. In some aspects, 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 aspects, the cleavable linker comprises the amino acid sequence set forth in SEQ ID NO: 18.

[0036] In some aspects, the polynucleotide comprises a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising SEQ ID NO: NO:12; and (b) armored protein, wherein the armored protein comprises an IL-2Rβ polypeptide, and the IL-2Rβ 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 with the amino acid sequence shown in SEQ ID NO:15.

[0037] In some aspects, the polynucleotide comprises a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising SEQ ID NO: NO:12; and (b) armored protein, wherein the armored protein comprises an IL-2Rβ polypeptide, and the IL-2Rβ polypeptide comprises the amino acid sequence shown in SEQ ID NO:15.

[0038] In some aspects, the polynucleotide does not comprise a nucleotide sequence encoding an IL-15Rα polypeptide or an IL-15 polypeptide.

[0039] In some aspects, the polynucleotide comprises a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising SEQ ID NO: NO:12; and (b) armorin, wherein the armorin 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 of 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 of SEQ ID NO:18; and (iii) 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 of SEQ ID NO:18. NO:17 has 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; 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 shown in SEQ ID NO:16.

[0040] In some aspects, the polynucleotide comprises a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising SEQ ID NO: NO:12; and (b) armored protein, wherein the armored protein comprises: (i) an IL-2Rβ polypeptide comprising the amino acid sequence shown in SEQ ID NO:15; (ii) a linker comprising the amino acid sequence shown 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 shown in SEQ ID NO:17; and the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:16.

[0041] Some aspects of the present disclosure relate to a vector or vector set comprising a polynucleotide disclosed herein. In some aspects, the vector comprises one or more promoters. In some aspects, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0042] Some aspects of the present disclosure relate to a polypeptide encoded by a polynucleotide disclosed herein.

[0043] Some aspects of the present disclosure relate to a host cell comprising a polynucleotide disclosed herein, a vector or vector collection disclosed herein, or a polypeptide disclosed herein.

[0044] In some aspects, the host cell is an immune cell. In some aspects, the immune cell is an innate lymphoid cell. In some aspects, the immune cell is selected from T cells, NK cells, B cells, or any combination thereof. In some aspects, the T cell is selected from γδT cells, αβT cells, or any combination thereof. In some aspects, the T cell is selected from Vδ1T cells and Vδ2T cells.

[0045] In some aspects, the host cell is obtained from a human subject. In some aspects, 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 aspects, the host cell is derived from an induced pluripotent stem cell (iPSC).

[0046] Some aspects of the present disclosure relate to a cell population comprising a host cell disclosed herein. In some aspects, 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 cell population comprise host cells.

[0047] Some aspects of the present disclosure relate to a pharmaceutical composition comprising a polynucleotide disclosed herein, a vector or a collection of vectors disclosed herein, a polypeptide disclosed herein, a host cell disclosed herein or a cell population disclosed herein, and a pharmaceutically acceptable excipient.

[0048] Some aspects of the present disclosure relate to a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a polynucleotide disclosed herein, a vector or vector collection disclosed herein, a polypeptide disclosed herein, a host cell disclosed herein, a cell population disclosed herein, or a pharmaceutical composition disclosed herein.

[0049] In some aspects, the disease or condition comprises cancer. In some aspects, cancer comprises lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, 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 myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney cancer or ureter cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos) or any combination thereof. In some aspects, the cancer comprises small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, mesothelioma or any combination thereof.

[0050] In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory or recurrent.

[0051] In some aspects, the method further comprises administering another anticancer agent. In some aspects, another anticancer therapy comprises chemotherapy, immunotherapy, radiotherapy, surgery or any combination thereof. In some aspects, another anticancer therapy comprises chemotherapy.

[0052] In some aspects, additional anticancer therapies include immune checkpoint inhibitors. In some aspects, additional anticancer therapies include PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, GITR antagonists, or any combination thereof. In some aspects, anticancer therapies include antibodies or antigen binding portions thereof that specifically bind to and inhibit PD-1. In some aspects, anticancer therapies include antibodies or antigen binding portions thereof that specifically bind to and inhibit PD-L1. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1A-1B :PoC of CAR-engineered cell functionality against Meso+ve targets. Figure 1A) Check the functionality of blood-derived γδT cells and non-engineered blood-derived Vδ1 cells ("GDX012") transduced with YP218 anti-mesothelin CAR constructs against solid tumor target OVCAR3 cells. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1:1, 1:1:2.5 and 1:5 and without cytokine supplementation for 18-24h. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay. The error bars show the mean ± SD of triplicate wells under each condition. These data show that genetic engineering of GDX012 to express mesothelin-CAR significantly enhances the inherent killing ability of Vδ1T cells against OVCAR3 cells. ( Figure 1B ) Blood-derived γδT cells transduced with the YP218 anti-mesothelin CAR construct (n=1) were cultured with Hela cells at an E:T ratio of 2:1 in a separate medium or a medium supplemented with IL-15 (10ng / ml). On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. These data indicate that genetic engineering of GDX012 to express mesothelin-CAR significantly enhances the intrinsic killing ability of Vδ1T cells against Hela tumor targets, and that in addition to CAR signaling, the survival and proliferation of mesothelin CAR-modified blood Vδ1T cells also require IL-15.

[0054] Figure 2 : As a proof-of-concept for targeting solid tumors, GDX012 was engineered with the YP218 anti-mesothelin CAR construct. Mesothelin was chosen as a proof-of-concept solid tumor target because this protein is overexpressed in various cancer indications and is an established target in various solid tumor models and clinical trials. Healthy tissues have minimal expression of mesothelin, making it an ideal solid tumor target. The anti-MSLN Vδ1 product was functionally tested to understand phenotype, cytotoxicity, proliferation, and in vivo activity to demonstrate solid tumor targeting and establish a framework for future engineered products.

[0055] Figure 3A-3B :GDX012, unlike skin-resident γδ T cells, exhibits modest intrinsic ability to target solid tumor targets, but can be improved through genetic engineering. Figure 3A ) examined GDX012 (n=3) and skin-resident γδ T cells (n=3) targeting mesothelin +Functionality of the target cell line Hela cells. Cryopreserved GDX012 and skin-resident γδT cells were thawed and immediately cultured with target cells at E:T ratios of 5:1, 2.5:1, and 1.25:1 in culture medium and without cytokine supplementation for 18-24h. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay. Error bars show the mean ± SD of triplicate wells under each condition. These data indicate that skin-resident γδT cells have an inherent killing capacity against solid tumor targets, with tumor target lysis >80% at a 5:1 E:T ratio, >60% at a 2.5 E:T ratio, and >50% at 1.25 E:T. GDX012 lysis of target cells did not exceed >20% lysis at all E:T ratios analyzed for Hela cells. ( Figure 3B ) Check the functionality of blood-derived γδT cells and GDX012 for OVCAR3 cells transduced with YP218 anti-mesothelin CAR constructs. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at an E:T ratio of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125:1 and 18-24h without supplementing cytokines. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay. Error bars show the mean ± SD of triplicate wells under each condition. These data demonstrate that genetically engineering GDX012 to express a mesothelin-CAR significantly enhanced the intrinsic killing capacity of Vδ1 T cells against OVCAR3 tumor targets, with >90% tumor target lysis at 5:1, >75% at 2.5:1, >50% at 1.1, and >20% at a 1:2.5 E:T ratio, while GDX012 lysis of target cells did not exceed 30% at all E:T ratios analyzed for OVCAR3 cells.

[0056] Figure 4A-4B :Mesothelin CAR modified blood Vδ1T cells showed anti-HeLa( Figure 4A ) and OVCAR3( Figure 4B) cells, and this cytotoxicity is comparable to CAR-modified αβT cells. The functionality of blood-derived γδT cells transduced with YP218 anti-mesothelin CAR constructs (n=3) and blood-derived αβT cells transduced with YP218 anti-mesothelin constructs (n=1) was examined for Hela and OVCAR3 tumor cells. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 and 18-24h without supplementing cytokines. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay. Error bars show the mean ± SD of triplicate wells under each condition.

[0057] Figure 5A-5B : Mesothelin CAR-modified blood Vδ1 T cells showed responses to HT29 ( Figure 5A ) and A549( Figure 5B ) has strong cytotoxicity, which is comparable to CAR-modified αβT cells. The functionality of blood-derived γδT cells transduced with YP218 anti-mesothelin CAR construct (n=2) and blood-derived αβT cells transduced with YP218 anti-mesothelin construct (n=1) was examined for HT29.MSLN. The functionality of blood-derived γδT cells transduced with YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβT cells transduced with YP218 anti-mesothelin construct (n=1) was tested for and A549.MSLN cells. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 and without cytokine supplementation for 18-24h. Specific cell lysis was detected by luciferase-based fluorocytometry. Error bars show mean ± SD of triplicate wells for each condition.

[0058] Figure 6: Experimental design for repeated antigen stimulation (RAS) assays for solid tumor targets. Effector cells were thawed and co-cultured with solid tumor target cancer cell lines at an E:T ratio of 2:1 in a medium (10ng / ml) supplemented with IL-15. Day +2: Supplement cytokines for the culture by replacing 50% of the culture medium. Day +4: Supplement targets and cytokines by replacing 50% of the culture medium. Day +7: Harvest culture. Effectors were counted, and an equal number of effectors inoculated at D0 were re-inoculated with target cells in a medium (10mg / ml) supplemented with IL-15 at an E:T ratio of 2:1. Additional effectors were used for phenotypic analysis by flow cytometry or inoculated with target cells in a 24hr killing assay. Overall, effector cells underwent 6 rounds of target cell killing and 21 days of culture within the assay, with the final 24hr killing assay showing the 7th round of continuous killing.

[0059] Figure 7A-7B : Mesothelin CAR-modified blood Vδ1T cells expanded after 6 rounds of tumor challenge with Hela cells ( Fig. 7A ) and enriched for CAR expression ( Figure 7B ). Blood-derived γδT cells transduced with the YP218 anti-mesothelin CAR construct (n=2) were cultured with Hela cells in a medium supplemented with IL-15 (10ng / ml) at an E:T ratio of 2:1. On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The multiple proliferation of effector cells at each time point was calculated by dividing the cell count of the harvested effector cells by the number of cells inoculated 7 days ago. The total multiple proliferation or cumulative multiple proliferation at each time point was calculated by multiplying the multiple proliferation by the previous multiple proliferation. The cells expanded 60 times over the course of 21 days. At days 0, 7, 14, and 21, CAR expression on Vδ1T cells was measured by flow cytometry. Vδ1T cells expressing CAR were enriched over the course of 21 days, increasing from <20% expression to >90% expression at the end of the assay. The data represent two independent experiments.

[0060] Figures 8A-8C : Mesothelin CAR-modified blood Vδ1 T cells expand in response to sequential killing of OVCAR3 and A549.MSLN cells in the RAS assay ( Fig. 8A ) and enrichment ( Figure 8B). Blood-derived γδT cells transduced with YP218 anti-mesothelin CAR constructs (n=3) were cultured with OVCAR3 and A549.MSLN cells in a medium supplemented with IL-15 (10 ng / ml) at an E:T ratio of 2:1. On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The multiple proliferation of effector cells at each time point was calculated by dividing the cell count of the harvested effector cells by the number of cells inoculated 7 days ago. The total (cumulative) multiple proliferation at each time point was calculated by multiplying the multiple proliferation by the previous multiple proliferation. Cells cultured with OVCAR3 cells expanded 20 times over the course of 21 days. Cells cultured with A549.MSLN cells expanded 400 times over the course of 21 days. CAR expression on Vδ1T cells was measured by flow cytometry on days 0, 7, 14, and 21. CAR-expressing Vδ1 T cells cultured with OVCAR3 and A549.MSLN cells were enriched over the course of 21 days, increasing to >95% expression on day 21. Figure 8C It is shown that the Vδ1 frequency remains constant during the analysis.

[0061] Figures 9A-9B :Mesothelin CAR-modified blood Vδ1 T cells remain cytotoxic even after 21 days of RAS targeting Hela tumor cells. Fig. 9A ) Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=2) were cultured with Hela cells in 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 2x10 5 Individual cells / well were inoculated and allowed to adhere for 4 h. Effectors were harvested, counted and cultured for 18-24 h in culture medium and without supplementation of cytokines with E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 for adherent target cells. Specific cell lysis was detected by fluorescence cell counting based on luciferase and data represent 7 rounds of continuous killing. Error bars show mean ± SD of triplicate wells under each condition. ( Fig. 9B ) Live CAR after 7, 14 and 21 days in RAS assay for Hela cells + Flow cytometric analysis of γδ T cells. Phenotypic analysis of Vδ1 T cells showed that the expression of NKG2D, CD56, NKp30, NKG2C, and PD1 was upregulated from day 0 to day 21, while CD27, representing the effector state, was downregulated by Vδ1 T cells from day 0 to day 21. Data are representative of two independent experiments.

[0062] Figures 10A-10F Figure 3: 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 transduced with the YP218 anti-mesothelin CAR construct (n=3) were compared with OVCAR3 ( Figures 10A-10C ) and A549.MSLN( Figures 10D-10F ) cells were cultured together in medium supplemented with IL-15 (10 ng / ml) at a ratio of 2:1 for 21 days. Fig. 10A and 10D )、Day 14( Fig. 10B and 10E ) and Day 21 ( Fig. 10C and 10F ), harvest fresh target cells, and obtain viable cell counts using an NC250 cell counter. Target cells were plated at 2x10 5 Individual cells / wells were inoculated and made to adhere for 4h. Effectors were harvested, counted and cultured with adherent target cells in culture medium with an E:T ratio of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 and without supplementing cytokines for 18-24h. Specific cell lysis was detected by fluorescence cell counting based on luciferase. Error bars show the mean ± SD of triplicate wells under each condition.

[0063] Figures 11A-11B : Compared with unmodified A549 ( Fig.11A ) tumor cells, the mesothelin CAR modified blood Vδ1T cells against A549 ( Fig.11A ) has greater cytotoxicity. Test the functionality of blood-derived γδT cells transduced with YP218 anti-mesothelin CAR construct (n=4) and blood-derived αβT cells transduced with YP218 anti-mesothelin construct (n=1) against A549 tumor cells and A549 tumor cells modified to overexpress MSLN. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at an E:T ratio of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 and cultured for 18-24h without supplementing cytokines. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay. Error bars show the mean ± SD of triplicate wells under each condition.

[0064] Figures 12A-12B: Skin-derived Vd1+γδT cells can be efficiently transduced with Meso-CAR. Skin-resident cells were thawed and immediately processed by MACS selection to remove αβT cells. The resulting negatively selected γδT cells were then transduced with vectors encoding P4 or YP218 anti-mesothelin CAR constructs. The transduced cells were then expanded for 14 days in the presence of IL-15 (80ng / ml) and IL21 (11.25ng / ml), then harvested and cryopreserved. The expansion factor of γδT cells was recorded on D14 of the expansion culture ( Fig. 12A The percentage of cells positive for each mesothelin-specific CAR was measured by flow cytometry at D14 ( Fig. 12B ).

[0065] Figures 13A-13B :Skin-derived γδT cells are cytotoxic to the mesothelin-positive target HeLa cell line. Figures 12A-12B The skin-resident γδ T cells were transduced and expanded. The cryopreserved cells were then thawed and immediately tested for mesothelin + Functionality of Hela cell lines. Cells were cultured for 17-20 h at different effector to target ratios without cytokine supplementation. Using the CellTitreGLO cell counting assay, P4 ( Fig.13A ) and YP218( Fig. 13B ) conjugates to detect specific cell lysis. White circles represent untransduced skin-resident γδT cells. Gray dots represent skin-resident γδT cells transduced with mesothelin-specific CAR.

[0066] Fig.14 Skin-derived γδ T cells are cytotoxic to mesothelin-positive target cells A459. Skin-resident γδ T cells transduced with the YP218 anti-mesothelin CAR construct were tested against mesothelin + Functionality of 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 cytokine supplementation. Specific cell lysis was detected by a luciferase-based fluorocytometry assay.

[0067] Figures 15A-15B : Mesothelin CAR-modified skin Vδ1 T cells are cytotoxic to HT29.MSLN and A549.MSLN tumor cells. Skin-resident γδ and αβ T cells transduced with YP218 anti-mesothelin CAR construct (n=1) were tested against mesothelin + Functionality of the A549 target cell line. Cryopreserved YP218+ expanded skin-resident γδ T cells were thawed and immediately incubated with the target HT29.MSLN ( Fig.15A ) and A549.MSLN( Fig. 15B ) cells were cultured together without cytokine supplementation for 18-24 h. Specific cell lysis was detected by luciferase-based fluorescence cell counting assay. Error bars show the mean ± SD of triplicate wells under each condition.

[0068] Figures 16A-16F Figure 1 is a graphic representation showing that unarmored P4 and YP218 CARγδ T cells are effective in the disseminated A549 tumor model in NSG mice. The CARs contain a "Binder Y" (YP218) or "Binder P" (P4) anti-mesothelin binding domain (as indicated), a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. Fig.16A The data presented show that γδ T cells transduced with either conjugate exhibited effective tumor control ("Conjugate Y" = YP218; "Conjugate P" = P4). Fig. 16B The data presented show that γδ T cells expressing the anti-MSLN CAR were able to control tumor growth when given as few as 1 million total cells ("Binding Y" = YP218; "Binding P" = P4). Figures 16C-16F The data presented show that total γδ T cells expressing either anti-MSLN conjugate were expressed in the bone marrow microenvironment evaluated ( Fig. 16C ),spleen( Fig.16D ),lung( Fig.16E ) and blood( Fig.16F ) were maintained at equal levels ("Conjugate Y" = YP218; "Conjugate P" = P4).

[0069] Figures 17A-17B There are two new armor constructs ( Fig.17A ) and the corresponding polycistronic gammaretroviral vectors generated, in which the chimeric antigen receptor (CAR) coding sequence is fused with the coding sequence for wild-type or IL-15 fusion (tethered) chain variants ( Fig. 17B ) were fused in frame to individual IL-15 receptor chain components.

[0070] Figures 18A-18D It is based on the P4α.15.β codon sequence ( Figures 18A-18B ) or YP218α.15.β codon sequence ( Figures 18C-18D ) Vδ1CAR derived from the codon optimization algorithm of the transduced drug product + Armor + The percentage of cells ( Fig.18A and 18C ) or armored maximum fluorescence intensity (MFI; Fig.18B and 18D ) in a bar chart. Fig.18ECytotoxicity of cells engineered with a mesothelin-targeting CAR that was unarmored (Meso-CAR) or armored with mbIL-15 and IL-15Rβ (Meso-CAR.α.15.β) is shown.

[0071] Figures 19A-19C are the results of a serial killing assay of cells transduced with the unarmored conjugate P construct or the P4β construct ( Fig.19A ), multiple amplification ( Fig.19B ) and the percentage of cells expressing CAR ( Fig.19C ) for a graphical display.

[0072] Fig. 20A and 20B Schematic diagram and results evaluating the in vivo performance of unarmored and MesoCAR-modified Vδ1 T cells armored 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 was engineered to express mesothelin (A549-Meso). Fig. 20A A schematic diagram showing the model used. Fig. 20B The in vivo efficacy of the investigated product is shown.

[0073] Figures 21A-21D Figure 2 is a graph evaluating the effects of in vitro survival and repeated antigen exposure after cryopreservation on two different Vδ1T modified with mesothelin-targeting CAR (YP218(1) or P4(2)) that were unarmored, armored with mbIL-15 and IL-15Rβ (CAR.α.15.β), or armored with IL-15Rβ (CAR.β). Figures 21A-21B Survival of unarmored and α.15.β armored CAR T cells is shown. Fig. 21C Shown is tumor confluence measured by tracking GFP fluorescence over time after treatment with different Meso-CAR T cell products. Fig.21D Fold expansion of CAR T cells during repeated antigen stimulation assays is shown. DETAILED DESCRIPTION

[0074] Some aspects of the present disclosure relate to an antigen binding portion that specifically binds to human mesothelin. Some aspects of the present disclosure relate to polynucleotides comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to human mesothelin. Some aspects of the present disclosure relate to a host cell comprising a polynucleotide disclosed herein, such as an immune cell, such as an innate lymphoid cell. Another aspect of the present disclosure relates to a method for treating a subject in need thereof, comprising administering a polynucleotide and / or a cell to the subject.

[0075] I. Terminology

[0076] In order to make this specification more easily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0077] It should be noted that the term "a" or "an" entity refers to one or more of the entities; for example, "nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a" (or "a"), "one or more / one or more" and "at least one / at least one" are used interchangeably herein.

[0078] Furthermore, “and / or” as used herein should be considered as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term “and / or” as used in phrases such as “A and / or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, the term “and / or” as used in phrases such as “A, B, and / or C” is intended to cover the following: 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 (alone); B (alone); and C (alone).

[0079] It should be understood that where aspects are described herein with the language "comprising," other similar aspects described with "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") will be understood to mean 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 "comprise," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms while retaining their ordinary meaning.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd edition, 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd edition, 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press provide a general dictionary of many of the terms used in the present disclosure for the skilled person.

[0081] Units, prefixes and symbols are expressed in a form accepted by the International System of Units (SI). Numerical ranges include numbers that define the range. It should be understood that when listing the range of values, each intermediate integer value and each fraction thereof between the listed upper and lower limits of the range and each subrange between such values ​​are also specifically disclosed. The upper and lower limits of any range may be independently included or not included in the range, and each range including any limit, not including any limit or including two limits is also encompassed in the present disclosure. Therefore, the ranges listed herein should be understood as abbreviations for all values ​​within the range, including the listed endpoints. For example, a range of 1 to 10 should be understood as including any number, combination of numbers or subranges of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

[0082] It is understood that when values ​​are explicitly listed, values ​​of quantities or amounts that are approximately the same as the listed values ​​are also within the scope of the present disclosure. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the present disclosure. Conversely, when different elements or groups of elements are disclosed individually, their combinations are also disclosed. When any element of the present disclosure is disclosed as having multiple alternatives, examples of the present disclosure are also disclosed herein, wherein each alternative is excluded singly or in any combination with other alternatives; more than one element of the present disclosure may have such exclusions, and all combinations of elements with such exclusions are disclosed herein.

[0083] Nucleotide is referred to by its generally accepted single-letter code.Unless otherwise indicated, nucleotide sequence is written from left to right in a 5' to 3' direction.Nucleotide is referred to herein by the common single-letter symbol recommended by the IUPAC-IUB Biochemical Nomenclature Committee.Therefore, "a" represents adenine, "c" represents cytosine, "g" represents guanine, "t" represents thymine, and "u" represents uracil.

[0084] Amino acid sequences are written left to right in the direction of amino to carboxyl.Amino acids are referred to herein by either the commonly used three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0085] The term "about" refers to a value or composition 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 manner in which the value or composition is measured or determined (i.e., the limitations of the measurement system). For example, "about" may mean within 1 standard deviation or greater than 1 standard deviation according to the practice in the art. Alternatively, "about" may mean a range of up to 10%. In addition, particularly for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value. When specific values ​​or compositions are provided in the present application and claims, unless otherwise indicated, the meaning of "about" should be assumed to be within an acceptable error range for the specific value or composition.

[0086] The terms "administration", "administering" and grammatical variations thereof refer to the introduction of a composition of the present invention (e.g., a polynucleotide encoding a CAR or a cell expressing a CAR) into a subject by a pharmaceutically acceptable route. The introduction of a composition of the present invention (e.g., a polynucleotide encoding a CAR or a cell expressing a CAR) into a subject is achieved by any suitable route, including intratumoral, oral, pulmonary, intranasal, parenteral (intravenous, intraarterial, intramuscular, intraperitoneal or subcutaneous), rectal, intralymphatic, intrathecal, periocular or local.

[0087] Administration includes self-administration and administration by others. Suitable routes of administration allow the composition or agent to exert its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into the vein of the subject.

[0088] The term "antigen" refers to a molecule that stimulates an immune response. This immune response may involve the production of antibodies or the activation of specific immunocompetent cells, or both. The skilled artisan will appreciate that any macromolecule, including nearly all proteins or peptides, may be used as an antigen. In addition, antigens may be derived from recombinant or genomic DNA.

[0089] As used herein, the term "epitope" refers to an antigen portion that specifically interacts with an antigen binding portion, such as a CAR or antibody molecule. Such portions, referred to herein as epitope determinants, typically include elements such as amino acid side chains or sugar side chains, or are part of the elements. Epitope determinants can be defined, for example, by methods known in the art, such as by crystallography or by hydrogen-deuterium exchange. At least one or some portions on an antibody molecule that specifically interacts with an epitope determinant are typically located in one or more CDRs. Typically, an epitope has specific three-dimensional structural features. Typically, an epitope has specific charge characteristics. Some epitopes are linear epitopes, while other epitopes are conformational epitopes.

[0090] The term "autologous" refers to any material derived from the same individual that is later reintroduced into that individual.

[0091] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct, which includes an extracellular antigen binding domain, a transmembrane domain, and an optional intracellular domain of an activation signal and / or a co-stimulatory signal that is activated to the cell. In some embodiments, CAR is included in an optional leader sequence at the N-terminus of the CAR fusion protein. In some embodiments, CAR lacks an intracellular (e.g., signal transduction) domain. The expression of 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 aspects, CAR is expressed by immune cells such as αβT cells, γδT cells, or NK cells. In some aspects, the antigen binding domain includes Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, vNAR, nano antibody (single domain antibody) or any combination thereof. In some aspects, the transmembrane domain includes a transmembrane domain selected from a transmembrane domain of CD8, CD4, or CD28. In some aspects, the intracellular domain includes a co-stimulatory domain or a portion thereof. In some aspects, the intracellular domain comprises a co-stimulatory domain selected from the group consisting of: a 4-1BB co-stimulatory domain, a CD3z, a CD28 co-stimulatory domain, a CD27 co-stimulatory domain, and an intracellular domain of any combination thereof. CAR may also include a "hinge" or "spacer" domain. Non-limiting examples of hinge / spacer domains include CD8 hinges or immunoglobulin hinge / spacer domains, such as IgG1 hinge domains and IgG2 hinge domains, IgG3 hinge domains, or IgG4 hinge domains.

[0092] As used herein, the term "intracellular signaling domain" refers to the intracellular portion of a molecule. The intracellular signaling domain can generate signals that promote the immune effector functions of CAR-containing cells (e.g., anti-ROR1 CAR T cells described herein). Non-limiting examples of immune effector functions, for example, in CAR T cells, include cytolytic activity and auxiliary activity, including the secretion of cytokines. In some aspects, the intracellular signaling domain is a protein portion that transduces effector function signals and instructs cells to perform special functions.

[0093] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid sequence within the antigen-binding variable region that confers antigen specificity and binding affinity. For example, typically, there are 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 precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Edition 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. According to the Kabat numbering scheme, in some aspects, 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). According to the Chothia numbering scheme, in some aspects, the CDR amino acids in VH are numbered 26-32 (VH-CDR1), 52-56 (VH-CDR2), and 95-102 (VH-CDR3); and the CDR amino acid residues in VL are numbered 26-32 (VL-CDR1), 50-52 (VL-CDR2), and 91-96 (VL-CDR3). In the combined Kabat and Chothia numbering scheme, in some aspects, CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some aspects, CDRs correspond to amino acid residues 26-35 (VH-CDR1), 50-65 (VH-CDR2), and 95-102 (VH-CDR3) in a VH, such as 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, such as a mammalian VL (e.g., a human VL).

[0094] In some aspects, 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 is connected to the transmembrane domain by a CAR hinge / spacer. In some aspects, CAR comprises a chimeric fusion protein comprising an antigen binding domain connected to a transmembrane domain by a CAR hinge / spacer and an intracellular signaling domain comprising a functional signaling domain derived from a co-stimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some aspects, CAR comprises a chimeric fusion protein comprising an antigen binding domain connected to a transmembrane domain by a CAR hinge / spacer and an intracellular signaling domain comprising two functional signaling domains derived from one or more co-stimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In some aspects, CAR comprises a chimeric fusion protein, the chimeric fusion protein comprising an antigen binding domain connected to a transmembrane domain by a CAR hinge / spacer and comprising at least two functional signaling domains derived from one or more costimulatory molecules and an intracellular signaling domain derived from a functional signaling domain of a stimulatory molecule. In some aspects, CAR is included in an optional leader sequence at the amino terminus (N-terminus) of CAR. In some aspects, CAR is also included in a leader sequence at the N-terminus of an antigen binding domain, wherein the leader sequence is optionally cut from an antigen binding domain (e.g., scFv) during CAR cell processing and localization to the cell membrane.

[0095] Although the present application often uses CAR to illustrate the different aspects of the disclosed subject matter, it will be apparent to a skilled person that the relevant disclosures provided herein may be equally applicable to other chimeric binding proteins. As used herein, the term "chimeric binding protein" refers to a protein that can bind one or more antigens (e.g., comprising an antigen binding moiety) and is produced by the connection of two or more heterologous polynucleotides, which initially encodes a single protein or protein fragment or a plurality of fragments of the same protein connected in a non-natural direction. Non-limiting examples of other chimeric binding proteins include T cell receptors (TCR) (e.g., engineered TCR), chimeric antibodies-T cell receptors (caTCR), chimeric signaling receptors (CSR), T cell receptor mimics (TCR mimics) and combinations thereof. Therefore, in some aspects, unless otherwise noted, the term CAR may encompass other types of chimeric binding proteins known in the art, such as those described herein.

[0096] As used herein, the term "affinity" refers to a measure of the strength of binding of an antigen or target (such as an epitope) to its cognate binding domain (such as a paratope). As used herein, the term "avidity" refers to the overall stability of the complex between an epitope group and a paratope group (i.e., an antigen and an antigen binding domain).

[0097] The term "binding to the same epitope" with respect to two or more antigen binding moieties means that the antigen binding moieties bind to the same stretch 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 completely) the binding of another antibody to the target.

[0098] As used herein, the terms "specific binding", "selective binding", "selectively binds", and "specifically binds" refer to an antigen binding moiety (e.g., CAR) that binds to an epitope on a predetermined antigen. Typically, the antigen binding moiety (e.g., CAR) (i) binds to an epitope on a predetermined antigen with a specific binding affinity of less than 10 -7 M, such as approximately less than 10 -8 M, 10 -9 M or 10 -10 M or lower equilibrium dissociation constant (KD), such as in 2000 instrument by, for example, surface plasmon resonance (SPR) technology, using a predetermined antigen (e.g., human mesothelin) as a target polypeptide and a CAR as a ligand, and (ii) the affinity of binding to the predetermined antigen is at least two times greater than its affinity for binding to a nonspecific antigen other than the predetermined antigen or a closely related antigen. Therefore, an antigen-binding portion (e.g., CAR) that "specifically binds to human mesothelin" refers to an antigen-binding portion (e.g., CAR) that binds to human mesothelin with a specific affinity of at least 10 -7 M or less, such as approximately less than 10 -8 M, less than 10 -9 M or less than 10 -10 The KD of M binds to the antigen binding portion of human mesothelin (e.g., CAR).

[0099] As used herein, the term "engineered γδ T cells" refers to γδ T cells that express a transgene (ie, a gene that has been transduced into the engineered γδ T cells or their parental cells).

[0100] As used herein, the term "primed γδT cells" refers to a starting population (e.g., an endogenous γδT cell population) that is affected by culture conditions. In some cases, the primed γδT cells have a different functional viral entry receptor profile relative to their unprimed counterparts prior to undergoing culture conditions. In some embodiments, the primed γδT cell population is an expanded γδT cell population.

[0101] As used herein, an "expanded γδ cell population" refers to a hematopoietic cell population including γδT cells, which is cultured under conditions and durations that induce γδ cell expansion, i.e., increase the number of γδ cells. Similarly, as used herein, an "expanded Vδ1T cell population" refers to a hematopoietic cell population including Vδ1T cells, which is cultured under conditions and durations that induce Vδ1T cell expansion, i.e., increase the number of Vδ1 cells. Similarly, as used herein, an "expanded Vδ2T cell population" refers to a hematopoietic cell population including Vδ2T cells, which is cultured under conditions and durations that induce Vδ2T cell expansion, i.e., increase the number of Vδ2 cells.

[0102] 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 ) γδT cells (e.g., engineered γδT cells). A population of a particular cell type (e.g., an endogenous γδT cell population, a primed γδT cell population, or an engineered γδT cell population) refers to that type of cell and not to different types of cells within a broader population. For example, if 10 8 If 10% of the starting population of T cells are γδT cells, then the starting population of γδT cells is 10 7 indivual.

[0103] As used herein, "armored protein" refers to a protein encoded by a transgene, which, when expressed by an immune cell, such as a NK cell or a γδT cell (e.g., a γδT cell expressing a CAR), improves the persistence and / or proliferation of immune cells, such as NK cells or γδT cells, and / or improves the cytotoxicity of immune cells, such as NK cells or γδT cells to target cells, to improve, for example, cell persistence, cell viability, activation, and other desired features. Armored protein can be a membrane-bound protein or a soluble protein. For example, armored proteins include membrane-bound proteins, such as 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), αβTCR, 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 In some aspects, armorin can be a soluble protein, such as 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 aspects, armorin is not antigen-specific. In some aspects, armorin comprises an IL-2Rβ polypeptide or a fragment thereof. In some aspects, the IL-2Rβ polypeptide is a recombinant polypeptide. In some aspects, the IL-2Rβ polypeptide is expressed in the absence of IL-2Rα and tethered to IL-15. In some aspects, the IL-2Rβ polypeptide is expressed in the absence of IL-2Rα and not tethered to IL-15.

[0104] In some aspects, armorin comprises two polypeptides. In some aspects, armorin comprises two fused polypeptides, i.e., as continuous polypeptides. In some aspects, armorin comprises two polypeptides that associate to form a dimer. In some aspects, armorin comprises a dimer comprising (i) a fusion protein containing IL-15Rα and IL-15 and (ii) IL-2Rβ.

[0105] As used herein, "IL-15" refers to natural or recombinant IL-15 or a variant thereof having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity over an equal length portion that acts as an agonist of one or more IL-15 receptor (IL-15R) subunits (e.g., mutant proteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). Like IL-2, IL-15 is a known T cell growth factor that can support the proliferation of the IL-2-dependent cell line CTLL-2. IL-15 was first reported by Grabstein et al. (Science 264.5161:965-969, 1994) as a mature protein of 114 amino acids. As used herein, the term "IL-15" means natural or recombinant IL-15 and its mutant proteins, analogs, subunits or complexes thereof (e.g., receptor complexes, e.g., sushi peptides, as described in PCT Publication No. WO2007 / 046006), and each of which can stimulate the proliferation of CTLL-2 cells. In a CTLL-2 proliferation assay, supernatants of cells transfected with recombinantly expressed precursors and in-frame fusions of mature forms of IL-15 can induce CTLL-2 cell proliferation. As used herein, the term "mbIL-15" refers to a membrane-bound IL-15Rα tethered to IL-15 (see, e.g., Fig.17A ).

[0106] 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 at and was assigned accession number 69245.

[0107] The amino acid sequence of human IL-15 (Gene ID 3600) is found in Genbank under accession locators 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 locator NP_001241676.1 GI: 363000984.

[0108] IL-15 may also refer to IL-15 derived from a variety of mammalian species, including, for example, human, monkey, cow, pig, horse and mouse. As referred to herein, an IL-15 "mutant" 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 comprise a conservative substitution sequence, meaning that a given amino acid residue is replaced by a residue having similar biochemical characteristics. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as substitutions of Ile, Val, Leu or Ala for each other, or substitutions of one polar residue for another polar residue, such as substitutions between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobic characteristics, are well known. Naturally occurring IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins produced by alternative mRNA splicing events or proteolytic cleavage of IL-15 proteins, in which IL-15 binding properties are maintained. Variable splicing of mRNA can produce truncated but biologically active IL-15 proteins. The variation attributable to proteolysis includes, for example, differences in the N or C termini after expression in different types of host cells due to proteolysis removing one or more terminal amino acids (usually 1-10 amino acids) from the IL-15 protein. In some embodiments, the ends of the protein can be modified, for example, with chemical groups such as polyethylene glycol to change its physical properties (Yang et al. Cancer 76: 687-694, 1995). In some embodiments, the ends or interior of the protein can be modified with additional amino acids (Clark-Lewis et al. PNAS 90: 3574-3577, 1993).

[0109] As used herein, "IL-2 receptor β subunit", "IL-2Rβ", "IL-15 receptor β subunit" and "IL-15Rβ" are used interchangeably herein to refer to natural or recombinant IL-15Rβ or a variant thereof having at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity over an equal length portion of a receptor that acts as one or more of IL-2 or IL-15 (e.g., a mutein, analog, fragment, isoform and peptidomimetic thereof). 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. As used herein, the term "IL-15Rβ" means natural or recombinant IL-2Rβ and its muteins, analogs, subunits or complexes thereof.

[0110] The amino acid sequence of human IL-2Rβ (Gene ID 3560) is found in Genbank under accession locators 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 locator NP_032394.

[0111] IL-15Rβ may also refer to IL-15Rβ derived from a variety of mammalian species, including, for example, human, simian, bovine, porcine, equine, and murine. As referred to herein, an IL-15Rβ "mutant" 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. A variant may comprise a conservative substitution sequence, meaning that a given amino acid residue is replaced by a residue having similar biochemical characteristics. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as substitutions of Ile, Val, Leu, or Ala for each other, or substitutions of one polar residue for another polar residue, such as substitutions between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions having similar hydrophobic characteristics, are well known. 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, wherein the IL-15Rβ binding properties are maintained. Alternative splicing of mRNA can produce truncated but biologically active IL-15Rβ proteins. Variations attributable to proteolysis include, for example, differences in the N or C termini after expression in different types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-15Rβ protein.

[0112] As used herein, "IL-15 receptor alpha subunit" and "IL-15Rα" refer to natural or recombinant IL-15Rα or a variant thereof that acts as an IL-15 receptor (e.g., a mutant protein, analog, fragment, isoform, and peptidomimetic thereof). 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. As used herein, the term "IL-15Rα" means natural or recombinant IL-15Rα and a mutant protein, analog, subunit, or complex thereof. Fragments of IL-15Rα include soluble fragments, such as sushi peptides, as described in PCT Publication No. WO 2007 / 046006. Soluble fragments (e.g., sushi peptides, such as fragments containing residues 31-95 of IL-15Rα or a variant thereof, which fragments have at least 70% (e.g., at least 80%, 85%, 90%, 95%, 97%, 99%, e.g., at least 85%) sequence identity over equal length portions) also include mutant proteins, analogs, fragments, isoforms, and peptidomimetics thereof. In some embodiments, the soluble fragment is attached to an Fc domain, e.g., Fc-sushi or sushi-Fc.

[0113] The amino acid sequence of human IL-15Rα (Gene ID 3601) is found in Genbank under accession locators 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 locator NP_001258426.

[0114] IL-15Rα may also refer to IL-15Rα derived from a variety of mammalian species, including, for example, humans, monkeys, cows, pigs, horses and mice. As referred to herein, an IL-15Rα "mutant" 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. The variant may comprise a conservative substitution sequence, meaning that a given amino acid residue is replaced by a residue with similar biochemical characteristics. Examples of conservative substitutions include substitutions of one aliphatic residue for another aliphatic residue, such as substitutions of Ile, Val, Leu or Ala for each other, or substitutions of one polar residue for another polar residue, such as substitutions between Lys and Arg; Glu and Asp; or Gln and Asn. Other such conservative substitutions, for example, substitutions of entire regions with similar hydrophobic characteristics, are well known. Naturally occurring IL-15Rα variants are also encompassed by the present invention. Examples of such variants are proteins produced by alternative mRNA splicing events or proteolytic cleavage of IL-15Rα proteins, in which IL-15Rα binding properties are maintained. Alternative splicing of mRNA can produce truncated but biologically active IL-15Rα proteins. Variations attributable to proteolysis include, for example, differences in the N or C termini after expression in different types of host cells due to proteolysis removing one or more terminal amino acids (usually 1-10 amino acids) from the IL-15Rα protein. In some embodiments, the ends of the protein may be modified, for example, with chemical groups such as polyethylene glycol to change its physical properties (Yang et al. Cancer 76: 687-694, 1995). In some embodiments, the ends or interiors of the protein may be modified with additional amino acids (Clark-Lewis et al. PNAS 90: 3574-3577, 1993).

[0115] "Polypeptide" refers to a chain of at least two consecutively linked amino acid residues, with no upper limit to the length of the chain. One or more amino acid residues in a protein may contain modifications, such as but not limited to glycosylation, phosphorylation or disulfide bond formation. A "protein" may contain one or more polypeptides.

[0116] As used herein, the term "mesothelin" or "CAK1 antigen" refers to a glycosylphosphatidylinositol-anchored membrane polypeptide involved in cell adhesion (see UNIPROT reference number Q13421). Overexpression of mesothelin has been observed in many types of cancer, making mesothelin a promising target for CAR-T therapy.

[0117] As used herein, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0118] "Conservative amino acid substitution" refers to replacing an amino acid residue with an amino acid residue with 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some aspects, the non-essential amino acid residues predicted in the mesothelin binding moiety (e.g., anti-mesothelin CAR) are replaced with another amino acid residue from the same side chain family.

[0119] As used herein, the term "tether" or "tethered" refers to a polypeptide that, when present, links two or more polypeptides (e.g., IL-15Rα or a fragment thereof (e.g., a sushi domain) and IL-15 or a variant thereof). In general, the tether may be about, for example, 1 to 100 (e.g., 5 to 50, 5 to 30, 5 to 20, such as 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 may be rich in glycine (G) and / or serine (S) residues, which may be present in the form of GS repeats. In some embodiments, the tether may connect the C-terminus of the first protein to the N-terminus of the second protein. In other embodiments, the tether can attach the C-terminus of the second protein to the N-terminus of the first protein.

[0120] As used herein, the term "percent identity (%)" refers to the percentage of amino acid residues of a candidate sequence (e.g., a VH, VL or IL-15Rβ variant) that are identical to 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 can be introduced in one or both of the candidate and reference sequences to achieve optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for determining percent identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid sequence identity of a given candidate sequence to, with, or against a given reference sequence (which can alternatively be expressed as a given candidate sequence having or including a certain percent amino acid sequence identity to, with, or against a given reference sequence) is calculated as follows:

[0121] 100x(A / B fraction)

[0122] Wherein A is the number of amino acid residues scored as identical in an alignment of the candidate sequence with the reference sequence, and wherein 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 between the candidate sequence and the reference sequence will not be equal to the percent amino acid sequence identity between the reference sequence and the candidate sequence.

[0123] As used herein, "2A peptide" refers to a class of 18-22 amino acid long peptides that share the core sequence motif of DxExNPGP, where X is any amino acid. The 2A peptide can be a foot-and-mouth disease virus 18 2A (F2A) peptide, an equine rhinitis A virus 2A (E2A) peptide, a porcine Teschovirus-12A (P2A) peptide, or a lycopodiella virus 2A (T2A) peptide.

[0124] "Suicide gene" refers to a gene that causes a cell to commit suicide. In some aspects, a suicide gene causes a cell to commit suicide by apoptosis. Non-limiting examples of suicide genes include viral thymidine kinase, cytosine deaminase, intracellular antibodies to antioxidant enzymes (AOE), bacterial nitroreductase, caspase, and DNA enzyme.

[0125] In one aspect, the suicide gene is a viral thymidine kinase (TK). Thymidine kinase is an ATP-thymidine 5'-phosphotransferase, which can convert deoxythymidine into deoxythymidine 5'-monophosphate, which is further phosphorylated to deoxythymidine diphosphate, and then phosphorylated to deoxythymidine triphosphate by viral thymidine kinase and nucleoside diphosphate kinase, respectively. Deoxythymidine triphosphate is incorporated into synthetic DNA molecules by DNA polymerase. Some dNTP analogs, such as ganciclovir (GCV), i.e., synthetic analogs of 2'-deoxyguanosine, can terminate DNA synthesis after it is incorporated into synthetic DNA. Synthetic termination triggers apoptosis signal transduction cascade. Although GCV is not recognized by human thymidine kinase, it is recognized as a substrate of some viral thymidine kinases, such as herpes simplex virus 1 thymidine kinase (HSV-TK). Therefore, human cells expressing HSV-TK convert GCV into GCV phosphate, which is further phosphorylated and incorporated into synthetic DNA, thereby causing synthesis termination and cell apoptosis. Although variants of HSV-TK are not limited, HSV-TK is, for example, TK007 (see Preuss et al., Hum Gene Ther. 2010 Aug;21(8):929-41).

[0126] In some aspects, the suicide gene is cytosine deaminase. Cytosine deaminase hydrolyzes cytosine to uracil and releases ammonia. Under physiological conditions, the modified site is recognized by the nuclease endonuclease, and then the phosphodiester bond in the DNA is destroyed and repair begins by incorporating new cytosine. However, cytosine deaminase can also convert 5-fluorocytosine into 5-fluorouracil (5-FU). Therefore, after providing the non-toxic prodrug 5-FC, cytosine deaminase converts it into the highly toxic 5-FU (suicide inhibitor of thymidylate synthase), thereby inhibiting cell growth and apoptosis.

[0127] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to a viral genome. Some vectors can replicate autonomously in the host cell introduced therein (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, some vectors can guide the expression of the gene operably connected thereto. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors used in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have the same function.

[0128] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell that contains a nucleic acid that is not naturally present in the cell, and may be a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the specific subject cell, but also to the progeny of the cell. Because certain modifications may occur in subsequent generations due to mutations 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.

[0129] "Immune response" is as understood in the art, and generally refers to a biological response in a vertebrate body to a foreign agent or abnormal substance (e.g., cancerous cells), which protects the organism from damage to these agents and diseases caused by them. 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 (including antibodies, cytokines, and complement) produced by any of these cells or the liver, resulting in selective targeting, binding, damage, destruction, and / or elimination of invading pathogens, cells or tissues infected by pathogens, cancerous cells, or other abnormal cells in vertebrates, or in the case of autoimmunity or pathological inflammation, normal human cells or tissues. Immune response includes, for example, activation or suppression of T cells, such as effector T cells, Th cells, CD4+ cells, CD8+T cells, or Treg cells, or activation or suppression of any other cells of the immune system, such as NK cells.

[0130] "Immunotherapy" refers to the treatment of a subject suffering from a disease or at risk of contracting a disease or suffering recurrence of a disease by methods that include inducing, enhancing, suppressing or otherwise altering the immune system or immune response.

[0131] As used herein, the terms "treat," "treatment," or "treatment of" when used in the context of treating a disease or condition (e.g., cancer) in a subject refer to reducing disease pathology, reducing or eliminating disease symptoms, promoting improved survival, and / or reducing discomfort. For example, treatment may refer to the ability of a therapy to reduce one or more disease symptoms, signs, or causes when administered to a subject. Treatment also refers to alleviating or reducing 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.

[0132] As used herein, "cancer" refers to a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0133] As used herein, the term "effective amount" or "therapeutically effective amount" of the administered therapeutic substance (such as an immune cell comprising a polynucleotide encoding a CAR) is an amount sufficient to achieve a specific specified or intended purpose (such as treating or treating cancer). An "effective amount" can be determined empirically in a conventional manner relative to a specified purpose.

[0134] As used herein, the term "subject", "individual" or "patient" refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis or treatment is desired. Mammalian subjects include, for example, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, bears, and the like.

[0135] As used herein, the terms "ug" and "uM" are used interchangeably with "μg" and "μM", respectively.

[0136] Various aspects described herein are described in further detail in the following subsections.

[0137] II. Compositions of the Disclosure

[0138] Some aspects of the present disclosure relate to an antigen binding portion that specifically binds to human mesothelin. Some aspects of the present disclosure relate to polynucleotides comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that binds to human mesothelin. Some aspects of the present disclosure relate to a host cell comprising a polynucleotide disclosed herein, such as an immune cell, such as an innate lymphoid cell. Another aspect of the present disclosure relates to a method for treating a subject in need thereof, comprising administering a polynucleotide and / or a cell to the subject.

[0139] 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.

[0140] 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 soluble 31-KD N-terminal protein called megakaryocyte potentiating factor (MPF) and mesothelin.

[0141] In one embodiment, the innate lymphoid cell is a natural killer (NK) cell. In an alternative embodiment, the innate lymphoid cell is a gamma delta (γδ) T cell. In another embodiment, the γδ T cell is a Vd1+γδ cell.

[0142] Reference herein to a "heterologous targeting construct" refers to any targeting construct that is not normally expressed by a cell.

[0143] In one embodiment, the heterologous targeting construct is a chimeric antigen receptor (CAR). CAR is composed of a single-chain variable fragment (scFv), which is composed of an antibody variable heavy chain (VH) and a variable light chain (VL) fused together by a peptide linker. This antigen-binding scFv of CAR is linked to the intracellular CD3ζ signaling domain. The first generation CAR contains only CD3ζ, while the second generation chimeric antigen receptor includes additional signaling domains, such as CD28 or 4-1BB. A third generation CAR including multiple costimulatory signaling modules has also been reported.

[0144] In another embodiment, CAR is a full-length CAR or a non-signaling CAR. "Full-length chimeric antigen receptor" or "full-length CAR" or "FL CAR" mentioned herein refers to a CAR comprising an intracellular signaling domain. "Non-signaling chimeric antigen receptor", "non-signaling CAR", "naked chimeric antigen receptor", "naked CAR" or "NKD CAR" mentioned herein refers to a CAR lacking the intracellular CD3 ζ signaling domain that is usually present in a CAR.

[0145] In another embodiment, CAR is an armored CAR. "Armored chimeric antigen receptor" or "armored CAR" mentioned herein refers to a CAR cell that has been engineered to secrete cytokines, or expresses ligands known to enhance endogenous immune cells or interact with endogenous immune cells, such as dendritic cells (DC), macrophages or regulatory T cells (Treg cells). 'Armor' can improve the cytotoxicity and specificity of CAR, escape immunosuppression, avoid host rejection and extend the therapeutic half-life of CAR.

[0146] II.A. Chimeric Antigen Receptors

[0147] Some aspects of the present disclosure relate to innate lymphoid cells (e.g., Vd1 derived from blood or skin) comprising a heterologous nucleic acid encoding a CAR. + γδ cells), wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin. In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. + γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin (e.g., wherein the antigen binding domain comprises a sequence of any one of SEQ ID NOs: 1–8, 41-48). In some aspects, the cells further comprise 4-1BB and CD3ζ domains. On the other hand, the cells relate to a γδ T cell population (e.g., Vd1 derived from blood or skin) + γδ cell population), comprising a γδT cell containing a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin (e.g., wherein the antigen binding domain comprises a sequence of any one of SEQ ID NOs: 1–8, 41-48), wherein the γδT cell further comprises a CD8 transmembrane domain. In another aspect, the cell relates to a γδT cell population (e.g., Vd1 derived from blood or skin +γδ cell population), which comprises γδT cells containing a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin (e.g., wherein the antigen binding domain comprises a sequence of any one of SEQ ID NOs: 1–8, 41-48), wherein the γδT cells further comprise (a) IL-15R-β or (b) (i) IL-15R-α tethered to IL-15 or a variant thereof and (ii) IL-15R-β. On the other hand, the γδT cells described herein comprise a heterologous nucleic acid encoding a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin (e.g., wherein the antigen binding domain comprises a sequence of any one of SEQ ID NOs: 1–8, 41-48) and may further comprise 4-1BB and a CD3ζ domain and / or a CD8 transmembrane domain and (a) IL-15R-β or (b) (i) IL-15R-α tethered to IL-15 or a variant thereof and (ii) IL-15R-β. In some aspects, the IL-2Rβ polypeptide is expressed in the absence of IL-15Rα and is tethered to IL- 15. In some aspects, the IL-15Rβ polypeptide is expressed in the absence of IL-2Rα and is not tethered to IL-15.

[0148] In some aspects, the antigen binding domain that specifically binds to human mesothelin comprises a variable heavy chain (VH) complementary determining region-1 (CDR1), VH-CDR2, VH-CDR3, a variable light chain (VL) CDR-1, VL-CDR2, and VL-CDR3. In some aspects, VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO:3. In some aspects, VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO:2. In some aspects, VH-CDR1 comprises the amino acid sequence shown in SEQ ID NO:1. In some aspects, VL-CDR1 comprises the amino acid sequence shown in SEQ ID NO:4. In some aspects, VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO:5. In some aspects, VL-CDR3 comprises the amino acid sequence shown in SEQ ID NO:6.

[0149] In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. +γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises: a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0150] In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. + γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein 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 shown 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 shown in SEQ ID NO:8, or (iii) both of (i) and (ii).

[0151] In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. + γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises (i) a VH comprising the amino acid sequence shown in SEQ ID NO:7, (ii) a VL comprising the amino acid sequence shown in SEQ ID NO:8, or (iii) both (i) and (ii).

[0152] In some aspects, an antigen binding domain that specifically binds human mesothelin comprises a P4 antigen binding domain (Table 1).

[0153] Table 1: Antigen binding domain amino acid sequence

[0154]

[0155]

[0156] In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. + γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises: a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41; a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42; a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43; a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44; a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45; and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46.

[0157] In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. + γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein 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 shown 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 shown in SEQ ID NO:48, or (iii) both of (i) and (ii).

[0158] In some aspects, the innate lymphoid cell is a blood-derived Vd1 comprising a heterologous nucleic acid molecule encoding a CAR. + γδ cells, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises (i) a VH comprising the amino acid sequence shown in SEQ ID NO:47, (ii) a VL comprising the amino acid sequence shown in SEQ ID NO:48, or (iii) both (i) and (ii).

[0159] II.A.1. Hinge region

[0160] In some aspects, CAR also includes a hinge region between an antigen binding domain and a transmembrane domain that specifically binds to mesothelin. In some aspects, hinges are derived from immunoglobulins (e.g., derived from hinge regions or loop regions). In certain aspects, these hinges include, for example, IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE or IgM hinge regions, fragments thereof (alone or by other sequences such as CH1 or CH2 region sequences capped), or a combination of fragments from IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE or IgM hinge regions. In some aspects, the hinge comprises, for example, an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM constant domain loop region, a fragment thereof (alone or capped by, for example, additional sequences from an adjacent β-strand), or a combination of fragments from an IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, or IgM loop region. In some aspects, the hinge of the present disclosure comprises a hinge region derived sequence, a loop region derived sequence, or a combination thereof.

[0161] In some aspects, the hinge region is selected from a CD8 hinge, a CD28 hinge, and an immunoglobulin hinge. In some aspects, the hinge includes a CD8 hinge. In some aspects, the CAR includes a hinge, and the hinge includes the amino acid sequence shown in SEQ ID NO:9 (Table 2).

[0162] Table 2: CAR sequences

[0163]

[0164] Therefore, in some aspects, the present disclosure provides a CAR (or a polynucleotide encoding a CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region comprising the hinge region of CD8, (iii) a transmembrane domain, and (iv) an intracellular domain. In some aspects, the present disclosure provides a CAR (or a polynucleotide encoding a CAR), wherein the CAR comprises (i) an antigen binding domain that specifically binds to mesothelin (e.g., an anti-mesothelin scFv), (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9, (iii) a transmembrane domain, and (iv) an intracellular domain.

[0165] II.A.2. Transmembrane domain

[0166] In some aspects, CAR also includes a transmembrane domain. The transmembrane domain can be derived from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. In some aspects, whenever the CAR of the present disclosure has been bound to a target, the transmembrane domain can conduct a signal to the intracellular domain.

[0167] In some aspects, the transmembrane domain can include at least the following transmembrane regions: for example, 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, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, L FA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD 229), 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.

[0168] In some aspects, the TM domain is derived from CD8, CD2, CD4, CD28, CD45, PD1, CD152, or any combination thereof. In some aspects, the TM domain is derived from CD8.

[0169] In some aspects, 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 aspects, the TM domain comprises the amino acid sequence set forth in SEQ ID NO: 10.

[0170] II.A.3. Co-stimulatory domain

[0171] In some aspects, CAR also includes a costimulatory domain. In some aspects, the costimulatory domain includes the following costimulatory domain: 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) missing due to Lck binding, BTLA, GITR, HVEM, LFA-1, LIGHT, NKG2C, PD-1, TILR2, TILR4, TILR7, TILR9, Fc receptor gamma chain, Fc receptor epsilon chain, and CD83 specifically bound ligand or any combination thereof. In some aspects, the CAR comprises a 4-1BB co-stimulatory domain.

[0172] In some aspects, 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 aspects, the TM domain comprises the amino acid sequence set forth in SEQ ID NO: 11.

[0173] II.A.4. Intracellular signaling domain

[0174] In some aspects, CAR also includes an intracellular signaling domain. In some aspects, the intracellular signaling domain includes a CD3 ζ activation domain, a CD3 δ activation domain, a CD3 ε activation domain, a CD3 η activation domain, a CD79A activation domain, a DAP 12 activation domain, a FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof. In some aspects, the intracellular signaling domain includes a CD3 ζ activation domain.

[0175] In some aspects, 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 aspects, the intracellular signaling domain comprises the sequence set forth in SEQ ID NO: 12.

[0176] In some aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 containing an amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 containing an amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 containing an amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 containing an amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 containing an amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 containing an amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some aspects, CAR also comprises a costimulatory domain. In some aspects, CAR also comprises a hinge region.

[0177] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown 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 aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 containing an amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 containing an amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 containing an amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 containing an amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 containing an amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 containing an amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain comprising an amino acid sequence shown in SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising a CD3 ζ activation domain. In some aspects, CAR also comprises a costimulatory domain. In some aspects, CAR also comprises a hinge region.

[0178] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a co-stimulatory domain; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a co-stimulatory 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 aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 1, a VH-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 2, a VH-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 3, a VL-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 4, a VL-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 5, and a VL-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a co-stimulatory domain comprising an amino acid sequence as shown in SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 ζ activation domain. In some aspects, CAR further comprises a hinge region.

[0179] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain 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; (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 aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0180] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0181] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of 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 of 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 of SEQ ID NO: 10; NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0182] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12.

[0183] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0184] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises 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 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 amino acid sequence set forth in SEQ ID NO:10. NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0185] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the CAR comprises a VH comprising the amino acid sequence shown in SEQ ID NO:7 and a VL comprising the amino acid sequence shown in SEQ ID NO:8; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:12.

[0186] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH comprising the amino acid sequence shown in SEQ ID NO:7 and a VL comprising the amino acid sequence shown in SEQ ID NO:8; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0187] In some aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 containing an amino acid sequence shown in SEQ ID NO: 41, a VH-CDR2 containing an amino acid sequence shown in SEQ ID NO: 42, a VH-CDR3 containing an amino acid sequence shown in SEQ ID NO: 43, a VL-CDR1 containing an amino acid sequence shown in SEQ ID NO: 44, a VL-CDR2 containing an amino acid sequence shown in SEQ ID NO: 45, and a VL-CDR3 containing an amino acid sequence shown in SEQ ID NO: 46; (ii) a transmembrane domain; and (iii) an intracellular signaling domain. In some aspects, CAR also comprises a costimulatory domain. In some aspects, CAR also comprises a hinge region.

[0188] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a transmembrane domain; and (iii) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain 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; (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 aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 containing an amino acid sequence shown in SEQ ID NO: 41, a VH-CDR2 containing an amino acid sequence shown in SEQ ID NO: 42, a VH-CDR3 containing an amino acid sequence shown in SEQ ID NO: 43, a VL-CDR1 containing an amino acid sequence shown in SEQ ID NO: 44, a VL-CDR2 containing an amino acid sequence shown in SEQ ID NO: 45, and a VL-CDR3 containing an amino acid sequence shown in SEQ ID NO: 46; (ii) a transmembrane domain comprising an amino acid sequence shown in SEQ ID NO: 10; and (iii) an intracellular signaling domain comprising a CD3 ζ activation domain. In some aspects, CAR also comprises a costimulatory domain. In some aspects, CAR also comprises a hinge region.

[0189] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a transmembrane domain; (iii) a co-stimulatory domain; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain 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; (ii) a transmembrane domain; (iii) a co-stimulatory 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 aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 41, a VH-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 42, a VH-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 43, a VL-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 44, a VL-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 45, and a VL-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 46; (ii) a transmembrane domain; (iii) a co-stimulatory domain comprising an amino acid sequence as shown in SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 ζ activation domain. In some aspects, CAR further comprises a hinge region.

[0190] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a hinge region; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; (ii) a hinge region comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 46. NO:9 has 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; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0191] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0192] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence of 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 of 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 of SEQ ID NO:9; NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0193] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:12.

[0194] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0195] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises 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 shown 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 shown 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 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 SEQ ID NO: 9. NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0196] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the CAR comprises a VH comprising the amino acid sequence shown in SEQ ID NO:47 and a VL comprising the amino acid sequence shown in SEQ ID NO:48; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:12.

[0197] In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 47 and a VL comprising the amino acid sequence shown in SEQ ID NO: 48; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0198] II.A.4. Armor

[0199] In some aspects, the modified immune cells disclosed herein (e.g., NK cells or γδT cells expressing the CAR disclosed herein) also express armorin. In some aspects, armorin increases the persistence of modified immune cells relative to similar modified immune cells that do not express armorin. In some aspects, armorin increases the proliferation of modified immune cells relative to similar modified immune cells that do not express armorin. In some aspects, armorin increases the cytotoxicity of modified immune cells relative to similar modified immune cells that do not express armorin.

[0200] In some aspects, armorin comprises a membrane-bound polypeptide. In some aspects, armorin comprises a membrane-bound receptor. In some aspects, a membrane-bound receptor comprises IL-15R (e.g., IL-15Rβ and IL-15Rα). In some aspects, a membrane-bound receptor comprises IL-2R (e.g., IL-2Rβ). In some aspects, a membrane-bound receptor comprises αβTCR. In some aspects, a membrane-bound receptor comprises a natural cytotoxic receptor (e.g., NKp30, NKp44 or NKp46). In some aspects, a membrane-bound receptor comprises a cytokine receptor (e.g., IL-12 receptor). In some aspects, a membrane-bound receptor comprises a chemokine receptor (e.g., CCR2 receptor). In some aspects, armorin comprises a membrane-bound ligand or a cytokine. In some aspects, a membrane-bound ligand or a cytokine comprises a membrane-bound IL-15, a membrane-bound IL-7, a membrane-bound CD40L, a membrane-bound 4-1BB, a membrane-bound 4-1BBL, a membrane-bound CCL19 or any combination thereof.

[0201] In some aspects, armorin comprises a soluble polypeptide. In some aspects, the soluble protein comprises a soluble ligand. In some aspects, the soluble protein comprises a cytokine. In some aspects, armorin comprises soluble IL-15, soluble IL-7, soluble IL-12, soluble CD40L, soluble 4-1BBL, soluble CCL19, or any combination thereof.

[0202] In some aspects, the armorin comprises a membrane-bound polypeptide or a soluble polypeptide.

[0203] In some aspects, armorin comprises an IL-2Rβ polypeptide. In some aspects, armorin 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 with the amino acid sequence shown in SEQ ID NO: 15 (Table 3). In some aspects, armorin comprises an amino acid sequence shown in SEQ ID NO: 15. In some aspects, armorin further comprises a signal peptide, wherein the signal peptide is cleaved after translation. Any signal peptide can be used. In some aspects, armorin comprises an IL-2Rβ polypeptide, and armorin does not comprise an IL-15Rα polypeptide or a construct containing an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0204] Table 3: Armoured proteins

[0205]

[0206]

[0207] In some aspects, the armorin comprises an IL-15Rα polypeptide. In some aspects, the armorin comprises a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some aspects, the armorin comprises (i) an IL-2Rβ polypeptide and (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some aspects, the armorin comprises (i) an IL-2Rβ polypeptide and (ii) a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0208] In some aspects, 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 aspects, the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In some aspects, the IL-15Rα polypeptide comprises a signal peptide sequence.

[0209] In some aspects, 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 aspects, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16. In some aspects, the IL-15 polypeptide comprises a signal peptide sequence.

[0210] In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker. In some aspects, the linker comprises one or more peptide bonds. In some aspects, the linker comprises a peptide linker. In some aspects, the peptide linker is a flexible linker. In some aspects, the linker is a rigid linker. In some aspects, the linker is a cleavable linker. In some aspects, the linker is a Gly-Ser linker. In some aspects, the linker comprises one or more repeats of the sequence GGGS, GGGS (SEQ ID NO: 19) or a combination thereof. In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker comprising the amino acid sequence shown in SEQ ID NO: 20. In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker comprising the amino acid sequence shown in SEQ ID NO: 21. In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker comprising the amino acid sequence shown in SEQ ID NO: 22. In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker comprising the amino acid sequence shown in SEQ ID NO: 23. In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker comprising the amino acid sequence set forth in SEQ ID NO: 24. In some aspects, the IL-15Rα polypeptide is tethered to the IL-15 polypeptide via a linker comprising the amino acid sequence set forth in SEQ ID NO:25.

[0211] In some aspects, the 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) N-terminus, (ii) 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) IL-15Rα polypeptide; (v) C-terminus. In some aspects, the 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) 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) C-terminus.

[0212] In some aspects, armorin is expressed as a single polypeptide. In some aspects, armorin 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 aspects, armorin 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 aspects, the cleavable linker comprises a P2A sequence. In some aspects, 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 with the amino acid sequence set forth in SEQ ID NO: 18. In some aspects, armorin 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.

[0213] In some aspects, armorin is expressed as a single polypeptide comprising (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-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: 18. NO:17 has 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; and 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 shown in SEQ ID NO:16.

[0214] In some aspects, armorin 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 tethered to an IL-15 polypeptide; wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO:17; and wherein the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:16.

[0215] II.B. Nucleic Acid Molecules

[0216] Some aspects of the present disclosure relate to a polynucleotide or polynucleotide set encoding a CAR disclosed herein, i.e., a CAR that specifically binds to mesothelin. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises: a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4; a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5; and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6.

[0217] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to 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 shown in SEQ ID NO: 7. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 7.

[0218] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises a VL, and 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 shown in SEQ ID NO: 8. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 8.

[0219] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to 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 shown 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 shown in SEQ ID NO:8.

[0220] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises (i) a VH comprising the amino acid sequence shown in SEQ ID NO:7, and (ii) a VL comprising the amino acid sequence shown in SEQ ID NO:8.

[0221] Some aspects of the present disclosure relate to a polynucleotide or polynucleotide set encoding a CAR disclosed herein, i.e., a CAR that specifically binds to mesothelin. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises: a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 41; a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42; a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 43; a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 44; a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 45; and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 46.

[0222] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to 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 shown in SEQ ID NO: 47. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises a VH comprising the amino acid sequence shown in SEQ ID NO: 47.

[0223] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises a VL that 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 shown in SEQ ID NO: 48. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises a VL comprising the amino acid sequence shown in SEQ ID NO: 48.

[0224] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to 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 shown 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 shown in SEQ ID NO:48.

[0225] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, and wherein the CAR comprises (i) a VH comprising the amino acid sequence shown in SEQ ID NO:47, and (ii) a VL comprising the amino acid sequence shown in SEQ ID NO:48.

[0226] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein; (ii) a transmembrane domain disclosed herein; and (iii) an intracellular signaling domain disclosed herein. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein; (ii) a CD8 transmembrane domain; and (iii) an intracellular signaling domain disclosed herein. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein; (ii) a transmembrane domain disclosed herein; and (iii) a CD3 ζ intracellular signaling domain. In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to mesothelin as disclosed herein; (ii) a CD8 transmembrane domain; and (iii) a CD3 ζ intracellular signaling domain.

[0227] In some aspects, the polynucleotide or polynucleotide set 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 with the amino acid sequence set forth in SEQ ID NO: 10; and (iii) an intracellular signaling domain disclosed herein. In some aspects, the polynucleotide or polynucleotide set 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 with the amino acid sequence set forth in SEQ ID NO: 12. In some aspects, the polynucleotide or polynucleotide set 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 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.

[0228] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR further comprises a hinge region disclosed herein. In some aspects, the hinge region comprises a CD8 hinge region. In some aspects, 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 shown in SEQ ID NO: 9. In some aspects, the hinge region comprises an amino acid sequence shown in SEQ ID NO: 9.

[0229] In some aspects, the polynucleotide or polynucleotide set encodes CAR, wherein CAR also comprises a co-stimulatory region disclosed herein. In some aspects, the co-stimulatory region comprises a 4-1BB co-stimulatory region. In some aspects, the co-stimulatory 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 shown in SEQ ID NO: 11. In some aspects, the co-stimulatory region comprises an amino acid sequence shown in SEQ ID NO: 11.

[0230] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a co-stimulatory domain; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a co-stimulatory 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 aspects, CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 1, a VH-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 2, a VH-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 3, a VL-CDR1 comprising an amino acid sequence as shown in SEQ ID NO: 4, a VL-CDR2 comprising an amino acid sequence as shown in SEQ ID NO: 5, and a VL-CDR3 comprising an amino acid sequence as shown in SEQ ID NO: 6; (ii) a transmembrane domain; (iii) a co-stimulatory domain comprising an amino acid sequence as shown in SEQ ID NO: 11; and (iv) an intracellular signaling domain comprising a CD3 ζ activation domain. In some aspects, CAR further comprises a hinge region.

[0231] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain 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; (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 aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0232] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0233] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 9; ID NO:10 having an amino acid sequence with 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; (iv) a costimulatory domain comprising an amino acid sequence with 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 shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising an amino acid sequence with 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 shown in SEQ ID NO:12.

[0234] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12.

[0235] In some aspects, the polynucleotides of the polynucleotide set encode a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO: 6; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 ζ activation domain.

[0236] In some aspects, the polynucleotides of the polynucleotide set encode a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises 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 shown 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 shown 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 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 SEQ ID NO:10. NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0237] In some aspects, the polynucleotides of the polynucleotide set encode CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the CAR comprises a VH comprising the amino acid sequence shown in SEQ ID NO:7 and a VL comprising the amino acid sequence shown in SEQ ID NO:8; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:12.

[0238] In some aspects, the polynucleotides of the polynucleotide set encode CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH comprising the amino acid sequence shown in SEQ ID NO:7 and a VL comprising the amino acid sequence shown in SEQ ID NO:8; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0239] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a transmembrane domain; (iii) a co-stimulatory domain; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a transmembrane domain; (iii) a co-stimulatory 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 aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a transmembrane domain; (iii) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (iv) an intracellular signaling domain comprising a CD3 zeta activation domain.In some aspects, the CAR further comprises a hinge region.

[0240] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a hinge region; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain. In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 46; (ii) a hinge region comprising a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 45, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 46. NO:9 has 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; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.In some aspects, the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain; (iv) a co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0241] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3 zeta activation domain.

[0242] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown 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 shown 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 shown in SEQ ID NO:9; NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0243] In some aspects, the polynucleotide or polynucleotide set encodes a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:12.

[0244] In some aspects, the polynucleotides of the polynucleotide set encode a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence shown in SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence shown in SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence shown in SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence shown in SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:46; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0245] In some aspects, the polynucleotides of the polynucleotide set encode a CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises 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 shown 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 shown 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 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 SEQ ID NO: 9. NO:10 having 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; (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 shown 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 shown in SEQ ID NO:12.

[0246] In some aspects, the polynucleotides of the polynucleotide set encode CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the CAR comprises a VH comprising the amino acid sequence shown in SEQ ID NO:47 and a VL comprising the amino acid sequence shown in SEQ ID NO:48; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:12.

[0247] In some aspects, the polynucleotides of the polynucleotide set encode CAR, wherein the CAR comprises (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH comprising the amino acid sequence shown in SEQ ID NO:47 and a VL comprising the amino acid sequence shown in SEQ ID NO:48; (ii) a CD8 hinge region; (iii) a CD8 transmembrane domain; (iv) a 4-1BB co-stimulatory domain; and (v) an intracellular signaling domain comprising a CD3ζ activation domain.

[0248] Some aspects of the present disclosure relate to a polynucleotide or a collection of polynucleotides encoding an armorin protein, i.e., an armorin protein disclosed herein. In some aspects, the polynucleotide or a collection of polynucleotides encodes an armorin protein, wherein the armorin protein comprises an IL-2Rβ polypeptide (e.g., a recombinant IL-2Rβ polypeptide) (e.g., wherein the armor comprises a recombinant IL-2Rβ polypeptide and the polynucleotide or polynucleotide collection does not express an IL-15Rα polypeptide tethered to an IL-15 polypeptide). In some aspects, the polynucleotide or a collection of polynucleotides encodes an armorin protein 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%, at least about 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 15. In some aspects, the polynucleotide or a collection of polynucleotides encodes an armorin protein comprising an amino acid sequence shown in SEQ ID NO: 15. In some aspects, the polynucleotide or collection of polynucleotides encodes armorin, wherein the armorin comprises (i) a signal peptide and (ii) an IL-2Rβ polypeptide, wherein the signal peptide is cleaved post-translationally. In some aspects, the armorin comprises an IL-2Rβ polypeptide, and the armorin does not comprise an IL-15Rα polypeptide or a construct comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0249] In some aspects, the polynucleotide or collection of polynucleotides encodes (i) an IL-2Rβ polypeptide disclosed herein, and (ii) does not encode an IL-15Rα polypeptide or a construct containing an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0250] In some aspects, a polynucleotide or collection of polynucleotides encodes (i) an IL-2Rβ polypeptide disclosed herein, and (ii) an IL-15Rα polypeptide disclosed herein or a construct containing an IL-15Rα polypeptide tethered to an IL-15 polypeptide disclosed herein.

[0251] In some aspects, the polynucleotide or collection of polynucleotides encodes an armorin comprising an IL-15Rα polypeptide. In some aspects, the polynucleotide or collection of polynucleotides encodes an armorin comprising an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some aspects, the polynucleotide or collection of polynucleotides encodes an armorin comprising (i) an IL-2Rβ polypeptide and (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

[0252] In some aspects, the polynucleotide or collection of polynucleotides encodes an armorin 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 aspects, the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17. In some aspects, the polynucleotide or collection of polynucleotides encodes a signal peptide immediately upstream of the sequence encoding the IL-15Rα polypeptide.

[0253] In some aspects, the polynucleotide or collection of polynucleotides encodes an armorin 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 aspects, the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16. In some aspects, the polynucleotide or collection of polynucleotides encodes a signal peptide immediately upstream of the sequence encoding the IL-15 polypeptide.

[0254] In some aspects, a single polynucleotide of the present disclosure encodes an IL-15Rα polypeptide tethered to an IL-15 polypeptide via a linker. In some aspects, the linker comprises one or more peptide bonds. In some aspects, the linker is a Gly-Ser linker. In some aspects, the linker comprises one or more repeats of the sequence GGGS, GGGS (SEQ ID NO: 19), or a combination thereof. In some aspects, a single polynucleotide of the present disclosure encodes an IL-15Rα polypeptide tethered to an IL-15 polypeptide via a linker, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 20-25.

[0255] In some aspects, a single polynucleotide of the present disclosure encodes (i) an IL-2Rβ polypeptide; (ii) a linker; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide. In some aspects, 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 aspects, the cleavable linker comprises a P2A sequence. In some aspects, 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 aspects, a single polynucleotide of the present 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.

[0256] In some aspects, a single polynucleotide of the present disclosure encodes (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-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: 18. NO:17 has 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; and 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 shown in SEQ ID NO:16.

[0257] In some aspects, a single polynucleotide of the present 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 tethered to an IL-15 polypeptide; wherein the IL-15Rα polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 17; and wherein the IL-15 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16.

[0258] In some aspects, (i) CAR and (ii) armored protein are both encoded by a single polynucleotide. In some aspects, the nucleic acid sequence of the single polynucleotide encoding CAR (i) and the nucleic acid sequence of the single polynucleotide encoding armored protein (ii) are separated by IRES. In some aspects, the nucleic acid sequence of the single polynucleotide encoding CAR (i) and the nucleic acid sequence of the single polynucleotide encoding armored protein (ii) are expressed under the control of the same promoter sequence. In some aspects, the nucleic acid sequence of the single polynucleotide encoding CAR (i) is expressed under the control of the first promoter, and the nucleic acid sequence of the single polynucleotide encoding armored protein (ii) is expressed under the control of the second promoter. In some aspects, the first promoter is the same as the second promoter. In some aspects, the first promoter is different from the second promoter.

[0259] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of 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 of 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 of SEQ ID NO: 10; NO:10 has 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; (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; and (B) an armored protein comprising (i) an IL-2Rβ polypeptide 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:13. NO:15 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 of 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 of SEQ ID NO:18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide; wherein the IL-15Rα polypeptide comprises an amino acid sequence identical to SEQ ID NO:18. The amino acid sequence set forth in NO:17 has 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;and 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. ;

[0260] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises 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. NO:10 has 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; (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; and (B) an armored protein comprising (i) an IL-2Rβ polypeptide 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:13. NO:15 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 of 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 of SEQ ID NO:18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide; wherein the IL-15Rα polypeptide comprises an amino acid sequence identical to SEQ ID NO:18. The amino acid sequence set forth in NO:17 has 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;and 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. ;

[0261] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence of SEQ ID NO: 9; (iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence of SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO: 12; and (B) an armored protein comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:15; (ii) a linker comprising the amino acid sequence shown 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 shown in SEQ ID NO:17; and wherein the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:16.

[0262] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:7 and a VL comprising the amino acid sequence of SEQ ID NO:8; (ii) a hinge region comprising the amino acid sequence of SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:12; and (B) an armorin comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:15; (ii) a linker comprising the amino acid sequence of 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 of SEQ ID NO:17; and wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:18. The amino acid sequence shown in NO:16.

[0263] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence of 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 of 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 of SEQ ID NO:9; NO:10 has 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; (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 shown 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 shown in SEQ ID NO:12; and (B) an armored protein comprising (i) an IL-2Rβ polypeptide 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 shown in SEQ ID NO:13. NO:15 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 of 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 of SEQ ID NO:18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide; wherein the IL-15Rα polypeptide comprises an amino acid sequence identical to SEQ ID NO:18. The amino acid sequence set forth in NO:17 has 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;and 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. ;

[0264] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises 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: NO:10 has 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; (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; and (B) an armored protein comprising (i) an IL-2Rβ polypeptide 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:13. NO:15 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 of 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 of SEQ ID NO:18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide; wherein the IL-15Rα polypeptide comprises an amino acid sequence identical to SEQ ID NO:18. The amino acid sequence set forth in NO:17 has 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;and 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. ;

[0265] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:41, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:42, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:43, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:44, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:45, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:46; (ii) a hinge region comprising the amino acid sequence of SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:10; (iv) a costimulatory domain comprising the amino acid sequence of SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:12. NO:12; and (B) armored protein, which comprises (i) an IL-2Rβ polypeptide, which comprises the amino acid sequence shown in SEQ ID NO:15; (ii) a linker, which comprises the amino acid sequence shown 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 shown in SEQ ID NO:17; and wherein the IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:16.

[0266] Some aspects of the present disclosure relate to a polynucleotide encoding: (A) a CAR comprising (i) an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO:47 and a VL comprising the amino acid sequence of SEQ ID NO:48; (ii) a hinge region comprising the amino acid sequence of SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO:10; (iv) a co-stimulatory domain comprising the amino acid sequence of SEQ ID NO:11; and (v) an intracellular signaling domain comprising the amino acid sequence of SEQ ID NO:12; and (B) an armorin comprising (i) an IL-2Rβ polypeptide comprising the amino acid sequence of SEQ ID NO:15; (ii) a linker comprising the amino acid sequence of 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 of SEQ ID NO:17; and wherein the IL-15 polypeptide comprises the amino acid sequence of SEQ ID NO:19. The amino acid sequence shown in NO:16.

[0267] In some aspects, the polynucleotide or polynucleotide set further comprises a suicide gene disclosed herein. In some aspects, the suicide gene encodes viral thymidine kinase, cytosine deaminase, intracellular antibody against antioxidant enzyme (AOE), bacterial nitroreductase, caspase and DNase.

[0268] Some aspects of the present disclosure relate to a vector comprising a polynucleotide or polynucleotide set disclosed herein. In some aspects, the present disclosure relates to a vector or vector set comprising a polynucleotide encoding a CAR as described herein. In some aspects, the vector set comprises a first vector and a second vector, wherein the first vector comprises a nucleic acid sequence encoding a CAR disclosed herein, and the second vector comprises a nucleic acid sequence encoding an armored protein disclosed herein. In some aspects, the present disclosure relates to a vector comprising (i) a nucleic acid sequence encoding a CAR disclosed herein and (ii) a nucleic acid sequence encoding an armored protein disclosed herein.

[0269] Any vector may be suitable for use in the present disclosure. In some aspects, the vector is a viral vector. In some aspects, the vector is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

[0270] II.C. Engineered cells

[0271] Some aspects of the present disclosure relate to an engineered cell 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 aspects, the engineered cell is an immune cell. In some aspects, the engineered cell is an innate lymphoid cell. In some aspects, the engineered cell is selected from a T cell, a NK cell, a B cell, or any combination thereof. In some aspects, the T cell is a γδT cell. In some aspects, the engineered cell is a Vδ1T cell. In some aspects, the engineered cell is a Vδ2T cell. In some aspects, the T cell is an αβT cell. In some aspects, the engineered cell is a tumor infiltrating lymphocyte (TIL).

[0272] Before the cell is engineered, the engineered cell can be obtained from any source. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from a human subject. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from a healthy human subject. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from a human subject suffering from a disease or condition. In some aspects, the disease or condition is related to the activation of the subject's immune response. In some aspects, the disease or condition includes cancer. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from a tumor sample from a human subject. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from peripheral blood from a human subject. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from a skin sample from a human subject. In some aspects, the cell before engineering, such as innate lymphoid cells, is obtained from an intestinal tissue sample obtained from a subject.

[0273] In some aspects, the engineered cells include T cells, wherein the T cells include polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR. In some aspects, the engineered cells include T cells, wherein the T cells include (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and (ii) polynucleotides encoding armorin disclosed herein.

[0274] In some aspects, the engineered cells include γδT cells, wherein the γδT cells include polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR. In some aspects, the engineered cells include γδT cells, wherein the γδT cells include (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and (ii) polynucleotides encoding armorin disclosed herein.

[0275] In some aspects, the engineered cells include Vδ1T cells, wherein the Vδ1T cells include polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR. In some aspects, the engineered cells include Vδ1T cells, wherein the Vδ1T cells include (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and (ii) polynucleotides encoding armorin disclosed herein.

[0276] In some aspects, the engineered cells include Vδ2T cells, wherein the Vδ2T cells include polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR. In some aspects, the engineered cells include Vδ2T cells, wherein the Vδ2T cells include (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and (ii) polynucleotides encoding armorin disclosed herein.

[0277] In some aspects, the engineered cells include NK cells, wherein the NK cells include polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR. In some aspects, the engineered cells include NK cells, wherein the NK cells include (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and (ii) polynucleotides encoding armorin disclosed herein.

[0278] In some aspects, engineered cells are derived from induced pluripotent stem cells (iPSC). In some aspects, iPSCs are differentiated into cells expressing innate lymphoid cells, such as T cells (e.g., γδT cells) or one or more markers of NK cells. In some aspects, iPSCs are transfected with the following before differentiating into cells expressing innate lymphoid cells, such as T cells (e.g., γδT cells) or one or more markers of NK cells: (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and / or (ii) polynucleotides encoding armored proteins disclosed herein. In some aspects, iPSCs are transfected with the following after differentiating into cells expressing innate lymphoid cells, such as T cells (e.g., γδT cells) or one or more markers of NK cells: (i) polynucleotides encoding CAR disclosed herein, such as anti-mesothelin CAR; and / or (ii) polynucleotides encoding armored proteins disclosed herein. In some aspects, 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 armorin disclosed herein during differentiation into cells expressing one or more markers of an innate lymphoid cell, e.g., a T cell (e.g., a γδ T cell) or a NK cell.

[0279] According to another aspect of the present 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 cell population comprising a plurality of engineered innate lymphoid cells as described herein.

[0280] In some embodiments, the engineered innate lymphoid cells may comprise greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90% of the total number of cells in an isolated cell population. In some aspects, 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 a cell population are engineered innate lymphoid cells as disclosed herein.

[0281] II.D. Pharmaceutical Compositions

[0282] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising an engineered innate lymphoid cell as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include: a buffer such as neutral buffered saline, phosphate buffered saline, etc.; a carbohydrate such as glucose, mannose, sucrose or dextran, mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. Cryopreservation solutions that can be used in the pharmaceutical compositions of the present invention include, for example, DMSO. The composition may be formulated, for example, for intravenous administration.

[0283] III. Methods of the Disclosure

[0284] According to another aspect of the present invention, a method of treating a patient in need thereof using an engineered innate lymphoid cell or pharmaceutical composition as described herein is provided. Some aspects of the present disclosure relate to a method of treating a disease or condition in a subject in need thereof, comprising administering to the subject an engineered innate lymphoid cell disclosed herein.

[0285] According to another aspect of the present invention, there is provided use of an engineered innate lymphoid cell or a pharmaceutical composition as described herein for treating a condition in a subject.

[0286] According to another aspect of the present invention, there is provided an engineered innate lymphoid cell or a pharmaceutical composition as described herein for use in therapy.

[0287] The engineered innate lymphoid cells or pharmaceutical compositions may be useful for treating cancer. As used herein, "cancer" refers to the abnormal growth or division of cells. Typically, cancer cells grow and / or live longer than, and out of harmony with, the normal cells and tissues around them. Cancer can be benign, precancerous, or malignant. Cancer occurs 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, testicles, 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.).

[0288] In some aspects, the disease or condition comprises cancer, e.g., the subject suffers from cancer. In some aspects, cancer comprises a solid tumor type of cancer. In some aspects, 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 or neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal region cancer, 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 In some aspects, the cancer is locally advanced. In some aspects, the cancer is metastatic. In some aspects, the cancer is refractory. In some aspects, the cancer is recurrent. In some aspects, the cancer is refractory or recurrent after one or more previous anticancer therapies. In some aspects, one or more previous anticancer therapies include standard of care therapy.

[0289] In some aspects, the compositions disclosed herein are administered in combination with other anticancer therapies. In some aspects, other anticancer therapies include chemotherapy, immunotherapy, radiotherapy, surgery, or any combination thereof. In some aspects, other anticancer therapies include chemotherapy. In some aspects, other anticancer therapies include immune checkpoint inhibitors. In some aspects, other anticancer therapies include PD-1 antagonists, PD-L1 antagonists, CTLA-4 antagonists, LAG-3 antagonists, GITR antagonists, or any combination thereof. In some aspects, anticancer therapies include antibodies or antigen binding portions thereof that specifically bind and inhibit PD-1. In some aspects, anticancer therapies include antibodies or antigen binding portions thereof that specifically bind and inhibit PD-L1.

[0290] In some aspects, the method also includes pre-treating the subject before administering the immune cell group. In some aspects, chemotherapy is administered to the subject before administering the immune cell group. In some aspects, immunodepletion chemotherapy is administered to the subject before administering the immune cell group. In some aspects, immunodepletion chemotherapy includes cyclophosphamide, fludarabine, or both.

[0291] In some aspects, the method comprises administering to a subject (i) an engineered innate lymphoid cell disclosed herein and (ii) a cytokine. In some aspects, the cytokine comprises IL-2, an analog thereof, a variant thereof, or a fragment thereof.

[0292] In some aspects, the cells of the present disclosure are expressed at a concentration of at least about 1 x 10 6 cells, at least about 2x10 6 cells, at least about 3x10 6 cells, at least about 4 x 10 6 cells, at least about 5 x 10 6 cells, 1x 10 7 cells, at least about 2 x 10 7 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, 1x 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, 1x 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 9cells or at least about 5 x 10 9 A dose of cells is administered to a subject.

[0293] Unless otherwise indicated, the practice of the present disclosure will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the scope of the art. Such techniques are fully explained in the literature. See, e.g., Sambrook et al., eds. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., eds. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover, ed. (1985) DNA Cloning, Vols. I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al., U.S. Pat. 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 (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al. (1987) Methods In Enzymology, Volumes 154 and 155; Mayer and Walker (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell (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, 2nd Edition CRC Press (2007) and Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.). .

[0294] All references cited above and all references cited herein are incorporated by reference in their entirety.

[0295] The following examples are offered by way of illustration and not limitation.

[0296] Example

[0297] Materials and methods

[0298] Cell culture

[0299] Retroviral transduction

[0300] Under a defined multiplicity of infection (MOI), amplified γδT cells were transduced with retroviral vectors in a cell expansion bag (PermaLife bag, OriGen Biomedical) coated with Retronectin (20 mg / mL). MOI refers to the number of infectious particles added to each cell during transduction (measured by flow cytometry). The viral vector was diluted in CTS OpTmizer culture medium. The transduction efficiency was determined by flow cytometry at regular intervals three days after transduction.

[0301] Flow cytometry

[0302] Immunophenotyping was performed using a BD FACSLyric™ flow cytometer. Dead cells were excluded using the LIVE / DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen Flow). Data were gated for real-time, single, Panγδ+, Vδ1+, and CAR+ events.

[0303] Transduction with a gamma-retroviral vector encoding an anti-MSLN chimeric antigen receptor

[0304] Cells were expanded in the presence of RetroNectin (20 μg / mL) and transduced with a γ-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 four to nine days after transduction.

[0305] Thawing of cryopreserved cell products

[0306] Frozen cryovials were thawed in a 37°C water bath and added to pre-warmed OpTmizer + 2.5% allogeneic plasma. Cells were centrifuged at 300 g for 7 minutes, counted and assessed for viability, and then resuspended at 2x106 cells per ml for phenotyping and downstream assays.

[0307] Survival assay

[0308] Cells were cultured in 96-well plates for 13 days in the absence of IL-15, and duplicate wells were harvested from each condition on days 3, 7, 10, and 13. Viable Pan-γδ+, CAR+ cells were quantified by flow cytometry using a BD FACS Lyric.

[0309] Repeated antigen stimulation assay

[0310] The drug product was thawed and co-cultured with A549-MLSN-fluc-gfp cells in 24-well plates (with or without 1ng / mL IL-15) at an E:T ratio of 2:1 (300,000 effectors to 150,000 targets) in Incucyte to track GFP fluorescence over time as a surrogate for tumor cell lysis. On the 2nd and 9th days of culture, cells were fed with fresh culture medium with or without 1ng / mL IL-15, and 50% of the pore volume was replaced by careful aspiration after centrifugation. On the 4th and 11th days of culture, cells were fed with fresh culture medium containing targets (150,000 per well) with or without 1ng / mL IL-15, and 50% of the pore volume was replaced by careful aspiration after centrifugation. On the 7th day, all wells were harvested, counted, and re-inoculated with an E:T of 2:1, as on the 0th day.

[0311] In vivo studies

[0312] Study 1 (Figure 16): 1x106 A549-MSLN-fluc-GFP cells were implanted into 6-8 week old NSG mice by intravenous (IV) injection. On the 19th day after implantation, mice were injected with vehicle (n=7, Cryostor CS5, Group 1), YP218γδCAR T cells (10 million, 3 million or 1 million total cells per mouse (n=7, Groups 2-4)) or P4γδCAR T cells (10 million total cells per mouse). Starting from the 19th day, mice were additionally given 1ug human IL-15 by IP injection every day. After intraperitoneal (IP) injection of 150mg / kg d-fluorescein, mice were imaged using an IVIS camera twice a week.

[0313] Study 2 (Figure 20): 1x106 A549-MSLN-fluc-GFP cells were implanted into 6-8 week old NSG mice by intravenous (IV) injection. On the 19th day after implantation, mice were injected with vehicle (n=7, Cryostor CS5, Group 1), P4 unarmored γδCAR T cells (10 million total cells per mouse (n=7, Group 2)), P4α.15.βγδCAR T cells (10 million total cells per mouse (n=7, Group 3)) or P4βγδCAR T cells (10 million total cells per mouse (n=7, Group 4)). After intraperitoneal (IP) injection of 150mg / kg d-fluorescein, mice were imaged using an IVIS camera twice a week. Mice in Group 4 were given 1μg IL-15 by IP injection every day.

[0314] Example 1

[0315] The functionality of blood-derived γδT cells and non-engineered blood-derived Vδ1 cells ("GDX012") transduced with the YP218 anti-mesothelin CAR construct against the solid tumor target OVCAR3 cells was examined. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1:1, 1:1:2.5 and 1:5 and without cytokine supplementation for 18-24h. Specific cell lysis was detected by luciferase-based fluorescence cell counting assay ( Figure 1A ). These data indicate that genetically engineering GDX012 to express mesothelin-CAR significantly enhances the intrinsic killing capacity of Vδ1 T cells against OVCAR3 cells.

[0316] Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=1) were cultured with Hela cells at an E:T ratio of 2:1 in either culture medium alone or culture 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 ( Figure 1B These data suggest that genetically engineering GDX012 to express a mesothelin-CAR significantly enhances 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.

[0317] As proof-of-concept for targeting solid tumors, GDX012 was genetically engineered with the YP218 anti-mesothelin CAR construct ( Figure 2 ). Mesothelin was selected as a proof-of-concept solid tumor target because this protein is overexpressed in various cancer indications and is an established target in various solid tumor models and clinical trials. Healthy tissues have minimal expression of mesothelin, making it an ideal solid tumor target. Anti-MSLN Vδ1 products were functionally tested to understand phenotype, cytotoxicity, proliferation, and in vivo activity, demonstrating solid tumor targeting and establishing a framework for future genetically engineered products.

[0318] GDX012, unlike skin-resident γδ T cells, exhibits modest intrinsic ability to target solid tumor targets, but this can be improved through genetic engineering. +Functionality of the target cell line Hela cells. Cryopreserved GDX012 and skin-resident γδ T cells were thawed and immediately cultured with target cells at an E:T ratio of 5:1, 2.5:1, and 1.25:1 in culture medium without cytokine supplementation for 18-24 h. Specific cell lysis was detected by luciferase-based fluorescence cell counting assay ( Figure 3A ). These data indicate that skin-resident γδT cells have an intrinsic killing capacity against solid tumor targets, with >80% tumor target lysis at a 5:1 E:T ratio, >60% at a 2.5 E:T ratio, and >50% at 1.25 E:T. GDX012 lysis of target cells did not exceed >20% lysis at all E:T ratios analyzed for Hela cells. The functionality of blood-derived γδT cells and GDX012 transduced with the YP218 anti-mesothelin CAR construct against OVCAR3 cells was examined. Cryopreserved CAR-modified blood Vδ1T 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 in culture medium and without cytokine supplementation for 18-24h. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay ( Figure 3B ). These data demonstrate that genetically engineering GDX012 to express the mesothelin-CAR significantly enhanced the intrinsic killing capacity of Vδ1 T cells against OVCAR3 tumor targets, with >90% tumor target lysis at 5:1, >75% at 2.5:1, >50% at 1.1, and >20% at 1:2.5 E:T ratios, while GDX012 lysis of target cells did not exceed 30% at all E:T ratios analyzed for OVCAR3 cells.

[0319] Mesothelin CAR-modified blood Vδ1T cells showed strong cytotoxicity against HeLa and OVCAR3 cells, and this cytotoxicity was comparable to that of CAR-modified αβT cells. Blood-derived γδT cells transduced with YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβT cells transduced with YP218 anti-mesothelin construct (n=1) were examined for HeLa ( Figure 4A ) and OVCAR3( Figure 4B ) Tumor cell functionality. Cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 and cultured for 18-24h without cytokine supplementation. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay.

[0320] Mesothelin CAR-modified blood Vδ1 T cells showed responses to HT29 cells ( Figure 5A ) and A549( Figure 5B ) has strong cytotoxicity, which is comparable to CAR-modified αβT cells. The functionality of blood-derived γδT cells transduced with YP218 anti-mesothelin CAR construct (n=2) and blood-derived αβT cells transduced with YP218 anti-mesothelin construct (n=1) was examined for HT29.MSLN. The functionality of blood-derived γδT cells transduced with YP218 anti-mesothelin CAR construct (n=3) and blood-derived αβT cells transduced with YP218 anti-mesothelin construct (n=1) was tested for and A549.MSLN cells. The cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 and without cytokine supplementation for 18-24h. Specific cell lysis was detected by a luciferase-based fluorocytometry assay.

[0321] Repeated Antigen Stimulation (RAS) Assays for Solid Tumor Targets.

[0322] Effector cells were thawed and co-cultured with solid tumor target cancer cell lines at an E:T ratio of 2:1 in medium supplemented with IL-15 (10 ng / ml). Figure 6 ). Day +2: supplement the culture with cytokines by replacing 50% of the culture medium. Day +4: supplement the target and cytokines by replacing 50% of the culture medium. Day +7: harvest the culture. Effectors were counted and an equal number of effectors seeded at D0 were re-seeded with target cells in a 2:1 E:T ratio in culture medium supplemented with IL-15 (10 mg / ml). Additional effectors were used for phenotyping by flow cytometry or seeded with target cells in a 24hr killing assay. Overall, effector cells underwent 6 rounds of intra-assay target cell killing and 21 days of culture, with the final 24hr killing assay demonstrating the 7th round of continuous killing.

[0323] Mesothelin CAR-modified blood Vδ1T cells were expanded and enriched for CAR expression after 6 rounds of tumor attack with Hela cells. Blood-derived γδT cells transduced with the YP218 anti-mesothelin CAR construct (n=2) were cultured with Hela cells in a medium supplemented with IL-15 (10ng / ml) at an E:T ratio of 2:1. On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The multiple proliferation of effector cells at each time point was calculated by dividing the cell count of the harvested effector cells by the number of cells inoculated 7 days ago. The total multiple proliferation or cumulative multiple proliferation at each time point was calculated by multiplying the multiple proliferation by the previous multiple proliferation. The cells expanded 60 times over the course of 21 days ( Fig. 7A ). CAR expression by Vδ1 T cells was measured by flow cytometry on days 0, 7, 14, and 21. Vδ1 T cells expressing CAR were enriched over the course of 21 days, increasing from <20% expression to >90% expression at the end of the assay ( Figure 7B ).

[0324] Mesothelin CAR modified blood Vδ1T cells were expanded and enriched in response to continuous killing of OVCAR3 and A549.MSLN cells in the RAS assay. Blood-derived γδT cells transduced with the YP218 anti-mesothelin CAR construct (n=3) were cultured with OVCAR3 and A549.MSLN cells in a medium (10ng / ml) supplemented with IL-15 at an E:T ratio of 2:1. On days 7, 14, and 21, cells were harvested and viable cells were counted using an NC250 cell counter. The multiple proliferation of effector cells at each time point was calculated by dividing the cell count of the harvested effector cells by the number of cells inoculated 7 days ago. The total (cumulative) multiple proliferation at each time point was calculated by multiplying the multiple proliferation by the previous multiple proliferation. Cells cultured with OVCAR3 cells expanded 20 times over the course of 21 days, while cells cultured with A549.MSLN cells expanded 400 times over the course of 21 days ( Fig. 8A ). CAR expression of Vδ1 T cells was measured by flow cytometry on days 0, 7, 14, and 21. CAR-expressing Vδ1 T cells cultured with OVCAR3 and A549.MSLN cells were enriched over the course of 21 days, increasing to >95% expression on day 21 ( Figure 8B ).

[0325] Blood-derived γδ T cells transduced with the YP218 anti-mesothelin CAR construct (n=2) were cultured with Hela cells in 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 plated at 2x10 5 Cells / well were inoculated and allowed to adhere for 4 h. Effectors were harvested, counted and cultured with adherent target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 without cytokine supplementation for 18-24 h. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay and data represent 7 rounds of serial killing ( Fig. 9A Live CAR was performed after 7, 14 and 21 days in the RAS assay against Hela cells. + Flow cytometric analysis of γδT cells. Phenotypic analysis of Vδ1T cells showed that the expression of NKG2D, CD56, NKp30, NKG2C, and PD1 was upregulated from day 0 to day 21, while CD27, which represents the effector state, was downregulated by Vδ1T cells from day 0 to day 21 ( Fig. 9B ).

[0326] Mesothelin CAR modified blood Vδ1T cells remain cytotoxic even after 21 days of RAS against OVCAR3 and A549.MSLN cells. Blood-derived γδT cells transduced with YP218 anti-mesothelin CAR constructs (n=3) were cultured with OVCAR3 and A549.MSLN cells in 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 counted at 2x10 5 Cells / well were inoculated and allowed to adhere for 4 h. Effectors were harvested, counted and cultured with adherent target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1 and 0.3125 without cytokine supplementation for 18-24 h. Specific cell lysis was detected by luciferase-based fluorescence cell counting assay ( Figures 10A-10F ).

[0327] Compared with unmodified A549 ( Fig.11A ) tumor cells, the mesothelin CAR modified blood Vδ1T cells against A549 ( Fig. 11B) has greater cytotoxicity. Blood-derived γδT cells transduced with YP218 anti-mesothelin CAR constructs (n=4) and blood-derived αβT cells transduced with YP218 anti-mesothelin constructs (n=1) were tested for functionality against A549 tumor cells and A549 tumor cells modified to overexpress MSLN. Cryopreserved CAR-modified blood Vδ1T cells were thawed and immediately cultured with target cells in culture medium at E:T ratios of 5:1, 2.5:1, 1.25:1, 0.625:1, and 0.3125 and without supplementing cytokines for 18-24h. Specific cell lysis was detected by luciferase-based fluorescent cell counting assay.

[0328] Skin-derived Vd1+γδT cells can be efficiently transduced with Meso-CAR. Skin-resident cells were thawed and immediately processed by MACS selection to remove αβT cells. The resulting negatively selected γδT cells were then transduced with vectors encoding 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 and then harvested and cryopreserved. The expansion factor of γδT cells was recorded on D14 of the expansion culture ( Fig. 12A The percentage of cells positive for each mesothelin-specific CAR was measured by flow cytometry at D14 ( Fig. 12B ).

[0329] Skin-derived γδT cells are cytotoxic to the mesothelin-positive target HeLa cell line ( Figures 13A-13B ).like Figures 12A-12B The skin-resident γδ T cells were transduced and expanded. The cryopreserved cells were then thawed and immediately tested for mesothelin + Functionality of the Hela cell line. Cells were cultured at different effector to target ratios for 17-20 h without cytokine supplementation. Specific cell lysis was detected using the CellTitreGLO cytometry assay. White circles represent non-transduced skin-resident γδ T cells, while gray dots represent skin-resident γδ T cells transduced with a mesothelin-specific CAR.

[0330] Skin-derived γδT cells are cytotoxic to mesothelin-positive target cells A459 ( Fig.14 ). Testing skin-resident γδ T cells transduced with the YP218 anti-mesothelin CAR construct against mesothelin + Functionality of 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 cytokine supplementation. Specific cell lysis was detected by a luciferase-based fluorocytometry assay.

[0331] Mesothelin CAR modified skin Vδ1T cells against HT29.MSLN ( Fig.15A ) and A549.MSLN( Fig. 15B ) tumor cells. Skin-resident γδ and αβ T cells transduced with the YP218 anti-mesothelin CAR construct (n=1) were tested for mesothelin + Functionality of the A549 target cell line. 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 cytokine supplementation. Specific cell lysis was detected by a luciferase-based fluorocytometry assay.

[0332] Example 2

[0333] Blood-derived Vd1+γδ is transduced with a transgene encoding an anti-mesothelin CAR comprising a P4 antigen binding domain (e.g., wherein the antigen binding domain comprises a sequence of any one of SEQ ID NOs: 1–8). Optionally, the cells may also be transduced to express 4-1BB and CD3ζ domains. Optionally, the cells may also be transduced to express the CD8 transmembrane domain and hinge region. The cells are also modified to express (a) IL-15R-β or (b) (i) IL-15R-α tethered to IL-15 or a variant thereof and (ii) IL-15R-β. Target cell binding and lysis of cells expressing CAR are assayed.

[0334] Example 3

[0335] The functionality of blood-derived γδ T cells transduced with YP218 or P4 anti-mesothelin CAR constructs (comprising an anti-mesothelin binder, a CD8 hinge region, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain) was evaluated in a disseminated A549 tumor model. Fig.16A It was shown that γδ T cells transduced with either conjugate exhibited effective tumor control. Fig. 16B It was shown that γδ T cells expressing the anti-MSLN CAR were able to control tumor growth when given as few as 1 million total cells. On day 18 after drug product administration, all mice were culled and bone marrow aspirates from both femurs, spleen, lungs, and peripheral blood (collected in lithium-heparin tubes) were harvested. Figures 16C-16F It was shown that γδ T cells expressing either anti-MSLN conjugate were maintained at equivalent levels in the microenvironments evaluated.

[0336] Example 4

[0337] To improve the survival of Vδ1γδCAR T cells in the presence or absence of low IL-15 concentrations, a novel collection of armored constructs was designed and evaluated using YP218 and P4 conjugates. Polycistronic gammaretroviral vectors were generated in which the chimeric antigen receptor (CAR) coding sequence was fused in frame to the IL-15 receptor chain components. Fig.17A Schematic diagram showing the armoring strategy for co-expression of CAR and armor moieties. Fig. 17B The domain structure of a conventional gammaretroviral vector encoding an armored CAR is shown. Cells transduced with IL-15Rβ alone will be referred to as "β", while cells transduced with IL-15Rβ + IL-15Rα tethered to IL-15 will be referred to as "α.15.β".

[0338] Example 5

[0339] For each of the P4 and YP218 conjugates armored with α.15.β, two different codon-optimized sequences generated using different optimization algorithms were explored to determine if this would affect the frequency and relative protein expression of each component of the armored cassette. Drug products were generated from 2 leukapheresis donors using each of 4 different vectors (P4α.15.β codon sequence 1, P4α.15.β codon sequence 2, YP218α.15.β codon sequence 1, YP218α.15.β codon sequence 2), with donor 1 represented by squares and donor 2 represented by circles. Drug products transduced with vectors encoding P4α.15.β codon sequences 1 and 2 showed significant differences in expression percentage ( Fig.18A ) and relative protein levels ( Fig.18B ) had approximately equal expression of each armor component. In contrast, the drug product transduced with YP218α.15.β codon sequence 2 exhibited a greater proportion of cells expressing each armor component than YP218α.15.β codon sequence 1 ( Fig.18C ). Compared with 1, the relative protein expression levels of IL15Rβ and IL15Rα with codon sequence 2 also increased ( Fig.18D ).

[0340] When targeting tumor targets expressing the antigen, significantly enhanced cytotoxicity of tethered IL-15 and IL-15Rβ co-expressing cells was immediately observed ( Fig.18E ).

[0341] Example 6

[0342] The response of the β-armored conjugate P compared to its unarmored counterpart to repeated antigen challenge was explored in the presence of 1 ng / mL IL-15. Tumor confluence was studied by measuring GFP fluorescence over time. The β-armored conjugate P4 construct demonstrated excellent tumor control over the course of the 14-day repeated challenge assay. These results suggest that the enhanced sensitivity to exogenous IL-15 provided by upregulation of IL-2Rβ enables the γδCAR T drug product to have excellent functionality ( Fig.19A This enhanced functionality was significantly associated with a greater proliferative potential in response to low levels of exogenous IL-15 (1 ng / mL) compared to unarmored counterparts, reflecting an enhanced sensitivity to soluble exogenous IL-15 ( Fig.19B ). Enrichment of CAR-modified cells increased throughout the assay, indicating that CAR-transduced cells represent the major population driving tumor control ( Fig.19C ).

[0343] Evaluation of the functionality of blood-derived γδ T cells transduced with P4α.15.β and P4β in a disseminated A549 tumor model ( Fig. 20A ). Fig. 20B The data in show that in the absence of IL-15 support, α.15.β armored γδCAR T cells provide better tumor control than unarmored counterparts, with P4α.15.β treated mice having an average of more than 10-fold lower BLI signal at the end of the study (day 19 after dosing) than mice given unarmored cells. Consistent with the in vitro experiments, in the presence of IL-15, β armored γδCAR T cells exhibited initial faster tumor elimination kinetics, but overall tumor control was comparable to the α.15.β armored (non-IL-15 supported) γδCAR T treated group.

[0344] Example 7

[0345] Blood-derived CARγδ T cells targeting mesothelin co-expressing IL-15Rα and IL-15Rβ tethered to IL-15 survived 14 days of cryopreservation, whereas the unarmored construct rapidly declined. Fig.21A ) and P4 CAR( Fig. 21B ) was indeed the case. Fig. 21Cshowed that in the absence of exogenous IL-15, Meso-CAR T cells armored with IL-15Rα and IL-15Rβ chains tethered to IL-15 can control tumors well during repeated antigen stimulation assays. In addition, this functionality was observed regardless of which CAR conjugate was used (YP218 or P4). In addition, the enhanced tumor control exhibited by CAR T cells armored with IL-15Rα and IL-15Rβ chains tethered to IL-15 is not necessarily associated with proliferation potential. Instead, proliferation potential appears to be a functionality driven by the CAR molecule and is specific to the conjugate used. Proliferation was observed when the YP218 conjugate was used, but not when the P4 conjugate was used ( Fig.21D ).

Claims

1. An engineered innate lymphoid cell comprising a heterologous targeting construct specific for mesothelin.

2. The engineered innate lymphoid cell of claim 1, wherein the lymphoid cell is a natural killer (NK) cell or a γδ T cell.

3. The engineered innate lymphoid cell of claim 2, wherein the γδ T cell is a Vd1+γδ cell.

4. The engineered innate lymphoid cell of any one of claims 1 to 3, wherein the heterologous targeting construct is a chimeric antigen receptor (CAR).

5. The engineered innate lymphoid cell of claim 4, wherein the CAR is a full-length CAR or a non-signaling CAR.

6. The engineered innate lymphoid cell of claim 4 or claim 5, wherein the CAR is an armored CAR.

7. A pharmaceutical composition comprising the engineered innate lymphoid cell of any one of claims 1 to 6, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.

8. A method of treating a patient in need thereof using the engineered innate lymphoid cell of any one of claims 1 to 6 or the pharmaceutical composition of claim 7.

9. Use of the engineered innate lymphoid cell of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 for treating a condition in a subject.

10. The engineered innate lymphoid cell of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 for use in therapy.

11. A method for preparing the engineered innate lymphoid cell according to any one of claims 1 to 6.

12. An isolated cell population comprising a plurality of the engineered innate lymphoid cells of any one of claims 1 to 6.

13. The engineered innate lymphoid cell of any one of claims 1-6, wherein the cell expresses: (a) IL-15R-β or (b) (i) IL-15R-α tethered to IL-15 or a variant thereof and (ii) IL-15R-β.

14. The engineered innate lymphoid cell of claim 13, wherein the cell expresses IL-15R-β.

15. The engineered innate lymphoid cell of claim 13, wherein the cell expresses (i) IL-15R-α and (ii) IL-15R-β tethered to IL-15 or a variant thereof.

16. A polynucleotide comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain that specifically binds to human mesothelin, wherein the antigen binding domain comprises a variable heavy chain (VH) domain and a variable light chain (VL) domain, wherein the VH comprises a VH-complementarity determining region 3 (CDR3), and the VH-complementarity determining region 3 comprises the amino acid sequence shown in SEQ ID NO:

3.

17. The polynucleotide of claim 16, wherein the CAR further comprises a VH-CDR2, wherein the VH-CDR2 comprises the amino acid sequence shown in SEQ ID NO:

2.

18. The polynucleotide of claim 16 or 17, wherein the CAR further comprises a VH-CDR3, and the VH-CDR3 comprises the amino acid sequence shown in SEQ ID NO:

3.

19. The polynucleotide of any one of claims 16 to 18, wherein the CAR further comprises a VL-CDR1 comprising the amino acid sequence shown in SEQ ID NO:

4.

20. The polynucleotide of any one of claims 16 to 19, wherein the CAR further comprises a VL-CDR2, wherein the VL-CDR2 comprises the amino acid sequence shown in SEQ ID NO:

5.

21. The polynucleotide of any one of claims 16 to 20, wherein the CAR further comprises a VL-CDR3 comprising the amino acid sequence shown in SEQ ID NO:

6.

22. A polynucleotide as described in any one of claims 16 to 21, wherein the CAR comprises: VH-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 1; VH-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 2; VH-CDR3, which comprises the amino acid sequence shown in SEQ ID NO: 3; VL-CDR1, which comprises the amino acid sequence shown in SEQ ID NO: 4; VL-CDR2, which comprises the amino acid sequence shown in SEQ ID NO: 5; and VL-CDR3, which comprises the amino acid sequence shown in SEQ ID NO:

6.

23. The polynucleotide of any one of claims 16 to 22, wherein 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 shown in SEQ ID NO:

7.

24. The polynucleotide of any one of claims 16 to 23, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:

7.

25. The polynucleotide of any one of claims 16 to 24, wherein 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 shown in SEQ ID NO:

8.

26. The polynucleotide of any one of claims 16 to 25, wherein the VL comprises the amino acid sequence shown in SEQ ID NO:

8.

27. The polynucleotide of any one of claims 16 to 26, wherein the VH comprises the amino acid sequence shown in SEQ ID NO:7, and wherein the VL comprises the amino acid sequence shown in SEQ ID NO:

8.

28. The polynucleotide of any one of claims 16 to 27, wherein the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a co-stimulatory domain, (iv) an intracellular signaling domain, or (v) any combination thereof.

29. The polynucleotide of any one of claims 16 to 28, wherein the CAR further comprises (i) a hinge region, (ii) a transmembrane domain, (iii) a co-stimulatory domain, and (iv) an intracellular signaling domain.

30. The polynucleotide of claim 28 or 29, wherein the hinge region comprises a hinge region derived from CD8, CD28 or an immunoglobulin.

31. The polynucleotide of claim 30, wherein the immunoglobulin is selected from the group consisting of IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, IgD, IgE, IgM, and any combination thereof.

32. The polynucleotide of any one of claims 28 to 31, wherein the hinge region is derived from the CD8 hinge region.

33. A polynucleotide as described in any one of claims 28 to 32, wherein 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 shown in SEQ ID NO:

9.

34. The polynucleotide of any one of claims 28 to 33, wherein the hinge region comprises the amino acid sequence shown in SEQ ID NO:

9.

35. The polynucleotide of any one of claims 28 to 34, wherein the transmembrane domain comprises a transmembrane domain derived from 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, IL2Rβ, IL2Rγ, IL7Rα, 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.

36. A polynucleotide as described in any one of claims 28 to 35, wherein the transmembrane domain is derived from a CD8 transmembrane domain.

37. A polynucleotide as described in any one of claims 28 to 36, wherein 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 shown in SEQ ID NO:

10.

38. The polynucleotide of any one of claims 28 to 37, wherein the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

10.

39. The polynucleotide of any one of claims 28 to 38, wherein the costimulatory domain comprises a costimulatory domain derived from 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 missing due to 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 to CD83, or any combination thereof.

40. The polynucleotide of any one of claims 28 to 39, wherein the co-stimulatory domain is derived from a 4-1BB co-stimulatory domain.

41. The polynucleotide of any one of claims 28 to 40, wherein 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 to the amino acid sequence shown in SEQ ID NO:

11.

42. The polynucleotide of any one of claims 28 to 41, wherein the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

11.

43. The polynucleotide of any one of claims 28 to 42, wherein 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 DAP 12 activation domain, a FCER1G activation domain, a DAP10 / CD28 activation domain, a ZAP70 activation domain, or any combination thereof.

44. The polynucleotide of any one of claims 28 to 43, wherein the intracellular signaling domain comprises a CD3 zeta activation domain.

45. The polynucleotide of any one of claims 28 to 44, wherein 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 shown in SEQ ID NO:

12.

46. ​​The polynucleotide of any one of claims 28 to 45, wherein the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

12.

47. A polynucleotide as described in any one of claims 16 to 46, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and (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 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; (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) to (b)(iv).

48. A polynucleotide as described in any one of claims 16 to 47, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of 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; (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 shown 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 shown in SEQ ID NO:

12.

49. A polynucleotide as described in any one of claims 16 to 48, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of 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; (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 shown 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 shown in SEQ ID NO:

12.

50. The polynucleotide of any one of claims 16 to 48, comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO:

12.

51. The polynucleotide of any one of claims 16 to 50, further comprising a second nucleotide sequence encoding armorin.

52. The polynucleotide of claim 51, wherein the armorin comprises an IL-15Rβ polypeptide, an IL-15Rα polypeptide, an IL-2Rβ polypeptide, an IL-15 polypeptide, or any combination thereof.

53. The polynucleotide of claim 51 or 52, wherein the armorin comprises an IL-2Rβ polypeptide.

54. The polynucleotide of any one of claims 51 to 53, wherein the armored 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 shown in SEQ ID NO:

15.

55. The polynucleotide of any one of claims 51 to 54, wherein the armored protein comprises the amino acid sequence shown in SEQ ID NO:

15.

56. The polynucleotide of any one of claims 51 to 55, wherein the armorin comprises an IL-2Rβ polypeptide, and wherein the armorin does not comprise an IL-15Rα polypeptide or an IL-15 polypeptide.

57. The polynucleotide of any one of claims 51 to 55, wherein the armorin comprises (i) an IL-15Rα polypeptide or (ii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

58. The polynucleotide of any one of claims 51 to 55 and 57, wherein the armored 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 shown in SEQ ID NO:

17.

59. The polynucleotide of any one of claims 51 to 55, 57 and 58, wherein the armorin comprises an IL-15Rα polypeptide comprising the amino acid sequence shown in SEQ ID NO:

17.

60. The polynucleotide of any one of claims 51 to 55 and 57 to 59, wherein the armorin comprises an IL-15 polypeptide.

61. The polynucleotide of any one of claims 51 to 55 and 57 to 60, wherein the armored 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 shown in SEQ ID NO:

16.

62. The polynucleotide of any one of claims 51 to 55 and 57 to 61, wherein the armorin comprises an IL-15 polypeptide comprising the amino acid sequence shown in SEQ ID NO:

16.

63. The polynucleotide of any one of claims 51 to 55 and 57 to 62, wherein the armorin comprises an IL-15Rα polypeptide tethered to an IL-15 polypeptide via a linker.

64. The polynucleotide of claim 63, wherein the linker comprises a peptide bond.

65. The polynucleotide of claim 63 or 64, wherein the linker comprises a Gly-Ser linker.

66. The polynucleotide of any one of claims 63 to 65, wherein the linker comprises an amino acid sequence selected from SEQ ID NOs: 20-25 or any combination thereof.

67. The polynucleotide of any one of claims 51 to 52 and 57 to 60, wherein the armorin comprises (i) an IL-2Rβ polypeptide; (ii) a cleavable linker; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide.

68. The polynucleotide of claim 67, wherein the cleavable linker comprises a P2A sequence.

69. The polynucleotide of claim 67 or 68, wherein 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 shown in SEQ ID NO:

18.

70. The polynucleotide of any one of claims 67 to 69, wherein the cleavable linker comprises the amino acid sequence shown in SEQ ID NO:

18.

71. The polynucleotide of any one of claims 16 to 70, comprising a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12; and (b) armored protein, wherein the armored protein comprises an IL-2Rβ polypeptide, and the IL-2Rβ 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 shown in SEQ ID NO:

15.

72. The polynucleotide of any one of claims 16 to 71, comprising a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12; and (b) Armorin, wherein the armorin comprises an IL-2Rβ polypeptide, and the IL-2Rβ polypeptide comprises the amino acid sequence shown in SEQ ID NO:

15.

73. The polynucleotide of claim 71 or 72, which does not comprise a nucleotide sequence encoding an IL-15Rα polypeptide or an IL-15 polypeptide.

74. A polynucleotide as described in any one of claims 16 to 55 and 57 to 72, comprising a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12; and (b) armored protein, wherein the armored 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-15Rα polypeptide tethered to an IL-15 polypeptide; in: 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 shown 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 shown in SEQ ID NO:

16.

75. A polynucleotide as described in any one of claims 16 to 55 and 57 to 72, comprising a nucleotide sequence encoding: (a) a chimeric antigen receptor (CAR), wherein the CAR comprises (i) an antigen-binding domain that specifically binds to human mesothelin, wherein the antigen-binding domain comprises a VH-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a VH-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a VH-CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VL-CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL-CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a VL-CDR3 comprising the amino acid sequence of SEQ ID NO: 6; (ii) a hinge region comprising the amino acid sequence shown in SEQ ID NO:9; (iii) a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 10; (iv) a costimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 11; and (v) an intracellular signaling domain comprising the amino acid sequence shown in SEQ ID NO: 12; and (b) armored protein, wherein the armored protein comprises: (i) an IL-2Rβ polypeptide comprising the amino acid sequence shown in SEQ ID NO: 15; (ii) a linker comprising the amino acid sequence shown in SEQ ID NO: 18; and (iii) an IL-15Rα polypeptide tethered to an IL-15 polypeptide; in: a. the IL-15Rα polypeptide comprises the amino acid sequence shown in SEQ ID NO: 17; and b. The IL-15 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

16.

76. A vector or a collection of vectors comprising the polynucleotide of any one of claims 16 to 75.

77. A vector or vector collection as described in claim 76, comprising one or more promoters.

78. The vector or vector collection of claim 76 or 77, which is a retroviral vector, a DNA vector, a murine leukemia virus vector, a SFG vector, a plasmid, an RNA vector, an adenoviral vector, a baculovirus vector, an Epstein-Barr virus vector, a papovavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector (AAV), a lentiviral vector, or any combination thereof.

79. A polypeptide encoded by the polynucleotide of any one of claims 16 to 75.

80. A host cell comprising the polynucleotide of any one of claims 16 to 75, the vector or vector collection of any one of claims 76 to 78, or the polypeptide of claim 79.

81. The host cell of claim 80, which is an immune cell.

82. The host cell of claim 81, wherein the immune cell is an innate lymphoid cell.

83. The host cell of claim 81 or 82, wherein the immune cell is selected from a T cell, a NK cell, a B cell, or any combination thereof.

84. The host cell of claim 83, wherein the T cell is selected from a γδ T cell, an αβ T cell, or any combination thereof.

85. The host cell of claim 83 or 84, wherein the T cell is selected from a Vδ1 T cell and a Vδ2 T cell.

86. A host cell as described in any one of claims 80 to 85, obtained from a human subject.

87. The host cell of any one of claims 80 to 86, 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.

88. The host cell of any one of claims 80 to 86, which is derived from an induced pluripotent stem cell (iPSC).

89. A cell population comprising the host cell of any one of claims 80 to 88.

90. A cell population as described in claim 89, wherein 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 cell population comprise the host cells.

91. A pharmaceutical composition comprising a polynucleotide as described in any one of claims 16 to 75, a vector or a collection of vectors as described in any one of claims 76 to 78, a polypeptide as described in claim 79, a host cell as described in any one of claims 80 to 88 or a cell population as described in claim 89 or 90, and a pharmaceutically acceptable excipient.

92. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a polynucleotide as described in any one of claims 16 to 75, a vector or vector collection as described in any one of claims 76 to 78, a polypeptide as described in claim 79, a host cell as described in any one of claims 80 to 88, a cell population as described in claim 89 or 90, or a pharmaceutical composition as described in claim 91.

93. The method of claim 92, wherein the disease or condition comprises cancer.

94. The method of claim 93, wherein the cancer comprises lung cancer, bone cancer, pancreatic cancer, skin cancer, head or 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, Disease), non-Hodgkin 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 myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors in children, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers (including those induced by asbestos), or any combination thereof.

95. The method of claim 93 or 94, wherein the cancer comprises small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, mesothelioma, or any combination thereof.

96. The method of any one of claims 93 to 94, wherein the cancer is locally advanced.

97. The method of any one of claims 93 to 95, wherein the cancer is metastatic.

98. The method of any one of claims 92 to 97, wherein the cancer is refractory or relapsed.

99. The method of any one of claims 92 to 98, further comprising administering an additional anticancer agent.

100. The method of claim 99, wherein the additional anti-cancer therapy comprises chemotherapy, immunotherapy, radiation therapy, surgery, or any combination thereof.

101. The method of claim 99 or 100, wherein the additional anti-cancer therapy comprises chemotherapy.

102. The method of any one of claims 99 to 101, wherein the additional anti-cancer therapy comprises an immune checkpoint inhibitor.

103. The method of any one of claims 98 to 101, wherein 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.

104. The method of any one of claims 99 to 103, wherein the anti-cancer therapy comprises an antibody, or an antigen-binding portion thereof, that specifically binds to and inhibits PD-1.

105. The method of any one of claims 99 to 104, wherein the anti-cancer therapy comprises an antibody, or antigen-binding portion thereof, that specifically binds to and inhibits PD-L1.

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