Immunoresponsive cells armoured with spatiotemporally restricted activity of cytokines of the il-1 superfamily

Abstract

To provide immunoresponsive cells having IL-1 superfamily activities with spatiotemporal restriction.SOLUTION: The present invention provides an immunoresponsive cell expressing a modified pro-cytokine of the IL-1 superfamily, where the modified pro-cytokine comprises, from N-terminus to C-terminus, (a) a pro-peptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase or proteinase 3; and (c) a fragment of a cytokine of the IL-1 superfamily.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. Background The tumor microenvironment imposes limitations on immune effector activity, including effector activity mediated by tumor-infiltrating lymphocytes, T cells engineered to express non-native T cell receptors (TCRs), and T cells engineered to express chimeric antigen receptors (CARs). To address such immune suppression within the tumor stroma, there is interest in modifying immunoresponsive cells to further express one or more pro-inflammatory cytokines, such as interleukin (IL)-12 and / or members of the IL-1 superfamily. [Background technology]

[0002] The IL-1 superfamily includes 11 members. See Baker et al., "IL-1 family members in cancer; two sides to every story," Front. Immunol. 10:Article 1197 (2019). Pro-inflammatory members include IL-1α, IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ. In contrast, antagonistic or anti-inflammatory properties are attributed to IL-1 receptor antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38. Importantly, some IL-1 superfamily members are synthesized in precursor forms that require proteolytic cleavage to exhibit biological activity. Examples of cytokines with antitumor activity regulated in this manner include IL-1β, IL-18, and IL-36α-γ.

[0003] Like IL-1β and IL-36α-γ, IL-18 lacks a conventional signal or leader sequence that directs the protein post-translationally to the secretory pathway, including the endoplasmic reticulum (ER) and Golgi apparatus. Instead, IL-18 is produced as a biologically inactive precursor (pro-IL-18) that is activated by cleavage of a 36-amino acid propeptide in its N-terminal region. This cleavage reaction is primarily mediated by caspase-1, which is found within an inducible multimolecular organelle known as the inflammasome. The pro-inflammatory IL-36 family members (IL-36α, IL-36β, and IL-36γ) are also synthesized as inactive precursors that are activated upon proteolytic cleavage of their N-terminal regions. Activating enzymes for pro-IL-36 cytokines include cathepsin G, elastase, and proteinase 3.

[0004] Several studies have engineered CAR- or TCR-modified T cells to express IL-18. Hu et al., "Augmentation of antitumor immunity by human and mouse CAR T cells secreting IL18," Cell Rep. 20(13):3025-3033 (2017); Chmielewski et al., "CAR T cells releasing IL-18 convert to T-Bet high FoxO1 loweffectors that exhibit augmented activity against solid tumors” Cell Rep.21(11):3205-3219(2017);Avanzi et al., “Engineered tumor-targeted T cells mediate enhanced anti-tumor efficacy both directly and through activation of the endogenous immune system” Cell Rep.23(7):2130-2141(2018);Kunert et al., “Intra-tumoral production of "IL18, but not IL12, by TCR-engineered T cells is non-toxic and counteracts immune evasion of solid tumors" Oncoimmunology 7(1):e1378842 (2017).

[0005] Hu et al. showed that constitutive expression of mature IL-18 by CAR T cells enhanced their T cell receptor-dependent amplification in vivo in addition to their antitumor activity. The study did not provide details on how IL-18 was engineered for secretion. Nevertheless, supplementary data show that IL-18 was both constitutively secreted (Figure S1b) and constitutively active (Figure S1c), suggesting that the mature (18 kD) form of IL-18 was fused to a conventional signal or leader peptide.

[0006] Avanzi et al. also demonstrated enhanced antitumor activity by IL-18-armored CAR T cells, accompanied by autocrine CAR T cell proliferation and persistence. A positive impact on endogenous immune surveillance was demonstrated by favorable regulation of cellular infiltration within tumors. Furthermore, epitope spreading occurred, resulting in enhanced antitumor activity of endogenous T cells. The use of IL-18 in this manner obviated the need for lymphocyte depletion to achieve antitumor activity. Macrophage depletion significantly prevented therapeutic benefit, supporting the important role of these cells in regulating the tumor microenvironment. Because native IL-18 lacks a conventional signal sequence, the IL-18 construct used in Avanzi's paper was constitutively expressed mature IL-18 driven by the IL-2 signal peptide.

[0007] Although expression of IL-18 in CAR-T cells has been shown to improve efficacy in various experiments, the safety and therapeutic benefits of constitutive expression of IL-18 have not been fully studied.

[0008] Given the strong association between IL-1 family members such as IL-18 and autoinflammatory syndromes such as macrophage activation syndrome (Weiss et al., "Interleukin-18 diagnostically distinguishes and pathogenically promotes human and murine macrophage activation syndrome," Blood 131(13):1442-1455 (2018)), there is concern that unregulated expression of mature IL-18 or other members of the IL-1 superfamily may be toxic. Therefore, strategies must be modified to "armor" immunocompetent cells against the suppressive effects of the tumor microenvironment without causing significant toxicity to noncancerous tissues.

[0009] Chmielewski et al. used an NFAT-responsive promoter in an attempt to restrict the release of mature IL-18 to activated CAR T cells. They showed that IL-18-producing CAR T cells modulated the tumor microenvironment to support a pro-inflammatory state that contributed to disease elimination. Tumor-specific T cells and NK cells increased at the site, while immunosuppressive M2-polarized macrophages and regulatory T cells decreased. Furthermore, the profile of costimulatory and co-inhibitory receptors expressed within the tumor was favorably altered. Nearly identical results were obtained by Kunert et al. with TCR-modified T cells. Conceptually, restricting mature IL-18 release to activated (NFAT-expressing) T cells should make the approach safer. However, implementation of this solution requires a cumbersome dual transduction procedure because CAR expression is constitutive (achieved using the first vector) while IL-18 expression is inducible (achieved using the second vector). A single vector containing both promoters might overcome this limitation, but would be difficult to produce given well-known problems with promoter interference. Furthermore, this inducible vector exhibited some degree of "leakyness," as indicated by toxicity seen in tumor-free mice in which IL-12 release is similarly regulated. Summary of the Invention [Problem to be solved by the invention]

[0010] 2. Summary of the Invention The present disclosure provides immunoresponsive cells having spatiotemporally restricted activity of IL-1 superfamily members with anti-tumor activity, particularly IL-18, IL-36α, IL-36β, and IL-36γ. In particular, immunoresponsive cells are provided that express a modified pro-cytokine of the IL-1 superfamily, wherein the modified pro-cytokine comprises, from N- to C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a biologically active cytokine fragment of the IL-1 superfamily.

[0011] CAR T cells—both αβ CAR-T cells and γδ CAR-T cells—have been produced that are further introduced with an exogenous polynucleotide encoding a pro-cytokine having a cleavage site recognized by a site-specific protease other than caspase-1, cathepsin G, elastase, or proteinase 3. In some experiments, the cells further expressed a site-specific protease. In particular, provided herein are pro-cytokines having a cleavage site recognized by the protease, granzyme B (GzB). Applicant has found that expression of IL-1 superfamily members with regulated activity can enhance T cell responses and the anti-tumor activity of CAR T cells in a controlled manner.

[0012] Pro-cytokines with regulated activity can be used in combination with various CAR T cells available in the art. For example, pCAR-T cells carrying parallel CAR (pCAR) constructs that bind to one or more antigens present on target cells can be further engineered to express a pro-cytokine with regulated activity. [Means for solving the problem]

[0013] Thus, according to some embodiments, provided herein are immunoresponsive cells that express a modified pro-cytokine of the IL-1 superfamily, wherein the modified pro-cytokine comprises, from N-terminus to C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a cytokine fragment of the IL-1 superfamily.

[0014] In some embodiments, the protease is granzyme B (GzB). In some embodiments, the cleavage site has the sequence of SEQ ID NO: 26. In some embodiments, the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 27. In some embodiments, the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO: 103 or 111.

[0015] In some embodiments, the protease is caspase-3. In some embodiments, the cleavage site has the sequence of SEQ ID NO: 28. In some embodiments, the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 29. In some embodiments, the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO: 109.

[0016] In some embodiments, the protease is caspase-8. In some embodiments, the cleavage site has the sequence of SEQ ID NO: 30. In some embodiments, the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 31. In some embodiments, the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO: 107.

[0017] In some embodiments, the protease is membrane-type matrix metalloproteinase 1 (MT1-MMP). In some embodiments, the cleavage site has the sequence of SEQ ID NO: 32. In some embodiments, the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 33. In some embodiments, the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO: 113.

[0018] In some embodiments, a cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24. In some embodiments, a cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24.

[0019] In some embodiments, the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25. In some embodiments, the propeptide is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25.

[0020] In some embodiments, the modified pro-cytokine is modified pro-IL-36α and has the sequence of SEQ ID NO: 37. In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42.

[0021] In some embodiments, the modified pro-cytokine is modified pro-IL-36β and has the sequence of SEQ ID NO: 39. In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 43.

[0022] In some embodiments, the modified pro-cytokine is modified pro-IL-36γ and has the sequence of SEQ ID NO: 41. In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 44.

[0023] In some embodiments, the immunoresponsive cell further comprises an exogenous polynucleotide encoding a protease.

[0024] In some embodiments, the immunoresponsive cells are αβ T cells, γδ T cells, or natural killer (NK) cells. In some embodiments, the T cells are αβ T cells. In some embodiments, the T cells are γδ T cells.

[0025] In some embodiments, the immunoresponsive cell further comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR is a second-generation chimeric antigen receptor (CAR), wherein the CAR comprises: (a) a signaling region; (b) a first costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen.

[0026] In some embodiments, the first epitope is an epitope on the MUC1 target antigen. In some embodiments, the first binding element comprises a CDR of an HMFG2 antibody. In some embodiments, the first binding element comprises a V H and V L In some embodiments, the first binding element comprises an HMFG2 single chain variable fragment (scFv).

[0027] In some embodiments, the immunoresponsive cell further comprises a chimeric costimulatory receptor (CCR), wherein the CCR comprises (a) a second costimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0028] In some embodiments, the second costimulatory domain is different from the first costimulatory domain. In some embodiments, the second target antigen comprising the second epitope is selected from the group consisting of ErbB homodimers and heterodimers. In some embodiments, the second target antigen is HER2. In some embodiments, the second target antigen is EGF receptor. In some embodiments, the second binding element comprises a binding portion of T1E, ICR12, or ICR62.

[0029] In some embodiments, the present disclosure provides immunoresponsive cells expressing modified pro-IL-18, wherein the modified pro-IL-18 is a polypeptide of SEQ ID NO: 27, and the cell further comprises: (a) an exogenous polynucleotide encoding GzB; (b) a chimeric antigen receptor (CAR) comprising: i. a signaling region; ii. a first costimulatory signaling region; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on a MUC1 target antigen; and (c) a chimeric costimulatory receptor (CCR) comprising: i. a second costimulatory signaling region; ii. a transmembrane domain; and iii. a second binding element that specifically interacts with a second epitope on a second target antigen.

[0030] In some embodiments, the present disclosure provides immunoresponsive cells expressing modified pro-IL-36α, pro-IL-36β, or pro-IL-36γ, wherein the modified pro-IL-36α, pro-IL-36β, or pro-IL-36γ is a polypeptide of SEQ ID NO: 37, 39, or 41, and the cell further comprises: (a) an exogenous polynucleotide encoding GzB; (b) a chimeric antigen receptor (CAR) comprising: i. a signaling region; ii. a first costimulatory signaling region; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on a MUC1 target antigen; and (c) a chimeric costimulatory receptor (CCR) comprising: i. a second costimulatory signaling region; ii. a transmembrane domain; and iii. a second binding element that specifically interacts with a second epitope on a second target antigen.

[0031] In another aspect, the disclosure provides a polynucleotide or set of polynucleotides comprising a first nucleic acid encoding a modified cytokine, wherein the modified pro-cytokine of the IL-1 superfamily comprises, from N-terminus to C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a cytokine fragment of the IL-1 superfamily.

[0032] In some embodiments, the protease is GzB. In some embodiments, the cleavage site has the sequence of SEQ ID NO: 26. In some embodiments, the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 27. In some embodiments, the polynucleotide or set of polynucleotides comprises the sequence of SEQ ID NO: 103 or 111.

[0033] In some embodiments, the protease is caspase-3. In some embodiments, the cleavage site has the sequence of SEQ ID NO: 28. In some embodiments, the modified cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 29. In some embodiments, the polynucleotide or set of polynucleotides comprises the sequence of SEQ ID NO: 109.

[0034] In some embodiments, the protease is caspase-8. In some embodiments, the cleavage site has the sequence of SEQ ID NO: 30. In some embodiments, the modified cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 31. In some embodiments, the polynucleotide or set of polynucleotides comprises the sequence of SEQ ID NO: 107.

[0035] In some embodiments, the protease is MT1-MMP. In some embodiments, the cleavage site has the sequence of SEQ ID NO: 32. In some embodiments, the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO: 33. In some embodiments, the polynucleotide or set of polynucleotides comprises the sequence of SEQ ID NO: 113.

[0036] In some embodiments, the polynucleotide or set of polynucleotides further comprises a second nucleic acid encoding a protease.

[0037] In some embodiments, the first nucleic acid and the second nucleic acid are in a single vector.

[0038] In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the cytokine fragment is capable of binding to and activating the IL-18 receptor when the cleavage site is cleaved. In some embodiments, the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25. In some embodiments, the propeptide is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25.

[0039] In some embodiments, the modified pro-cytokine is modified pro-IL-36α and has the sequence of SEQ ID NO: 37. In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42.

[0040] In some embodiments, the modified pro-cytokine is modified pro-IL-36β and has the sequence of SEQ ID NO: 39. In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 43. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 43.

[0041] In some embodiments, the modified pro-cytokine is modified pro-IL-36γ and has the sequence of SEQ ID NO: 41. In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 44. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 44.

[0042] In some embodiments, the polynucleotide or set of polynucleotides comprises a first nucleic acid encoding a modified pro-IL-36 α, β, or γ, wherein the modified pro-IL-36 α, β, or γ comprises, from N-terminus to C-terminus, (a) a propeptide; (b) a cleavage site recognized by a protease other than cathepsin G, elastase, or proteinase 3; and (c) an IL-36 α, β, or γ fragment.

[0043] In some embodiments, the protease is Granzyme B (GzB). In some embodiments, the cleavage site has the sequence of SEQ ID NO: 26. In some embodiments, the modified pro-IL-36 α, β, or γ comprises the sequence of SEQ ID NO: 37, 39, or 41.

[0044] In some embodiments, the polynucleotide or set of polynucleotides further comprises a second nucleic acid encoding a protease. In some embodiments, the first nucleic acid and the second nucleic acid are in a single vector.

[0045] In some embodiments, an IL-36 fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42, 43 or 44. In some embodiments, an IL-36 fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42, 43 or 44. In some embodiments, an IL-36 fragment is capable of binding to and activating the IL-36 receptor when the cleavage site is cleaved.

[0046] In some embodiments, the polynucleotide or set of polynucleotides further comprises a third nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR is a second-generation chimeric antigen receptor (CAR) comprising: (a) a signaling region; (b) a first costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen.

[0047] In some embodiments, the first epitope is an epitope on the MUC1 target antigen. In some embodiments, the first binding element comprises a CDR of an HMFG2 antibody. In some embodiments, the first binding element comprises a V H and V L In some embodiments, the first binding element comprises an HMFG2 single chain variable fragment (scFv).

[0048] In some embodiments, the polynucleotide or set of polynucleotides further comprises a fourth nucleic acid encoding a chimeric costimulatory receptor (CCR), wherein the CCR comprises (a) a second costimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0049] In some embodiments, the second target antigen comprising the second epitope is selected from the group consisting of ErbB homodimers and heterodimers. In some embodiments, the second target antigen is HER2. In some embodiments, the second target antigen is EGF receptor. In some embodiments, the second binding element comprises a binding portion of T1E, ICR12, or ICR62.

[0050] In some embodiments, the third nucleic acid and the fourth nucleic acid are in a single vector.

[0051] In some embodiments, the polynucleotide or set of polynucleotides comprises: (a) a first nucleic acid encoding a modified pro-IL-18 (the modified pro-IL-18 is the polypeptide of SEQ ID NO: 27); (b) a second nucleic acid encoding GzB; (c) a third nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising: i. a signaling region; ii. a first costimulatory signaling region; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on a MUC1 target antigen; and (d) a fourth nucleic acid encoding a chimeric costimulatory receptor (CCR), the CCR comprising: i. a second costimulatory signaling region; ii. a transmembrane domain; and iii. a second binding element that specifically interacts with a second epitope on a second target antigen. In some embodiments, the polynucleotide or set of polynucleotides comprises the polynucleotide of SEQ ID NO: 103.

[0052] In some embodiments, the polynucleotide or set of polynucleotides comprises: (a) a first nucleic acid encoding a modified pro-IL-36 (the modified pro-IL-36 is a polypeptide of SEQ ID NO: 37, 39, or 41); (b) a second nucleic acid encoding GzB; (c) a third nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising: i. a signaling region; ii. a first costimulatory signaling region; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on a MUC1 target antigen; and (d) a fourth nucleic acid encoding a chimeric costimulatory receptor (CCR), the CCR comprising: i. a second costimulatory signaling region; ii. a transmembrane domain; and iii. a second binding element that specifically interacts with a second epitope on a second target antigen.

[0053] In some embodiments, the first nucleic acid and the third nucleic acid are in a single vector. In some embodiments, the first nucleic acid and the fourth nucleic acid are expressed from a single vector. In some embodiments, the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid are expressed from a single vector.

[0054] In one aspect, the present invention provides a method of preparing an immunoresponsive cell, the method comprising transfecting or transducing a polynucleotide or set of polynucleotides provided herein into the immunoresponsive cell.

[0055] In another aspect, the present disclosure provides methods of directing a T cell-mediated immune response to target cells in a patient in need thereof, the method comprising administering to the patient an immunoresponsive cell provided in the present disclosure. In some embodiments, the target cell expresses MUC1.

[0056] In yet another aspect, the present disclosure provides methods of treating cancer, the methods comprising administering to a patient an effective amount of an immunoresponsive cell provided in the present disclosure. In some embodiments, the patient's cancer cells express MUC1. In some embodiments, the patient has a cancer selected from the group consisting of breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, myeloma, non-Hodgkin's lymphoma, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, and renal cell carcinoma. In some embodiments, the patient has breast cancer. In some embodiments, the patient has ovarian cancer.

[0057] In one aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (a) Second-generation chimeric antigen receptors (CARs), including: i. signaling region; ii.Co-stimulatory signaling region; iii. transmembrane domain; iv. a first binding element that specifically interacts with a first epitope on a first target antigen; and (b) a chimeric costimulatory receptor (CCR) comprising: v. a costimulatory signaling region distinct from that of ii; vi. a transmembrane domain; and vii. a second binding element that specifically interacts with a second epitope on a second target antigen. and providing γδ T cells expressing

[0058] In some embodiments, the first target antigen is the same as the second target antigen.

[0059] In some embodiments, the first target antigen is a MUC antigen. In some embodiments, the first binding element comprises a CDR of an HMFG2 antibody. In some embodiments, the first binding element comprises a V H and V L In some embodiments, the first binding element comprises an HMFG2 single chain variable fragment (scFv).

[0060] In some embodiments, the second target antigen comprising the second epitope is selected from the group consisting of ErbB homodimers and heterodimers. In some embodiments, the second target antigen is HER2. In some embodiments, the second target antigen is EGF receptor. In some embodiments, the second binding element comprises T1E, ICR12, or ICR62. In some embodiments, the second binding element is T1E. In some embodiments, the second target antigen is αvβ6 integrin. In some embodiments, the second binding element is A20 peptide.

[0061] In yet another aspect, the present disclosure provides a method for producing an immunoresponsive cell, comprising introducing a transgene. In some embodiments, the transgene encodes a CAR or pCAR. In some embodiments, the transgene encodes a modified pro-cytokine of the IL-1 superfamily, wherein the modified pro-cytokine comprises, from N-terminus to C-terminus, (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a cytokine fragment of the IL-1 superfamily. In some embodiments, the method further comprises the preceding step of activating the γδ T cells with an anti-γδ TCR antibody. In some embodiments, the anti-γδ TCR antibody is immobilized.

[0062] 3. Brief description of the drawings The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the present invention. [Brief explanation of the drawings]

[0063] [Figure 1]A schematic diagram showing the salient features of certain second-generation CAR and pCAR constructs used in the experiments described herein is provided. The cell membrane is represented as parallel horizontal lines, with the extracellular domain shown above the membrane and the intracellular domain shown below the membrane. For pCAR, the name of the chimeric costimulatory receptor (CCR) is listed first, and the CAR is identified to the right of the slash or stroke mark ( / ). H2 is a second-generation CAR originally described in Wilkie et al., J. Immunol. 180:4901-9 (2008) (incorporated herein by reference in its entirety). From the extracellular domain to the intracellular domain, it contains a human MUC1-targeting HMFG2 single-chain antibody (scFv) domain, a CD28 transmembrane and costimulatory domain, and a CD3z signaling region. Cell transduction with H2 alone is a standard second-generation CAR-T cell with specificity for the MUC1 tumor-associated glycoform recognized by HMFG2 scFv. TBB / H is a pCAR. It utilizes a MUC1-targeted second-generation "H2" CAR, but has a co-expressed chimeric costimulatory receptor (CCR). The CCR in TBB / H pCAR has a T1E binding domain and a 4-1BB costimulatory domain fused to the CD8α transmembrane domain. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF), and is a promiscuous ErbB ligand. See Wingens et al., "Structural analysis of an epidermal growth factor / transforming growth factor-alpha chimera with unique ErbB binding specificity," J. Biol. Chem. 278:39114-23 (2003), and Davies et al., "Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells," the disclosures of which are incorporated herein by reference in their entireties. [Figure 2]1 is a diagram illustrating the modifications of pro-IL-18 in various constructs used herein. IL-18 is secreted as inactive pro-IL-18. In native pro-IL-18, activation requires caspase-1 cleavage at the cleavage site between the propeptide and the mature IL-18 protein fragment. However, caspase-1 is not expressed in T cells. Caspase-3 and caspase-8 are upregulated in the cytoplasm of activated T cells (Alam et al., "Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells," J. Exp. Med 190(12):1879-1890 (1999); Chun et al., "Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency," Nature 419(6905):395-9 (2002)). In the constructs shown below, the native caspase-1 cleavage site in pro-IL-18 has been replaced by either a caspase-3 or caspase-8 cleavage site (GzB or MT1-MMP cleavage site). These modified derivatives are referred to as pro-IL-18(casp3), pro-IL-18(casp8), pro-IL-18(GzB), and pro-IL-18(MT1-MMP), respectively. Comparisons are made with a constitutively active form of IL-18 (referred to as "constitutive IL-18") in which mature IL-18 is placed downstream of the CD4 signal peptide. [Figure 3]Figure 1 provides flow cytometry (FACS) results confirming co-expression of the second-generation H2 CAR ("H28z") and TBB CCR ("TIE") (together TBB / H pCAR) and IL-18 variants in T cells transfected with a retroviral vector encoding both the second-generation TBB / H pCAR and the IL-18 variants (identified at the top of the figure). Transfected T cells were analyzed for expression of the two components of the pCAR using FACS to measure expression of the H28z CAR (H-2) and TIE-4-1BB CCR separately. [Figure 4] Figure 4A shows the secretion of pro-IL-18 or modified pro-IL-18 in transduced T cells analyzed by ELISA, and Figure 4B shows the functional activity of secreted IL-18 measured by an IL-18-responsive colorimetric reporter assay. [Figures 5A-5D] Figure 5 shows the percentage viability of MDA-MB-468 breast cancer cells after co-culture of cancer cells with pCAR T cells expressing pro-IL-18 or modified pro-IL-18 at different effector:target (T cell:tumor cell) ratios (x-axis) (pro-IL-18 (Figure 5A); constitutive IL-18 (Figure 5B); pro-IL-18(casp8) (Figure 5C); and pro-IL-18(casp3) (Figure 5D)). [Figure 6] T cell numbers (FIG. 6A) and percentage viability of MDA-MB-468 breast cancer cells (FIG. 6B) are provided after the indicated number of restimulation cycles with T cells expressing TBB / H pCAR and pro-IL-18 or modified pro-IL-18 (constit IL-18, pro-IL-18(casp8) or pro-IL-18(casp3)). [Figure 7]IL-18 secretion levels (Figure 7A) and IL-18 functional activity (Figure 7B) detected by ELISA are provided for CAR T cells expressing TBB / H MUC1 pCAR alone, TBB / H and pro-IL-18 (GzB), or TBB / H and constit IL-18, unstimulated or stimulated with anti-CD3 / CD28 antibodies. [Figure 8] To compare the percentage viability of MDA-MB-468 breast cancer cells after co-culture of cancer cells with untransduced T cells, TBB / H pCAR T cells, TBB / H pCAR T cells expressing pro-IL-18, or TBB / H pCAR T cells co-expressing pro-IL-18 (GzB) and additional granzyme B. [Figure 9] The levels of IL-18 (Figure 9A) and IFN-γ (Figure 9B) secreted by TBB / H pCAR T cells are provided. Comparisons are made between TBB / H alone (not expressing exogenous IL-18) and TBB / H pCAR T cells co-expressing pro-IL-18 or pro-IL-18 (GzB) and additional granzyme B. [Figure 10] The percentage viability of MDA-MD-468 cells (Figure 10A) and BxPC-3 cells (Figure 10B) after T cell restimulation cycles is provided. Comparisons are made between untransduced T cells, TBB / H pCAR T cells (not expressing exogenous IL-18), and TBB / H pCAR T cells co-expressing either pro-IL-18, constit IL-18, or a combination of pro-IL-18 (GzB) with additional granzyme B. [Figures 11A-11B]Figure 11 shows the number of successful cycles of antigen stimulation of CAR-T cells with MDA-MD-468 tumor target cells (Figure 11A) or BxPC-3 tumor target cells (Figure 11B). The cells tested were TBB / H pCAR T cells (TBB / H) that do not express exogenous IL-18, or TBB / H pCAR T cells that express pro-IL-18 or pro-IL-18 (GzB) together with additional granzyme B. Restimulation resulting in cytotoxicity of more than 20% of target tumor cells was considered successful. [Figure 12] The number of T cells at the fourth restimulation cycle is provided for pCAR T cells (TBB / H) that do not express exogenous IL-18, or TBB / H pCAR T cells that express pro-IL-18 or pro-IL-18 (GzB) together with additional granzyme B. [Figure 13] Bioluminescence emission ("total flux") is graphed in tumor-injected mice treated with PBS or pCAR T cells that do not express exogenous IL-18 (TBB / H), or TBB / H pCAR T cells that express pro-IL-18, constit IL-18, or pro-IL-18 (GzB) together with additional granzyme B. [Figure 14] FACS data are provided showing T cell expression of pCAR (top) or γδ TCR (bottom) in γδ T cells transduced with retroviral vectors encoding TBB / H pCAR alone (TBB / H) or TBB / H pCAR together with one of four IL-18 mutants (pro-IL-18+pCAR; pro-IL-18(GzB)+pCAR; constit IL-18+pCAR; or pro-IL-18(GzB)+pCAR together with additional granzyme B). [Figure 15]The percentage viability of MDA-MD-468 cells (Figure 15A) and BxPC-3 cells (Figure 15B) is provided after co-culture with either non-transduced T cells or TBB / H pCAR T cells not expressing exogenous IL-18 (TBB / H), or TBB / H pCAR T cells expressing IL-18 variants (either pro-IL-18, constit IL-18, pro-IL-18(GzB) or pro-IL-18(GzB) plus additional granzyme B) at different effector:target ratios. [Figure 16] 1 provides a diagram illustrating the construction of a construct encoding pro-IL-18 with a cleavage site recognized by MT1-MMP (MMP14). [Figures 17A-17C] Shown are bioluminescence emissions ("total intensity") in SKOV-3 tumor-injected mice treated with 0.5 million T4 CAR T cells (Figure 17A), T1NA CAR T cells (a T4 signaling-deficient endodomain-truncated control, Figure 17B), or T4 plus T cells co-expressing pro-IL-18 (MT1-MMP) (Figure 17C). [Figure 18] 1 provides a diagram illustrating the construction of the SFG retroviral construct encoding TBB / H pCAR and pro-IL-18. [Figure 19] FIG. 1 provides a diagram illustrating the construction of the SFG retroviral construct encoding TBB / H pCAR and modified pro-IL-18 with a GzB cleavage site (referred to as pro-IL-18(GzB)). [Figure 20] A diagram illustrating the construction of the SFG retroviral construct encoding TBB / H pCAR and constitutively active IL-18 (referred to as constit IL-18) is provided. [Figure 21] 1 provides a diagram illustrating the construction of the SFG retroviral construct encoding TBB / H pCAR and modified pro-IL-18 with a caspase-8 cleavage site (referred to as pro-IL-18(casp8)). [Figure 22]1 provides a diagram illustrating the construction of the SFG retroviral construct encoding TBB / H pCAR and modified pro-IL-18 with a caspase-3 cleavage site (referred to as pro-IL-18(casp3)). [Figure 23] A diagram illustrating the structure of the SFG retroviral construct encoding TBB / H pCAR, a modified pro-IL-18 with a GzB cleavage site and additional granzyme B (referred to as pro-IL-18(GzB)+granzyme B) is provided. [Figure 24] 1 provides a diagram illustrating the construction of the SFG retroviral construct encoding T4 pCAR and modified pro-IL-18 with an MP1-MMP cleavage site (referred to as pro-IL-18(MT1-MMP)). [Figure 25] A description of various first generation CARs, costimulatory chimeric receptors, and second generation CARs that can be used in various embodiments of the immunoresponsive cells disclosed herein is provided. [Figure 26] A description of various third generation CARs and cis and trans costimulatory chimeric receptors that can be used in various embodiments of the immunoresponsive cells disclosed herein is provided. [Figure 27] A description of various dual-targeting CARs, inhibitory CARs / NOT gates, combinatorial CARs / AND gates, and TanCARs that can be used in various embodiments of the immunoresponsive cells disclosed herein is provided. [Figure 28] A description of Go-CART, Trucks, Armoured CARs, and CARs with modified costimulation that can be used in various embodiments of the immunoresponsive cells disclosed herein is provided. [Figure 29] A description of SynNotch / sequential AND gated CARs and parallel (p)CARs that can be used in various embodiments of the immunoresponsive cells described herein is provided. [Figure 30]Figure 30A graphs the total luminosity in tumor-injected mice treated with PBS or 10 million TBB / H pCAR-αβ T cells that do not express exogenous IL-18 (TBB / H) or TBB / H pCAR-αβ T cells that express additional granzyme B together with pro-IL-18 or pro-IL-18(GzB). Figure 30B graphs the total luminosity in tumor-injected mice treated with PBS or 8 million TBB / H pCAR-γδ T cells that do not express exogenous IL-18 (TBB / H) or TBB / H pCAR-γδ T cells that express additional granzyme B together with pro-IL-18 or pro-IL-18(GzB). Figure 30C graphs the total luminosity in tumor-injected mice treated with PBS or 4 million TBB / H pCAR-γδ T cells that do not express exogenous IL-18 (TBB / H), or TBB / H pCAR-γδ T cells that express pro-IL-18 or pro-IL-18 (GzB) together with additional granzyme B. All graphs show pooled data from three individual mice. [Figure 31] The total light intensity is graphed in three individual tumor-injected mice treated with PBS as a control. [Figure 32A-32B] Total luminosity in individual tumor-injected mice treated with 8×10 TBB / H pCAR-γδ T cells (FIG. 32A) or 4×10 TBB / H pCAR-γδ T cells (FIG. 32B) is provided. In each case, the T cells were devoid of exogenous IL-18 expression. [Figure 33A-33B] Total luminosity in individual tumor-injected mice treated with 8×10 TBB / H pCAR-γδ T cells (FIG. 33A) or 4×10 TBB / H pCAR-γδ T cells (FIG. 33B) is provided. In each case, T cells also produced exogenous pro-IL-18. [Figure 34A-34B]Total luminosity in individual tumor-injected mice treated with 8×10 TBB / H pCAR-γδ T cells (FIG. 34A) or 4×10 TBB / H pCAR-γδ T cells (FIG. 34B) is provided. In each case, T cells also produced exogenous pro-IL-18 (GzB) and exogenous granzyme B. [Figure 35] Figure 1 shows IL-18 activity measured in αβ T cell cultures after stimulation with MUC1+ MDA-MB-468 breast cancer cells ("+468") or beads coated with anti-CD3 and anti-CD28 antibodies ("aCD3 / 28 beads"). Tested αβ T cells were untransduced or transduced to express (i) TBBH, (ii) TBBH and pro-IL-18 (GzB), (iii) TBBH and pro-IL-18 (GzB), (iv) TBBH, pro-IL-18 (GzB) and granzyme B, or (iv) TBBH and constit IL-18. [Figures 36A-36F] Bioluminescence emission ("total intensity") in tumor-injected mice treated with or without αβ T cells is graphed. The graphs show the results for mice treated with PBS (FIG. 36A), or with αβ T cells expressing TBB / H (FIG. 36B), αβ T cells expressing TBB / H + pro-IL-18 (FIG. 36C), αβ T cells expressing TBB / H + pro-IL-18(GzB) (FIG. 36D), αβ T cells expressing TBB / H + constit IL-18 (FIG. 36E), or αβ T cells expressing TBB / H + pro-IL-18(GzB) + granzyme B (FIG. 36F). [Figure 37] Shown are survival curves of tumor-injected mice treated with αβ TBB / H pCAR T cells or αβ TBB / H pCAR T cells additionally expressing pro-IL-18 (GzB), constit IL-18, or pro-IL-18 (GzB) together with granzyme B. [Figure 38]The number of successful restimulation cycles of TBB / H pCAR-T cells (TBB / H) that do not express exogenous IL-18, or TBB / H pCAR T cells that express pro-IL-18, pro-IL-18(GzB), pro-IL-18(GzB) together with additional granzyme B, or constit IL-18, is provided. pCAR T cells were cultured with MDA-MD-468 tumor target cells (Figure 38A) or BxPC-3 tumor target cells (Figure 38B). A restimulation that resulted in more than 30% cytotoxicity against the target tumor cells was considered a successful restimulation cycle. [Figure 39] IL-18 activity measured in γδ T cell cultures after stimulation with MUC1+ MDA-MB-468 breast cancer cells ("+468") or beads coated with anti-CD3 and anti-CD28 antibodies ("aCD3 / 28 beads"). γδ T cells were untransduced or transduced to express (i) TBBH, (ii) TBBH and pro-IL-18 (GzB), (iii) TBBH and pro-IL-18 (GzB), (iv) TBBH, pro-IL-18 (GzB) and granzyme B, or (iv) TBBH and constit IL-18. [Figures 40A-40F] Bioluminescence emission ("total luminosity") in tumor-injected mice treated with or without γδ T cells is shown. Graphs show results for mice treated with PBS (FIG. 40A) or with γδ T cells expressing TBB / H (FIG. 40B), γδ T cells expressing TBB / H+pro-IL-18 (FIG. 40C), γδ T cells expressing TBB / H+pro-IL-18(GzB) (FIG. 40D), γδ T cells expressing TBB / H+constit IL-18 (FIG. 40E), and γδ T cells expressing TBB / H+pro-IL-18(GzB)+granzyme B (FIG. 40F). [Figure 41]1 shows survival curves of tumor-injected mice treated with γδ TBB / H pCAR T cells or γδ TBB / H pCAR T cells additionally expressing granzyme B together with pro-IL-18 (GzB), constit IL-18, or pro-IL-18 (GzB). [Figure 42] The percentage viability of MDA-MD-468 LT cells (Figure 42A) and BxPC-3 LT cells (Figure 42B) after restimulation cycles with TBB / H pCAR T cells is provided. A comparison is made between TBB / H pCAR T cells (not expressing exogenous IL-36) and TBB / H pCAR T cells co-expressing a combination of either granzyme B with pro-IL-36γ or granzyme B with pro-IL-36γ (GzB). [Figure 43] The number of T cells at each restimulation cycle in assays targeting MDA-MB-468 cells (Figure 43A) or BxPC-3 cells (Figure 43B) is provided for pCAR T cells not expressing exogenous IL-36 (TBB / H), TBB / H pCAR T cells expressing granzyme B with pro-IL36γ, or TBB / H pCAR T cells expressing granzyme B with pro-IL-36γ (GzB). [Figure 44] Figures 44A and 44B provide the levels of IFN-γ secreted from TBB / H pCAR T cells co-cultured with MDA-468-LT cells (Figure 44A) or BxPC3-LT cells (Figure 44B). A comparison is made between TBB / H pCAR T cells (not expressing exogenous IL-36) and TBB / H pCAR T cells co-expressing a combination of either granzyme B with pro-IL-36γ or granzyme B with pro-IL-36γ (GzB). [Figure 45]The percentage viability of MDA-MB-468-LT cells after co-culture of cancer cells with non-transduced T cells, TBB / H pCAR T cells, or TBB / H pCAR T cells additionally expressing pro-IL-36γ and granzyme B, or pro-IL-36γ (GzB) and granzyme B, at a range of initial effector:target cell ratios (E:T) is compared. [Figure 46] The percentage viability of BxPC3-LT cells after co-culture of cancer cells with non-transduced T cells, TBB / H pCAR T cells, or TBB / H pCAR T cells additionally expressing pro-IL-36γ and granzyme B, or pro-IL-36γ (GzB) and granzyme B, at a range of initial effector:target cell ratios (E:T) will be compared. [Figures 47A-47D] Bioluminescence emission ("total intensity") in tumor-injected mice treated with or without αβ T cells is graphed. The graphs show the results for mice treated with PBS (FIG. 47A), TBB / H (FIG. 47B), TBB / H + pro-IL-36γ + granzyme B (FIG. 47C), or TBB / H + pro-IL-36γ (GzB) + granzyme B (FIG. 47D). [Figure 48A-48B] Flow cytometry (FACS) results confirming expression of the TBB CCR ("TIE") (in TBB / H pCAR) and γδ TCR in untransduced (Figure 48A) or TBB / H pCAR γδ T cells (Figure 48B) are provided. [Figure 49] Figure 49A provides the fold cell expansion after 15 days of culture of untransduced or TBB / H pCAR γδ T cells. Figure 49B provides the number of cells obtained and cultured from three individual donors at three different time points (day 1, day 8, and day 15). [Figure 50A-50B] Provided are the % viability of MDA-MB-468 tumor cells (Figure 50A) or BxPC-3 tumor cells (Figure 50B) after culture with untransduced or TBB / H pCAR-γδ T cells (1:1 ratio) compared to tumor cells cultured alone. [Figure 51] Figures 51A-51B provide the number of successful restimulation cycles of untransduced or TBB / H pCAR γδ T cells. T cells were cultured with MDA-MD-468 tumor target cells (Figure 51A) or BxPC-3 tumor target cells (Figure 51B). Figures 51C-51D provide the % viability of MDA-MB-468 tumor cells (Figure 51C) or BxPC-3 tumor cells (Figure 51D) over successive restimulation cycles with untransduced or TBB / H pCAR-γδ T cells. [Figure 52] 1 provides bioluminescence emission ("total intensity") over time in BxPC-3 tumor-injected NSG mice treated with PBS, untransduced γδ T cells ("UT"), or TBB / H pCAR γδ T cells ("TBBH"). [Figure 53] 1 provides bioluminescence emission ("total intensity") over time in MDA-MB-468 tumor-injected SCID Beige mice treated with PBS or TBB / H pCAR γδ T cells ("TBBH"). DETAILED DESCRIPTION OF THE INVENTION

[0064] 4. Detailed Description Details of various embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will be apparent from the specification and drawings, and from the claims.

[0065] 4.1.Definition Unless otherwise defined herein, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0066] The term "IL-1 family member" refers to a member of the IL-1 family, which includes seven proteins with pro-inflammatory activity (IL-1α and IL-1β, IL-18, IL-33, IL-36α, IL-36β, and IL-36γ) and four proteins with anti-inflammatory activity (IL-1 receptor antagonist (IL-1Ra), IL-36Ra, IL-37, and IL-38). In some embodiments, the IL-1 family member is IL-18, IL-36α, IL-36β, or IL-36γ. IL-36α, IL-36β, and IL-36γ are collectively referred to as "IL-36."

[0067] The term "pro-cytokine" refers to an inactive precursor of an IL-1 family member. A pro-cytokine generally comprises (i) a propeptide, (ii) a cleavage site recognized by a protease, and (iii) a mature, biologically active cytokine fragment. The activity of the cytokine fragment can be modulated by processing of the cleavage site. In a preferred embodiment, the pro-cytokine is pro-IL-18, pro-IL-36α, pro-IL-36β, or pro-IL-36γ.

[0068] The term "pro-IL-18" refers to the native 24 kDa inactive precursor of IL-18. Pro-IL-18 contains, from N- to C-terminus, (i) the propeptide, (ii) the cleavage site recognized by caspase 1, and (iii) the mature, biologically active IL-18 protein fragment. In a preferred embodiment, pro-IL-18 refers to human pro-IL-18, a 193 aa, 24.2 kDa protein. The cDNA sequence of human pro-IL-18 is provided by GenBank / EBI Data Bank accession number AF077611 (nucleotides 1-579). The protein sequence of human pro-IL-18 is provided by GenBank accession number AAC27787.

[0069] The term "pro-IL-36α" refers to the native 17.7 kDa inactive precursor of IL-36α. Pro-IL-36α contains, from N- to C-terminus, (i) a propeptide, (ii) a cleavage site recognized by neutrophil proteases, including cathepsin G and elastase, and (iii) a mature, biologically active IL-36α protein fragment. In a preferred embodiment, pro-IL-36α refers to human pro-IL-36α, a 158-aa, 17.7 kDa protein. The cDNA sequence of human pro-IL-36α is provided by GenBank / EBI Data Bank accession number AF201831.1 (nucleotides 1-477). The protein sequence of human pro-IL-36α is provided by GenBank accession number AAY14988.1 and is also provided herein as SEQ ID NO: 36.

[0070] The term "pro-IL-36β" refers to the native 18.5 kDa inactive precursor of IL-36β. Pro-IL-36β contains, from N- to C-terminus, (i) a propeptide, (ii) a cleavage site recognized by neutrophil proteases, including cathepsin G, and (iii) a mature, biologically active IL-36β protein fragment. In a preferred embodiment, pro-IL-36β refers to human pro-IL-36β, an 164-aa, 18.5 kDa protein. The cDNA sequence of human pro-IL-36β is provided by GenBank / EBI Data Bank accession number AF200494.1 (nucleotides 1-1190). The protein sequence of human pro-IL-36β is provided by GenBank accession number NP_055253 and is also provided herein as SEQ ID NO: 38.

[0071] The term "pro-IL-36γ" refers to the native 18.7 kDa inactive precursor of IL-36γ. Pro-IL-36γ contains, from N- to C-terminus, (i) the propeptide, (ii) a cleavage site recognized by neutrophil proteases, including proteinase 3 and elastase, and (iii) the mature, biologically active IL-36γ protein fragment. In a preferred embodiment, pro-IL-36γ refers to human pro-IL-36γ, a 169-aa, 18.7 kDa protein. The cDNA sequence of human pro-IL-36γ is provided by GenBank / EBI Data Bank accession number AF200492 (nucleotides 1-1183). The protein sequence of human pro-IL-36γ is provided by GenBank accession number NP_062564 and is also provided herein as SEQ ID NO: 40.

[0072] As used herein, the term "modified pro-cytokine" refers to a protein produced by the insertion, deletion, and / or substitution of one or more amino acids of a pro-cytokine protein. In preferred embodiments, the modified pro-cytokine contains a new cleavage site that is recognized and cleaved by a protease other than the protease that cleaves the unmodified pro-cytokine to release the cytokine fragment.

[0073] As used herein, the term "modified pro-IL-18" refers to a protein produced by the insertion, deletion, and / or substitution of one or more amino acids of the pro-IL-18 protein. In a preferred embodiment, the modified pro-IL-18 contains a new cleavage site recognized by a protease other than caspase-1, such that the modified pro-IL-18 can be cleaved by a protease other than caspase-1 to release biologically active IL-18 protein fragments.

[0074] As used herein, the term "modified pro-IL-36" refers to a protein produced by the insertion, deletion, and / or substitution of one or more amino acids of the pro-IL-36 protein. In a preferred embodiment, the modified pro-IL-36 contains a new cleavage site recognized by a protease other than cathepsin G, elastase, and proteinase 3, such that the modified pro-IL-36 can be cleaved by a protease other than cathepsin G, elastase, or proteinase 3 to release a biologically active IL-36 protein fragment.

[0075] As used herein, the term "pro-IL-18([protease])" refers to modified pro-IL-18 containing a cleavage site recognized by the protease specified in parentheses. For example, proIL-18(GzB) refers to modified pro-IL-18 containing a cleavage site cleavable by granzyme B (GzB), pro-IL-18(casp3) refers to modified pro-IL-18 containing a cleavage site cleavable by caspase-3, and pro-IL-18(casp8) refers to modified pro-IL-18 containing a cleavage site cleavable by caspase-8.

[0076] As used herein, the term "pro-IL-36(GzB)" refers to a modified pro-IL-36 that contains a cleavage site recognized by GzB.

[0077] As used herein, the term "cleavage site" refers to a sequence of amino acids that can be recognized by a protease. As used herein, a cleavage site "recognized by" a protease is an amino acid sequence that is cleavable by the protease under conditions that exist or are achievable in vivo.

[0078] As used herein, the terms "biologically active cytokine fragment" and "cytokine fragment" refer to a biologically active polypeptide produced by cleavage of a pro-cytokine by a protease that recognizes a cleavage site upstream (N-terminal) of the cytokine fragment. "Biologically active" means that the cytokine fragment is capable of binding to and activating its corresponding receptor. A cytokine fragment may be a native cytokine protein fragment or a modification thereof. In some embodiments, a cytokine fragment has improved biological activity compared to the native mature cytokine. In some embodiments, a cytokine fragment refers to an IL-18 fragment or an IL-36 fragment, as defined below.

[0079] As used herein, the terms "IL-18 fragment" and "IL-18 protein fragment" refer to a biologically active IL-18 polypeptide produced by cleavage of pro-IL-18 with a protease that recognizes a cleavage site upstream (N-terminal) of the IL-18 fragment. "Biologically active" means that the IL-18 fragment is capable of binding to and activating the IL-18 receptor. The IL-18 fragment may be a native mature IL-18 protein fragment or a modification thereof. In some embodiments, the IL-18 fragment has improved biological activity compared to native mature IL-18.

[0080] As used herein, the terms "IL-36 fragment" and "IL-36 protein fragment" refer to a biologically active IL-36 polypeptide produced by cleavage of pro-IL-36 with a protease that recognizes a cleavage site upstream (N-terminal) of the IL-36 fragment. "Biologically active" means that the IL-36 fragment is capable of binding to and activating the IL-36 receptor. An IL-36 fragment may be a native mature IL-36 protein fragment or a modification thereof. In some embodiments, an IL-36 fragment has improved biological activity compared to native mature IL-36. An IL-36 fragment may refer to the mature IL-36 α, β, or γ protein.

[0081] As used herein, the term "IL-18 variant" refers collectively to pro-IL-18 protein, modified pro-IL-18 protein, and IL-18 fragments, including native mature IL-18 fragments.

[0082] As used herein, the term "IL-36 variant" collectively refers to pro-IL-36 protein, modified pro-IL-36 protein, and IL-36 fragments, including native mature IL-36 α, β, or γ fragments.

[0083] As used herein with reference to binding elements of engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs) and immunoresponsive cells engineered to express such TCRs or CARs, the terms "recognize," "specifically bind," "specifically bind to," "specifically interact with," "specifically specific for," "selectively bind to," "selectively interact with," and "selective for" a particular antigen or epitope thereof (which may be a protein antigen, glycopeptide antigen, or peptide-MHC complex) refer to binding that is measurably different from nonspecific or nonselective interactions (e.g., with non-target molecules). Specific binding can be measured, for example, by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the recognized epitope on the target molecule.

[0084] 4.2. Other interpretive practices In the claims, articles such as "a," "an," and "the" may mean one or more than one, unless indicated otherwise or clear from the context. A claim or specification containing "or" between one or more members of a group is considered to be satisfied when one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process, unless indicated otherwise or clear from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0085] It should also be noted that the term "comprising" is intended to be open, permitting but not requiring the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of" is also included and disclosed.

[0086] When ranges are given, the endpoints are included. Furthermore, unless otherwise indicated, or otherwise apparent from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can, in different embodiments of the invention, be extrapolated to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0087] All cited sources, e.g., references, publications, databases, database entries, and art cited herein, are incorporated by reference into this application, even if not expressly stated in the citation. In the event of a conflict between the statements in a cited source and this application, the statements in this application will control.

[0088] Section and table headings are not intended to be limiting.

[0089] 4.3. Immunoresponsive cells In a first aspect, an immunoresponsive cell is provided that expresses a modified pro-cytokine of the IL-1 superfamily, wherein the modified pro-cytokine comprises, from N-terminus to C-terminus, (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a cytokine fragment of the IL-1 superfamily.

[0090] In some embodiments, the immunoresponsive cells express modified pro-IL-18, wherein the modified pro-IL-18 comprises, from N-terminus to C-terminus, (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1; and (c) a biologically active IL-18 fragment.

[0091] In some embodiments, the immunoresponsive cells express a modified pro-IL-36, wherein the modified pro-IL-36 comprises, from N-terminus to C-terminus, (a) a propeptide; (b) a cleavage site recognized by a protease other than cathepsin G, elastase, and proteinase 3; and (c) a biologically active IL-36 α, β, or γ fragment.

[0092] 4.3.1.Cells In an exemplary embodiment, the immunoresponsive cell is a T cell.

[0093] In certain embodiments, the immunoresponsive cells are αβ T cells. In certain embodiments, the immunoresponsive cells are cytotoxic αβ T cells. In certain embodiments, the immunoresponsive cells are αβ helper T cells. In certain embodiments, the immunoresponsive cells are regulatory αβ T cells (Tregs).

[0094] In certain embodiments, the immunoresponsive cells are γδ T cells. In certain embodiments, the immunoresponsive cells are Vδ2 T cells. + In a specific embodiment, the immunoresponsive cells are V52 T cells. - In a specific embodiment, V52 - T cells are Vδ1 + It is a cell.

[0095] In certain embodiments, the immunoresponsive cells are natural killer (NK) cells.

[0096] In some embodiments, the immunoresponsive cells do not express additional exogenous proteins. In other embodiments, the immunoresponsive cells are modified to express additional exogenous proteins, such as modified T cell receptors (TCRs) or chimeric antigen receptors (CARs). Immunoresponsive cells that further express modified TCRs and CARs are further described below.

[0097] In some embodiments, the immunoresponsive cells are obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the immunoresponsive cells are obtained from a tumor. In certain embodiments, the immunoresponsive cells obtained from a tumor are tumor-infiltrating lymphocytes (TILs). In specific embodiments, the TILs are αβ T cells. In other specific embodiments, the TILs are γδ T cells, particularly Vδ2 T cells. - γδ T cells.

[0098] Modified pro-IL-18 In some embodiments, the immunoresponsive cells express modified pro-IL-18.

[0099] The modified pro-IL-18 comprises, from the N-terminus to the C-terminus, (i) a propeptide, (ii) a cleavage site recognized by a protease other than caspase-1, and (iii) an IL-18 fragment. The modified pro-IL-18 can be cleaved by a protease that recognizes the cleavage site to release the propeptide and a biologically active IL-18 fragment.

[0100] Propeptide In typical embodiments, the propeptide is the unmodified native propeptide of the pro-IL-18 protein. In particular embodiments, the propeptide is the unmodified native propeptide of the human pro-IL-18 protein.

[0101] In other embodiments, the propeptide is modified from the native propeptide of the pro-IL-18 protein. In certain embodiments, the modified propeptide comprises one or more amino acid modifications compared to the native pro-IL-18 propeptide. In certain embodiments, the propeptide is a propeptide derived from a non-pro-IL-18 protein. In certain embodiments, the propeptide has a non-naturally occurring, synthetic amino acid sequence.

[0102] In some embodiments, the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25. In some embodiments, the propeptide is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 25.

[0103] 4.3.2.2. Cutting site The cleavage site within the modified pro-IL-18 is recognized by proteases other than caspase-1.

[0104] In typical embodiments, only a single cleavage site recognized by a protease other than caspase-1 is present in the modified pro-IL-18. In other embodiments, multiple cleavage sites recognized by proteases other than caspase-1 are introduced. In such embodiments, the multiple cleavage sites may be cleavage sites recognized by the same or different proteases other than caspase-1.

[0105] In various embodiments, a cleavage site recognized by a protease other than caspase-1 is introduced (a) between the propeptide and the caspase-1 cleavage site, (b) at a predetermined position in the caspase-1 cleavage site, or (c) between the caspase-1 cleavage site and the IL-18 fragment.

[0106] In some embodiments, the cleavage site replaces the caspase-1 cleavage site of pro-IL-18, hi some embodiments, the cleavage site is in addition to the caspase-1 cleavage site.

[0107] In typical embodiments, the cleavage site in the modified pro-IL-18 is selected from protease cleavage sites known in the art, hi typical embodiments, the protease is a protease known to be expressed in activated T cells or NK cells. In certain embodiments, the cleavage site is recognized by granzyme B (GzB), caspase-3, caspase-8, or membrane-type matrix metalloproteinase 1 (MT1-MMP, also known as MMP14), alternative tumor-associated matrix metalloproteinase (MMP1-13), a disintegrin and metalloproteinase (ADAM) family member (particularly ADAM10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-related peptidase (KLK), e.g., KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, hepsin, or urokinase plasminogen activator (Dudani et al., "Harnessing protease activity to improve cancer care," Annu. Rev. Cancer Biol., 2:353-76 (2018). In certain embodiments, the cleavage site is recognized by granzyme B (GzB). In certain embodiments, the cleavage site is recognized by caspase-3. In certain embodiments, the cleavage site is recognized by caspase-8. In certain embodiments, the cleavage site is recognized by MT1-MMP.

[0108] In some embodiments, the cleavage site comprises a sequence selected from SEQ ID NOs: 26, 28, 30, and 32. In some embodiments, the modified pro-IL-18 comprises a sequence selected from SEQ ID NOs: 27, 29, 31, and 33.

[0109] In other embodiments, the cleavage site is a synthetic cleavage site of non-natural origin.

[0110] IL-18 Fragments

[0111] In various embodiments, the IL-18 fragment is a native IL-18 fragment. In a preferred embodiment, the native IL-18 fragment is a human IL-18 fragment.

[0112] In other embodiments, the IL-18 fragment is modified from a native IL-18 fragment but retains the ability to bind to and activate the IL-18 receptor when cleaved from the modified pro-IL-18 by protease cleavage at the cleavage site. In various embodiments, the IL-18 fragment has biological activity similar to, less than, or better than, the native mature IL-18 protein.

[0113] In some embodiments, the IL-18 fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the IL-18 fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the modified pro-IL-18 protein is expressed from an exogenous sequence introduced into the T cell. In some embodiments, the exogenous sequence is selected from the group consisting of SEQ ID NOs: 102, 103, 105, 107, 109, 111, and 113. In some embodiments, the exogenous sequence is a coding sequence cloned into an expression vector, e.g., a viral or non-viral vector.

[0114] Modified pro-IL-36 In some embodiments, the immunoresponsive cells express a modified pro-IL-36 α, β, or γ protein.

[0115] The modified pro-IL-36 comprises, from the N-terminus to the C-terminus, (i) a propeptide, (ii) a cleavage site recognized by a protease other than cathepsin G, elastase, and proteinase 3, and (iii) an IL-36 fragment. The modified pro-IL-36 can be cleaved by a protease that recognizes the cleavage site to release the propeptide and biologically active IL-36α, β, or γ fragment.

[0116] Propeptide In typical embodiments, the propeptide is the unmodified native propeptide of the pro-IL-36 α, β, or γ protein. In a specific embodiment, the propeptide is the unmodified native propeptide of the human pro-IL-36 protein.

[0117] In other embodiments, the propeptide is modified from the native propeptide of the pro-IL-36 protein. In certain embodiments, the modified propeptide comprises one or more amino acid modifications compared to the native pro-IL-36 propeptide. In certain embodiments, the propeptide is a propeptide derived from a non-pro-IL-36 protein. In certain embodiments, the propeptide has a non-naturally occurring, synthetic amino acid sequence.

[0118] In some embodiments, the propeptide is derived from pro-IL-36α (SEQ ID NO: 45). In some embodiments, the propeptide is derived from modified pro-IL-36α (SEQ ID NO: 46). In some embodiments, the propeptide is derived from pro-IL-36β (SEQ ID NO: 47). In some embodiments, the propeptide is derived from modified pro-IL-36β (SEQ ID NO: 48). In some embodiments, the propeptide is derived from pro-IL-36γ (SEQ ID NO: 49). In some embodiments, the propeptide is derived from modified pro-IL-36γ (SEQ ID NO: 50).

[0119] 4.3.3.2. Cutting site The cleavage site within the modified pro-IL-36 is recognized by proteases other than cathepsin G, elastase, and proteinase 3.

[0120] In typical embodiments, only a single cleavage site recognized by a protease other than cathepsin G, elastase, and proteinase 3 is present in the modified pro-IL-36. In other embodiments, multiple cleavage sites are introduced that are recognized by proteases other than cathepsin G, elastase, and proteinase 3. In such embodiments, the multiple cleavage sites may be cleavage sites recognized by the same or different proteases other than cathepsin G, elastase, and proteinase 3.

[0121] In various embodiments, a cleavage site recognized by a protease other than cathepsin G, elastase, and proteinase 3 is introduced (a) between the propeptide and the cathepsin G, elastase, or proteinase 3 cleavage site, (b) at a predetermined position in the cathepsin G, elastase, or proteinase 3 cleavage site, or (c) between the cathepsin G, elastase, or proteinase 3 cleavage site and the IL-36 fragment.

[0122] In some embodiments, the cleavage site replaces the cathepsin G, elastase, or proteinase 3 cleavage site naturally present in pro-IL-36 α, β, or γ. In some embodiments, the cleavage site is in addition to the cathepsin G, elastase, and / or proteinase 3 cleavage site naturally present in pro-IL-36 α, β, or γ.

[0123] In exemplary embodiments, the cleavage site within the modified pro-IL-36 is selected from protease cleavage sites known in the art, hi exemplary embodiments, the protease is a protease known to be expressed in activated T cells or NK cells. In certain embodiments, the cleavage site is recognized by granzyme B (GzB), caspase-3, caspase-8, or membrane-type matrix metalloproteinase 1 (MT1-MMP, also known as MMP14), alternative tumor-associated matrix metalloproteinase (MMP1-13), a disintegrin and metalloproteinase (ADAM) family member (particularly ADAM10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-related peptidase (KLK), e.g., KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, hepsin, or urokinase plasminogen activator (Dudani et al., "Harnessing protease activity to improve cancer care," Annu. Rev. Cancer Biol., 2:353-76 (2018). In certain embodiments, the cleavage site is recognized by granzyme B (GzB). In certain embodiments, the cleavage site is recognized by caspase-3. In certain embodiments, the cleavage site is recognized by caspase-8. In certain embodiments, the cleavage site is recognized by MT1-MMP.

[0124] In some embodiments, the cleavage site comprises a sequence selected from SEQ ID NOs: 26, 28, 30, and 32. In some embodiments, the modified pro-IL-36 comprises a sequence selected from SEQ ID NOs: 37, 39, and 41.

[0125] In other embodiments, the cleavage site is a synthetic cleavage site of non-natural origin.

[0126] IL-36 Fragments In various embodiments, the IL-36 fragment is a native IL-36 α (SEQ ID NO: 42), β (SEQ ID NO: 43), or γ (SEQ ID NO: 44) fragment. In a preferred embodiment, the native IL-36 fragment is a human IL-36 fragment.

[0127] In other embodiments, the IL-36 fragment is modified from a native IL-36 fragment but retains the ability to bind to and activate the IL-36 receptor when cleaved from the modified pro-IL-36 by protease cleavage at the cleavage site. In various embodiments, the IL-36 fragment has biological activity similar to, less than, or better than, the native mature IL-36 α, β, or γ protein.

[0128] In some embodiments, the IL-36 α, β, or γ fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42, 43, or 44, respectively. In some embodiments, the IL-36 α, β, or γ fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42, 43, or 44, respectively. In some embodiments, the modified pro-IL-36 protein is expressed from an exogenous sequence introduced into the T cell. In some embodiments, the exogenous sequence is a coding sequence cloned into an expression vector, e.g., a viral or non-viral vector.

[0129] 4.3.4. Expressed Proteases In some embodiments, the immunoresponsive cells are modified to further express a protease that recognizes a cleavage site in the co-expressed modified pro-IL-18 or modified pro-IL-36.

[0130] In some embodiments, the protease is selected from the group consisting of GzB, caspase-3, caspase-8 and MT1-MMP.

[0131] In certain embodiments, the expressed protease is GzB. In preferred embodiments, the expressed protease is human GzB. In specific embodiments, the expressed protease comprises SEQ ID NO: 20 or a modification thereof.

[0132] In certain embodiments, the expressed protease is caspase-3. In preferred embodiments, the expressed protease is human caspase-3. In specific embodiments, the expressed protease comprises SEQ ID NO:21 or a modification thereof.

[0133] In certain embodiments, the expressed protease is caspase-8. In preferred embodiments, the expressed protease is human caspase-8. In specific embodiments, the expressed protease comprises SEQ ID NO: 22 or a modification thereof.

[0134] In certain embodiments, the expressed protease is MT1-MMP. In preferred embodiments, the expressed protease is human MT1-MMP. In specific embodiments, the expressed protease comprises SEQ ID NO: 23 or a modification thereof.

[0135] In some embodiments, the expressed protease is an alternative tumor-associated matrix metalloproteinase (MMP1-13), a disintegrin and metalloproteinase (ADAM) family member (particularly ADAM10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-related peptidase (KLK) such as KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, hepsin, or urokinase plasminogen activator (see Dudani et al., "Harnessing protease activity to improve cancer care," Annu. Rev. Cancer Biol., 2:353-76 (2018)).

[0136] The expressed protease is expressed from an exogenous sequence introduced into the immunoresponsive cell in an expression vector. In some embodiments, the immunoresponsive cell expresses the modified pro-cytokine and the protease from a single expression vector. In some embodiments, the immunoresponsive cell expresses the modified pro-cytokine and the protease from multiple expression vectors. In certain embodiments, the immunoresponsive cell expresses the modified pro-cytokine from a first expression vector and the protease from a second expression vector.

[0137] 4.3.5.CAR In an exemplary embodiment, the immunoresponsive cells are modified to further express a chimeric antigen receptor (CAR).

[0138] 4.3.5.1.CAR specificity In typical embodiments, the CAR is specific for at least one antigen present in cancer. In typical embodiments, the CAR is specific for at least one antigen present in solid tumors.

[0139] In various embodiments, the antigen is human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B1 (CYP1B), HER2 / neu, Wilms tumor gene 1 (WT1), livin, alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, or cyclin D1. For example, the target antigen is hTERT or survivin. In some embodiments, the target antigen is CD38. In some embodiments, the target antigen is B-cell maturation antigen (BCMA, BCM). In some embodiments, the target antigen is BCMA, B-cell activating factor receptor (BAFFR, BR3), and / or transmembrane activator and interactor of CAML (TACI), or related proteins thereof. For example, in some embodiments, the target antigen is or is related to BAFFR or TACI. In some embodiments, the target antigen is CD33 or TIM-3. In some embodiments, it is CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or CD362.

[0140] In some embodiments, the CAR is selected from the group consisting of alpha folate receptor, 5T4, alpha v beta 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, CMV, EBV, EGFR, EGFR family including ErbB2 (HER2), ErbB family homo- and heterodimers, EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FcA ... R.α., GD2, GD3, Glypican-3(GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, HPV, IL-11R.α., Specific for IL-13R.α.2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, or VEGFR2.

[0141] In some embodiments, the CAR is selected from the group consisting of TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, EphrinB2, IGF-I receptor, CAIX, LMP2, gp100, b cr-abl, tyrosinase, EphA2, fucosyl-GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-1a, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutSpecific for hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1.

[0142] In some embodiments, the CAR is specific for the MUC1 target antigen. In certain embodiments, the CAR is specific for a tumor-associated MUC1 epitope. In a specific embodiment, the targeting domain of the CAR comprises the CDRs of the HMFG2 antibody. See Wilkie et al., "Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor," J. Immunol. 180(7):4901-4909 (2008), incorporated herein by reference in its entirety. In some embodiments, the CAR is specific for the V nucleotides of the HMFG2 antibody. H and V L In some embodiments, the CAR comprises an HMFG2 single chain variable fragment (scFv).

[0143] In some embodiments, the CAR is specific for ErbB homo- and / or heterodimers. In certain embodiments, the targeting domain of the CAR comprises a promiscuous ErbB peptide ligand, T1E. T1E is a chimeric peptide derived from transforming growth factor-alpha (TGF-α) and epidermal growth factor (EGF). See Wingens et al., "Structural analysis of an epidermal growth factor / transforming growth factor-alpha chimera with unique ErbB binding specificity," J. Biol. Chem. 278:39114-23 (2003) and Davies et al., "Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells," Mol. Med. 18:565-576 (2012), the disclosures of which are incorporated herein by reference in their entireties.

[0144] CAR Format In some embodiments, the CAR is a first-generation CAR. First-generation CARs most commonly use CD3 zeta (CD3z or CD3ζ) or Fcεr1γ intracellular signaling domains to provide TCR-like signals, thereby resulting in tumoricidal function. However, binding of the CD3z chain fusion receptor may be insufficient to result in substantial IL-2 secretion and / or T cell proliferation in the absence of a concomitant costimulatory signal. In physiological T cell responses, optimal lymphocyte activation may require binding of one or more costimulatory receptors, such as CD28 or 4-1BB.In some embodiments, the first generation CARs disclosed in Eshhar et al., "Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors," PNAS 90(2):720-4 (1993), or Alvarez-Vallina et al., "Antigen-specific targeting of CD28-mediated T cell co-stimulation using chimeric single-chain antibody variable fragment-CD28 receptors," Eur. J. Immunol. 26(10):2304-9 (1996) and Krause et al., "Antigen-dependent CD28 signaling selectively enhances survival and proliferation in genetically modified activated human primary T The costimulatory chimeric receptors disclosed in "Cell Signaling in Immunocompetent Cells," J. Exp. Med. 188(4):619-26 (1998) are expressed in the immunoresponsive cells described herein (Figure 25); both of these references are incorporated herein by reference in their entirety.

[0145] In some embodiments, the CAR is a second-generation CAR. Second-generation CARs can transmit functional antigen-dependent costimulatory signals in human primary T cells in addition to antigen-dependent TCR-like signals, enabling T cell proliferation in addition to tumoricidal activity. Second-generation CARs most commonly provide costimulation using costimulatory domains (synonymously, costimulatory signaling regions) derived from CD28 or 4-1BB. The combined delivery of costimulation and CD3 zeta signals can make second-generation CARs functionally superior to their first-generation counterparts.Exemplary second-generation CARs that can be usefully expressed in the immunoresponsive cells described herein are described in U.S. Pat. No. 7,446,190; Finney et al., "Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product," J. Immunol 161(6):2791-7 (1998); Maher et al., "Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRzeta / CD28 receptor," Nat. Biotechnol. 20(1):70-5 (2002); Finney et al., "Activation of resting human primary T cells with chimeric receptors: costimulation from CD28, inducible costimulator, CD134, and CD137 in series with signals from the TCR zeta chain," J. Immunol. 172(1):104-13 (2004); and Imai et al. al., "Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia," Leukemia 18(4):676-84 (2004), which are incorporated herein by reference in their entirety.

[0146] Further exemplary second generation CARs that can be usefully expressed in the immunoresponsive cells described herein are provided in Figure 25.

[0147] The examples herein provide additional second-generation CARs that can be usefully expressed in the immunoresponsive cells described herein. In certain embodiments, a second-generation CAR (referred to as "H," "H2," or "H28z") is used. The H2 CAR comprises, from the extracellular to intracellular domain, the MUC-1-targeting HMFG2 scFv, the CD28 transmembrane and costimulatory domains, and the CD3z signaling region. See Figure 1. The H2 CAR is described in Wilkie et al., "Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor," J. Immunol. 180:4901-9 (2008), which is incorporated herein by reference in its entirety. In certain embodiments, a second-generation CAR (referred to as T1E28z) is used. The T1E28z CAR contains, from the extracellular to the intracellular domain, an ErbB-targeting T1E peptide, a CD28 transmembrane and costimulatory domain, and a CD3z signaling region. See Figure 1. The T1E28z second generation CAR is described in Davies, "Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells," Mol. Med. 18:565-576 (2012), which is incorporated herein by reference in its entirety.

[0148] In some embodiments, third-generation CARs are used. Third-generation CARs can further enhance efficacy by combining multiple costimulatory domains (synonymously, costimulatory signaling regions) with TCR-like signaling domains (synonymously, signaling regions) in cis (e.g., CD28+4-1BB+CD3z or CD28+OX40+CD3z). In some embodiments, third-generation CARs comprise tandemly arranged costimulatory domains within the CAR endodomain (generally located upstream of CD3z or equivalent). Some exemplary third-generation CARs that can be usefully expressed in the immunoresponsive cells described herein are disclosed in Pule et al., "A chimeric T cell antigen receptor that augments cytokine release and supports clonal expansion of primary human T cells," Mol Ther. 12(5):933-41 (2005); Geiger et al., "Integrated src kinase and costimulatory activity enhances signal transduction through single-chain chimeric receptors in T lymphocytes," Blood 98:2364-71 (2001); and Wilkie et al., "Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor," J. Immunol. 180(7):4901-9 (2008), the disclosures of which are incorporated herein by reference in their entireties, as well as in FIG. 26.In some embodiments, a CAR is used that employs both cis and trans costimulatory signals, as disclosed in Stephan et al., "T cell-encoded CD80 and 4-1BBL induce auto- and transcostimulation, resulting in potent tumor rejection," Nat. Med. 13(12)1440-9 (2007), incorporated herein by reference, and provided in FIG. 26.

[0149] Other CAR formats available and known in the art can be expressed in various embodiments of the immunoresponsive cells described herein. In particular, Figures 27-29 disclose additional CAR formats that can be expressed in the immunosuppressive cells of the present disclosure, including Wilkie et al., "Dual Targeting of ErbB2 and MUC1 in Breast Cancer Using Chimeric Antigen Receptors Engineered to Provide Complementary Signaling," J. Clin. Immunol. 32(5)1059-70 (2012); Fedorov et al., "PD-1- and CTLA-4-based inhibitory chimeric antigen receptors (iCARs) diverte off-target immunotherapy responses," Sci. Transl. Med. 5(215)215ra172 (2013); Kloss et al., "Combinatorial antigen recognition with balanced signaling promotes selective tumor eradication by engineered T cells," Nat. Biotechnol. 31(1):71-6 (2013); Grada et al., "TanCAR: A Novel Bispecific Chimeric Antigen Receptor for "Cancer Immunotherapy" Mol.Ther.Nucleic Acids.2:e105(2013);Foster et al. "Regulated Expansion and Survival of Chimeric Antigen Receptor-Modified T Cells Using Small Molecule-Dependent Inducible MyD88 / CD40" Mol.Ther.25(9):2176-2188(2017);Chmielewski et al. al.「IL-12 release by engineered T cells expressing chimeric antigen receptors can effectively muster an antigen-independent macrophage response on tumor cells that have shut down tumor antigen expression」Cancer Research,71:5697-5706(2011);Pegram et al.,「Tumor-targeted T cells modified to secrete IL-12 eradicate systemic tumors without need for prior conditioning」Blood 119:4133-4141(2012);Curran et al.「Enhancing antitumor efficacy of chimeric antigen receptor T cells through constitutive CD40L expression」Mol.Ther.23(4):769-78(2015);Zhao et al.,「Structural design of engineered costimulation determines tumor rejection kinetics and persistence of CAR T cells」Cancer Cell 28:415-28(2015);Roybal et al.,「Precision tumor recognition by T Cells with combinatorial antigen-sensing circuits,Cell 164:770-9(2016);Whilding et al., "CAR T-Cells targeting the integrin alphavbeta6 and co-expressing the chemokine receptor CXCR2 demonstrate enhanced homing and efficacy against several solid malignancies," Cancers 11(5), 674 (2019), and Kosti et al., "Perspectives on Chimeric Antigen Receptor T-Cell immunotherapy for solid tumors," Front Immunol 9:1104, (2018), which are incorporated herein by reference in their entireties.

[0150] 4.3.5.2.1.pCAR Format In certain embodiments, parallel CARs (pCARs) are expressed in immunoresponsive cells.

[0151] In pCAR embodiments, immunoresponsive cells are engineered to express two constructs (a second-generation CAR and a chimeric costimulatory receptor (CCR)) in parallel. The second-generation CAR comprises, from the intracellular to the extracellular domain, (a) a signaling region; (b) a first costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen. The CCR comprises, from the intracellular to the extracellular domain, (a) a costimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen. Typically, the CCR lacks a TCR-like signaling region (e.g., CD3z). In some embodiments, the costimulatory domain of the CCR (the second costimulatory domain) is different from the costimulatory domain of the CAR (the first costimulatory domain). In some embodiments, the second epitope is different from the first epitope. Parallel CAR (pCAR) modified T cells have been shown to have superior activity and resistance to depletion compared to first-, second-, and third-generation CAR-T cells. See U.S. Pre-Grant Publication 2019 / 0002521, incorporated herein by reference in its entirety.

[0152] In some embodiments, the second target antigen is different from the first target antigen. In some embodiments, the second target antigen is the same as the first target antigen.

[0153] In some embodiments, the first antigen is a MUC1 antigen. In certain embodiments, the first epitope is a tumor-associated epitope on the MUC1 target antigen. In some embodiments, the first binding element comprises a CDR of an HMFG2 antibody. In some embodiments, the first binding element comprises a V H and V L In some embodiments, the first binding element comprises an HMFG2 single chain variable fragment (scFv).

[0154] In certain embodiments, the CAR is an H2 second-generation CAR, which comprises, from the extracellular to the intracellular domain, a MUC-1-targeting HMFG2 scFv, a CD28 transmembrane and costimulatory domain, and a CD3z signaling region. See Figure A. The H2 CAR is described in Wilkie et al., "Retargeting of human T cells to tumor-associated MUC1: the evolution of a chimeric antigen receptor," J. Immunol. 180:4901-9 (2008), which is incorporated herein by reference in its entirety.

[0155] In certain embodiments, the CAR is a T1E28z second generation CAR, which comprises, from the extracellular to the intracellular domain, an ErbB-targeting T1E peptide, a CD28 transmembrane and costimulatory domain, and a CD3z signaling region. See Figure A. The T1E28z second generation CAR is described in Davies, "Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells," Mol. Med. 18:565-576 (2012), which is incorporated herein by reference in its entirety.

[0156] In some embodiments, the second target antigen is selected from the group consisting of ErbB homodimers and heterodimers. In certain embodiments, the second target antigen is HER2. In certain embodiments, the second target antigen is EGF receptor. In some embodiments, the second binding element comprises a binding portion of T1E, ICR12, or ICR62.

[0157] In some embodiments, pCAR "TBB / H" or "I12BB / H" are expressed in immunoresponsive cells. These pCARs utilize a MUC1-targeting second generation "H" (synonymously, "H2") CAR, but the co-expressed CCR is different. The CCR in the TBB / H pCAR is fused to the CD8α transmembrane domain. T1E-binding domain and 4-1 BB It has a costimulatory domain. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) and is a promiscuous ErbB ligand. See Wingens et al., "Structural analysis of an epidermal growth factor / transforming growth factor-alpha chimera with unique ErbB binding specificity," J. Biol. Chem. 278:39114-23 (2003), and Davies et al., "Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells," Mol. Med. 18:565-576 (2012), the disclosures of which are incorporated herein by reference in their entirety. The CCR in I12BB / H pCAR is fused to the CD8α transmembrane domain. I CR 12 Binding domain and 4-1 BB It has a costimulatory domain. ICR12 is a HER2 (ErbB2)-targeting scFv domain. See Styles et al., "Rat monoclonal antibodies to the external domain of the product of the C-erbB-2 proto-oncogene," Int. J. Cancer 45(2):320-24 (1990), incorporated herein by reference in its entirety. In some embodiments, "TBB / H" or other pCARs described in PCT / GB2020 / 050590 (incorporated herein by reference in their entirety) can be used.

[0158] In some embodiments, ABB / H and I62BB / H pCARs are used. The CAR in both ABB / H and I62BB / H is a MUC1-targeting second generation "H" CAR. The CCR in ABB / H pCAR is fused to the CD8α transmembrane domain. A 20 peptides and 4-1 BB The A20 peptide binds to the αvβ6 integrin. See DiCara et al., "Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands," J Biol Chem. 282(13):9657-9665 (2007), which is incorporated herein by reference in its entirety. The CCR in I62BB / H pCAR is fused to the CD8α transmembrane domain. I CR 62 Binding domain and 4-1 BB ICR62 has a costimulatory domain. ICR62 is an EGFR-targeting scFv domain. See Modjtahedi et al., "Antitumor activity of combinations of antibodies directed against different epitopes on the extracellular domain of the human EGF receptor," Cell Biophys. 22(1-3):129-146 (1993), which is incorporated herein by reference in its entirety.

[0159] In some embodiments, the immunoresponsive cells express the modified pro-cytokine (e.g., modified pro-IL-18 or modified pro-IL-36), the optional expressed protease, and the optional CAR or pCAR from a single expression construct. In some embodiments, the immunoresponsive cells express the modified pro-cytokine (e.g., modified pro-IL-18 or modified pro-IL-36), the optional expressed protease, the CAR or pCAR from multiple separate constructs.

[0160] Signaling Area The CAR construct comprises a signaling region (i.e., a TCR-like signaling region). In some embodiments, the signaling region comprises an immunoreceptor tyrosine-based activation motif (ITAM), as reviewed, for example, by Love et al., "ITAM-mediated signaling by the T-cell antigen receptor," Cold Spring Harbor Perspect. Biol 2(6)1 a002485 (2010). In some embodiments, the signaling region comprises the intracellular domain of the human CD3 zeta chain, as described, for example, in U.S. Pat. No. 7,446,190 (incorporated herein by reference), or a variant thereof. In certain embodiments, the signaling region comprises the domain spanning amino acid residues 52-163 of the full-length human CD3 zeta chain. The CD3 zeta chain has multiple known polymorphic forms (e.g., sequence IDs: gb|AAF34793.1 and gb|AAA60394.1), all of which are useful herein and are set forth as SEQ ID NOS: 1 and 2, respectively:

[0161] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:1);

[0162] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2).

[0163] Alternative signaling regions to the CD3 zeta domain include, for example, FceR1γ, CD3ε, and multi-ITAM. Eshhar Z et al., "Specific activation and targeting of cytotoxic lymphocytes through chimeric single chains consisting of antibody-binding domains and the gamma or zeta subunits of the immunoglobulin and T-cell receptors" Proc Natl Acad Sci USA 90:720-724 (1993); Nolan et al., "Bypassing immunization: optimized design of 'designer T cells' against carcinoembryonic antigen(CEA)-expressing tumors, and lack of suppression by soluble CEA" Clin Cancer Res 5:3928-3941(1999); Zhao et al., "A herceptin-based chimeric antigen receptor with modified signaling domains leads to enhanced survival of transduced T lymphocytes and antitumor activity" J Immunol 183:5563-5574(2009); and James JR, "Tuning ITAM multiplicity on See "T cell receptors can control potency and selectivity to ligand density," Sci Signal 11(531)eaan1088 (2018), the disclosures of which are incorporated herein by reference in their entirety.

[0164] 4.3.5.2.3. Costimulatory Signaling Region In CARs, the costimulatory signaling region is appropriately positioned between the signaling region and the transmembrane domain, and separate from the binding element.

[0165] In CCRs, the costimulatory signaling region is appropriately positioned adjacent to the transmembrane domain and distant from the binding element.

[0166] Suitable costimulatory signaling regions are well known in the art and include costimulatory signaling regions of B7 / CD28 family members, e.g., B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2, or PDCD6; or ILT / CD85 family proteins, e.g., LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, or LILRB4; or tumor necrosis factor (TNF) superfamily members, e.g., 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, lymphotoxin-α, OX40, RELT, TACI, TL1A, TNF-α, or TNF RII; or a member of the SLAM family, e.g., 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A or SLAM; or a member of the TIM family, e.g., TIM-1, TIM-3 or TIM-4; or other costimulatory molecules, e.g., CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3 or TSLP R. Mondino A et al., "Surface proteins involved in T cell costimulation" J Leukoc Biol.55:805-815(1994);Thompson CB "Distinct roles for the costimulatory ligands B7-1 and B7-2 in T helper cell differentiation?" Cell.81:979-982(1995);Somoza C and Lanier LL "T-cell costimulation via "CD28-CD80 / CD86 and CD40-CD40 ligand interactions" Res Immunol. 146:171-176 (1995); Rhodes DA et al.,「Regulation of immunity by butyrophilins」Annu Rev Immunol.34:151-172(2016);Foell J et al.,「T cell costimulatory and inhibitory receptors as therapeutic targets for inducing anti-tumor immunity」Curr Cancer Drug Targets.7:55-70(2007);Greenwald RJ et al.,Annu Rev Immunol.,「The B7 family revisited」23:515-548(2005);Flem-Karlsen K et al.,「B7-H3 in cancer-beyond immune regulation」Trends Cancer.4:401-404(2018);Flies DB et al.,「The new B7s:playing a pivotal role in tumor immunity」J Immunother.30:251-260(2007);Gavrieli M et al.,「BTLA abd HVEM cross talk regulates inhibition and costimulation」Adv Immunol.92:157-185(2006);Zhu Y et al.,「B7-H5 costimulates human T cells via CD28H」Nat Commun.4:2043(2013);Omar HA et al.,「Tacking molecular targets beyond PD-1 / PD-L1:Novel approaches to boost patients’response to cancer immunotherapy」Crit Rev Oncol Hematol.135:21-29(2019);Hashemi M et al.,「Association of PDCD6 polymorphisms with the risk of cancer:Evidence from a meta-analysis」Oncotarget.9:24857-24868 (2018);Kang Immunol.23:23-68(2005);Bryceson YT et al., "Activation, coactivation, and costimulation of resting human natural killer cells" Immunol Rev.214:73-91(2006);Sharpe AH, "Analysis of lymphocyte costimulation in vivo using transgenic and 'knockout' mice" Curr Opin Immunol.7:389-395(1995);Wingren AG et al., “T cell activation See "Pathways: B7, LFA-3, and ICAM-1 Shape Unique T Cell Profiles," Crit Rev Immunol. 15:235-253 (1995), the disclosures of which are incorporated herein by reference in their entirety.

[0167] The costimulatory signaling region can be selected depending on the particular use intended for the immunoresponsive cell. In particular, the costimulatory signaling region can be selected to act additively or synergistically. In some embodiments, the costimulatory signaling region is selected from the costimulatory signaling regions of CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, and CD40.

[0168] In a particular embodiment, one costimulatory signaling region of the pCAR is the costimulatory signaling region of CD28 and the other is the costimulatory signaling region of 4-1BB.

[0169] 4.3.5.2.4. Transmembrane Domain The transmembrane domains of the CAR and CCR constructs may be the same or different. In a currently preferred embodiment, when the CAR and CCR constructs are expressed from a single vector, the transmembrane domains of the CAR and CCR are different to ensure the separation of the constructs on the cell surface. Because the inclusion of direct repeat nucleic acid sequences in viral vectors involves the deletion of sequences between the direct repeats, making them prone to rearrangement, selecting different transmembrane domains can also increase the stability of the expression vector. In embodiments where the transmembrane domains of the CAR and CCR of pCAR are selected to be identical, this risk can be reduced by modifying or "wobbling" the selected codons to encode the same protein sequence.

[0170] Suitable transmembrane domains are known in the art, and include, for example, CD8α, CD28, CD4, or CD3z transmembrane domains. Selecting CD3z as the transmembrane domain may result in the association of CAR or CCR with other elements of the TCR / CD3 complex. This association may recruit more ITAMs, but may also result in competition between CAR / CCR and endogenous TCR / CD3.

[0171] 4.3.5.2.5. Costimulatory Signaling Domains and Transmembrane Domains In embodiments where the costimulatory signaling region of a CAR or CCR is or includes the costimulatory signaling region of CD28, the CD28 transmembrane domain represents a suitable, and often preferred, choice for the transmembrane domain. The full-length CD28 protein is a 220 amino acid protein of SEQ ID NO: 3, with the transmembrane domain shown in bold: JPEG2025161810000001.jpg34166

[0172] In some embodiments, one of the costimulatory signaling regions is based on the hinge region, and suitably also the transmembrane domain and endodomain of CD28. In some embodiments, the costimulatory signaling region comprises amino acids 114-220 of SEQ ID NO:3, shown below as SEQ ID NO:4: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 4).

[0173] In certain embodiments, one of the costimulatory signaling regions is a modified version of SEQ ID NO:4, which includes a c-myc tag of SEQ ID NO:5: EQKLISEEDL (SEQ ID NO: 5).

[0174] The c-myc tag may be added to the costimulatory signaling region by insertion within the ectodomain or by substitution of a region within the ectodomain, thus within the region of amino acids 1-152 of SEQ ID NO:3.

[0175] In a particularly preferred embodiment, the c-myc tag replaces the MYPPPY motif in the CD28 sequence. This motif represents a potentially dangerous sequence. It is responsible for the interaction between CD28 and its natural ligands (CD80 and CD86), and therefore provides the potential for off-target toxicity when CAR-T cells or pCAR-T cells encounter target cells expressing either of these ligands. By replacing this motif with a tag sequence as described above, the potential for unwanted side effects is reduced. Thus, in a specific embodiment, the costimulatory signaling region of the CAR construct comprises SEQ ID NO:6: IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 6).

[0176] Furthermore, the inclusion of the c-myc epitope facilitates detection of pCAR-T cells using monoclonal antibodies against the c-myc epitope, which is extremely useful since flow cytometry detection has proven unreliable with some available antibodies.

[0177] Furthermore, provision of a c-myc epitope tag can facilitate antigen-independent proliferation of targeted CAR-T cells, for example, by cross-linking the CAR with a suitable monoclonal antibody in solution or immobilized on a solid phase (e.g., a bag).

[0178] Furthermore, expression of an epitope for the anti-human c-myc antibody (9e10) within the variable region of the TCR has previously been shown to be sufficient to enable antibody- and complement-mediated cytotoxicity both in vitro and in vivo (Kieback et al. Proc. Natl. Acad. Sci. USA "A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer" 105(2)623-8(2008)). Thus, provision of such an epitope tag can also be used as a "suicide system," whereby the antibody can be used to deplete pCAR-T cells in vivo in the event of toxicity.

[0179] Combined Elements The binding elements of the CAR and CCR constructs of pCAR bind to a first epitope and a second epitope, respectively.

[0180] In an exemplary embodiment, the binding elements of the CAR and CCR constructs are different from each other.

[0181] In various embodiments, the binding elements of the CAR and CCR specifically bind to a first epitope and a second epitope of the same antigen. In certain of these embodiments, the binding elements of the CAR and CCR specifically bind to the same, overlapping, or different epitopes of the same antigen. In embodiments in which the first and second epitopes are the same or overlapping, the binding elements on the CAR and CCR can compete for their binding.

[0182] In various embodiments, the binding elements of the CAR and CCR constructs of the pCAR bind to different antigens. In certain embodiments, the antigens may be different but associated with the same disease (e.g., the same specific cancer).

[0183] Thus, a suitable binding element can be any element that provides the pCAR with the ability to recognize a target of interest. The target to which the pCAR of the present invention is directed can be any target of clinical interest to which it is desirable to direct a T cell response.

[0184] In various embodiments, the binding elements used in the CARs and CCRs of the pCARs described herein are antibody antigen binding sites (ABS). In exemplary embodiments, the ABS used as binding elements are formatted into single-chain antibodies (scFv) or are single-domain antibodies derived from camelids, humans, or other species.

[0185] Alternatively, the binding element of pCAR may comprise a ligand that binds to a surface protein of interest.

[0186] In some embodiments, the binding element is associated with a leader (signal peptide) sequence that facilitates expression on the cell surface. Many leader sequences are known in the art, including, but not limited to, the CD8α leader sequence, the immunoglobulin kappa light chain sequence, the macrophage colony-stimulating factor receptor (FMS) leader sequence, or the CD124 leader sequence.

[0187] MUC1 pCAR In certain embodiments, at least one of the binding elements specifically interacts with an epitope on the MUC1 target antigen. In some embodiments, the binding element of the CAR specifically interacts with an epitope on the MUC1 antigen. In some embodiments, the binding element of the CCR specifically interacts with an epitope on the MUC1 target antigen or with an alternative tumor-associated molecule, such as an NKG2D ligand, αvβ6 integrin, or ErbB homo- or heterodimer. In certain embodiments, the binding element of the CAR specifically interacts with an epitope on the MUC1 antigen and the binding element of the CCR specifically interact with the same, overlapping, or different epitopes on the MUC1 target antigen.

[0188] In currently preferred embodiments, the binding element of the CAR specifically interacts with a first epitope on the MUC1 target antigen. In some embodiments, the CAR binding element comprises the antigen-binding site of the HMFG2 antibody. In certain embodiments, the CAR binding element comprises the CDRs of the HMFG2 antibody. The CDR sequences of the HMFG2 antibody were determined using tools provided at www.abysis.org and are shown below as SEQ ID NOS: 8-13:

[0189] VH CDR1 GFTFSNY (SEQ ID NO: 8); VH CDR2 RLKSNNYA (SEQ ID NO: 9); VH CDR3 GNSFAY (SEQ ID NO: 10); VL CDR1 RSSTGAVTTSNYAN (SEQ ID NO: 11); VL CDR2 GTNNRAP (SEQ ID NO: 12); VL CDR3 ALWYSNHWV (SEQ ID NO: 13).

[0190] In certain embodiments, the CAR binding element is a V H and V L V domain of HMFG2 antibody H and V L The domain sequences are shown below as SEQ ID NOs: 14-15:

[0191] EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSS (SEQ ID NO: 14)

[0192] QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE (SEQ ID NO: 15).

[0193] In a particularly preferred embodiment, the CAR binding element is H -Spacer-V L or V L -Spacer V H In certain embodiments, the amino acid sequence of the scFv of the HMGF2 antibody is 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identical to SEQ ID NO: 16 shown below: EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGS GGGGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE (SEQ ID NO: 16).

[0194] In one particular embodiment, the nucleic acid encoding the scFv of the HMGF2 antibody is SEQ ID NO: 17, shown below: GAGGTGCAGCTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCCGTGGTCACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGATCAGAG (SEQ ID NO: 17).

[0195] In some embodiments, the CCR binding element is ICR12, which binds to HER2. See Styles et al., "Rat monoclonal antibodies to the external domain of the product of the C-erbB-2 proto-oncogene," Int. J. Cancer 45(2):320-24 (1990), incorporated herein by reference in its entirety. In some embodiments, the CCR binding element is ICR62, which binds to EGFR. See Modjtahedi et al., "Antitumor activity of combinations of antibodies directed against different epitopes on the extracellular domain of the human EGF receptor," Cell Biophys. 22(1-3):129-46 (1993), incorporated herein by reference in its entirety. In some embodiments, the CCR binding element is the A20 peptide, which binds to αvβ6 integrin. See DiCara et al., "Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands," J Biol Chem. 282(13):9657-9665 (2007), incorporated herein by reference in its entirety.

[0196] In some embodiments, the CCR binding element is a T1E peptide that binds to ErbB homo- and heterodimers. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF) and is a promiscuous ErbB ligand. The T1E peptide is a chimeric fusion protein consisting of the entire mature human EGF protein, except for the five most N-terminal amino acids (amino acids 971-975 of the pro-epidermal growth factor precursor (NP001954.2)), which are replaced with the seven most N-terminal amino acids of the mature human TGF-α protein (amino acids 40-46 of the pro-transforming growth factor α isoform 1 (NP003227.1)). See Wingens et al., "Structural analysis of an epidermal growth factor / transforming growth factor-alpha chimera with unique ErbB binding specificity," J. Biol. Chem. 278:39114-23 (2003), and Davies et al., "Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells," Mol. Med. 18:565-576 (2012), the disclosures of which are incorporated herein by reference in their entireties. The sequence of T1E is shown below as SEQ ID NO: 18: VVSHFNDCPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 18).

[0197] In one particular embodiment, the nucleic acid encoding the T1E sequence is SEQ ID NO: 19, shown below: GTGGTGAGCCACTTCAACGACTGCCCTCTGAGCCACGACGGCTACTGCCTGCACGACGGCGTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAACTGCGTGGTGGGCTACATCGGCGAGAGATGCCAGTACAGAGACCTGAAGTGGTGGGAGCTGAGA (SEQ ID NO: 19).

[0198] The protein sequence of TBB / H pCAR is shown below as SEQ ID NO: 7. TBB / H pCAR comprises a CCR containing a T1E binding domain and a 4-1 BB costimulatory domain ("TBB") fused to a CD8α spacer and transmembrane domain, and a second-generation CAR containing a human MUC1-targeting HMFG2 domain ("H"). The CCR and CAR are linked by a furin cleavage site, a Ser-Gly linker (SGSG), and a T2A ribosomal skipping peptide. The VH and VL sequences of the HMFG2 sequence are underlined and in bold: JPEG2025161810000002.jpg96166

[0199] In some embodiments, one of the binding elements of pCAR is specific for a marker associated with various types of cancer, including, for example, one or more ErbB homodimers or heterodimers, such as EGFR and HER2. In some embodiments, the binding element binds to a marker associated with prostate cancer (e.g., using a binding element that binds to prostate-specific membrane antigen (PSMA)), breast cancer (e.g., using a binding element that targets HER2 (also known as ErbB2)), or neuroblastoma (e.g., using a binding element that targets GD2), melanoma, small cell or non-small cell lung carcinoma, sarcoma, brain tumor, ovarian cancer, pancreatic cancer, colorectal cancer, gastric cancer, bladder cancer, myeloma, non-Hodgkin's lymphoma, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, or renal cell carcinoma.

[0200] 4.3.5.3. Chimeric Cytokine Receptors In a further series of embodiments, cells expressing a CAR and a CCR are engineered to co-express a chimeric cytokine receptor, particularly a 4αβ chimeric cytokine receptor (Figure 1). In 4αβ, the ectodomain of the IL-4 receptor-α chain is linked to the transmembrane and endodomain of the IL-2 / 15 receptor-β. This allows for selective expansion and enrichment of genetically engineered T cells ex vivo by culturing these cells in an appropriate support medium, which in the case of 4αβ contains IL-4 as the only cytokine support. See Wilkie et al., "Selective expansion of chimeric antigen receptor-targeted T-cells with potent effector function using interleukin-4," J. Biol. Chem. 285(33):25538-44 (2010) and Schalkwyk et al., "Design of a Phase 1 clinical trial to evaluate intratumoural delivery of ErbB-targeted chimeric antigen receptor T-cells in locally advanced or recurrent head and neck cancer," Human Gene Ther. Clin. Devel. 24:134-142 (2013), which are incorporated herein by reference in their entireties.

[0201] Similarly, this system can be used with chimeric cytokine receptors in which the ectodomain of the IL-4 receptor-α chain is linked to the transmembrane and endodomain of another receptor to which the cytokine naturally binds, also binding to the common γ chain.

[0202] 4.3.6. Modified TCR In some embodiments, the immunoresponsive cells are modified to further express an altered (non-native) T cell receptor (TCR).

[0203] Modified TCRs that can be usefully expressed in the immunoresponsive cells described herein are described in U.S. Patent Nos. 9,512,197; 9,822,163; and 10,344,074, the disclosures of which are incorporated herein by reference in their entireties. Modified TCRs that can be usefully expressed in the immunoresponsive cells described herein are described in U.S. Pregrant Publication Nos. 2019 / 0161528; 2019 / 0144521; 2019 / 0135892; 2019 / 0127436; 2018 / 0218043; 2017 / 0088599; 2016 / 0159771; and 2016 / 0137715, the disclosures of which are incorporated herein by reference in their entireties.

[0204] 4.3.7. Nucleic Acids and Methods for Producing pCAR-T Cells Also provided herein is a polynucleotide or set of polynucleotides comprising a first nucleic acid encoding a modified pro-cytokine, wherein the modified pro-cytokine comprises, from N-terminus to C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a cytokine fragment. The cleavage site is a specific sequence recognized by the protease.

[0205] In some embodiments, the first nucleic acid encodes a modified pro-IL-18, wherein the modified pro-IL-18 comprises, from N-terminus to C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1; and (c) an IL-18 fragment. The cleavage site is a specific sequence recognized by the protease. In some embodiments, the cleavage site is downstream, upstream, or at a predetermined position of the caspase-1 recognition site of pro-IL-18. In some embodiments, the cleavage site is followed by a stop codon. The cleavage site in the modified pro-IL-18 can be selected from a variety of protease cleavage sites known in the art. For example, the cleavage site can be recognized by granzyme B (GzB), caspase-3, caspase-8, MT1-MMP (MMP14), alternative tumor-associated matrix metalloproteinase (MMP1-13), a disintegrin and metalloproteinase (ADAM) family member (e.g., ADAM10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-related peptidase (KLK), e.g., KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, hepsin, or urokinase plasminogen activator (Dudani et al., "Harnessing protease activity to improve cancer care," Annu. Rev. Cancer Biol., 2:353-76 (2018). In some embodiments, the cleavage site comprises a sequence selected from SEQ ID NOs: 26, 28, 30, and 32. In some embodiments, the modified pro-IL-18 comprises a polypeptide of a sequence selected from SEQ ID NOs: 27, 29, 31, and 33. In certain embodiments, the modified pro-IL-18 comprises a polypeptide of the sequence of SEQ ID NO: 27.

[0206] In some embodiments, the first nucleic acid is selected from the group consisting of SEQ ID NOs: 102, 103, 105, 107, 109, 111, and 113. In particular embodiments, the first nucleic acid comprises the polynucleotide of SEQ ID NO: 103. In some embodiments, the first nucleic acid is a coding sequence cloned into an expression vector, e.g., a viral or non-viral vector.

[0207] Alternatively, the modified pro-cytokine is a modified pro-IL-36α, β, or γ protein, wherein the modified pro-IL-36 comprises, from N-terminus to C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than cathepsin G, elastase, and proteinase 3; and (c) an IL-36 fragment. The cleavage site is a specific sequence recognized by a protease. In some embodiments, the cleavage site is downstream, upstream, or at a predetermined position of the cathepsin G, elastase, and / or proteinase 3 recognition site of pro-IL-36α, β, or γ. In some embodiments, the cleavage site is followed by a stop codon. The cleavage site within the modified pro-IL-36 can be selected from a variety of protease cleavage sites known in the art. For example, the cleavage site can be recognized by granzyme B (GzB), caspase-3, caspase-8, MT1-MMP (MMP14), alternative tumor-associated matrix metalloproteinase (MMP1-13), a disintegrin and metalloproteinase (ADAM) family member (e.g., ADAM10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-related peptidase (KLK), e.g., KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, hepsin, or urokinase plasminogen activator (Dudani et al., "Harnessing protease activity to improve cancer care," Annu. Rev. Cancer Biol., 2:353-76 (2018). In some embodiments, the cleavage site comprises a sequence selected from SEQ ID NOs: 26, 28, 30, and 32. In some embodiments, the modified pro-IL-36 α, β, and γ comprise a polypeptide of a sequence selected from SEQ ID NOs: 37, 39, and 41, respectively.

[0208] In some embodiments, the polynucleotide or set of polynucleotides further comprises a second nucleic acid encoding a protease that recognizes a cleavage site on the first nucleic acid. The protease can be granzyme B (GzB), caspase-3, caspase-8, MT1-MMP (MMP14), alternative tumor-associated matrix metalloproteinase (MMP1-13), a disintegrin and metalloproteinase (ADAM) family member (particularly ADAM10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-related peptidase (KLK), e.g., KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, hepsin, or urokinase plasminogen activator (see Dudani et al., "Harnessing protease activity to improve cancer care," Annu. Rev. Cancer Biol., 2:353-76 (2018)). In some embodiments, the first nucleic acid and the second nucleic acid are in a single vector or two different vectors.

[0209] In some embodiments, the polynucleotide or set of polynucleotides further comprises a third nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR is a second-generation CAR as described above, and comprises: (a) a signaling region; (b) a first costimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen.

[0210] In some embodiments, the polynucleotide or set of polynucleotides further comprises a fourth nucleic acid encoding the above-mentioned CCR. In some embodiments, the CCR comprises (a) a second costimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0211] As mentioned above, for convenience, the combination of CAR and CCR is referred to herein as pCAR in the singular, although the CAR and CCR are separate co-expressed proteins. The third and fourth nucleic acids can be expressed from a single vector or from two or more vectors. The appropriate sequence for the nucleic acid will be apparent to those skilled in the art based on the above description of CAR and CCR. The sequence may be optimized for use in the required immunoresponsive cells. However, in some cases, as mentioned above, the codons may be suboptimal or "wobbled" to avoid repeat sequences. Specific examples of such nucleic acids encode the preferred embodiments described above.

[0212] To achieve transduction, the nucleic acid encoding pCAR is suitably introduced into one or more vectors, such as a plasmid or a retroviral or lentiviral vector. Such vectors, including plasmid vectors, or cell lines containing them, form further aspects of the present invention.

[0213] In typical embodiments, immunoresponsive cells are subjected to genetic modification, e.g., by retroviral or lentiviral-mediated transduction, to introduce the first, second, third, and / or fourth nucleic acids into the host T cell genome, thereby enabling stable expression of a modified pro-cytokine (e.g., modified pro-IL-18 or modified pro-IL-36), a protease, a CAR, and / or a CCR, respectively. The first, second, third, and / or fourth nucleic acids can be introduced as a single vector or as multiple vectors, each containing one or more nucleic acids. They can then be reintroduced into the patient (possibly after expansion) to provide a beneficial therapeutic effect, as described below.

[0214] In some embodiments, the immunoresponsive cells are γδ T cells, and the γδ T cells are activated with an anti-γδ TCR antibody prior to genetic modification, hi some embodiments, an immobilized anti-γδ TCR antibody is used for activation.

[0215] The first and second nucleic acids encoding the modified pro-cytokine (e.g., modified pro-IL-18 or modified pro-IL-36) and the protease can be expressed from the same vector or multiple vectors. The third and fourth nucleic acids encoding the CAR and CCR can be expressed from the same vector or multiple vectors. In one embodiment, the first, second, third, and fourth nucleic acids are expressed from the same vector. The vector or multiple vectors comprising them can be combined in a kit provided to produce the immunoresponsive cell of the first aspect disclosed herein.

[0216] In some embodiments in which T cells are engineered to co-express a chimeric cytokine receptor, such as 4αβ, the expansion step may include ex vivo culture in a medium containing the cytokine (e.g., a medium containing IL-4 as the sole cytokine support in the case of 4αβ). Alternatively, the chimeric cytokine receptor may comprise the ectodomain of the IL-4 receptor-α chain linked to an endodomain used by a common gamma cytokine with distinct properties, such as IL-7. Growth of cells in IL-4 may result in less cell differentiation than use of IL-7. In this way, selective expansion and enrichment of genetically engineered T cells with the desired differentiation state can be ensured.

[0217] 4.4. Treatment Method As described above, immunoresponsive cells expressing modified pro-cytokines (e.g., modified pro-IL-18 or modified IL-36) are useful in therapy for directing a T cell-mediated immune response to target cells with reduced immunosuppression. Accordingly, in another aspect, a method is provided for directing a T cell-mediated immune response to target cells in a patient in need thereof. The method comprises administering to the patient a population of immunoresponsive cells as described above, wherein the binding element is specific for the target cells. In an exemplary embodiment, the target cells express MUC1.

[0218] In another aspect, a method for treating cancer in a patient in need thereof is provided. The method comprises administering to the patient a population of immunoresponsive cells as described above, wherein the binding element is specific for the target cell. In an exemplary embodiment, the target cell expresses MUC1. In various embodiments, the patient has breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, myeloma, non-Hodgkin's lymphoma, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, or renal cell carcinoma. In some embodiments, the patient has breast cancer.

[0219] In various embodiments, a therapeutically effective number of immunoresponsive cells are administered to a patient. In certain embodiments, the immunoresponsive cells are administered by intravenous infusion. In certain embodiments, the immunoresponsive cells are administered by intratumoral injection. In certain embodiments, the immunoresponsive cells are administered by peritumoral injection. In certain embodiments, the immunoresponsive cells are administered by intraperitoneal injection. In certain embodiments, the immunoresponsive cells are administered by multiple routes selected from intravenous infusion, intratumoral injection, and peritumoral injection.

[0220] In another aspect, the present disclosure provides immunoresponsive cells, polynucleotides, or γδ T cells for use in therapy or as a medicament. The present disclosure further provides immunoresponsive cells, polynucleotides, or γδ T cells for use in treating a pathological disorder. The present disclosure also provides the use of immunoresponsive cells, polynucleotides, or γδ T cells in the manufacture of a medicament for treating a pathological disorder. In some embodiments, the pathological disorder is cancer. [Example]

[0221] 5. Working Example Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0222] 5.1. Method Cell line culture All tumor cells and 293T cells were grown in DMEM supplemented with L-glutamine and 10% FBS (D10 medium). Where indicated, tumor cells were transduced to express the firefly luciferase-tdTomato (LT) SFG vector, followed by fluorescence-activated cell sorting (FACS) for red fluorescent protein (RFP) expression. MDA-MB-468-HER2 ++ Cells were produced by transduction of MDA-MB-468-LT cells with an SFG retroviral vector encoding human HER2. Transduced cells were FACS sorted using ICR12 rat anti-human HER2 antibody and goat anti-rat PE.

[0223] Retrovirus production 293T cells were triple transfected in GeneJuice (MilliporeSigma, Merck KGaA, Darmstadt, Germany) with (i) SFG retroviral vectors encoding modified pro-IL-18, protease, and / or CAR / pCAR as indicated, (ii) RDF plasmid encoding RD114 envelope, and (iii) Peq-Pam plasmid encoding gag-pol, as recommended by the manufacturer. 1.5 × 10 cells in 100 mm plates were transfected. 6For transfection of 293T cells, 4.6875 μg of SFG retroviral vector, 4.6875 μg of Peq-Pam plasmid, and 3.125 μg of RDF plasmid were used. Media containing viral vectors was collected 48 and 72 hours posttransfection, flash-frozen, and stored at -80°C. In some cases, stable packaging cell lines were generated by transduction of 293VEC GALV cells with transiently produced retroviral vectors encoding modified pro-IL-18, protease, and / or CAR / pCAR. Viruses prepared from either source were used interchangeably for transduction of target cells.

[0224] αβ T cell culture and transduction Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood samples by density gradient centrifugation using Ficoll-Paque (ethical approval number 18 / WS / 0047). T cells were cultured in RPMI containing GlutaMax supplemented with 5% human AB serum. T cell activation was achieved by culturing for 24–48 hours in the presence of 5 μg / mL phytohemagglutinin leukoagglutinin (PHA-L). Cells were then expanded in IL-2 (100 U / mL) for an additional 24 hours before transduction. T cell transduction was achieved using RetroNectin (Takara Bio)-coated plates according to the manufacturer's protocol. Activated PBMCs (1 × 10) were cultured per well of a RetroNectin-coated 6-well plate. 6 Then, 3 mL of medium containing the retrovirus was added per well along with 100 U / mL of IL-2.

[0225] γδ T cell expansion and transduction To generate γδ T cells, 9 × 10 cells were cultured per well in a 6-well plate coated with 2.4 μg of activating anti-γ / δ-1 TCR antibody (BD biosciences) per well. 6PBMCs were activated. After 24 hours, the cells were expanded for an additional 48 hours in 100 U / mL IL-2 and 5 ng / mL TGF-β. 3 × 10 cells were injected per well of a RetroNectin-coated 6-well plate pre-coated with 3 mL of retrovirus-containing medium. 6 Activated PBMCs were added. Cells were grown for 14 days in 100 U / mL IL-2 and 5 ng / mL TGF-β (R&D Systems). Fold expansion compared to the starting number of PBMCs was calculated.

[0226] Cytotoxicity assay MDA-MB-468 or BxPC-3 tumor cells were cultured at 1 × 10 4 Cells were seeded at densities of 1000 cells / well and incubated with T cells for 72 hours at effector:target ratios ranging from 4 to 0.03 (see Figures 3A-3D). Destruction of tumor cell monolayers by T cells was quantified using an MTT assay. MTT (Sigma) was added at 500 μg / ml in D10 medium for 2 hours at 37°C and 5% CO2. After removal of the supernatant, formazan crystals were resuspended in 100 μL of DMSO. Absorbance was measured at 560 nm. Tumor cell viability was calculated as (absorbance of monolayers cultured with T cells / absorbance of untreated monolayers only) × 100%.

[0227] Detection of IFN-γ and IL-2 Supernatants were collected from the cocultures of CAR-T / pCAR-T cells and MDA-MB-468 tumor cells described above at 24 hours. Cytokine levels were quantified using human IFN-γ (Bio-Techne) or human IL-2 ELISA kits (Invitrogen) according to the manufacturer's protocol. Data represent the mean ± SEM cytokines detected from six independent experiments (each performed in duplicate wells).

[0228] Detection of active human IL-18 T cells were harvested, washed, and cultured for 48 hours in the absence of stimuli or cytokines. T cells were then stimulated for 24 hours with either a 10:1 effector:tumor ratio or a 200:1 T cell:anti-CD3 / 28 bead ratio. Supernatants were then harvested and plated at 5x10 cells / well in 96-well plates. 4 The cells were cultured with 1000 HEK blue IL-18 cells / well for 24 hours, after which 20 μl of supernatant was removed from the co-culture and added to 180 μl of QUANTI-Blue solution, and the absorbance was measured at 620-650 nm.

[0229] Repeated antigen stimulation assay MDA-MB-468 tumor cells were co-cultured with CAR-T / pCAR-T cells at an initial effector:target ratio of 1 CAR-T / pCAR-T cell:1 tumor cell or 1 CCR+ / γδ TCR+ T cell:1 tumor cell for 72–96 hours. All T cells were then removed, centrifuged at 400 g for 5 minutes, resuspended in 3 ml of fresh RPMI supplemented with GlutaMax and 5% human serum, and added to a fresh tumor cell monolayer. Remaining tumor cell viability was assessed by MTT assay after each co-culture. If >20% (or >30% for γδ T cells) of tumor cells were killed compared to untreated cells, T cells were added to a fresh tumor cell monolayer. Data represent the mean ± SEM of the number of rounds of antigen stimulation. Cell counts were performed by pooling triplicate wells and counting the total number of cells.

[0230] Alternatively, tumor cell lines were plated in triplicate at 1 x 10 cells per well in 24-well culture plates. 5Cells were plated 24 hours before the addition of T cells. CAR-T / pCAR-T cells were added at a 1:1 effector:target ratio. Tumor cell killing was measured 72 hours later using a luciferase assay (D-luciferin (PerkinElmer) was added at 150 mg / mL immediately before luminescence reading). If >20% tumor cell death compared to untreated cells was observed, all T cells were restimulated by adding them to a new tumor cell monolayer. Tumor cell viability was calculated as (luminescence of monolayer cultured with T cells / luminescence of untreated monolayer only) × 100%.

[0231] In vivo testing PBMCs from healthy donors were either engineered to express the indicated CAR / pCAR or left untransduced. After 11 days (αβ T cells) or 14 days (γδ T cells) of expansion in IL-2 (100 U / mL, added every 2–3 days) or IL-2 + TGF-β, cells were analyzed by flow cytometry for expression of CCR or CCR and γδ TCR.

[0232] Female severe combined immunodeficiency (SCID) Beige mice were given 1 × 10 6 MDA-MB-468 LT cells were injected into the mice (Figure 13). Twelve days after tumor cell injection, mice received 10x10 6 CCR-positive or CCR, γδ TCR double-positive (or untransduced) T cells were injected i.p., or PBS alone was injected as a control. Tumor status was monitored by bioluminescence imaging performed under isoflurane anesthesia 20 minutes after injection of StayBrite™ D-luciferin, potassium salt (150 mg / kg) in 200 μl of PBS. Images were acquired at the indicated time points using an IVIS® Lumina III (PerkinElmer) with Living Image software (PerkinElmer) set for automatically optimized exposure time, binning, and F / stop. Animals were humanely killed when the experimental endpoint was reached.

[0233] Female NOD SCID gamma null (NSG) mice via the intraperitoneal (i.p.) route, 0.5 × 10 6 SKOV3 ovarian cancer cells were injected into the mice (Figure 15). 18 days after tumor cell injection, the mice were each injected with 0.5 x 10 cells in 200 μl of PBS. 6 CAR T cells were injected i.p. Tumor status was monitored by bioluminescence imaging as described above. Animals were humanely killed when the experimental endpoint was reached.

[0234] Female NSG mice received 1 × 10 5 10 x 10 BxPC-3 LT cells were injected into the mice. Nine days after tumor cell injection, 10 x 10 BxPC-3 LT cells were injected into the mice in 200 μl of PBS. 6 Mice were injected i.p. with CCR / γδ TCR double-positive (or untransduced) T cells or PBS alone as a control. Tumor status was monitored by bioluminescence imaging as described above. Animals were humanely killed when the experimental endpoint was reached.

[0235] 5.2. Example 1: Generation of IL-18-expressing CAR / pCAR T cells The vector containing the coding sequence of TBB / H pCAR (SEQ ID NO: 7) described above was modified to further contain the coding sequences of various human IL-18 constructs.

[0236] A construct encoding TBB / H and pro-IL-18 (FIG. 18; SEQ ID NO: 102) was produced by inserting a synthetic polynucleotide (SEQ ID NO: 101) into the unique Kfl and Xhol restriction sites in the TBB / H vector, replacing the 224-bp fragment between the Kfl and Xhol restriction sites. The insertion site for the pro-IL-18 sequence is downstream of the second wobbling T2A and is followed by a stop codon. Because cleavage of the propeptide requires caspase-1, which is not expressed in T cells, this construct is predicted not to express active IL-18 in T cells.

[0237] A construct encoding TBB / H and modified pro-IL-18 (pro-IL-18(GzB)) (FIG. 19; SEQ ID NO: 103) was synthesized by substituting GAC GAC GAG AAC CTG GAG AGC GAC TAC (SEQ ID NO: 34) of MUC1-13 with GAC GAC GAG AAC A T C GAG CC C GAC TAC (SEQ ID NO: 35; changes are underlined). This modified pro-IL-18 replaces the native caspase-1 cleavage site between the IL-18 propeptide and the mature IL-18 protein (LESD) with a granzyme B (GzB) cleavage site (IEPD).

[0238] A construct encoding TBB / H and constitutive IL-18 (Figure 20; SEQ ID NO: 105) was produced by inserting a synthetic polynucleotide (SEQ ID NO: 104) into the unique Kfl and Xhol restriction sites in the TBB / H vector, replacing the 224 bp fragment between the Kfl and Xhol restriction sites. The IL-18 insertion site is downstream of the CD4 leader and followed by a stop codon. The IL-18 insert encodes mature IL-18 protein without the IL-18 propeptide. This construct is predicted to express constitutively active IL-18 protein in T cells.

[0239] A construct encoding TBB / H and modified pro-IL-18 (pro-IL-18(casp8)) (FIG. 19; SEQ ID NO: 107) was produced by inserting a synthetic polynucleotide (SEQ ID NO: 106) into the unique Kfl and Xhol restriction sites in the TBB / H construct, replacing the 224-bp fragment between the Kfl and Xhol restriction sites. The insertion site for the modified pro-IL-18 sequence is downstream of the second wobbling T2A and is followed by a stop codon. This modified proIL-18 replaces the native caspase-1 cleavage site between the IL-18 propeptide and the mature IL-18 protein (LESD) with a caspase-8 cleavage site (IETD).

[0240] A construct encoding TBB / H and modified pro-IL-18 (pro-IL-18(casp3)) (FIG. 22; SEQ ID NO: 109) was produced by inserting a synthetic polynucleotide (SEQ ID NO: 108) into the unique Kfl1 and Xho1 restriction sites in the TBB / H construct, replacing the 224-bp fragment that had been removed. The insertion site for the modified pro-IL-18 sequence is downstream of the second wobbling T2A and is followed by a stop codon. Modified pro-IL-18 replaces the native caspase-1 cleavage site between the propeptide and the mature protein with a caspase-3 cleavage site (DEVD).

[0241] A construct encoding TBB / H with modified pro-IL-18 (GzB) and additional granzyme B (Figure 23; SEQ ID NO: 111) was produced by inserting a synthetic polynucleotide (SEQ ID NO: 110) into the unique Ale1 and Xho1 restriction sites in the TBB / H GzB Pfn construct (encoding granzyme B, perforin, and TBBH; SEQ ID NO: 112) to replace the removed 1,788 bp fragment.

[0242] A construct (SEQ ID NO: 113) encoding T4 and modified pro-IL-18 (MT1-MMP) was produced by inserting a synthetic polynucleotide (SEQ ID NO: 32) of the MT1-MMP cleavage site into the caspase-1 site of pro-IL-18 (Figures 16 and 24).

[0243] SFG retroviral vectors containing the coding sequences of the constructs were produced as described above and then transduced into PBMCs. T cells were expanded from PBMCs in the presence of IL-2 as described above. T cells expressed modified pro-IL-18. IL-18 activity depended on the expression in T cells of a protease that recognized the cleavage site in the modified pro-IL-18.

[0244] 5.3. Example 2: In vitro antitumor activity of pCAR T cells armoured with IL-18 T cells transfected with SFG retroviral vectors encoding TBB / H pCAR and one of the IL-18 variants described in Example 1 were analyzed for expression of the IL-18 variant (FIG. 4A) and pCAR using flow cytometry to separately measure expression of the H28z CAR (H-2) and TIE-4-1BB CCR (FIG. 3). The results provided show that the majority of transduced T cells express both components of TBB / H pCAR.

[0245] IL-18 secretion by transfected T cells was analyzed by ELISA (Fig. 4A), and the functional activity of expressed IL-18 was tested by reporter assay (Fig. 4B) (a commercially available reporter cell line was used to detect functional IL-18 (i.e., active IL-18 fragments produced after propeptide cleavage)).

[0246] IL-18 secretion (Figure 4A) was detected in naive T cells modified by retroviral transduction to express each of the IL-18 mutants tested: (native) pro-IL-18; constit IL-18; pro-IL-18(casp8); and pro-IL-18(casp3). However, IL-18 activity was detected only in T cells transduced with the constitutive mutant ("constit IL-18"), in which the mature IL-18 fragment was placed downstream of the CD4 signal peptide (Figure 4B). Active IL-18 was not detected in conditioned medium produced by unstimulated pCAR T cells expressing pro-IL-18 or modified pro-IL-18 in which the cleavage site has been altered to be recognized by caspase-3 (pro-IL-18(casp3)) or caspase-8 (pro-IL-18(casp8)).

[0247] T cells co-expressing TBB / H pCAR and each IL-18 variant were co-cultured in vitro with MDA-MB-468 breast cancer cells for 72 hours. The effector:target (modified T cell:tumor cell) ratio ranged from 4 to 0 (including 4, 2, 1, 0.5, 0.25, 0.125, 0.06, and 0.03). The remaining viable cancer cells present after the end of co-culture were quantified by MTT assay. The percentage of viable MDA-MB-468 breast cancer cells after co-culture with pCAR-T cells is shown in Figures 5A-5D. MDA-MB-468 breast cancer cells express dimers of MUC-1 and ErbB and very low levels of HER2. As shown in Figures 5A-5D, T cells expressing TBB / H pCAR and each IL-18 mutant exhibited higher cytotoxic antitumor activity at effector:target ratios of 4 and 2 compared with effector:target ratios of 1 or 0.5. No clear differences were detected between T cells expressing different IL-18 mutants.

[0248] T cells expressing TBB / H pCAR and IL-18 mutants were then transfected with MUC1 +These CAR T cells were subjected to repeated restimulation with MDA-MB-468 breast cancer cells (Figures 6A-6B). Constitutive expression of active IL-18 fragments allowed pCAR T cells to undergo more restimulation cycles while maintaining cytotoxic activity, whereas this was not observed with pro-IL-18 or caspase-3-cleavable (pro-IL-18(casp3)) or caspase-8-cleavable (pro-IL-18(casp8)) derivatives. Constitutive IL-18, but not pro-IL-18 or caspase-3 / 8-cleavable derivatives, mediated a significant increase in CAR T cell proliferation (Figure 6A). Based on these data, we concluded that neither the caspase-3-cleavable nor the caspase-8-cleavable IL-18 muteins were activated upon CAR T cell stimulation. Without wishing to be bound by theory, the most likely explanation for this is that neither protein was able to access the cellular substrate found in activated T cells as active caspase-3 and caspase-8 (Alam et al., "Early activation of caspases during T lymphocyte stimulation results in selective substrate cleavage in nonapoptotic cells," J. Exp. Med 190(12):1879-1890 (1999); Chun et al., "Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency," Nature 419(6905):395-9 (2002)).

[0249] Next, we tested a GzB-cleavable mutant of pro-IL-18 (MUC1-13b) (hereafter referred to as "pro-IL-18(GzB)") as described above. Unlike the caspase-3-cleavable or caspase-8-cleavable pro-IL-18 muteins, pro-IL-18(GzB) was functionally active when T cells were activated but not in the unstimulated state (Figures 7A-7B). This was confirmed by stimulating CAR T cells with a combination of anti-CD3 and anti-CD28 antibodies (Figure 7B). Nevertheless, when T cells coexpressing pCAR and IL-18(GzB) were tested in restimulation assays, they exhibited inferior antitumor activity compared to T cells with constitutive IL-18 activity.

[0250] We found that GzB is primarily expressed in CD8 T cells, whereas autocrine stimulation by IL-18 is primarily mediated by CD4 T cells, which normally express significantly less GzB. + Considering its role in T cells, we reasoned that GzB itself might be a limiting factor. To address this, we engineered TBB / H pCAR T cells to co-express native GzB in addition to IL-18 (GzB). PBMCs were transduced with this retroviral construct and co-cultured with MDA-MB-468 tumor cells at an effector:target ratio of 1:1. Antitumor activity was measured after 72 hours.

[0251] T cells engineered to co-express TBB / H and proIL-18, or a combination of TBB / H, proIL-18 (GzB), and an additional granzyme B protease, induced similar tumor cell killing. Figure 8 provides data from five independent donors, each performed in triplicate.

[0252] IL-18 (Figure 9A) and IFN-γ (Figure 9B) production was examined in T cells expressing TBB / H + pro-IL-18 or TBB / H + pro-IL-18 (GzB) + granzyme B. Supernatants from T cell cultures were harvested at 72 hours, and IL-18 and IFN-γ concentrations were measured.

[0253] Unstimulated T cells co-expressing TBB / H and pro-IL-18 or a combination of TBB / H, pro-IL-18 (GzB), and granzyme B secreted similar levels of IL-18 as detected by ELISA (Figure 9A). However, upon activation with target-expressing tumor cells, T cells expressing TBB / H, pro-IL-18 (GzB) + granzyme B produced significantly greater amounts of IFN-γ than T cells expressing TBB / H and pro-IL-18 (Figure 9B). Data from four independent donors, each performed in triplicate, are shown (**p=0.008).

[0254] Transduced T cells were further subjected to successive rounds of antigen stimulation in the absence of exogenous IL-2. Cells were cultured at an initial effector:target ratio of 1:1 with either MDA-MD-468 cells (Figure 10A) or BxPC-3 cells (Figure 10B) as the target population. Tumor cell survival was measured twice weekly by MTT assay after 72–96 hours. Using MDA-MD-468 cells as the target population, T cells co-expressing TBB / H and constitutive IL-18 or a combination of TBB / H, pro-IL-18 (GzB), and granzyme B were successfully restimulated for significantly more cycles than T cells expressing TBB / H alone or together with pro-IL-18 (Figure 10A). A similar pattern was observed using BxPC-3 cells as the target population (Figure 10B). Data shown were generated from one donor for Figure 10A and one donor for Figure 10B, each performed in triplicate.

[0255] The number of successful restimulations for each pCAR T cell population was measured. Data are provided in Figures 11A and 11B. If greater than 20% cytotoxicity was observed, pCAR T cells were advanced to the next round of stimulation. Cells were cultured at an effector-to-target ratio of 1 using either MDA-MD-468 cells (Figure 11A) or BxPC-3 cells (Figure 11B) as the target population. Using MDA-MD-468 cells as the target population, T cells that co-expressed TBB / H+pro-IL-18 (GzB)+granzyme B were successfully restimulated for more cycles than T cells that co-expressed TBB / H+pro-IL-18 (Figure 11A). A similar pattern was seen using BxPC-3 cells as the target population (Figure 11B). Data shown are from five independent donors, each performed in triplicate ( * p=0.039).

[0256] The number of T cells in each culture was also counted at the start of each restimulation cycle. TBB / H+pro-IL-18(GzB)+granzyme B co-expressing T cells proliferated significantly more than control TBB / H pCAR T cells, but TBB / H+pro-IL-18 did not. Counts shown are from the fourth restimulation cycle, each performed in triplicate from three independent donors (Figure 12; * p=0.014).

[0257] 5.4. Example 3: In vitro antitumor activity of IL-18-armed pCAR αβ T cells Using the method described in Example 1, αβ T cells were engineered to express TBB / H pCAR alone or TBB / H pCAR in combination with pro-IL-18, pro-IL-18 (GzB), constit IL-18, or pro-IL-18 (GzB) and granzyme B. αβ T cells were assayed for IL-18 activity using a reporter cell line (a commercially available reporter cell line was used to detect functional IL-18). The results presented in Figure 35 show that IL-18 activity was detected in TBB / H pCAR αβ T cells co-expressing constit IL-18, but not in other TBB / H pCAR αβ T cells in the absence of stimulation. However, αβ T cells were engineered to express MUC1 + TBB / H pCAR αβ T cells coexpressing pro-IL18 (GzB) and granzyme B also had IL-18 activity when stimulated with MDA-MB-468 breast cancer cells ("+468") or beads coated with anti-CD3 and anti-CD28 antibodies ("aCD3 / 28 beads"). TBB / H pCAR αβ T cells coexpressing pro-IL18 (GzB) and granzyme B had higher IL-18 activity than stimulated TBB / H pCAR αβ T cells expressing only pro-IL18 (GzB).

[0258] 5.5. Example 4: In vivo antitumor activity of IL-18-armed pCAR-αβ T cells The antitumor activity of CAR-αβ T and pCAR-αβ T cells was evaluated in vivo in a tumor xenograft mouse model.

[0259] 1 x 10 expressing luciferase 6 Established xenograft models were developed by injecting 1 × 10 MDA-MB-468 tumor cells into the peritoneal cavity (ip) of female SCID Beige mice. 11 or 12 days after tumor injection, 1 × 10 cells expressing or not expressing IL-18 were injected into the peritoneal cavity (ip) of female SCID Beige mice. 7CAR-αβ T cells were injected i.p. The pooled bioluminescence emission ("total light intensity") from the tumors was measured for each treatment. As presented in Figure 13 and Figures 36A-36F, SCID Beige mice treated with αβ T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B showed a significantly greater reduction in tumor-derived total light intensity compared to SCID Beige mice treated with TBB / H pCAR T cells. T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B also showed a trend toward improved tumor control when compared to T cells co-expressing TBB / H and constit IL-18 (Figure 13, Figure 36E, and Figure 36F). Data shown in Figure 13 is pooled from six mice. Data shown in Figure 36B is from 10 mice, data shown in Figure 36C is from 10 mice, data shown in Figure 36D is from 6 mice, data shown in Figure 36E is from 5 mice, and data shown in Figure 36F is from 5 mice.

[0260] Figure 37 shows survival data for mice treated after tumor injection with PBS, αβ T cells expressing only TBB / H, or αβ T cells expressing TBB / H in combination with const. IL-18, pro-IL-18 (GzB), or pro-IL-18 (GzB) together with granzyme B. The results show improved survival in mice treated with αβ T cells co-expressing TBB / H, pro-IL-18 (GzB), and granzyme B.

[0261] 5.6. Example 5: In vitro antitumor activity of pCAR-γδ T cells γδ T cells were activated with 2.4 ng of immobilized anti-γδ TCR antibody per well of a 6-well non-TC-treated plate and, 48 hours later, engineered to express TBB / H pCAR by retroviral transduction. Non-transduced γδ T cells and TBB / H pCAR γδ T cells were cultured and expanded (Figures 49A and 49B). Co-expression of the second-generation H2 CAR ("H28z") and TBB CCR ("TIE") (together, TBB / H pCAR) was confirmed in non-transduced (Figure 48A) or TBB / H pCAR γδ T cells (Figure 48B) using flow cytometry.

[0262] The antitumor effects of untransduced γδ T cells and TBB / H pCAR δγ T cells were evaluated by co-culturing them with MDA-MB-468 breast cancer cells (Figure 50A) or BxPC-3 cells (Figure 50B) at a 1:1 effector:target (γδ T cells:tumor cells) ratio for 72 hours. Tumor cell viability (%) was measured by MTT assay at the first stimulation cycle and compared to tumor cells cultured without γδ T cells. As shown in Figure 50A and Figure 50B, TBB / H pCAR δγ T cells had a cytotoxic effect on tumor cells.

[0263] Non-transduced γδ T cells and TBB / H pCAR δγ T cells were further subjected to successive rounds of antigen stimulation. Cells were cultured for 72 to 96 hours with either MDA-MD-468 cells (Figure 51A) or BxPC-3 cells (Figure 51B) as the target population at an initial effector:target ratio of 1:1. The cytotoxicity of γδ T cells against tumor cells was determined by MTT assay with successive monolayer challenges. A restimulation resulting in greater than 25% cytotoxicity against target tumor cells was considered a successful restimulation cycle. When greater than 25% cytotoxicity was observed, T cells proceeded to the next stimulation round. The number of successful restimulations for each transduced γδ T cell population was measured. The data are presented in Figures 51A and 51B. The results show that TBB / H pCAR δγ T cells were successfully restimulated for more cycles than δγ T cells.

[0264] The percent tumor cell viability measured over multiple stimulation cycles is provided in Figure 51C and Figure 51D. The data show the cytotoxic activity of TBB / H pCAR δγ T cells against MDA-MD-468 tumor cells (Figure 51C) or BxPC-3 tumor cells (Figure 51D) over restimulation cycles.

[0265] 5.7. Example 6: In vivo antitumor activity of pCAR-γδ T cells The antitumor activity of TBB / H pCAR δγ T cells was measured in vivo in a tumor xenograft mouse model.

[0266] For the BxPC3-NSG mouse model, 1 x 10 luciferase-expressing cells were used. 5 Established xenograft models were developed by injecting 1 × 10 BxPC3-LT tumor cells into the peritoneal cavity (ip) of NSG mice. For the 468s-SCID Beige mouse model, 1 × 10 luciferase-expressing BxPC3-LT tumor cells were injected into the peritoneal cavity (ip) of NSG mice. 6 MDA-MB-468 tumor cells were injected into the peritoneal cavity (ip) of female SCID Beige mice to develop an established xenograft model.

[0267] 11 days after tumor injection, 1 × 10 7 1 x 10 untransduced δγ T cells 7 TBB / H pCAR δγ T cells or PBS were injected i.p. into each animal model. Pooled bioluminescence emission from the tumors ("total light intensity") was measured for each treatment. As presented in Figure 52 (BxPC3-NSG) and Figure 53 (468s-SCID Beige), in both tumor xenograft mouse models, TBB / H pCAR δγ T cells induced a significant reduction in tumor-derived total light intensity compared to non-transduced δγ T cells or PBS controls, demonstrating antitumor activity.

[0268] 5.8. Example 7: In vitro antitumor activity of IL-18-armed pCAR-γδ T cells γδ T cells were activated with immobilized anti-γδ TCR antibodies and engineered by retroviral transduction to express TBB / H pCAR alone or pro-IL-18, pro-IL-18(GzB), constit IL-18, or pro-IL-18(GzB) together with granzyme B. Using flow cytometry, expression of pCAR was determined after incubation with anti-EGF antibodies (detecting CCR; Figure 14, upper panel), and enrichment of γδ T cells was also confirmed (Figure 14, lower panel).

[0269] The antitumor effects of γδ T cells were evaluated by 72-hour coculture with MDA-MB-468 breast cancer cells (Figure 15A) or BxPC-3 cells (Figure 15B). The effector:target (γδ T cell:tumor cell) ratio ranged from 128 to 1 (including 128, 64, 32, 16, 8, 4, 2, and 1). The remaining viable cancer cells after coculture were quantified by MTT assay. As shown in Figures 15A and 15B, γδ T cells expressing only TBB / H pCAR or any IL-18 variant (pro-IL-18; constit IL-18; pro-IL-18(GzB) or pro-IL-18(GzB) + granzyme B) together with TBB / H pCAR exhibited greater cytotoxic effects against tumor cells compared with untransduced γδ T cells.

[0270] Transduced γδ T cells were subjected to successive rounds of antigen stimulation in the absence of exogenous IL-2. Cells were cultured for 72–96 hours using either MDA-MD-468 cells (Figure 38A) or BxPC-3 cells (Figure 38B) as the target population at an initial effector:target ratio of 1:1. T cells proceeded to the next round of stimulation when greater than 30% cytotoxicity was observed. The number of successful restimulations for each transduced γδ T cell population was measured. Data are presented in Figures 38A and 38B. Using MDA-MD-468 cells as the target population, T cells co-expressing TBB / H+pro-IL-18 (GzB)+granzyme B were successfully restimulated for more cycles than T cells co-expressing TBB / H+pro-IL-18 (Figure 38A). A similar pattern was observed using BxPC-3 cells as the target population (Figure 38B). * p<0.05 ** p<0.01).

[0271] γδ T cells engineered to express TBB / H pCAR alone or in combination with pro-IL-18, pro-IL-18(GzB), or pro-IL-18(GzB) plus granzyme B were assayed for IL-18 activity using a reporter cell line. IL-18 activity was measured without stimulation or with MUC1 stimulation. + IL-18 activity was measured with or without stimulation with MDA-MB-468 breast cancer cells ("+468") or beads coated with anti-CD3 and anti-CD28 antibodies ("aCD3 / 28 beads"). The results, presented in Figure 39, show that IL-18 activity is dependent on the stimulation of transduced γδ T cells. Stimulation of T cells co-expressing TBB / H, pro-IL-18 (GzB), and granzyme B resulted in higher IL-18 activity than stimulated T cells co-expressing TBB / H and pro-IL-18 (GzB) alone or TBB / H and pro-IL-18 alone (Figure 39).

[0272] 5.9. Example 8: In vivo antitumor activity of IL-18-armed pCAR-γδ T cells The antitumor activity of pCAR-γδ T cells was evaluated in vivo in a tumor xenograft mouse model.

[0273] 1 x 10 expressing luciferase 6 1 × 10 MDA-MB-468 tumor cells were injected into the peritoneal cavity (ip) of female SCID Beige mice to develop established xenograft models. 11 days after tumor injection, 1 × 10 cells expressing or not expressing IL-18 were injected into the peritoneal cavity (ip) of female SCID Beige mice. 7 TBB / H pCAR-γδ T cells were injected i.p. into SCID Beige mice. The pooled bioluminescence emission ("total light intensity") from the tumors was measured for each treatment. As presented in Figures 40A-40F, SCID Beige mice treated with γδ T cells co-expressing TBB / H pro-IL-18 (GzB) granzyme B showed a significantly greater reduction in tumor-derived total light intensity compared to SCID Beige mice treated with TBB / H pCAR T cells. γδ T cells co-expressing TBB / H pro-IL-18 (GzB) granzyme B also showed a trend toward improved tumor control when compared to γδ T cells co-expressing TBB / H and constit IL-18 (Figures 40E and 40F). Data shown in Figure 40B is from 5 mice, data shown in Figure 40C is from 4 mice, data shown in Figure 40D is from 5 mice, data shown in Figure 40E is from 4 mice, and data shown in Figure 40F is from 3 mice.

[0274] Figure 41 shows survival data for mice treated after tumor injection with PBS, γδ T cells expressing TBB / H alone, or γδ T cells expressing TBB / H in combination with const. IL-18, pro-IL-18 (GzB), or pro-IL-18 (GzB) together with granzyme B. The results show improved survival in mice treated with γδ T cells co-expressing TBB / H, pro-IL-18 (GzB), and granzyme B.

[0275] 5.10. Example 9: In vivo antitumor activity of IL-18-armed pCAR αβ or γδ T cells The antitumor activity of pCAR-T cells was evaluated in vivo in a tumor xenograft mouse model.

[0276] 1 x 10 expressing luciferase 6 MDA-MB-468 tumor cells were injected into the peritoneal cavity (ip) of female SCID Beige mice to develop an established xenograft model. Eleven days after tumor cell injection, TBB / H pCAR T cells (1 × 10 7 pCAR-αβ or -γδ T cells, or 8 x 10 6 pCAR-γδ T cells, or 4 × 10 6 pCAR-γδ T cells) without exogenous IL-18 expression ("TBB / H"), or with exogenous expression of granzyme B together with pro-IL-18 alone or pro-IL-18 (GzB) were injected i.p. The pooled bioluminescence emission from the tumors ("total intensity") was measured from each treated animal.

[0277] The total light intensity measured in animals within each treatment group was pooled and presented in Figures 30A, 30B, and 30C. As shown in the graphs, SCID Beige mice treated with TBB / H pCAR-T cells co-expressing pro-IL-18 (GzB) and granzyme B showed a significantly greater reduction in tumor-derived total light intensity compared to mice in other groups treated with PBS, TBB / H pCAR T cells, or TBB / H pCAR T cells co-expressing pro-IL-18. This effect was observed in both αβ T cells (Figure 30A) and γδ T cells (Figures 30B and 30C).

[0278] 5.11. Example 10: Antitumor Activity of IL-18-Armored Second-Generation CAR-T Cells 5 x 10 expressing luciferase 5SKOV-3 tumor cells were injected intraperitoneally (ip) into female SCID Beige mice to develop a SKOV-3 xenograft model. Eighteen days after tumor cell injection, CAR-T cells were administered ip to three groups of mice. Group 1 received CAR-T cells engineered to co-express the T1E28z ErbB-targeted second-generation CAR and the 4αβ chimeric cytokine receptor. This combination is referred to as "T4" (see Schalkwyk et al., "Design of a Phase 1 clinical trial to evaluate intratumoural delivery of ErbB-targeted chimeric antigen receptor T-cells in locally advanced or recurrent head and neck cancer," Human Gene Ther. Clin. Devel. 24:134-142 (2013)). A second group of mice received T4-modified T cells co-expressing an MT1-MMP (MMP14)-cleavable pro-IL-18 mutant (pro-IL18(MT1)) (schematically depicted in Figure 16). Tumor cells express high levels of the MT1-MMP (MMP14) protease. A third control group received an endodomain-truncated, signaling-inactive version of the T1E-28z CAR (T1NA- T1 E N o A The researchers administered T cells expressing a specific domain called the activation domain.

[0279] Treatment with low doses (0.5 million) of second-generation CAR T cells or CAR T cells expressing T1NA (an endodomain-truncated control) was ineffective in these models. In contrast, CAR T cells co-expressing a T4 CAR and MT1-MMP (MMP14)-cleavable pro-IL-18 led to tumor elimination in 1 / 5 mice and disease regression in an additional 2 animals (Figure 17C). This provides an alternative approach to restricting IL-18 activation to the tumor microenvironment.

[0280] 5.12. Example 11: In vitro antitumor activity of IL-36-armed pCAR-T cells Constructs encoding TBB / H and the mature IL-36 fragment (pro-IL-36γ) were produced according to the method described above. Constructs encoding TBB / H and modified pro-IL-36γ were then produced by adding a cleavage site recognized by granzyme B (GzB) into the construct encoding TBB / H and pro-IL-36γ. Constructs encoding TBB / H + pro-IL-36 (GzB) + granzyme B were also produced by inserting the coding sequence for granzyme B into the construct encoding TBB / H and modified pro-IL-36γ.

[0281] T cells were transfected with TBB / H pCAR and SFG retroviral vectors encoding pro-IL-36γ or modified pro-IL-36γ (GzB).

[0282] T cells expressing TBB / H, or T cells co-expressing TBB / H, pro-IL-36γ, and granzyme B, or T cells co-expressing a combination of TBB / H, pro-IL-36γ (GzB), and granzyme B protease, were subjected to repeated stimulation with MDA-MB-468 breast cancer cells or BxPC-3 pancreatic cancer cells. The effector:target (modified T cell:tumor cell) ratio ranged from 2 to 0.03 (including 1, 0.5, 0.25, 0.125, and 0.06). The remaining viable cancer cells present after the end of coculture were quantified by MTT assay. The results, shown in Figure 42A (MDA-MB-468 cells) and Figure 42B (BxPC-3 cells), demonstrate significant cytotoxic activity of TBB / H cells expressing pro-IL-36γ and granzyme B, or pro-IL-36γ (GzB) and granzyme B. T cells co-expressing TBB / H, pro-IL-36γ (GzB), and granzyme B expanded significantly over the restimulation cycle (Figures 43A and 43B). IFN-γ production (Figures 44A and 44B) was also significantly higher in T cells expressing TBB / H + pro-IL-36γ + granzyme B or TBB / H + pro-IL-36γ (GzB) + granzyme B compared with TBB / HT cells.

[0283] T cells engineered to co-express TBB / H+proIL-36γ+granzyme B or TBB / H+proIL-36γ (GrzB)+granzyme B induced tumor cell killing of both MDA-MB-468 cells (Figure 45) and BxPC-3 cells (Figure 46) at effector:target (engineered T cell:tumor cell) ratios ranging from 2 to 0.03 (including 1, 0.5, 0.25, 0.125, and 0.06) (all experiments performed in triplicate).

[0284] 5.13. Example 12: In vivo antitumor activity of IL-36-armed pCAR-T cells The antitumor activity of IL-36-armed pCAR-T cells was further tested in vivo. 61 × 10 MDA-MB-468 tumor cells were injected into the peritoneal cavity (ip) of female SCID Beige mice to develop established xenograft models. 12 days after tumor injection, 1 × 10 IL-36-expressing non-IL-36-expressing tumor cells were injected into the peritoneal cavity (ip) of female SCID Beige mice. 7 TBB / H pCAR-T cells, or TBB / H pCAR-T cells co-expressing pro-IL36γ and granzyme B or pro-IL36γ (GzB) and granzyme B, were injected i.p.

[0285] The pooled bioluminescence emission from the tumors ("total light intensity") was measured for each treatment. Mice treated with TBB / H+pro-IL-36γ(GzB)+granzyme B co-expressing T cells show a significantly greater reduction in tumor-derived total light intensity compared to mice treated with TBB / H pCAR T cells (Figures 47A-47D).

[0286] 6. Arrays JPEG2025161810000003.jpg226166JPEG2025161810000004.jpg227166JPEG2025161810000005.jpg226166JPEG2025161810000006.jp g226166JPEG2025161810000007.jpg224166JPEG2025161810000008.jpg224166JPEG2025161810000009.jpg224166JPEG2025161810000 010.jpg224166JPEG2025161810000011.jpg224166JPEG2025161810000012.jpg224166JPEG2025161810000013.jpg226166JPEG2025161 810000014.jpg224166JPEG2025161810000015.jpg224166JPEG2025161810000016.jpg225166JPEG2025161810000017.jpg222166JPEG2 025161810000018.jpg223166JPEG2025161810000019.jpg224166JPEG2025161810000020.jpg226166JPEG2025161810000021.jpg2261 66JPEG2025161810000022.jpg225166JPEG2025161810000023.jpg226166JPEG2025161810000024.jpg224166JPEG2025161810000025.j pg225166JPEG2025161810000026.jpg225166JPEG2025161810000027.jpg224166JPEG2025161810000028.jpg224166JPEG2025161810000029.jpg224166JPEG2025161810000030.jpg224166JPEG2025161810000031.jpg224166JPEG2025161810000032.jpg1571667. Equivalents and Range Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments according to the invention described herein. The scope of the invention is not intended to be limited to the above description, but rather is as defined in the appended claims.

Claims

1. 1. An immunoresponsive cell that expresses a modified pro-cytokine of the IL-1 superfamily, comprising: The modified pro-cytokine comprises, from the N-terminus to the C-terminus: (a) propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a fragment of the cytokine of the IL-1 superfamily. Including, Immunoresponsive cells.

2. The immunoresponsive cell of claim 1 , wherein the protease is granzyme B (GzB).

3. The immunoresponsive cell of claim 2 , wherein the cleavage site has the sequence of SEQ ID NO:

26.

4. 4. The immunoresponsive cell of claim 3, wherein the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO:

27.

5. 5. The immunoresponsive cell of claim 4, wherein the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO: 103 or 111.

6. The immunoresponsive cell of claim 1, wherein the protease is caspase-3.

7. The immunoresponsive cell of claim 6 , wherein the cleavage site has the sequence of SEQ ID NO:

28.

8. 8. The immunoresponsive cell of claim 7, wherein the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO:

29.

9. 9. The immunoresponsive cell of claim 8, wherein the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO:

109.

10. The immunoresponsive cell of claim 1, wherein the protease is caspase-8.

11. The immunoresponsive cell of claim 10, wherein the cleavage site has the sequence of SEQ ID NO:

30.

12. 12. The immunoresponsive cell of claim 11, wherein the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO:

31.

13. 13. The immunoresponsive cell of claim 12, wherein the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO:

107.

14. The immunoresponsive cell of claim 1, wherein the protease is MT1-MMP.

15. The immunoresponsive cell of claim 14 , wherein the cleavage site has the sequence of SEQ ID NO:

32.

16. 16. The immunoresponsive cell of claim 15, wherein the modified pro-cytokine is modified pro-IL-18 and has the sequence of SEQ ID NO:

33.

17. 17. The immunoresponsive cell of claim 16, wherein the modified pro-IL-18 is expressed from the polynucleotide of SEQ ID NO:

113.

18. 18. The immunoresponsive cell of any one of claims 1 to 17, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

24.

19. The immunoresponsive cell of any one of claims 1 to 17, wherein the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

25.

20. 2. The immunoresponsive cell of claim 1, wherein the modified pro-cytokine is modified pro-IL-36α and has the sequence of SEQ ID NO:

37.

21. The immunoresponsive cell of claim 20, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

42.

22. 2. The immunoresponsive cell of claim 1, wherein the modified pro-cytokine is modified pro-IL-36β and has the sequence of SEQ ID NO:

39.

23. The immunoresponsive cell of claim 22, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

43.

24. 2. The immunoresponsive cell of claim 1, wherein the modified pro-cytokine is modified pro-IL-36γ and has the sequence of SEQ ID NO:

41.

25. The immunoresponsive cell of claim 24, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

44.

26. The immunoresponsive cell of any one of claims 1 to 25, further comprising an exogenous polynucleotide encoding the protease.

27. The immunoresponsive cell of any one of claims 1 to 26, wherein the immunoresponsive cell is an αβ T cell, a γδ T cell, or a natural killer (NK) cell.

28. 28. The immunoresponsive cell of claim 27, wherein the T cell is an αβ T cell.

29. 28. The immunoresponsive cell of claim 27, wherein the T cell is a γδ T cell.

30. The immunoresponsive cell of any one of claims 1 to 29, further comprising a chimeric antigen receptor (CAR).

31. The CAR is signaling region; a first costimulatory signaling domain; a transmembrane domain; and a first binding element that specifically interacts with a first epitope on a first target antigen; The immunoresponsive cell of claim 30, which is a second-generation chimeric antigen receptor (CAR) comprising:

32. 32. The immunoresponsive cell of claim 31, wherein the first epitope is an epitope on a MUC1 target antigen.

33. 33. The immunoresponsive cell of claim 32, wherein the first binding element comprises a CDR of an HMFG2 antibody.

34. The first binding element is a V Ｈ and V Ｌ The immunoresponsive cell of claim 32, comprising a domain.

35. 33. The immunoresponsive cell of claim 32, wherein the first binding element comprises an HMFG2 single-chain variable fragment (scFv).

36. 36. The immunoresponsive cell of any one of claims 1 to 35, further comprising a chimeric costimulatory receptor (CCR), The CCR is a second costimulatory signaling domain; a transmembrane domain; and a second binding element that specifically interacts with a second epitope on a second target antigen; Including, Immunoresponsive cells.

37. 37. The immunoresponsive cell of claim 36, wherein the second costimulatory domain is different from the first costimulatory domain.

38. 38. The immunoresponsive cell of any one of claims 36 to 37, wherein the second target antigen comprising the second epitope is selected from the group consisting of ErbB homodimers and heterodimers.

39. 36. The immunoresponsive cell of claim 35, wherein the second target antigen is HER2.

40. 36. The immunoresponsive cell of claim 35, wherein the second target antigen is an EGF receptor.

41. The immunoresponsive cell of any one of claims 36 to 40, wherein the second binding element comprises a binding portion of T1E, ICR12, or ICR62.

42. The immunoresponsive cell of any one of claims 1 to 41, the cells express modified pro-IL-18; the modified pro-IL-18 is the polypeptide of SEQ ID NO: 27; The cells GzB, expressed from an exogenous polynucleotide; A chimeric antigen receptor (CAR) comprising: signaling region; i. a first costimulatory signaling domain; ii. a transmembrane domain; and iii. a first binding element that specifically interacts with a first epitope on the MUC1 target antigen; and A chimeric costimulatory receptor (CCR) comprising: iv. a second costimulatory signaling domain; v. a transmembrane domain; and vi. a second binding element that specifically interacts with a second epitope on a second target antigen; immunoresponsive cells that further express

43. A polynucleotide or set of polynucleotides comprising a first nucleic acid encoding a modified pro-cytokine, The modified pro-cytokine comprises, from the N-terminus to the C-terminus: (a) propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) cytokine fragments of the IL-1 superfamily Including, A polynucleotide or set of polynucleotides.

44. 44. The polynucleotide or set of polynucleotides of claim 43, wherein the protease is Granzyme B (GzB).

45. 45. The polynucleotide or set of polynucleotides of claim 44, wherein the cleavage site has the sequence of SEQ ID NO:

26.

46. 46. ​​The polynucleotide or set of polynucleotides of claim 45, wherein the modified pro-cytokine is modified pro-IL-18 and comprises the sequence of SEQ ID NO:

27.

47. 47. The polynucleotide or set of polynucleotides of claim 46, comprising the sequence of SEQ ID NO: 103 or 111.

48. 44. The polynucleotide or set of polynucleotides of claim 43, wherein the protease is caspase-3.

49. 49. The polynucleotide or set of polynucleotides of claim 48, wherein the cleavage site has the sequence of SEQ ID NO:

28.

50. 50. The polynucleotide or set of polynucleotides of claim 49, wherein the modified cytokine is modified pro-IL-18 and comprises the sequence of SEQ ID NO:

29.

51. 51. The polynucleotide or set of polynucleotides of claim 50, comprising the sequence of SEQ ID NO:

109.

52. 44. The polynucleotide or set of polynucleotides of claim 43, wherein the protease is caspase-8.

53. 53. The polynucleotide or set of polynucleotides of Claim 52, wherein the cleavage site has the sequence of SEQ ID NO:

30.

54. 54. The polynucleotide or set of polynucleotides of claim 53, wherein the modified cytokine is modified pro-IL-18 and comprises the sequence of SEQ ID NO:

31.

55. 55. The polynucleotide or set of polynucleotides of claim 54, comprising the sequence of SEQ ID NO:

107.

56. The polynucleotide or set of polynucleotides according to claim 43, wherein the protease is MT1-MMP.

57. 57. The polynucleotide or set of polynucleotides of claim 56, wherein the cleavage site has the sequence of SEQ ID NO:

32.

58. 58. The polynucleotide or set of polynucleotides of claim 57, wherein the modified cytokine is modified pro-IL-18 and comprises the sequence of SEQ ID NO:

33.

59. 59. The polynucleotide or set of polynucleotides of claim 58, comprising the sequence of SEQ ID NO:

113.

60. 60. The polynucleotide or set of polynucleotides of any one of claims 43 to 59, further comprising a second nucleic acid encoding said protease.

61. 61. The polynucleotide or set of polynucleotides of Claim 60, wherein said first nucleic acid and said second nucleic acid are in a single vector.

62. 62. The polynucleotide or set of polynucleotides of any one of claims 43 to 61, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

24.

63. 63. The polynucleotide or set of polynucleotides of any one of claims 43 to 62, wherein the cytokine fragment is capable of binding to and activating an IL-18 receptor when the cleavage site is cleaved.

64. 64. The polynucleotide or set of polynucleotides of any one of claims 43 to 63, wherein the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

25.

65. 44. The polynucleotide or set of polynucleotides of claim 43, wherein the modified pro-cytokine is modified pro-IL-36α and has the sequence of SEQ ID NO:

37.

66. 66. The polynucleotide or set of polynucleotides of claim 65, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

42.

67. 44. The polynucleotide or set of polynucleotides of claim 43, wherein the modified pro-cytokine is modified pro-IL-36β and has the sequence of SEQ ID NO:

39.

68. 68. The polynucleotide or set of polynucleotides of claim 67, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

43.

69. 44. The polynucleotide or set of polynucleotides of claim 43, wherein the modified pro-cytokine is modified pro-IL-36γ and has the sequence of SEQ ID NO:

41.

70. 70. The polynucleotide or set of polynucleotides of claim 69, wherein the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:

44.

71. A polynucleotide or set of polynucleotides comprising a first nucleic acid encoding a modified pro-IL-36 α, β, or γ, The modified pro-IL-36α, β, or γ has from the N-terminus to the C-terminus: (a) propeptide; (b) a cleavage site recognized by a protease other than cathepsin G, elastase, or proteinase 3; and (c) IL-36 fragment Including, A polynucleotide or set of polynucleotides.

72. 72. The polynucleotide or set of polynucleotides of claim 71, wherein the protease is Granzyme B (GzB).

73. 73. The polynucleotide or set of polynucleotides of Claim 72, wherein the cleavage site has the sequence of SEQ ID NO:

26.

74. 73. The polynucleotide or set of polynucleotides of claim 72, wherein the modified pro-IL-36 α, β, or γ comprises the sequence of SEQ ID NO: 37, 39, or 41.

75. 75. The polynucleotide or set of polynucleotides of any one of claims 71 to 74, further comprising a second nucleic acid encoding said protease.

76. 76. The polynucleotide or set of polynucleotides of Claim 75, wherein said first nucleic acid and said second nucleic acid are in a single vector.

77. 77. The polynucleotide or set of polynucleotides of any one of claims 71 to 76, wherein the IL-36 fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 42, 43 or 44.

78. The polynucleotide or set of polynucleotides according to any one of claims 65 to 71, wherein the IL-36 fragment is capable of binding to and activating the IL-36 receptor when the cleavage site is cleaved.

79. 79. The polynucleotide or set of polynucleotides of any one of claims 43 to 78, further comprising a third nucleic acid encoding a chimeric antigen receptor (CAR).

80. The CAR is signaling region; a first costimulatory signaling domain; a transmembrane domain; and a first binding element that specifically interacts with a first epitope on a first target antigen; 80. The polynucleotide or set of polynucleotides of Claim 79, which is a second generation chimeric antigen receptor (CAR), comprising:

81. 81. The polynucleotide or set of polynucleotides of claim 80, wherein the first epitope is an epitope on a MUC1 target antigen.

82. 81. The polynucleotide or set of polynucleotides of claim 80, wherein the first binding element comprises a CDR of an HMFG2 antibody.

83. The first binding element is a V of an HMFG2 antibody. Ｈ and V Ｌ 81. The polynucleotide or set of polynucleotides of Claim 80, comprising a domain.

84. 81. The polynucleotide or set of polynucleotides of claim 80, wherein the first binding element comprises an HMFG2 single chain variable fragment (scFv).

85. A polynucleotide or set of polynucleotides according to any one of claims 43 to 84, further comprising a fourth nucleic acid encoding a chimeric costimulatory receptor (CCR); The CCR is a second costimulatory signaling domain; a transmembrane domain; and a second binding element that specifically interacts with a second epitope on a second target antigen; Including, A polynucleotide or set of polynucleotides.

86. 86. The polynucleotide or set of polynucleotides of claim 85, wherein the second target antigen comprising the second epitope is selected from the group consisting of ErbB homodimers and heterodimers.

87. 86. The polynucleotide or set of polynucleotides of claim 85, wherein the second target antigen is HER2.

88. 86. The polynucleotide or set of polynucleotides of claim 85, wherein the second target antigen is an EGF receptor.

89. 89. The polynucleotide or set of polynucleotides of any one of claims 43 to 88, wherein the second binding element comprises a binding portion of T1E, ICR12, or ICR62.

90. 90. The polynucleotide or set of polynucleotides of any one of claims 85 to 89, wherein the third nucleic acid and the fourth nucleic acid are in a single vector.

91. A polynucleotide or set of polynucleotides according to any one of claims 43 to 90, a first nucleic acid encoding a modified pro-IL-18; wherein said modified pro-IL-18 is the polypeptide of SEQ ID NO: 27; a second nucleic acid encoding GzB; a third nucleic acid encoding a chimeric antigen receptor (CAR); wherein the CAR comprises: i. signaling region; ii. a first costimulatory signaling domain; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on the MUC1 target antigen; a fourth nucleic acid encoding a chimeric costimulatory receptor (CCR); wherein the CCR comprises: v. a second costimulatory signaling domain; vi. a transmembrane domain; and vii. a second binding element that specifically interacts with a second epitope on a second target antigen. Including, A polynucleotide or set of polynucleotides.

92. 92. The polynucleotide or set of polynucleotides of Claim 91, comprising the polynucleotide of SEQ ID NO:

103.

93. 93. The polynucleotide or set of polynucleotides of any one of claims 43 to 92, wherein the first nucleic acid and the third nucleic acid are in a single vector.

94. 93. The polynucleotide or set of polynucleotides of any one of claims 43 to 92, wherein the first nucleic acid and the fourth nucleic acid are expressed from a single vector.

95. 93. The polynucleotide or set of polynucleotides of any one of claims 43 to 92, wherein the first nucleic acid, the second nucleic acid, the third nucleic acid, and the fourth nucleic acid are expressed from a single vector.

96. A polynucleotide or set of polynucleotides according to any one of claims 43 to 95, a first nucleic acid encoding a modified pro-IL-36; wherein said modified pro-IL-36 is the polypeptide of SEQ ID NO: 37, 39 or 41; a second nucleic acid encoding GzB; a third nucleic acid encoding a chimeric antigen receptor (CAR); wherein the CAR comprises: i. signaling region; ii. a first costimulatory signaling domain; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on the MUC1 target antigen; a fourth nucleic acid encoding a chimeric costimulatory receptor (CCR); wherein the CCR comprises: v. a second costimulatory signaling domain; vi. a transmembrane domain; and vii. a second binding element that specifically interacts with a second epitope on a second target antigen. Including, A polynucleotide or set of polynucleotides.

97. (a) a second generation chimeric antigen receptor (CAR) comprising: i. signaling region; ii. a costimulatory signaling region; iii. a transmembrane domain; and iv. a first binding element that specifically interacts with a first epitope on a first target antigen; and (b) a chimeric costimulatory receptor (CCR) comprising: v. a costimulatory signaling region different from that of (ii); vi. a transmembrane domain; and vii. a second binding element that specifically interacts with a second epitope on a second target antigen. expressing γδ T cells.

98. 98. The γδ T cell of claim 97, wherein the first target antigen is the same as the second target antigen.

99. 98. The γδ T cell of claim 97, wherein the first target antigen is a MUC antigen.

100. 98. The γδ T cell of claim 97, wherein the first binding element comprises the CDRs of an HMFG2 antibody.

101. The first binding element is a V of an HMFG2 antibody. Ｈ and V Ｌ 100. The γδ T cell of claim 99, comprising a domain.

102. 102. The γδ T cell of any one of claims 97-101, wherein the first binding element comprises an HMFG2 single chain variable fragment (scFv).

103. 103. The γδ T cell of any one of claims 97-102, wherein the second target antigen comprising the second epitope is selected from the group consisting of ErbB homodimers and heterodimers.

104. The γδ T cell of any one of claims 97 to 103, wherein the second target antigen is HER2.

105. 105. The γδ T cell of claim 104, wherein the second target antigen is an EGF receptor.

106. The γδ T cell of any one of claims 97-105, wherein the second binding element comprises T1E, ICR12, or ICR62.

107. The γδ T cell of claim 106, wherein the second binding element is a T1E.

108. The γδ T cell of any one of claims 97 to 107, wherein the second target antigen is αvβ6 integrin.

109. 109. The γδ T cell of claim 108, wherein the second binding element is an A20 peptide.

110. A method for preparing the immunoresponsive cells of any one of claims 1 to 42, comprising: The method comprises transfecting or transducing an immunoresponsive cell with a polynucleotide or set of polynucleotides according to any one of claims 43 to 96, method.

111. 1. A method for directing a T cell-mediated immune response to a target cell in a patient in need thereof, comprising: The method comprises: administering to said patient a therapeutically effective number of immunoresponsive cells of any one of claims 1 to 42 or γδ T cells of any one of claims 97 to 109. method.

112. 112. The method of claim 111, wherein the target cell expresses MUC1.

113. 1. A method of treating cancer, comprising: The method comprises: administering to a patient an effective amount of an immunoresponsive cell according to any one of claims 1 to 42 or a γδ T cell according to any one of claims 97 to 109. method.

114. 110. The immunoresponsive cell of any one of claims 1 to 42, the polynucleotide of any one of claims 43 to 96, or the γδ T cell of any one of claims 97 to 109 for use (i) in therapy or as a medicament, or (ii) in the treatment of a cancer patient.

115. 115. The immunoresponsive cell, polynucleotide, or γδ T cell of claim 113, wherein the patient's cancer cells express MUC1.

116. 115. The immunoresponsive cell, polynucleotide, or γδ T cell of claim 113, wherein the patient has a cancer selected from the group consisting of breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal carcinoma, thyroid carcinoma, renal cell carcinoma, multiple myeloma, and non-Hodgkin's lymphoma.

117. 117. The method or immunoresponsive cell, polynucleotide, or γδ T cell of claim 116, wherein the patient has breast cancer.

118. 117. The method of claim 116 or the immunoresponsive cell, polynucleotide, or γδ T cell, wherein the patient has ovarian cancer.

119. 110. Use of an immunoresponsive cell according to any one of claims 1 to 42, a polynucleotide according to any one of claims 43 to 96, or a γδ T cell according to any one of claims 97 to 109 in the manufacture of a medicament for the treatment of a pathological disorder.

120. A method for producing immunoresponsive cells, comprising the step of introducing a transgene.

121. 121. The method of claim 120, wherein the transgene encodes a CAR or pCAR.

122. The transgene encodes a modified pro-cytokine of the IL-1 superfamily, the modified pro-cytokine comprising, from N-terminus to C-terminus: (a) propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or proteinase 3; and (c) a fragment of a cytokine of the IL-1 superfamily Including, The method of claim 120.

123. 123. The method of any one of claims 120 to 122, further comprising the preceding step of activating γδ T cells with an anti-γδ TCR antibody.

124. 124. The method of claim 123, wherein the anti-γδ TCR antibody is immobilized.

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