Molecular engineering to enhance chimeric antigen receptor-mediated tumor immunotherapy

Abstract

The present disclosure provides a chimeric antigen receptor (CAR) immunotherapy for treating a hematologic cancer comprising (a) genetically modifying a population of immune effector cells comprising T cell receptors (TCRs) to stably express at least one CAR, wherein the ectodomain of the CAR specifically binds a cancer antigen; (b) expanding the population of CAR-containing immune effector cells in the presence of one or more cytokines in vitro to achieve a therapeutic dose, wherein the population of CAR-containing immune effector cells exhibits a nonexhausted memory T cell phenotype; and (c) infusing eligible subjects with the CAR-containing immune effector memory T cell phenotype population of cells as needed until the hematologic cancer in the body is destroyed. The intracellular signal transduction domain of the CAR includes 4-1BB, a CD3zeta T cell activation chain and a genetically engineered 'TLE3, which can be ectopically expressed.

Description

MOLECULAR ENGINEERING TO ENHANCE CHIMERIC ANTIGEN RECEPTOR- MEDIATED TUMOR IMMUNOTHERAPY CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. provisional application 63 / 707,903 (filed October 16, 2024) and U.S. provisional application 63 / 765,909 (filed March 3, 2025). The contents of each of these applications are incorporated herein by reference. FIELD OF THE INVENTION

[0002] The present invention relates to methods for improving persistence and decreasing exhaustion of genetically engineered immune effector cells for treating hematological cancers. BACKGROUND OF THE INVENTION The relationship between the immune system and cancer

[0003] A tumor originates from a normal cell that has undergone tumorigenic transformation. This transformed cell is the cell-of-origin (COO) for the tumor. Tumorigenesis consists of four stages [Bi, Q. J. Immunology Res. (2022) (2022) article 3128933, citing Balani, S. et al. Nature Communic. (2017) 8 (1): article 15422; Chaffer, CL and Weinberg, RA. Cancer Discovery (2015) 5 (10): 22-24; Loeb, LA and Harris, CC. Cancer Res. (2008) 68 (17): 6863- 71]: (1) tumor initiation, the initial stage of tumorigenesis, is the stage in which normal cells undergo irreversible genetic alterations under the influence of oncogenic factors, thus transforming into COOs with the possibility of malignant transformation; (2) tumor promotion is the period during which COOs clone selectively and transform into premalignant cells under the influence of protumor factors and other specific conditions; (3) malignant conversion is the stage in which premalignant cells start expressing malignant phenotypes; and (4) tumor progression is the final stage of tumorigenesis, in which premalignant cells develop into real tumor cells, obtain a series of new biological characteristics (including sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing or accessing vasculature, activating invasion and metastasis, deregulating cellular metabolism, avoiding immune destruction, and unlocking phenotypic plasticity, nonmutational epigeneticreprogramming, polymorphic microbiomes, and senescent cells) [Id., citing Hanahan, D. Cancer Discovery (2022) 12 (1): 31-46], and undergo more invasion and metastasis. These characteristics are the result of the superposition of various factors, particularly the tumor microenvironment (TME).

[0004] Tumors are edited by the immune system as they evolve and can escape rejection in many ways. In the elimination phase, when tumors arise in a tissue, a number of immune cells (e.g., NK cells, CD4 T cells, CD8 T cells, and γδT cells) can recognize and destroy potential tumor cells. If elimination is not completely successful, what follows is an equilibrium phase, in which, according to the cancer immunoediting hypothesis, tumor cells undergo changes or mutations that aid their survival because of the selection pressure imposed by the immune system so that a variety of different tumor cells develop. In the escape phase, tumor cells that have acquired the ability to elude the attentions of the immune system, by either escaping the killing mechanism or recruiting regulatory cells to protect it, spread unchallenged and become clinically detectable. [Janeway’s Immunobiology, 9thEd. Kenneth Murphy & Casey Weaver, Garland Science, New York, NY (2017), at pp 717].

[0005] Tumors can avoid stimulating an immune response or can evade it when it occurs by numerous mechanisms. For example, spontaneous tumors may initially lack mutations that produce new tumor-specific antigens that elicit T cell responses. Even when a tumor specific antigen is expressed and is taken up and presented by APCs, if co-stimulatory signals are absent the APC will tend to treat the tumor as self, rather than activating T cells. Some tumors lose the expression of a particular MHC class I molecule perhaps through immune selection by T cells specific for a peptide presented by that MHC class I molecule; such tumors that have lost such antigens are no longer eliminated by the immune system, a process termed “antigenic modulation”. Tumors also seem able to evade immune attack by creating a generally immunosuppressive microenvironment. Many tumors make immunosuppressive cytokines, such as transforming growth factor beta (TGFβ), which tends to suppress the inflammatory T cell responses and cell mediated immunity needed to control tumor growth. For example, TGFβ induces the development of inducible regulatory T cells (Tregs). Some tumors, such as melanoma, ovarian carcinoma, and B-cell lymphoma, produce the immunosuppressive cytokine IL-10, which can reduce dendritic cell activity and inhibit T cell activation. The microenvironment of some tumors also contains populations of myeloid-derived suppressor cells(MDSCs), which can inhibit T-cell activation within the tumor. Some tumors express immune checkpoints, which directly inhibit immune responses. Tumors also can produce enzymes that act to suppress local immune responses. For example, the enzyme indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan, an essential amino acid, to produce the immunosuppressive metabolite kynurenine. Tumor cells also can produce materials, such as collagen, which create a physical barrier around the tumor, preventing interaction with cells of the immune system. [Janeway’s Immunobiology, 9thEd. Kenneth Murphy & Casey Weaver, Garland Science, New York, NY (2017), at pp 718-19]. An established clinical tumor can be sustained by subpopulations of self-renewing cancer cells operationally termed cancer stem cells (CSCs) that can generate, intraclonally, both tumorigenic and non-tumorigenic cells. [Rycaj, K. and Tang, DG, Cancer Res. (2015) 75 (19): 4003-11].

[0006] Immune checkpoints. During immune homeostasis, a crucial mechanism of peripheral tolerance is the regulation of an effector T-cell response via immune checkpoints on cytotoxic lymphocytes (CTLs) and activated CD4+ T cells to protect tissue from inflammatory damage. For example, two checkpoint molecules, CTLA-4 and PD-1, act as negative regulators of T-cell function and have been associated with immune evasion in cancer [Gonzalez, H. et al. Genes & Development (2018) 32: 1267-84, citing Pardoll, DM (2012) Nat Rev Cancer 12: 252– 264]. The involvement of CTLA-4 signaling in cancer has been described in melanoma ([Id., citing Bouwhuis, MG et al. (2010) Cancer Immunol Immunother 59: 303–312], lung ([Id., citing Khaghanzadeh, N. et al. (2010) Cancer Genet Cytogenet 196: 171–174], breast ([Id., citing Erfani N. et al. Cancer Genet. Cytogenet. (2006) 165 (2): 114-20], gastric [Id., citing Hadinia, A. et al. (2007) J Gastroenterol Hepatol 22: 2283–2287], and colorectal ([Id., citing Hadinia, A. et al. (2007) J Gastroenterol Hepatol 22: 2283–2287; Dilmec, F. et al. (2008). Int J Immunogenet 35: 317–321] cancers. Furthermore, the engagement of PD-1 with its coreceptor, PD-L1 (expressed by other immune cells, mesenchymal cells, vascular cells, and cancer cells), results in the down-regulation of T-cell activity, which inhibits their anti-tumor activities, such as T-cell migration, proliferation, secretion of cytotoxic mediators, and restriction of cell killing ([Id., citing Topalian, SL et al. (2015) Cancer Cell 27: 450–461]. The use of immune checkpoint inhibitors such as anti-PD-1 (e.g., lambrolizumab, pembrolizumab (KEYTRUDA®) and nivolumab (OPDIVO®), anti-PD-L1 (MPDL3280A, Atezolizumab, TECENTRIQ®), and anti- CTLA4 (ipilimumab, YERVOY®) has had success enhancing the effector anti-tumor responsein different malignancies ([Id., citing Gotwals, P. et al. (2017). Nat Rev Cancer 17: 286–301], especially in melanoma and lung cancer ([Id., citing Hamid, O. et al. (2013) N Engl J Med 369: 134–144; Herbst, RS et al. (2014). Nature 515: 563–567; Topalian, SL et al. (2015) Cancer Cell 27: 450–461].

[0007] As the tumor grows and the TME changes, new antigens are produced, and the ability of the immune system to prime new repertoires of T cells and direct them toward the tumor changes, thus altering the efficacy of tumor containment. As the immune system functions to stall tumor growth, cancer cells and the TME simultaneously suppress anti-tumor function by engaging immune checkpoints and the recruitment of regulatory CD4+ T cells (Tregs). Tregs are responsible for suppressing the priming, activation, and cytotoxicity of other effector immune cells, such as TH1 CD4 T cells, CTLs, macrophages, NK cells, and neutrophils ([Id., citing Ward-Hartstonge KA, Kemp RA. (2017). Clin Transl Immunology 6: e154]. The Treg-mediated immunosuppression is orchestrated by contact-dependent mechanisms such as the expression of PD-L1, LAG-3, CD39 / 73, CTLA-4, or PD-1, with CTLA-4 and PD-1 even enhancing suppressive activity ([Id., citing Walker LS, Sansom DM. (2015) Trends Immunol 36: 63–70], and by contact-independent mechanisms, which involve the sequestration of IL-2 and production of immune-suppressive molecules such as IL-10, TGF-β, prostaglandin E2, adenosine, and galectin-1 [Id., citing Francisco, LM et al. (2009). J Exp Med 206: 3015–3029; Campbell, DJ (2015) Eur J Immunol 195: 2507–2513]. In squamous cell carcinoma, the inhibition of focal adhesion kinase (FAK)—a cell contact-independent mechanism—results in CCL5 secretion by cancer cells that induces the recruitment of Tregs to the tumor site, where they suppress cytotoxic anti-tumor CD8+ T cells ([Id., citing Serrels, A. et al. (2015) Cell 163: 160–173]. In breast and lung adenocarcinoma, Tregs suppress T-cell activation and the anti-tumor immune response in tumor-associated tertiary structures. Specific Treg depletion results in tumor cell death and increased production of IFN-γ ([Id., citing Bos, PD et al. (2013) J Exp Med 210: 2435–2466; Joshi, NS et al. (2015) Immunity 43: 579–590]. In breast cancer, infiltration of Tregs was correlated with worse patient outcome [Id., citing Allaoui, R. et al. (2017) Cancer Biomark 20: 395–409].

[0008] In metastasis, CTLs exert an anti-metastatic effect in bone metastasis [Id., citing Bidwell, BN et al. (2012) Nat Med 18: 1224–1231], while prospective analyses of lung and breast cancer patients established an opposite correlation between the level of circulating cancercells and T cells in peripheral blood [Id., citing Mego, M et al. (2016) Circulating tumor cells (CTC) are associated with defects in adaptive immunity in patients with inflammatory breast cancer. J Cancer 7: 1095–1104; Sun, WW et al. (2017) Onco Targets Ther 10: 2413–2424].

[0009] These data extend to clinical trials reporting the therapeutic efficacy of immune checkpoint inhibition in metastatic carcinomas [Id., citing Di Giacomo, AM et al. (2012) Lancet Oncol 13: 879–886; Queirolo, P. et al. (2014) J Neurooncol 118: 109–116; Motzer, RJ et al. (2015) N Engl J Med 373: 1803–1813; Furudate, S. et al. (2016) Case Rep Oncol 9: 644–649; Goldberg, SB et al. (2016) Lancet Oncol 17: 976–983; Pai-Scherf, L. et al. (2017) Oncologist 22: 1392–1399]. Checkpoint inhibitors are significantly effective in treating brain metastatic tumors from melanoma and lung cancer [Id., citing Queirolo, P. et al. (2014). J Neurooncol 118: 109– 116; Goldberg, SB et al. (2016) Lancet Oncol 17: 976–983; Di Giacomo, AM et al. (2017) Cytokine Growth Factor Rev 36: 33–38]. Evidence suggests that the effectiveness of checkpoint inhibition in melanoma brain metastasis depends on extracranial disease and peripheral activation of CD8+ T cells [Id., citing Taggart, D. et al. (2018) Proc Natl Acad Sci 115: E1540– E1549]. On the other hand, a high level of circulating Tregs has been associated with a higher risk of metastasis in non-small lung carcinoma patients. [Id., citing Erfani, N. et al. (2012) Lung Cancer 77: 306–311]. Similar associations have been described in breast cancer [Id., citing Metelli, A. et al. (2016) Cancer Res 76: 7106–7117], colorectal carcinoma metastasis [Id., citing Wang, Q. et al. (2014) Cell Immunol 287: 100–105, and hepatocellular carcinoma [Id., citing Ye, LY et al. (2016) Cancer Res 76: 818–830]. Chimeric antigen receptor immunotherapy

[0010] T cell receptors (TCRs) expressed on the surface of T lymphocytes can only recognize the peptide antigens presented to them through major histocompatibility complexes (MHCs) by antigen-presenting cells (APCs). For example, human T cells genetically engineered to express a high-avidity TCR that targets HPV-16 E7 through recognition of the E7(11-19)epitope complexed with HLA-A*02:01 demonstrated clinical activity in a phase 1 clinical trial for the treatment of metastatic human papilloma virus-associated epithelial cancers (NCT02858310). [Naagarasheth, NB et al. Nature Med. (2021) 27 (30: 419-425). However, transcriptional loss of the specific HLA genes presenting the targeted viral epitope under CD8+ T cell pressure leading to a late acquired resistance to immunotherapy has been reported in metastatic Merkel cell carcinoma. [Paulson, KG et al. Nature Communic. (2018) 9: 3868].

[0011] Monoclonal antibodies can recognize and bind cell surface-expressed antigens that are not presented by MHCs. This ability has been utilized to redirect the cytotoxicity of various kinds of immune cells toward tumor surface-expressed antigens of interest that can be either tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). [Kozani, PS et al. Front. Immunol. (2022) 13: 795164].

[0012] The term “chimeric antigen receptor” or “CAR” as used herein refers to a synthetic MHC-independent receptor that targets T cells to a chosen antigen and reprograms T cell function, metabolism, and persistence [Riviere, I. and Sadelain, M. Mol. Ther. (2017) 25 (5): 1117-24, citing Eshhar, Z. et al. Springer Semin. Immunopathol. (1996) 18: 199-209; Sadalain, M. et al. Nat. Rev. Cancer (2003) 3: 35-45]. A CAR is mainly composed of three parts: an extracellular antigen recognition domain, usually a single-chain variable fragment (scFv) derived from a monoclonal antibody; a spacer / hinge region and transmembrane domain; and an intracellular signal transduction domain.

[0013] In first generation CARs, the intracellular signal transduction domain included a CD3ζ chain. In a second-generation CAR, the intracellular signal transduction domain included a CD3 ζ chain and one costimulatory molecule. In a third generation CAR, the intracellular signal transduction domain included a CD3ζ chain and two costimulatory molecules. The intracellular signal transduction domain of a fourth generation CAR includes a CD3 ζ chain and one costimulatory molecule and expresses a cytokine, such as IL-12.

[0014] An optimal CAR will specifically bind an antigen expressed in high abundance in tumor cells to form an effective immunological synapse leading to downstream T-cell signaling and a potent and specific anti-tumor effect.

[0015] Through their extracellular domain, CARs bind cell surface molecules independently of the major histocompatibility complex (MHC), in contrast to the physiological T cell receptor, which engages MHC / peptide complexes. CARs may thus target proteins, carbohydrates, or glycolipids and function independently of patient HLA haplotype. Binding to antigen triggers T cell activation, which is commonly mediated by the cytoplasmic domain of the CD3-ζ chain. [Riviere, I. and Sadelain, M. Mol. Ther. (2017) 25 (5): 1117-24, citing Irving, BA and Weiss, A. Cell (1991) 64: 891-901; Romeo, C., Seed, B. Cell (1991) 64: 1037-46; Letourneur, F. and Klausner, RD. Proc. Natl. Acad. Sci. USA (1991) 88: 8905-9; Eshhar, Z. etal. Proc. Natl Acad. Sci. USA (1993) 90: 720-24; Brocker, T. et al. Eur. J. Immunol. (1993) 23: 1435-1439].

[0016] Merely providing T cell activation is, however, not sufficient to direct a productive immune response. [Id., citing Brocher, T. and Karjalainen, K. J. Exp. Med. (1995) 181: 1653-1659; Gong, MMC et al. Neoplasia (1999) 1: 123-7; Brocker, T. Blood (2000) 96: 1999-2001]. The CARs that have provided tangible clinical benefits incorporate a costimulatory domain [Id., citing Krause, A. et al. J. Exp. Med. (1998) 188: 619-26;] which enables T cells to expand and retain their functionality upon repeated exposure to antigen [Id., citing Maher, J. et al. Nat. Biotechnol. (2002) 20: 70-75]. These receptors have been dubbed second generation CARs [Id., citing Sadelain, M. et al. Curr. Opinion. Immunol. (2009) 21: 215-233] and are key to the design of persisting engineered T cells that can attack tumors as long as they retain their functionality. Several reviews have addressed CAR design [Id., citing Jensen, MC and Riddell, SR. Curr. Opinion. Immunol. (2015) 33: 9-15; van der Stegan, SJ et al. Nat. Rev. Drug Discov. (2015) 14: 499-509; Sadelain, M. J. Clin. Invest. (2015) 125: 3392-3400; Maus, MV and June, CH. Clin. Cancer Res. (2016) 22: 1875-1884], CAR prospects for solid tumors [Id., citing Hinrichs, CS and Restifo, NP Nat. Biotechnol. (2013) 999-1008; Morello, A. et al. Cancer Discov. (2016) 6: 133-46], and T cell manufacturing [Id., citing Wang, X. and Riviere I., Mol. Ther. Oncolytics. (2016) 3: 16015; Levine, BL et al. Mol. Ther. Methods Clin. Dev. (2016) 4: 92-101; Wang, X., Riviere, I. Cancer Gene Ther. (2015) 22: 85-94].

[0017] Clinical trials of CAR-T cells targeting the B cell antigen CD19 have shown sustained complete responses in patients with refractory relapsed B cell cancers, although with associated toxicity [Gavriil, A. et al. Cancers (2020) 12: 2326, citing Maude, SL et al. N. Engl. J. Med. (2014) 371: 1507-17; Titov, A. et al. Cell Death Dis. (2018) 9: 897]. Solid tumors have been generally refractory for diverse reasons, including physical barriers to access of T cells, the presence of suppressive immune cells and tumor cells, the variability of expression of the target antigen, and in many tumors, low mutational burden limiting antigen spreading and generation of memory responses [Id., citing Majzner, RG and Mackall, CL. Nat. Med. (2019) 25: 1341-1355; Martinez, M. et al. Front. Immunol. (2019) 10: 128; Yu, W-L et al. Cancers (2019) 11: 47]. Tumor Escape from CAR-T cell therapy.

[0018] To date, antigen loss, immune dysfunction, exhaustion and microenvironment- mediated upregulation of anti-apoptotic pathways have been identified as major modes of escape by solid tumors from CAR-T cell immunotherapy.

[0019] Antigen loss. Loss of expression of the antigen on tumor cells targeted by the CAR extracellular domain via the selective immune pressure of CAR-T cells is a major mechanism of CAR-T cell therapy failure. While antigen downregulation or dim expression is a well-known event in lymphoma and myeloma treated with therapeutic IgG antibodies [Rasche, L. et al. N. Kroger, et al, eds. Chapter 4 in The EBMT / EHA CAR-T Cell Handbook (2002) doi.org / 10.1007 / 978-3-030-94353-0_4, citing Plesner, T. et al. Cell (2020) 9 (2): 378; Jilani, I. et al. (2003) Blood (2003) 102 (10): 3514-20], complete target loss is a phenomenon typically occurring after T-cell based therapy, such as CAR-T cell or T cell engaging bispecific antibodies (TCE) therapy, and rarely after treatment with antibody-drug conjugates. For example, antigen loss is a key mechanism of resistance to B cell immunotherapies targeting CD19, CD20 and CD22. [Id., citing Orlando, EJ, et al. Nat. Med. (2018) 24 (10): 1504-6; Bannerji, R. et al. Blood (2018) 132 (Suppl. 1): 1690, Paul, MR et al. J. Pediatr. Hematol. Oncol. (2019) 41 (8): e546-e9]. Case reports also have described irreversible B cell maturation antigen (BCMA) loss after anti- BCMA CAR-T cell treatment of multiple myeloma (MM) [Id., citing Da Via, MC et al. Nat. Med. (2021) 27: 616-619; Leblay, N. et al. Blood (2020) 136 (Suppl.1) 11-2].

[0020] In addition to antigen loss, there are other mechanisms that limit or abrogate the effective recognition of cancer cells by CAR-T cells; these mechanisms are either conveyed directly by tumor cells or through a rewiring of the microenvironment. In preclinical models, especially in solid tumors, it was shown that tumor-infiltrating CAR-T cells undergo rapid loss of functionality, limiting their therapeutic efficacy. This hyporesponsiveness appears to be reversible when the T cells are isolated away from the tumor and is associated with upregulation of intrinsic T cell inhibitory enzymes [diacylglycerol kinase and SHP-1, an SH2 domain- containing protein tyrosine phosphatase] and with the expression of surface inhibitory receptors (e.g., PD-1, LAG3, TIM3, and 2B4) [Id., citing Moon, EK et al. Clin. Cancer Res. (2014) 20 (16): 4262-4273].

[0021] Dysfunctional T cell states

[0022] Although TCR and CAR signaling are different, most of the framework for understanding the influence of CAR signaling on CAR-T function and differentiation is derived from work in the TCR field.

[0023] TCRs have a hierarchical threshold of antigen density for induction of cell lysis, proliferation and cytokine product [Watanabe, K., et al. Front. Immunol. (2018) 9: 2486, citing Au-Yeung, BB et al. Proc. Nat. Acad. Sci. USA (2014) 111: E3679-88], where less antigen density is required for cell lysis than for cytokine production. CAR-T cells can recognize target cells with considerably lower levels of target antigen and have hierarchical T cell signaling thresholds for cell lysis, proliferation and individual cytokine production. Watanabe demonstrated that the target antigen density required to induce T cell proliferation and cytokine production was higher than that required to induce CAR mediated lysis. [Id., citing Watanabe, K. et al. J. Immunol. (2015) 194: 911-920].

[0024] Functional T cells perform a range of activities upon stimulation with their cognate antigen. They can (1) expand, (2) secrete effector cytokines, and lyse target cells; (3) survive after removal of antigen stimulation and (4) do all the above upon secondary antigen challenge. A T cell is considered dysfunctional if at least one of these activities is not met.

[0025] Some studies suggest that the efficacy of immunotherapy is limited by the generation of dysfunctional T cells in the tumor microenvironment (TME).

[0026] T cell exhaustion

[0027] T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells.

[0028] Exhausted T cells become dysfunctional via a progressive loss of functionality that is mainly mediated by upregulation of multiple inhibitory receptors, including the inhibitory pathways mediated by PD-1 in response to binding of PD1 ligand 1 (PD-L1) and / or PD-L2. [Wherry EJ and Kurachi, M. Nature (2015) 15: 486-499, citing Okazaki T, et al., Nature Immunol. (2013) 14:1212–1218, Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965, Araki K, et al. Cold Spring Harb. Symp. Quant. Biol. (2013) 78:239–247]. Exhausted T cells can co-express PD-1 together with lymphocyte activation gene 3 protein (LAG3), 2B4 (also known as CD244), CD160, T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM3; encoded by HAVCR2), CTLA-4 and many other inhibitory receptors [Id., citingBlackburn SD, et al. Nat. Immunol. (2009) 10:29–37]. Typically, the higher the number of inhibitory receptors co-expressed by exhausted T cells, the more severe the exhaustion. It has been suggested that inhibitory receptors such as PD-1 might regulate T cell function in several ways [Id., citing Schietinger A, Greenberg PD. Trends Immunol. (2014) 35:51–60; Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965], e.g., first, by ectodomain competition, which refers to inhibitory receptors sequestering target receptors or ligands and / or preventing the optimal formation of microclusters and lipid rafts (for example, CTLA-4); second, through modulation of intracellular mediators, which can cause local and transient intracellular attenuation of positive signals from activating receptors such as the TCR and co-stimulatory receptors [Id., citing Parry RV, et al. Molec. Cell. Biol. (2005) 25:9543–9553; Yokosuka T, et al. J. Exp. Med. (2012) 209:1201–1217; Clayton KL, et al. J. Immunol. (2014) 192:782–791]; and third, through the induction of inhibitory genes [Id., citing Quigley M, et al. Nat. Med. (2010) 16:1147–1151]. Co-stimulatory receptors also are involved in T cell exhaustion [Id., citing Odorizzi PM, Wherry EJ. J. Immunol. (2012) 188:2957–2965.]. It has also been possible to exploit the potential beneficial role of co-stimulation to reverse exhaustion by combining agonistic antibodies to positive co-stimulatory pathways with blockade of inhibitory pathways. 4-1BB (also known as CD137, encoded by TNFRSF9) is a TNFR family member and positive co-stimulatory molecule that is expressed on activated T cells. Combining PD-1 blockade and treatment with an agonistic antibody to 4-1BB dramatically improved exhausted T cell function and viral control [Id, citing Vezys V, et al. J. Immunol. (2011) 187:1634–1642]. Soluble molecules are a second class of signals that regulate T cell exhaustion; these include immunosuppressive cytokines such as IL-10 and transforming growth factor-β (TGFβ) and inflammatory cytokines, such as type I interferons (IFNs) and IL-6. [Wherry EJ and Kurachi, M. Nature (2015) 15: 486-499.]

[0029] Antigen-independent (tonic) signaling of a CAR-T cell also can result in an exhausted phenotype. [Gavriil, A. et al. Cancers (2020) 12: 2326, citing Long, AH et al. Nat. Med. (2015) 21]. Long et al showed that second generation CAR-T cells with scFvs targeting 2- ganglioside (G(D2)) derived from antibody 14g2a, CD22, and ErbB2 plus a CD28-CD3ζ endodomain, in contrast to CD19 CARs, signal constitutively during their in vitro expansion. These CARs had enhanced T cell exhaustion and diminished anti-tumor activity in vivo. The authors demonstrated that scFv clustering, due to interaction between scFv framework regions,was responsible. Similarly, Frigault et al. have shown that high level expression of certain scFv- containing CARs leads to long term antigen-independent signaling and functional exhaustion. [Id., citing Frigault, MJ et al., Cancer Immunol. Res. (2015) 3: 356-67].

[0030] Beltra, et al. [Beltra, JC, et al. Immunity (2020) 19: 825-41] identified a four-cell stage developmental framework for T cell exhaustion during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice with complementary analysis in human tumors. A similar pattern of four Tex subsets was found in mouse B16 tumors and for tumor- infiltrating lymphocytes from human melanoma. The four distinct Tex subsets were based on Ly208 (Slamf6) and CD69 expression. Two interconverting TCF-1+ progenitor states were identified, one quiescent and blood inaccessible and a second that initiated robust cell cycling and gained access to circulation. This second TCF-1+Tex subset gave rise to a TCF-1- Tbethiintermediate Tex subset that (re)acquired some effector-like features. These intermediate Tex cells ultimately became terminally differentiated, losing T-bet (and gaining Eomes) and permanently exiting the cell cycle. This final transition was coordinated by TOX-mediated antagonism of T-bet. PD-1 pathway blockage preferentially expanded the second progenitor and the T-bethiintermediate Tex subsets. These cell subset transitions were regulated by the transcription factors TCF-1, T-bet and TOX in a hierarchical developmental pathway. The level and duration of chronic antigen stimulation and infection seemed to be key factors that lead to T cell exhaustion and correlated with the severity of dysfunction during chronic infection. T cell senescence

[0031] Another dysfunctional state is senescence, which occurs when T cells permanently arrest their cell cycle and proliferation while retaining cytotoxic capability. Senescent T cells have a distinct phenotype including downregulated expression of the costimulatory molecules CD27 and CD28, and high expression of CD57, killer cell lectin-like receptor subfamily G member 1 (KLRG-1), and CD45RA [Zhang, J. et al. EBioMedicine (2021) 68: 103409, citing Barbarin, A. et al. Front. Immunol. (2017) 8: 316; Covre, LP et al. Aging Cell (2020) 19: e13272; Liu, W. et al. Cancers (2020):12]. Senescent T cells also show a terminally differentiated phenotype with downregulation of chemokine receptors CCR7 and CD45RO but upregulation of CD45RA [Id., citing Henson, SM et al. J. Clin. Invest. (2014) 124: 4004-4016; Huang, B. et al. J. Immunotherapy Cancer (2020) 8; Kunert, A. et al. J. Immunother. Cancer (2019) 7: 149]. In senescent T cells, the efficiency of TCR signaling is compromised in that theexpression of key components of TCR signaling (CD3, Lck, Zap70, SLP-76, LAT, and PLCγ1) [Id., citing Pereira, BI et al. Nature Immunol. (2020) 21: 684-694; Lanna, A. et al. Nature Immunol. (2014) 15: 965-972], as well as the costimulatory molecules CD27 and CD28 [Id., citing Ye, J. et al. Blood (2012) 120: 2021-2031; Lanna, A. et al. J. Immunol. (2013) 191: 3744- 3752] is decreased. In addition, the phosphorylation of Zap70 is impaired following CD3 activation [Id., citing Pereira, BI et al. Nature Immunol. (2020) 21: 684-694; Lanna, A. et al. Nature Immunol. (2014) 15: 965-72]; phosphorylation of Zap-70 is required to initiate T cell receptor signaling [Yan, Q. et al. Mol. Cell Biol. (2013) 33 (11): 2188-2201]. Senescent T cells, when stimulated with anti-CD3 plus anti-CD28, especially those isolated from old individuals, produce significantly lower amounts of IL-2, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and granzyme B than do other types of T cells [Zhang, J. et al. EBioMedicine (2021) 68: 103409, citing Henson, SM et al. Eur. J. Immunol. (2015) 45: 1441-1451; Song, Y. et al. Aging Cell (2018) 17]. Moreover, TCRβ chain diversity roughly declines linearly with age, especially in senescent CD8+ T cells [Id., citing Britanova, OV et al. J. Immunol. (2014) 192: 2689-2698; Bjorkstrom, NK et al. Blood (2012) 120: 3455-3465]. These findings led to the conclusion that senescent T cells probably dampen TCR-dependent antigen-specific killing.

[0032] Senescent T cells also upregulate the expression of NK cell receptors (NKRs), including NKG2A / C and killer cell lectin like receptor G1 (KLRG-1) [Id., citing Barbarin, A. et al. Front. Immunol. (2017) 8: 316; Pereira, BI et al. Nature Immunol. (2020) 21: 684-694]. On NK cells, NKG2C delivers activating signals through immunoreceptor tyrosine-based activation motifs at the cytoplasmic tails of DAP12 transmembrane proteins [Ma, M. et al. Front. Immunol. (2017) 8: 1176, citing Call, ME et al. Nat. Immunol. (2010) 11: 1023-1029], while NKG2A appears to inhibit NK activation via immunoreceptor tyrosine-based inhibitory motifs in the cytoplasm [Id., citing Lanier, LL. Annu. Rev. Immunol. (1998) 16: 359-63]. KLRG1 plays an inhibitory role in human NK cells and T cells [Yang, X. et al. BMC Cancer (2021) 21: 752].

[0033] Senescent T cells display higher levels of CD107a, granzyme B, and perforin than do other subsets with no stimulation [Zhang, J. et al. EBioMedicine (2021) 68: 103409, citing Song, Y. et al. Aging Cell (2018) 17] and under NKG2D stimulation [Id., citing Pereira, BI et al. Nature Immunol. (2020) 21: 684-694]. In vitro experiments have shown that senescent T cells kill tumor cells independent of TCR, with the same efficiency as natural killer (NK) cells [Id., citing Pereira, BI et al. Nature Immunol. (2020) 21: 684-694]. This evidence led to theconclusion that although antigen specific killing is lost, senescent T cells with strong nonspecific killing potential fight antitumor immunity to some extent. T cell anergy

[0034] Anergy, which is generally described as an induced hyporesponsive state with low IL-2 production or incomplete activation to which naïve T cells fall upon low co-stimulatory and / or high co-inhibitory stimulation, is another dysfunctional state. [Crespo, J. et al. Curr. Opin Immunol. (2013) 25 (2): 214-221].

[0035] The evidence for T cell anergy in the tumor context has been indirect. A core problem has been a lack of positive markers to characterize a loss of function state of T cells. Nonetheless, the following observations support that T cell anergy can be an important phenomenon in cancer:

[0036] First, there is an active imbalance between stimulatory and inhibitory B7 family members in the tumor microenvironment [Id., citing Zou, W. Nature Rev. Cancer (2005) 5: 263- 74; Pardoll, DM. Nat. Rev. Cancer (2012) 12: 252-264; Zou W. and Chen, L. Nat. Rev. Immunol. (2008) 8: 467-477; Blank, C. et al. Cancer Res. (2004) 64: 1140-1145]. Human tumors and tumor associated antigen presenting cells (APCs) often express high levels of B7-H1 (CD274 or PD-L1), B7-H2 (CD275 or ICOS-L), B7-H3 (CD276), B7-H4 (B7S1 or B7x), and B7-DC (CD273 or PD-L2) with low-to-absent expression of B7.1 (CD80) and B7.2 (CD86) [Id., citing Zou W. and Chen, L. Nat. Rev. Immunol. (2008) 8: 467-77; Blank, C. et al. Cancer Res. (2004) 64: 1140-1145; Curiel, TJ et al. Nat. Med. (2003) 9: 562-567; Kryczek, I. et al. J. Exp. Med. (2006) 203: 871-881]. This indicates a poor co-stimulatory, high co-inhibitory and therefore anergy-promoting environment.

[0037] Second, animal model studies have shown that introduction of B7.1 into tumors by transfection or blockade of inhibitory B7 family members can reduce tumor growth or result in spontaneous tumor rejection in vivo [Id., citing Zou W. and Chen, L. Nat. Rev. Immunol. (2008) 8: 467-77; Blank, C. et al. Cancer Res. (2004) 64: 1140-1145; Curiel, TJ et al. Nat. Med. (2003) 9: 562-567; Kryczek, I. et al. J. Exp. Med. (2006) 203: 871-881; Chen, L. et al. Cell (1992) 71: 1093-1102; Gajewski, TF. J. Immunol. (1996) 156: 465-72].

[0038] Third, homeostatic proliferation of anti-tumor T cells in a lymphopenic host both reverses anergy and promotes tumor rejection in vivo [Id., citing Brown, IE et al. J. Immunol. (2006) 177: 4521-4529].

[0039] Fourth, there is evidence indicating antigen specific, T cell-intrinsic dysfunction in the tumor microenvironment [Id., citing Nazareth, MR et al. J. Immunol. (20007) 178: 5552- 5562; Broderick, L. et al. Clin. Immunol. (2006) 118: 159-169].

[0040] Based on this evidence, it has been suggested that T cell anergy may be a functional mechanism in patients with cancer. However, the relative impact of T cell anergy on tumor immunity remains to be defined.

[0041] Cellular and molecular mechanisms controlling T cell anergy are insufficiently understood. It is generally accepted that T cells that are presented antigen along with suboptimal CD28 co-stimulation [Id., citing Schwartz, RH. Science (1990) 248: 1349-1356; Schwartz, RH. (2003) 21: 305-334] and / or high co-inhibition [Id., citing Greenwald, RJ et al. Immunity (2001) 14: 145-55] result in anergic phenotypes, as characterized by their low IL-2 production and cell cycle arrest at the G1 / S phase. Early growth response gene 2 (Egr2) may be a central transcription factor that regulates the T cell anergic state [Id., citing Zeng, Y. et al. J. Exp. Med. (2012) 209 (12): 2157-2163].

[0042] It has been suggested that the anergy program is initiated by improper mTOR and Ras / MAPK signaling in the cell, a pathway which lies directly downstream of TCR / CD28 engagement. Specifically, the sole binding of TCR by MHC promotes Ca2+ imbalance on T cells and retention of active-RAP-1 in the cytosol, an imbalance that would normally be corrected by co-stimulation through CD28 (Ras / MAPK) [Id., citing Boussiotis, VA et al. Science (1997) 278: 124-128; Dolmetsch, RE et al. Nature (1998) 392: 93393-6]. The effects of this imbalance on the genetic reprogramming of these cells have been hypothesized to be mediated by NFAT homodimer formation and transcription of anergy-inducing genes [Id., citing Anandasabapathy, N. et al. Immunity (2003) 18: 535-547; Soto-Nieves, N. et al. J. Exp. Med. (2009) 206: 867- 876]. The E3 ubiquitin ligase family can affect PI3K, mTOR, and Ras / MAPK signaling pathways and help to actively maintain anergy [Id., citing Anandasabapathy, N. et al. Immunity (2003) 18: 535-547; Jeon, MS et al. Immunity (2004) 21: 167-177; Mueller, DL. Nat. Immunol. (2004) 5: 883-890]. Epigenetic factors such as IKAROS (through acetylation), an inhibitory C- type lectin with an intracellular immunoreceptor tyrosine-based inhibitory motif (ITIM), and Sirt1 are involved in histone modifications that promote T cell anergy [Id., citing Gao, B. et al Proc. Natl Acad. Sci. USA (2012) 109: 899-904; Bandyopadhyay, S. et al. Blood (2007) 109: 2878-2886]. Thus, anergy may be the combined result of factors that negatively regulateproximal TCR-coupled signal transduction, together with a program of active transcriptional silencing that is reinforced through epigenetic mechanisms [Id., citing Wells, AD. J. Immunol. (2009) 182: 7331-7341]. Tumor microenvironment

[0043] The TME comprises cellular components, noncellular components and signaling molecules [Arneth, B. Medicina (2020) 56: 15, citing Spill, F. et al. Curr. Opin. Biotechnol. (2016) 40: 41-48; Del Prete, A. et al. Curr. Opin. Pharmacol. (2017) 35: 40-47]. The cellular components include endothelial cells, which play a role in tumor development and tumor cell protection from the immune system; immune cells, such as granulocytes, lymphocytes and macrophages, which are involved in various immune responses and activities, such as inflammatory reactions orchestrated by the tumor to promote survival; and fibroblasts, which allow cancer cells to migrate from the primary tumor location into the bloodstream for systemic metastasis and provide a passage for endothelial cells undergoing angiogenesis in the tumor. The extracellular matrix (ECM), which is mainly secreted by cancer-associated fibroblasts (CAFs), which produce more ECM proteins than normal fibroblasts, is composed of various macromolecules, including collagens, glycoproteins (fibronectin and laminins), proteoglycans and polysaccharides with different physical and biological properties. [Brassart-Pasco, S. et al. Front. Oncology (2020) 10: 397]. Interstitial matrix, primarily synthesized by stromal cells, is rich in fibrillary collagens and proteoglycans. CAF secretome analyses show an increased secretion of bone morphogenetic protein (BMP) 1, thrombospondin-1 and elastin interface 2 [Id. citing Santi, A. et al. Proteomics (2018) 18: e1700167; Socovich, AM and Naba, A. Semin. Cell Dev. Biol. (2019) 89: 157-166]. Several splice variants of fibronectin ED-A and ED-B and tenascins C and W may be secreted by CAFs. [Id., citing Grahovac, J. and Wells, A. Lab Investig. (2014) 94: 31-40].

[0044] Tumor cells can influence the microenvironment through the release of extracellular paracrine signals that induce peripheral immune tolerance and support tumor angiogenesis [Arneth, B. Medicina (2020) 56: 15, citing Korneev, KV et al. Cytokine (2017) 89: 127-135]. The composition and structure of the TME is known to vary among cancer types and among patients. [Id., citing Bussard, K. et al. Breast Cancer Res. (2016) 18: 84].

[0045] Cellular interactions in the TME also affect cancer development and progression. Within the heterogeneous TME, T cells are a major part of the immune infiltrate. [Zhang, Z. etal. Frontiers Cell & Devel. Biol. (2020) 8: 17]. The intratumoral T cell populations comprise naïve T cells, memory T cells, effector T cells, and Tregs. [Id., citing Hashimoto, M. et al. Annu. Rev. Med. (2018) 69: 301-18]. In a murine model, the tumor-specific T cell dysfunctional exhaustion state was found to be initiated early after tumor initiation and antigen encounter. [Id., citing Schietinger, A.et al., Immunity (2016): 45: 389-401]. Dysfunctional CD8+ T cells are characterized by a loss of effector functions, such as cytotoxicity and proliferation. In addition, the upregulation of immune checkpoints and changes in transcriptional and metabolic molecules have been described as hallmarks of T cell dysfunction. Emerging evidence indicates that epigenetic states, including DNA methylation and histone modifications, and chromatin landscapes are closely associated with the functional state of dysfunctional or exhausted CD8+ T cells. [Id., citing Pauken, KE et al. Science (2016) 342: 1242454; Sen, DR et al. Science (2016) 354: 1165-1169; Kartikasari, AER et al. Front. Immunol. 9: 3109].

[0046] In many hematological cancers, the bone marrow tumor microenvironment (BMME) is known to upregulate anti-apoptotic mechanisms in tumor cells through tight cross- talk between MSCs and tumor cells; tumor cell lysis by T and NK cells also is largely mediated via activation of extrinsic and intrinsic apoptotic pathways [Rasche, L. et al. N. Kroger, et al, eds. Chapter 4 in The EBMT / EHA CAR-T Cell Handbook (2002) doi.org / 10.1007 / 978-3-030- 94353-0_4, citing Hanabuchi, S. et al. Proc. Natl Acad. Sci.199491(1)]: 4930-4; Falschlehner, C. et al. Immunology (2009) 127 (2): 145-54; Carneiro, BA and El-Deiry, WS. Nat. Rev. Clin. Oncol. (2020) 17 (7): 395-417; Culen, SP et al. Cell Death Differ. (2010) 17 (4): 616-23; Sutton, VR et al. J. Exp. Med. (2000) 192 (10): 1403-1414]. Myeloma cell-bone marrow mesenchymal stromal cell (BMMSC) interactions have been reported to protect myeloma cells from conventional cytotoxic T cells and from daratumumab (DARZALEX®) redirected NK cells [Id., citing McMillin, DW et al. Blood (2012) 119 (15): e131-8; de Haart, SJ et al. Clin. Cancer Res. (2013) 19 (20): 5591-601; de Haart, SJ et al. Haematologica (2016) 101 (8): e339-e342].

[0047] The importance of the tumor stroma in the efficacy of CAR-T cells. These studies were extended to CAR-T cells by testing a panel of nine different myeloma-reactive CAR-T cells that were reactive to three different myeloma-associated antigens (CD138, B Cell Maturation Antigen (BCMA), and CD38) with different target affinities and with different costimulatory domains (CD28, 4-1BB, or CD28 plus 4-1BB [Id., citing Holthof, L. et al. Clin. Cancer Res. (2021) 27 (13): 3793-803]. In the absence of BMMSCs, BCMAbb2121CAR-T cells,high affinity CD38 CAR-T cells, and intermediate affinity CD38 CAR-T cells containing CD28 costimulatory domains showed high levels of anti-myeloma cell lysis, whereas other CAR-T cells showed moderate cytotoxic activity against myeloma cells. BMMSCs did not modulate the lytic activity of highly lytic CAR-T cells but readily protected myeloma cells against all other CAR-T cells with intermediate killing capacity. Overall, a strong inverse correlation was demonstrated between the lytic capacity of the CAR-T cells and the extent of BMMSC-mediated protection. Furthermore, the BMMSC-mediated protection of myeloma cells from these CAR-T cells was readily abrogated by inhibition of survivin (an apoptosis inhibitor), myeloid cell leukemia 1 (MCL-1), and X-linked inhibitor of apoptosis (XIAP) using the small molecule surviving inhibitor FL118. These results confirmed that BMMSC-mediated immune resistance was mediated by negative regulation of apoptotic pathways. In addition, in a solid tumor mouse model, destruction of the tumor stroma contributed to eradication of large tumors by HER2- specific CAR-T cells (Id., citing Textor, A. et al. Cancer Res. (2014) 74 (23): 6796-6805). Current Challenges of CAR-T therapy

[0048] Toxicities with CAR-T cells can be classified as on-target (likely to achieve the intended outcome at the target) or off-target (adverse effects resulting from modulation of other targets). The longest running clinical experience in the CAR-T cell field has been with CD19- directed CAR T cells, for which two on-target toxicities, cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) have been described. [Finck, AV et al. Nat. Med. (2022) 28 (4): 678-689, citing Lee, DW et al. Biol. Blood Marrow Transpl. (2019) 25: 625-638]. CRS and ICANS toxicities, while now regarded as a class effect of CAR-T cells as they have been observed in patients treated with CAR-T cells targeting CD19, BCMA, and many other cell surface structures [Id., citing Lateau, CA et al. Cancer Cell (2021) 39: 1553- 1557] may be more of a feature of increasingly potent therapies. Also, the expression of targets in healthy patients may not be the same as in patients with cancer.

[0049] Cytokine release syndrome (CRS) is a potentially life-threatening, systemic inflammatory response observed following administration of antibodies and adoptive T cell therapies, including CAR-T cell therapy. With immune activation, an associated increase in a wide array of systemic proinflammatory cytokines, such as IL-6, C-reactive protein (CRP), ferritin, and IL-2, occurs, which coincides with peak-T cell expansion. Severity can vary from mild symptoms including fever, myalgia, and fatigue, to severe symptoms including but notlimited to acute respiratory distress syndrome, hypotension, disseminated intravascular coagulation, and / or renal and liver toxicities. [Shalabi, H. et al. Chapter 12 in Novel Designs of Early Phase Trials for Cancer Therapeutics (2018) Academic Press, pages 175-191].

[0050] ICANS was initially thought to result from a cascade of off-target effects due to systemic inflammation and cytokine release [Finck, AV et al. Nat. Med. (2022) 28 (4): 678-689, citing Taraseviciute, A. et al. Cancer Discov. (2018) 8: 750-63]; however, some aspects of the syndrome may be related to the expression of CD19 in pericytes in the central nervous system [Id., citing Parker, KR et al. Cell (2020) 183: 126-142], which was unexpected since CD19 initially was described as a B-lineage-restricted protein.

[0051] On-target, off-tumor toxicity can occur because most tumor-associated antigens are not tumor-specific but are only overexpressed on tumor cells. Although target antigens are expressed on both tumor and normal cells, not all CAR-T cell therapies exhibit observable on- target off-tumor effects. [Han, et al. J. Hematol. & Oncol. (2919) 12: 128, citing Brown, CE et al. Clin. Cancer Res. (2015) 21 (18): 4062-4072; Katz, SC et al. Clin. Cancer Res. (2015) 21 (14): 3149-3150; Klampatsa, A. et al. Cancers (Basel) (2017) 9 (9): 115; Kakarla, S. et al. Mol. Ther. (2013) 21 (8): 1611-1620; Wang, LC et al. Cancer Immunol. Res. (2014) 2 (2): 154-166]. During solid tumor treatment, on-target, off-tumor toxicities following the infusion of CAR-T cells can cause serious adverse events or be lethal. [Han, X. et al. J. Hematol & Oncol. (2019) 12: 128, citing Morgan, RA et al. Mol. Ther. (2010) 18 (4): 843-851; Lamers, CH et al. J. Clin. Oncol. (2006) 24 (13): 20-22; Tran, E. et al. J. Exp. Med. (2013) 210 (6): 1125-1135].

[0052] Disease relapse after CAR-T cell infusions. There are two main forms of disease relapse: antigen-positive relapse in the early phase and antigen escape relapse in a later phase. The disease relapse of antigen-positive cells is closely related to CAR-T cell persistence, meaning how long the functional CAR-T cells exist after infusion into the subject. CAR-T cell persistence is a major challenge. Preclinical and clinical trials have shown that the 4-1BB (CD137) costimulatory domain induced longer-term persistence of CAR-T cells than did CD28 [Id., citing Zhao, Z. et al. Cancer Cell (2015) 28 (4): 415-428; Kachenderfer, JN et al. J. Clin. Oncol. (2015) 33 (6): 540-549].

[0053] Antigen escape, including antigen loss or down regulation, also can drive disease relapse following CAR-T immunotherapy. Complete antigen loss is not necessary for CAR-T cell resistance, and in some cases, a reduction in antigen density is sufficient for tumor cells toevade CAR-T cells [Id., citing Fry, TJ et al. Nat. Med. (2018) 24 (10: 20-28]. Investigators have shown that combinatorial multi-antigen targeted strategies could effectively prevent the tumor escape caused by low antigen density that results from CAR-T cell trogocytosis [Id., citing Hamieh, M. et al. Nature (2019) 568 (7750): 112-116]. Immune escape caused by low antigen level and / or heterogeneous expression is a cause of solid tumor resistance to CAR-T cells.

[0054] Optimization of CAR design. Optimization of the CAR design is equally important for better persistence and overall treatment efficacy.

[0055] The concepts of Boolean Logic gates have been applied to CAR-T cell design primarily as a tool for increasing specificity by integrating signals from two or more target antigens. Such studies have focused on AND gates (where two target antigens are required for full activation to limit toxicity), OR gates (involving two equally powered CARs against alternate target antigens in the same tumor) and NOT gates (an off-signal associated with a target antigen present on normal tissue as a means to limit off-target toxicity). A CAR with an intracellular domain providing signal 2 but no signal 1 is also referred to as a chimeric co- stimulatory receptor (CCR) and is said to provide “co-stimulation in trans,” thereby enhancing proliferation and survival without an effect on cytotoxicity. [Gavriil, A. et al. Cancers (2020) 12: 2326, citing Kloss, CC et al. Nat. Biotechnol. (2013) 31: 71-75; Lanitis, E. et al. Cancer Immunol. Res. (2013) 1: 43-53].

[0056] Several paired target antigens have been evaluated using AND gate approaches in preclinical models [Gavriil, A. et al. Cancers (2020) 12: 2326, citing Wilkie, S. et al. J. Clin. Immunol. (2012) 32: 1059-1070; Kloss, CC et al. Nat. Biotechnol. (2013) 31: 71-75]. Several groups have further designed bi-specific CARS, which utilize the OR-gate circuit. [Id., citing Zah, E. et al. Cancer Immunol. Res. (2016) 4: 498-508; Munter, SD et al. Int. J. Mol. Sci. (2018) 19: 403]. The NOT gate logic has been applied to generate CARs coupled with an immune- inhibitory signal (iCAR), such as PD-1 or CTLA-4, to divert off-target immunotherapy responses [Id., citing Fedorov, VD, et al. Sci. Transl. Med. (2013) 5: 215ra172].

[0057] The basic logic principles above have been extended to the concept of the sequential AND gate, whereby engagement with one target antigen triggers the expression of a CAR against a second target antigen. Since the first antigen does not provide activating T cell signals, and the CAR against the second antigen is not expressed until the T cell reaches the tumor, this arrangement potentially can limit both tonic signaling and exhaustion, as well as off-target toxicity. [Id., citing Morsut, L. et al. Cell (2016) 164: 780-791; Roybal, KT et al. Cell (2016) 167: 419-432; Roybal, KT et al. Cell (2016) 164: 770-779].

[0058] Immune checkpoint blockade is another approach to overcome tumor-associated immune suppression [Finck, AV et al. Nat. Med. (2022) 28 (4): 678-89, citing Rafiq, S. et al. Nat. Biotechnol. (2018) 36: 847-856] and revitalize T cells [Id., citing Xia, Y. et al. Blood Rev. (2016) 30: 189-200]. In clinical trials, the combination of the PD-1 immune checkpoint inhibitor pembrolizumab (KEYTRUDA®) with lesothelin-targeting CAR-T cells further enhanced the persistence and function of the latter in patients with malignant pleural diseases [Id., citing Adusumillli, PS et al. Cancer Discov. (2021) 11: 2748-2763]. One study demonstrated that two CARs, one targeting EGFRvIII and the other targeting IL-13Ra2, preferred different checkpoint blockades within the same tumor model [Id., citing Yin, Y. et al. Mol. Ther. Oncolytics (2018) 11: 20-38]. An alternative approach is to engineer CAR-T cells to produce bispecific antibodies, as shown in a preclinical study for the treatment of glioblastoma. [Id., citing Choi, BD et al. Nat. Biotechnol. (2019) 37: 1049-1058].

[0059] Adoptive cellular immunotherapy with engineered chimeric antigen receptor (CAR)-T cells has transformed the treatment of hematological malignancies [Weber, EW et al. Cell (2020) 181: 46-62; Lim, WA and June, CH. Cell (2017) 168: 724-740]. However, relapse remains a major challenge to over 50% of patients who initially respond to CAR-T cell therapies. In addition, CAR-T cells are less effective in treating solid tumors [Cappell, KM and Kochenderfer, JN. Nat. Rev. Clin. Oncol. (2023) 20: 359-371]. The diminished efficacy of CAR- T therapy can be ascribed to long-term exposure of CAR-T cells to tumor antigen and tumor microenvironment, leading to T cell dysfunction / exhaustion Ruella, MN. et al. Nat. Rev. Drug Discov. (2023) 22: 976-995]. CAR-T cell exhaustion is manifested as induction of inhibitory mechanisms, attenuated effector function, failure to acquire memory cell characteristics, and poor in vivo persistence. Drivers of T cell exhaustion include key transcription factors (TFs), such as nuclear factor of activated T cell (NFAT), thymus high-mobility group box protein (TOX), and nuclear receptor group 4a (NR4A), which act together to induce the expression of multiple inhibitory receptors, such as programmed cell death 1 (PD1), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin domain and mucin domain 3 (TIM3) and cytotoxic T lymphocyte antigen 4 (CTLA4) [Mognol, GP et al. Proc. Natl Acad. Sci. USA (2017) 114:E2776-E2785].. Therefore, there is an urgent need to identify approaches to prevent or mitigate CAR-T cell exhaustion, enhance CAR-T cell function, and improve CAR-T cell persistence.

[0060] Efforts to enhance CAR-T function have focused on DNA sequence-specific transcription factors (TFs) or epigenetic regulators that modify DNA per se or histones. Loss-of- function approaches such as triple deletion of NT4A family members (Nr4a1, Nr4a2, and Nr4a3) [Chen, J. et al. Nature (2019) 567: 530-534]. or ablation of BLIMP1 TF [Yung, IY. Sci. Transl. Med. (2022) 14: eabn7336], TET2 (a methylcytosine dioxygenase that facilitates DNA demethylation) [Fraietta, JA et al., Nature (2018) 558: 307-312; Jain, N. et al. Nature (2023) 615: 315-322] DNMT3A (de novo DNA methyl transferase that catalyzes DNA methylation) [Prinzing, B. et al. Sci. Transl. Med. (2021) 13: eabh0272, or SUV39H1 (a histone methyl transferase that catalyzes H3K9me3) [Jain, N. et al. Cancer Discov. (2024) 14: 142-157] in CAR-T cells result in enhanced tumor control.

[0061] Conversely, gain-of-function via overexpression of TFs, including c-JUN [Lynn, RC et al. Nature (2019) 576: 293-300], BATF [Seo, H. et al. Nat. Immunol. (2021) 22: 983-995] and FOXO1 Chan, JD et al. Nature (2024) 629: 201-210; Doan, AE et al. Nature (2024) 629: 211-218] in CAR-T cells also show beneficial effects on curtailing tumor growth. The potency of each approach differed depending on the models used, and it remains unknown if these strategies could be effectively translated into clinic therapeutics and patient care. Such uncertainties necessitate novel approaches to boost CAR-T cell functions.

[0062] For example, studies show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR-T cells. [Doan, AE et al. Nature (2024) 629: 211-218]. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the anti-tumor activity of CAR-T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR-T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation and exhibited enhanced persistence and tumor control in vivo. [Id.]

[0063] Overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR-T cells. TCF1OEand FOXO1OEcells that were serially rechallenged with Nalm6 leukemia both exhibited enhanced cytokine secretion compared withcontrols, but only FOXO1OEincreased CD8 proliferation and memory marker expression while suppressing the levels of TOX. In contrast, TCF1OEincreased the expression of TOX and CD39 relative to controls expressing the truncated rat nerve growth factor receptor (tNGFR) marker alone, consistent with a more exhausted or effector-like phenotype.

[0064] Tle proteins

[0065] TFs do not act alone and require cofactors to achieve regulatory specificity, stability, and transcriptional output; this is particularly important for TFs such as Runx3, which are stably expressed throughout the CD8+ T cell differentiation process [Zhao, X. et al. Nature Immunol. (2024) 25: 294-306, citing Pipkin, ME. Immunol. Rev. (300): 100-124; Shan, Q. et al. Nature Immunol. (2017) 18: 931-939]. One such cofactor, Transducin-Like Enhancer of split (Tle) protein, interacts with many immune function-related transcription factors including Tcf1 / Lef1, Runx1 / Runx3, Myc and Blimp1 [Id., citing Orian, A. et al. Proc. Natl Acad. Sci. USA (2007) 104: 15771-15776; Ren, B. et al. Genes & Development (1999) 13: 125-137; Zhao, X. et al. Nat. Rev. Immunol. (2022) 22: 147-157; Seo, W. and Taniuchi, I. Mol Cells (2020) 43: 107-113].

[0066] Tle proteins are the mammalian homologues of Drosophila Groucho transcriptional repressor. There are four mammalian Tle genes, Tle1–4, which encode full-length Tle proteins; these Tle proteins have both unique and redundant functions in development of multiple organs, including hematopoiesis [Id., citing Turki-Judeh, W. and Courey, AJ. Curr Top. Dev. Biol. (2012) 98: 65-96; Buscarlet, M. and Stifani, S. Trends in Cell Biol. (2007) 17: 353- 361; Jennings, BH and Ish-Horowicz, D., Genome Biol. (2008) 9: 205; Gasperowicz, M. and Otto, Fl. J. cell Biochem. (2005) 95: 670-687]. In T lineage cells, TLE3 is most abundantly expressed, followed by Tle4 and Tle1, with Tle2 at a barely detectable level [Id., citing Xing, S. et al. J. Exp. Med. (2018) 215: 2211-2226]. Tle1, TLE3, and Tle4 are critically required for CD8+ T cell lineage choice during thymic development, in a gene-dose dependent manner [Id., citing Xing, S. et al. J. Exp. Med. (2018) 215: 2211-2226]. Functional analyses collectively demonstrated that TLE3 supports effector memory T cell fate and targeting TLE3 instead promotes central memory T cell formation.

[0067] The Tle proteins contain five defined domains, with the N-terminal glutamine- rich domain (namely, Q domain) and C-terminal tryptophan-aspartate (WD)–repeat domain responsible for most of protein–protein interactions (Xing, S. et al. Exp. Med. (2018) 215 (8):2211-2226, citing Buscarlet , M. and Stifani,, S. Trends Cell Biol. (2007) 17: 353-361; Turki- Judeh, W. and Courey, A.J. Curr. Top. Dev. Biol. (2012) 98: 65-96]. The full-length Tcf1 and Lef1 contain an N-terminal β-catenin binding domain, C-terminal high-mobility-group (HMG) DNA binding domain, and a context-dependent regulatory (CRD) domain that harbors histone deacetylase activity (Xing, S. et al. Nat. Immunol. (2016a) 17: 695-703]. Both CRD and HMG domains in Tcf1 and Lef1 contribute to interaction with the Q domain in Tle proteins [Id., citing (Arce, L. et al., (2009) BMC Cancer 9: 159; Chodaparambil, JV et al. EMBO J. (2014) 33: 719- 731]. On the other hand, both Runx1 and Runx3 use their conserved C-terminus VWRPY motifs to interact with the WD-repeat domain in Tle proteins (Id., citing Levanon, D. et al. Proc. Natl Acad. Sci. USA (1998) 95: 11590-11595].

[0068] All Tle proteins interact with Tcf1 and Lef1 downstream of the Wnt signaling pathway [Xing, S. et al. Exp. Med. (2018) 215 (8): 2211-2226, citing Brantjes, HJ., et al., Nucleic Acids Res. (2001) 29: 1410-1419; Daniels, DL and Weis, WI. Nat. Struct. Mol. Biol. (2005) 12: 364-371; Staal, FJT and Sen, JM Eur. J. Immunol. (2008) 38: 1788-1794)] suggestive of involvement in T cell development and function [Id., citing Xue, HH and Zhao, DM Ann. N.Y. Acad. Sci. (2012) 1247: 16-33; Steinke, FC and Xue, HH, Immunol. Res. (2014) 59: 45- 55]. Tle1 has been shown to bind Runx1 [Id., citing Levanon, D. et al. Proc. Natl Acad. Sci. USA (1998) 95: 11590-11595], which is essential for the generation and maintenance of hematopoietic stem / progenitor cells (HSPCs) [Id., citing Cai, X. et al. Cell Stem Cell (2015) 17: 165-177]. In line with such broadly interacting partners, germline deletion of Tle4 in mice causes a profound reduction in cellularity of hematopoietic cells including HSPCs and B cells (Id., citing Wheat, JC et al. PLoS One (2014) 9: e105557].

[0069] In a recent study [Zhao, X. et al. Nature Immunol. (2024) 25: 294-306) our group analyzed Tle genes in CD8+ T cell responses to acute viral infection. While ablating all three Tle 1, 3, 4 genes abrogates differentiation of effector CD8+ T (Teff) cells, TLE3 shows non- redundant roles in regulating memory CD8+ T (Tmem) lineage stability without affecting Tmem cell pool size. Induced deletion of TLE3 accelerates formation of CD62L+ central Tmem (TCM) at the expense of CD62L- effector Tmem (TEM) cells. This effect is not a phenocopy of ablating any of its partner TFs (Tcf1, Runx3 or Tbet), and likely results from specific disruption of Tle-sensitive TF complexes, highlighting unique integrative functions by TLE3. On the mechanistic side, key observations include: (1) besides its known co-repressor roles, TLE3functions as a transcriptional coactivator, where TLE3 establishes / maintains a chromatin open state to induce its target gene expression; 2) TLE33 engages Tbet as a novel partner TF in Teff and Tmem cells, to form complexes with its known partners, such as Runx3; (3) while its expression is relatively stable, TLE3 is dynamically redistributed in the Teff and Tmem cell genome, representing a novel means / pattern of gene regulation.

[0070] Building on these findings, we reasoned that gain-of-function for TLE3 may confer advantages to anti-tumor immunity. Because ablation of TLE3 showed both ‘preferred” and ‘unwanted’ regulatory effects and Tle3 has distinctive functional domains, we further reasoned that TLE functions can be uncoupled through molecular engineering and utilized to improve anti-tumor immunity. The present disclosure is a result of this approach. SUMMARY OF THE INVENTION

[0071] According to one aspect, the present disclosure provides a chimeric antigen receptor (CAR) immunotherapy for treating a hematologic cancer comprising: (a) genetically modifying a population of immune effector cells comprising T cell receptors (TCRs) to stably express at least one CAR, wherein the ectodomain of the CAR specifically binds a cancer antigen; (b) expanding the population of CAR-containing immune effector cells in the presence of one or more cytokines in vitro to achieve a therapeutic dose, wherein the population of CAR- containing immune effector cells expresses a nonexhausted memory T cell phenotype; and (c) infusing eligible subjects with the CAR-containing immune effector memory T cell phenotype population of cells as needed until all cancer in the body is destroyed.

[0072] According to some embodiments of the CAR immunotherapy, the hematologic cancer is a leukemia, a lymphoma or a myeloma.

[0073] According to some embodiments of the CAR immunotherapy, the CAR comprises an extracellular antigen recognition domain, a spacer / hinge region and transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain of the CAR comprises 4-1BB; a CD3ζ T cell activation chain; and a genetically engineered TLE3 molecule.

[0074] According to some embodiments of the CAR immunotherapy, the engineered TLE3 molecule is derived from a wild type TLE3 comprising, in order, an N-terminal Q domain, a GP domain, a CcN domain, an SP domain and a C terminal WDR domain; or the engineeredTLE3 molecule is a truncated TLE3 protein; or the engineered TLE3 molecule is a C-terminus truncated TLE3 (TLE3ΔWDR) protein.

[0075] According to some embodiments of the CAR immunotherapy, expression of the engineered TLE molecule is ectopic.

[0076] According to some embodiments of the CAR immunotherapy, the target antigen of the CAR is CD19; the target antigen of the CAR is CD20; the target antigen of the CAR is CD22; the target antigen of the CAR is NKG2D-1; the target antigen of the CAR is CD33; the target antigen of the CAR is BCMA, or the target antigen of the CAR is CD123.

[0077] According to some embodiments of the CAR immunotherapy, the CAR- containing population of immune effector cells recognizes two or more tumor associated antigens simultaneously.

[0078] According to some embodiments of the CAR immunotherapy, the immune effector cell population engineered to express the CAR comprising a TLE3 construct by lentiviral transduction is a CD3+ T cell population; and the T cell population comprises CD4+ T cells, CD8+ T cells, or both.

[0079] According to some embodiments of the CAR immunotherapy, the immune effector cell engineered to express the CAR comprising a TLE3 construct by lentiviral transduction adapted from the CAR-T design is an NK cell.

[0080] According to some embodiments of the CAR immunotherapy, the source of the immune effector cell is peripheral blood or umbilical cord blood; or the population of immune effector cells is autologous to the subject; or the population of immune effector cells is allogeneic to the subject.

[0081] According to some embodiments of the CAR immunotherapy, when the engineered TLE3 molecule is a C-terminus truncated TLE3 (TLE3ΔWDR) protein, compared to a no-TLE3 CAR immune effector control, expression of the TLE3ΔWDR construct in CAR- containing immune effector cells enhances CAR-immune effector cell function despite chronic antigen stimulation; and / or expression of the TLE3ΔWDR construct in stimulated CAR- containing immune effector cells promotes activation of genes that promote anti-tumor activity and also represses genes involved in excessive effector function, exhaustion or both; and / or expression of the TLE3ΔWDR construct in stimulated CAR-containing immune effector cells reduces expression of at least one inhibitory receptor comprising PD-1, CTLA-4, LAG3, TIGIT,or a combination thereof; and / or expression of the CAR-TLE3ΔWDR construct in stimulated CAR-containing immune effector cells induces transcriptomic changes comprising differential expression of genes that enhance the anti-tumor response and / or promotes proliferation of the CAR-containing immune effector cells; enhances persistence of the CAR-containing immune effector cells and maintains immune homeostasis despite chronic antigen stimulation; and / or expression of the CAR-TLE3ΔWDR construct in stimulated CAR-containing immune effector cells reprograms the immune effector cells to acquire stem-like, memory cell characteristics including production of IFN-γ and TNF, expression of TCF-1 or both, and / or expression of the TLE3ΔWDR construct in CAR-containing immune effector cells enhances CAR-immune effector cell function after rechallenge with the cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0083] FIG.1A and FIG. 1Bdepict generation of CAR-T cells expressing engineered TLE3. FIG.1A is a diagram showing TLE protein structure (Taken from Buscarlet and Stifani, Trends in Cell Biol. (2007) 17: 353-361], designated domains in WT and engineered TLE proteins. FIG.1B is a diagram showing human CD19-directed chimeric antigen receptor (CAR) protein, linked to engineered TLE3 protein via P2A peptide, which allows simultaneous translation of both proteins.

[0084] FIG.2A, FIG. 2B, FIG.2C, and FIG.2D show that TLE3ΔWDR expression enhances leukemia control by CAR-T cells and promotes survival. FIG. 2A is a diagram showing key steps of the generation of engineered CAR-T cells from healthy donors. FIG. 2B shows the study design using a xenograft of Raji leukemic and CAR-T cells into highly immunodeficient NSG mice. FIG.2C shows longitudinal tracking of leukemic burden in NSG recipients via in vivo imaging. Images are representative of 6 independent experiments. FIG. 2D shows a Kaplan-Meier survival curve of NSG recipients. Data are pooled results from 2 independent experiments, and the p values were obtained by log-rank (Mantel-Cox) statistical analysis.

[0085] FIG.3A, FIG. 3B, FIG.3C, and FIG.3D show that TLE3ΔWDR expression confers growth and survival advantages to CAR-T cells. FIG.3A shows immunoblotting of endogenous (Endo.) WT TLE3 and engineered TLE3ΔWDR in CAR-T cells, with detection of β-actin as a loading control. FIG. 3B shows tracking CAR-T cell accumulation over extended culture ex vivo. Established control or TLE3ΔWDR+ CAR-T cells (12 days after initial activation) were sort-purified and seeded in 24-well plate at 5x10E5 cell / well in the presence of IL-2 (10 ng / ml), IL-7 (5 ng / ml) and IL-15 (5 ng / ml). FIG.3C shows the result of PCA of RNA- seq libraries of CAR-T cells from two healthy donors. The established CAR-T cells were cultured with or without Raji cells (at 1:9 ratio) for 48 hrs and then sort-purified for RNA-seq analysis. FIG.3D shows numbers of differentially expressed genes (DEGs) that were common to both donor-derived CAR-T cells. Up- and down-regulated genes in TLE3ΔWDR+ CAR-T cells were defined as TLE3ΔWDR-induced and -repressed genes, respectively. FIG. 3E and FIG.3F are heatmaps showing select TLE3ΔWDR-induced (FIG.3E) and -repressed (FIG. 3F) genes, with key functional annotation marked on the right. Color scale represents z-score transformed transcript levels.

[0086] FIG.4A and FIG. 4B show that TLE3ΔWDR expression amplifies CD8+ CAR- T cell response in vivo. FIG.4A shows detection of CAR-T cells in peripheral blood cells of NSG recipients at the indicated time windows. FIG.4B shows detection of CD4+ vs CD8+ CAR-T cell distribution in the peripheral blood cells. Data are means ± s.d. from two independent experiments, with individual data points shown. **, p<0.01; ***, p<0.001 by student’s t-test. NRD, not reliably detected, because of the low frequency of CAR-T cells.

[0087] FIG.5A, FIG. 5B, and FIG. 5C show that TLE3ΔWDR limits CAR-T cell exhaustion at an early response stage in vivo. On 12 days post-tumor implantation (dpt), splenocytes were harvested from NSG recipients, and analyzed for CD4+ and CD8+ CAR-T cell numbers (FIG. 5A), and expression of PD1, TIM3 and LAG3 in the CAR-T cells (FIG.5B). FIG.5C. Cumulative data on frequency of TIM3hiPD1+, TIM3loPD1+, and PD1¬– subsets in splenic CAR-T cells. Data in A and C are means ±s.d. from two independent experiments, with individual data points shown. *, p<0.05; **, p<0.01; ***, p<0.001; ns, not statistically significant by student’s t-test.

[0088] FIG.6A, FIG. 6B, FIG.6C and FIG.6D show that TLE3ΔWDR sustains repression of CAR-T cell exhaustion at late response stage in vivo. FIG.6A shows theoccurrence of high leukemia burden in NSG recipient mice on 30 days post-tumor implantation (dpt), as detected with in vivo imaging. For examples, please refer to FIG 2B, where all recipients of control CAR-T and the 5th recipient (on the far right of the panel) of TLE3ΔWDR+ CAR-T cells were considered to have high leukemia burden, while leukemia in the rest of TLE3ΔWDR+ CAR-T cell recipients were below detection limited and considered ‘cured’. Data are cumulative from 3 independent experiments. FIG 6B shows CAR-T cell numbers in recipient spleens on 30 dpt. The differences between control and TLE3ΔWDR+ CAR-T cell numbers were not statistically significant and therefore unmarked. FIG.6C shows detection of PD1 and TIM3 expression in CAR-T cells, where TLE3ΔWDR+ CAR-T cells were from the ‘leukemia- cured’ recipients. FIG. 6D shows cumulative data of relative expression of coinhibitory receptors in CD8+ (top) and CD4+ CAR-T cells (bottom), where the average of geometric mean fluorescence intensity (gMFI) for each receptor in control CAR-T cells was set as 1, and all samples were then normalized to the average in the same experiment. This approach allows direct comparison across different experiments. Data in FIG. 6B and FIG.6D are means ±s.d. from two independent experiments **, p<0.01; ***, p<0.001 by student’s t-test.

[0089] FIG.7A, FIG. 7B, and FIG. 7C show that TLE3ΔWDR expression promotes stemness and memory T cell features in CAR-T cells. FIG. 7A shows detection of TOX and TCF1 expression CAR-T cells via intranuclear staining. FIG. 7B shows detection of cytokine production by CAR-T cells. On 30 days post-tumor implantation (dpt), splenocytes were stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of Golgi plug and Golgi stop for 5 hours, and detected for IFN-γ, TNF-α and IL-2 via intracellular staining. IFN-γ+ cells were then gated for further analysis of TNF-α and IL-2 production. FIG.7C shows profiles of cytokine production in CAR-T cells, where cells producing all three cytokines are marked as "triple", those producing IFN-γ and TNF-α as "double", and those producing IFN-γ only as "single" producers. Data are means ±s.d. from two independent experiments.

[0090] FIG.8A, FIG. 8B and FIG.8C show TLE3ΔWDR-expressing CAR-T cells protect against secondary leukemia challenge. The leukemia-bearing NSG mice were infused with control or TLE3ΔWDR+CAR-T cells as in FIG.2B. The recipients that survived to 36 days post-tumor implantation (dpt) were rechallenged with 10-20 fold higher dose of Raji leukemic cells. FIG. 8A shows a Kaplan-Meier survival curve of the rechallenged recipients. Data are pooled results from 2-3 independent experiments, and the p values were determined with log-rank (Mantel-Cox) statistical analysis. FIG.8B shows longitudinal tracking of leukemia burden in rechallenged mice harboring TLE3ΔWDR+CAR-T cells via in vivo imaging. Data are representative from 3 experiments. FIG. 8C. Tracking of frequency of leukemia-responding TIM3hiPD1+ subset in TLE3ΔWDR+CAR-T cells in the peripheral blood cells. The day of injection of high-dose Raji cells was designated as day 0 in the graph.

[0091] FIG.9 is a schematic of gene expression regulation by genomic enhancers, which contain binding sites for sequence-specific transcription factors (TFs). The transcription factors recruit cofactors (COFs) that mediate the regulatory communication between the core promoter and the enhancer. Core-promoters typically contain characteristic core-promoter elements or motifs, for example, TATA box, Initiator or Downstream Promoter Element (SPE). Core promoters encompass short sequences of about 100 base pairs surrounding the transcription start site, where Polymerase II (Pol II) assembles and initiates transcription. [Taken from Zabidi, MA and Stark, A. Trends Genet. (2016) 32 (12): 801-14].

[0092] FIG.10 is a diagram summarizing the beneficial impact on CAR-T cells by ectopic expression of TLE3ΔWDR, where + and – signs denote positive and negative regulation, respectively.

[0093] FIG.11 shows that TLE3ΔWDR reprograms CAR-T cells to acquire molecular features of memory T cells from an early response stage in vivo. On 12dpt, control or TLE3ΔWDR+ CAR-T cells were sort-purified from NSG recipient spleens and were analyzed with CITE-seq. FIG.11A is a uniform manifold approximation and projection (UMAP) plot of scRNA-seq data on CD8+ CAR-T cells, with each dot representing a single cell and all five clusters identified with Seurat in distinct colors. FIG.11B is a bar graph showing distribution of control and TLE3ΔWDR+ CAR-T cells in each cluster defined in A. FIG.11C is a heat map showing expression of select characteristic genes for indicated functional programs, with each column corresponding to a single cell and the color scale representing z-score transformed transcript levels. DETAILED DESCRIPTION OF THE INVENTION Glossary

[0094] The terms “4-1BB” or “CD137” or “tumor necrosis factor receptor super family 9” as used herein refer to an inducible costimulatory receptor expressed on activated Tand natural killer (NK) cells. [Chester, C. et al. Blood (2018) 131 (1): 49-57]. There is widespread expression of 4-1BB throughout the hematopoietic and nonhematopoietic compartments. 4-1BB is expressed on activated monocytes and NK cells, dendritic cells (DCs), neutrophils, eosinophils and mast cells. [Id., citing Wilcox, RA et al. J. Immunol. (2002) 168 (9): 4262-4267; Heinisch IV, et al. Eur. J. Immunol. (2000) 39 (120): 3441-3446; Schwarz, H. et al. Blood (1995) 85 (4): 1043-1052].

[0095] On T cells, 4-1BB is transiently expressed after T-cell receptor engagement and, when 4-1BB is engaged by its natural or artificial ligand, it provides CD28-independent costimulation resulting in enhanced proliferation and TH1 cytokine production. [Id., citing DeBenedette, MA e al. J. Immunol. (1997) 158 (2): 551-9; Saoullli, K. et al. J. Exp. Med. (1998) 187 (11): 1849-1862]. The major biological ligand of 4-1BB, 4-1BBL, is expressed on activated APCs, including dendritic cells (DCs) and macrophages and B cells [Id., citing Goodwin, RG et al. Eur. J. Immunol. (1993) 23 (10): 2631-2641; Alderson, MR et al. Eur. J. Immunol. (1994) 24 (9): 2219-2227; Futagawa, T. et al. Intl Immunol. (2002) 14 (3): 275-286; Pollok, KE et al. Eur. J. Immunol. (1994) 24 (2): 367074]. Ligation of 4-1BB recruits TNFR- associated factor (TRAF) 1 and TRAF2 and induces signaling through the master transcription factor NF-κB and MAPKs. [Id., citing Lee, DY et al. PLoS One (2013) 8 (7): e69677; Vinay, DS and Kwon, BS. Semin. Immunol. (1998) 10 (6): 481-489]. Upon ligation with agonist mAbs, 4-1BB rapidly internalizes to an endosomal compartment from which it keeps signaling through this pathway. [Id., citing Bradley, JR and Pober, JS. Oncogene (2001) 20 (44): 6482- 6491; Kim, CM et al. Sci Rep. (2016) 6 (1): 25526]. 4-1BB signaling ultimately contributes to the secretion of IL-2 and IFN-γ and upregulation of the antiapoptotic Bcl-2 family members Bcl- xL and Bfl-1, which provide strong protection against activation-induced T cell death. [Id., citing Hurtado, JC et al. J. Immunol. (1997) 158 (6): 2600-2609 see. Lee, H-W et al. J. Immunol. (2002) 169 (9): 4882-4888; Maus, MV et al. Nat. Biotechnol. (2002) 20 (2): 143-148; Takahashi, C. et al. J. Immunol. (1999) 162 (9): 5037-5040]. Even though 4-1BB and CD28 costimulation are said to be functionally independent, CD28 costimulation is a powerful stimulus for 4-1BB upregulation.

[0096] Upon Fc-receptor triggering, NK cells upregulate 4-1BB and increase cytotoxic function in response to 4-1BB agonism, but 4-1BB agonism on resting NK cells may reduce NK cell frequency and compromise NK cell cytotoxic function. [Id., citing Choi, BK et al. J.Immunol. (2010) 185 (30): 1404-1411; Lee, S-H and Hahm, D. J. Immunol. (2016) 196 (1 suppl.) Abstract 61.4; Chester, C. et al. Cancer Immunol. Immunother. (2016) 65 (10): 1243- 1248]. Endothelial cells also can upregulate 4-1BB following stimulation with TNF alpha, lipopolysaccharide, and IL-1beta. Expression of 4-1BB along blood vessel walls at the sites of inflammation, chiefly including tumor microvasculature and atherosclerotic areas, suggests that 4-1BB may mediate leukocyte extravasation and migration. [Id., citing Drenkard, D. et al. FASEB J. (2007) 21 (20: 456-463; Broll, K. et al. Am J. Clin. Pathol. (2001) 115 (4): 543-549; Teijeira, A. et al. FASEB J. (2012) 26(8): 3380-3392]. Tregs also express 4-1BB, but its role on this immune subset remains poorly understood. [Id., citing Smith, SE et al. Cancer Immunol. Immunother. (2011) 60 (12): 1775-87; Zhang, P. et al. Scand. J. Immunol. (2007) 66 (4): 435- 440].

[0097] Endothelial cells in hypoxic blood vessels within tumors can upregulate 4-1BB in a hypoxia-inducible factor 1-alpha mediated fashion. [Id., citing Palazon, A. et al. Cancer Res. (2011) 71 (30: 801-11]. In this compartment, anti-4-1BB antibodies give rise to an increased expression of homing receptors for T-cell infiltration.4-1BB signaling can break and reverse established anergy in cytotoxic T lymphocytes. 4-1BB signaling also plays a role in restoring the functionality of exhausted CD8+ T cells. [Id., citing Williams, JB et al. J. Exp. Med. (2017) 214 (2): 381-400]. On NK cells, 4-1BB signaling can increase antibody-dependent cell mediated cytotoxicity. Agonistic monoclonal antibodies targeting 4-1BB can lead to tumor clearance and durable antitumor immunity. [Id. citing Kohrt, HE et al. Blood (2011) 117 (8): 2423-2432; Kohrt, HE et al. J. Clin. Invest. (2012) 122 (3): 1066-1075; Kohrt, HE et al. J. Clin. Invest. (2014) 124 (6): 2668-2682].

[0098] The term “adaptive response” refers to an immune response mediated by uniquely specific recognition of a non-self entity by lymphocytes whose activation leads to elimination of the entity and the production of specific memory lymphocytes.

[0099] The term “adoptive immunotherapy” also known as “cellular immunotherapy” is a type of immunotherapy in which T cells are given to a recipient patient to help the body fight diseases. Types of adoptive cell therapy include chimeric antigen receptor (CAR) T-cell therapy and tumor-infiltrating lymphocyte (TIL) therapy.

[0100] The term “adaptor protein” as used herein refers to a protein that contains a series of protein-binding sites that link respective interaction partners to each other and facilitate the generation of larger signaling complexes.

[0101] The term “adverse event” or “AE” as used herein refers to an unfavorable medical event that occurs in a subject who is given a therapeutic product but does not necessarily have a causal relationship with the treatment. An adverse event may be any adverse and unwanted sign (including an abnormal laboratory result), symptom, or temporary illness associated with the use of the product, whether or not it is related to the product. The correlation between adverse events and test medications is either affirmative, likely related, may be relevant, may be irrelevant, and certainly not relevant. The severity of adverse events according to the National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) ranges from Mild (Grade 1), to Moderate (grade 2); to Severe (Grade 3), to Life-Threatening (Grade 4); to Death (grade 5).

[0102] The term “allele” as used herein refers to any of one or more alternative forms of a given gene.

[0103] The term “allogeneic” as used herein refers to being derived from a genetically different individual of the same species.

[0104] The term “amplicon” as used herein refers to the end product of a replicated or amplified piece of DNA.

[0105] The term “anergy” as used herein refers to a state of lymphocyte nonresponsiveness to specific antigen induced by an encounter of the lymphocyte with cognate antigen under less-than-optimal conditions, such as in the absence of costimulation.

[0106] As used herein, the term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term "antibody" includes polyclonal antibodies and monoclonal antibodies, and fragments thereof. Furthermore, the term "antibody" includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof.

[0107] As used herein, the term “antibody” is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, antibody fragments, chimeric antibodies and wholly synthetic antibodies as long as they exhibit the desired antigen-binding activity. In nature, antibodies are serum proteins the molecules of which possess small areas of their surface that are complementary tosmall chemical groupings on their targets. These complementary regions (referred to as the antibody combining sites or antigen binding sites) of which there are at least two per whole antibody molecule, and in some types of antibody molecules ten, eight, or in some species as many as 12, may react with their corresponding complementary region on an antigen (the antigenic determinant or epitope) to link several molecules of multivalent antigen together to form a lattice. The basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N-terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface.

[0108] The basic structural unit of a whole antibody molecule consists of four polypeptide chains, two identical light (L) chains (each containing about 220 amino acids) and two identical heavy (H) chains (each usually containing about 440 amino acids). The two heavy chains and two light chains are held together by a combination of noncovalent and covalent (disulfide) bonds. The molecule is composed of two identical halves, each with an identical antigen-binding site composed of the N-terminal region of a light chain and the N-terminal region of a heavy chain. Both light and heavy chains usually cooperate to form the antigen binding surface.

[0109] Human antibodies show two kinds of light chains, κ and λ; individual molecules of immunoglobulin generally are only one or the other. In mammals, there are five classes of antibodies, IgA, IgD, IgE, IgG, and IgM, each with its own class of heavy chain. All five immunoglobulin classes differ from other serum proteins in that they show a broad range of electrophoretic mobility and are not homogeneous. This heterogeneity - that individual IgG molecules, for example, differ from one another in net charge - is an intrinsic property of the immunoglobulins.

[0110] The principle of complementarity, which often is compared to the fitting of a key in a lock, involves relatively weak binding forces (hydrophobic and hydrogen bonds, van der Waals forces, and ionic interactions), which are able to act effectively only when the tworeacting molecules can approach very closely to each other and indeed so closely that the projecting constituent atoms or groups of atoms of one molecule can fit into complementary depressions or recesses in the other. Antigen-antibody interactions show a high degree of specificity, which is manifested at many levels. Brought down to the molecular level, “specificity” means that the combining sites of antibodies to an antigen have a complementarity not at all similar to the antigenic determinants of an unrelated antigen. Whenever antigenic determinants of two different antigens have some structural similarity, some degree of fitting of one determinant into the combining site of some antibodies to the other may occur; this phenomenon gives rise to cross-reactions. Cross reactions are of major importance in understanding the complementarity or specificity of antigen-antibody reactions. Immunological specificity or complementarity makes possible the detection of small amounts of impurities / contaminations among antigens.

[0111] Monoclonal antibodies (mAbs) can be generated by fusing mouse spleen cells from an immunized donor with a mouse myeloma cell line to yield established mouse hybridoma clones that grow in selective media. A hybridoma cell is an immortalized hybrid cell resulting from the in vitro fusion of an antibody-secreting B cell with a myeloma cell. In vitro immunization, which refers to primary activation of antigen-specific B cells in culture, is another well-established means of producing mouse monoclonal antibodies.

[0112] Diverse libraries of immunoglobulin heavy (VH) and light (VL=Vκand Vλ) chain variable genes from peripheral blood lymphocytes also can be amplified by polymerase chain reaction (PCR) amplification. Genes encoding single polypeptide chains in which the heavy and light chain variable domains are linked by a polypeptide spacer (single chain Fv or scFv) can be made by randomly combining heavy and light chain V-genes using PCR. A combinatorial library then can be cloned for display on the surface of filamentous bacteriophage by fusion to a minor coat protein at the tip of the phage.

[0113] The technique of guided selection is based on human immunoglobulin V gene shuffling with rodent immunoglobulin V genes. The method entails (i) shuffling a repertoire of human VLchains with the heavy chain variable region (VH) domain of a mouse monoclonal antibody reactive with an antigen of interest; (ii) selecting half-human Fabs on that antigen (iii) using the selected VLgenes as “docking domains” for a library of human heavy chains in a second shuffle to isolate clone Fab fragments having human light chain genes; (v) transfectingmouse myeloma cells by electroporation with mammalian cell expression vectors containing the genes; and (vi) expressing the V genes of the Fab reactive with the antigen as a complete IgG1 antibody molecule in the mouse myeloma.

[0114] The term antibody may include an oligoclonal antibody, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody, and an antibody that can be labeled in soluble or bound form, as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques.

[0115] An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the invention. Binding fragments of an antibody can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and di-sulfide stabilized variable region (dsFv). Structural and functional domains can be identified by comparison of the nucleotide and / or amino acid sequence data to public or proprietary sequence databases. For example, computerized comparison methods can be used to identify sequence motifs or predicted protein conformation domains that occur in other proteins of known structure and / or function. Methods to identify protein sequences that fold into a known three-dimensional structure are known. See, for example, Bowie et al. Science 253:164 (1991), which is incorporated by reference in its entirety. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical.

[0116] The term “antibody construct” as used herein refers to a polypeptide comprising one or more the antigen-binding portions linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen-binding portions. Such linker polypeptides are well known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123). An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art. Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques.

[0117] The term “antigen” as used herein refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host’s immune-system to make a humoral and / or cellular antigen-specific response. The term is used interchangeably with the term “epitope.” Normally, a B-cell epitope will include at least about 5 amino acids but can be as small as 3-4 amino acids. A T-cell epitope, such as a CTL epitope, will include at least about 7-9 amino acids, and a helper T-cell epitope will include at least about 12-20 amino acids. Normally, an epitope will include between about 7 and 15 amino acids, such as, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. The term includes polypeptides which include modifications, such as deletions, additions and substitutions (generally conservative in nature) as compared to a native sequence, as long as the protein maintains the ability to elicit an immunological response, as defined herein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens.

[0118] The term “antigen binding site” as used herein refers to the structure formed at the amino-terminal ends (variable domains) of the light and heavy chains of an antibody, which are folded to form 3-dimensional (globular) variable domains, VHand VL. The antigen binding site makes physical contact with the antigen and binds it noncovalently. The antigen specificity of the site is determined by its shape and the amino acids present.

[0119] The term “antigen presentation” as used herein, generally refers to the display to a T cell of antigen on the surface of a cell, e.g., in the form of peptide fragments bound to MHC molecules.

[0120] As used herein, the term “antigen presenting cell (APC)” refers to a class of cells capable of displaying on its surface (“presenting”) one or more antigens in the form of peptide- MHC complexes recognizable by specific effector cells of the immune system, and thereby inducing an effective cellular immune response against the antigen or antigens being presented. Examples of professional APCs are dendritic cells and macrophages, although any cell expressing MHC Class I or II molecules can potentially present a peptide antigen. An APC can be an “artificial APC,” meaning a cell that is engineered to present one or more antigens. Before a T cell can recognize a foreign protein, the protein must be processed inside an antigenpresenting cell or target cell so that it can be displayed as peptide-MHC complexes on the cell surface.

[0121] As used herein the term “antigen processing” refers to the intracellular degradation of foreign proteins into peptides that can bind to MHC molecules for presentation to T cells.

[0122] The term “AP-1” as used herein refers to a heterodimeric transcription factor formed as one of the outcomes of intracellular signaling via the antigen receptors of lymphocytes and the toll like receptors (TLRs) of cells of innate immunity. Most often AP-1 contains one Fos-family member and one Jun-family member. AP-1 mainly activates the expression of genes for cytokines and chemokines.

[0123] The term “apheresis” as used herein refers to a medical technology in which the blood of a donor or patient is passed through an apparatus that separates out one particular constituent and returns the remainder back to the donor or patient’s circulation. Leukapheresis is one type of apheresis where leukocytes (white blood cells) are selectively removed.

[0124] The terms “apoptosis” or “programmed cell death” refer to a highly regulated and active process that contributes to biologic homeostasis comprising a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane, such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation, without damaging the organism.

[0125] Apoptotic cell death is induced by many different factors and involves numerous signaling pathways, some dependent on caspase proteases (a class of cysteine proteases) and others that are caspase independent. It can be triggered by many different cellular stimuli, including cell surface receptors, mitochondrial response to stress, and cytotoxic T cells, resulting in activation of apoptotic signaling pathways.

[0126] The caspases involved in apoptosis convey the apoptotic signal in a proteolytic cascade, with caspases cleaving and activating other caspases that then degrade other cellular targets that lead to cell death. The caspases at the upper end of the cascade include caspase-8 and caspase-9. Caspase-8 is the initial caspase involved in response to death domain (DD) containing receptors like Fas.

[0127] Receptors in the TNF receptor family are associated with the induction of apoptosis, as well as inflammatory signaling. The Fas receptor (CD95) mediates apoptotic signaling by Fas-ligand expressed on the surface of other cells. The Fas-FasL interaction plays an important role in the immune system and lack of this system leads to autoimmunity, indicating that Fas-mediated apoptosis removes self-reactive lymphocytes. Fas signaling also is involved in immune surveillance to remove transformed cells and virus infected cells. Binding of Fas to oligimerized FasL on another cell activates apoptotic signaling through a cytoplasmic domain termed the death domain (DD) that interacts with signaling adaptors including FAF, FADD and DAX to activate the caspase proteolytic cascade. Caspase-8 and caspase-10 first are activated to then cleave and activate downstream caspases and a variety of cellular substrates that lead to cell death.

[0128] Mitochondria participate in apoptotic signaling pathways through the release of mitochondrial proteins into the cytoplasm. Cytochrome c, a key protein in electron transport, is released from mitochondria in response to apoptotic signals, and activates Apaf-1, a protease released from mitochondria. Activated Apaf-1 activates caspase-9 and the rest of the caspase pathway. Smac / DIABLO is released from mitochondria and inhibits inhibitor of apoptosis (IAP) proteins that normally interact with caspase-9 to inhibit apoptosis. Apoptosis regulation by Bcl-2 family proteins occurs as family members form complexes that enter the mitochondrial membrane, regulating the release of cytochrome c and other proteins. TNF family receptors that cause apoptosis directly activate the caspase cascade, but can also activate Bid, a Bcl-2 family member, which activates mitochondria-mediated apoptosis. Bax, another Bcl-2 family member, is activated by this pathway to localize to the mitochondrial membrane and increase its permeability, releasing cytochrome c and other mitochondrial proteins. Bcl-2 and Bcl-xL prevent pore formation, blocking apoptosis. Like cytochrome c, AIF (apoptosis-inducing factor) is a protein found in mitochondria that is released from mitochondria by apoptotic stimuli. While cytochrome c is linked to caspase-dependent apoptotic signaling, AIF release stimulates caspase- independent apoptosis, moving into the nucleus where it binds DNA. DNA binding by AIF stimulates chromatin condensation, and DNA fragmentation, perhaps through recruitment of nucleases.

[0129] The mitochondrial stress pathway begins with the release of cytochrome c from mitochondria, which then interacts with Apaf-1, causing self-cleavage and activation of caspase-9. Caspase-3, -6 and-7 are downstream caspases that are activated by the upstream proteases and act themselves to cleave cellular targets.

[0130] Granzyme B and perforin proteins released by cytotoxic T cells induce apoptosis in target cells, forming transmembrane pores, and triggering apoptosis, perhaps through cleavage of caspases, although caspase-independent mechanisms of Granzyme B mediated apoptosis have been suggested.

[0131] Fragmentation of the nuclear genome by multiple nucleases activated by apoptotic signaling pathways to create a nucleosomal ladder is a cellular response characteristic of apoptosis. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase- activated DNase (CAD). DFF / CAD is activated through cleavage of its associated inhibitor ICAD by caspases proteases during apoptosis. DFF / CAD interacts with chromatin components such as topoisomerase II and histone H1 to condense chromatin structure and perhaps recruit CAD to chromatin. Another apoptosis activated protease is endonuclease G (EndoG). EndoG is encoded in the nuclear genome but is localized to mitochondria in normal cells. EndoG may play a role in the replication of the mitochondrial genome, as well as in apoptosis. Apoptotic signaling causes the release of EndoG from mitochondria. The EndoG and DFF / CAD pathways are independent since the EndoG pathway still occurs in cells lacking DFF.

[0132] Hypoxia, as well as hypoxia followed by reoxygenation, can trigger cytochrome c release and apoptosis. Glycogen synthase kinase (GSK-3) a serine-threonine kinase ubiquitously expressed in most cell types, appears to mediate or potentiate apoptosis due to many stimuli that activate the mitochondrial cell death pathway. [Loberg, RD, et al., J. Biol. Chem. (2002)277 (44): 41667-41673]. It has been demonstrated to induce caspase 3 activation and to activate the proapoptotic tumor suppressor gene p53. It also has been suggested that GSK-3 promotes activation and translocation of the proapoptotic Bcl-2 family member, Bax, which, upon aggregation and mitochondrial localization, induces cytochrome c release. Akt is a critical regulator of GSK-3, and phosphorylation and inactivation of GSK-3 may mediate some of the antiapoptotic effects of Akt.

[0133] The term “assay for transposase-accessible chromatin with sequencing” or “ATAC-Seq” as used herein refers to a popular method for determining chromatin accessibility across the genome. By sequencing regions of open chromatin, ATAC-Seq can help uncover how chromatin packaging and other factors affect gene expression.

[0134] The term “autologous” as used herein refer to being present in or derived from an individual’s own tissues.

[0135] The term “B7 molecules” as used herein refers to cell surface proteins on specialized APCs, such as dendritic cells, which are the major costimulatory molecules for T cells. B7.1 (CD80) and B7.2 (CD86) are closely related members of the immunoglobulin superfamily, and both bind to CD28 and CTLA-4 proteins on T cells.

[0136] The term “basic leucine zipper ATF-like transcription factor” (“Batf) refers to a bZIP transcription factor that plays an important role in regulating differentiation and function in many lymphocyte lineages [Kurachi, M. et al. Nature Immunol. (2014) 15 (4): 373-383, citing Schraml BU, et al. Nature.2009;460(7253):405–409. Betz BC, et al. J Exp Med. 2010;207(5):933–942, 16; Ise W, et al. Nat Immunol.2011;12(6):536–543, Murphy TL, et al. Nat Rev Immunol.2013;13(7):499–509, Grigoryan G, et al. Nature. 2009;458(7240):859–864]. In the CD8+ T cell lineage, increased expression of BATF in exhausted CD8+ T cells suppresses their effector function [Quigley M, et al. Nat Med. 2010;16(10):1147–1151]. In the CD4+ T cell lineage, BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17) [Id., citing Schraml BU, et al. Nature. 2009;460(7253):405–409], where it binds co- operatively with the transcription factor IRF4 [Glasmacher E, et al. Science. 2012;338(6109):975–980, Li P., et al. Nature.2012;490(7421):543–546, Ciofani M, et al. Cell. 2012;151(2):289–303] and its dimerization partners c-Jun, JunB and JunD [Id., citing Grigoryan G, et al. Nature.2009;458(7240):859–864]. BATF is also important for the development of follicular helper T cells (TFH) by regulating the transcription factors Bcl-6 and c-Maf [Id., citing Betz BC, Jordan-Williams KL, Wang C, Kang SG, Liao J, Logan MR, et al. Batf coordinates multiple aspects of B and T cell function required for normal antibody responses. J Exp Med. 2010;207(5):933–942, Ise W, Kohyama M, Schraml BU, Zhang T, Schwer B, Basu U, et al. The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Nat Immunol. 2011;12(6):536–543]. In addition, BATF is required for class- switch recombination in B cells and to regulate activation-induced cytidine deaminase [Id., citing Ise W, Kohyama M, Schraml BU, Zhang T, Schwer B, Basu U, et al. The transcription factor BATF controls the global regulators of class-switch recombination in both B cells and T cells. Nat Immunol.2011;12(6):536–543] as well as DNA damage checkpoint in hematopoietic stem cell (HSC) self-renewal [Id., citing Wang J, Sun Q, Morita Y, Jiang H, Groß A, Lechel A, et al.A differentiation checkpoint limits hematopoietic stem cell self-renewal in response to DNA damage. Cell.2012;148(5):1001–101423.

[0137] The term “binding” and its various grammatical forms means a lasting attraction between chemical substances.

[0138] The term “binding specificity” as used herein involves both binding to a specific partner and not binding to other molecules. Functionally important binding may occur at a range of affinities from low to high, and design elements may suppress undesired cross-interactions. Post-translational modifications also can alter the chemistry and structure of interactions. “Promiscuous binding” may involve degrees of structural plasticity, which may result in different subsets of residues being important for binding to different partners. “Relative binding specificity” is a characteristic whereby in a biochemical system a molecule interacts with its targets or partners differentially, thereby impacting them distinctively depending on the identity of individual targets or partners.

[0139] The term "biocompatible" as used herein refers to causing no clinically relevant tissue irritation, injury, toxic reaction, or immunological reaction to living tissue.

[0140] The term "biodegradable" as used herein refers to material that will break down actively or passively over time by simple chemical processes, by action of body enzymes or by other similar biological activity mechanisms.

[0141] As used herein, the term “biomarker” (or "biosignature") refers to a peptide, protein, nucleic acid, antibody, gene, metabolite, or any other substance used as an indicator of a biologic state. It is a characteristic that is measured objectively and evaluated as a cellular or molecular indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Once a proposed biomarker has been validated, it may be used to diagnose disease risk, presence of disease in an individual, or to tailor treatments for the disease in an individual (choices of treatment or administration regimes). In evaluating potential therapies, a biomarker may be used as a surrogate for a natural endpoint, such as survival or irreversible morbidity. If a treatment alters the biomarker, and that alteration has a direct connection to improved health, the biomarker may serve as a surrogate endpoint for evaluating clinical benefit. Clinical endpoints are variables that can be used to measure how patients feel, function or survive. Surrogate endpoints are biomarkers that are intended tosubstitute for a clinical endpoint; these biomarkers are demonstrated to predict a clinical endpoint with a confidence level acceptable to regulators and the clinical community.

[0142] The terms “cancer” or “malignancy” as used herein refer to diseases in which abnormal cells divide without control and can invade nearby tissues. Cancer cells also can spread to other parts of the body through the blood and lymph systems.

[0143] The term “CD19” as used herein refers to a 95 kd transmembrane glycoprotein belonging to the immunoglobulin (Ig) superfamily. It is encoded by the 7.41 kilobyte CD19 gene located on the short arm of chromosome 16, 16p11.2. The gene contains 15 exons and codes for the CD19 molecule with 556 amino acids. Structurally, the gene contains an unusually short 5'- untranslated region. The proximal cd19 promoter lacks a TATA box, and its major start sites are found within 50 bp of the initiation codon. CD19 is classified as a type I transmembrane protein, with a single transmembrane domain, a cytoplasmic C-terminus, and extracellular N-terminus. No significant homology exists between CD19 and other known proteins. The extracellular element contains two C2-type Ig-like domains divided by a smaller potential disulfide linked non-Ig-like domain, as well as N-linked carbohydrate addition sites. The highly conserved cytoplasmic domain consists of 242 amino acids with nine tyrosine residues near the C-terminus [9–11]. Multiple studies have come to suggest that the biologic functions of CD19 are dependent on three cytoplasmic tyrosine residues – Y391, Y482 and Y513. CD19 is critically involved in establishing intrinsic B cell signaling thresholds through modulating both B cell receptor (BCR)- dependent and independent signaling. It plays roles in the antigen-independent development as well as the immunoglobulin-induced activation of B cells. [Wang, K. et al. Exptl Hematology & Oncology (2012) 1: art. 36].

[0144] The term “CD20” as used herein refers to a 33-37 kDa non-glycosylated protein expressed on the surface of normal and malignant B lymphocytes. It belongs to the MS4A (membrane-spanning 4-domain family A) protein family. CD20 protein consists of four hydrophobic transmembrane domains, one intracellular and two extracellular domains (large and small loops) with both N- and C- termini residing within the cytosol. Three CD20 isoforms (33, 35 and 37 kDa) resulting from different phosphorylation have been identified, and CD20 phosphorylation was reported to be higher in proliferating malignant B cells than in resting B cells. Normally, CD20 does not form hetero-oligomers,18 but exists on the cell surface as homodimeric and homo-tetrameric oligomers associated with other cell-surface and cytoplasmicproteins contributing to the signal transduction. CD20 is also known to be physically coupled to major histocompatibility complex class II (MHCII), CD40 molecule, BCR, and the C-terminal src kinase-binding protein (CBP) that interacts with Src kinases such as LYN, FYN, and LCK. [Pavlasova, G. and Mraz, M. Haematologica (2020) 105 (6): 1494-1506]. It may be found in higher than normal amounts in patients with certain types of B-cell lymphomas and leukemias.

[0145] The term “CD22” as used herein refers to a surface molecule expressed early during the ontogeny of B cells in the bone marrow and spleen that can be found on B cells isolated from the different lymphoid compartments in humans. CD22 is an inhibitory coreceptor of the B-cell receptor (BCR) and plays a critical role in establishing signaling thresholds for B- cell activation. CD22 is expressed by most blasts from the majority (60–90%) of B-cell acute lymphoblastic leukemia (B-ALL). [Lanza,. et al. Cancers (Basel) (2020) 12 (20: 303].

[0146] The term “CD33” as used herein refers to a myeloid cell surface antigen that is not expressed on blood stem cells or within the hematopoietic system, but that can be expressed on the surface of natural B-lymphocytes, activated T-lymphocytes, and natural killer (NK) cells.

[0147] The term “CD123” as used herein refers to the α chain of the interleukin 3 receptor, which is a cytokine receptor overexpressed in multiple hematolymphoid neoplasms, including acute myeloid leukemia, blastic plasmacytoid dendritic cell neoplasm, acute lymphoblastic leukemia, hairy cell leukemia, and systemic mastocytosis. CD123 expression is upregulated in leukemic stem cells relative to non-neoplastic hematopoietic stem cells.

[0148] The terms “cell line” and “cultured cell line” are used interchangeably to refer to cells of a single type that have been adapted to grow continuously in the laboratory.

[0149] As used herein, the term “cell growth” is the process by which cells accumulate mass and increase in physical size. There are many different examples in nature of how cells can grow. In some cases, cell size is proportional to DNA content. For instance, continued DNA replication in the absence of cell division (called endoreplication) results in increased cell size. Megakaryoblasts, which mature into granular megakaryocytes, the platelet-producing cells of bone marrow, typically grow this way. By a different strategy, adipocytes can grow to approximately 85 to 120 μm by accumulating intracellular lipids. In contrast to endoreplication or lipid accumulation, some terminally differentiated cells, such as neurons and cardiac muscle cells, cease dividing and grow without increasing their DNA content. These cells proportionately increase their macromolecule content (largely protein) to a point necessary toperform their specialized functions. This involves coordination between extracellular cues from nutrients and growth factors and intracellular signaling networks responsible for controlling cellular energy availability and macromolecular synthesis. Perhaps the most tightly regulated cell growth occurs in dividing cells, where cell growth and cell division are clearly separable processes. Dividing cells generally must increase in size with each passage through the cell division cycle to ensure that a consistent average cell size is maintained. For a typical dividing mammalian cell, growth occurs in the G1 phase of the cell cycle and is tightly coordinated with S phase (DNA synthesis) and M phase (mitosis). The combined influence of growth factors, hormones, and nutrient availability provides the external cues for cells to grow. [Guertin, D.A., Sabatini, D.M., “Cell Growth,” in The Molecular Basis of Cancer (4th Edn) Mendelsohn, J. et al Eds, Saunders (2015), 179-190].

[0150] As used herein, the term “cell proliferation” refers to the process that results in an increase of the number of cells and is defined by the balance between cell divisions and cell loss through cell death or differentiation.

[0151] The term “chemical bond” as used herein refers to an attractive force between atoms strong enough to permit the combined aggregate to function as a unit.

[0152] As used herein, the term “chemokine” refers to a class of chemotactic cytokines that orchestrate migration and positioning of immune cells within the tissues. Chemokines bind to seven transmembrane G protein-coupled receptors that trigger intracellular signaling that drives cell polarization, adhesion, and migration [Vilgelm, AE and Richmond, A. Front. Immunol. (2019) doi.org / 10.3389 / fimmu.2019.00333, citing Griffith, JW et al. Annu. Rev. Immunol. (2014) 32: 659-702; Nagarsheth, N. et al. Nat. Rev. Immunol. (2017) 17: 559-572]. They are divided into four families based upon structure: CXC, CC, CX3C, and C chemokines. The receptors follow a similar nomenclature system, based upon the family of chemokines to which they bind. In addition, there is a family of atypical chemokine receptors that do not directly couple to G proteins but are reported to have a variety of roles in development, homeostasis, inflammatory disease, infection, and cancer [Id., citing Nibbs, RJ, Graham, GJ. Nat. Rev. Immunol. (2013) 13: 815-829].

[0153] The term “CCR7” as used herein refers to a receptor for the constitutive chemokines CC chemokine ligand (CCL)-19 and CCL21, which are produced by stomal cells in the T cell zone of the spleen, lymph nodes and Peyer’s patches. [Unsoeld, H. et al. J. Immunol.(2002) 169 (2): 638-641, citing Butcher, EC et al. Adv. Immunol. (1999) 72: 209; Cyster, JG. Science (1999) 286: 2098; Sallusto, F., et al. Annu. Rev. Immunol.1 (2000) 18: 593] This enables naïve T cells to migrate to T cell areas of lymphoid organs in search of antigen presented by dendritic cells. [Id., citing Gunn, MD et al. J. Exp. Med. (12999) 189: 451; Forster, R. et al. Cell (1999) 99: 23] In humans, CCR7 has been described as a defining factor for two different types of memory T cells, termed central and effector memory T cells [Id., citing Sallusto, F. et al. Nature (1999) 401: 708]. Central memory cells express CCR7 and represent a nonpolarized Ag- experienced cell population that lacks immediate effector cell functions. In contrast, effector memory cells have down-regulated CCR7 and are capable of immediately producing cytokines after Ag recognition.

[0154] The term “CD62L” as used herein refers to L-selectin, a leukocyte adhesion molecule associated with differentiating naïve and activated / memory T cells. It has been demonstrated that naive T cells express a CD62LhiCD44lophenotype, whereas memory T cells exhibit a CD62LloCD44hiphenotype. [Gerberick, GF et al. Toxicology and Applied Pharmacol. (1997) 146 (1): 1-10].

[0155] The term “CITE-Seq” or “cellular indexing of transcriptomes and epitopes” as used herein refers to a sequencing-based method that uses unique oligonucleotide-tagged antibodies to identify surface proteins. The oligo-conjugated antibodies combined with single- cell library preparation solutions use barcodes to link proteome and transcriptome profiles to single cells.

[0156] The term “clonal expansion” as used herein refers to rapid proliferation of a specific cell type.

[0157] The term “cluster analysis” as used herein in the context of clonal expansion, is a computational method used to group together cells or DNA sequences that share a high degree of similarity, allowing researchers to identify populations of cells originating from a single progenitor cell (a clone), indicating clonal expansion.

[0158] The term “CTLA-4” or “cytotoxic T-lymphocyte-associated antigen 4” or “CD152” as used herein refers to an inhibitory receptor and immune checkpoint that aids in maintaining self-antigen immunity by dampening T cell responses. It is a counterreceptor to costimulatory molecule CD28.

[0159] The term “cofactor” as used herein refers to a regulatory protein factor that typically is unable to bind to DNA itself and is recruited to enhancers by sequence-specific transcription factors (TFs). Cofactors can have enzymatic functions (for example, they can catalyze post-translational modifications of proteins) and can mediate the regulatory function of the enhancers. [Zabidi, MA and Stark, A. Trends Genet. (2016) 32 (12): 801-14].

[0160] The term “cognate” signifies two biomolecules that typically interact, e.g., a receptor and its cognate ligand.

[0161] The term "compatible" as used herein refers to components of a composition that are capable of being combined with each other in a manner such that there is no interaction that would substantially reduce the efficacy of the composition under ordinary use conditions.

[0162] The terms “complete response” or “complete remission” or “CR” as used herein refer to the disappearance of all signs of cancer in response to treatment. This does not always mean the cancer has been cured.

[0163] The term “component” as used herein, is meant to refer to a constituent part, element or ingredient.

[0164] The term “composition” as used herein, is meant to refer to a material formed by a mixture of two or more substances.

[0165] As used herein, the term “condition” as used herein, is meant to refer to a variety of health states and is meant to include disorders or diseases caused by any underlying mechanism or disorder.

[0166] As used herein, the term “contact” and its various grammatical forms is meant to refer to a state or condition of touching or of immediate or local proximity. Contacting a composition to a target destination may occur by any means of administration known to the skilled artisan.

[0167] The term “core-promoter” or “minimal promoter” as used herein refers to a short sequence around the transcription start site (TSS) that can direct the recruitment of RNA Polymerase II (Pol II) and transcription initiation. Core-promoters typically have low basal activities in the absence of enhancers. [Zabidi, MA and Stark, A. Trends Genet. (2016) 32 (12): 801-814].

[0168] The term “costimulation” as used herein refers to the second signal required for completion of lymphocyte activation and prevention of anergy, which is supplied by engagement of CD 28 by CD 80 and CD 86 (T cells) and of CD 40 by CD 40 Ligand (B cells).

[0169] The term “costimulatory molecule” as used herein refers to molecules that are displayed on the cell surface that have a role in enhancing the activation of a T cell that is already being stimulated through its TCR. For example, HLA proteins, which present foreign antigen to the T cell receptor, require costimulatory proteins which bind to complementary receptors on the T cell’s surface to result in enhanced activation of the T cell. Co-stimulatory molecules are highly active immunomodulatory proteins that play a critical role in the development and maintenance of an adaptive immune response (Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy (2007) Chpt 5, 67-121). The two signal hypothesis of T cell response involves the interaction between an antigen bound to an HLA molecule and with its cognate T cell receptor (TCR), and an interaction of a co-stimulatory molecule and its ligand. Specialized APCs, which are carriers of a co-stimulatory second signal, can activate T cell responses following binding of the HLA molecule with TCR. In contrast, somatic tissues do not express the second signal and thereby induce T cell unresponsiveness [Kaufman and Wolchok eds., General Principles of Tumor Immunotherapy (2007) Chpt 5, 67-121]. Many of the co-stimulatory molecules involved in the two-signal model can be blocked by co-inhibitory molecules that are expressed by normal tissue (Id.). In fact, many types of interacting immunomodulatory molecules expressed on a wide variety of tissues may exert both stimulatory and inhibitory functions depending on the immunologic context (Id.).

[0170] The term “co-stimulatory receptor” as used herein refers to a cell surface receptor on naïve lymphocytes through which they receive signals in addition to those received through the antigen receptor, and which are necessary for the full activation of the lymphocyte. Examples are CD 30 and CD 40 on B cells and CD 27 and CD 28 on T cells.

[0171] The term “cross-dressing” as used herein refers to a pathway for cross- presentation. In cross-dressing, dendritic cells acquire preformed MHC class I molecules in complex with antigens from other cells by the process of trogocytosis [Yewdell, JW and Dolan, BP, “Cross-dressers turn on T cells”. Nature (2011) 471 (7340): 581-582, citing Joly, E. and Hudrisier, D. Nature Immunol. (2003) 4: 815; Herrera OB et al. J. Immunol. (2004) 173: 4828- 4837] or through gap junctions. This allows antigen presentation by acceptor dendritic cells tooccur immediately, without any processing. Cross-dressing is used to activate memory T cells, but not naïve T cells, in response to viral infection [Id., citing Wakins, LM and Bevan, MJ. Nature (2011) 471: 629-632].

[0172] The term “cross-presentation” as used herein refers to a process by which proteins taken up by dendritic cells from the extracellular milieu can give rise to peptides presented by MHC class I molecules. It enables antigens from extracellular sources to be presented by MHC class I molecules and to activate CD8 T cells.

[0173] The term “cross-priming” as used herein refers to activation of CD8 T cells by dendritic cells in which the antigenic peptide presented by MHC class I molecules is derived from an exogenous protein (i.e., by cross-presentation), rather than produced within the dendritic cells directly (cf. direct presentation).

[0174] The terms “CCTC-binding factor” or “CTCF” as used herein refers to a highly conserved zinc finger protein that can function as a transcriptional activator, a repressor or an insulator protein, blocking the communication between enhancers and promoters. CTCF can also recruit other transcription factors while bound to chromatin domain boundaries. The three- dimensional organization of the eukaryotic genome dictates its function, and CTCF serves as one of the core architectural proteins that help establish this organization. The mapping of CTCF- binding sites in diverse species has revealed that the genome is covered with CTCF-binding sites. [Kim, S. et al. Exp. & Molec. Med. (2015) 47: e166].

[0175] The term “culture” and its other grammatical forms as used herein, is meant to refer to a process whereby a population of cells is grown and proliferated on a substrate in an artificial medium.

[0176] The term "cytokine" as used herein refers to small soluble protein substances secreted by cells which have a variety of effects on other cells. Cytokines mediate many important physiological functions including growth, development, wound healing, and the immune response. They act by binding to their cell-specific receptors located in the cell membrane, which allows a distinct signal transduction cascade to start in the cell, which eventually will lead to biochemical and phenotypic changes in target cells. Generally, cytokines act locally. They include type I cytokines, which encompass many of the interleukins, as well as several hematopoietic growth factors; type II cytokines, including the interferons and interleukin- 10; tumor necrosis factor ("TNF")-related molecules, including TNFα and lymphotoxin;immunoglobulin super-family members, including interleukin 1 ("IL-1"); and the chemokines, a family of molecules that play a critical role in a wide variety of immune and inflammatory functions. The same cytokine can have different effects on a cell depending on the state of the cell. Cytokines often regulate the expression of, and trigger cascades of, other cytokines. Non- limiting examples of cytokines include e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 / IL-23 P40, IL13, IL-15, IL-15 / IL15-RA, IL-17, IL-18, IL-21, IL-23, TGF-β, IFNγ, GM-CSF, Groα, MCP-1 and TNF-α.

[0177] The term “cytotoxic T lymphocytes” (CTLs) as used herein refers to effector CD8+ T cells. Cytotoxic T cells kill by inducing their targets to undergo apoptosis / programmed cell death via extrinsic and intrinsic pathways.

[0178] The term “CX3CR1” as used herein refers to CX3C chemokine receptor 1, which is a marker of T cell differentiation. It was found that CX3CR1+CD8+T cells were the progeny of CX3CR1-CD8+T cells and exhibited robust cytotoxicity in anti-viral immunity [Yamauchi, T. et al. Nature Communic. (2021) 12: article 1402, citing Bottcher, JP et al. Nat. Commun. (2015) 6: 8306; Gerlach, C. et al. Immunity (2016) 45: 1270-1284].

[0179] The term “dendritic cells” (or “DCs”) as used herein refers to professional antigen presenting cells, which induce naïve T cell activation and effector differentiation. [Patente, TA, et al., Frontiers Immunol. (2019) doi.org / 10.3389 / fimmu.2018.03176]. Human DCs are identified by their high expression of major histocompatibility complex (MHC) class II molecules (MHC-II) and of CD11c, both of which are found on other cells, like lymphocytes, monocytes and macrophages [Id., citing Carlens J, et al. J Immunol. (2009) 183:5600–7; Drutman SB, et al. J Immunol. (2012) 188:3603–3610; Hochweller K, Set al. Eur J Immunol. (2008) 38:2776–2783; Huleatt JW, Lefrançois L. J Immunol. (1995) 154:5684–5693; Rubtsov AV, et al. Blood (2011) 118:1305–1315; Probst HC, et al. Clin Exp Immunol. (2005) 141:398– 404; Vermaelen K, Pauwels R. Cytometry (2004) 61A:170–177]. DCs express many other molecules which allow their classification into various subtypes. Although some of the DC subtypes were originally described as macrophages, DC and macrophages have distinct characteristics [Id., citing Delamarre L, Science (2005) 307:1630–1634; Geissmann F, et al. Science (2010) 327:656–661; van Montfoort N, et al. Proc Natl Acad Sci USA. (2009) 106:6730–6735] and ontogeny, so that, currently, little doubt remains that they belong to distinct lineages [Id., citing Haniffa M, et al. (2013) 120:1–49; Hashimoto D, et al. Immunity (2013)38:792–804; Hettinger J, et al. Nat Immunol. (2013) 14:821–830; McGovern N, et al. Immunity (2014) 41:465–477; Naik SH, et al. Nature (2013) 496:229–232; Schulz C, et al. Science (2012) 336:86–90; Schraml BU, et al. Cell (2013) 154:843–858; Wang J, et al. Mol Med Rep. (2017) 16:6787–6793; Yona S, et al. Immunity (2013) 38:79–91].

[0180] The term “derived from” as used herein encompasses any method for receiving, obtaining, or modifying something from a source of origin.

[0181] The term “detectable marker” encompasses both selectable markers and assay markers. The term “selectable markers” refers to a variety of gene products to which cells transformed with an expression construct can be selected or screened, including drug-resistance markers, antigenic markers useful in fluorescence-activated cell sorting, adherence markers such as receptors for adherence ligands allowing selective adherence, and the like. “Assay markers” are measurable components whose presence or absence can be detected and correlated to a particular detectable response.

[0182] The term “detectable response” as used herein, is meant to refer to any signal or response that may be detected in an assay, which may be performed with or without a detection reagent. Detectable responses include, but are not limited to, radioactive decay and energy (e.g., fluorescent, ultraviolet, infrared, visible) emission, absorption, polarization, fluorescence, phosphorescence, transmission, reflection or resonance transfer. Detectable responses also include chromatographic mobility, turbidity, electrophoretic mobility, mass spectrum, ultraviolet spectrum, infrared spectrum, nuclear magnetic resonance spectrum and x-ray diffraction. Alternatively, a detectable response may be the result of an assay to measure one or more properties of a biologic material, such as melting point, density, conductivity, surface acoustic waves, catalytic activity or elemental composition. A “detection reagent” is any molecule that generates a detectable response indicative of the presence or absence of a substance of interest. Detection reagents include any of a variety of molecules, such as antibodies, nucleic acid sequences and enzymes. To facilitate detection, a detection reagent may comprise a marker.

[0183] The term “direct presentation” as used herein refers to the process by which proteins produced within a given cell give rise to peptides presented by MHC class I molecules. This may refer to APCs (such as dendritic cells), or to nonimmune cells that will become the targets of CTLs.

[0184] The term “detection limit” as used herein refers to the smallest amount or concentration of an analyte in a test sample that can reliably be distinguished from the baseline.

[0185] The term “differential gene expression” as used herein refers to an observed difference or change in expression level of a gene between two experimental conditions that is statistically significant.

[0186] The term “differentiate” and its various grammatical forms as used herein refer to the process of development with an increase in the level of organization or complexity of a cell or tissue, accompanied with a more specialized function.

[0187] The terms “disease progression” or “progressive disease” as used herein refers to a cancer that continues to grow or spread.

[0188] The term “domain” as used herein refers to a region of a protein with a characteristic tertiary structure and function and to any of the three-dimensional subunits of a protein that together make up its tertiary structure formed by folding its linear peptide chain.

[0189] The term “dose” as used herein, is meant to refer to the quantity of a therapeutic substance prescribed to be taken at one time. The term “maximum tolerated dose” as used herein is meant to refer to the highest dose of a drug or treatment that does not cause unacceptable side effects.

[0190] The term “dose escalation study” as used herein refers to a type of study where enrolled patients receive different doses of an investigational agent to determine the recommended phase 2 dose.

[0191] The term “dose limiting toxicities” as used herein refers to side effects of a treatment that are serious enough to prevent an increase in dose of that treatment.

[0192] The term “ectopic gene expression” refers to expression of a gene in a place or at a time in which it is not expressed in nature.

[0193] The term “effective dose” as used herein generally refers to that amount of therapeutic agent sufficient to induce a therapeutic effect. An effective dose may refer to the amount of the therapeutic agent sufficient to delay or minimize the onset of symptoms. An effective dose may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease, disorder or condition. Further, an effective dose is the amount with respect to a therapeutic agent alone, or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of a disease. Aneffective dose may also be the amount sufficient to enhance the subject’s (e.g., a human’s) own immune response. Levels of immunity can be monitored, e.g., by measuring amounts of neutralizing secretory and / or serum antibodies, e.g., by plaque neutralization, complement fixation, enzyme-linked immunosorbent, or microneutralization assay. In the case of a vaccine, an “effective dose” is one that prevents disease and / or reduces the severity of symptoms.

[0194] For any therapeutic agent described herein the effective dose may be initially determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose may also be determined from human data. The applied dose may be adjusted based on the relative bioavailability and potency of the administered agent. Adjusting the dose to achieve maximal efficacy based on the methods described above and other well-known methods is within the capabilities of the ordinarily skilled artisan.

[0195] General principles for determining therapeutic effectiveness, which may be found in Chapter 1 of Goodman and Gilman’s The Pharmacological Basis of Therapeutics, 10th Edition, McGraw-Hill (New York) (2001), incorporated herein by reference, are summarized below.

[0196] Pharmacokinetic principles provide a basis for modifying a dosage regimen to obtain a desired degree of therapeutic efficacy with a minimum of unacceptable adverse effects. In situations where an agent’s plasma concentration can be measured and related to the therapeutic window, additional guidance for dosage modification can be obtained.

[0197] The term “effector cell” as used herein refers to a cell that carries out a final response or function. The main effector cells of the immune system, for example, are activated lymphocytes and phagocytes.

[0198] The term “effector functions” as used herein refers to the actions taken by effector cells and antibodies to eliminate foreign entities, and includes, without limitation, cytokine secretion, cytotoxicity, and antibody-mediated clearance.

[0199] The term “eligible subject” as used herein refers to a subject that satisfies the requirements to be treated with the immunotherapy of the present disclosure under the professional judgment of the patient’s physician. Eligibility criteria may include the subject’s age, type and stage of cancer, current health status, medical history, and previous treatments.

[0200] The term “enhancer” as used herein refers to a sequence of several tens to hundreds of base pairs that boosts transcription from a target core-promoter in a cell-type-specific manner and independent of the enhancer’s relative orientation and distance. Enhancers and core-promoters are two major classes of cis-regulatory elements. [Zabidi, MA and Stark, A. Trends Genet. (2016) 32 (12): 801-814].

[0201] The term “enrich” as used herein refers to increasing the proportion of a desired substance, for example, to increase the relative frequency of a subtype of cell compared to its natural frequency in a cell population. Positive selection, negative selection, or both are generally considered necessary to any enrichment scheme. Exemplary selection methods include, without limitation, magnetic separation and FACS.

[0202] The term “epigenetic marks” as used herein refer to DNA methylation, histone modifications, chromatin remodeling and microRNA.

[0203] The term “epigenetics” as used herein refers to a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence.

[0204] The term “exclusion criteria” as used herein refers to specify characteristics that disqualify subjects from clinical studies and often include factors such as comorbidities or concomitant treatment or factors that could mask the effect of the study treatment.

[0205] The term “exon” as used herein refers to nucleic acid coding sequences of an RNA transcript, or the DNA encoding it, that are translated into protein.

[0206] The terms “expand” or “amplify” as used herein with respect to cells refers to increasing in cell number.

[0207] As used herein, the term “expression” and its other grammatical forms refers to production of an observable phenotype by a gene, usually by directing the synthesis of a protein. It includes the biosynthesis of mRNA, polypeptide biosynthesis, polypeptide activation, e.g., by post-translational modification, or an activation of expression by changing the subcellular location or by recruitment to chromatin.

[0208] The term “flow cytometry” as used herein, is meant to refer to a tool for interrogating the phenotype and characteristics of cells. It senses cells or particles as they move in a liquid stream through a laser (light amplification by stimulated emission of radiation) / light beam past a sensing area. The relative light-scattering and color-discriminated fluorescence of the microscopic particles is measured. Flow analysis and differentiation of the cells is based on size, granularity, and whether the cell is carrying fluorescent molecules in the form of either antibodies or dyes. As the cell passes through the laser beam, light is scattered in all directions,and the light scattered in the forward direction at low angles (0.5°-10°, inclusive) from the axis is proportional to the square of the radius of a sphere and so to the size of the cell or particle. Light may enter the cell; thus, the 90 ° light (right-angled, side) scatter may be labeled with fluorochrome-linked antibodies or stained with fluorescent membrane, cytoplasmic, or nuclear dyes. Thus, the differentiation of cell types, the presence of membrane receptors and antigens, membrane potential, pH, enzyme activity, and DNA content may be facilitated. Flow cytometers are multiparameter, recording several measurements on each cell; therefore, it is possible to identify a homogeneous subpopulation within a heterogeneous population (Marion G. Macey, Flow cytometry: principles and applications, Humana Press, 2007). Fluorescence-activated cell sorting (FACS), which allows isolation of distinct cell populations too similar in physical characteristics to be separated by size or density, uses fluorescent tags to detect surface proteins that are differentially expressed, allowing fine distinctions to be made among physically homogeneous populations of cells.

[0209] The term “forkhead box, subgroup O (FOXO) transcription factors” as used herein refers to a subfamily of a conserved FOX protein family which regulates gene expression, consisting of FOXO1, FOXO3, FOXO4, and FOXO6 in mammals. They bind to DNA through the FoxO-recognized element in the C-terminal basic region of the forkhead DNA-binding domain. Following forkhead binding to DNA, target gene expression is repressed or activated through multiple protein-DNA contacts with the primary recognition site located at α-helix H3. [Dai, S. et al. Nucleic Acids Res. (2021) 49 (18): 10235-10249]. FOXOs control cellular homeostasis, such as stress resistance, cell cycle, cell differentiation, apoptosis, proteostasis, intracellular signaling, metabolism, and autophagy. FOXOs are well known for their functions in oxidative and redox regulation downstream of the insulin / insulin-like growth factor-1 (IGF-1) signaling (IIS) pathway. The pI3K pathway is an important regulator of FOXO activity. FOXOs also play critical functions in DNA damage repair.

[0210] The term “gene” as used herein refers to a region of DNA that includes the entire functional unit encompassing coding DNA sequences, noncoding regulatory DNA sequences, and introns, which controls a discrete hereditary characteristic, usually corresponding to a single protein or RNA.

[0211] The term “gene activator protein” as used herein refers to a gene regulatory protein that when bound to its regulatory sequence in DNA activates transcription.

[0212] The term “gene editing” as used herein refers to alteration of the genetic material of a living organism by inserting, replacing, or deleting a DNA sequence, typically with the goal of improving some characteristic of the organism.

[0213] The term “gene expression” as used herein refers to the process by which information encoded in a gene is turned into a function. This mostly occurs via the transcription of RNA molecules that code for proteins or non-coding RNA molecules that serve other functions. Eukaryotic gene expression is regulated by genomic enhancers that recruit transcription factors and cofactors to activate transcription from target core-promoters.

[0214] The term “gene fusion” or “fusion gene” as used herein refers to a hybrid gene that is formed from the abnormal combination of two chromosomes The formation of fusion genes in cells can occur through multiple mechanisms. In the most common scenario, a fusion gene is formed via somatic chromosomal rearrangement. The four basic types of chromosomal rearrangement are deletions, translocations, tandem duplications, and inversions. For example, in hematologic cancers, in chronic myelogenous leukemia patients an interchromosomal translocation produces a chimeric BCR-ABL1 transcript that encodes a constitutively active form of ABL kinase; an interchromosomal translocation in acute lymphocytic leukemia produces the fusion gene ETV6-RUNX1; and an interchromosomal translocation in acute myeloid leukemia produces a RUNX1-ETO chimeric protein. [Annala, M. et al. Cancer Lett. (2013) 340 (2): 192-200].

[0215] The term “gene regulatory protein” as used herein refers to any protein that binds to a specific DNA sequence to alter the expression of a gene.

[0216] The term “gene repressor protein” as used herein refers to a gene regulatory protein that prevents the initiation of transcription.

[0217] The term “general transcription factor” as used herein refers to any of the proteins whose assembly around the TATA box indicates where a genetic sequence can be read and decoded, which is required for the initiation of transcription of most eukaryotic genes.

[0218] The term “graft versus host disease “or “GVHD” as used herein refers to an attack on the tissues of a graft recipient by mature T cells from a nonidentical donor, which can cause a variety of symptoms, sometimes severe.

[0219] The term “graft versus tumor effect” as used herein refers to immune reactivity mediated by donor T cells against the recipient’s tumor cells.

[0220] The term “granzyme B” or “GZMB” as used herein refers to a caspase-like serine proteinase that is released by recently activated CD8+ T cells. Resting CD8+ memory cells are granzyme B negative. [Nowacki, TM et al. Cell Immunol. (2007) 247 (1): 36-48].

[0221] The term “haploidentical” as used herein refers to half-matched donors. A haploidentical donor is usually an individual’s mother, father, or child. Parents are always a half- match for their children. Siblings (brothers or sisters) have a 50% (1 out of 2) chance of being a half-match for each other.

[0222] The term “hashtag antibody” as used herein refers to an antibody targeting cell surface proteins that is conjugated to a barcode allowing researchers to identify and pool different samples of cells for analysis simultaneously that can be de-multiplexed during analysis.

[0223] The term “healthy subject” or “healthy donor” or “healthy adult donor” as used herein refers to a subject having no signs or symptoms of a cancer.

[0224] The term “hematologic cancer” as used herein refers to a cancer that begins in blood-forming tissue. Examples of hematologic cancer are a cancer that begins in blood -forming tissue, such as the bone marrow (e.g. a leukemia), a cancer that begins in cells of the immune system (a lymphoma), and a cancer that arises in plasma cells (e.g., multiple myeloma).

[0225] The term “IL7R” as used herein refers to the receptor for the cytokine interkeukin-7. One of the principal roles of IL-7 is to promote the survival of both naïve and memory T cells. Expression of the IL7R following infection is thought to contribute to the dynamic changes in the quantitative T cell response during the expansion and contraction phases of an immune response [Colpitts, SL et al. J. Immunol. (2009) 182 (9): 5702-5711, citing Schluns, KS et al. Nat. Immunol. (2000) 1: 426-432; Schluns, KS and Lefrancois, L. Nat. Rev. Immunol. (2003) 3: 269-279]. For example, at the peak of the expansion phase to an acute viral or bacterial infection, the majority of the antigen-specific cells are IL7Rlow, and they are destined to be a short-lived population that can execute effector function and need not be maintained after the pathogen is cleared [Id., citing Schluns, KS et al. Nat. Immunol. (2000) 1: 426-432; Kaech, SM et al. Nat. Immunol. (2003) 4: 1191-1198; Huster, KM et al. Proc. Natl Acad. Sci. USA (2004) 101: 5610-5615]. On the other hand, after contraction occurs, the remaining antigen-specific T cells express high levels of the IL7R, which promotes their continued maintenance.

[0226] As used herein, the term “immune checkpoints” refers to the array of inhibitory pathways necessary for maintaining self-tolerance and that modulate the duration and extent of immune responses to minimize damage to normal tissue. In T cells, the ultimate amplitude and quality of the immune response, which is initiated through antigen recognition by the TCR, is regulated by a balance between co-stimulatory and inhibitory signals (immune checkpoints). [Pardoll, DM. Nat. Rev. Cancer (2012) 12(4): 252-264]. Immune checkpoint molecules such as PD-1, PD-L1, and CTLA-4 are cell surface signaling receptors that play a role in modulating the T-cell response in the tumor microenvironment. Tumor cells have been shown to utilize these checkpoints to their benefit by up-regulating their expression and activity. With the tumor cell’s ability to commandeer some immune checkpoint pathways as a mechanism of immune resistance, it has been hypothesized that checkpoint inhibitors that bind to molecules of immune cells to activate or inactivate them may relieve their inhibition of an immune response. Immune checkpoint inhibitors have been reported to block discrete checkpoints in an active host immune response allowing an endogenous anti-cancer immune response to be sustained. Recent discoveries have identified immune checkpoints or targets, like PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, LAG-3, CCR4, OX40, OX40L, IDO, and A2AR, as proteins responsible for immune evasion.

[0227] The terms “immune escape” or “immune evasion” as used herein refers to a strategy to evade a host’s immune response. It is characterized by the inability of the immune system to eliminate transformed cells prior to and after tumor development. The host’s contribution is manifested by its inability to recognize antigens expressed by tumor cells, a phenomenon known as “host ignorance.” It happens because of defects in both the innate and adaptive arms of the immune system. The tumor’s contribution is manifested by the adaptation of tumor cells to evade the immune system or by developing a microenvironment that suppresses the immune system. [Qian J. et al. (2011) Immune Escape. In: Schwab M. (eds) Encyclopedia of Cancer. Springer, Berlin, Heidelberg. https: / / doi.org / 10.1007 / 978-3-642-16483-5_2975].

[0228] The term “immune homeostasis” refers to the delicate and finely regulated balance of appropriate immune activation and suppression in tissues and organs, driven by a myriad of cellular players and chemical factors. [da Gama Duarte, J. et al. Immunology and Cell Biology (2018) 96: 497-506]

[0229] The terms “immune response” and “immune-mediated” are used interchangeably herein to refer to any functional expression of a subject’s immune system, against either foreign or self-antigens, whether the consequences of these reactions are beneficial or harmful to the subject. The term “immunological response” to an antigen or composition as used herein is meant to refer to the development in a subject of a humoral and / or a cellular immune response to an antigen. For purposes of the present disclosure, a “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and / or other white blood cells. One aspect of cellular immunity involves an antigen-specific response by cytolytic T lymphocytes (“CTL’s”). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves an antigen-specific response by helper T-cells. Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. The term “cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and / or other white blood cells, including those derived from CD4+ and CD8+ T-cells. Hence, an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and / or the activation of T cells, suppressor T-cells and / or γδ T-cells directed specifically to an antigen or antigens present in the composition of interest. These responses may serve to neutralize infectivity, and / or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host. Such responses can be determined using standard immunoassays and neutralization assays, well known in the art.

[0230] The term “immune phenotype” or “immunotype” as used herein refers to the collective frequency of various immune cell populations and their functional responses to stimuli (cell signaling and antibody responses). [See Kaczorowski, KJ et al. Proc. Nat. Acad. Sci. USA (2017) doi / 10.1073 / pnas.1705065114]

[0231] The terms “immune surveillance” and “immunological surveillance” are used interchangeably to refer to a monitoring process by the immune system to detect and destroy infected and neoplastically transformed cells in the body.

[0232] The term “immune system” as used herein refers to the body’s system of defenses against disease, which comprises the innate immune system and the adaptive immune system. The innate immune system provides a non-specific first line of defense against pathogens. It comprises physical barriers (e.g. the skin) and both cellular (granulocytes, natural killer cells) and humoral (complement system) defense mechanisms. The reaction of the innate immune system is immediate, but unlike the adaptive immune system, it does not provide permanent immunity against pathogens. The adaptive immune response is the response of the vertebrate immune system to a specific antigen that typically generates immunological memory.

[0233] The term “immunological synapse” (“IS”) as used herein refers to a highly structured body that functions to concentrate T cell signaling in a defined area. It is associated with the selective recruitment of signaling molecules and exclusion of negative regulators. The synapse is stabilized by a ring of adhesion molecules, including, for example, LFA1, which binds to ICAM1 on the APC. The immune synapse modulates TCR signaling by several mechanisms. In the earliest stages of immune synapse formation, TCR-containing microclusters are recruited to the central molecular cluster. The TCR responds to two distinct pMHC ligands (agonist and self-peptide-MHC) in co-agonism rather than nonspecific TCR-MHC interactions, which adds to the overall binding strength of the TCR-p-MHC complex. The strength of the TCR-p-MHC interactions has a role in determining the influence of coreceptors that are recruited to the immune synapse (e.g., CD8). The immune synapse also modulates TCR signaling by regulating interactions between kinases in the TCR pathway and their substrates. [Morris, G. and Allen, PM. Nature Immunol. (2012) doi:10.1038 / nm.2190].

[0234] Studies have shown that expression of CD5 increases according to the magnitude of the signal delivered by the TCR. Consequently, CD5 is an activation marker of T cells, and CD5 expression reflects the heterogeneity of the signal strength associated with each individual TCR within a polyclonal T cell population. [Voisinne, G. et al. Front. Immunol. (2018) 9:2900]. In homeostasis, high expression of CD5 is considered a surrogate for TCR affinity for self-p- MHC and is positively correlated with expression of the IL-7 receptor. [Morris, GP and Allen, PM. Nature Immunol. (2012) doi: 10.1038 / ni.2190]. Another model, termed “coreceptor tuning”proposes a mechanism for the adjustment of homeostasis in the periphery whereby expression of the Il-7 receptor and CD8 are reciprocally regulated. [Morris, GP and Allen, PM. Nature Immunol. (2012) doi: 10.1038 / ni.2190].

[0235] The T cell signaling pathway includes proximate signaling (including phosphorylation of the invariant signaling protein CD3 and early signaling molecules, calcium- mediated signaling (release of intracellular Ca2+ stores and influx of extracellular Ca2+), and GTPase Ras-MAPK signaling. [Morris, GP and Allen, PM. Nature Immunol. (2012) doi: 10.1038 / ni.2190]

[0236] T cell activation is mediated through highly organized and dynamic interaction of TCRs with MHC-peptide complexes at the IS. A mature IS is an aggregation of TCR-based signalosomes (meaning multimolecular complexes) that induce T cell responses and is defined by three concentric rings of clustered molecules. The inner circle is termed the central supramolecular activation cluster (cSMAC) where TCR signaling takes place. The cSMAC contains most of the TCR-MHC peptide complexes, CD28, PKC-theta and Lck, whereas the peripheral SMAC (pSMAC) contains proteins involved in cell adhesion, such as integrin LFA-1, cytoskeletal linker talin, and ICAM1. Larger molecules, such as CD43 and CD45, are excluded from the pSMAC and make up the distal SMAC (dSMAC). Inhibitory and costimulatory molecules, such as PD-1, CTLA-4, and ICOS also are aggregated at the region of the IS and play crucial roles in the regulation of T cell activation. [Watanabe, K. et al. Front. Immunol. (2018) 9: 2486, citing Yokosuka, T. et al. Immunity (2010) 33: 326-39]. Secretion of lytic granules occurs within a secretary synapse between CTLs and target cells. The secretory synapse has two separate and distinct domains in cSMAC: one is a signaling domain, which contains the signaling proteins, and another is a secretory domain for exocytosis of cytokines, perforins and granzymes. The transient polarization and docking of the centrosome to the plasma membrane, which is controlled by Lck signaling, has an important role in the mechanism of directing this secretion. [Id., citing Stinchcombe, JC et al. Nature (2006) 443: 462-465; Tsun, A. et al. J. Cell Biol. (2011) 192: 663-674].

[0237] The intracellular signaling downstream of CARs and the mechanisms of the IS formed by CARS have not been extensively studied. It has been demonstrated that CAR clustering, ZAP70 recruitment to IS, and exclusion of CD45 outside of IS occur between CD19- specific CAR-T cells and target cells that is similar to TCR activation. Downstream signalingmolecules of the TCR, such as CD3zeta, LAT, Lck and ZAP70 are phosphorylated after CD19- CAR-T cell activation by autologous CD19 B cells [Id., citing Karlsson, H. et al. PLoS ONE (2015) 10: e0144787]. The CAR IS does not present a systematic bull’s eye structure, which is a characteristic feature of TCR IS. Organization of the actin ring is poor, and actin may not be completely diminished at the center of CAR IS [Id., citing Xiong, W. et al. Mol. Ther. (2018) 26: 963-975]. LFA-1 is disorganized and CAR-tumor antigen complexes form microclusters that are randomly distributed at the CAR immunological synapse [Id., citing Davenport, AJ et al. Proc. Natl Acad. Sci. USA (2018) 115: E2068-76]. While the TCR IS requires 5-10 min to form the bull’s eye structure, the CAR IS might not need to form these stable structures because the disorganized multifocal pattern of the CAR IS is sufficient to rapidly induce proximal signaling, which occurs within a short period of time (<2 min). The rapid but short duration of proximal signaling of the CAR IS also induces a rapid microtubule organizing center (MTOC) to the IS and accelerates the delivery of cytotoxic granules including perforin and granzymes, to the IS [Id., citing Davenport, AJ et al. Proc. Natl Acad. Sci. USA (2018) 115: E2068-E2076].

[0238] The term “immunotherapy” as used herein refers to measures taken using immunological methods and principles to target the hyper or hyo-immune state of an organism, intervene or adjust the organism’s immune function artificially, and strengthen or attenuate the immune response to treat disease. It enhances the immune system’s ability to recognize, target and eliminate cancer cells in the body. [Zhang, Z. et al. Front. Immunol. (2021) 12: 672356; Barbari, C. et al. Intl J. Mol. Sci. (2020) 21: 5009]. Some types of immunotherapy only target certain cells of the immune system. Others affect the immune system in a general way.

[0239] The term “inflammation” as used herein refers to the physiologic process by which vascularized tissues respond to injury. See, e.g., FUNDAMENTAL IMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers, Philadelphia (1999) at 1051-1053, incorporated herein by reference. During the inflammatory process, cells involved in detoxification and repair are mobilized to the compromised site by inflammatory mediators. Inflammation is often characterized by a strong infiltration of leukocytes at the site of inflammation, particularly neutrophils (polymorphonuclear cells). These cells promote tissue damage by releasing toxic substances at the vascular wall or in uninjured tissue. Traditionally, inflammation has been divided into acute and chronic responses.

[0240] The term “acute inflammation” as used herein refers to the rapid, short-lived (minutes to days), relatively uniform response to acute injury characterized by accumulations of fluid, plasma proteins, and neutrophilic leukocytes. Examples of injurious agents that cause acute inflammation include, but are not limited to, pathogens (e.g., bacteria, viruses, parasites), foreign bodies from exogenous (e.g. asbestos) or endogenous (e.g., urate crystals, immune complexes), sources, and physical (e.g., burns) or chemical (e.g., caustics) agents.

[0241] The term “chronic inflammation” as used herein refers to inflammation that is of longer duration and which has a vague and indefinite termination. Chronic inflammation takes over when acute inflammation persists, either through incomplete clearance of the initial inflammatory agent or as a result of multiple acute events occurring in the same location. Chronic inflammation, which includes the influx of lymphocytes and macrophages and fibroblast growth, may result in tissue scarring at sites of prolonged or repeated inflammatory activity.

[0242] The term “inhibitor of DNA binding protein 2” or “Id2” as used herein refers to a small helix-loop-helix transcription factor that transcriptionally and epigenetically regulates the generation of Slamf6+progenitor exhausted (Texprog) CD8+T cells and their conversion to Tim-3+terminally exhausted (Textem) CD8+T cells. [Li, Y. et al. Cellular & Molecular Immunol. (2024) 21: 292-308].

[0243] The term “inhibitor receptor lymphocyte activation gene-3” or “LAG-3” as used herein refers to a member of the immunoglobulin superfamily (“IgSF”) that binds to major histocompatibility complex (MHC) class II. LAG-3 expression on tumor infiltrating lymphocytes (TILs) is associated with tumor-mediated immune suppression.

[0244] The term “innate response” as used herein refers to non-specific and broadly specific mechanisms that deter entry or promote elimination of foreign entities. These include physical, chemical and molecular barriers that exclude antigens in a totally nonspecific way; and soluble and membrane bound factors that recognize a limited number of molecular patterns that are common to a wide variety of pathogens or produced by host cells under stress.

[0245] The term “insulator” as used herein refers to DNA elements that can block regulatory enhancer-core-promoter communication, typically by the recruitment of insulator proteins, such as CTCF, and thus demarcate the range of enhancer activity and define TAD borders. [Zabidi, MA and Stark, A. Trends Genet. (2016) 32 (12): 801-814].

[0246] The term “intron” as used herein refers to a noncoding region of a eukaryotic gene that is transcribed into an RNA molecule but is then excised by RNA splicing and does not remain in the final mature mRNA molecule following transcription of that gene.

[0247] The term “Kaplan-Meier survival curve” or “survival curve” as used herein refers to the probability of surviving in a given length of time while considering time in many small intervals. It is commonly used to analyze time-to-event (survival) data, such as the time until death or the time until a specific event occurs. Time is plotted on the x-axis and the survival rate is plotted on the y-axis. Each subject is characterized by three variables: (1) their serial time; (2) their status at the end of their serial time (occurrence of an event of interest or censored); and (3) the study group they are in. The term “serial time” refers to the clinical course duration for each subject having a beginning and an end along the timeline of the complete study. An “interval”, which is graphed as a horizontal line, is the serial time duration of known survival. An interval therefore is terminated only by the event of interest. “Censoring” means the total survival time for that subject cannot be accurately determined; this can happen when something negative for the study occurs, such as the subject drops out, is lost to follow-up, or required data is not available, or, conversely, something good happens, such as the study ends before the subject had the event of interest occur. Censoring can occur within the study or terminally at the end. Censored subjects are indicated as tick marks; these do not terminate the interval. [Rich, JT., et al. Otolaryngol. Head Neck Surg. (2010) 143 (3): 331-336]. The Kaplan Meier plot assumes that: (i) at any time subjects who are censored (i.e., lost) have the same survival prospects as subjects who continue to be followed; (ii) the survival probabilities are the same for subjects recruited early and late in the study; and (iii) the event (e.g., death) happens at the time specified. Probabilities of occurrence of an event are computed at a certain point of time with successive probabilities multiplied by any earlier computed probabilities to get a final estimate. The survival probability at any particular time is calculated as the number of subjects surviving divided by the number of subjects at risk. Subjects who have died, dropped out, or have been censored from the study are not counted as at risk.

[0248] The term “killer cell lectin-like receptor subfamily G, member 1” or “KLRG1”as used herein refers to a signaling molecule acquired as T cells become terminally differentiated. [Gattinoni, L. et al. Nature Reviews Cancer (2012) 12: 671-684].

[0249] The term “leukemia” is a broad term for cancers of the blood cells. The type of leukemia depends on the type of blood cell that becomes cancerous and whether it grows quickly or slowly.

[0250] Like other cancers, leukemia is characterized by a succession of mutations in genes that regulate the processes of cellular division, death and differentiation leading to the progressive shift of cells from normal to malignant state [Sak, K. & Evaraus, H., Curr. Genomics (2017) 18 (1): 3-26]. Hematopoietic cancers often emerge in consequence of the uncontrolled growth and accumulation of immature blasts, since cellular differentiation is typically blocked at a particular maturation stage leading to the deficiency of normal functional blood cells and causing numerous serious symptoms [Id.].

[0251] Leukemia consists of a heterogeneous group of hematological malignancies affecting the cells of all hematopoietic lineages [Id., citing Mahbub, A.A. et al. Anticancer Agents med. Chem. (2013) 13 (10): 1601-1613; Qin, Y. et al. Eur. J. Pharm. Sci. (2012) 45 (5): 648-656; Kikuchi, H. et al. Intl J. Oncol. (2013) 43(6): 1976-1984]. Examples of leukemias include, without limitation, acute lymphoblastic leukemia (also called acute lymphocytic leukemia and ALL), acute myelogenous leukemia (also called acute myeloblastic leukemia, acute myeloid leukemia, acute nonlymphocytic leukemia, AML, and ANLL), acute myeloid leukemia with myelodysplasia, acute promyelocytic leukemia (also called APL), chronic eosinophilic leukemia; chronic granulocytic leukemia (also called chronic myelogenous leukemia, chronic myeloid leukemia, and CML), erythroleukemia; mast cell leukemia; and hairy cell leukemia.

[0252] The term “linker for activation of T cells” or “LAT” as used herein refers to a signaling adaptor protein at the immune synapse. Once phosphorylated by ZAP70, LAT recruits cytosolic adaptors including SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP76), growth factor receptor-bound protein 2 (GRB2), GRB2-related adaptor protein (GRAP2), as well as enzymes like Phospholipase C (PLCγ1) and IL-2 inducible T cell kinase (ITK).

[0253] The term “Lck” or “p56Ick” as used herein refers to a Src family kinase recruited to phosphorylate CD3 ITAMs during immune synapse maturation. [Ehrlich, L.I.R. et al. Immunity (2002) 17 (6): 809-822].

[0254] The term “lymphocyte” refers to a small white blood cell formed in lymphatic tissue throughout the body and in normal adults making up about 22-28% of the total number of leukocytes in the circulating blood that plays a large role in defending the body against disease. Individual lymphocytes are specialized in that they are committed to respond to a limited set of structurally related antigens. This commitment, which exists before the first contact of the immune system with a given antigen, is expressed by the presence on the lymphocyte’s surface membrane of receptors specific for determinants (epitopes) on the antigen. Each lymphocyte possesses a population of receptors, all of which have identical combining sites. One set, or clone, of lymphocytes differs from another clone in the structure of the combining region of its receptors and thus differs in the epitopes that it can recognize. Lymphocytes differ from each other not only in the specificity of their receptors, but also in their functions. Lymphocytes are much more common in the lymphatic system, and include B cells, T cells, natural killer T (NKT) cells, and natural killer (NK) cells. There are two broad categories of lymphocytes, namely T cells and B cells. T-cells are responsible for cell-mediated immunity whereas B-cells are responsible for humoral immunity (relating to antibodies). T-cells are so-named because these lymphocytes mature in the thymus; B-cells mature in bone marrow. B cells make antibodies that bind to pathogens to enable their destruction. CD4+ (helper) T cells coordinate the immune response. CD8+ (cytotoxic) T cells, NKT, and Natural Killer (NK) cells are able to kill cells of the body that are, e.g., infected by a virus or display a recognized antigenic sequence.

[0255] The term “lymphocyte activation” or “activation” refers to stimulation of lymphocytes by specific antigens, nonspecific mitogens, or allogeneic cells resulting in synthesis of RNA, protein and DNA and production of lymphokines, the soluble product of lymphocytes; it is followed by proliferation and differentiation of various effector and memory cells. For example, a mature B cell can be activated by an encounter with an antigen that expresses epitopes that are recognized by its cell surface immunoglobulin (Ig). The activation process may be a direct one, dependent on cross-linkage of membrane Ig molecules by the antigen (cross- linkage-dependent B cell activation) or an indirect one, occurring most efficiently in the context of an intimate interaction with a helper T cell (“cognate help process”). T-cell activation is dependent on the interaction of the TCR / CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. Full responsiveness of a T cell requires, inaddition to receptor engagement, an accessory cell-delivered costimulatory activity, e.g., engagement of CD28 on the T cell by CD80 and / or CD86 on the antigen presenting cell (APC).

[0256] The term “lymphodepletion” as used herein refers to a short course of chemotherapy to kill T cells. It creates a favorable immune environment for CAR T cells, which improves their expansion, persistence and clinical activity while reducing the potential for anti- CAR immune responses. [Wagner, DL et al. Nat. Rev. Clin. Oncol. (2021) 18 (6): 379-393].

[0257] The term “lymphoid enhancer binding factor” or “Lef1” as used herein refers to a gene that encodes a transcription factor that shares homology with high mobility group (HMG) protein -1. The HMG proteins are known to interact with a minor groove of the DNA double- helix and thus bend DNA to modulate DNA structure. [Shan, Q. et al. Nature Communic. (2021) 12: 5863, citing Grosschedl, R. et al. Trends Genet. (1994) 10: 94-100]. Lef1 binds a minimal TCRα enhancer and induce a sharp bend in DNA in vitro; the DNA bending facilitates interaction of Ets and Runx family transcription factors that bind to sequences flanking the Lef1 site. {Id., citing Giese, K. et al. Genes Dev. (1995) 9: 995-1008; Love, JJ et al. Nature (1995) 376: 791-795].

[0258] The term “lymphoma” as used herein refers to a cancer of the lymphatic system, which includes the lymph nodes, spleen, thymus gland, and bone marrow that initiates from the malignant transformation of a single lymphocyte. Examples include Hodgkin's lymphoma, non- Hodgkin's lymphoma, cutaneous B cell lymphoma; cutaneous T cell lymphoma and Waldenstrom macroglobulinemia. Lymphomas almost always depend on surrounding stromal cells for survival and growth factors as well as vital intercellular contacts and so are generally restricted to sites within tissues. From its initiation site, a lymphoma tends to spread to additional secondary lymphoid tissues and eventually to non-lymphoid organs. Occasionally, a lymphoma cell undergoes additional mutations that allow it to survive and circulate in the blood, i.e., it becomes a leukemic cell. The disease then may be called a “leukemia / lymphoma.” [Mak, TW et al. Ch. 20 Hematopoietic cancers, in Primer to the Immune response (2014) Elsevier, Inc., pp. 573-574]. The progression of any lymphoma can be described in four stages:

[0259] In Stage I, one or more diseased lymph nodes are present in a single group of lymph nodes in a lymphoid tissue of the body.

[0260] In stage 2, diseased lymph nodes are present in more than one group of lymph nodes, but all diseased nodes are contained either above or below the diaphragm. Tumor cells may also be present in a single organ near an affected node.

[0261] In stage III, diseased lymph nodes are present in two or more groups on both sides of the diaphragm. Tumor cells also may be present in the spleen and / or another organ near an affected node.

[0262] In stage IV, there is wide dissemination of tumor cells into multiple lymph nodes, bone marrow, liver and multiple organs. [Mak, TW et al. Ch. 20 Hematopoietic cancers, in Primer to the Immune response (2014) Elsevier, Inc., pp. 573-574]

[0263] Lymphomas display a tumor microenvironment (TME), with huge differences amongst the various forms. Hodgkin lymphoma (HL), both classic HL and nodular lymphocyte predominant HL, as well as several T cell lymphoma entities, such as angioimmunoblastic T-cell lymphomas (AITL), predominantly (>80% of the tumor mass) consist of TME cells. In indolent B-cell lymphomas, such as follicular lymphoma (FL) or marginal zone lymphomas, the TME constitutes about 50% of the cellular mass. In aggressive lymphomas, such as diffuse large B- cell lymphomas (CLBCL), the proportion of the TME varies and is generally lower. In Burkitt lymphoma, plasmablastic lymphoma and lymphoblastic T cell and B cell lymphomas, the TME is barely existent. [Menter, T. & Tzankov, A. Pathobiology (2019) 86: 225-36].

[0264] The term “macrophage” as used herein refers to a mononuclear, actively phagocytic cell arising from monocytic stem cells in the bone marrow. These cells are widely distributed in the body and vary in morphology and motility. Phagocytic activity is typically mediated by serum recognition factors, including certain immunoglobulins and components of the complement system, but also may be nonspecific. Macrophages also are involved in both the production of antibodies and in cell-mediated immune responses, particularly in presenting antigens to lymphocytes. They secrete a variety of immunoregulatory molecules.

[0265] The terms “Major Histocompatibility Complex (MHC), MHC-like molecule” and “HLA” are used interchangeably herein to refer to cell-surface molecules that display a molecular fraction known as an epitope or an antigen and mediate interactions of leukocytes with other leukocyte or body cells. MHCs are encoded by a large gene group and can be organized into three subgroups- class I, class II, and class III. In humans, the MHC gene complex is called HLA (“Human leukocyte antigen”); in mice, it is called H-2 (for “histocompatibility”). Bothspecies have three main MHC class I genes, which are called HLA-A, HLA-B, and HLA-C in humans, and H2-K, H2-D and H2-L in the mouse. These encode the α chain of the respective MHC class I proteins. The other subunit of an MHC class I molecule is β2-microglobulin. The class II region includes the genes for the α and β chains (designated A and B) of the MHC class II molecules HLA-DR, HLA-DP, and HLA-DQ in humans. Also in the MHC class II region are the genes for the TAP1:TAP2 peptide transporter, the PSMB (or LMP) genes that encode proteasome subunits, the genes encoding the DMα and BMβ chains (DMA and DMB), the genes encoding the α and β chains of the DO molecule (DOA and DOB, respectively), and the gene encoding tapasin (TAPBP). The class II genes encode various other proteins with functions in immunity. The DMA and DMB genes encoding the subunits of the HLA-DM molecule that catalyzes peptide binding to MHC class II molecules are related to the MHC class II genes, as are the DOA and DOB genes that encode the subunits of the regulatory HLA-DO molecule. [Janeway’s Immunobiology.9th ed., GS, Garland Science, Taylor & Francis Group, 2017. pps. 232-233]. In humans, there are three MHC class II isotypes, HLA-DR, HLA-DP, and HLA-DQ, encoded by α and β chain genes within the Human Leukocyte Antigen (HLA) locus on chromosome 6 [Wosen, JE et al. Front. Immunol. (2018) doi.10.3389 / fimmu.2018.02144].

[0266] The term “MHC restriction” as used herein refers to the requirement that APCs or target cells express MHC molecules that a T cell recognizes as self for the T cell to respond to the antigen presented by that APC or target cell (T cells will only recognize antigens presented by their own MHC molecules). For example, CD8 T cells bind class I MHCs which are expressed on most cells in the body, and CD4 T cells bind class II MHCs which are only expressed on specialized APCs.

[0267] As used herein, the terms “marker” or “cell surface marker” are used interchangeably herein to refer to an antigenic determinant or epitope found on the surface of a specific type of cell. Cell surface markers can facilitate the characterization of a cell type, its identification, and eventually its isolation. Cell sorting techniques are based on cellular biomarkers where a cell surface marker(s) may be used for either positive selection or negative selection, i.e., for inclusion or exclusion, from a cell population.

[0268] The term “marker gene analysis” as used herein refers to amplicon-based sequencing or metabarcoding where the transcript levels of a gene in a specific cluster issufficiently higher than those in other clusters so that the gene can be used as a “marker” to identify the cluster.

[0269] The term “mediated”, and its various grammatical forms as used herein refers to depending on, acting by or connected through some intervening agency.

[0270] The term” memory cells” as used herein refers to B and T lymphocytes generated during a primary immune response that remain in a quiescent state until fully activated by a subsequent exposure to specific antigen (secondary immune response). Memory cells generally are more sensitive than naïve lymphocytes to antigen and respond rapidly on re-exposure to the antigen that originally induced them. During an immune response, naïve T cells (TN) are primed by antigen-presenting cells (APCs). Depending on the strength and quality of stimulatory signals, proliferating T cells progress along a differentiation pathway that culminates in the generation of terminally differentiated short-lived effector T (TEFF) cells. When antigenic and inflammatory stimuli cease, primed T cells become quiescent and enter the memory stem cell (TSCM), central memory (TCM) cell or effector memory (TEM) cell pools, depending on the signal strength received. TSCMcells possess stem cell-like attributes to a greater extent than any other memory lymphocyte population. Although both TCMand TEMcells can also undergo self- renewal, the capacity to form diverse progeny is progressively restricted, so that only TSCMcells can generate all three memory subsets and TEFFcells; TCMcells can give rise to TCM, TEMand TEFFcells, and TEMcells can only produce themselves and TEFFcells. [Gattinoni, L. et al. Nature Revs. Cancer 12 (2012) 671-684].

[0271] The term “modulate” and its other grammatical forms as used herein means to regulate, alter, adapt, or adjust to a certain measure or proportion.

[0272] The term “molecule” as used herein refers to a chemical unit composed of one or more atoms connected by chemical bonds. The term “myeloma” as used herein refers to a plasma cell tumor that secretes large quantities of an Ig protein of usually unknown specificity. When tumors are present in multiple body sites, the disease is referred to as multiple myeloma (MM). Normal plasma cells cannot divide and so die soon after secreting antigen-specific antibody. In contrast, cancerous plasma cells divide uncontrollably and express huge quantities of antibodies or single Ig chains of unknown antigenic specificity.

[0273] As used herein, the term "mutation" refers to a change of the DNA sequence within a gene or chromosome of an organism resulting in the creation of a new character or traitnot found in the parental type, or the process by which such a change occurs in a chromosome, either through an alteration in the nucleotide sequence of the DNA coding for a gene or through a change in the physical arrangement of a chromosome. Three mechanisms of mutation include substitution (exchange of one base pair for another), addition (the insertion of one or more bases into a sequence), and deletion (loss of one or more base pairs).

[0274] The term “naïve T cell” as used herein refers to a T cell that has not previously been exposed to an antigen. Naïve T cells are conventionally defined by co-expression of the RA isoform of the transmembrane phosphatase CD45, the lymph node homing molecules L- selectin (CD62L) and CCR7, and the costimulatory receptors CD27 and CD28. [De Rosa, SC et al. Nature Med. (2001) 7: 245-248].

[0275] The term “natural killer (NK) cells” as used herein refers to lymphocytes in the same family as T and B cells, classified as group I innate lymphocytes. They can kill tumor cells without any priming or prior activation, in contrast to cytotoxic T cells, which need priming by antigen presenting cells. NK cells secrete cytokines such as IFNγ and TNFα, which act on other immune cells, like macrophages and dendritic cells, to enhance the immune response. Activating receptors on the NK cell surface recognize molecules expressed on the surface of cancer cells and infected cells and switch on the NK cell. Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHCI receptors, which mark them as “self.” Inhibitory receptors on the surface of the NK cell recognize cognate MHCI, which switches off the NK cell, preventing it from killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell. Natural killer reactivity, including cytokine secretion and cytotoxicity, is controlled by a balance of several germline encoded inhibitory and activating receptors such as killer immunoglobulin- like receptors (KIRs) and natural cytotoxicity receptors (NCRs). The presence of the MHC Class I molecule on target cells serves as one such inhibitory ligand for MHC Class I-specific receptors, the Killer cell Immunoglobulin-like Receptor (KIR), on NK cells. Engagement of KIR receptors blocks NK activation and, paradoxically, preserves their ability to respond to successive encounters by triggering inactivating signals. Therefore, if a KIR can sufficiently bind to MHC Class I, this engagement may override the signal for killing and allows the target cell to live. In contrast, if the NK cell is unable to sufficiently bind to MHC Class I on the target cell, killing of the target cell may proceed. Consequently, those tumors which express low MHCClass I and which are thought to be capable of evading a T-cell-mediated attack may be susceptible to an NK cell-mediated immune response instead.

[0276] The term “neoantigens” as used herein refers to newly formed antigens generated by tumor cells as a result of various tumor-specific alterations, such as genomic mutation, dysregulated RNA splicing, disordered post-translational modification, and integrated viral open reading frames. Leukemia and sarcoma (which are among the cancers with the lowest predicted single nucleotide variant (SNV) burden) express gene fusions and splice variant transcripts shared across multiple tumors. [Smith, CC et asl. Nature Reviews Cancer (2019) 19: 465-478].

[0277] The term “Next Generation Sequencing” or “NGS” as used herein refers to a method of parallel sequencing. For instance, a nucleic acid (e.g., DNA) sample is obtained and prepared into a library (meaning a collection of nucleic acid fragments from the sample). The library is prepared by fragmenting the DNA or RNA sample. Fragmentation can be performed by physical (e.g., sheared by acoustics, nebulization, centrifugal force, needles, or hydrodynamics) or enzymatic (e.g., site-specific or non-specific nucleases) methods. According to some embodiments, the fragments are about 200 bp, about 20 bp, about 300 bp, or about 350 bp in length. The DNA or RNA samples are repaired at the ends (e.g., blunt-ended) and then A-tailed (e.g., an adenosine is added to the 3’ end resulting in an overhang). Adapters (which are short pieces of DNA around 80 bases in length) are ligated to each end to flank either side of the sequence of interest. Adapters include sequences, such as barcodes to uniquely tag each molecule in a given sample library, restriction sites, and primer sequences to bind general sequencing primers.

[0278] The term “NKG2D” as used herein refers to an activating receptor expressed by all NK cells and subsets of T cells (γδ T cells, CD8+ T cells and CD4+ T cells) in humans. It is encoded by the KLRK1 gene (killer cell lectin-like receptor subfamily K, member 1). The NKG2D receptor functions as an activating receptor by virtue of its interactions with the signaling adaptor dimer DAP10 in humans and with DAP10 and DAP12 in mice [Raulet, DH et al. Annu. Rev. Immunol. (2013) 31: 4123-4141, citing Champsaur, M. and Lanier, LL. Immunol. Rev. (2010) 235: 267-285; Wu, J. et al. Science (1999) 285: 730-732]. When the receptor is ligated, DAP10 provides signals that recruit the p85 subunit of phosphatidylinositol 3- kinase (PI3K) and a complex of GRB2 and VAV1. Engagement of NKG2D on NK cells inducesdegranulation and cytokine production. However, engagement of NKG2D provides an enhancing or co-stimulatory signal for the activation of CD8+ T cells and probably other T cells.

[0279] NKG2D binds to several different ligands, all of which are homologous to MHC class I molecules but have no known role in antigen presentation [Id., citing Raulet, DH. Nat. Rev. Immunol. (2003) 3: 781-790; Champsaur, M. and Lanier, LL. Immunol. Rev. (2010) 235: 267-285; Eagle, RA and Trowsdale, J. Nat. Rev. Immunol. (2007) 7: 737-744; Machuldova, A. et al. Front. Immunol. (2021) 12: 651751, citing Stephens, HA. Trends Immunol. (2001) 22 (7): 378-385]. Like MHC proteins, they exhibit considerable allelic variation. In humans, the NKG2D ligands include MHC class I chain-related protein A (MICA) and MHC class I chain- related protein B (MICB), both encoded by genes in the MHC, and up to six different proteins called Unique long (UL)16-binding proteins (ULBPs), also known as retinoic acid early transcript 1 (RAET1) proteins. Like MHC proteins, the NKG2D ligands exhibit considerable allelic variation.

[0280] All NKG2D ligands are encoded by distinct genes in the host’s own genome, i.e., the ligands are self-proteins. NKG2D ligands are expressed poorly or not at all by most normal cells but are upregulated in transformed / cancer cells, virus-infected cells, and, in some cases, stressed cells. This type of recognition process, in which self-coded ligands for activating receptors are induced on unhealthy cells, has been termed “induced self-recognition [Id. citing Diefenbach, A. and Raulet, DH. Immunol. Rev. (2001) 181: 170-184], which is distinct from “missing self-recognition”, a phenomenon in which loss of MHC ligands for NK inhibitory receptors sensitizes cells for elimination by NK cells. Various cellular pathways activated because of cellular stress, infection, or tumorigenesis regulate expression of the NKG2D ligands.

[0281] The structures of NKG2D–ligand complexes indicate that NKG2D binds diagonally over the α1 and α2 helices of the ligands, much as T-cell receptors bind over MHC molecules. Despite the poor homology of different ligands, some of the key residues that interact with NKG2D are conserved, and the NKG2D residues involved in binding are similar in the different structures.

[0282] The term “non-expanded” as used herein, is meant to refer to a cell population that has not been grown in culture (in vitro) to increase the number of cells in the cell population.

[0283] The term “nuclear factor of activated T cells proteins” or “NFAT proteins” refers to a family of transcription factors whose activation is controlled by calcineurin, a calciumdependent phosphatase. During periods of sustained elevations of calcium, calcineurin dephosphorylates NFATC1–C4, allowing NFAT to translocate to the nucleus. This nuclear translocation is blocked by cyclosporine A (CSA), which blocks calcineurin activity. Once in the nucleus, NFAT binds to consensus DNA sites and controls gene transcription. Originally identified in T cells as inducers of cytokine gene expression, NFAT proteins play varied roles in cells outside of the immune system. [Horsley, V. and Pavlath, GK. J. Cell Biol. (2002) 156 (5): 771-774].

[0284] The term “objective response rate” or “ORR” as used herein refers to the percentage of people in a study or treatment group who have a partial response or complete response to the treatment within a certain period of time.

[0285] The term “overall survival” as used herein refers to the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive.

[0286] The term “PD-1” or “programmed cell death protein 1” as used herein refers to an inhibitory receptor expressed on the surface of activated T cells. Its ligands, PD-L1 and PD-L2, are expressed on the surface of DCs or macrophages. PD-1 and its ligands PD-L1 / PL-L2 act as co-inhibitory factors that can limit the development of the T cell response. PD-L1 is overexpressed on tumor cells or on non-transformed cells in the tumor microenvironment [Pardoll, DM. Nat. Rev. Cancer (2012) 12: 252-264]. PD-L1 expressed on the tumor cells binds to PD-1 receptors on the activated T cells, which leads to the inhibition of the cytotoxic T cells. These deactivated T cells remain inhibited in the tumor microenvironment.

[0287] The term “partial response” or “PR” as used herein refers to a decrease in the size of a tumor or in the extent of cancer in the body in response to treatment.

[0288] The term “peptide” is used herein to designate a series of amino acid residues, connected one to the other typically by peptide bonds between the alpha-amino and carbonyl groups of the adjacent amino acids. Peptides are typically 9 amino acids in length but can be as short as 8 amino acids in length, and as long as 14 amino acids in length. A series of amino acids are considered an “oligopeptide” when the amino acid length is greater than about 14 amino acids in length, typically up to about 30 to 40 residues in length. When the amino acid residue length exceeds 40 amino acid residues, the series of amino acid residues is termed a “polypeptide”.

[0289] As used herein, the term “perforin” as used herein refers to a molecule that can insert into the membrane of target cells and promote lysis of those target cells. Perforin- mediated lysis is enhanced by enzymes called granzymes.

[0290] The terms "peripheral blood mononuclear cells" or "PBMCs" are used interchangeably herein to refer to blood cells having a single round nucleus such as, for example, a lymphocyte or a monocyte. PBMCs are a critical component in the immune system's responses to infections.

[0291] The term “phenotype” as used herein refers to qualitative and quantitative observable characteristics of cells. A cell’s phenotype is the culmination of several cellular processes through a complex network of molecular interactions that ultimately result in a unique morphological signature. Clinical, biochemical and imaging methodologies can be used to refine and characterize a phenotype.

[0292] The term “PRDM1” or “PR domain-containing 1 with ZNF domain” or “BLIMP- 1” as used herein refers to a gene that encodes a transcriptional regulator of effector T cell differentiation. [Gattinoni, L. et al. Nature Reviews Cancer (2012) 12: 671-684].

[0293] The term “pre-initiation complex “or “PIC” as used herein refers to an assembly of proteins, including polymerase II and general transcription factors, that first nucleates at the core-promoter before transcription initiation and makes polymerase II transcription competent.

[0294] The terms “posttranslational modification”, “PTMs” or “covalent modifications” as used herein refers to the breaking or generation of covalent bonds on the backbones or amino acid side chains of proteins. More than 650 types of protein modifications, such as the most well‐ known phosphorylation, acetylation, methylation, ubiquitination, glycosylation, acylation, cysteine oxidation, SUMOylation, ADP‐ribosylation, neddylation, citrullination, and carbamylation, have been described, and the inventory is still increasing. [Zhong, Q. et al. Med Comm. (2020) 4 (30): e261, citing Ramazi, S. and Zhiri, J. Database (Oxford) (2021) 2021: baab012; Wang, H. et al. Cancer Gene Ther. (2023) 30 (4): 529-47].

[0295] Most PTMs are dynamically reversible, and the addition and removal of the PTMs are enzymatically regulated. [Id., citing Macek, B. et al. Nat. Rev. Microbiol. (2019) 17 (11): 651-664] These protein modifications occur faster than the synthesis of new proteins, which allows cells or organisms to respond rapidly to changes in the surrounding environment [Id., citing Conibear, AC Nat. Rev. Chem. (2020) 4 (120: 674-695]. By changing proteinconformation, activity, charges and stability and interactions with DNA, RNA, and other proteins within and between cells, PTMs ultimately alter the phenotypes and biological functions of cells [Id., citing Liddy, KA et al. Genome Med. (2013) 5 (2): 20] and participate in the regulation of numerous cellular processes and pathways, such as cell cycle [Id., citing Chen, L. et al. Signal Transduct. Target Ther. (2020) 5 (1): 90] cell differentiation [Id., citing Shvedunova, M. and Akhtar, A. Nat. Rev. Mol. Cell Biol. (*2022) 23 (50: 329-49], transcriptional regulation, [Id., citing Nozaki, T. and Kanai, M. Acc. Che. Res. (2021) 54 (9): 2313-2322], cell metabolism [Id., citing Humphrey, SH et al. Trends Endocrinol. Metab. (2015) 26 (12): 676-87], immunity [Liu, J. et al. Immunity (2016) 45 (1): 15-30; signal transduction [Id., citing Guccione, E. and Richard, S. Nat. Rev. Mol. Cell Biol. (2019) 20 (10): 642-657], and autophagy [[Id., citing Polletta, L. et al. Autophagy (2015) 11 (20: 253-270]. For example, phosphorylation is involved in cell signal transduction and the cell cycle [Id., citing Ubersax, JA and Ferrell, JE, Jr. Nat. Rev. Mol. Cell Biol. (2007) 8 (70: 530-541] ; acetylation and methylation are associated with transcriptional regulation and cell metabolism [Id., citing Choudhary, C. et al. Science (2009) 325 (5942): 834- 840; Wu, Q. et al. Nat. Rev. Drug Discov. (2021) 20 (7): 509-530]; glycosylation plays an important role in protein folding and cell adhesion [Id., citing Ohtsubo, K. and Marth, JD. Cell (2006) 126(5): 855-867]; and ubiquitination regulates protein degradation and localization [Id., citing Meyer-Schwesinger, C. Nat. Rev. Nephrol. (2019) 15 (7): 393-411].

[0296] The term “priming” as used herein refers to the first encounter with a given antigen, which generates a primary adaptive immune response. The term “unprimed cells” (also referred to as virgin, naïve, or inexperienced cells) as used herein refers to T cells and B cells that have generated an antigen receptor (TCR for T cells, BCR for B cells) of a particular specificity, but have never encountered the antigen. For example, before helper T cells and B cells can interact to produce specific antibody, the antigen-specific T cell precursors must be primed.

[0297] Priming involves several steps: antigen uptake, processing, and cell surface expression bound to class II MHC molecules by an antigen presenting cell, recirculation and antigen-specific trapping of helper T cell precursors in lymphoid tissue, and T cell proliferation and differentiation. [Janeway, CA, Jr., “The priming of helper T cells, Semin. Immunol. (1989) 1(1): 13-20]. Helper T cells express CD4, but not all CD4 T cells are helper cells. Id. The signals required for clonal expansion of helper T cells differ from those required by other CD4 Tcells. The critical antigen-presenting cell for helper T cell priming appears to be a macrophage; and the critical second signal for helper T cell growth is the macrophage product interleukin 1 (IL-1). Id. If the primed T cells and / or B cells receive a second, co-stimulatory signal, they become activated T cells or B cells.

[0298] The term “progression” as used herein refers to the course of disease as it becomes worse or spreads in the body.

[0299] The term “progression-free survival” or PFS” as used herein refers to the length of time during and after the treatment of the disease that a patient lives with the disease, but it does not get worse.

[0300] The term “proliferate” and its various grammatical forms as used herein is meant to refer to the process that results in an increase of the number of cells, and is defined by the balance between cell division and cell loss through cell death or differentiation.

[0301] The term “promoter” as used herein refers to a sequence several kbs upstream of TSSs (transcription start signals) that can autonomously drive transcription. This functionality and the fact that some promoters can activate transcription from a distal core-promoter in reporter assays is in line with promoters consisting of a core-promoter and a proximal or overlapping enhancer.

[0302] The term “promoter-proximal tethering elements” as used herein refers to sequences proximal to the core-promoter that enable or facilitate its interaction with enhancers.

[0303] The term “promoter-targeting sequences” as used herein refers to sequences proximal to enhancers that enable or facilitate their interactions with core-promoters.

[0304] The term “recurrent cancer” or “recurrence” means a cancer that has come back, usually after a period during which the cancer could not be detected. The cancer may come back to the same place as the primary tumor or to another place in the body.

[0305] The term “refractory cancer” or “resistant cancer” means a cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment, or it may become resistant during treatment.

[0306] The term “relapse” refers to the return of a disease or the signs and symptoms of a disease after a period of improvement.

[0307] The terms “relapse-free survival” (RFS) or “disease-free survival” (DFS) mean the length of time after primary treatment for a cancer ends that the subject or patient survives without any signs or symptoms of that cancer.

[0308] The term “repertoire” as used herein refers to the collection of immune cells that respond to a particular foreign substance (e.g., pathogen). The repertoire recognizing a molecule would be the sum of the repertoires responding against all the component epitopes of the molecule. Likewise, the repertoire against an organism or cell would be the sum of all the repertoires against all the molecules from the pathogen or cell.

[0309] The term “RNA-seq” as used herein refers to a process of creating short sequencing reads from RNA molecules. The steps consist of first converting the RNA into cDNA; then (optionally) amplifying the cDNA by PCR; and finally fragmenting the cDNA into short pieces / fragments. After the sequencing library is prepared, the fragments are used as input for next-generation sequencing. The resulting sequence reads contained in FASTQ files are then aligned to a known reference sequence. [Deshpande, D., et al. Frontiers Genetics (2023) 14: 997383].

[0310] The term “RUNX” as used herein refers to an evolutionary conserved family of transcription factors (TFs) that are best known for their roles in the regulation of the expression of genes involved in embryonic development and cell differentiation. All three Runx genes have two alternative promoters, proximal (P2) and distal (P1), and the transcripts also undergo alternative splicing, giving rise to multiple isoforms and producing diffuse bands on Western blots. The patterns of the isoform expression are cell type–specific, time-dependent and nonredundant. As with other TFs, Runx proteins are subject to post-translational modifications that affect Runx cellular localization, stability, DNA-binding affinity and ability to interact with other proteins—sumoylation [Korinfskaya, S. et al. Front. Immunol. (2021) 12: 701924, citing Lee, JW et al. in Advances in Exptl Med & Biol. New York LLC: Springer (2017) pp.321-332], acetylation, phosphorylation [Id., citing Tanaka, T. et al. Mol. Cell Biol. (1996) 16: 3967-3979; Kim, HR et al. J. Cell Biochem. (2008) 105: 1048-1058; Goh, YM et al. J. Biol. Chem. (2010) 285: 10122-10129], ribosylation [Id., citing Tay, LS et al. Cell Rep. (2018) 24: 1747-1755], methylation [Id., citing Zhao, X. et al. Genes Dev. (2008) 22: 640-653; Herglotz, J. et al. Oncogene (2013) 32: 2565-2575] and ubiquitination [Id., citing Zhang, M. et al. J. Cell Sci. (2009) 122: 1382-9]. For example, methylated RUNX1 has increased transcription activationpotency [Id., citing Zhao, X et al. Genes Dev. (2008) 22: 640-53], whereas poly (ADP)- ribosylation of RUNX1 and RUNX3 enables interaction with the helicase BLM (Bloom syndrome protein) in response to DNA damage in the context of Fanconi anemia [Id., citing Tay, LS et al. Cell Rep. (2018) 24: 1747-1755]. Runx proteins share the ability to dimerize with the transcription factor CBF beta (CBFβ) [Id., citing Wang, S. et al. Mol. Cell Biol. (1993) 13: 3324- 3339]. Heterodimerization of Runx proteins increases their ability to bind DNA and protects them from ubiquitination [Id., citing Tahirov, TH and Bushweller, J. Mol Cell Biol. (1993) 13: 3324-39]. Additionally, CBFβ controls the translation of RUNX1 [Id., citing Malik, N. et al. Nat. Commun. (2019) 10: 1-15]. CBFβ does not have DNA binding activity itself but is instead recruited to DNA by all members of the Runx family through physical interactions [Id., citing Huang, G. et al. EMBO J. (2001) 20: 723-733]. Among the TFs that cooperate and / or compete with the Runx proteins upon DNA binding are members of the ETS family, which positively and / or negatively regulates the expression of genes involved in signaling pathways, development, cell proliferation, differentiation, migration, apoptosis, invasion and metastasis [Id., citing Mao, S. et al. Mol. Cell Biol. (1999) 19: 3635-3644; Kar, A. and Gutierrez-Hartman, Crit. Rev. Biochem. Mol. Biol. (2013) 48 (6): 522-43], Kruppel-like factor 4 [KLF4], an evolutionarily conserved zinc finger-containing transcription factor that regulates diverse cellular processes such as cell growth, proliferation, and differentiation [Id., citing Liu, S. et al. Haematologica (2019) 104: 1597-1607; Ghaleb, AM and Yang, VW. Gene (2017) 611: 27-37], nuclear factor of activated T cell [NFAT; Id., citing Gabriel, CH et al. J. Biol. Chem. (2016) 291: 24172-24187], activator protein-1 [AP-1; Id., citing D’Alonzo, RC et al. J. Biol. Chem (2002) 277: 816-822; Hess, J. et al. J. Biol. Chem. (2001) 276: 20029-200238], STAT5 [Ogawa, S. et al. Biochem. (2008) 143: 695-709] and many others. Runx proteins can repress or activate genes through direct and / or indirect interactions with chromatin modifiers Id., citing Taniuchi, I. and Littman, DR. Oncogene (2004) 23: 4341-4345].

[0311] The term “scFv” as used herein refers to a class of engineered functional antibodies generated by the fusion of the variable heavy (VH) and variable light (VH) domains of an immunoglobulin through a short polypeptide linker.

[0312] The term “sign” as used herein refers to a healthcare provider’s evidence of disease.

[0313] The term “Signal transducer and activator of transcription 3 “or “STAT3” as used herein refers to a transcription factor that is activated downstream of a large range of cell surface receptors. It forms part of a family of proteins that also includes STAT1, 2, 4, 5A, 5B, and 6, which are activated in a similar manner downstream of surface receptors. Binding of their ligand by these receptors leads to the activation of receptor-associated Janus activating kinases (JAKs). The activated JAKs then phosphorylate the receptor providing docking sites for STATs, which in turn become tyrosine phosphorylated. This leads to the formation of homodimers or heterodimers, followed by translocation to the nucleus where the dimers bind to DNA and induce transcription of a broad range of target genes [Deenick, EK., et al. Front. Immunol. (2018) 9: doi.org / 10.3389 / fimmu.2018.00168, O’Shea, JJ., et al. N. Engl. J. Med. (2013) 368 (20): 161- 170; Shuai, K. and Liu, B. Nat. Rev. Immunol. (2003) 3 (11): 900-911]. Many of the cytokine receptors that lead to STAT3 activation are expressed by lymphocytes including those for IL-6, IL-10, IL-21, IL-23, and IFNs. STAT3 plays a central role in regulation of immune responses. Loss of function (LOF) mutations in STAT3 cause the primary immunodeficiency autosomal dominant hyper IgE syndrome (ADHIES), which is characterized by defects in both T and B cells [Id., citing Holland, SM., et al. N. Engl. J. Med. (2007) 357 (16): 1608-1619; Minegishi, Y., et al. Nature (2007) 448 (7157): 1058-1062]. Gain of function (GOF) mutations in STAT3 have been identified in patients who presented with early onset autoimmunity as well as immunodeficiency [Id., citing Flanagan, SE., et al. Nat. Genet. (2014) 46 (8): 812-814; Haapaniemi, EM et al. Blood (2015) 125 (4): 639-648; Milner, JD., et al. Blood (2015) 125 (40): 59159-9].

[0314] The term “solid tumor” as used herein refers to an abnormal mass of tissue that usually does not contain cysts or liquid areas. Solid tumors may be benign (a growth that does not invade nearby tissue or spread to other parts of the body) or malignant (meaning to grow in an uncontrolled way; malignant tumors can invade nearby tissues and spread to other parts of the body through the blood and lymph system). Different types of solid tumors are named for the type of cells that form them. Types of solid tumors are sarcomas, carcinomas, and lymphomas; leukemias (cancers of the blood) generally do not form solid tumors.

[0315] A carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. An adenocarcinoma is a cancer that forms in the glandular tissue that lines certain internal organs and makes and releases substances in the body, such as mucus, digestivejuices, and other fluids. Most cancers of the breast, lung, esophagus, stomach, colon, rectum, pancreas, prostate, and uterus are adenocarcinomas.

[0316] A lymphoma is a malignant solid neoplasm of the lymphoid system, which produces immune cells. Abnormal lymphocytes become lymphoma cells, which multiply and collect in the lymph nodes. Over time, these cancerous cells impair the immune system. There are two categories of lymphomas: Hodgkin lymphoma and non-Hodgkin lymphoma. About 12 percent of people with lymphoma have Hodgkin lymphoma. Most non-Hodgkin lymphomas are B-cell lymphomas, and either grow quickly (high-grade) or slowly (low-grade). There are over a dozen types of B-cell non-Hodgkin lymphomas. The rest are T-cell lymphomas.

[0317] Hodgkin’s lymphoma is a lymphoma in which the tumor mass is made up of a reactive infiltrate of nontransformed lymphocytes, macrophages and fibroblasts plus scattered, malignant Reed-Sternberg cells. Reed-Sternberg cells are large, abnormal lymphocytes that may contain more than one nucleus, are clonal in their growth and are the tumor cells of the malignant mass. While of the B-cell lineage, they lack common B-cell specific surface markers such as CD19 and CD79a as well as Ig gene transcripts [Hertel, CB et al. Oncogene (2002) 21: 4908- 4920]. Hodgkin lymphoma most commonly affects lymph nodes in the neck or in the area between the lungs and behind the breastbone. It can also begin in groups of lymph nodes under an arm, in the groin, or in the abdomen or pelvis. If it spreads, it may spread to the lung, spleen liver, bone marrow or bone.

[0318] There are two major categories of Hodgkin lymphoma: classical Hodgkin lymphoma, which is divided into 4 subtypes based on the appearance of lymph node structure and cells, and nodular lymphocyte-predominant Hodgkin lymphoma.

[0319] Classical Hodgkin lymphoma (cHL) represents about 95% of cases of Hodgkin lymphoma. It is diagnosed when characteristic abnormal lymphocytes, (Reed-Sternberg cells) are found. There are 4 subtypes of cHL. Nodular sclerosis Hodgkin lymphoma, the most common subtype of cHL, affects up to 80% of people diagnosed with cHL. It is most common in young adults, especially women. In addition to Reed-Sternberg cells, there are bands of connective tissue (called fibrosis) found in the lymph node. This type of lymphoma often affects the lymph nodes in the mediastinum.

[0320] Non-Hodgkin’s lymphoma (NHL) is a heterogeneous group of lymphomas in which the solid tumor mass consists almost entirely of malignant lymphocytes. [Mak, TW et al.Ch.20 Hematopoietic cancers, in Primer to the Immune response (2014) Elsevier, Inc., pp. 573- 574] NHL may be described by how quickly the cancer is growing. An “indolent or low-grade lymphoma’ is a type of lymphoma that tends to grow and spread slowly and has few symptoms. When indolent lymphoma is in stages 1 and 2, it is called localized disease. An “aggressive lymphoma” “high-grade lymphoma” or intermediate-grade lymphoma” is a type of lymphoma that grows and spreads quickly and has severe symptoms. In children, aggressive non-Hodgkin lymphoma is more common. Some types of lymphoma cannot be easily classified as indolent or aggressive.

[0321] There are more than 60 NHL subtypes of NHL that tend to mimic stages of normal B cell differentiation. [Mancuso, S. et al. Immunity & Aging (2018) 15: 22]. Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma representing about 30% of NHL in the US. It is an aggressive form of NHL that involves organs other than the lymph nodes about 40% of the time. DLBCL includes germinal center and non-germinal center DLBCL. Follicular lymphoma (FL) is the second most common form of lymphoma in the United States and Europe, representing about 20% of people with NHL. It usually begins in the lymph nodes, is most often indolent, and grows very slowly. Mantle cell lymphoma represents about 5-7% of individuals with NHL. It is an aggressive (fast-growing) type of B-cell non- Hodgkin lymphoma that usually occurs in middle-aged or older adults is marked by small- to medium-size cancer cells that may be in the lymph nodes, spleen, bone marrow, blood, and gastrointestinal system.

[0322] Small lymphocytic lymphoma, which represents about 5% of individuals with NHL, is the same disease as B-cell chronic lymphocytic leukemia (CLL), without a significant amount of disease in the blood. It is considered an indolent lymphoma.

[0323] Burkitt lymphoma / Burkitt cell leukemia is a very rare and aggressive form of lymphoma that occurs most often in children and young adults. The disease may affect the jaw, central nervous system, bowel, kidneys, ovaries, or other organs. There are three forms: endemic (which occurs commonly in Africa, appears most often in the jawbones of children, and is usually associated with infection with EBV); sporadic (which occurs throughout the world), and immunodeficiency-related (which is most often seen in AIDS patients). This subtype often has abnormalities involving the MYC gene, which can contribute to cancer growth.

[0324] T cell lymphomas make up approximately 10%1 - 15% of lymphoid malignancies. [Armitage, J.O. Am. J. Hematol. (2017) 92 (7): 706-715]. The frequency of these lymphomas varies geographically, with the highest incidence in parts of Asia. T‐cell lymphomas can be divided into those of precursor T‐cells (i.e., precursor T‐lymphoblastic lymphoma) and those arising in more mature T‐cells, with the latter termed peripheral T‐cell lymphomas (PTCLs). The aggressive PTCLs are associated with a short survival. These can be subdivided into those of primarily nodal origin and those that typically present in specific extranodal sites and are often associated with characteristic clinical syndromes. [Armitage, J.O. Am. J. Hematol. (2017) 92 (7): 706-715]

[0325] Adult T-cell lymphoma / leukemia (human T cell lymphotropic virus type I positive) is caused by the human T-cell lymphotropic virus type 1. It is an aggressive disease that often involves the bone and skin. Lymphoma cells are often found in the blood.

[0326] A sarcoma is a type of cancer that begins in bone or in the soft tissues of the body, including cartilage, fat, muscle, blood vessels, fibrous tissue, or other connective or supportive tissue. Bone and soft tissue sarcomas are the main types of sarcoma. Soft tissue sarcomas can develop in soft tissues like fat, muscle, nerves, fibrous tissues, blood vessels, or deep skin tissues. They can be found in any part of the body. Most of them start in the arms or legs. They can also be found in the trunk, head and neck area, internal organs, and the retroperitoneum. The different types of sarcoma are based on where the cancer forms. For example, osteosarcoma forms in bone, liposarcoma forms in fat, and rhabdomyosarcoma forms in muscle.

[0327] The term “splice-site variant” or “splice-site mutation” as used herein refers to a genetic alteration in the DNA sequence that occurs at the boundary of an exon and an intron (splice site). This change can disrupt RNA splicing resulting in the loss of exons or the inclusion of introns and an altered protein-coding sequence.

[0328] As used herein, the term “stimulate” in any of its grammatical forms as used herein is meant to refer to inducing activation or increasing activity.

[0329] The term “stimulate an immune cell” or “stimulating an immune cell” as used herein is meant to refer to a process (e.g., involving a signaling event or stimulus) causing or resulting in a cellular response, such as activation and / or expansion, of an immune cell, e.g. a CD8+ T cell.

[0330] The terms "subject" or "individual" or "patient" are used interchangeably to refer to a member of an animal species of mammalian origin, including but not limited to, mouse, rat, cat, goat, sheep, horse, hamster, ferret, pig, dog, guinea pig, rabbit and a primate, such as, for example, a monkey, ape, or human.

[0331] The phrase “subject in need thereof” as used herein refers to an eligible patient that (i) will be administered an immunotherapy according to the present disclosure, (ii) is receiving at least one immunotherapy according to the present disclosure; or (iii) has received at least one immunotherapy according to the present disclosure, unless the context and usage of the phrase indicates otherwise.

[0332] The term “symptom” as used herein refers to a patient’s subjective evidence of disease.

[0333] The terms “T cell” or “T lymphocyte” are used interchangeably to refer to cells that mediate a wide range of immunologic functions, including the capacity to help B cells develop into antibody-producing cells, the capacity to increase the microbicidal action of monocytes / macrophages, the inhibition of certain types of immune responses, direct killing of target cells, and mobilization of the inflammatory response. These effects depend on their expression of specific cell surface molecules and the secretion of cytokines. T cells recognize antigens on the surface of antigen presenting cells (APCs) and mediate their functions by interacting with, and altering, the behavior of these APCs. T cells can also be classified based on their function as helper T cells; T cells involved in inducing cellular immunity; suppressor T cells; and cytotoxic T cells. T-cell activation is dependent on the interaction of the TCR / CD3 complex with its cognate ligand, a peptide bound in the groove of a class I or class II MHC molecule. The molecular events set in motion by receptor engagement are complex. Among the earliest steps appears to be the activation of tyrosine kinases leading to the tyrosine phosphorylation of a set of substrates that control several signaling pathways. These include a set of adaptor proteins that link the TCR to the ras pathway, phospholipase Cγ1, the tyrosine phosphorylation of which increases its catalytic activity and engages the inositol phospholipid metabolic pathway, leading to elevation of intracellular free calcium concentration and activation of protein kinase C, and a series of other enzymes that control cellular growth and differentiation. The ultimate amplitude and quality of the T cell immune response, which isinitiated through antigen recognition by the TCR, is regulated by a balance between co- stimulatory and inhibitory signals (immune checkpoints).

[0334] Although the lineage relationship between T cell subsets remains controversial, T cells cluster in populations that can be arranged as a progressive continuum based on phenotypic, functional and transcriptional attributes. T lymphocytes transition through progressive stages of differentiation that are characterized by a stepwise loss of functional and therapeutic potential in an order from naive T (TN) cells to T memory stem cells (TSCM) (the most immature antigen- experienced T cells), to T central memory (TCM) cells, which patrol central lymphoid organs, to T effector memory (TEM) cells, which patrol peripheral tissues. In contrast to TNcells, memory T cells are capable of rapidly releasing cytokines on restimulation. TCMcells more efficiently secrete IL-2 and TEMhave an increased capacity for IFNγ release and cytotoxicity. All antigen- experienced T cells upregulate the common IL-2 and IL-15β receptor (IL-2Rβ) conferring the ability to undergo homeostatic proliferation in response to IL-15, and also display high amounts of CD95 (also known as FAS), a receptor that provides either costimulatory or pro-apoptotic signals depending on the efficiency of CD95 signaling complex formation and on which particular intracellular signaling proteins are part of the complex. [Gattinoni, L. et al. Nature Revs. Cancer 12: 671-684].

[0335] The term “T cell antigen” as used herein is meant to refer to a protein, lipid (for CD1) or fragment thereof which can be processed into a peptide that can bind to either Class I MHC, Class II MHC, non-classical MHC, or CD1 family molecules (collectively antigen presenting molecules), and, in this combination, can engage a T cell receptor on a T cell.

[0336] The term “T cell epitope” as used herein is meant to refer to a short peptide molecule that binds to a class I or II MHC molecule and that is subsequently recognized by a T cell. T cell epitopes that bind to class I MHC molecules are typically 8-14 amino acids in length, and most typically 9 amino acids in length. T cell epitopes that bind to class II MHC molecules are typically 12-20 amino acids in length. In the case of epitopes that bind to class II MHC molecules, the same T cell epitope may share a common core segment, but differ in the length of the carboxy- and amino-terminal flanking sequences due to the fact that ends of the peptide molecule are not buried in the structure of the class II MHC molecule peptide-binding cleft as they are in the class I MHC molecule peptide-binding cleft.

[0337] The term “T cell factor 1” or (TCF-1), encoded by Tcf7, as used herein refers to the key transcription factor of the canonical Wnt signaling pathway. TCF1 and its homolog LEF1 are known as effector transcription factors downstream of the WNT signaling pathway and are essential for early T cell development. TCF1 is required for the self-renewal of stem-like CD8+ T cells generated in response to viral or tumor antigens, and for preserving heightened responses to checkpoint blockade immunotherapy. In the helper T cell lineages, TCF1 is indispensable for the differentiation of T follicular helper and T follicular regulatory cells and crucially regulates immunosuppressive functions of regulatory T cells. Mechanistic investigations have also identified TCF1 as the first transcription factor that directly modifies histone acetylation, with the capacity to bridge transcriptional and epigenetic regulation. [Shan, Q. et al. Nature Communic. (2021) 12: 5863, citing Zhao, X. et al. Nat. Rev. Immunol. (2022) 22: 147-157].

[0338] The term “T cell receptor” (TCR) as used herein refers to a complex of integral membrane proteins that participates in the activation of T cells in response to an antigen. The TCR expressed by the majority of T cells consisting of a heterodimer of α and β chains. A small group of T cells express receptors made of γ and δ chains. Among the α / β T cells are two sublineages: those that express the coreceptor molecule CD4 (CD4+ cells), and those that express CD8 (CD8+ cells). These cells differ in how they recognize antigen and in their effector and regulatory functions. The TCR is composed of four distinct signal transducing subunits (CD3-gamma (γ), -delta (δ), -epsilon-(ε), and zeta (ζ)) that share a common functional sequence, the immunoreceptor tyrosine-based activation motif (ITAM) [Shores, EW et al. J. Exp. Med. (1997) 185 (5): 893-900, citing Robey, E. and Fowlkes, BJ. Annu. Rev. Immunol. (1994) 12: 675-705] within their intracytoplasmic domains [Id., citing Reth, M. Nature (Lond) (1989): 338: 383-384; Samelson, LE and Klausner, RD. J Biol. Chem. (1992) 267: 24913-24916]. After TCR engagement, phosphorylation of ITAMs leads to the recruitment of SH2 domain–containing proteins (e.g., tyrosine kinases) to the TCR complex and initiation of the T cell activation cascade [Id., citing Samelson, LE and Klausner, RD. J Biol. Chem. (1992) 267: 24913-24916; Weiss, A., and Littman, DR. Cell (1994) 76: 263-274; Irving, BA and Weiss, A. Cell (1991) 64: 891-901; Romeo, C. et al. Cell (1992) 68: 889-897]. The CD3 subunits each contain a single ITAM, whereas ζ contains three ITAMs within its longer cytoplasmic tail. ITAM sequences are conserved but nonidentical. Irving et al constructed a chimeric protein linking the extracellularand transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain and demonstrated that the CD8 / zeta chimera was expressed independently of the TCR and was capable of transducing signals that, by criteria of early and late activation, were indistinguishable from those generated by the intact TCR. [Irving, BA and Weiss, A. Cell (1991) 64: 891-901]. Their data showed that CD8 / zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggested that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms.

[0339] The term “helper T cells” or “TH” cells as used herein refers to effector CD4 T cells that stimulate or “help” B cells to make antibody in response to antigenic challenge. TH2, TH1 and the TFHsubsets of effector CD4 T cells can perform this function.

[0340] The term “T follicular helper (TFH) cells” as used herein refers to a distinct subset of CD4+ T lymphocytes, specialized in B cell help and in regulation of antibody responses. They develop within secondary lymphoid organs (SLO) and can be identified based on their unique surface phenotype, cytokine secretion profile, and signature transcription factor. They support B cells to produce high-affinity antibodies toward antigens to develop a robust humoral immune response and are crucial for the generation of B cell memory. They are essential for infectious disease control and optimal antibody responses after vaccination. Stringent control of their production and function is critically important, both for the induction of an optimal humoral response against thymus-dependent antigens but also for the prevention of self-reactivity. [Gensous, N. et al. Front. Immunol. (2018) doi.org / 10.3389 / fimmu.2018.01637).

[0341] The term “TH1 cells” as used herein refers to a lineage of CD4+ effector T cells that promotes cell-mediated immune responses and is required for host defense against intracellular viral and bacterial pathogens. They are mainly involved in activating macrophages but can also help stimulate B cells to produce antibody. TH1 cells secrete IFN-gamma, IL-2, IL- 10, and TNF-alpha / beta. IL-12 and IFN-γ make naive CD4+ T cells highly express T-bet and STAT4 and differentiate to TH1 cells. [Zhang, Y. et al. Adv. Exp. Med. Bio. (2014) 841: 15-44].

[0342] The term “TH2 cells” as used herein refers to a lineage of CD4+ effector T cells that secrete IL-4, IL-5, IL-9, IL-13, and IL-17E / IL-25. These cells are required for humoral or antibody-mediated immunity and play an important role in coordinating the immune response to large extracellular pathogens. IL-4 makes naive CD4+ T cells highly express STAT6 andGATA3 and differentiate to TH2 cells. (Zhang, Y. et al. Adv. Exp. Med. Bio. (2014) 841: 15- 44).

[0343] The term “TH17 cells” as used herein refers to a CD4+ T-cell subset characterized by production of interleukin-17 (IL-17). IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues, resulting in production of inflammatory cytokines and recruitment of leukocytes, especially neutrophils, thus creating a link between innate and adaptive immunity. [Tesmer, LA, et al., Immunol. Rev. (2008) 223: 87-113]. The key transcription factor in TH17 cell development is RORγt.

[0344] The term “TATA box” as used herein refers to an A / T rich sequence located about 25-30 nucleotides upstream of the transcription start site in the promoter region of many eukaryotic genes involved in binding RNA polymerase via a TATA binding protein.

[0345] The term “T-bet” as used herein refers to a TH1 cell transcription factor. Differential expression of the TH1 cell transcription factor T bet and a closely related T-box family transcription factor, factor particularly in CD8+ T cells, Eomesodermin (Eomes) facilitates the cooperative maintenance of the pool of antiviral CD8+ T cells during chronic viral infection. [Paley, MA et a., Science (2012) 338: 1220-1225]. During chronic infections, T-bet is reduced in virus-specific CD8+ T cells; this reduction correlates with T cell dysfunction. In contrast, Eomes mRNA expression is up-regulated in exhausted CD8+ T cells during chronic infection. [Id.]

[0346] The term “Tpex” as used herein refers to progenitors or precursors of exhausted T cells. [Utzschneider, DT et al. Nature Immunol. (2020) 21: 1256-1266].

[0347] The term “Treg” or “regulatory T cells” as used herein refers to effector CD4 T cells that inhibit T cell responses and are involved in controlling immune reactions and preventing autoimmunity. The natural regulatory T cell lineage that is produced in the thymus is one subset. The induced regulatory T cells that differentiate from naïve CD4 T cells in the periphery in certain cytokine environments is another subset. Tregs are most commonly identified as CD3+CD4+CD25+FoxP3+ cells in both mice and humans. Additional cell surface markers include CD39, 5’ Nucleotidase / CD73, CTLA-4, GITR, LAG-3, LRRC32, and Neuropilin-1. Tregs can also be identified based on the secretion of immunosuppressive cytokines including TGF-beta, IL-10, and IL-35. Cell surface molecules CTLA-4, LAG-3, and neuropilin-1 (Nrp1) impair dendritic cell (DC)-mediated conventional T cell activation: CTLA-4 and LAG-3 outcompete CD28 and T cell receptor expressed on conventional T cells for binding to CD80 / 86 and MHC class II on DCs, and Nrp1 stabilizes DC–Treg contact, thereby preventing antigen presentation to conventional T cells [Ikebuchi, R. et al. Front. Immunol. (2019) doi.org / 10.3389 / fimmu.2019.01098].

[0348] The term “therapeutic effect” as used herein is meant to refer to a consequence of treatment, the results of which are judged to be desirable and beneficial. A therapeutic effect can include, directly or indirectly, the arrest, reduction, or elimination of a disease manifestation. A therapeutic effect can also include, directly or indirectly, the arrest reduction or elimination of the progression of a disease manifestation.

[0349] The term “therapeutic window” as used herein is meant to refer to a concentration range that provides therapeutic efficacy without unacceptable toxicity. In a drug context, following administration of a dose of a therapeutic agent / drug, its effects usually show a characteristic temporal pattern. A lag period is present before the drug concentration exceeds the minimum effective concentration (“MEC”) for the desired effect. Following onset of the response, the intensity of the effect increases as the drug continues to be absorbed and distributed. This reaches a peak, after which drug elimination results in a decline in the effect’s intensity that disappears when the drug concentration falls back below the MEC. Accordingly, the duration of a drug’s action is determined by the time period over which concentrations exceed the MEC. The therapeutic goal is to obtain and maintain concentrations within the therapeutic window for the desired response with a minimum of toxicity. Drug response below the MEC for the desired effect will be subtherapeutic, whereas for an adverse effect, the probability of toxicity will increase above the MEC. Increasing or decreasing drug dosage shifts the response curve up or down the intensity scale and is used to modulate the drug’s effect. Increasing the dose also prolongs a drug’s duration of action but at the risk of increasing the likelihood of adverse effects. Accordingly, unless the drug is nontoxic, increasing the dose is not a useful strategy for extending a drug’s duration of action.

[0350] The treatment of solid tumors by CAR-T cells is complex, multifactorial and has a narrower therapeutic window than does the targeting of CD19 for the treatment of B cell leukemia and non-Hodgkin lymphoma. [Watanabe, et al. Front. Immunol. (2018) 9: 2486]. Possible approaches to expand the therapeutic window of CAR-T cell therapy include:

[0351] (1) optimizing CAR affinity and sensing (with the caveat that increasing the affinity of CAR-T cells may reduce or prevent serial killing, promote T cell exhaustion, decrease the generation and persistence of TCMand TEFF[Watanabe, citing Caserta, S. et al. J. Immunol. (2010) 185: 6545-6554] or increase the loss of T cells through activation-induced cell death [Id., citing Engels, B. et al. Mol. Ther. (2012) 20: 652-660]; and / or combinatorial antigen recognition through two different antigens on tumor cells. In one such example, CAR-1 can drive only the activation signal (signal 1 of CD3ζ, and CAR2 can drive only co-stimulation (signal 2) through costimulatory molecules (AND logic gated CAR) or OR logic gated CAR [Id., citing Wilkie, S. et al. J. Clin. Immunol. (2012) 32: 105-170; Kloss, CC et al. Nat. Biotechnol. (2013) 31: 71-75; Grada, Z. et al. Mol. Ther. Nucleic Acids (2013) 2: e105; Hegde, M. et al. Mol Ther. (2013) 21: 2087-2101; Hegde, M. et al. J. Clin. Invest. (2016) 126: 3036-3052].

[0352] (2) optimizing immunological synapse formation, e.g., CD28 plus 4-1BB based third generation CARs have been found superior to CD28 based second generation CARs as measured by immunosynapse structure, signaling and function [Id., citing Xiong, W. et al. Mol. Ther. (2018) 26: 963-975].

[0353] (3) combination therapies for overcoming tumor heterogeneity and thereby expanding the therapeutic window using oncolytic viruses [Id., citing Watanabe, K. et al. JCI Insight (2018) 3: 99573; Nishio, N. et al. Cancer Res. (2014) 4: 5195-5205; Rosewell, Shaw, A. et al. Mol. Ther. (2017) 25: 2440-2451; Wing, A. et al. Cancer Immunol. Res. (2018) 6: 605- 616].

[0354] (4) local delivery of CAR-T cells and therapeutic agents into the tumor bed [Id., citing Tchou, J. et al. Cancer Immunol. Res. (2017) 5: 1152-1161].

[0355] (5) induction of target antigen expression [Id., citing Garnett, CT et al. Cancer Res. (2004) 64: 7985-7994; Hiraga, J. et al. Blood (2009) 113: 4885-4893]; or

[0356] (6) other modifications to enhance safety.

[0357] The term “TIGIT” as used herein refers to a member of the Ig super family and an immune inhibitory receptor.

[0358] The term “TIM-3” as used herein refers to a transmembrane protein and immune checkpoint receptor. It is associated with tumor-mediated immune suppression.

[0359] The term “tissue-resident memory T cell” or “TRM” as used herein refers to memory lymphocytes that do not migrate after taking up residence in barrier tissues, where theyare retained long term. They appear to be specialized for rapid effector function after restimulation with antigen or cytokines at sites of pathogen entry.

[0360] The term “tolerance” as used herein refers to a failure to respond to a particular antigen. Tolerance mechanisms that operate in the thymus before the maturation and circulation of T cells are referred to as “central tolerance.” Not all antigens of which T cells need to be tolerant are expressed in the thymus, and therefore central tolerance mechanisms alone are insufficient. Additional tolerance mechanisms exist to restrain the numbers and or function of T cells that are reactive to developmental or food antigens, which are not typically expressed. Tolerance acquired by mature circulating T cells in the peripheral tissues is called “peripheral tolerance.”

[0361] The term “toll-like receptors” or “TLR” as used herein refers to membrane bound pattern recognition receptors (PRRs) that bind to pathogen products.

[0362] The terms “Topically Associating Domains” or “TADs” refers to self-associating, loop-like domains that contain interacting cis-regulatory elements and target genes. The term “TAD boundaries” as used herein refers to insulator elements that restrict interactions of cis- regulatory sequences, such as enhancers, to target genes. [McArthur, E. and Capra, JA. Am. J. Hum. Genet. (2021) 108 (2): 269-283].

[0363] The terms “TOX” or “thymocytes selection-associated HMG BOX” as used herein refers to a member of a family of transcriptional factors that contain the highly conserved high mobility group box (HMG-box) region. Increasing studies have shown that TOX is involved in maintaining tumors and promoting T cell exhaustion. [Liang, C. et al. Biomark Res. (2021) 9: 20].

[0364] The term “toxicity” as used herein refers to the degree to which a substance can harm humans or animals. Acute toxicity involves harmful effects in an organism through a single or short-term exposure.

[0365] The terms “transcription” or “DNA transcription” are used interchangeably herein to refer to copying of one strand of DNA into a complementary RNA sequence by the enzyme RNA polymerase II (Pol II). Transcription initiates within core-promoters, short sequences of around 100 base pairs (bps) surrounding the transcription start sites (TSSs) at the 5’ start of genes. Core-promoters recruit Pol II, assemble the Pre-Initiation Complex (PIC) and dictate the accurate position of initiation and direction of transcription [Zabidi, MA and Stark, A. TrendsGenet. (2016) 32 (12): 801-814, citing Roeder, RG. Trends in Biochemical Sci. (1996) 21: 327- 335]. Typically, however, core-promoters on their own cannot support efficient transcription and exhibit only low basal activities. Instead, their cell-type-specific activities are typically determined by enhancers, the second key type of transcriptional regulatory elements [Id., citing Spitz, F. Furlong, EEM. Nat. Rev. Genet. (2012) 13: 613-626; Shlyueva, D. et al. Nat. Rev. Genet. (2014) 13: 613-626]. Enhancers are genomic DNA elements of up to several hundred bps in length, which contain short transcription factor (TF) recognition sequences or binding sites. Through these sites, combinations of TFs are recruited to enhancers and in turn recruit cofactors with a variety of biochemical functions [FIG.9, taken from Zabidi, MA and Stark, A. Trends Genet. (2016) 32 (12): 801-814]. Through the combined activating or repressive cues of the different TFs and cofactors, enhancers exert their overall regulatory function to control transcription from target core-promoters irrespective of their orientation and distance [Id., citing Banerji, J. et al. Cell (1981) 27: 299-308]. Since enhancers can act over short and long distances, i.e. their positions with respect to their target core-promoters can be arbitrary, they do not always regulate the nearest gene.

[0366] The term “transcriptional control” as used herein refers to control of gene expression by controlling when and how often a gene is transcribed.

[0367] The term “transcription factor” as used herein refers to a protein required to initiate or regulate transcription in eukaryotes. This includes both gene regulatory proteins as well as the general transcription factors.

[0368] The term “transduction” as used herein refers to a process whereby foreign DNA is introduced into another cell via a viral vector.

[0369] The term “transfection” as used herein refers to the process of introducing a foreign DNA molecule into a eukaryotic cell by nonviral methods.

[0370] The terms “treat” or “treating” include abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristicof the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s).

[0371] The term “trogocytosis” as used herein refers to a cellular process whereby a cell acquires a membrane fragment from another cell in a contact-dependent manner allowing for the transfer of surface proteins with functional integrity. [Mattei, F. et al. Science (2022) 25 (10): 105110; doi.org / 10.1016 / j.isci.2022.105110].

[0372] The term “tumor associated antigen” or “TAA” refers to a protein or other molecule that has elevated levels on tumor cells but that is also expressed at lower levels on healthy cells.

[0373] The term “tumor associated macrophages” or “TAMs” as used herein refers to an immunosuppressive macrophage subtype found in the tumor microenvironment that is involved in the progression and metastasis of cancer. TAMs are broadly considered M2-like, which can be further classified into the M2a phenotype (induced by IL-4 or IL-13), M2b phenotype (IL-10 high, IL-12 low) and M2c phenotype (TNF-α low) according to distinct signal stimuli. They produce abundant growth factors, extracellular matrix (ECM) remodeling molecules and cytokines for the regulation of cancer proliferation via noncoding RNAs, exosomes and epigenetics [Yan, S. and Wan, G. The FEBS Journal (2021) 288 (21): 6174-6186, citing Qian, BZ and Pollard, JW. (Cell (2010) 141: 39-51]. Activated M2 macrophages distinctively express arginase 1 (ARG1). TAMs can demonstrate direct inhibition on the cytotoxicity of T- lymphocytes through multiple mechanisms and characteristics of tumor evolution, including immune checkpoint engagement via expression, production of inhibitory cytokines [such as IL- 10 and transforming growth factor (TGF)-β] and metabolic activities consisting of depletion of l- arginine (or other metabolites) and the production of reactive oxygen species (ROS). The suppressive immune response renders cancer cells capable of escaping from immune surveillance.

[0374] The term “tumor infiltrating lymphocytes” or “TILs” as used herein refers to a heterogeneous lymphocyte population mainly composed of T lymphocytes that may consist of numerous antitumor effector and / or regulatory T cells (Tregs) that have invaded a tumor tissue; TILs are key players in the host’s immune response to a tumor. [Wang, J. et al. BMC Cancer (2020) 20: 731].

[0375] The term “tumor microenvironment” or “TME” as used herein refers to the dynamic and complex ecosystem in which tumor cells exist.

[0376] The term “TME macrophages” as used herein refers to macrophages that arise primarily from bone marrow-derived monocytes that are recruited by tumor or stroma-derived chemokines such as colony-stimulating factor 1 (CSF1; also known as M-CSF) and CCL2, to the tumor microenvironment. M1 and M2 phenotypes are differentiated in response to different signal stimuli and are polarized according to the TME, exhibiting strong plasticity, such that macrophages adopt context-dependent phenotypes when stimulated [Yan, S. and Wan, G. The FEBS Journal (2021) 288 (21): 6174-6186, citing Murry, PJ and Wynn, TA. Nat. Rev. Immunol. (2011) 11: 723-737]. Antitumorigenic M1 macrophages express high levels of tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS; also known as NOS2) and major histocompatibility complex (MHC) class II molecules, whereas pro-tumorigenic M2 macrophages are marked with high levels of arginase 1 (ARG1), interleukin (IL)-10, CD163, CD204 or CD206 expression. The activation of primary macrophages into M1 or M2 phenotype is mainly induced by interferon-regulatory factor / signal transducer and activator of transcription (STAT) signaling pathways [Id., citing Waqas, SFH et al. in Nuclear Receptors: Methods and Experimental Protocols, MZ Badr. Ed., Springer, New York, NY, pp. 211-224].

[0377] The term “tumor specific antigens” or (“TSA”) refers to a protein or other molecule found on cancer cells only.

[0378] The terms “tumorigenesis” “oncogenesis” and “carcinogenesis” are used interchangeably to refer to the transformation of normal cells into cells-of-origin (COOs) and the development of cells-of-origin into tumors.

[0379] The term “Uniform Manifold Approximation and Projection” or “UMAP” as used herein refers to a dimensionality reduction technique that constructs a high dimensional graph representation of data and then creates a low-dimensional graph to be as structurally similar as possible. This results in the creation of two new parameters UMAP 1 and UMAP 2. UMAP captures local relationships within a cluster as well as global relationships between distinct clusters.

[0380] The term “variable (V) domain” as used herein refers to the structural unit of an immunoglobulin or TCR chain that is encoded by the corresponding variable (V) exon.

[0381] The term “variable (V) region” as used herein refers to the highly variable N- terminal portion of an Ig or TCR molecule composed of the variable domain that contains the antigen binding site.

[0382] The term “wild-type” as used herein refers to the typical form of an organism, strain, gene, protein, nucleic acid, or characteristic as it occurs in nature. “Wild-type” refers to the most common phenotype in the natural population. The terms "wild-type" and "naturally occurring" are used interchangeably.

[0383] The term “zeta-chain-associated protein kinase 70” or “ZAP-70” as used herein refers to a non-src family protein kinase that associates with phosphorylated CD3 zeta chain and plays an important role in TCR-CD3 complex signaling. The main substrate of ZAP70 is the transmembrane adaptor LAT.

[0384] Embodiments

[0385] According to one aspect, the present disclosure provides a chimeric antigen receptor (CAR) immunotherapy for treating a hematologic cancer comprising

[0386] (a) genetically modifying a population of immune effector cells comprising T cell receptors (TCRs) to stably express at least one chimeric antigen receptor (CAR), wherein the ectodomain of the CAR specifically binds a cancer antigen;

[0387] (b) expanding the population of CAR-containing immune effector cells in the presence of one or more cytokines in vitro to achieve a therapeutic dose, wherein the population of CAR-containing immune effector cells exhibits a nonexhausted memory T cell phenotype;

[0388] (c) infusing eligible subjects with the CAR-containing immune effector memory T cell phenotype population of cells as needed until the hematologic cancer in the body is destroyed.

[0389] TCRs expressed on the surface of T lymphocytes can only recognize a peptide antigen presented to them through MHCs by antigen-presenting cells (APCs). However, monoclonal antibodies can recognize and bind cell surface-expressed antigens that are not presented by MHCs.

[0390] According to some embodiments, the hematologic cancer is a leukemia, a lymphoma or a myeloma.

[0391] According to some embodiments, the hematologic cancer is a leukemia. Examples include acute lymphoblastic leukemia (also called acute lymphocytic leukemia andALL), acute myelogenous leukemia (also called acute myeloblastic leukemia, acute myeloid leukemia, acute nonlymphocytic leukemia, AML, and ANLL), acute myeloid leukemia with myelodysplasia, acute promyelocytic leukemia (also called APL), chronic eosinophilic leukemia; chronic granulocytic leukemia (also called chronic myelogenous leukemia, chronic myeloid leukemia, and CML), erythroleukemia; mast cell leukemia; and hairy cell leukemia.

[0392] According to some embodiments, the hematologic cancer is a lymphoma. Examples include Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous B cell lymphoma; cutaneous T cell lymphoma and Waldenstrom macroglobulinemia. According to some embodiments, the hematologic cancer is a leukemia / lymphoma.

[0393] According to some embodiments, the hematologic cancer is a myeloma. According to some embodiments, the hematologic cancer is a monoclonal gammopathy of undetermined significance, a smoldering multiple myeloma, or a multiple myeloma (MM). The progression of MM, a B cell malignancy, begins with the precursor pathogenic state of monoclonal gammopathy of undetermined significance (“MGUS”). In the absence of clinical symptoms, MGUS is diagnosed by quantifying the amount of immunoglobulin (Ig) present in both the bloodstream and bone marrow (BM), specifically with a plasma cell population of <10% in the BM. The Ig protein produced by the malignant plasma cell is called a paraprotein, IgM paraprotein, para-IgM or M protein. Evidence indicates that MGUS, previously characterized by myeloma cell growth without bone destruction or other organ involvement, is in fact associated with alterations in the bone. Epidemiologic evidence has shown that patients with MGUS suffer from a significantly increased fracture risk, and that the prevalence of MGUS is increased in patients with osteoporosis. [Fairfield, H. et al., Ann. NY Acad. Sci. (2016) 1364 (1): 32-51, citing Drake, MT. J. Bone Miner. Res. (2014) 29: 2529-33] It has been demonstrated that the onset of MGUS is concurrent with the deterioration of both auxiliary and appendicular microarchitecture leading to skeletal fragility [Id., citing Drake, MT. J. Bone Miner. Res. (2014) 29: 2529-33]. The disease state transitions into either smoldering MM (SMM) or MM (if clinically manifested), with a plasma cell content exceeding 10%, [Fairfield, H. et al., Ann. NY Acad. Sci. (2016) 1364 (1): 32-51., citing Berenson, JR et al. Br. J. Haematol. (2010) 150: 28- 38].

[0394] Smoldering multiple myeloma (SMM) is an intermediate clinical stage between MGUS and MM. It is classified as having high serum or urinary monoclonal protein as well asclonal BM plasma cells in the range of 10–60%, in the absence of additional myeloma-defining events [Id., citing Glavey, SV & Ghobrial, IM. Expert Rev. Hematol. (2015) 8: 273-5] such as hypercalcemia, renal insufficiency, anemia, or bone lesions. [Id., citing Rajkumar, SV et al. Lancet Oncol. (2014) 15: e538-3548]. MGUS progresses to MM at a rate of 1–2% of patients per year; this transition is likely influenced by the presence of mutational diversity or clonality of MM cell populations as well as changes in the local BM and other systemic factors [Id., citing Jemal, A. et al. CA Cancer J. Clin. (2009) 59: 1-25; Pawlyn, C. et al. Blood (2015) 125 (5): 831- 840; Barlogie, B. et al. Blood (2004) 103: 20-32; Rollig, C. et al. Lancet (2014) 385]. MM cells are thought to initially create a plasmacytoma, a single tumor, and then develop into multiple lesions to form the disease of multiple myeloma. [Id., citing Lorsbach, RB et al. Am. J. Clin. Pathol. (2011) 136: 168-182].

[0395] MM results in osteolytic lesions and fractures. In the disease state, bone healing is limited due to increased osteoclastic and decreased osteoblastic activity, as well as an MM- induced forward-feedback cycle where bone-embedded growth factors further enhance tumor progression as bone is resorbed. [Fairfield, H. et al., Ann. NY Acad. Sci. (2016) 1364 (1): 32- 51]. MM is characterized by dissemination of multiple tumor cells throughout the bone marrow (BM). A hallmark of MM is heterogeneous chromosomal aberrations and numerous mutations in a range of genes, both of which make the disease very difficult to target therapeutically. [Id., citing Morgan, GJ et al. Nat. Rev. Cancer (2012) 12: 335-348].

[0396] According to some embodiments, the CAR comprises an extracellular antigen recognition domain, a spacer / hinge region and transmembrane domain, and an intracellular signal transduction domain. According to some embodiments, for each antigen, the extracellular antigen recognition domain comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody specific for that antigen. According to some embodiments, each CAR is specific for a tumor associated antigen or a tumor specific antigen. According to some embodiments, the tumor specific antigen is a tumor neoantigen.

[0397] According to some embodiments, the intracellular signal transduction domain of the CAR comprises 4-1BB. According to some embodiments, the intracellular signal transduction domain comprises a CD3ζ T cell activation chain. According to some embodiments, the intracellular signal transduction domain of the CAR comprises 4-1BB and a CD3ζ T cell activation chain. According to some embodiments, the intracellular signaltransduction domain comprises an engineered TLE3. According to some embodiments, the engineered TLE3 is ectopically expressed. According to some embodiments, the CD3ζ T cell activation domain comprises a bicistronically linked green fluorescent protein (GFP) as an expression marker.

[0398] According to some embodiments, the TLE3 molecule is a wild type TLE3 comprising, in order, an N-terminal Q domain, a GP domain, a CcN domain, an SP domain and a C terminal WDR domain. According to some embodiments, an engineered TLE3 molecule is a truncated TLE3 protein. According to some embodiments, the engineered TLE3 molecule is a C-terminus truncated TLE3 (TLE3ΔWDR) protein.

[0399] According to some embodiments, the target antigen is CD19. According to some embodiments, the target antigen is CD20. According to some embodiments, the target antigen is CD22. According to some embodiments, the target antigen is NKG2D-1. According to some embodiments, the target antigen is CD33. According to some embodiments, the target antigen is BCMA. According to some embodiments, the target antigen is CD123. According to some embodiments, the target antigen is a cancer neoantigen. According to some embodiments, the target antigen comprises a splice-site mutation. According to some embodiments, the target antigen comprises a gene fusion mutation.

[0400] Approved commercial CD19-CAR-T cell products include Kymriah® for treating relapsed and refractory ALL and diffuse large B cell lymphoma (DLBCL), a form of non- Hodgkin lymphoma; Yescarta® (axicabtagene ciloleucel), for treating certain types of large B- cell lymphoma and follicular lymphoma in adults; and Tecartus® (Brexucabtagene autoleucel) for treating adults with mantle cell lymphoma or acute lymphoblastic leukemia.

[0401] According to some embodiments, populations of CAR-containing immune effector cells that target different tumor antigens can be mixed. According to some embodiments, the CAR containing population of immune effector cells recognizes two or more tumor associated antigens simultaneously, for example, CD19 and CD20, CD19 and CD22, CD19 and NKG2D-1; CD19 and CD33, CD19 and BCMA, CD19 and CD123; CD20 and CD22; CD20 and NKG2D-1; CD20 and CD33; CD20 and BCMA, CD20 and CD123; CD22 and CD33; CD22 and BCMA; CD22 and NKG2D-1; CD22 and CD123; etc. According to some embodiments, the anti-cancer specificity of the CAR-containing immune effector cell populationis enhanced by activating the cells in response to cancer antigens while inhibiting the cells in response to HLA-DR. [Fei, F. et al. Molecular Therapy (2022) 30 (3): 1215-1226].

[0402] An optimal CAR will specifically bind an antigen expressed exclusively in tumor cells to form an effective immunological synapse leading to downstream T-cell signaling and a potent and specific anti-tumor effect.

[0403] According to some embodiments, the immune effector cell population engineered to express the CAR comprising a TLE3 construct by lentiviral transduction is a CD3+ T cell population. According to some embodiments, the T cell population comprises CD4+ T cells, CD8+ T cells, or both.

[0404] According to some embodiments, the immune effector cell engineered to express the CAR comprising a TLE3 construct by lentiviral transduction adapted from the CAR-T design is an NK cell.

[0405] NK cells are innate effector lymphocytes that also can exhibit features of memory-like or adaptive response [Valeri, A. et al. Front. Immunol. (2022) 13: 953849, citing [Foley, B. et al. Blood (2012) 119 (11) 2665-2674; Romee, R. et al. Sci. Transl. Med. (2016) 8 (357): 357ra123; Sun JC et al. Nature (92009) 457 (7229): 557-561]. NK cells identify and rapidly discriminate and kill virally infected, stressed, or senescent cells and control several types of tumor cells and metastases [Id., citing Pereira, BI et al. Nat. Commun. (2019) 10 (10): 2387; Lopez-Soto, A. et al. Cancer Cell (2017) 32 (2): 135-154; Shimasaki, N. et al. Nat. Rev. Drug Discovery (2020) 19 (3): 200-18]. Traditionally, human NK cells have been subclassified into immature immunomodulatory NK cells (CD56brightCD16- / dim) and the mature NK cell (CD56dimCD16bright) subset, which mediates the cytolytic function [Id., citing Freud, AG et al. Immunity (2017) 475): 820-33; Melsen, JE, et al. Front. Immunol. (2016) 7: 262].

[0406] NK cells have a short lifespan in vivo and release a different spectrum of cytokines and growth factors (e.g, TNF-α, IFN-γ, GM-CSF, and IL-3) during NK cell killing than do T cells. [Id., citing Zhang, Y. et al. Immunology (2007) 121 (2): 258-265; Kilingemann, H. Oncoimmunology (2014) 3: e28147]. In contrast to T cells, adoptive NK or CAR-NK therapy does not cause serious adverse events, such as on-target off-tumor toxicities, cytokine release syndrome (CRS), or immune effector cell‐associated neurotoxicity syndrome (ICANS), which may increase hospitalization length and raise therapy cost [Id., citing Olson, JA et al. Blood(2010) 115 (21): 4293-4301; Shah, N. et al. Br. J. Haematol. (2017) 177 (3): 457-466; Liu, E. et al. N. Engl. J. Med. (2020) 382 (6): 545-553].

[0407] CAR-NK cells have potential advantages. For example, allogeneic NK products surpass the expensive and lengthy procedure of autologous CAR-T manufacturing [Id., citing Tyagarajan, S. et al. Mol. Ther. Methods Clin. Dev. (2020) 16: 136-144]. Allogenic NK and CAR-NK cells constitute an “off‐the‐shelf” product for immunotherapy that can be applied to different patients and generated from multiple sources [Id., citing Olson, JA, et al. Blood (2010) 115 (21) 4293-4301; Shah, N. et al. Br. J. Haematol. (2017) 177 (3): 457-466; Liu, E. et al. N. Engl. J. Med. (2020) 382 (6): 545-453; Ruggeri, L. et al. Science (2002) 295 (5562): 2097-2100]. This potential arises due to their minimal risk to cause graft‐versus‐host disease (GvHD). Furthermore, NK cells are functionally similar to CD8+ T cells but do not require prior sensitization and lack TCR expression, thereby their responses are not human leukocyte antigen (HLA)-restricted. Instead, NK cell function depends on the balance between activating and inhibitory signaling generated by several germline‐encoded receptors [Id., citing Sivori, S. et al. Cell Mol. Immunol. (2019) 16 (5): 430-441; Xie, G. et al. EBioMedicine (2020) 59: 102975]. NK cells retain CAR-independent killing capacity through these innate receptors even in a tumor escape scenario characterized by CAR antigen loss or down-regulation. NK cells could eliminate tumor cells through CD16-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), direct target killing by cytolytic granules, (e.g. perforin and granzymes), or via engagement of death receptors (e.g. FASL or TRAIL) [Id., citing Prager, I. et al. J. Exp. Med. (2019) 216 (9): 2113-2127]. Additionally, NK cells efficiently produce cytokines and chemokines that modulate other immune mediators of cytotoxicity [Id., citing Dunn, GP et al. Nat. Rev. Immunol. (2006) 6 (11): 836-848].

[0408] CAR-NK immunotherapy is not without drawbacks. For example, NK cell ex vivo expansion can entail fratricide, by which cells recognize receptors or ligands on the surface of their siblings and trigger a cytotoxic activity against them. Because several mechanisms can lead to fratricide during NK or CAR-NK cell expansion, strategies are being developed to avoid fratricidal events that result in lower yields and diminished efficacy of CAR-NK cells in vivo and to prevent host system rejection. Since donor NK cell recognition and rejection by the host immune system may potentially reduce allogenic CAR-NK cell persistence in the clinical setting and low persistence in vivo could cause early relapses due to the disappearance of CAR-NKtherapy, an extension in the effector longevity may be beneficial to boost CAR-NK cell efficacy. Similarly, because a short lifespan limits NK cell proliferation and expansion ex vivo during manufacturing, making it harder to achieve sufficient cell numbers for immunotherapy doses and diminishing the time for NK cell optimization by genetic engineering, an extension in the effector longevity may be beneficial. It is known that tumor cells release soluble ligands that can bind activating and inhibitory receptors expressed in NK cells to promote their dysfunction. For example, soluble NKG2D-L (sNKG2D-L) generated by proteolytic shedding decreases the expression of NKG2D, reducing NK cell antitumor potency and well-known soluble factors present in the TME of most cancers, such as certain interleukins, enzymes, and metabolites impact NK cell effectiveness. [Hilpert, J. et al. J. Immunol. (2012) 189 (3): 1360-1371; Jinushi, M. et al. Proc. Natl Acad. Sci. USA (2008) 105(4): 1285-1290].

[0409] Kong, et al. Front. Immunol. (2024) 15: 1384039 reported the following 36 ongoing CAR-NK clinical trials focusing on hematological malignances:

[0410] Preparation of mononuclear immune effector cells

[0411] Human PBMCs can be isolated from the buffy coat layer of a whole blood sample using Ficoll-Paque® density gradient centrifugation. Specific cell types can be enriched by negative cell selection. For example, according to some embodiments, CD3+ T cells can be isolated by negative selection, e.g., using Miltenyi Pan T-cell Isolation Kit, human (130-096- 535). Non-target cells, i.e., monocytes, neutrophils, eosinophils, B cells, stem cells, dendritic cells, NK cells, granulocytes, and erythroid cells are labeled using a cocktail of biotin-conjugated antibodies. The cocktail contains antibodies against antigens of the non-target cells, e.g., CD14 (macrophages), CD15 (human myeloid cells; granulocytes, macrophages), CD16 (NK cells), CD19 (B cells), CD34 (HSCs, EPCs, ECs), CD36 (macrophages), CD56 (NK cells), CD123 (pDCs, basophils, myeloid progenitors), and CD235a (glycophorin A on RBC precursors). Subsequently, non-target cells are magnetically labelled with the Pan T Cell MicroBead Cocktail. The non-T cells then are retained in a MAXS Column placed in a MACS Separator, while the unlabeled T cells pass through the column and are collected as the enriched, unlabeled T cell fraction.

[0412] Similarly, according to some embodiments, CD16+ CD56+ NK cells can be isolated from human PBMCs by negative selection. Non-target cells, i.e., monocytes, neutrophils, eosinophils, B cells, stem cells dendritic cells, T cells, granulocytes, and erythroid cells are labeled using a cocktail of biotin-conjugated antibodies. The cocktail contains antibodies against CD3 (T cells), CD14 (macrophages), CD15 (human myeloid cells, granulocytes, macrophages), CD34 (HSCs, EPCs, ECs), CD36 (macrophages), CD123 (pDCs, basophils, myeloid progenitors), CD199 (naïve CD8+ T cells), and CD235a (glycophorin A on RBC precursors). Subsequently, non-target cells are magnetically labelled with the Pan T Cell MicroBead Cocktail. The non-NK cells are retained in a MAXS Column placed in a MACS Separator, while the unlabeled NK cells pass through the column and are collected as the enriched, unlabeled NK cell fraction.

[0413] According to some embodiments, the immune effector cell is derived from peripheral blood of the subject. According to some embodiments, the immune effector cell is derived from and is therefore autologous to the subject. According to some embodiments, the immune effector cell is derived from a normal healthy subject and is therefore allogeneic to the subject.

[0414] According to some embodiments, the immune effector cell is derived from umbilical cord blood (UCB). More than 85% of UCB derived T cells have a naive phenotype, allowing them to induce fewer graft versus host disease during transplantation [Kwoczek, J. et al. Transfusion (2018) 58: 88-99]. Moreover, they express significantly lower markers of exhaustion (PD1, LAG3, TIM3) in comparison to PB-derived T cells, allowing them to have long-term persistence and efficiency. [Cael, B. et al., Cancers (2022) 14: 3168, citing Lin, Y. et al. Stem Cell Invest. (2019) 6: 35].

[0415] According to some embodiments, expression of the TLE3ΔWDR construct in CAR-containing immune effector cells enhances CAR-immune effector cell function despite chronic antigen stimulation. According to some embodiments, expression of the TLE3ΔWDR construct in stimulated CAR-containing immune effector cells in vitro promotes activation of genes that promote anti-tumor activity and also represses genes involved in excessive effector function, exhaustion or both. According to some embodiments, ectopic expression of the TLE3ΔWDR construct in stimulated CAR-containing immune effector cells reduces expressionof at least one inhibitory receptor comprising PD-1, CTLA-4, LAG3; TIGIT, or a combination thereof compared to a CAR-immune effector cell that does not overexpress Tle3 (control). According to some embodiments, expression of the CAR-TLE3ΔWDR construct in stimulated CAR-containing immune effector cells induces transcriptomic changes comprising differential expression of genes that enhance the anti-tumor response comprising proliferation of the CAR- containing immune effector cells; enhances persistence of the CAR-containing immune effector cells while maintaining immune homeostasis despite chronic antigen stimulation.

[0416] According to some embodiments, expression of the CAR-TLE3ΔWDR construct in stimulated CAR-containing immune effector cells reprograms the immune effector cells to acquire stem-like, memory cell characteristics including production of IFN and TNF, expression of TCF-1 or both.

[0417] According to some embodiments, expression of the TLE3ΔWDR construct in CAR-containing immune effector cells enhances CAR-immune effector cell function after rechallenge with the cancer, compared to a no-TLE3-CAR immune effector cell control.

[0418] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0419] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials have been described. All publications mentioned herein are incorporated herein by reference to disclose and described the methods and / or materials in connection with which the publications are cited.

[0420] It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural references unless the context clearly dictates otherwise.

[0421] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and each is incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. EXAMPLES

[0422] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0423] Example 1.

[0424] Building on our long-standing interest in Tcf1 / Lef1 and Runx3 TFs in T cells (16- 19), we investigated their cofactors, Transducin-Like Enhancer of split (TLE) proteins, the mammalian homologs of Drosophila Groucho transcriptional corepressor [Turki-Judeh, A. and Courey, J. Curr. Top. Dev. Biol. (2012) 98: 65-96; Buscarlet, M. and Stifani, S. Trends in Cell Biol. (2007) 17: 353-361; Jennings, BH and Ish-Horowicz, D. Genome Biol. (2008) 9: 205; Gasperowicz, M. and Otto, F. J. Cellular Biochem. (2005) 95: 670-687].

[0425] TLE cofactors interact with 1) Tcf / Lef TFs through its N-terminal glutamine-rich Q domain to counter Wnt activity, 2) Runx TFs through its C-terminal WD-repeat (WDR) domain, and 3) Hdac1 through the Gly-Pro-rich GP domain to exert its repressive function[Turki-Judeh, W. and Courey, AJ. Curr. Top. Dev. Biol. (2012) 98: 65-96; Chen, G. et al. Genes & Development (1999) 13: 2218-2230] (FIG. 1A). In mammals, there are 4 TLE genes (TLE1- 4) that encode full-length proteins. In T cells, TLE3 is most abundantly expressed, followed by TLE4 and TLE1. In our previous study [Xing, S. et al. J. Exp. Med. (2018) 215: 2211-2226], we demonstrated that TLE cofactors are differentially partitioned into distinct TF complexes, with TLE-Runx3 complex instructing CD8+ lineage choice during thymic development and TLE- Tcf / Lef complex protecting naïve CD8+ T cell identity, where TLE genes showed redundant and gene-dose effect, with TLE3 exerting a more dominant function.

[0426] Recently, we analyzed TLE genes in CD8+ T cell responses to acute viral infection [Zhao, X. et al. Nature Immunol. (2024) 25: 294-306]. While ablating all three TLE1,3,4 genes abrogates differentiation of effector CD8+ T (Teff) cells, TLE3 shows non- redundant roles in regulating memory CD8+ T (Tmem) lineage stability without affecting Tmemcell pool size. Induced deletion of TLE3 accelerates formation of CD62L+ central Tmem (Tcm) at the expense of CD62L– effector Tmem (Tem) cells. Notably, this effect is not a phenocopy of ablating any of its partner TFs (Tcf1, Runx3 or Tbet), and likely results from specific disruption of TLE3-sensitive TF complexes. On the mechanistic side, key observations include: 1) besides its known corepressor roles, TLE3 functions as a transcriptional coactivator, where TLE3 establishes / maintains chromatin open state to induce its target gene expression; 2) TLE3 engages Tbet as a novel partner TF in Teffand Tmemcells, to form complexes with its known partners such as Runx3) while its expression is relatively stable, TLE3 is dynamically redistributed in Teffand Tmemcell genome, representing a novel means / pattern of gene regulation. We propose that the TLE transcription cofactors exert their unique regulatory roles in T cell biology by integrating the action of multiple TF pathways. Generation of CAR-T cells expressing engineered TLE proteins.

[0427] Building on the concept that TLE cofactors function as TF integrators, we reasoned that gain-of-function for TLE3 may reprogram the fate of activated T cells, boosting Tmem formation, persistence and recall capacity. We further reasoned that molecular engineering of TLE3 may skew partner TF utilization, leading to its function uncoupling and boosting its preferred regulatory effects in CAR-T cells. Therefore, we engineered human TLE3 to make TLE3ΔQ protein that lacks N-terminal Tcf1-interacting Q domain and TLE3ΔWDRprotein that lacks C-terminal Runx3-interacting WDR domain. The domain structure of wild type TLE3 protein and the resulting engineered TLE molecules are shown in FIG.1A. TLE3 utilizes different functional domains to engage distinct partner TFs. The N-terminal side (the Q domain) interacts with TCF-1, the GP domain interacts with Hdac1, and the C-terminal WDR domain interacts with Runx3. The Ccn domain functions in nuclear localization and phosphorylation, the SP domain functions in phosphorylation.

[0428] Using a codon-optimization method, we synthesized CD19-BBz-CAR that consists of anti-hCD19 single-chain variable fragment (scFv), a 4-1BB costimulatory domain and a CD3ζ T cell activation domain along with bicistronically linked green fluorescent protein (GFP) as an expression marker [Id., citing Porter, DL et al. New Engl. J. Med. (2011) 365: 725- 733]. We further engineered the hCD19-CAR to co-express TLE3ΔQ or TLE3ΔWDR via P2A peptide in the same lentiviral expression construct. A schematic of the construct is shown in FIG.1B.

[0429] We then activated T cells from healthy donors using anti-CD3 and anti-CD28 antibodies, followed by lentiviral transduction to express a control CAR without TLE3, TLE3ΔQ or TLE3ΔWDR CAR in T cells as shown schematically in FIG.2A. The CAR-T cells were enriched by sorting for GFP+ cells and further expanded in the present of cytokines including IL- 2, IL-7, and IL-15 to obtain CAR-T cells for in vivo assessment.

[0430] Example 2. Ectopic expression of TLE3-ΔWDR but not TLE3-ΔQ improves leukemia control.

[0431] As shown schematically in FIG.2B, we used these CAR-T cells in a xenograft mouse model. We first inoculated Raji Burkitt’s lymphoma cells i.v. into NSG mice (0.25 million lymphoma cells / mouse), and 3 days later, infused control CAR-T cells or those expressing Tle3ΔQ or Tle3ΔWDR (0.5 million CAR-T cells / mouse).

[0432] The Raji cells express firefly luciferase, allowing tracking of tumor growth in live mice through in vivo imaging. Conventional CAR T cells curtailed tumor growth by 15 day post tumor inoculation (dpt), compared with the no CAR-T group, but did not fully control tumor expansion at later time points (28 dpt). In contrast, TLE3ΔWDR+ CAR-T cells kept the tumors under the detection limit in most cases till 28 dpt, while TLE3ΔQ+ CAR-T cells showed impaired anti-tumor activity (FIG. 2C). By 36 dpt, while recipients of control and TLE3ΔQ+CAR-T cells had succumbed to the lymphoma, most recipients of TLE3ΔWDR+ CAR-T cells survived (FIG. 2D), without overt signs of xeno-GVHD. These data demonstrate that ectopic expression of TLE3ΔWDR but not Tle3ΔQ in CAR-T cells enhanced their anti-tumor activity and conferred robust protection against leukemia growth.

[0433] Example 3. TLE3-ΔWDR promotes CAR-T cell expansion and survival ex vivo.

[0434] To investigate the beneficial effect of ectopic TLE3ΔWDR expression, we first validated TLE3 expression in CAR-T cells by immunoblotting. As shown in FIG. 3A, endogenous TLE3 was detected in both control and TLE3ΔWDR+ CAR-T cells, while the C- terminus-truncated TLE3ΔWDR was only detected in the latter.

[0435] Because CD8+ CAR-T cells outgrew CD4+ CAR-T cells in vivo, especially after TLE3ΔWDR expression (see below), we focused our ex vivo analysis on CD8+ CAR-T cells. Prolonged ex vivo culture of established CAR-T cells showed that TLE3ΔWDR+ CAR-T cells continued to expand over-time, while control CAR-T cells had limited numerical gain (FIG.3B), suggesting that TLE3ΔWDR expression conferred growth advantages to CAR-T cells ex vivo.

[0436] To determine if and how TLE3ΔWDR expression reprograms CAR-T cells on a molecular level, we performed RNA-seq analysis of control and TLE3ΔWDR+ CAR-T cells before and after ex vivo stimulation with Raji cells for 48 hrs.

[0437] To extract reproducible transcriptomic changes induced by TLE3ΔWDR expression, CAR-T cells were generated from two healthy donors, each in 2-3 technical replicates. Principal component analysis (PCA) reduces the number of dimensions in large datasets to principal components that retain most of the original information. It does this by transforming potentially correlated variables into a smaller set of variables, called principal components.

[0438] Principal component analysis showed that ex vivo stimulation with Raji cells causes strong transcriptomic diversification in CAR-T cells, and the differences between control and TLE3ΔWDR+ CAR-T cells were more evident after stimulation in both donors (FIG.3C). By the criteria of ≥1.25 fold changes and false discovery rate <0.1, we identified differentially expressed genes (DEGs) between control and TLE3ΔWDR+ CAR-T cells before and afterstimulation. Upon extracting common DEGs between the two donors, there were higher numbers of DEGs after stimulation than those before stimulation (FIG.3D). TLE3ΔWDR+ CAR-T cells had higher TLE3 expression as expected (FIG.3E, top row).

[0439] Common methods for gene (protein) functional analysis include metabolic signaling pathway analysis and Gene Ontology (GO) analysis. Gene ontology (GO) analysis categorizes gene functions into three major classes: Biological Process (BP), Molecular Function (MF), and Cellular Component (CC). Functional annotation is the process of attaching biological information to sequences of genes or proteins. The basic level of annotation is using sequence alignment tool BLAST for finding similarities, and then annotating genes or proteins based on that. Functional annotation consists of three main steps: identifying portions of the genome that do not code for proteins; identifying elements on the genome, a process called gene prediction; and attaching biological information to these elements. Functional annotation analysis involves annotating genes with GO terms and pathway information.

[0440] Functional annotation analysis showed that TLE3ΔWDR expression induced expression of several transcription regulators including EPAS1 (encoding hypoxia-inducible factor 2α, HIF2α), which promotes anti-tumor activity by tumor-infiltrating CD8+ T cells [Likanan, I. et al. J. Clin. Invest. (2021) 131] (FIG 3E). TLE3ΔWDR induced cell cycle regulators, such as E2F2 that promotes G1-S phase transition [Johnson, DG and Schneier- Broussard, R. Front. Biosci. (1998) 3: d447-448]. and SMACA2 that encodes BRM in SWI / SNF chromatin remodeling complex and contributes to self-renewal of neural stem cells [Gao, F. et al. Molecular Cell (2019) 75: 891-904] (FIG. 3E). The SWI / SNF chromatin remodeling complex functions by displacing nucleosomes near important regulatory sites which can facilitate transcription factor binding and thus promote gene activation in both uni- and multi-cellular eukaryotes [Church, MC and Workman, JL. Biochem. Soc. Trans. (2024) 52 (30: 1327-37, citing [Owen-Hughes, T. et al. Science (1996) 273: 513-16; Cote, J. et al. Science (1994) 265: 53-60; Hirschhorn, JN et al. Genes Dev. (1992) 6: 2288-98; Kruger, W. et al. Genes Dev. (1995) 9: 2770-9; Imbalzano, AN et al. Nature (1994) 370: 481-85; Kwon, H. et al. Nature (1994) 370: 477-81; Wang, W. et al. EMBO J. (1996) 15: 5370-82; Khavari, PA et al. Nature (1993) 366: 170-74; Hodges, C. et al. Cold Spring Harb. Perspect. Med. (2016) 6: a026930]. TLE3ΔWDR also induced several genes in the PI3K pathway, which is intimately linked to a pro-survival effect [Pungsrinont, T. et al. Int. J. Mol. Sci. (2021) 22] (FIG.3E).

[0441] On the other hand, TLE3ΔWDR expression repressed other genes that are involved in apoptosis, effector functions and lipid metabolism (FIG. 3F). Of particular interest was BCL2L11, which encodes BIM proapoptotic protein [Sionov, RV et al. Oncotarget (2015) 6: 23058-23134], which was the sole gene that was repressed in TLE3ΔWDR+ CAR-T cells before stimulation. TBX21 (encoding T-BET) and ID2 promote effector T cell differentiation [Yang, CY et al. Nature Immunol. (2011) 12: 1221-1229; Intlekofer, AM et al. Nature Immunol. (2005) 6: 1236-1244; Kaech, SM and Cui, W. Nat. Rev. Immunol. (2012) 12: 749-761], and their repression by TLE3ΔWDR likely prevented excessive effector function and / or CAR-T cell exhaustion. Collectively, these data suggest that TLE3ΔWDR expression reprogrammed CAR-T cells to gain advantages in proliferation and survival, while maintaining more balanced effector functions. Example 4. TLE3ΔWDR amplifies CAR-T cell responses in vivo:

[0442] To further determine the beneficial effect of ectopic TLE3ΔWDR expression in vivo, we tracked CAR-T cells in the peripheral blood of NSG recipient mice. Following the study design depicted in FIG. 2B, we chose 3 time-windows, including early time point 10-12 days post-tumor transplantation (dpt) when the leukemia burden was similar between recipients of control and TLE3ΔWDR+ CAR-T cells, 20-22 dpt and 28-30 dpt when the leukemia was largely cleared in recipients of TLE3ΔWDR+ CAR-T cells, whereas leukemic cells continued to expand in control CAR-T cells (FIG.2B).

[0443] At 10- and 20-days post-tumor transplantation (dpt), control CAR-T cells were barely detectable, frequently at <0.1% of viable blood cells (FIG. 4A). In contrast, TLE3ΔWDR+ CAR-T cells were more readily detectable in all recipients examined, at approximately 15- and 150-fold higher frequency than control CAR-T cells at 10 and 20 dpt, respectively (FIG.4A). These data validated the robust growth / survival advantages of TLE3ΔWDR+ CAR-T cells in vivo. On 30 dpt, control CAR-T cells became more detectable, likely as a result of responding to persisting stimulation by leukemic cells (FIG. 4A); TLE3ΔWDR+ CAR-T cells were detected at lower frequencies on 30 dpt compared with 20 dpt, suggesting that they were undergoing contraction because of greatly diminished leukemia burden in these recipients (FIG. 4A). Contraction is a physiological phenomenon for T cell responses to bacterial or acute viral infection [Haring, JS et al. Immunity (2006) 25: 19-29].

[0444] These data suggest that TLE3ΔWDR+ CAR-T cells did not undergo uncontrolled expansion in numbers, in spite of the growth / survival advantages over control CAR-T cells and retained the ability of physiological contraction in the absence of antigen stimulation. Collectively, TLE3ΔWDR expression amplified the magnitude and changed the kinetics of CAR-T cell response to leukemia.

[0445] In the CAR-T production process, the CD4+ and CD8+ T cell ratio could vary due to donor-derived differences and / or introduction of engineered TLE3 molecules. To avoid such pre-existing bias, we generated CD4+ and CD8+ CAR-T cells in parallel and mixed those in a 1:1 ratio for recipient infusion (FIG.4A). Control CAR-T cells largely maintained the CD4+ / CD8+ ratio on 20 dpt but showed biased expansion of CD8+ CAR-T cells on 30 dpt (FIG. 4B). In contrast, TLE3ΔWDR+ CAR-T cells exhibited clear bias toward the CD8+ lineage, with an average CD4+ / CD8+ ratio of 1:3 from the early time point, and the CD8+-bias was further amplified over time, showing a CD4+ / CD8+ ratio of 1:5.5 on 20 dpt and that of 1:7 on 30 dpt (FIG.4B). These data indicate that TLE3ΔWDR expression preferentially amplified CD8+ CAR-T cell responses. Example 5. TLE3DWDR ameliorates CAR-T cell exhaustion in vivo:

[0446] CAR-T cell exhaustion is one of the major factors that compromise their anti- tumor activities. We next examined the expression pattern of coinhibitory receptors in CAR-T cells from the spleens of NSG recipients. We first analyzed 12 dpt when the leukemia burden was similar between recipients of control and TLE3ΔWDR+ CAR-T cells. At this timepoint, control CD8+ CAR-T cells did not gain growth advantage over control CD4+ CAR-T cells in the spleens, while TLE3ΔWDR+ CD8+ CAR-T cells were consistently detected at higher numbers than TLE3ΔWDR+ CD4+ and control CD8+ CAR-T cells (FIG.5A). The vast majority of control CD8+ CAR-T cells expressed high level of PD1, with a substantial portion co-expressed TIM3 or LAG3; in contrast, a substantially smaller portion of TLE3ΔWDR+ CD8+ CAR-T cells expressed PD1, and both TIM3 and LAG3 expression were greatly diminished in TLE3ΔWDR+ CD8+ CAR-T cells (FIG.5B, FIG. 5C). On the other hand, TIM3 and LAG3 expression was less pronounced in control CD4+ compared with CD8+ CAR-T cells, and PD1 was expressed at high levels in >50% of CD4+ CAR-T cells; nonetheless, all these coinhibitory receptors showed further reduced expression in TLE3ΔWDR+ CD4+ CAR-T cells (FIG.5B, FIG.5C). Thesedata demonstrate that TLE3ΔWDR prevented excessive induction of co-inhibitory receptors in CAR-T cells during the early response stage.

[0447] To further determine if Tle3ΔWDR suppressed CAR-T cell exhaustion in long- term, we performed similar analysis at a later time window. By 30 dpt, all NSG recipients of control CAR-T cells showed high leukemic burden, while leukemia was below the detection limit in over 80% of recipients of TLE3ΔWDR+ CAR-T cells (FIG.6A). In this context, control CAR-T cells showed continued expansion in response to the high leukemia burden, albeit at varied levels (FIG. 6B). On the other hand, TLE3ΔWDR+ CAR-T cells in 'leukemia-cured' recipients were sustained at a readily detectable level, with CD8+ CAR-T cells showing consistently higher numbers and abundance compared with CD4+ CAR-T cells (FIG.6B).

[0448] Phenotypic analysis further showed that control CAR-T cells continued to express high levels of PD1 and TIM3, with control CD8+ CAR-T cells adopting almost uniform TIM3hiPD1+phenotype, as a result of persisting leukemic antigen (FIG. 6C). In key contrast, TLE3ΔWDR+ CAR-T cells had minimal expression of PD1, TIM3 and LAG3 in both CD8+ and CD4+ lineages (FIG. 6C, FIG. 6D). We also analyzed CAR-T cells in the bone marrow at both time windows and observed similar suppression of exhaustion markers by TLE3ΔWDR expression (data not shown). These data indicate that TLE3ΔWDR had the long-term effect of ameliorating CAR-T cell exhaustion. Example 6. TLE3ΔWDR promotes generation of CAR-T cells with memory T cell characteristics:

[0449] In a productive immune response where antigen is cleared, memory T cells are generated and provide long-term protection Martin, MD and Badovinac, VP. Front. Immunol. (2018) 9: 2692; Jameson, SC and Masopust, D. Immunity (2018) 48: 214-226]. Our previous studies demonstrated that the transcription factor TCF1 is necessary and sufficient to promote memory CD8+ T cell longevity and recall capacity upon rechallenge with the same antigen [Zhou, X. et al. Immunity (2010) 33: 229-240; Shan, Q. et al. Nature Immunol. (2022) 23: 386- 398]. In T cell response to persisting antigens, TCF1 expression demarcates a subset of exhausted T (Tex) cells that has stem-like, self-renewal capacity and expands rapidly in response to immune checkpoint blockade therapy, known as Tex-stem cells [Zhao, X. et al. Nat. Rev. Immunol. (2022) 22: 147-157]. We and others showed that TCF1 is necessary and sufficient forthe Tex-stem cell pool in chronic viral infections and tumor microenvironment [Zhao, X. et al. Nat. Rev. Immunol. (2022) 22: 147-157; (Im, SJ et al. Nature (2016) 537: 417-4212; Siddiqui, I. et al. Immunity (2019) 50: 195-211 e110; Chen, Z. et al. Immunity (2019) 51: 840-855 e845; Shan, Q. et al. Cell Mol. Immunol. (2021) 18: 1262-1277].

[0450] We therefore examined the expression of TCF1 together with Tex-characteristic TOX in CAR-T cells via intracellular staining. On 30 dpt, almost all control and TLE3ΔWDR+ CAR-T cells were TOXhi, consistent with their exposure to persisting antigens (FIG. 7A). Whereas few CD8+ CAR-T cells expressed TCF1, over 1 / 3 of TLE3ΔWDR+ CD8+ CAR-T cells expressed high levels of TCF1 (FIG. 7A). In addition, TCF1+ population was expanded in TLE3ΔWDR+ CD4+ CAR-T cells compared to their control counterparts (FIG. 7A). These observations suggest that TLE3ΔWDR expression reprogrammed CAR-T cells to acquire stem- like, memory T cell characteristics.

[0451] Among memory T cells, IL-2 production is a key feature for central memory T cells which persist longer and show more robust recall response than other memory subsets (Martin, MD and Badovinac, VP. Front. Immunol. (2018) 9: 2692; Jameson, SC and Masopust, D. Immunity (2018) 48: 214-226]. A hall mark for T cell exhaustion is the diminished capacity of poly-cytokine production, showing early failure to produce IL-2, followed by gradual decline in TNF-α and then IFN-γ production [Blank, CU et al. Nat. Rev. Immunol. (2019) 19: 665-674; McLane, LM et al. Annu. Rev. Immunol. (2019) 37: 457-495]. On 30 dpt, both control and TLE3ΔWDR+ CAR-T cells retained the capacity of producing IFN-γ. However, among the IFN- γ-producing CAR-T cells, control CD8+ CAR-T cells partially retained the ability of producing TNF-α but failed to generate IL-2; in contrast, IFN-γ-producing TLE3ΔWDR+ CAR-T cells retained robust capacity of generating both cytokines (FIG. 7B, left panels). On the other hand, while control TLE3ΔWDR+ CD4+ CAR-T cells were high producers of TNF-α, only the latter robustly generated IL-2 (FIG.7B, right panels). As summarized in FIG. 7C, control CAR-T cells were rarely poly-cytokine producers, with approximately 1 / 3 as IFN-γ+TNF-α+ double cytokine producers, while TLE3ΔWDR expression greatly expanded the proportion of double and poly cytokine producers in CAR-T cells. These data collectively demonstrate that TLE3ΔWDR-expressing CAR-T cells acquired central memory T cell characteristics for long- term persistence and better-preserved functionalities.

[0452] Example 7. TLE3-ΔWDR+ CAR-T cells provide long-term protection against leukemia:

[0453] Memory T cells mount robust recall response when re-encountering the same antigen. To test if the memory-like TLE3ΔWDR+ CAR-T cells provided prolonged protection against leukemia, we performed rechallenge assay by injecting 10-20 times higher dose of Raji lymphoma cells over the initial inoculated dose in the primary response (2.5-5 million / mouse). At 36 dpt, only ~1 / 4 of the recipients of control CAR-T cells survived, and the surviving mice all had high leukemia burden (FIG.2C, FIG. 2D). When rechallenged with excess dose of leukemic cells, the mice rapidly succumbed within 5 days (FIG.8A). In contrast, most of the recipients of TLE3ΔWDR+ CAR-T cells showed prolonged survival (FIG.8A). In the rechallenged mice harboring TLE3ΔWDR+ CAR-T cells, leukemia was either below detection limit by in vivo imaging or transiently expanded to detectable levels but was diminished to undetectable levels within 2-3 weeks (FIG.8B). When monitored in peripheral blood, TLE3ΔWDR+ CAR-T cells showed a transient increase in the TIM3hiPD1+ subset, demonstrating that CAR-T cells responded to leukemia restimulation (FIG 8C). It is of interest to note that the TIM3hiPD1+ subset fell back to a similar level at later time points as that before rechallenge (FIG.8C), concordant with leukemia clearance. These observations support the notion that the memory-like TLE3ΔWDR+ CAR-T cells remained highly responsive to secondary challenge and could effectively curtail leukemia relapse.

[0454] Conclusion

[0455] Preventing or reversing T cell exhaustion has been a long sought-after goal, which has profound bearings in improving cancer immunotherapy. Through molecular engineering of the TLE3 transcription cofactor, we identified a TLE3ΔWDR molecule that reprogrammed CAR-T cells and enhanced their anti-tumor activities in a xenograft leukemia model. Ectopic expression of TLE3ΔWDR showed multifaceted beneficial effects on CAR-T cells (FIG.10). The foremost impact was the suppression of multiple coinhibitory receptors, preventing their downstream signaling leading to CAR-T cell exhaustion; this effect was evident during the early stage of an anti-leukemia response, and was further sustained for long-term after the leukemia burden dropped below the detection limit on in vivo imaging. Secondly, TLE3ΔWDR redirected the CAR-T cell along a different path from exhaustion to memory cell lineage, as featured byelevated expression of TCF1 that promotes stem-like self-renewal and longevity, and by retaining the poly-cytokine producing capacity. These memory-like TLE3ΔWDR-expressing CAR-T cells provided long-term protection over leukemia rechallenge and may hence maintain leukemia remission. Thirdly, TLE3ΔWDR conferred on CAR-T cells advantages in proliferation and survival, which allowed their early and sustained expansion upon encountering leukemia cells. The beneficial effects of overexpressing engineered TLE3ΔWDR were more robust than those of overexpressing c-Jun, Foxo1, and Batf3 reported in the literature [Lynn, RC et al. Nature (2019) 576: 293-300; Seo, H. et al. Nat. Immunol. (2021) 22: 983-995; Chan, JD et al. Nature (2024) 629: 201-210; Doan, A.E. et al. Nature (2024) 629: 211-218]. Collectively, our comprehensive functional and molecular analyses demonstrated that TLE3ΔWDR enhances CAR-T cell function in tumor control, long-term persistence, and preventing exhaustion. Our invention represents a novel approach to enhance CAR-T cell function in tumor immunotherapy.

[0456] Example 8. TLE3ΔWDR promotes acquisition of memory T cell features in CAR-T cells in vivo:

[0457] To better define the molecular mechanisms by which TLE3ΔWDR enhances CAR-T cell function, we perform single cell RNA-seq on sort-purified control or TLE3ΔWDR+ CAR-T cells from the spleens of NSG recipient mice on 12 dpt (day post tumor inoculation) when the leukemia burden was similar between recipients of control and TLE3ΔWDR+ CAR-T cells. We took the CITE-seq approach (1) so that each cell type was distinguishable via hashtag antibodies and both cell types were processed in the same reaction for library preparation. This approach avoided batch effect and ensured fair comparison of transcriptomes of control and TLE3ΔWDR+ CAR-T cells. We first focused on CD8+ CAR-T cells, which were resolved into five distinct clusters (FIG.11A). Marker gene analysis revealed that cluster 1 (C1) and C2 cells were proliferative, expressing high levels of MKI67 (encoding Ki-67, a nuclear marker of cell proliferation) (2) and CDK1 (encoding cyclin-dependent kinase 1) (FIG. 11C), which was particularly evident in C1 cells. C3 cells had active expression of genes in the exhaustion program (including TOX and PRDM1 transcription factors, LAG3 and HAVCR3 / TIM3 co-inhibitory receptors) (3) and genes in the effector program (including TBX21 / T-BET, ID2, and RUNX3 transcription factors, GZMB / granzyme B cytotoxic molecule, and KLRG1 and CX3CR1 markers) (4) (FIG.11C), hence named Tex-eff cells. In contrast, C5 cells expressed central memory T (Tcm) cell-characteristic genes, including TCF7 / TCF1, LEF1,and EOMES transcription factors, CCR7, SELL / CD62L and IL7R markers (5, 6) (FIG.11C), hence named Tcm-like cells. On the other hand, C4 cells had modest features of both C3 Tex-effand C5 Tcm-like cells and were thus considered as effector memory T (Tem)-like cells.

[0458] Analysis of CAR-T cell distribution showed that control CAR-T cells were predominantly in C1 and C2 proliferative and C3 Tex-effclusters, with minor contribution to Tem- like C4 cells (FIG. 11B). In contrast, TLE3ΔWDR+ CAR-T cells sustained the pool of C2 proliferative cells, greatly expanded the C4 Tem-like and C5 Tcm-like at the expense of Tex-effcells (FIG. 11B). These data demonstrated that ectopic expression of TLE3ΔWDR resulted in robust molecular rewiring and reprogrammed CAR-T cells to rapidly acquire memory-like features, since the early response stage. References 1. M. Stoeckius et al., Simultaneous epitope and transcriptome measurement in single cells. Nat Methods 14, 865-868 (2017). 2. K. Stamatiou et al., Ki-67 is necessary during DNA replication for fork protection and genome stability. Genome Biol 25, 105 (2024). 3. C. U. Blank et al., Defining 'T cell exhaustion'. Nat Rev Immunol 19, 665-674 (2019). 4. M. Philip, A. Schietinger, CD8(+) T cell differentiation and dysfunction in cancer. Nat Rev Immunol 22, 209-223 (2022). 5. M. D. Martin, V. P. Badovinac, Defining Memory CD8 T Cell. Front Immunol 9, 2692 (2018). 6. X. Zhao, Q. Shan, H. H. Xue, TCF1 in T cell immunity: a broadened frontier. Nat Rev Immunol 22, 147-157 (2022).

[0459] While the present invention has been described with reference to the specific embodiments thereof it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adopt a particular situation, material, composition of matter, process, process step or steps, to the objective spirit and scopeof the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A chimeric antigen receptor (CAR) immunotherapy for treating a hematologic cancer comprising (a) genetically modifying a population of immune effector cells comprising T cell receptors (TCRs) to stably express at least one chimeric antigen receptor (CAR), wherein the ectodomain of the CAR specifically binds a cancer antigen; (b) expanding the population of CAR-containing immune effector cells in the presence of one or more cytokines in vitro to achieve a therapeutic dose, wherein the population of CAR-containing immune effector cells expresses a nonexhausted memory T cell phenotype; and (c) infusing eligible subjects with the CAR-containing immune effector memory T cell phenotype population of cells as needed until all cancer in the body is destroyed.

2. The CAR-immunotherapy according to claim 1, wherein the hematologic cancer is a leukemia, a lymphoma or a myeloma.

3. The CAR immunotherapy according to claim 1, wherein the CAR comprises an extracellular antigen recognition domain, a spacer / hinge region and transmembrane domain, and an intracellular signal transduction domain, wherein the intracellular signal transduction domain of the CAR comprises 4-1BB; a CD3ζ T cell activation chain; and an engineered TLE3 molecule.

4. The CAR immunotherapy according to claim 3, wherein (a) the engineered TLE3 molecule is derived from a wild type TLE3 comprising, in order, an N-terminal Q domain, a GP domain, a CcN domain, an SP domain and a C terminal WDR domain; or(b) the engineered TLE3 molecule is a truncated TLE3 protein; or (c) the engineered TLE3 molecule is a C-terminus truncated TLE3 (TLE3ΔWDR) protein.

5. The CAR immunotherapy according to claim 4, wherein expression of the engineered TLE molecule is ectopic.

6. The CAR immunotherapy according to claim 1, wherein a) the target antigen of the CAR is CD19; b) the target antigen of the CAR is CD20; c) the target antigen of the CAR is CD22; d) the target antigen of the CAR is NKG2D-1; e) the target antigen of the CAR is CD33; f) the target antigen of the CAR is BCMA, or g) the target antigen of the CAR is CD123.

7. The CAR immunotherapy according to claim 6, wherein the CAR-containing population of immune effector cells recognizes two or more tumor associated antigens simultaneously.

8. The CAR immunotherapy according to claim 1 wherein: a) the immune effector cell population engineered to express the CAR comprising a TLE3 construct by lentiviral transduction is a CD3+ T cell population; and b) the T cell population comprises CD4+ T cells, CD8+ T cells, or both.

9. The CAR immunotherapy according to claim 1, wherein the immune effector cell engineered to express the CAR comprising a TLE3 construct by lentiviral transduction adapted from the CAR-T design is an NK cell.

10. The CAR immunotherapy according to claim 1, wherein a) a source of the immune effector cell is peripheral blood or umbilical cord blood; b) the population of immune effector cells is autologous to the subject; or c) the population of immune effector cells is allogeneic to the subject.

11. The CAR immunotherapy according to claim 1, wherein when the engineered TLE3 molecule is a C-terminus truncated TLE3 (TLE3ΔWDR) protein, compared to a no-TLE3 CAR immune effector control, a. expression of the TLE3ΔWDR construct in CAR-containing immune effector cells enhances CAR-immune effector cell function despite chronic antigen stimulation; and / or b. expression of the TLE3ΔWDR construct in stimulated CAR-containing immune effector cells promotes activation of genes that promote anti-tumor activity and also represses genes involved in excessive effector function, exhaustion or both; and / or c. expression of the TLE3ΔWDR construct in stimulated CAR-containing immune effector cells reduces expression of at least one inhibitory receptor comprising PD-1, CTLA-4, LAG3; TIGIT, or a combination thereof; and / or d. expression of the CAR-TLE3ΔWDR construct in stimulated CAR-containing immune effector cells induces transcriptomic changes comprising differential expression of genes that enhance the anti-tumor response comprising proliferation of the CAR-containing immune effector cells; enhances persistence of the CAR-containing immune effector cells and maintains immune homeostasis despite chronic antigen stimulation; and / or e. expression of the CAR-TLE3ΔWDR construct in stimulated CAR-containing immune effector cells reprograms the immune effector cells to acquire stem-like, memory cell characteristics including production of IFN and TNF, expression of TCF-1 or both, and / or f. expression of the TLE3ΔWDR construct in CAR-containing immune effector cells enhances CAR-immune effector cell function after rechallenge with the cancer.

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