IL-1 superfamily spatiotemporally restricted activity cytokine-armed immune response cells

Abstract

Provided herein are immune responsive cells with spatiotemporally restricted IL-1 superfamily activity. The immune responsive cells can further express a protease that modulates IL-1 superfamily activity and a chimeric antigen receptor (CAR) or a parallel CAR. Also provided herein are methods of making the immune responsive cells and methods of using the immune responsive cells to direct T cell-mediated immune responses.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an immune response cell armored with IL-1 superfamily spatiotemporally restricted active cytokines. Background Technology

[0002] The tumor microenvironment imposes restrictions on the activity of immune effectors, including those mediated by tumor-infiltrating lymphocytes, T cells engineered to express non-natural T cell receptors (TCRs), and T cells engineered to express chimeric antigen receptors (CARs). To address this immunosuppression within the tumor stroma, there is interest in engineering immune-response cells to further express one or more pro-inflammatory cytokines, such as interleukin (IL)-12 and / or members of the IL-1 superfamily.

[0003] The IL-1 superfamily consists of 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, IL-1 receptor antagonists (IL-1Ra), IL-36Ra, IL-37, and IL-38 have antagonistic or anti-inflammatory effects. Importantly, some IL-1 superfamily members are synthesized in precursor forms, which require proteolytic cleavage to demonstrate their biological activity. Cytokines with antitumor activity regulated in this manner include IL-1β, IL-18, and IL-36α-γ.

[0004] Like IL-1β and IL-36α-γ, IL-18 lacks a conventional signaling or lead sequence that would prevent post-translational guidance of the protein to secretory pathways involving the endoplasmic reticulum (ER) and Golgi apparatus. Instead, IL-18 is produced as a bioactive precursor (pro-IL-18), activated by cleavage of a 36-amino acid propeptide in its N-terminal region. This cleavage is primarily mediated by caspase-1, which is present in inducible multimolecular organelles known as inflammasomes. Pro-inflammatory IL-36 family members (IL-36α, IL-36β, IL-36γ) are also synthesized as inactive precursors, activated upon proteolytic cleavage in their N-terminal regions. Activating enzymes of pro-IL-36 cytokines include cathepsin G, elastase, and protease 3.

[0005] Many laboratories have engineered CAR- or TCR-based 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 low Avanzi et al., "Engineeredtumor-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 solidtumors," Oncoimmunology 7(1):e1378842 (2017).

[0006] Hu et al. demonstrated that, in addition to antitumor activity, constitutive expression of mature IL-18 by CAR T cells enhanced their in vivo T cell receptor-dependent expansion. This study did not detail how IL-18 was engineered for secretion. Nevertheless, supplemental data indicated both constitutive release (Fig. S1b) and constitutive activity of IL-18 (Fig. S1c), suggesting that the mature (18kD) form of IL-18 is fused with a conventional signal peptide or lead peptide.

[0007] Avanzi et al. also demonstrated enhanced antitumor activity of IL-18-coated CAR T cells, accompanied by the proliferation and persistence of autocrine CAR T cells. Favorable regulation of intratumoral cell infiltration suggests a positive impact on endogenous immune surveillance. Furthermore, epitope diffusion led to enhanced antitumor activity of endogenous T cells. Using IL-18 in this manner avoids the need for lymphocyte depletion to achieve antitumor activity. Macrophage depletion significantly hindered therapeutic efficacy, confirming the important role of these cells in the regulation of the tumor microenvironment. Because native IL-18 lacks a conventional signaling sequence, the IL-18 structure used in Avanzi's article was a constitutively expressed mature IL-18 with an IL-2 signal peptide.

[0008] Although IL-18 expression in CAR-T cells has been shown to improve therapeutic efficacy in various experiments, the safety and therapeutic benefits of constitutive IL-18 expression have not been fully studied.

[0009] Given the close 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 are concerns that unregulated expression of mature IL-18 or other members of the IL-1 superfamily may be toxic. Therefore, there is a need for improved strategies that “armor” immune-response cells against the inhibitory effects of the antitumor microenvironment, without causing significant toxicity to non-cancerous tissues.

[0010] Chmielewski et al. used the NFAT-responsive promoter to attempt to restrict the release of mature IL-18 into activated CAR T cells. They found that IL-18-producing CAR T cells modulated the tumor microenvironment, favoring an inflammatory state that could contribute to disease elimination. Tumor-specific T cells and NK cells increased at this site, while immunosuppressive M2-polarized macrophages and regulatory T cells decreased. Furthermore, the distribution of co-stimulatory and co-inhibitory receptors expressed in the tumor was also favorablely altered. Kunert et al. obtained largely similar results in TCR-engineered T cells. Conceptually, restricting the release of mature IL-18 into activated (NFAT-expressing) T cells should make this approach safer. However, achieving this solution requires a cumbersome double-conversion process. This is 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 its production would be challenging given the well-known promoter interference problem. Furthermore, this inducible vector exhibited a degree of "leakage," and the toxicity observed in tumor-free mice suggests that IL-12 release is similarly regulated. Summary of the Invention

[0011] This invention provides immune response cells with spatiotemporally restricted activity of IL-1 superfamily members with antitumor activity, particularly IL-18, IL-36α, IL-36β, and IL-36γ. Specifically, this invention provides immune response cells expressing modified procytokines of the IL-1 superfamily, wherein the modified procytokines comprise, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3; and (c) a biologically active cytokine fragment of the IL-1 superfamily.

[0012] CAR T cells – including αβCAR-T cells and γδCAR-T cells – are generated from an exogenous polynucleotide encoding a procytokine whose cleavage site is recognized by a site-specific protease other than caspase-1, cathepsin G, elastase, or protease 3. In some experiments, the cells further express the site-specific protease. In particular, the procytokine provided in this application contains a cleavage site recognized by protease granzyme B (GzB). The applicant has found that the expression of regulatory IL-1 superfamily members can enhance T cell responses and the antitumor activity of CAR T cells in a controlled manner.

[0013] Pre-cytokines with regulatory activity can be used in combination with various CAR T cells in the prior art. For example, pCAR-T cells with a parallel CAR (pCAR) construct that binds to one or more antigens present on target cells can be further modified to express pre-cytokines with regulatory activity.

[0014] Therefore, according to some embodiments, this article provides an immune-responding cell that expresses: a modified procytokine of the IL-1 superfamily, wherein the modified procytokine comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3; and (c) a cytokine fragment of the IL-1 superfamily.

[0015] In some embodiments, the protease is granzyme B (GzB). In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:26. In some embodiments, the modified procytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:26. In some embodiments, the modified pro-IL-18 is expressed by the polynucleotide shown in SEQ ID NO:103 or 111.

[0016] In some embodiments, the protease is caspase-3. In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:28. In some embodiments, the modified procytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:29. In some embodiments, the modified pro-IL-18 is expressed by the polynucleotide shown in SEQ ID NO:109.

[0017] In some embodiments, the protease is caspase-8. In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:30. In some embodiments, the modified procytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:31. In some embodiments, the modified pro-IL-18 is expressed by the polynucleotide shown in SEQ ID NO:107.

[0018] In some embodiments, the protease is a membrane type 1 matrix metalloproteinase (MT1-MMP). In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:32. In some embodiments, the modified procytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:33. In some embodiments, the modified pro-IL-18 is expressed by the polynucleotide shown in SEQ ID NO:113.

[0019] In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:24.

[0020] In some embodiments, the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:25.

[0021] In some embodiments, the modified pro-cytokine is modified pro-IL-36α and has the sequence shown in 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 with SEQ ID:42. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:42.

[0022] In some embodiments, the modified pro-cytokine is a modified pro-IL-36β having the sequence shown in 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 with SEQ ID:43. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:43.

[0023] In some embodiments, the modified pro-cytokine is a modified pro-IL-36γ having a sequence such as 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 with SEQ ID:44. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:44.

[0024] In some implementations, the immune response cells further include exogenous polynucleotides encoding proteases.

[0025] In some embodiments, the immune response cells are αβT cells, γδT cells, or natural killer (NK) cells. In some embodiments, the T cells are αβT cells. In some embodiments, the T cells are γδT cells.

[0026] In some embodiments, the immune-responding cells further include a chimeric antigen receptor (CAR). In some embodiments, the CAR is a second-generation chimeric antigen receptor (CAR), wherein the CAR includes: (a) a signaling region; (b) a first co-stimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen.

[0027] 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 the HMFG2 antibody. In some embodiments, the first binding element comprises a V of the HMFG2 antibody. H and V L Structural domain. In some embodiments, the first binding element includes an HMFG2 single-chain variable fragment (scFv).

[0028] In some embodiments, the immune response cells also include a chimeric co-stimulatory receptor (CCR), wherein the CCR includes: (a) a second co-stimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0029] In some embodiments, the second costimulatory domain differs from the first costimulatory domain. In some embodiments, the second target antigen including 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 an EGF receptor. In some embodiments, the second binding element includes a binding portion of T1E, ICR12, or ICR62.

[0030] In some embodiments, the present invention provides an immune-response cell expressing a modified pro-IL-18, wherein the modified pro-IL-18 is the polypeptide of SEQ ID NO:27, wherein the cell further comprises: (a) an exogenous polynucleotide encoding GzB; (b) a chimeric antigen receptor (CAR) comprising: i. a signaling region; ii. a first co-stimulatory 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 co-stimulatory receptor (CCR) comprising: i. a second co-stimulatory 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 some embodiments, the present invention provides an immune-responding cell expressing a 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 represented by SEQ ID NO: 37, 39, or 41, wherein the cell further comprises: (a) an exogenous polynucleotide encoding GzB; (b) a chimeric antigen receptor (CAR) comprising: i. a signaling region; ii. a first co-stimulatory 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 co-stimulatory receptor (CCR) comprising: i. a second co-stimulatory signaling region; ii. a transmembrane domain; and iii. a second binding element that specifically interacts with a second epitope on a second target antigen.

[0032] In another aspect, the present invention provides a polynucleotide or a group of polynucleotides comprising a first nucleic acid encoding a modified cytokine, wherein the modified procytokine of the IL-1 superfamily comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3; and (c) a cytokine fragment of the IL-1 superfamily.

[0033] In some embodiments, the protease is GzB. In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:26. In some embodiments, the modified procytokine is modified pro-IL-18 having the sequence shown in SEQ ID NO:27. In some embodiments, the polynucleotide or group of polynucleotides comprises the sequence shown in SEQ ID NO:103 or 11.

[0034] In some embodiments, the protease is caspase-3. In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:28. In some embodiments, the modified cytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:29. In some embodiments, the polynucleotide or a group of polynucleotides includes the sequence shown in SEQ ID NO:109.

[0035] In some embodiments, the protease is caspase-8. In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:30. In some embodiments, the modified cytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:31. In some embodiments, the polynucleotide or group of polynucleotides includes the sequence shown in SEQ ID NO:107.

[0036] In some embodiments, the protease is MT1-MMP. In some embodiments, the cleavage site has the sequence shown in SEQ ID NO:32. In some embodiments, the modified procytokine is modified pro-IL-18 and has the sequence shown in SEQ ID NO:33. In some embodiments, the polynucleotide or group of polynucleotides includes the sequence shown in SEQ ID NO:113.

[0037] In some implementations, the polynucleotide or a group of polynucleotides further includes a second nucleic acid encoding a protease.

[0038] In some implementations, the first and second nucleic acids are contained in a single vector.

[0039] In some embodiments, the cytokine fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:24. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:24. In some embodiments, the cytokine fragment can bind to and activate 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 with SEQ ID:25. In some embodiments, the propeptide is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:25.

[0040] In some embodiments, the modified pro-cytokine is modified pro-IL-36α and has the sequence shown in 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 with SEQ ID:42. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:42.

[0041] In some embodiments, the modified pro-cytokine is a modified pro-IL-36β having the sequence shown in 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 with SEQ ID:43. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:43.

[0042] In some embodiments, the modified pro-cytokine is a modified pro-IL-36γ and includes the sequence shown in 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 with SEQ ID:44. In some embodiments, the cytokine fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:44.

[0043] In some embodiments, the polynucleotide or group of polynucleotides includes a first nucleic acid encoding a modified pro-IL-36α, β, or γ, wherein the modified pro-IL-36α, β, or γ includes, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than cathepsin G, elastase, or protease 3; and (c) an IL-36α, β, or γ fragment.

[0044] In some embodiments, the protease is granzyme B (GzB). In some embodiments, the cleavage site has a sequence as shown in SEQ ID NO:26. In some embodiments, the modified pro-IL-36α, β, or γ includes a sequence as shown in SEQ ID NO:37, 39, or 41.

[0045] In some embodiments, the polynucleotide or group of polynucleotides further includes a second nucleic acid encoding the protease. In some embodiments, the first and second nucleic acids are in a single vector.

[0046] In some embodiments, the IL-36 fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:42, 43, or 44. In some embodiments, the IL-36 fragment is a polypeptide having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:42, 43, or 44. In some embodiments, when the cleavage site is cleaved, the IL-36 fragment can bind to and activate the IL-36 receptor.

[0047] In some embodiments, the polynucleotide or group of polynucleotides further includes 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 co-stimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen.

[0048] 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 the HMFG2 antibody. In some embodiments, the first binding element comprises a V of the HMFG2 antibody. H and V L Structural domain. In some embodiments, the first binding element includes an HMFG2 single-chain variable fragment (scFv).

[0049] In some embodiments, the polynucleotide or group of polynucleotides further includes a fourth nucleic acid encoding a chimeric co-stimulatory receptor (CCR), wherein the CCR includes: (a) a second co-stimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0050] In some embodiments, the second target antigen including 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 an EGF receptor. In some embodiments, the second binding element includes a binding portion of T1E, ICR12, or ICR62.

[0051] In some implementations, the third and fourth nucleic acids are contained in a single vector.

[0052] In some embodiments, the polynucleotide or group of polynucleotides comprises: (a) a first nucleic acid encoding a modified pro-IL-18, wherein the modified pro-IL-18 is the polypeptide shown in SEQ ID NO:27; (b) a second nucleic acid encoding GzB; (c) a third nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: i. a signaling region; ii. a first co-stimulatory 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 co-stimulatory receptor (CCR), wherein the CCR comprises: i. a second co-stimulatory 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 group of polynucleotides comprises a polynucleotide as shown in SEQ ID NO:103.

[0053] In some embodiments, the polynucleotide or group of polynucleotides includes: (a) a first nucleic acid encoding a modified pro-IL-36, wherein the modified pro-IL-36 is a polypeptide represented by 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), wherein the CAR includes: i. a signaling region; ii. a first co-stimulatory 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 co-stimulatory receptor (CCR), wherein the CCR includes: i. a second co-stimulatory signaling region; ii. a transmembrane domain; and iii. a second binding element that specifically interacts with a second epitope on a second target antigen.

[0054] In some embodiments, the first nucleic acid and the third nucleic acid are located 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.

[0055] In one aspect, the present invention provides a method for preparing immune-response cells, the method comprising transfecting or transducing the polynucleotides or a set of polynucleotides provided herein into immune-response cells.

[0056] In another aspect, the present invention provides a method for directing a T-cell-mediated immune response to target cells in a patient who requires T cells, the method comprising administering immune response cells provided in the present invention to the patient.

[0057] In some implementations, the target cells express MUC1.

[0058] In another aspect, the present invention provides a method for treating cancer, the method comprising administering to a patient an effective amount of the immune-response cells provided in the present invention. In some embodiments, the patient's cancer cells express MUC1.

[0059] In some implementations, the patient has breast cancer, ovarian cancer, pancreatic cancer, colorectal cancer, lung cancer, stomach cancer, bladder cancer, multiple myeloma, non-Hodgkin's lymphoma, prostate cancer, esophageal cancer, endometrial cancer, hepatobiliary cancer, duodenal cancer, thyroid cancer, and renal cell carcinoma. In some implementations, the patient has breast cancer. In some implementations, the patient has ovarian cancer.

[0060] In one aspect, the present invention provides a method for γδT cell expression:

[0061] (a) Second-generation chimeric antigen receptors (CARs), including

[0062] i. Signal area;

[0063] ii. Co-stimulatory signal region;

[0064] iii. Transmembrane domains;

[0065] iv. A first binding element that specifically interacts with a first epitope on a first target antigen; and

[0066] (b) A chimeric co-stimulatory receptor (CCR), comprising

[0067] v. A co-stimulatory signal region different from ii;

[0068] vi. transmembrane domain; and

[0069] vii. The second binding element, which specifically interacts with the second epitope on the second target antigen.

[0070] In some implementations, the first target antigen is the same as the second target antigen.

[0071] 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 of an HMFG2 antibody. H and V L Structural domain. In some embodiments, the first binding element includes an HMFG2 single-chain variable fragment (scFv).

[0072] In some embodiments, the second target antigen including 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 an EGF receptor. In some embodiments, the second binding element includes 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 an A20 peptide.

[0073] In another aspect, this disclosure provides a method for preparing immune-response cells, including the step of introducing a transgene. In some embodiments, the transgene encodes a CAR or pCAR. In some embodiments, the transgene encodes a modified procytokine of the IL-1 superfamily, wherein the modified procytokine comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3; and (c) a cytokine fragment of the IL-1 superfamily. In some embodiments, the method further includes a preceding step of activating γδT cells with an anti-γδTCR antibody. In some embodiments, the anti-γδTCR antibody is immobilized. Attached Figure Description

[0074] The accompanying drawings are not necessarily drawn to scale, but are intended to illustrate the principles of various embodiments of the present invention.

[0075] Figure 1 A schematic diagram is provided showing the distinctive features of some of the second-generation CAR and pCAR constructs used in the experiments described herein. The cell membrane is shown as parallel horizontal lines, with the extracellular domain shown above the membrane and the intracellular domain shown below. For pCARs, the chimeric co-stimulatory receptor (CCR) is named first, and the CAR is identified to the right of the slash or forward slash ( / ) marker.

[0076] H2 is a second-generation CAR originally described by Wilkie et al., J. Immunol. 180:4901-9 (2008), the entire contents of which are incorporated herein by reference. From extracellular to intracellular, it consists of a human MUC1-targeting HMFG2 single-chain antibody (scFv) domain, a CD28 transmembrane domain and a co-stimulatory domain, and a CD3z signaling region. Cells transduced with H2 alone are standard second-generation CAR-T cells, specific for the MUC1 tumor-associated glycoform recognized by the HMFG2 single-chain antibody.

[0077] TBB / H is a pCAR. It utilizes MUC1 to target a second-generation "H2" CAR, but with a co-expressed chimeric co-stimulatory receptor (CCR). The CCR in the TBB / H pCAR has a fusion into the CD8α transmembrane domain. T 1E combines a structural domain and a 4-1 BB Co-stimulatory domain. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF), and is a hybrid 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 contents of which are incorporated herein by reference in their entirety.

[0078] Figure 2Cartoon illustrations show modified pro-IL-18 in the various constructs used in this paper. IL-18 is secreted in the form of inactive pro-IL-18. Native pro-IL-18 requires caspase-1 cleavage at a cleavage site between the propeptide and the mature IL-18 protein fragment for activation. 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 at the bottom, the native caspase-1 cleavage site within pro-IL-18 has been replaced by either a caspase-3 cleavage site, a caspase-8 cleavage site, a GzB cleavage site, or an MT1-MMP cleavage site. These modified derivatives are named pro-IL-18(casp 3), pro-IL-18(casp 8), pro-IL-18(GzB), and pro-IL-18(MT1-MMP), respectively. Comparing these to the constitutively active form of IL-18, referred to as "constitutive IL-18," where the mature IL-18 is located downstream of the CD4 signal peptide, further clarifies this distinction.

[0079] Figure 3 Flow cytometry (FACS) results are provided to confirm the co-expression of the second-generation H2 CAR (“H28z”) and TBB CCR (“TIE”) (together, TBB / H pCAR) and the IL-18 variant in T cells transfected with a retroviral vector encoding the second-generation TBB / H pCAR and the IL-18 variant identified at the top of the figure. The expression of both pCAR components in the transfected T cells was analyzed, with the expression of H28z CAR (H-2) and TIE-4-1BB CCR measured separately using FACS.

[0080] Figure 4AThe results showed that transduced T cells secreted pro-IL-18 or modified pro-IL-18 as analyzed by ELISA. Figure 4B The functional activity of secreted IL-18 was demonstrated by the IL-18 responsive colorimetric report assay.

[0081] Figures 5A-5D Provide MDA-MB-468 breast cancer cells with pro-IL-18 or modified pro-IL-18 ( Figure 5A For pro-IL-18; Figure 5B The percentage of survival of pCAR T cells co-cultured with constitutive IL-18; Figure 5C For pro-IL-18 (casp 8); Figure 5D The expression of pro-IL-18 (casp 3) in the x-axis under different effector:target cell (T cell:tumor cell) ratios.

[0082] Figure 6A Provide T cell count, Figure 6B Provides the percentage of survival of MDA-MB-468 breast cancer cells after a specified number of restimulation cycles with T cells expressing TBB / H pCAR and pro-IL-18 or modified pro-IL-18 (consisting of IL-18, pro-IL-18(casp 8) or pro-IL-18(casp3)).

[0083] Figure 7A Provides IL-18 secretion levels as detected by ELISA. Figure 7B Provides IL-18 functional activity in CAR T cells expressing only TBB / H MUC1 pCAR, TBB / H and pro-IL-18 (GzB) or TBB / H and constituent IL-18, whether without stimulation (unstimulated) or with stimulation using an anti-CD3 / CD28 antibody.

[0084] Figure 8 Compare the survival rates of MDA-MB-468 breast cancer cells with untransformed 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) with added granzyme B.

[0085] Figure 9A Provides IL-18 level, Figure 9BProvides the IFN-γ levels secreted by TBB / H pCAR T cells. TBB / H pCAR T cells alone (without expressing exogenous IL-18) and those co-expressing pro-IL-18 or co-expressing pro-IL-18 with additional granzyme B (GzB) were compared.

[0086] Figure 10A Provide the survival percentage of MDA-MD-468 cells. Figure 10B The percentage of BxPC-3 cells that survived after T cell restimulation cycling was provided. Comparisons were made between untransformed T cells, TBB / H pCAR T cells (not expressing exogenous IL-18), and TBB / HpCAR T cells co-expressing pro-IL-18, constituent IL-18, or pro-IL-18 (GzB) combined with added granzyme B.

[0087] Figure 11A-11B Provides tumor target cells with MDA-MD-468 ( Figure 11A ) or BxPC-3 tumor target cells ( Figure 11B The number of successful cycles of antigen stimulation of CAR-T cells. Test cells were TBB / HpCAR T cells that did not express exogenous IL-18 (TBB / H), or TBB / H pCAR T cells that expressed pro-IL-18 or pro-IL-18 (GzB) and additional granzyme B. A successful restimulation cycle was defined as one that resulted in more than 20% cytotoxicity to the target tumor cells.

[0088] Figure 12 The number of T cells provided in the 4th restimulation cycle: pCAR T cells that do not express exogenous IL-18 (TBB / H) or TBB / H pCAR T cells that express pro-IL-18 or pro-IL-18 (GzB) and additional granzyme B.

[0089] Figure 13 Bioluminescent emission (“total flux”) of 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, constituent IL-18 or pro-IL-18 (GzB) and additional granzyme B.

[0090] Figure 14FACS data are provided showing T cell expression of pCAR (upper part) or γδTCR (lower part) in γδT- cells transduced with a retroviral vector encoding TBB / H pCAR (TBB / H) alone or with one of the four IL-18 variants (pro-IL-18+pCAR; pro-IL-18(GzB)+pCAR; composed IL-18+pCAR; or pro-IL-18(GzB)+pCAR and additional granzyme B).

[0091] Figure 15A The survival percentage of MDA-MD-468 cells was provided. Figure 15B The percentage of BxPC-3 cells that survived after co-culturing with TBB / H pCAR T cells at different effector:target ratios at different concentrations were presented. These cells were either uninduced T cells or TBB / H cells that did not express exogenous IL-18 (TBB / H) or TBB / H cells that expressed IL-18 variants (pro-IL-18, constituent IL-18, pro-IL-18(GzB), or pro-IL-18(GzB) and additional granzyme B).

[0092] Figure 16 A structural diagram illustrating the encoding of the pro-IL-18 construct having a cleavage site recognized by MT1-MMP (MMP14) is provided.

[0093] Figures 17A-17C The demonstration used 500,000 T4 CAR T cells ( Figure 17A ), T1NA CAR T cells (T4 signaling defect intracellular domain truncated control, Figure 17B T cells that co-express T4+pro-IL-18 (MT1-MMP) or T cells that co-express T4+pro-IL-18 (MT1-MMP) Figure 17C Bioluminescence emission (“total flux”) in SKOV-3 tumor-injected mice treated with α-phosphorus phosphoprotein (SKOV-3) tumors.

[0094] Figure 18 Structural diagrams illustrating the SFG retroviral constructs encoding TBB / H pCAR and pro-IL-18 are provided.

[0095] Figure 19 Structural diagrams are provided illustrating the SFG retroviral construct encoding TBB / H pCAR and modified pro-IL-18 with a GzB cleavage site, named pro-IL-18(GzB).

[0096] Figure 20 Structural diagrams are provided illustrating the SFG retroviral construct encoding TBB / H pCAR and constitutively active IL-18, named constitutive IL-18.

[0097] Figure 21 Structural diagrams are provided illustrating the SFG retroviral construct encoding TBB / H pCAR and modified pro-IL-18 with a caspase-8 cleavage site, named pro-IL-18(casp 8).

[0098] Figure 22 Structural diagrams are provided illustrating the SFG retroviral construct encoding TBB / H pCAR and modified pro-IL-18 with a caspase-3 cleavage site, named pro-IL-18(casp 3).

[0099] Figure 23 A structural diagram of the SFG retroviral construct is provided, which encodes TBB / H pCAR, a modified pro-IL-18 with a GzB cleavage site, and an additional granzyme B, named pro-IL-18(GzB)+granzyme B.

[0100] Figure 24 Structural diagrams of an SFG retroviral construct encoding T4 pCAR and modified pro-IL-18 with an MP1-MMP cleavage site are provided, named pro-IL-18(MT1-MMP).

[0101] Figure 25 Illustrations of various first-generation CARs, co-stimulatory chimeric receptors, and second-generation CARs are provided for various implementations of the immune-response cells disclosed in this application.

[0102] Figure 26 Illustrations of various third-generation CARs and cis- and trans-co-stimulatory chimeric receptors are provided for use in various embodiments of the immune-response cells disclosed in this application.

[0103] Figure 27 Illustrations of various dual-targeting CARs, inhibitory CARs / NOT gates, combined CARs / AND gates, and TanCARs are provided for use in various implementations of the immune response cells disclosed in this application.

[0104] Figure 28 Illustrations are provided of Go-CART, Trucks, armored CAR, and engineered co-stimulatory CAR in various implementations of the immune response cells disclosed in this application.

[0105] Figure 29 Illustrations of SynNotch / continuous AND-gate CAR and parallel (p)CAR in various implementations that can be used with the immune response cells described in this application are provided.

[0106] Figure 30AThe total flux is shown in tumor-injected mice treated with PBS or 1 million TBB / H pCAR-αβT cells (TBB / H) that do not express exogenous IL-18, TBB / H pCAR-αβT cells that express pro-IL-18 or pro-IL-18 (GzB), and TBB / H pCAR-αβT cells with added granzyme B. Figure 30B The total throughput is shown in tumor-injected mice treated with PBS or 800,000 TBB / H pCAR-γδT cells (TBB / H) that do not express exogenous IL-18, TBB / H pCAR-γδT cells that express pro-IL-18 or pro-IL-18 (GzB), and TBB / H pCAR-γδT cells with added granzyme B. Figure 30C The total flux is shown in tumor-injected mice treated with PBS or 400,000 TBB / H pCAR-γδT cells (TBB / H) that do not express exogenous IL-18, TBB / H pCAR-γδT cells that express pro-IL-18 or pro-IL-18 (GzB), and TBB / H pCAR-γδT cells with added granzyme B. All figures show pooled data from 3 mice.

[0107] Figure 31 The total throughput is shown in three individual tumor-injected mice treated with PBS as a control.

[0108] Figures 32A-32B Provided with 8×10 6 TBB / H pCAR-γδT cells ( Figure 32A ) or 4×10 6 TBB / H pCAR-γδT cells ( Figure 32B Total flux of a single tumor-injected mouse was measured. In each case, T cells did not express exogenous IL-18.

[0109] Figures 33A-33B Provided using 8×10 6 TBB / H pCAR-γδT cells ( Figure 33A ) or 4×10 6 TBB / H pCAR-γδT cells ( Figure 33B Total flux of a single tumor-injected mouse treated with the drug. In each case, T cells produced exogenous pro-IL-18.

[0110] Figures 34A-34B Provided with 8×10 6 TBB / H pCAR-γδT cells ( Figure 34A ) or 4×10 6 TBB / H pCAR-γδT cells ( Figure 34BTotal flux of a single tumor-injected mouse treated with the drug. In each case, T cells produced exogenous pro-IL-18 (GzB) and exogenous granzyme B.

[0111] Figure 35 It shows the use of MUC1 + IL-18 activity was measured in αβT cell culture after stimulation with MDA-MB-468 breast cancer cells (“+468”) or beads coated with anti-CD3 and anti-CD28 antibodies (“aCD3 / 28 beads”). The αβT cells tested were untranslated or untransduced 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 IL-18.

[0112] Figures 36A-36F Bioluminescent emission (“total flux”) in tumor mice with or without αβ T cells. The figure shows the bioluminescence emission (“total flux”) using PBS ( Figure 36A ) or express TBB / H ( Figure 36B ), TBB / H+pro-IL-18 ( Figure 36C ), TBB / H+pro-IL-18(GzB)( Figure 36D IL-18 composed of TBB / H+ Figure 36E ) or TBB / H+pro-IL-18(GzB)+granulase B( Figure 36F Results of treating mice with αβT cells.

[0113] Figure 37 Survival curves of tumor-injected mice treated with αβTBB / H pCAR T cells or αβTBB / H pCAR T cells further expressing pro-IL-18 (GzB), constituent IL-18 or pro-IL-18 (GzB) and granzyme B are shown.

[0114] Figure 38 shows the number of successful restimulation cycles for TBB / H pCAR-T cells (TBB / H) that do not express exogenous IL-18 or for TBB / H pCAR T cells that express pro-IL-18, pro-IL-18(GzB), pro-IL-18(GzB) and additional granzyme B or constituent IL-18. pCAR T cells with MDA-MD-468 tumor target cells ( Figure 38A ) or BxPC-3 tumor target cells ( Figure 38B They are cultured together. A restimulation cycle that produces more than 30% cytotoxicity to the target tumor cells is considered a successful restimulation cycle.

[0115] Figure 39 Display using MUC1 + IL-18 activity was measured after stimulating γδT cells with MDA-MB-468 breast cancer cells (“+468”) or beads coated with anti-CD3 and anti-CD28 antibodies (“aCD3 / 28 beads”). γδT cells expressed (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 IL-18 as a whole without translation or transduction.

[0116] Figure 40A-40F The figure shows bioluminescent emission (“total flux”) in mice injected with tumors with or without γδT cells. The figure also shows the effect of PBS (…). Figure 40A ) or express TBB / H ( Figure 40B ), TBB / H+pro-IL-18 ( Figure 40C ), TBB / H+pro-IL-18(GzB)( Figure 40D IL-18 composed of TBB / H+ Figure 40E ) and TBB / H+pro-IL-18(GzB)+granzyme B ( Figure 40F Results of treating mice with γδT cells.

[0117] Figure 41 Survival curves of tumor-injected mice treated with γδTBB / H pCAR T cells or γδTBB / H pCAR T cells further expressing pro-IL-18 (GzB), constituent IL-18 or pro-IL-18 (GzB) and granzyme B are shown.

[0118] Figure 42A Provide the survival percentage of MDA-MD-468LT cells. Figure 42B The percentage of BxPC-3 LT cells viable after cycle restimulation with TBB / H pCAR T cells is provided. Comparisons were made between TBB / H pCAR T cells (non-expressing exogenous IL-36) and TBB / HpCAR T cells co-expressing pro-IL-36γ and granzyme B, or co-expressing pro-IL-36γ (GzB) and granzyme B.

[0119] Figure 43 shows MDA-MB-468 cells targeting pCAR T cells (TBB / H) that do not express exogenous IL-36, TBB / H pCAR T cells that express pro-IL36γ and granzyme B, or TBB / H pCAR T cells that express pro-IL36γ (GzB) and granzyme B. Figure 43A ) or BxPC-3 cells ( Figure 43B The test provides the number of T cells for each restimulation cycle.

[0120] Figure 44A and Figure 44B Provided with MDA-468-LT cells ( Figure 44A ) or BxPC3-LT cells ( Figure 44B The IFN-γ levels secreted by TBB / H pCAR T cells co-cultured were compared. Comparisons were made between TBB / H pCAR T cells (without expressing exogenous IL-36) and TBB / H pCAR T cells co-expressing pro-IL-36γ and granzyme B, or co-expressing pro-IL-36γ (GzB) and granzyme B.

[0121] Figure 45 The survival rate of MDA-MB-468-LT cells was compared within a certain range of initial effector to target cell ratios (E:T), after co-culturing cancer cells with untransformed T cells, TBB / H pCAR T cells, or TBB / H pCAR T cells expressing pro-IL-36γ and granzyme B or pro-IL-36γ (GzB) and granzyme B.

[0122] Figure 46 The survival rate of BxPC3-LT cells was compared within a certain range of initial effector to target cell ratios (E:T) after co-culturing cancer cells with untransformed T cells, TBB / H pCAR T cells, or TBB / H pCAR T cells further expressing pro-IL-36γ and granzyme B or pro-IL-36γ (GzB) and granzyme B.

[0123] Figures 47A-47D The bioluminescent emission (“total flux”) of tumor mice injected with or without αβ T cells is shown. The figure shows the bioluminescent emission (“total flux”) using PBS ( Figure 47A ), TBB / H ( Figure 47B ), TBB / H+pro-IL-36γ+ granzyme B ( Figure 47C ) or TBB / H+pro-IL-36γ(GzB)+granulase B( Figure 47D Results of mice treated with )

[0124] Figures 48A-48B This provides confirmation of TBB CCR (“TIE”) expression (within TBB / H pCAR), as well as expression in the unconverted ( Figure 48A ) or expression of γδTCR in TBB / H pCARγδT cells ( Figure 48BFlow cytometry (FACS) results.

[0125] Figure 49A The cell expansion fold is provided after 15 days of culturing untransformed or TBB / H pCARγδT cells. Figure 49B The number of cells obtained and cultured from three individual donors at three different time points (day 1, day 8, and day 15) is provided.

[0126] Figures 50A-50B MDA-MB-468 tumor cells were provided. Figure 50A ) or BxPC-3 tumor cells ( Figure 50B Survival rate (%) of tumor cells cultured alone compared with untransformed or TBB / H pCAR-γδT cells (at a 1:1 ratio).

[0127] Figures 51A-51B The number of successful restimulation cycles for untransformed or TBB / H pCARγδT cells was provided. T cells versus MDA-MD-468 tumor target cells (… Figure 51A ) or BxPC-3 tumor target cells ( Figure 51B We will cultivate them together. Figure 51C-51D MDA-MB-468 tumor cells were provided. Figure 51C ) or BxPC-3 tumor cells ( Figure 51D Survival rate (%) of untransformed or TBB / H pCAR-γδT cells during consecutive successful restimulation cycles.

[0128] Figure 52 Bioluminescent emission (“total flux”) over time was provided in NSG mice injected with BxPC-3 tumors treated with PBS, untransduced γδT cells (“UT”), or TBB / H pCARγδT cells (“TBBH”).

[0129] Figure 53 Bioluminescent emission (“total flux”) over time was provided in SCID Beige mice injected with MDA-MB-468 tumors treated with PBS or TBB / H pCARγδT cells (“TBBH”).

[0130] 4. Detailed description

[0131] Details of various embodiments of the present invention are set forth in the following description. Other features, objects, and effects of the invention will be apparent from the specification, the accompanying drawings, and the claims.

[0132] 4.1. Definition

[0133] 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 pertains. As used herein, the following terms have the following meanings.

[0134] The term "IL-1 family member" refers to members of the IL-1 family, including 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".

[0135] The term "procytokine" refers to an inactive precursor of a member of the IL-1 family. A procytokine typically 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 the cleavage site. In a preferred embodiment, the procytokine is pro-IL-18, pro-IL-36α, pro-IL-36β, or pro-IL-36γ.

[0136] The term "pro-IL-18" refers to the natural 24-kDa inactive precursor of IL-18. From the N-terminus to the C-terminus, pro-IL-18 comprises (i) a propeptide, (ii) a cleavage site recognized by cysteine ​​protease 1, and (iii) a mature, biologically active IL-18 protein fragment. In a preferred embodiment, pro-IL-18 is human pro-IL-18, a 24.2 kDa protein of 193 aa. The cDNA sequence of human pro-IL-18 is provided by GenBank / EBI accession number AF077611 (nucleotides 1-579). The protein sequence of human pro-IL-18 is provided by GenBank accession number AAC27787.

[0137] The term "pro-IL-36α" refers to the natural 17.7 kDa inactive precursor of IL-36α. Pro-IL-36α comprises, from the N-terminus to the 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, which is a 158aa, 17.7 kDa protein. The cDNA sequence of human pro-IL-36α is provided by GenBank / EBI database accession number AF201831.1 (nucleotides 1-477). The protein sequence of human pro-IL-36α is provided by GenBank accession number AAY14988.1, as shown herein in SEQ ID NO:36.

[0138] The term "pro-IL-36β" refers to the natural 18.5 kDa inactive precursor of IL-36β. Pro-IL-36β comprises, from the N-terminus to the 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-36β protein fragment. In a preferred embodiment, pro-IL-36β refers to human pro-IL-36β, which is a 164 aa, 18.5 kDa protein. The cDNA sequence of human pro-IL-36β is provided by GenBank / EBI accession number AF200494.1 (nucleotides 1-1190). The protein sequence of human pro-IL-36β is provided by GenBank accession number NP_055253, as shown herein in SEQ ID NO:38.

[0139] The term "pro-IL-36γ" refers to the natural 18.7 kDa inactive precursor of IL-36γ. Pro-IL-36γ comprises, from the N-terminus to the C-terminus, (i) a propeptide, (ii) a cleavage site recognized by neutrophil proteases including protease 3 and elastase, and (iii) a mature, biologically active IL-36γ protein fragment. In a preferred embodiment, pro-IL-36γ refers to human pro-IL-36γ, which is a 169aa, 18.7 kDa protein. The cDNA sequence of human pro-IL-36γ is provided by GenBank / EBI accession number AF200492 (nucleotides 1-1183). The protein sequence of human pro-IL-36γ is provided by GenBank accession number NP_062564, as shown herein in SEQ ID NO:40.

[0140] As used herein, the term "modified precytokine" refers to a protein produced by inserting, deleting, and / or replacing one or more amino acids of a precytokine protein. In a preferred embodiment, the modified precytokine includes a novel cleavage site recognized and cleaved by a protease other than a protease that cleaves an unmodified cytokine progenitor to release a cytokine fragment.

[0141] 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 includes a novel cleavage site recognized by a protease other than caspase-1, and the modified pro-IL-18 is cleavable by a protease other than caspase-1 to release a biologically active IL-18 protein fragment.

[0142] 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 includes a novel cleavage site recognized by proteases other than cathepsin G, elastase, and protease 3, and the modified pro-IL-36 can be cleaved by proteases other than cathepsin G, elastase, or protease 3 to release a biologically active IL-36 protein fragment.

[0143] As used herein, the term "pro-IL-18 ([protease])" refers to a modified pro-IL-18 containing a cleavage site recognized by the protease as specified in parentheses. For example, pro-IL-18(GzB) refers to a modified pro-IL-18 containing a cleavage site that can be cleaved by granzyme B (GzB), pro-IL-18(casp-3) refers to a modified pro-IL-18 containing a cleavage site that can be cleaved by caspase-3, and pro-IL-18(casp-8) refers to a modified pro-IL-18 containing a cleavage site that can be cleaved by caspase-8.

[0144] As used in this article, the term "pro-IL-36(GzB)" refers to modified pro-IL-36 containing a cleavage site recognized by GzB.

[0145] As used in this article, the term "cleavage site" refers to an amino acid sequence that can be recognized by a protease. As used herein, a cleavage site that a protease "recognizes" is an amino acid sequence that can be cleaved by a protease under conditions present in vivo or achievable.

[0146] As used herein, the terms "bioactive cytokine fragment" and "cytokine fragment" refer to a bioactive polypeptide derived from a protease that recognizes a cleavage site upstream (at its N-terminus) of the cytokine fragment. Biological activity means that the cytokine fragment can bind to and activate the corresponding receptor. The cytokine fragment can be a fragment of a natural cytokine protein or a modified fragment thereof. In some embodiments, the cytokine fragment exhibits improved biological activity compared to the natural mature cytokine. In some embodiments, the cytokine fragment refers to the IL-18 fragment or the IL-36 fragment as defined below.

[0147] As used herein, the terms "IL-18 fragment" and "IL-18 protein fragment" refer to biologically active IL-18 peptides produced by the cleavage of protease protease at a cleavage site upstream (N-terminus) of the IL-18 fragment. Biological activity means that the IL-18 fragment can bind to and activate the IL-18 receptor. The IL-18 fragment can be a naturally occurring, mature IL-18 protein fragment or a modified version thereof. In some embodiments, the IL-18 fragment exhibits improved biological activity compared to naturally occurring, mature IL-18.

[0148] As used herein, the terms "IL-36 fragment" and "IL-36 protein fragment" refer to biologically active IL-36 polypeptides produced by the cleavage of protease protease at a cleavage site upstream (N-terminus) of the IL-36 fragment. Biological activity means that the IL-36 fragment can bind to and activate the IL-36 receptor. The IL-36 fragment can be a naturally occurring, mature IL-36 protein fragment or a modification thereof. In some embodiments, the IL-36 fragment exhibits improved biological activity compared to naturally occurring, mature IL-36. The IL-36 fragment can refer to the mature IL-36α, β, or γ protein.

[0149] The term “IL-18 variant” used in this article refers collectively to pro-IL-18 protein, modified pro-IL-18, and IL-18 fragments, including naturally occurring mature IL-18 fragments.

[0150] The term “IL-36 variant” as used in this article refers collectively to pro-IL-36 protein, modified pro-IL-36 protein, and IL-36 fragments, including naturally occurring mature IL-36α, β, or γ fragments.

[0151] As described herein, regarding the binding elements of engineered T-cell receptors (TCRs) or chimeric antigen receptors (CARs), and immune-responding cells engineered to express such TCRs or CARs, the terms “recognition,” “specific binding,” “specific binding to,” “specific interaction,” “specific to,” “selective binding,” “selective interaction,” and “selective to” refer to a specific antigen or its epitope—which can be a protein antigen, a glycopeptide antigen, or a peptide MHC complex—that binds differently within a measurable range to a nonspecific or nonselective interaction (e.g., with a non-target molecule). For example, specific binding can be measured by measuring binding to a target molecule and comparing it to binding to a non-target molecule. Specific binding can also be determined by competing with a control molecule that mimics the epitope recognized on a target molecule.

[0152] 4.2. Other Interpretive Conventions

[0153] In the claims, the article "a" or similar can refer to one or more, unless otherwise indicated or clearly apparent from the context. A claim or description containing "or" among one or more members of a group is considered satisfied if one, more than one, or all members of that group are present in, used in, or otherwise associated with a given product or process, unless otherwise indicated or clearly apparent from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The invention includes embodiments in which multiple or all members of a group are present in, used in, or otherwise associated with a given product or process.

[0154] It should also be noted that the term "comprising" is intended to be open-ended, allowing 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.

[0155] If a range is given, the endpoints are included. Furthermore, it should be understood that, unless otherwise indicated from the context and understanding of one of ordinary skill in the art or otherwise apparent, in different embodiments of the invention, values ​​represented as ranges may take any particular value or subrange within the range, up to one-tenth of the lower limit of the range, unless the context explicitly specifies otherwise.

[0156] All sources cited, such as references, publications, databases, database entries, and techniques cited herein, are incorporated herein by reference, even if not explicitly stated in the citation. In the event of any conflict between the statements of cited sources and this application, the statements in this application shall prevail.

[0157] There are no restrictions on chapter and table titles.

[0158] 4.3. Immune Response Cells

[0159] In a first aspect, an immune-response cell is provided. The immune-response cell expresses a modified procytokine of the IL-1 superfamily, wherein the modified procytokine comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3; and (c) a cytokine fragment of the IL-1 superfamily.

[0160] In some embodiments, immune response cells express modified pro-IL-18, wherein the modified pro-IL-18 comprises, from the N-terminus to the 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.

[0161] In some embodiments, immune-responding cells express modified pro-IL-36, wherein the modified pro-IL-36 comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by proteases other than cathepsin G, elastase, and protease 3; and (c) a biologically active IL-36α, β, or γ fragment.

[0162] 4.3.1. Cells

[0163] In a typical implementation, the immune response cells are T cells.

[0164] In some embodiments, the immune response cells are αβ T cells. In a particular embodiment, the immune response cells are cytotoxic αβ T cells. In a particular embodiment, the immune response cells are αβ helper T cells. In a particular embodiment, the immune response cells are regulatory αβ T cells (Tregs).

[0165] In some embodiments, the immune response cells are γδT cells. In a particular embodiment, the immune response cells are Vδ2+γδT cells. In a particular embodiment, the immune response cells are Vδ2–T cells. In a particular embodiment, the Vδ2–T cells are Vδ1. + cell.

[0166] In some implementations, the immune response cells are natural killer (NK) cells.

[0167] In some embodiments, the immune response cells do not express additional exogenous proteins. In other embodiments, the immune response cells are engineered to express additional exogenous proteins, such as engineered T-cell receptors (TCRs) or chimeric antigen receptors (CARs). Immune response cells that further express engineered TCRs and CARs will be described further below.

[0168] In some embodiments, the immune response cells are obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, the immune response cells are obtained from a tumor. In a particular embodiment, the immune response cells obtained from the tumor are tumor-infiltrating lymphocytes (TILs). In a particular embodiment, the TILs are αβT cells. In other particular embodiments, the TILs are γδT cells, especially Vδ2–γδT cells.

[0169] 4.3.2. Modified pro-IL-18

[0170] In some implementations, immune response cells express modified pro-IL-18.

[0171] 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 the bioactive IL-18 fragment.

[0172] 4.3.2.1. Propeptide

[0173] In a typical embodiment, the propeptide is an unmodified natural propeptide of the pro-IL-18 protein. In a specific embodiment, the propeptide is an unmodified natural propeptide of the human pro-IL-18 protein.

[0174] In other embodiments, the propeptide is modified from the native propeptide of the pro-IL-18 protein. In some embodiments, the modified propeptide contains one or more amino acid modifications compared to the native pro-IL-18 propeptide. In some embodiments, the propeptide is a propeptide derived from a non-pro-IL-18 protein. In some embodiments, the propeptide has a non-naturally synthesized amino acid sequence.

[0175] In some embodiments, the propeptide is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:25.

[0176] 4.3.2.2. Cutting site

[0177] The cleavage site in the modified pro-IL-18 is recognized by proteases other than caspase-1.

[0178] In a typical embodiment, the modified pro-IL-18 contains only a single cleavage site recognized by proteases other than caspase-1. In other embodiments, multiple cleavage sites recognized by proteases other than caspase-1 are introduced. In these embodiments, the multiple cleavage sites can be cleavage sites recognized by the same or different proteases other than caspase-1.

[0179] In various embodiments, a cleavage site recognized by a protease other than caspase-1 is introduced between (a) the propeptide of caspase-1 and the cleavage site, (b) in place of the cleavage site of caspase-1, or (c) between the cleavage site of caspase-1 and the IL-18 fragment.

[0180] In some embodiments, the cleavage site replaces the caspase-1 cleavage site of pro-IL-18. In some embodiments, the cleavage site is a site other than the caspase-1 cleavage site.

[0181] In a typical embodiment, the cleavage site in the modified pro-IL-18 is selected from protease cleavage sites known in the art. In a typical embodiment, the protease is a protease known to be expressed in activated T cells or NK cells. In some embodiments, the cleavage site is composed of granzyme B (GzB), caspase-3, caspase-8, or membrane type 1 matrix metalloproteinase (MT1-MMP, also known as MMP14), alternative tumor-associated matrix metalloproteinases (MMP1-13), detegrin and metalloproteinase (ADAM) family members (especially ADAM 10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-associated peptidase (KLK), such as KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, heparin, or urokinase plasminogen activator (Dudani et al., “Harnessing protease activity to improve cancercare,” Annu. Rev. Cancer Biol., 2:353-76 (2018)). In a particular embodiment, the cleavage site is recognized by granzyme B (GzB). In a particular embodiment, the cleavage site is recognized by caspase-3. In a particular embodiment, the cleavage site is recognized by caspase-8. In a particular embodiment, the cleavage site is recognized by MT1-MMP.

[0182] In some embodiments, the cleavage site comprises sequences selected from SEQ ID NO:26, 28, 30, and 32. In some embodiments, the modified pro-IL-18 comprises sequences selected from SEQ ID NO:27, 29, 31, and 33.

[0183] In other implementations, the cleavage site is a synthetic cleavage site that is not naturally occurring.

[0184] 4.3.2.3.IL-18 fragment

[0185] In various embodiments, the IL-18 fragment is a natural IL-18 fragment. In a preferred embodiment, the natural IL-18 fragment is a human IL-18 fragment.

[0186] In other embodiments, the IL-18 fragment is modified from the 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 via a protease cleavage site. In various embodiments, the IL-18 fragment has biological activity similar to, less than, or superior to that of the native mature IL-18 protein.

[0187] In some embodiments, the IL-18 fragment is a polypeptide having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID: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 with SEQ ID:24. In some embodiments, the modified pro-IL-18 protein is expressed from a foreign sequence introduced into T cells. In some embodiments, the foreign sequence is selected from the group consisting of sequences SEQ ID NO:102, 103, 105, 107, 109, 111, and 113. In some embodiments, the foreign sequence is a coding sequence cloned in an expression vector (e.g., a viral vector or a non-viral vector).

[0188] 4.3.3. Modified pro-IL-36

[0189] In some implementations, immune response cells express modified pro-IL-36α, β, or γ proteins.

[0190] 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 protease 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 the bioactive IL-36α, β, or γ fragment.

[0191] 4.3.3.1. Propeptide

[0192] In a typical embodiment, the propeptide is an unmodified natural propeptide of the pro-IL-36α, β, or γ protein. In a specific embodiment, the propeptide is an unmodified natural propeptide of the human pro-IL-36 protein.

[0193] In other embodiments, the propeptide is modified from the native propeptide of the pro-IL-36 protein. In some embodiments, the modified propeptide contains one or more amino acid modifications compared to the native pro-IL-36 propeptide. In some embodiments, the propeptide is a propeptide derived from a non-pro-IL-36 protein. In some embodiments, the propeptide has a non-naturally synthesized amino acid sequence.

[0194] 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).

[0195] 4.3.3.2. Cutting site

[0196] The cleavage site in the modified pro-IL-36 is recognized by proteases other than cathepsin G, elastase, and protease 3.

[0197] In a typical embodiment, the modified pro-IL-36 contains only a single cleavage site recognized by proteases other than cathepsin G, elastase, and protease 3. In other embodiments, multiple cleavage sites recognized by proteases other than cathepsin G, elastase, and protease 3 are introduced. In these embodiments, the multiple cleavage sites can be cleavage sites recognized by the same or different proteases other than cathepsin G, elastase, and protease 3.

[0198] In various embodiments, a cleavage site recognized by a protease other than cathepsin G, elastase, and protease 3 is introduced between (a) the propeptide of cathepsin G, elastase, or protease 3 and the cleavage site, (b) in lieu of the cleavage site of cathepsin G, elastase, or protease 3, or (c) between the cleavage site of cathepsin G, elastase, or protease 3 and the IL-36 fragment.

[0199] In some embodiments, the cleavage site replaces the cleavage site of cathepsin G, elastase, or protease 3 naturally present in pro-IL-36α, β, or γ. In some embodiments, the cleavage site is a complement to the cleavage site of cathepsin G, elastase, and / or protease 3 naturally present in pro-IL-36α, β, or γ.

[0200] In a typical embodiment, the cleavage site in the modified pro-IL-36 is selected from protease cleavage sites known in the art. In a typical embodiment, the protease is a protease known to be expressed in activated T cells or NK cells. In some embodiments, the cleavage site is composed of granzyme B (GzB), caspase-3, caspase-8, or membrane type 1 matrix metalloproteinase (MT1-MMP, also known as MMP14), alternative tumor-associated matrix metalloproteinases (MMP1-13), detegrin and metalloproteinase (ADAM) family members (especially ADAM 10 or ADAM17), cathepsin B, L, or S, fibroblast activation protein (FAP), kallikrein-associated peptidase (KLK), such as KLK2, 3, 6, or 7, dipeptidyl peptidase (DPP) 4, heparin, or urokinase plasminogen activator (Dudani et al., “Harnessing protease activity to improve cancercare,” Annu. Rev. Cancer Biol., 2:353-76 (2018)). In a particular embodiment, the cleavage site is recognized by granzyme B (GzB). In a particular embodiment, the cleavage site is recognized by caspase-3. In a particular embodiment, the cleavage site is recognized by caspase-8. In a particular embodiment, the cleavage site is recognized by MT1-MMP.

[0201] In some embodiments, the cleavage site comprises sequences selected from SEQ ID NO:26, 28, 30, and 32. In some embodiments, the modified pro-IL-36 comprises sequences selected from SEQ ID NO:37, 39, and 41.

[0202] In other implementations, the cleavage site is a synthetic cleavage site that is not naturally occurring.

[0203] 4.3.3.3.IL-36 fragment

[0204] In various embodiments, the IL-36 fragment is a natural IL-36α (SEQ ID NO:42), β (SEQ ID NO:43), or γ (SEQ ID NO:44) fragment. In a preferred embodiment, the natural IL-36 fragment is a human IL-36 fragment.

[0205] In other embodiments, the IL-36 fragment is modified from the 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 via a protease cleavage site. In various embodiments, the IL-36 fragment has biological activity similar to, less than, or superior to that of the native mature IL-36α, β, or γ protein.

[0206] In some embodiments, the IL-36α, β, or γ fragments are polypeptides having at least 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:42, 43, or 44, respectively. In some embodiments, the IL-36α, β, or γ fragments are polypeptides having at least about 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity with SEQ ID:42, 43, or 44, respectively. In some embodiments, the modified pro-IL-36 protein is expressed from a foreign sequence introduced into T cells. In some embodiments, the foreign sequence is a coding sequence cloned in an expression vector (e.g., a viral vector or a non-viral vector).

[0207] 4.3.4. Expression of protease

[0208] In some implementations, immune response cells are engineered to further express a protease that recognizes the cleavage site of co-expressed modified pro-IL-18 or modified pro-IL-36.

[0209] In some implementations, the protease is selected from the group consisting of GzB, caspase-3, caspase-8 and MT1-MMP.

[0210] In a particular embodiment, the expressed protease is GzB. In a preferred embodiment, the expressed protease is human GzB. In a particular embodiment, the expressed protease includes, for example, SEQ ID NO:20 or its modifications.

[0211] In a particular embodiment, the expressed protease is caspase-3. In a preferred embodiment, the expressed protease is human cysteine ​​protease-3. In a particular embodiment, the expressed protease includes, for example, SEQ ID NO:21 or its modifications.

[0212] In a particular embodiment, the expressed protease is caspase-8. In a preferred embodiment, the expressed protease is human cysteine ​​protease-8. In a particular embodiment, the expressed protease includes, for example, SEQ ID NO:22 or its modifications.

[0213] In a particular embodiment, the expressed protease is MT1-MMP. In a preferred embodiment, the expressed protease is human MT1-MMP. In a particular embodiment, the expressed protease includes, for example, SEQ ID NO:23 or its modifications.

[0214] In some implementations, the expressed protease is an alternative tumor-associated matrix metalloproteinase (MMP1-13), a detegrin and metalloproteinase (ADAM) family member (especially ADAM 10 or ADAM17), cathepsin B, L or S, fibroblast activating protein (FAP), kallikrein-associated peptidase (KLK), such as KLK2, 3, 6 or 7, dipeptidyl peptidase (DPP) 4, heparin or urokinase plasminogen activator (Dudani et al., “Harnessing protease activity to improve cancer care,” Annu. Rev. Cancer Biol., 2:353-76 (2018)).

[0215] The expressed protease is expressed from a foreign sequence introduced into an expression vector into immune response cells. In some embodiments, immune response cells express modified procytokines and proteases from a single expression vector. In some embodiments, immune response cells express modified procytokines and proteases from multiple expression vectors. In a particular embodiment, immune response cells express modified procytokines from a first expression vector and proteases from a second expression vector.

[0216] 4.3.5.CAR

[0217] In a typical implementation, immune response cells are engineered to further express chimeric antigen receptors (CARs).

[0218] 4.3.5.1. CAR Specificity

[0219] In a typical implementation, the CAR is specific to at least one antigen present in cancer. In a typical implementation, the CAR is specific to at least one antigen present in solid tumors.

[0220] In various embodiments, the antigen is human telomerase reverse transcriptase (hTERT), survivin, mouse dual-microsome 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 activating factor and CAML interacting factor (TACI) or related proteins. For example, in some embodiments, the target antigen is or is associated with BAFFR or TACI. In some embodiments, the target antigen is CD33 or TIM-3. In some implementations, it is CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, or CD362.

[0221] In some implementations, CAR targets α-folate receptor 5T4, αvβ6 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 homologs and heterodimers, EGFRvIII, EGP2, EGP40, EPCAM, EPA2, EPCAM, FAP, fetal AchR, and FR. alph. a. GD2, GD3, phosphatidylinositol proteoglycan-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, IL-13R.α2, Lambda, Lewis-Y, Kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEMs, or VEGFR2 are specific.

[0222] In some implementations, CAR targets TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRIII, 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, PDGFRβ, SSEA-4, CD20, folate receptor α, ERB2 (Her2 / neu), MUC1, EGFR, NCAM, prostaglandins, PAP, ELF2M, and Ep... hrinB2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucose 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, MAGEA1, ETV6-AML, Sperminin 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-associated antigen 1, p53, p53 mutant, prostaglandins, staminatin and telomerase, PCTA-1 / Galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, 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, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, or IGLL1 are specific.

[0223] In some embodiments, the CAR is specific for the MUC1 target antigen. In a particular embodiment, the CAR is specific for the tumor-associated MUC1 epitope. In a particular embodiment, the targeting domain of the CAR includes the CDR 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), the entire contents of which are incorporated herein by reference. In some embodiments, the CAR includes the V of the HMFG2 antibody. H and V L Structural domain. In some implementations, the CAR includes HMFG2 single-stranded variable fragments (scFv).

[0224] In some implementations, the CAR is specific for ErbB homologs and / or heterodimers. In specific implementations, the targeting domain of the CAR includes various ErbB peptide ligands, T1E. T1E is a chimeric peptide derived from transforming growth factor-α (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 entire contents of which are incorporated herein by reference.

[0225] 4.3.5.2. CAR Configuration

[0226] In some implementations, the CAR is a first-generation CAR. First-generation CARs can provide TCR-like signaling, most commonly using the intracellular signaling domains of CD3 zeta (CD3z or CD3ζ) or Fcer1g, thereby stimulating tumor-killing function. However, without accompanying co-stimulatory signals, the involvement of the CD3z chain fusion receptor may be insufficient to induce substantial IL-2 secretion and / or T cell proliferation. In physiological T cell responses, optimal lymphocyte activation may require the involvement of one or more co-stimulatory receptors, such as CD28 or 4-1BB. In some implementations, such as the first-generation CAR disclosed by Eshhar et al., “Specific activation and targeting of cytotoxic lymphocytes through chimeric singlechains 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 the co-stimulatory chimeric receptor disclosed by 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 lymphocytes,” J. Exp. Med. 188(4):619-26 (1998), expressed in the immune response cells described herein ( Figure 25 ); The entire contents of these two references are incorporated into this paper.

[0227] In some implementations, the CAR is a second-generation CAR. In addition to antigen-dependent TCR-like signaling, second-generation CARs can transduce functional antigen-dependent co-stimulatory signals in primary human T cells, thereby allowing T cells to proliferate while maintaining tumor-killing activity. Second-generation CARs typically use co-stimulatory domains (synonyms, co-stimulatory signaling regions) derived from CD28 or 4-1BB to provide co-stimulation. The combined delivery of co-stimulation and CD3 zeta signaling can make second-generation CARs functionally superior to first-generation CARs. US Patent No. 7446190 discloses an exemplary second-generation CAR that can be effectively expressed in the immune response cells described herein; 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 inseries with signals from the TCR zeta chain,” J. Immunol. 172(1):104-13 (2004); and Imai et al., “Chimeric receptors with 4-1BB signaling capacity provokepotent cytotoxicity against acute lymphoblastic leukemia,”Leukemia 18(4):676-84(2004), incorporated herein by reference.

[0228] Figure 25 This paper provides yet another exemplary second-generation CAR that can be effectively expressed in the immune response cells described herein.

[0229] The examples in this article provide additional second-generation CARs that can be effectively expressed in the immune response cells described herein. In certain embodiments, second-generation CARs named "H," "H2," or "H28z" are used. The H2 CAR consists of a MUC-1 targeting an HMFG2 single-chain antibody, a CD28 transmembrane and co-stimulatory domain, and a CD3z signaling region, from extracellular to intracellular. See [link to documentation]. Figure 1 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), describe the H2 CAR. This article is incorporated herein by reference in its entirety. In a particular implementation, a second-generation CAR called T1E28z is used. The T1E28z CAR consists of an ErbB-targeting T1E peptide, a CD28 transmembrane and co-stimulatory domain, and a CD3z signaling region, extending from the extracellular to the intracellular space. See [link to relevant documentation]. Figure 1 The T1E28z second-generation CAR is described in Davies, “Flexible targeting of ErbB dimers that drive tumourigenesis by using genetically engineered T cells,” Mol. Med. 18:565-576 (2012). This article is incorporated herein by reference in its entirety.

[0230] In some implementations, a third-generation CAR is used. A third-generation CAR can combine multiple co-stimulatory domains (synonyms, co-stimulatory signal regions) with TCR-like signal domains (synonyms, signal regions) in the CIS, such as CD28+4-1BB+CD3z or CD28+OX40+CD3z, to further enhance potency. In some implementations, the third-generation CAR includes co-stimulatory domains arranged in tandem within the CAR's inner domain, typically located upstream of CD3z or its equivalent. 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), disclose some exemplary third-generation CARs that can be effectively expressed in the immune response cells described herein; Geiger et al., “Integrated src kinase and costimulatory activity enhances signal transduction through single-chain chimeric receptors in Tlymphocytes,” Blood 98:2364-71 (2001); Wilkie et al., “Retargeting of human T cells to tumor-associated MUC1: the evolution of achimeric antigen receptor,” J. Immunol. 180(7):4901-9 (2008), the disclosures of which are incorporated herein by reference in their entirety. Figure 26 In some implementations, CARs using cis and trans co-stimulatory signals are employed, as described 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), which is incorporated herein by reference. Figure 26 Provided by China.

[0231] Other CAR formats available and known in the art can be expressed in various implementations of the immune response cells described herein. In particular Figure 27-29Other CAR forms that can be expressed in the immunosuppressive cells disclosed herein have been disclosed, 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) divert off-target immunotherapy responses,” Sci. Transl. Med. 5(215): 215ra172 (2013); Kloss et al., “Combinatorial antigen recognition with balanced signaling promotes selective tumoreradication by engineered T cells,” Nat. Biotechnol. 31(1): 71-6 (2013); Grada et al., “TanCAR: A Novel Bispecific Chimeric Antigen Receptor for CancerImmunotherapy,” Mol. Ther.25(9):2176-2188(2017); Chmielewski et 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), incorporated herein by reference.

[0232] 4.3.5.2.1. pCAR configuration

[0233] In a particular implementation, parallel CAR (pCAR) is expressed in immune-responding cells.

[0234] In the pCAR implementation, immune-responding cells are engineered to express two constructs in parallel: a second-generation CAR and a chimeric co-stimulatory receptor (CCR). The second-generation CAR, from intracellular to extracellular domains, includes: (a) a signaling region; (b) a first co-stimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen. From intracellular to extracellular, the CCR includes: (a) a co-stimulatory 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, such as CD3z. In some implementations, the co-stimulatory domain of the CCR (the second co-stimulatory domain) differs from the co-stimulatory domain of the CAR (the first co-stimulatory domain). In some implementations, the second epitope differs from the first epitope. Compared to first-generation, second-generation, and third-generation CAR-T cells, parallel CAR (pCAR) engineered T cells exhibit higher activity and anti-exhaustion capabilities. See US pre-grant publication 2019 / 0002521, which is incorporated herein by reference in its entirety.

[0235] In some implementations, the second target antigen is different from the first target antigen. In some implementations, the second target antigen is the same as the first target antigen.

[0236] In some embodiments, the first antigen is the MUC1 antigen. In a particular embodiment, the first epitope is a tumor-associated epitope on the MUC1 target antigen. In some embodiments, the first binding element comprises a CDR of the HMFG2 antibody. In some embodiments, the first binding element comprises a V of the HMFG2 antibody. H and V L Structural domain. In some implementations, the first binding element includes an HMFG2 single-chain variable fragment (scFv).

[0237] In a specific implementation, the CAR is an H2 second-generation CAR whose extracellular to intracellular domains include MUC-1 targeting the HMFG2 single-chain antibody, the CD28 transmembrane and co-stimulatory domains, and the CD3z signaling region. See Figure A. Wilkie et al., “Retargeting of human T cells to tumor-associated MUC1: the evolution of achimeric antigen receptor,” J. Immunol. 180:4901-9 (2008), is incorporated herein by reference in its entirety.

[0238] In a specific implementation, the CAR is a second-generation T1E28z CAR, whose extracellular to intracellular domains include ErbB-targeting T1E peptides, CD28 transmembrane and co-stimulatory domains, and CD3z signaling domains. See Figure A. The second-generation T1E28z CAR is described in Davies, “Flexible targeting of ErbB dimers that drive tumourigenesis by using genetically engineered T cells,” Mol. Med. 18:565-576 (2012), which is incorporated herein by reference in its entirety.

[0239] In some embodiments, the second target antigen is selected from the group consisting of ErbB homodimers and heterodimers. In a particular embodiment, the second target antigen is HER2. In a particular embodiment, the second target antigen is an EGF receptor. In some embodiments, the second binding element includes a binding portion of T1E, ICR12, or ICR62.

[0240] In some implementations, pCARs “TBB / H” or “I12BB / H” are expressed in immune-responding cells. These pCARs utilize MUC1 to target second-generation “H” (synonymous with “H2”) CARs, but have different co-expressed CCRs. The CCR in the TBB / H pCAR has a T1E-binding domain fused to the CD8α transmembrane domain and a 4-1 BBCo-stimulatory domain. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF), and is a hybrid 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 ErbBdimers that drive tumourigenesis by using genetically engineered T cells,” Mol. Med. 18:565-576 (2012), the entire contents of which are incorporated herein by reference. The CCR in I12BB / H pCAR has an ICR12 binding domain fused to the CD8α transmembrane domain and the 4-1BB co-stimulatory domain. ICR12 is HER2 (ErbB2) targeting the single-chain antibody 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), which is incorporated herein by reference in its entirety. In some implementations, “TBB / H” or other PCARs as described in PCT / GB2020 / 050590 may be used, which is incorporated herein by reference in its entirety.

[0241] In some implementations, ABB / H and I62BB / H pCARs are used. Both ABB / H and I62BB / H CARs are second-generation “H”-based MUC1 CARs. The CCR in the ABB / H pCAR contains an A20 peptide fused to a CD8α transmembrane domain and a 4-1BB co-stimulatory domain. The A20 peptide binds to αvβ6 integrin. See DiCara et al., “Structure-function analysis of Arg-Gly-Asp helix motifs in alpha v beta 6 integrin ligands,” JBiol Chem. 282(13):9657-9665 (2007), incorporated herein by reference in its entirety. The CCR in the I62BB / H pCAR has an ICR62 binding domain fused to both a CD8α transmembrane domain and a 4-1BB co-stimulatory domain. ICR62 is an EGFR-targeting single-chain antibody 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.

[0242] In some embodiments, immune-responding cells express modified procytokines (e.g., modified pro-IL-18 or modified pro-IL-36) from a single expression construct, optionally expressed proteases, and optionally CAR or pCAR. In some embodiments, immune-responding cells express modified procytokines (e.g., modified pro-IL-18 or modified pro-IL-36), optionally proteases, CAR, or pCAR from multiple different constructs.

[0243] 4.3.5.2.2. Signal Area

[0244] The CAR construct includes a signaling region (i.e., a TCR-like signaling region). In some embodiments, the signaling region contains an immune receptor tyrosine activation motif (ITAM), as described in Love et al., “ITAM-mediated signaling by the T-cell antigen receptor,” Cold Spring Harbor Perspect. Biol 2(6)1a002485 (2010). In some embodiments, the signaling region includes an intracellular domain of the human CD3 zeta chain (as described in U.S. Patent No. 7,446,190, which is incorporated herein by reference) or a variant thereof. In a particular embodiment, the signaling region includes a domain spanning amino acid residues 52-163 of the full-length human CD3 zeta chain. The CD3 zeta chain contains many known polymorphic forms (e.g., Sequence IDs: gb|AAF34793.1 and gb|AAA60394.1), all of which are useful herein and are shown as SEQ ID NOs: 1 and 2, respectively.

[0245] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 1);

[0246] RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 2).

[0247] Alternative signal regions for the CD3 zeta domain include, for example, FceR1γ, CD3ε, and multiple ITAM. See Eshhar Z et al., "Specific activation and targeting of cytotoxic lymphocytes throughchimeric single chains consisting of antibody-binding domains and the gammaor zeta subunits of the immunoglobulin and T-cell receptors," Proc Natl AcadSci USA 90:720-724 (1993); Nolan et al., "Bypassing immunization: optimized design of "designer T cells" against carcinoembryonic antigen(CEA)-expressingtumors, 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 ITAMmultiplicity on T “Cell receptors can control potency and selectivity to ligand density,” Sci Signal 11(531)eaan1088(2018), the entire contents of which are incorporated herein by reference.

[0248] 4.3.5.2.3. Co-stimulation signal region

[0249] In CAR, the co-stimulatory signaling region is appropriately located between the signaling region and the transmembrane structural domain, and is far from the binding element.

[0250] In CCR, the co-stimulatory signal region is appropriately located near the transmembrane structural domain and far from the binding element.

[0251] Suitable co-stimulatory signaling regions are well known in the art and include co-stimulatory signaling regions of B7 / CD28 family members, such as 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, such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3, or LILRB4; or tumor necrosis factor (TNF) superfamily members, such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, lymphotoxin α, OX40, RELT, TACI, TL1A, TNFα, or TNF. RII; or SLAM family members, such as 2B4, BLAME, CD2, CD2F-10, CD48, CD8, CD84, CD229, CRACC, NTB-A, or SLAM; or TIM family members, such as TIM-1, TIM-3, or TIM-4; or other co-stimulatory molecules, such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, integrin α4β1, integrin α4β7 / LPAM-1, LAG-3, or TSLP R. See Mondino A et al., "Surface proteins involved in T cell costimulation," J LeukocBiol. 55:805-815 (1994); Thompson CB, "Distinct roles for the costimulatoryligands 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 andCD40-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 therapeutictargets 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 pivotalrole in tumor immunity," J Immunother. 30:251-260(2007); Gavrieli M et al., "BTLAabd 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," NatCommun. 4:2043(2013); Omar HA et al., "Tacking molecular targets beyond PD-1 / PD-L1:Novel approaches to boost patients’response to cancer immunotherapy," CritRev Oncol Hematol. 135:21-29(2019); Hashemi M et al., "Association of PDCD6polymorphisms with the risk of cancer:Evidence from a meta-analysis," Oncotarget. 9:24857-24868(2018); Kang X et al., "Inhibitory leukocyteimmunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors," Cell Cycle. 15:25-40 (2016); Watts TH, "TNF / TNFR family members incostimulation of T cell responses," Annu Rev Immunol. 23:23-68 (2005); BrycesonYT 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 OpinImmunol. 7:389-395 (1995); Wingren AG et al., "T cell activation pathways: B7, LFA-3, and ICAM-1shape unique T cell profiles,” Crit Rev Immunol. 15:235-253 (1995), the entirety of which is incorporated herein by reference.

[0252] The co-stimulatory signaling region can be selected based on the specific purpose of the immune response cells. Specifically, the co-stimulatory signaling region can be selected for additional or synergistic action. In some embodiments, the co-stimulatory signaling region is selected from the co-stimulatory signaling regions of CD28, CD27, ICOS, 4-1BB, OX40, CD30, GITR, HVEM, DR3, and CD40.

[0253] In a particular implementation, one co-stimulatory signal region of pCAR is the co-stimulatory signal region of CD28, and the other is the co-stimulatory signal region of 4-1BB.

[0254] 4.3.5.2.4. Transmembrane domain

[0255] The transmembrane domains of the CAR and CCR constructs may be the same or different. In the currently preferred embodiment, when the CAR and CCR constructs are expressed by a single vector, the transmembrane domains of the CAR and CCR are different to ensure construct segregation on the cell surface. Choosing different transmembrane domains can also enhance the stability of the expression vector, as the inclusion of direct repeat nucleic acid sequences in the viral vector facilitates rearrangement and the deletion of sequences between the direct repeat sequences. In embodiments where the transmembrane domains of the pCAR and CCR are chosen to be the same, this risk can be mitigated by modifying or “wobbling” the selection of codons encoding the same protein sequence.

[0256] Suitable transmembrane domains known in the art include, for example, CD8α, CD28, CD4, or CD3z. Choosing CD3z as the transmembrane domain may result in the CAR or CCR binding to other elements of the TCR / CD3 complex. This association may recruit more ITMs, but it may also lead to competition between the CAR / CCR and endogenous TCR / CD3.

[0257] 4.3.5.2.5. Co-stimulatory signal domain and transmembrane structural domain

[0258] In embodiments where the co-stimulatory signaling domain of CAR or CCR is or includes the co-stimulatory signaling domain of CD28, the CD28 transmembrane domain represents a suitable, generally preferred, transmembrane domain. The full-length CD28 protein is a 220-amino acid protein as shown in SEQ ID NO:3, wherein the transmembrane domain is shown in bold.

[0259]

[0260] In some embodiments, one of the co-stimulatory signaling domains is based on the hinge region, and suitably also on the transmembrane and intracellular domains of CD28. In some embodiments, the co-stimulatory signaling domain includes amino acids 114-220 as in SEQ ID NO:3, as shown in SEQ ID NO:4 below:

[0261] IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 4).

[0262] In a particular embodiment, one of the co-stimulatory signal domains is a modified SEQ ID NO:4, which includes the c-myc tag of SEQ ID NO:5:

[0263] EQKLISEEDL (SEQ ID NO:5).

[0264] The c-myc tag can be added to the co-stimulatory signaling region by inserting it into the extracellular domain or by replacing a region in the extracellular domain, thus the region being located within the amino acid 1-152 of SEQ ID NO:3.

[0265] 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 potential off-target toxicity when CAR-T cells or pCAR-T cells encounter target cells expressing one of these two ligands. By replacing this motif with a tag sequence as described above, the likelihood of unwanted side effects is reduced. Therefore, in a particular embodiment, the co-stimulatory signaling region of the CAR construct includes the sequence shown in SEQ ID NO:6:

[0266] IEVEQKLISEEDLLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 6).

[0267] Furthermore, the inclusion of the c-myc epitope facilitates the detection of pCAR-T cells using monoclonal antibodies containing the c-myc epitope. This is extremely useful because flow cytometry detection has proven unreliable when using some of the available antibodies.

[0268] In addition, providing c-myc epitope tags can promote antigen-independent amplification of CAR-T cells, for example by crosslinking CARs in solution or immobilized on a solid phase (e.g., a bag) using appropriate monoclonal antibodies.

[0269] Furthermore, the expression of epitope 9e10 of the anti-human c-myc antibody within the variable region of the TCR has previously been shown to be sufficient to achieve antibody-mediated and complement-mediated cytotoxicity 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)). Therefore, providing such an epitope tag could also serve as a “suicide system” that could deplete pCAR-T cells in vivo using antibodies.

[0270] 4.3.5.2.6. Combining components

[0271] The binding elements of the pCAR CAR and CCR constructs bind to the first and second epitopes, respectively.

[0272] In a typical implementation, the bonding elements of the CAR and CCR constructs are different from each other.

[0273] 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 some 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 where the first and second epitopes are the same or overlapping, the binding elements on the CAR and CCR may compete for their binding.

[0274] In various implementations, the binding elements of the pCAR CAR and CCR constructs bind to different antigens. In some implementations, the antigens are different but may be associated with the same disease, such as the same specific cancer.

[0275] Therefore, a suitable binding element can be any element that provides the pCAR with the ability to recognize targets of interest. The target of the pCAR of the present invention can be any clinical target for which a T-cell response is desired.

[0276] In various embodiments, the binding element used in the CAR and CCR of the pCAR described herein is the antigen-binding site (ABS) of the antibody. In a typical embodiment, the ABS used as the binding element forms a single-chain antibody (scFv) or a single-domain antibody derived from camels, humans, or other species.

[0277] Alternatively, the binding element of a pCAR may include a ligand that binds to a surface protein of interest.

[0278] In some embodiments, the binding element is associated with a lead (signal peptide) sequence that promotes cell surface expression. Many lead sequences are known in the art, including but not limited to the CD8α lead sequence, the immunoglobulin κ light chain sequence, the macrophage colony-stimulating factor receptor (FMS) lead sequence, or the CD124 lead sequence.

[0279] MUC1 pCARs

[0280] In certain embodiments, at least one binding element 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 or alternative tumor-associated molecule (e.g., NKG2D ligand, αvβ6 integrin, or ErbB homologous or heterodimer) on the MUC1 target antigen. In some embodiments, the binding element of the CAR specifically interacts with an epitope on the MUC1 antigen, and the binding element of the CCR specifically interacts with the same, overlapping, or different epitopes on the MUC1 target antigen.

[0281] In the present preferred embodiment, the CAR binding element specifically interacts with a first epitope on the MUC1 target antigen. In some embodiments, the CAR binding element comprises an antigen-binding site of the HMFG2 antibody. In some embodiments, the CAR binding element comprises a CDR of the HMFG2 antibody. The CDR sequence of the HMFG2 antibody is determined using tools available at www.abysis.org. As shown in the following SEQ ID NO:8-13:

[0282] VH CDR1 GFTFSNY (SEQ ID NO:8);

[0283] VH CDR2 RLKSNNYA (SEQ ID NO:9);

[0284] VH CDR3 GNSFAY (SEQ ID NO:10);

[0285] VL CDR1RSSTGAVTTSNYAN(SEQ ID NO:11);

[0286] VL CDR2 GTNNRAP (SEQ ID NO:12);

[0287] VL CDR3 ALWYSNHWV (SEQ ID NO: 13).

[0288] In some embodiments, the CAR binding element includes the V of the HMFG2 antibody. H and V L Domain. V of HMFG2 antibody H and V L The domain sequences are shown in SEQ ID NO:14-15 below:

[0289] EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSS(SEQ ID NO:14)

[0290] QAVVTQESALTSPGETVTTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE (SEQ ID NO: 15).

[0291] In a particularly preferred embodiment, the CAR-binding element includes an antigen-binding site for an HMFG2 antibody formed as a single-chain antibody, with V H -Interval Zone-V L or V L -Interval zone V H The sequence configuration. In some embodiments, the amino acid sequence of the scFv of the HMGF2 antibody has 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% identity with SEQ ID NO:16 shown below:

[0292] EVQLQQSGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRLKSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYCTFGNSFAYWGQGTTVTVSSGGGGSGG GGSGGGGSQAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQEKPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGAQTEDEAIYFCALWYSNHWVFGGGTKLTVLGSE(SEQ ID NO:16).

[0293] In some embodiments, the nucleic acid of the single-chain antibody encoding the HMGF2 antibody is as shown in SEQ ID NO:17 below:

[0294] GAGGTGCAGCTGCAGCAGTCTGGAGGAGGCTTGGTGCAACCTGGAGGATCCATGAAACTCTCCTGTGTTGCCTCTGGATTCACTTTCAGTAACTACTGGATGAACTGGGTCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGGTTGCTGAAATTAGATTGAAATCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAGGGAGGTTCACCATCTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAACTTAAGAGCTGAAGACACTGGCATTTATTACTGTACCTTTGGTAACTCCTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGCCGTGGTCACTCAGGAATCTGCACTCACCACATCACCTGGTGAAACAGTCACACTCACTTGTCGCTCAAGTACTGGGGCTGTTACAACTAGTAACTATGCCAACTGGGTCCAAGAAAAACCAGATCATTTATTCACTGGTCTAATAGGTGGTACCAACAACCGAGCACCAGGTGTTCCTGCCAGATTCTCAGGCTCCCTGATTGGAGACAAGGCTGCCCTCACCATCACAGGGGCACAGACTGAGGATGAGGCAATATATTTCTGTGCTCTATGGTACAGCAACCATTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTAGGATCAGAG(SEQ ID NO:17)。

[0295] In some embodiments, the CCR binding element is ICR12, which binds to HER2. See Styles et al., “Ratmonoclonal 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 motifsin alpha v beta 6 integrin ligands,” J Biol Chem. 282(13):9657-9665 (2007), which is incorporated herein by reference in its entirety.

[0296] In some embodiments, the CCR binding element is a T1E peptide that binds ErbB homodimers and heterodimers. T1E is a chimeric peptide derived from transforming growth factor-α (TGF-α) and epidermal growth factor (EGF), and is a hybrid ErbB ligand. The T1E peptide is a chimeric fusion protein composed of the complete mature human EGF protein, excluding the five terminal amino acids (amino acids 971-975 of the pre-epidermal growth factor precursor (NP 001954.2)), which have been replaced by the seven N-terminal amino acids of the mature human TGF-α protein (amino acids 40-46 of the transforming growth factor α isoform 1 (NP 003227.1)). 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 ErbBdimers 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 sequence of T1E is shown in SEQ ID NO:18 below:

[0297] VVSHFNDCPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWELR (SEQ ID NO: 18).

[0298] In some implementations, the nucleic acid sequence encoding T1E is shown in SEQ ID NO:19 below:

[0299] GTGGTGAGCCACTTCAACGACTGCCCTCTGAGCCACGACGGCTACTGCCTGCACGACGGCGTTGTGCATGTACATCGAGGCCCTGGACAAGTACGCCTGCAACTGCGTGGTGGGCTACATCGGCGAGAGATGCCAGTACAGAGACCTGAAGTGGTGGGAGCTGAGA (SEQ ID NO: 19).

[0300] The protein sequence of TBB / H pCAR is shown in SEQ ID NO:7. TBB / H pCAR comprises a CCR including a T1E binding domain fused to the CD8α spacer region and a transmembrane domain, and a 4-1BB co-stimulatory domain (“TBB”), and a second-generation CAR including a human MUC1-targeting HMFG2 domain (“H”). The CCR and CAR are linked via a furin cleavage site, a serine-glycine linker (SGSG), and a T2A ribosomal jumping peptide. The VH and VL sequences of the HMFG2 sequence are bolded and underlined.

[0301]

[0302] In some implementations, a binding element of the pCAR is specific for markers associated with various types of cancer, including, for example, one or more ErbB homodimers or heterodimers, such as EGFR and HER2. In some implementations, the binding element binds to markers 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 cancer, 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 cancer, thyroid cancer, or renal cell carcinoma.

[0303] 4.3.5.3. Chimeric cytokine receptors

[0304] In another series of implementations, cells expressing CAR and CCR are engineered to co-express chimeric cytokine receptors, particularly the 4αβ chimeric cytokine receptor (CAR). Figure 1In 4αβ, the outer domain of the IL-4 receptor α chain is linked to the transmembrane and intracellular domains of the IL-2 / 15 receptor β. This allows for the selective in vitro expansion and enrichment of these genetically engineered T cells by culturing them in a suitable supporting medium, for 4αβ, in which IL-4 will be the sole 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 entirety.

[0305] Similarly, this system can be used with chimeric cytokine receptors, in which the outer domain of the α chain of the IL-4 receptor is linked to the transmembrane and intracellular domains of another receptor that is naturally bound by cytokines that also bind to the common γ chain.

[0306] 4.3.6. Engineered TCR

[0307] In some implementations, immune response cells are engineered to further express engineered (non-natural) T cell receptors (TCRs).

[0308] Engineered TCRs that can be efficiently expressed in the immune-response cells described herein are described in U.S. Patents 9,512,197; 9,822,163 and 10,344,074, the entire disclosures of which are incorporated herein by reference. Engineered TCs that can be efficiently expressed in the immune-response cells described herein are described in U.S. pre-grant publications 2019 / 0161528; 2019 / 0144521; 2019 / 0135892; 2019 / 0127436; 2018 / 0218043; 2017 / 0088599; 2016 / 0159771 and 2016 / 0137715, the entire disclosures of which are incorporated herein by reference.

[0309] 4.3.7. Nucleic Acids and Methods for Preparing pCAR-T Cells

[0310] This document also provides a polynucleotide or a group of polynucleotides comprising a first nucleic acid encoding a modified procytokine, wherein the modified procytokine comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3; and (c) a cytokine fragment. The cleavage site is a specific sequence recognized by the protease.

[0311] In some embodiments, the first nucleic acid encodes a modified pro-IL-18, wherein the modified pro-IL-18 comprises, from the N-terminus to the 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 located downstream, upstream, or replacing 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 various protease cleavage sites known in the art. For example, the cleavage site may be recognized by granzyme B (GzB), caspase-3, caspase-8, MT1-MMP (MMP14), alternative tumor-associated matrix metalloproteinases (MMP1-13), deintegrin and metalloproteinase (ADAM) family members (especially ADAM 10 or ADAM17), cathepsin B, L or S, fibroblast activating protein (FAP), kallikrein-associated peptidase (KLK), such as KLK2, 3, 6 or 7, dipeptidyl peptidase (DPP) 4, heparin 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 cleavage site includes sequences selected from SEQ ID NO:26, 28, 30 and 32. In some embodiments, the modified pro-IL-18 includes sequences selected from SEQ ID NO:26, 28, 30 and 32. The polypeptides with sequences shown in NO:27, 29, 31 and 33. In a particular embodiment, the modified pro-IL-18 comprises the polypeptide as shown in SEQ ID NO:27.

[0312] In some embodiments, the first nucleic acid is selected from the group consisting of SEQ ID NO:102, 103, 105, 107, 109, 111, and 113. In a particular embodiment, the first nucleic acid comprises a polynucleotide such as SEQ ID NO:103. In some embodiments, the first nucleic acid is a coding sequence cloned in an expression vector (e.g., a viral vector or a non-viral vector).

[0313] Alternatively, the modified procytokine is a modified pro-IL-36α, β, or γ protein, wherein the modified pro-IL-36 comprises, from the N-terminus to the C-terminus: (a) a propeptide; (b) a cleavage site recognized by a protease other than cathepsin G, elastase, and protease 3; and (c) an IL-36 fragment. The cleavage site is a specific sequence recognized by the protease. In some embodiments, the cleavage site is located downstream, upstream, or replaces the cathepsin G, elastase, and / or protease 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 in the modified pro-IL-36 can be selected from various protease cleavage sites known in the art. For example, the cleavage site may be generated by granzyme B (GzB), caspase-3, caspase-8, MT1-MMP (MMP14), alternative tumor-associated matrix metalloproteinases (MMP1-13), deintegrin and metalloproteinase (ADAM) family members (especially ADAM 10 or ADAM17), cathepsin B, L or S, fibroblast activating protein (FAP) recognition, kallikrein-associated peptidase (KLK), such as KLK2, 3, 6 or 7, dipeptidyl peptidase (DPP) 4, heparin 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 cleavage site includes sequences selected from SEQ ID NO:26, 28, 30 and 32. In some embodiments, the modified pro-IL-36α, β or γ includes sequences selected from SEQ ID NO:26, 28, 30 and 32 respectively. The polypeptides shown in the sequences of NO:37, 39 and 41.

[0314] In some embodiments, the polynucleotide or group of polynucleotides further includes a second nucleic acid encoding a protease that recognizes a cleavage site on the first nucleic acid. The protease may be granzyme B (GzB), caspase-3, caspase-8, MT1-MMP (MMP14), alternative tumor-associated matrix metalloproteinases (MMP1-13), deintegrin and metalloproteinase (ADAM) family members (especially ADAM 10 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, heparin, or urokinase plasminogen activator (see Dudani et al., “Harnessing protease activity to improve cancercare,” Annu. Rev. Cancer Biol., 2:353-76 (2018). In some embodiments, the first and second nucleic acids are in a single vector or two different vectors.

[0315] In some embodiments, the polynucleotide or the group of polynucleotides further includes a third nucleic acid encoding a chimeric antigen receptor (CAR). In some embodiments, the CAR is a second-generation CAR as described above, comprising (a) a signaling region; (b) a first co-stimulatory signaling region; (c) a transmembrane domain; and (d) a first binding element that specifically interacts with a first epitope on a first target antigen.

[0316] In some embodiments, the polynucleotide or group of polynucleotides further includes a fourth nucleic acid encoding the CCR as described above. In some embodiments, the CCR includes: (a) a second co-stimulatory signaling region; (b) a transmembrane domain; and (c) a second binding element that specifically interacts with a second epitope on a second target antigen.

[0317] As described above, for convenience, the combination of CAR and CCR will be referred to as pCAR, even though CAR and CCR are separate, co-expressed proteins. The third and fourth nucleic acids can be expressed from a single vector or two or more vectors. Based on the description of CAR and CCR above, suitable sequences of nucleic acids will be obvious to those skilled in the art. These sequences can be optimized for use in the desired immune response cells. However, in some cases, as mentioned above, codons may deviate from optimal values ​​or “wobble” to avoid repetitive sequences. Specific instances of such nucleic acids will encode the preferred embodiments described above.

[0318] To achieve transduction, the nucleic acid encoding pCAR is appropriately introduced into one or more vectors, such as plasmids, retroviral vectors, or lentiviral vectors. Such vectors, including plasmid vectors or cell lines containing them, constitute another aspect of the present invention.

[0319] In a typical implementation, immune-responding cells undergo genetic modification, such as through retroviral or lentiviral-mediated transduction, to introduce first, second, third, and / or fourth nucleic acids into the host T cell genome, thereby allowing stable expression of modified procytokines (e.g., modified pro-IL-18 or modified pro-IL-36), proteases, CARs, and / or CCRs, respectively. The first, second, third, and / or fourth nucleic acids can be introduced as a single vector or multiple vectors, each vector comprising one or more nucleic acids. They can then be selectively reintroduced into the patient after expansion to provide beneficial therapeutic effects, as described below.

[0320] In some embodiments, the immune response cells are γδT cells, and the γδT cells are activated by anti-γδ-TCR antibodies prior to genetic modification. In some embodiments, immobilized anti-γδ-TCR antibodies are used for activation.

[0321] The first and second nucleic acids encoding modified procytokines (e.g., modified pro-IL-18 or modified pro-IL-36) and proteases can be expressed from the same vector or multiple vectors. The third and fourth nucleic acids encoding CARs and CCRs 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. One or more vectors containing them can be combined in a kit provided for the production of immune response cells of the first aspect disclosed herein.

[0322] In some implementations, when T cells are engineered to co-express a chimeric cytokine receptor (e.g., 4αβ), the expansion step may include an in vitro culture step in a medium containing cytokines, such as a medium including IL-4 as the sole cytokine carrier in the case of 4αβ. Alternatively, the chimeric cytokine receptor may include the α-external domain of the IL-4 receptor, which is linked to an inner domain used by a common gamma cytokine with unique properties (e.g., IL-7). Expansion of cells in IL-4 may result in less cell differentiation compared to the use of IL-7. In this way, selective expansion and enrichment of genetically engineered T cells with the desired differentiation state can be ensured.

[0323] 4.4. Treatment methods

[0324] As described above, immune-responding cells expressing modified pro-cytokines (e.g., modified pro-IL-18 or modified IL-36) can be used to direct T-cell-mediated immune responses to immunosuppressed target cells for treatment. Therefore, in another aspect, a method is provided for directing T-cell-mediated immune responses to target cells in patients in need. This method includes administering to a patient a population of immune-responding cells as described above, wherein the binding element is specific to the target cells. In a typical embodiment, the target cells express MUC1.

[0325] On the other hand, a method for treating cancer in patients in need is provided. The method includes administering to a patient an immune-response cell population as described above, wherein the binding element is specific to the target cells. In a typical embodiment, the target cells express 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 cancer, thyroid cancer, or renal cell carcinoma. In some embodiments, the patient has breast cancer.

[0326] In various embodiments, a therapeutically effective number of immune-response cells are administered to the patient. In some embodiments, the immune-response cells are administered via intravenous infusion. In some embodiments, the immune-response cells are administered via intratumoral injection. In some embodiments, the immune-response cells are administered via intraperitoneal injection. In some embodiments, the immune-response cells are administered via multiple routes selected from intravenous infusion, intratumoral injection, and peritumoral injection.

[0327] In another aspect, the present invention provides immune-response cells, polynucleotides, or γδT cells for treatment or as medicaments. The present invention also provides immune-response cells, polynucleotides, or γδT cells for treating pathological diseases. The present invention further provides the use of immune-response cells, polynucleotides, or γδT cells in the manufacture of medicaments for treating pathological diseases. In some embodiments, the pathological disease is cancer. Detailed Implementation

[0328] The following are examples of specific embodiments for carrying out the present invention. The examples provided are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Every effort has been made to ensure the accuracy of the figures used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should of course be taken into account.

[0329] 5.1. Methods

[0330] Cell line culture

[0331] All tumor cells and 293T cells were cultured in DMEM supplemented with L-glutamine and 10% FBS (D10 medium). Tumor cells were transduced to express the firefly luciferase (LT) SFG vector as instructed, followed by fluorescence-activated cell sorting (FACS) to express red fluorescent protein (RFP). MDA-MB-468-HER2 ++ Cells were generated by transducing MDA-MB-468-LT cells using an SFG retroviral vector encoding human HER2. FACS classification of the transduced cells was performed using ICR12 rat anti-human HER2 antibody and goat anti-mouse PE.

[0332] Retrovirus production

[0333] 293T cells were triple-transfected in GeneJuice (MilliporeSigma, Merck KGaA, Darmstadt, Germany) with: (i) an SFG retroviral vector encoding a modified pro-IL-18, a protease, and / or CAR / pCAR; (ii) an RDF plasmid encoding the RD114 envelope; and (iii) a Peq Pam plasmid encoding gag-pol. 1.5 × 10⁻⁶ cells were transfected onto 100 mm plates. 6 For 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. Culture medium containing the viral vector was collected at 48 and 72 hours post-transfection, rapidly frozen, and stored at -80°C. In some cases, stable packaging cell lines were generated by transducing 293 VEC GALV cells with transiently generated retroviral vectors encoding modified pro-IL-18 (a protease) and / or CAR / pCAR. Viruses prepared from either source can be used interchangeably for transduction of target cells.

[0334] Culture and transduction of αβT cells

[0335] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor peripheral blood samples using Ficoll Paque (Ethical approval no. 18 / WS / 0047) via density gradient centrifugation. T cells were cultured in RPMI containing 5% human AB serum and glutamine. Activation of T cells was achieved by culturing for 24–48 hours in the presence of 5 μg / mL phytohemagglutinin-leukocyte agglutinin (PHA-L), followed by a further 24-hour culture in IL-2 (100 U / mL) prior to gene transfer. T cell transduction was performed using a reverse transcription connexin (Takara Bio) coated plate according to the manufacturer's protocol. Activated PBMCs (1 × 10⁻⁶) were then transferred to the plate.6 (1 cell) was added to each well of a 6-well plate coated with reverse transcription linker protein. Then, 3 mL of culture medium containing retrovirus was added to each well, along with 100 U / mL IL-2.

[0336] Expansion and transduction of γδT cells

[0337] To generate γδT cells, 2.4 μg of activating anti-γ / δ-1TCR antibody (BDbiosciences) was coated onto each well in a 6-well plate, with 9 × 10⁹ cells activated per well. 6 PBMCs. After 24 hours, cells were regenerated for 48 hours in 100 U / mL IL-2 and 5 ng / mL TGF-β. 3 × 10⁻⁶ cells were added to each well. 6 Activated PBMCs were pre-coated with 3 mL of retrovirus-containing medium in 6-well plates coated with reverse transcription linker protein. Cells were cultured for 14 days in 100 U / mL IL-2 and 5 ng / mL TGF-β (R&D system). Fold growth was calculated based on the initial number of PBMCs.

[0338] Cytotoxicity analysis

[0339] MDA-MB-468 tumor cells or BxPC-3 tumor cells were injected at a dose of 1×10⁻⁶. 4 Cells were seeded at a density in 96-well plates and incubated with T cells for 72 hours at an effector-to-target cell ratio ranging from 4 to 0.03 (e.g., Figure 3 (A-3D). The destruction of tumor cell monolayers by T cells was quantified using the MTT assay. MTT(σ) was added to D10 medium at a concentration of 500 μg / ml and cultured at 37°C and 5% CO2 for 2 hours. After removing the supernatant, formazan crystals were resuspended in 100 μL DMSO. Absorbance was measured at 560 nm. Tumor cell viability was calculated as (absorbance of monolayers cultured with T cells / absorbance of untreated monolayers alone) × 100%.

[0340] Detection of IFN-γ and IL-2

[0341] Supernatant was collected from the co-culture of MDA-MB-468 tumor cells and the aforementioned CAR-T / pCAR-T cells at 24 hours. Cytokine levels were quantified using a human IFN-γ (Bio-Techne) or human IL-2 ELISA kit (Invitrogen) according to the manufacturer's protocol. Data are presented as mean ± SEM cytokine levels from six independent experiments, each experiment repeated twice.

[0342] Detection of active human IL-18

[0343] T cells were collected, washed, and cultured for 48 hours without stimulation or cytokines. Then, T cells were stimulated for 24 hours with an effector-to-tumor ratio of 10:1 or a T cell-to-anti-CD3 / 28 magnetic bead ratio of 200:1. The supernatant was then collected and mixed with 5 × 10⁻⁶ cells / mL of anti-CD3 / 28 magnetic beads. 4 HEK blue IL-18 cells / well were cultured in 96-well plates for 24 hours. Then, 20 μl of supernatant was extracted from the co-culture and added to 180 μl of QUANTI-Blue solution, and the absorbance was measured at 620–650 nm.

[0344] Repeated antigen stimulation test

[0345] 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+ / γδTCR+ T cell:1 tumor cell for 72–96 hours. All T cells were then removed, centrifuged at 400g for 5 min, resuspended in 3 ml of fresh RPMI supplemented with glutamine and 5% human serum, and added to a new tumor cell monolayer. Residual tumor cell viability was assessed by the MTT assay after each co-culture. T cells were added to a fresh tumor cell monolayer if the proportion of tumor cells killed was greater than 20% (or the proportion of γδT cells greater than 30%) compared to untreated cells. Data were presented as mean ± SEM of the number of antigen stimulation rounds. Cell counting was performed by pooling three replicate wells and counting the total number of cells.

[0346] Alternatively, 24 hours before adding T cells, tumor cell lines are added at a concentration of 1 × 10⁶ cells per well. 5 Cells were seeded in triplicate in 24-well plates. CAR-T / pCAR-T cells were added at an effector:target ratio of 1:1. After 72 hours, tumor cell killing was assessed using a luciferase assay, with D-luciferin (Perkinlemer) added immediately before luminescence readings. If the proportion of tumor cells killed was greater than 20% compared to untreated cells, all T cells were restimulated by adding a new tumor cell monolayer. Tumor cell survival was calculated as (luminescence of monolayers cultured with T cells / luminescence of untreated monolayers) × 100%.

[0347] In vivo studies

[0348] PBMCs from healthy volunteers were either engineered to express the specified CARs / PCARs or left unengineered. After expansion in IL-2 (100 U / mL, added every 2–3 days) or IL-2+TGF-β for 11 days (αβT cells) or 14 days (αβT cells), the expression of CCR or CCR and αβTCR in the cells was analyzed by flow cytometry.

[0349] Female severely combined immunodeficiency (SCID) Beige mice were injected intraperitoneally (ip) with 1×10 6 MDA-MB-468LT cells ( Figure 13 Twelve days after tumor cell injection, mice were intraperitoneally injected with 200 μl of PBS containing 10 × 10⁻⁶ cells. 6 CCR-positive or CCR and γδTCR double-positive (or untransformed) T cells, or PBS alone as a control. Twenty minutes after injection of StayBrite™ D-fluorescein potassium in 200 μl PBS (150 mg / kg), tumor status was monitored by bioluminescence imaging under isoflurane anesthesia. Use at designated time points. Images were acquired using Lumina III (Perkinlemer) and real-time imaging software (Perkinlemer), which was set to automatically optimize exposure time, stops, and F / stop. The animals were humanely euthanized at the end of the experiment.

[0350] NOD SCID gamma is transmitted to females via the intraperitoneal (IP) route. null (NSG) mice were injected with 0.5 × 10 6 SKOV3 ovarian cancer cells (Figure 15). Eighteen days after tumor cell injection, mice were intraperitoneally injected with 200 μl of 0.5 × 10⁻⁶ PBS. 6 CAR T cells. Tumor status was monitored using bioluminescence imaging, as described above. Animals were humanely euthanized upon reaching the experimental endpoint.

[0351] Female NSG mice were injected with 1×10 via intraperitoneal (ip) injection. 5 BxPC-3 LT cells. Nine days after tumor cell injection, mice were intraperitoneally injected with 200 μl of PBS containing 10 × 10⁻⁶ cells. 6 CCR / γδTCR double-positive (or untransformed) T cells, or PBS alone as a control. Tumor status was monitored using bioluminescence imaging, as described above. Animals were humanely euthanized upon reaching the experimental endpoint.

[0352] 5.2. Example 1: Generation of CAR / pCAR T cells expressing IL-18

[0353] Vectors including the coding sequence of TBB / H pCAR (SEQ ID NO:7) as described above are modified to further include coding sequences of various human IL-18 constructs.

[0354] Encoding TBB / H and pro-IL-18 ( Figure 18 The construct (SEQ ID NO:102) was generated by inserting a synthetic polynucleotide (SEQ ID NO:101) into the unique Kfl1 and Xho1 restriction sites in the TBB / H vector, replacing the 224 bp fragment between the Kfl1 and Xho1 restriction sites. The pro-IL-18 insertion site is located downstream of the second swing of T2A, followed by a stop codon. This construct is not expected to express active IL-18 in T cells because the cleavage of the propeptide requires cysteine ​​protease-1, which is not expressed in T cells.

[0355] Encoding TBB / H and modified pro-IL-18 (pro-IL-18(GzB)) Figure 19 The construct of SEQ ID NO:103 is obtained by replacing 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 This modified pro-IL-18 is generated by C GAC TAC (SEQ ID NO:35; changed to underscore). The modified pro-IL-18 replaces the native caspase-1 cleavage site between the IL-18 precursor and the mature IL-18 protein (LESD) with a granzyme B (GzB) cleavage site (IEPD).

[0356] Encoding TBB / H and Constitt IL-18 ( Figure 20 The construct (SEQ ID NO: 105) was generated by inserting a synthetic polynucleotide (SEQ ID NO: 104) into the unique Kfl1 and Xho1 restriction sites in the TBB / H vector, replacing the 224 bp fragment between the Kfl1 and Xho1 restriction sites. The IL-18 insertion site is located downstream of the CD4 leader, followed by a stop codon. The IL-18 insert encodes mature IL-18 protein without the IL-18 propeptide. It is predicted that this construct can express constitutively active IL-18 protein in T cells.

[0357] Encoding TBB / H and modified pro-IL-18 (pro-IL-18(casp 8))( Figure 19The construct (SEQ ID NO: 107) was generated by inserting a synthetic polynucleotide (SEQ ID NO: 106) into the Kfl1 and Xho1 restriction sites unique to the TBB / H construct, replacing the 224 bp fragment between the Kfl1 and Xho1 restriction sites. The insertion site of the modified pro-IL-18 is located downstream of the second swing of T2A, followed by a stop codon. 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 caspase-8 cleavage site (IETD).

[0358] Encoding TBB / H and modified pro-IL-18 (pro-IL-18(casp 3))( Figure 22 The construct (SEQ ID NO: 109) was generated by inserting a synthetic polynucleotide (SEQ ID NO: 108) into the Kfl1 and Xho1 restriction sites unique to the TBB / H construct, replacing the removed 224 bp fragment. The insertion site of the modified pro-IL-18 sequence is located downstream of the second swing T2A, followed by a stop codon. The 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).

[0359] The construct encoding TBB / H with modified pro-IL-18 (GzB) and additional granzyme B ( Figure 23 ;SEQ ID NO:111) is obtained by inserting a synthetic polynucleotide (SEQ ID NO:110) into the TBB / H GzB Pfn construct (encoding granzyme B, perforin and TBBH; SEQ ID NO:112), replacing the removed 1788bp fragment.

[0360] The construct encoding T4 and modified pro-IL-18 (MT1-MMP) (SEQ ID NO:113) was generated by replacing the caspase-1 site of pro-IL-18 with a synthetic polynucleotide inserted into the MT1-MMP cleavage site (SEQ ID NO:32). Figure 16 and 24 ).

[0361] An SFG retroviral vector, including the construct coding sequence, was generated as described above and then transduced into PBMCs. T cells were then expanded from PBMCs in the presence of IL-2, as described above. The T cells expressed modified pro-IL-18. IL-18 activity depended on the expression of a protease in the T cells that recognized the cleavage site in the modified pro-IL-18.

[0362] 5.3. Example 2: In vitro antitumor activity of IL-18-armored pCAR T cells

[0363] Analysis of IL-18 variants using T cells transfected with an SFG retroviral vector encoding TBB / H pCAR and one of the IL-18 variants described in Example 1. Figure 4A The expression of H28z CAR (H-2) and TIE-4-1BB CCR was measured by flow cytometry. Figure 3 The results showed that most transduced T cells expressed both components of TBB / H pCAR.

[0364] ELISA analysis of IL-18 secreted by transfected T cells ( Figure 4A ), and tested the functional activity of expressed IL-18 through reporting experiments. Figure 4B The study used commercially available reporter cell lines to detect functional IL-18 (i.e., the active IL-18 fragment produced after propeptide cleavage).

[0365] IL-18 secretion was detected in unstimulated T cells. Figure 4A The T cells have been engineered via retroviral transduction to express each of the tested IL-18 variants: (natural) pro-IL-18; constituent IL-18; pro-IL-18 (casp-8); and pro-IL-18 (casp-3). However, IL-18 activity was only detected in T cells transduced with the constituent variant (“constitutive IL-18”), where the mature IL-18 fragment is located downstream of the CD4 signal peptide. Figure 4B Active IL-18 was not detected in conditioned medium generated from unstimulated pCAR T cells expressing pro-IL-18 or modified pro-IL-18, where the cleavage site had been switched to a position recognized by caspase-3 (pro-IL-18(casp3)) or caspase-8 (pro-IL-18(casp8)).

[0366] T cells co-expressing TBB / H pCAR and each IL-18 variant were co-cultured with MDA-MB-468 breast cancer cells in vitro for 72 hours. The effector:target cell (engineered T cells:tumor cells) 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 after co-culture were quantified using the MTT assay. The survival rate of MDA-MB-468 breast cancer cells co-cultured with pCAR-T cells is shown in the figure. Figures 5A-5D As shown, MDA-MB-468 breast cancer cells simultaneously express MUC-1 and ErbB dimers, and have very low HER2 levels. Figures 5A-5D As shown, T cells expressing TBB / H pCAR and each IL-18 variant exhibited greater cytotoxic antitumor activity at effector:target cell ratios of 4 and 2 compared to effector:target cell ratios of 1 or 0.5. No significant differences were observed between T cells expressing different IL-18 variants.

[0367] Using MUC1 + MDA-MB-468 breast cancer cells were repeatedly restimulated by T cells expressing TBB / H pCAR and IL-18 variants. Figures 6A-6B While constitutive expression of the active IL-18 fragment enables pCAR T cells to undergo more restimulation cycles and retain cytotoxic activity, this was not observed in pro-IL-18 or cleavable caspase-3 (pro-IL-18(casp 3)) or cleavable caspase-8 (pro-IL-18(casp 8)) derivatives. Constitutive IL-18 (but not pro-IL-18 or caspase 3 / 8 cleavable derivatives) mediates a significant increase in CAR T cell proliferation. Figure 6ABased on these data, we conclude that neither the IL-18 mutant protein that cleaves caspase 3 nor caspase 8 was activated under CAR T cell stimulation. Not wanting to be bound by theory, the most likely explanation is that when active caspase 3 and caspase 8 are found in activated T cells, neither protein can enter the cytoplasm (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)).

[0368] As mentioned above, the GzB cleavable variant of pro-IL-18 (MUC1-13b) (hereinafter referred to as "pro-IL-18(GzB)") will be tested next. Unlike the mutant protein modified with pro-IL-18 that cleaves caspase 3 or caspase 8, pro-IL-18(GzB) is functionally active when T cells are activated, but inactive 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 co-expressing pCAR and IL-18 (GzB) were tested in restimulation assays, their antitumor activity was lower than that of constitutive T cells with IL-18 activity.

[0369] We infer that GzB itself may be a limiting factor, as it is primarily expressed in CD8 T cells, while the autocrine stimulation of IL-18 mainly occurs in CD4 cells. + GzB plays a crucial role in T cells, which naturally express much less GzB. To address this issue, we designed TBB / H pCAR T cells that co-express natural GzB in addition to IL-18 (GzB). This retroviral construct was introduced into PBMCs, which were then co-cultured with MDA-MB-468 tumor cells at a 1:1 effector:target cell ratio. Antitumor activity was measured after 72 hours.

[0370] Engineered T cells co-expressing TBB / H and pro-IL-18, or combinations of TBB / H, pro-IL-18 (GzB), and other granzyme B proteases, can induce similar tumor cell killing. Figure 8 Data was provided from five independent donors, with three copies for each donor.

[0371] Detection of IL-18 in T cells expressing TBB / H+pro-IL-18 or TBB / H+pro-IL-18(GzB)+granzyme B. Figure 9A ) and IFN-γ Figure 9B The production of IL-18 and IFN-γ was observed. At 72 hours, the supernatant of the T cell culture was collected, and the concentrations of IL-18 and IFN-γ were measured.

[0372] ELISA analysis showed that unstimulated T cells co-expressing TBB / H and pro-IL-18, or a combination of TBB / H, pro-IL-18 (GzB), and granzyme B, had similar levels of IL-18 secreted. Figure 9A However, after activation with targeted expression of tumor cells, T cells expressing TBB / H, pro-IL-18(GzB)+ granzyme B produced significantly higher levels of IFN-γ than T cells expressing TBB / H and pro-IL-18. Figure 9B The data shown comes from four independent donors, with three copies from each donor. (**p = 0.008).

[0373] In the absence of exogenous IL-2, the transduced T cells were further subjected to several rounds of antigen stimulation. MDA-MD-468 cells were used. Figure 10A ) or BxPC-3 cells ( Figure 10B Using MDA-MD-468 cells as the target cell population, cells were cultured at a 1:1 ratio of initial effectors to target cells. Tumor cell viability was measured twice weekly using the MTT assay after 72–96 hours. Compared to T cells expressing TBB / H alone or in combination with pro-IL-18, T cells co-expressing TBB / H and IL-18 or a combination of TBB / H, pro-IL-18 (GzB), and granzyme B were successfully restimulated with significantly more cell cycles (Cyc). Figure 10A A similar pattern can also be observed when using BxPC-3 cells as the target population. Figure 10B The data shown is from... Figure 10A One donor and Figure 10B One donor is generated, and each donor is made in triplicate.

[0374] The number of successful restimulations for each pCAR T cell population was measured, and the data are as follows: Figure 11A and11B As shown. If more than 20% cytotoxicity is observed, pCAR T cells proceed to the next round of stimulation. MDA-MD-468 cells were used ( Figure 11A ) or BxPC-3 cells ( Figure 11B Using MDA-MD-468 cells as the target cell population, cells were cultured at an effector-to-target cell ratio of 1. Compared to T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B, T cells co-expressing TBB / H+pro-IL-18 successfully restimulated for more cell cycles. Figure 11A Similar patterns can also be observed when using BxPC-3 cells as the target population. Figure 11B The data shown comes from 5 independent donors, with three copies from each donor. (*p = 0.039).

[0375] At the start of each restimulation cycle, the number of T cells in each culture was also counted. T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B but not expressing TBB / H+pro-IL-18 proliferated significantly more than control TBB / HpCAR T cells. The counts shown are from the fourth restimulation cycle, from three independent donors, in triplicate from each donor. Figure 12 *p = 0.014).

[0376] 5.4. Example 3: In vitro antitumor activity of pCARαβT cells coated with IL-18

[0377] Using the method described in Example 1, αβT cells were engineered to express TBB / H pCAR alone, or to express TBB / H pCAR in combination with pro-IL-18, pro-IL-18(GzB), constitutive IL-18, or pro-IL-18(GzB) with granzyme B. IL-18 activity in αβT cells was detected using reporter cell lines, with commercially available reporter cell lines used to detect functional IL-18. Figure 35 The results provided showed that IL-18 activity was detected in TBB / H pCARαβT cells co-expressing constitutive IL-18 without stimulation, but not in other TBB / H pCARαβT cells. However, when MUC1 was used... +When αβT cells were stimulated with MDA-MB-468 breast cancer cells (“+468”) or beads coated with anti-CD3 and anti-CD28 antibodies (“aCD3 / 28 beads”), TBB / H pCARαβT cells co-expressing pro-IL18 (GzB) and granzyme B also exhibited IL-18 activity. TBB / H pCARαβT cells co-expressing pro-IL18 (GzB) and granzyme B showed higher IL-18 activity than stimulated TBB / H pCARαβT cells expressing only pro-IL18 (GzB).

[0378] 5.5. Example 4: In vivo antitumor activity of pCAR-αβT cells coated with IL-18

[0379] The antitumor activity of CAR-αβT and pCAR-αβT cells was evaluated in an in vivo xenograft mouse model.

[0380] 1×10 6 MDA-MB-468 tumor cells expressing luciferase were injected intraperitoneally (ip) into female SCID Beige mice to establish a xenograft model. Eleven or twelve days after tumor injection, 1 × 10⁻⁶ mcg of luciferase-expressing MDA-MB-468 tumor cells were injected intraperitoneally. 7 CAR-αβ T cells with or without IL-18 expression. Total bioluminescence emission of the tumor (“total flux”) was measured at each treatment. Figure 13 and Figures 36A-36F As shown, compared with SCID Beige mice treated with TBB / H pCAR T cells, SCID Beige mice treated with αβ T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B had significantly reduced tumor-derived total flux. T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B also showed a trend toward improved tumor control compared with T cells co-expressing TBB / H and IL-18. Figure 13 , 36E and 36F). Figure 13 The data shown comes from 6 mice. Figure 36B The data shown are from 10 mice. Figure 36C From 10 mice, Figure 36D From 6 mice, Figure 36E From 5 mice, Figure 36F From 5 mice.

[0381] Figure 37Survival data for mice treated with PBS, αβT cells expressing TBB / H alone, or αβT cells co-expressing const TBB / H were presented. Following tumor injection, IL-18, pro-IL-18 (GzB), or pro-IL-18 (GzB) was administered co-injected with granzyme B. Results showed that αβT cells co-expressing TBB / H, pro-IL-18 (GzB), and granzyme B improved mouse survival.

[0382] 5.6. Example 5: In vitro antitumor activity of pCAR-γδT cells

[0383] γδT cells were activated with 2.4 ng of immobilized anti-γδTCR antibody per well on 6-well non-TC treated plates and expressed TBB / H pCAR after 48 hours via retroviral transduction. Untransformed γδT cells and TBB / HpCAR γδT cells were cultured and expanded. Figure 49A and Figure 49B ). Using flow cytometry in untransformed ( Figure 48A ) or TBB / H pCARγδT cells ( Figure 48B The study confirmed the co-expression of the second-generation H2 CAR (“H28z”) and TBB CCR (“TIE”) (collectively referred to as TBB / H pCAR).

[0384] By interacting with MDA-MB-468 breast cancer cells ( Figure 50A ) or BxPC-3 cells ( Figure 50B The antitumor effects of untransduced γδT cells and TBB / H pCARδγT cells were evaluated after co-culturing them at a 1:1 effector:target cell ratio (γδT cells:tumor cells) for 72 hours. The survival rate (%) of tumor cells compared to tumor cells without γδT cells was determined by the MTT assay during the first stimulation cycle. Figure 50A and Figure 50B As shown, TBB / H pCARδγT cells have cytotoxic effects on tumor cells.

[0385] Untransformed γδT cells and TBB / H pCARδγT cells were further subjected to several rounds of antigen stimulation. MDA-MD-468 cells were used ( Figure 51A ) or BxPC-3 cells ( Figure 51BAs the target cell population, cells were cultured at a 1:1 ratio of initial effectors to target cells for 72-96 hours. In sequential monolayer stimulation, the cytotoxicity of γδT cells against tumor cells was measured using the MTT assay. A successful restimulation cycle was defined as generating more than 25% cytotoxicity against the target tumor cells. If more than 25% cytotoxicity was observed, the T cells proceeded to the next round of stimulation. The number of successful restimulations for each transduced γδT cell population was measured, and the data are as follows: Figure 51A and 51B As shown in the figure. The results indicate that TBB / H pCARδγT cells were successfully restimulated for more cycles than δγT cells.

[0386] Figure 51C and Figure 51D Tumor cell survival (%) measured across multiple stimulation cycles is provided. Data shows that, during restimulation cycles, TBB / H pCARδγT cells significantly reduced the survival rate of MDA-MD-468 tumor cells (…). Figure 51C ) or BxPC-3 tumor cells ( Figure 51D It has cytotoxic activity.

[0387] 5.7. Example 6: In vivo antitumor activity of pCAR-γδT cells

[0388] Evaluation of the antitumor activity of TBB / H pCARδγT cells in a mouse model of tumor xenograft.

[0389] For the BxPC3 NSG mouse model, 1×10⁻⁶ mice expressing luciferase will be used. 5 BxPC3 LT tumor cells were injected intraperitoneally (ip) into NSG mice to establish a xenograft model. For the 468s SCID Beige mouse model, female SCID Beige mice were injected intraperitoneally with 1×10-1 6 MDA-MB-468 tumor cells expressing luciferase were used to establish a xenograft model.

[0390] Eleven days after tumor injection, 1×10⁻⁶ cells were injected intraperitoneally into each animal model. 7 10 untransformed δγT cells, 1×10 7 TBB / H pCARδγT cells or PBS. Measure the total bioluminescent emission of the tumor (“total flux”) at each treatment. Figure 52 (BxPC3 NSG) and Figure 53 As shown in (468s-SCID Beige), in both tumor xenograft mouse models, the tumor-derived total flux induced by TBB / H pCARδγT cells was significantly reduced compared with untransformed δγT cells or PBS controls, demonstrating antitumor activity.

[0391] 5.8. Example 7: In vitro antitumor activity of pCAR-γδT cells coated with IL-18

[0392] γδT cells were activated by immobilized anti-γδTCR antibody and expressed TBB / HpCAR via retroviral transduction, whether expressed alone or in combination with pro-IL-18, pro-IL-18(GzB), constitutive IL-18 or pro-IL-18(GzB) and granzyme B. pCAR expression was measured by flow cytometry after incubation with anti-EGF antibody (CCR detection). Figure 14 The above figure also confirms the enrichment of γδT cells. Figure 14 (See image below).

[0393] By interacting with MDA-MB-468 breast cancer cells ( Figure 15A ) or BxPC-3 cells ( Figure 15B The antitumor effect of γδT cells was evaluated by co-culturing the cells for 72 hours. The effector:target cell (γδT cell:tumor cell) ratio ranged from 128 to 1, including 128, 64, 32, 16, 8, 4, 2, and 1. The residual surviving cancer cells after co-culture were quantified using the MTT assay. Figure 15A and 15B As shown, γδT cells expressing TBB / H pCAR alone or TBB / H pCAR with any IL-18 variant (pro-IL-18; constitutive IL-18; pro-IL-18(GzB) or pro-IL-18(GzB) + granzyme B) showed greater cytotoxicity against tumor cells compared to untransfected γδT cells.

[0394] In the absence of exogenous IL-2, transduced γδT cells were subjected to several rounds of antigen stimulation. MDA-MD-468 cells were used. Figure 38A ) or BxPC-3 cells ( Figure 38B As the target cell population, cells were cultured at a 1:1 ratio of initial effectors to target cells for 72–96 hours. If cytotoxicity exceeding 30% was observed, the T cells proceeded to the next round of stimulation. The number of successful restimulations for each transduced γδT cell population was measured, and the data are as follows: Figure 38A and 38B As shown. Using MDA-MD-468 cells as the target cell population, compared with T cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B, T cells co-expressing TBB / H+pro-IL-18 successfully restimulated for more cell cycles (GzB+). Figure 38A Similar patterns can also be observed when using BxPC-3 cells as the target population. Figure 38B(*p<0.05**p<0.01).

[0395] IL-18 activity in engineered γδT cells expressing TBB / H pCAR alone or in combination with pro-IL-18, pro-IL-18(GzB), or pro-IL-18(GzB) + granzyme B was analyzed using reporter cell lines. IL-18 activity was measured without stimulation or with MUC1+MDA-MB-468 breast cancer cells (“+468”) or beads coated with anti-CD3 and anti-CD28 antibodies (“aCD3 / 28 beads”). Figure 39 The results provided indicate that IL-18 activity is dependent on stimulation by transduced γδT cells. This is in contrast to co-expression of only TBB / H and pro-IL-18 (GzB) or TBB / H and pro-IL-18 (GzB). Figure 39 Compared to stimulated T cells, T cells co-expressing TBB / H, pro-IL-18 (GzB), and granzyme B produce higher IL-18 activity.

[0396] 5.9. Example 8: In vivo antitumor activity of pCAR-γδT cells coated with IL-18

[0397] The antitumor activity of pCAR-γδT cells was evaluated in a mouse model of tumor xenograft.

[0398] 1×10 6 MDA-MB-468 tumor cells expressing luciferase were intraperitoneally (ip) injected into female SCIDBeige mice to establish a xenograft model. Eleven days after tumor injection, 1×10⁻⁶ MDA-MB-468 tumor cells were injected intraperitoneally. 7 TBB / H pCAR-γδT cells (regardless of IL-18 expression). Total bioluminescence emission of the tumor (“total flux”) was measured at each treatment. Figure 40A-40F As shown, compared with SCIDBeige mice treated with TBB / H pCAR T cells, SCIDBeige mice treated with γδT cells co-expressing TBB / H+pro-IL-18(GzB)+granzyme B had significantly reduced tumor-derived total flux. γδT cells co-expressing TBB / H and constitutive IL-18 also showed a trend toward improved tumor control compared with γδT cells co-expressing both TBB / H and pro-IL-18(GzB)+granzyme B. Figure 40E and 40F ). Figure 40B The data shown are from 5 mice. Figure 40C From 4 mice, Figure 40D From 5 mice, Figure 40E From 4 mice, Figure 40FFrom 3 mice.

[0399] Figure 41 Survival data for mice treated with PBS, γδT cells expressing TBB / H alone, or in combination with constitutively TBB / H-expressing γδT cells are presented. Following tumor injection, IL-18, pro-IL-18 (GzB), or pro-IL-18 (GzB) was administered co-injected with granzyme B. The results indicate that γδT cells co-expressing TBB / H, pro-IL-18 (GzB), and granzyme B improved mouse survival.

[0400] 5.10. Example 9: In vivo antitumor activity of pCARαβ or γδ T cells coated with IL-18

[0401] Evaluation of the antitumor activity of pCAR-T cells in a mouse model of tumor xenograft.

[0402] 1×10 6 MDA-MB-468 tumor cells expressing luciferase were intraperitoneally (ip) injected into female SCIDBeige mice to establish a xenograft model. Eleven days after tumor cell injection, TBB / H pCAR T cells (1×10⁻⁶) were injected intraperitoneally. 7 pCAR-αβ or -γδ T cells, or 8 × 10 6 pCAR-γδT cells, or 4 × 10 6 pCAR-γδT cells, without exogenous IL-18 expression (“TBB / H”) or with only exogenous expression of pro-IL-18 or pro-IL-18 (GzB) along with granzyme B. Mixed bioluminescent emission (“total flux”) of tumors was measured from each treated animal.

[0403] Total flux measured in animals in each treatment group was pooled together and... Figure 30A , 30B Provided in 30C. As shown in the figure, SCID Beige mice treated with TBB / H pCAR-T cells co-expressing pro-IL-18 (GzB) and granzyme B had significantly reduced tumor-derived total flux compared to mice in other groups (PBS, TBB / H pCAR T cells, or TBB / H pCAR T cells co-expressing pro-IL-18). In αβT cells ( Figure 30A ) and γδT cells ( Figure 30B and Figure 30C This effect was observed in all of them.

[0404] 5.11. Example 10: Antitumor activity of second-generation CAR-T cells coated with IL-18

[0405] 5 × 10⁻⁶ luciferase-expressing 5 SKOV-3 tumor cells were intraperitoneally (ip) injected into female SCID Beige mice to establish an SKOV-3 xenograft model. Eighteen days after tumor cell injection, three groups of mice were injected with CAR-T cells via intraperitoneal injection. The first group received CAR-T cells engineered to co-express T1E28zErbB-targeting second-generation CARs with a 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 chimericantigen receptor T-cells in locally advanced or recurrent head and neck cancer,” Human Gene Ther. Clin. Devel. 24:134-142 (2013)). The second group of mice received T4-engineered T cells co-expressing a cleavable MT1-MMP (MMP14) pro-IL-18 variant (pro-IL18(MT1)). Figure 16 (As shown). Tumor cells expressed high levels of the MT1-MMP (MMP14) protease. The third control group consisted of T cells expressing truncated intracellular domains and emitting inactive signals, specifically the T1E-28z CAR (referred to as T1NA–T1E non-activating domain).

[0406] In this model, treatment with low-dose (500,000) second-generation CAR T cells or CAR T cells expressing T1NA (an intima-truncation control) was ineffective. In contrast, CAR T cells co-expressing T4 CAR and cleavable MT1-MMP (MMP14) pro-IL-18 resulted in tumor elimination in 1 / 5 of the mice and disease regression in 2 other animals. Figure 17C This provides another approach to limiting the activation of IL-18 in the tumor microenvironment.

[0407] 5.12. Example 11: In vitro antitumor activity of IL-36-armored pCAR-T cells

[0408] Constructs encoding TBB / H and the mature IL-36 fragment (pro-IL-36γ) were generated according to the above method. Then, by adding a cleavage site recognized by granzyme B (GzB) to the constructs encoding TBB / H and pro-IL-36γ, a construct encoding TBB / H and modified pro-IL-36γ was generated. Furthermore, by inserting the granzyme B coding sequence into the constructs encoding TBB / H and modified pro-IL-36γ, a construct encoding TBB / H+pro-IL-36(GzB)+granzyme B was also generated.

[0409] T cells were transfected with an SFG retroviral vector encoding TBB / H pCAR and pro-IL-36γ or a modified pro-IL-36γ (GzB).

[0410] T cells expressing TBB / H or co-expressing TBB / H, pro-IL-36γ, and granzyme B, or TBB / H, pro-IL-36γ (GzB), and granzyme B protease, were iteratively stimulated using MDA-MB-468 breast cancer cells or BxPC-3 pancreatic cancer cells. The effector:target cell (engineered T cell:tumor cell) ratio ranged from 2 to 0.03, including 1, 0.5, 0.25, 0.125, and 0.06. Residual viable cancer cells remaining after termination of co-culture were quantified using the MTT assay. Figure 42A (MDA-MB-468 cells) and Figure 42B The results shown in (BxPC-3 cells) indicate that TBB / HT cells expressing pro-IL-36γ and granzyme B or pro-IL-36γ (GzB) and granzyme B exhibit significant cytotoxic activity. During restimulation cycles, T cells co-expressing TBB / H, pro-IL-36γ (GzB), and granzyme B significantly proliferated. Figure 43A and 43B Compared to TBB / HT cells, in T cells expressing TBB / H+pro-IL-36γ+granzyme B or TBB / H+pro-IL-36γ(GzB)+granzyme B, IFN-γ ( Figure 44A and Figure 44B The production of ) also increased significantly.

[0411] T cells co-expressing TBB / H+pro-IL-36γ+granzyme B or TBB / H+pro-IL-36γ(GrzB)+granzyme B induced MDA-MB-468 cells ( Figure 45 ) and BxPC-3 cells ( Figure 46Tumor cells were killed at effector:target cell (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 were performed in triplicate).

[0412] 5.13. Example 12: In vivo antitumor activity of pCAR-T cells coated with IL-36

[0413] The antitumor activity of IL-36-encapsulated pCAR-T cells was further investigated in vivo. 1×10 6 MDA-MB-468 tumor cells expressing luciferase were intraperitoneally (ip) injected into female SCID Beige mice to establish a xenograft model. Twelve days post-tumor injection, 1×10⁻⁶ MDA-MB-468 tumor cells not expressing IL-36 were intraperitoneally injected. 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.

[0414] Total bioluminescence emission (“total flux”) of the tumor was measured at each treatment. Mice treated with T cells co-expressing TBB / H+pro-IL-36γ(GzB)+granzyme B showed a significant reduction in tumor-derived total flux compared to mice treated with TBB / H pCAR T cells. Figures 47A-47D ).

[0415] 6. Sequence

[0416]

[0417]

[0418]

[0419]

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431]

[0432]

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442]

[0443]

[0444]

[0445]

[0446]

[0447]

[0448]

[0449]

[0450]

[0451]

[0452]

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459] 7. Equivalents and scope

[0460] Those skilled in the art will recognize, or be able to determine, many equivalents of the specific embodiments of the invention described herein using only conventional experiments. The scope of the invention is not limited to the foregoing description, but rather as set forth in the appended claims.

Claims

1. An immune-responding cell expressing a modified procytokine of the IL-1 superfamily and comprising a chimeric antigen receptor (CAR), wherein the modified procytokine comprises, from the N-terminus to the C-terminus: (a) Propeptide; (b) A non-natural cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3, wherein the protease is granzyme B (GzB); and (c) Biologically active IL-18 fragments; The modified procytokine can be cleaved by granzyme B (GzB) to release the propeptide and the bioactive IL-18 fragment. The modified pro-cytokine is modified pro-IL-18, the sequence of which is shown in SEQ ID NO: 27, and the immune response cells are αβ T cells or γδ T cells.

2. The immune response cells of claim 1, wherein the modified pro-IL-18 is expressed by a polynucleotide as shown in SEQ ID NO:103 or 111.

3. The immune response cell of claim 1, wherein the bioactive IL-18 fragment is a polypeptide with the sequence shown in SEQ ID NO:

24.

4. The immune response cell of claim 1, further comprising an exogenous polynucleotide encoding the protease.

5. The immune response cell of claim 1, wherein the CAR is a second-generation chimeric antigen receptor (CAR) comprising: Signal area; First co-stimulation signal region; Transmembrane domain; and The first binding element that specifically interacts with the first epitope on the first target antigen.

6. The immune response cell of claim 5, wherein the first epitope is an epitope on the MUC1 target antigen.

7. The immune response cell of claim 6, wherein the first binding element comprises a CDR of the HMFG2 antibody.

8. The immune response cell of claim 6, wherein the first binding element comprises the VH and VL domains of the HMFG2 antibody.

9. The immune response cell of claim 6, wherein the first binding element comprises an HMFG2 single-chain variable fragment (scFv).

10. The immune-response cell of claim 5, further comprising a chimeric co-stimulatory receptor (CCR), wherein the CCR comprises: Second co-stimulation signal region; Transmembrane domain; and A second binding element that specifically interacts with a second epitope on a second target antigen.

11. The immune response cell of claim 10, wherein the second co-stimulation signal region is different from the first co-stimulation signal region.

12. The immune response cell of claim 10, wherein the second target antigen of the second epitope is selected from the group consisting of ErbB homodimers and heterodimers.

13. The immune response cell of claim 10, wherein the second target antigen is HER2.

14. The immune response cell of claim 10, wherein the second target antigen is an EGF receptor.

15. The immune response cell of claim 10, wherein the second binding element comprises a binding portion of T1E, ICR12, or ICR62.

16. The immune response cells according to any one of claims 1-15, wherein the cells express modified pro-IL-18, wherein the modified pro-IL-18 is the polypeptide shown in SEQ ID NO: 27, and wherein the cells further express: GzB expressed by exogenous polynucleotides; A chimeric antigen receptor (CAR) includes: Signal area; i. First common stimulation signal region; ii. Transmembrane domains; and iii. A first binding element that specifically interacts with a first epitope on the MUC1 target antigen; and A chimeric co-stimulatory receptor (CCR) includes: iv. Second co-stimulatory signal region; v. transmembrane domain; and vi. A second binding element that specifically interacts with a second epitope on a second target antigen.

17. Use of the immune response cells as described in any one of claims 1-16 in the preparation of a medicament for treating cancer; wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, and pancreatic cancer.

18. The use as described in claim 17, wherein the cancer is breast cancer.

19. The use as described in claim 17, wherein the cancer is ovarian cancer.

20. A method for preparing immune-response cells, characterized in that, The method includes the step of introducing a genetically modified organism; the method is for non-therapeutic purposes. The transgene encodes a modified procytokine of the IL-1 superfamily, wherein the modified procytokine comprises, from the N-terminus to the C-terminus: (a) Propeptide; (b) A non-natural cleavage site recognized by a protease other than caspase-1, cathepsin G, elastase, or protease 3, wherein the protease is granzyme B (GzB); and (c) Biologically active IL-18 fragments; The modified procytokine can be cleaved by granzyme B (GzB) to release the propeptide and the bioactive IL-18 fragment. The modified procytokine is a modified pro-IL-18 with the sequence shown in SEQ ID NO:

27. The immune response cells also include a chimeric antigen receptor (CAR) and the immune response cells are αβ T cells or γδ T cells.

21. The method of claim 20, further comprising the preceding step of activating γδ T cells with an anti-γδ TCR antibody.

22. The method of claim 21, wherein the anti-γδ TCR antibody is immobilized.

Images