Use of LMO4 to bolster stemness and antitumor efficacy of t-cells

Abstract

The present invention relates to the identification of LMO4 as a key regulator for augmenting stem- like behavior in CD8-positive T cells. Ectopic overexpression of LMO4 increases stem-like T-cell numbers, thereby enhancing polyfunctionality, recall capacity, and antitumor immunity. In respective models, the overexpression of LMO4 resulted in enhanced tumor clearance and prolonged survival.

Description

[0001] Use of LMO4 to bolster sternness and antitumor efficacy of T-cells

[0002] Statement regarding funding

[0003] This project was supported by the National Heart, Lung, and Blood Institute, National Institutes of Health Intramural Research Program (support to Warren J. Leonard, M.D.). The Government of the United States has certain rights in the invention.

[0004] Background

[0005] T cell-based therapies are transforming medical oncology, treating previously incurable hematologic cancers, but their efficacy against solid tumors remains disappointing. Preclinical models and retrospective analyses of T-cell therapies have emphasized the crucial role of stem-like memory T cells in mediating successful antitumor responses. Therefore, significant research endeavors have been directed toward unraveling the pathways regulating the formation of these adult stem cells (Nat Rev Cancer 12, 671-684 (2012)). Knockout studies have been instrumental in identifying key transcriptional regulators that physiologically support long-term immune responsiveness and memory function such as c-MYB (Nat Immunol 20, 337-349 (2019)), T cell factor 1 (TCF1; Proc Natl Acad Sci U S A 107, 9777- 9782 (2010)), forkhead box protein 01 (FOXO1; J Exp Med 210, 1189-1200 (2013)), BTB Domain And CNC Homolog 2 (BACH2; Nat Immunol 17, 851-860 (2016)), DNA-binding protein inhibitors ID3 (Nat Immunol 12, 1230-1237 (2011)), and signal transducer and activator of transcription 3 (STAT3; Immunity 35, 792-805 (2011)). Synthetic biology offers the opportunity to impart new functions to a cell or to enhance desired features through genetic manipulation of genes that do not normally operate in a specific cell under physiological conditions. To this end, CRISPR activation (CRISPRa) gain- of-function screens are powerful tools for uncovering genes that might not be active under normal conditions but have the potential to promote phenotypes of interest. Herein, by conducting a CRISPRa screen analysis, we identified LMO4 among the top transcriptional regulators that can be harnessed to augment stem-like behavior in CD8-positive T cells.

[0006] LMO4 belongs to the family of LIM-domain-only (LMO) proteins, which are transcriptional coregulators known to play pivotal roles in the processes of cell fate determination, cell growth and differentiation, and organ development (Trends Genet 14, 156-162 (1998)). Much of our understanding of LMO proteins in T cells has been derived from studies focusing on their oncogenic properties (Nat Rev Cancer 13, 111-122 (2013)). For instance, LMO1 and LMO2 were initially identified due to their involvement in translocations associated with acute T cell leukemia (T-ALL). LMO4, on the other hand, has been demonstrated to play an essential role in normal thymus development (Dev Dyn 239, 1988-1994 (2010)). The co-expression of LMO4 with FEZF2 was also reported to be useful for the reprogramming of neuronal cells (W02017 / 081033). LMO4 is the only LMO family member expressed in mature post-thymic T cells (Proc Natl Acad Sci U S A 95, 11257-11262 (1998)), but the expression is very low when compared to innate immune cell populations, opening a window to exploiting its therapeutic potential through synthetic biology.

[0007] Here, it is demonstrated that LMO4 can be repurposed in CD8-positive T cells to augment their sternness. Instead of directly influencing gene transcription, LMO4 primarily modulates cytokine signaling, specifically related to interleukin-21 (IL-21). IL-21 is a pleiotropic cytokine with broad actions including promoting T follicular helper cell (Nat Rev Drug Discov 13, 379-395 (2014)) and terminal B cell differentiation (Science 298, 1630-1634 (2002)), but it is also known to cooperatively expand CD8- positive T cells (J Exp Med 201, 139-148 (2005)) and to promote adoptive transfer-mediated antitumor activity (Proc Natl Acad Sci U S A 117, 6047-6055 (2020); Blood 111, 5326-5333 (2008)). By boosting the IL-21-STAT3 axis, LMO4 promotes the expression of key memory-related factors to enhance the formation of memory T cells while curbing their terminal differentiation. Lastly, the therapeutic effect of LMO4 is demonstrated: enforcing LMO4 expression in adoptively transferred CD8-positive T cells significantly boosts CD8-positive T-cell antitumor immunity, resulting in enhanced tumor clearance and prolonged survival of tumor-bearing animals.

[0008] Summary of the invention

[0009] The present disclosure relates to a recombinant T cell overexpressing a recombinant nucleic acid encoding a functional LMO4 polypeptide for use in the treatment of a cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant T cell overexpressing a recombinant nucleic acid encoding a functional LMO4 polypeptide compared to a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, said functional LMO4 polypeptide is ectopically expressed. In certain embodiments, the present disclosure relates to a recombinant T cell ectopically overexpressing a recombinant nucleic acid encoding a functional LMO4 polypeptide compared to a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant T cell transgenically overexpressing a recombinant nucleic acid encoding a functional LM04 polypeptide compared to a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, said recombinant nucleic acid is operably linked to a promoter. In certain embodiments, said recombinant nucleic acid is encoded on an expression vector, preferably a viral vector or a plasmid. In certain embodiments, said expression vectors triggers the overexpression of said recombinant nucleic acid encoding said functional LM04 polypeptide. In certain embodiments, said recombinant nucleic acid encoding said functional LM04 polypeptide is different from a genomic copy of LM04 in said host cell. In certain embodiments, said functional LM04 polypeptide comprises the amino acid sequence of SEQ ID No. 1. In certain embodiments, said recombinant nucleic acid encoding the functional LM04 polypeptide comprises the nucleic acid sequence of SEQ ID No. 2.

[0010] The present disclosure also relates to a recombinant T cell overexpressing a recombinant nucleic acid encoding a functional LM04 polypeptide for use in the treatment of a cancer or a tumor, wherein said T cell further comprises a nucleic acid encoding a chimeric antigen receptor or a T cell receptor. In certain embodiments, said T cell receptor is specific for a cancer antigen. In certain embodiments, said cancer or tumor is a solid cancer or a hematological cancer.

[0011] The present disclosure also relates to a recombinant T cell overexpressing a recombinant nucleic acid encoding a functional LM04 polypeptide for use in the treatment of a cancer or a tumor, wherein said recombinant T cell is a CD8-positive T cell. In certain embodiments, said T cell is an isolated or purified T cell, preferably an isolated or purified human T cell.

[0012] The present disclosure also relates to a population of cells comprising at least one recombinant T cell according as disclosed herein.

[0013] The present disclosure also relates to a composition comprising at least one recombinant T cell or a population of cells as disclosed herein, and a pharmaceutical carrier.

[0014] Definitions

[0015] The term "LM04" as used herein refers to a cysteine-rich protein that contains two LIM domains but lacks a DNA-binding homeodomain. It is also known as '"LMO-4", "LIM Domain Only 4", "LIM Domain Only Protein 4" or "LIM-Only 4 Protein". The amino acid sequence of LM04 is as follows (UniProt: P61968): MVNPGSSSQPPPVTAGSLSWKRCAGCGGKIADRFLLYAMDSYWHSRCLKCSCCQAQ LGDIGTSCYTKSGMILCRNDYIRLFGNSGACSACGQS IPASELVMRAQGNVYHLKC FTCSTCRNRLVPGDRFHYINGSLFCEHDRPTALINGHLNSLQSNPLLPDQKVC ( SEQ ID No . 1 )

[0016] The nucleic acid encoding LMO4 used in this study has the following nucleic acid sequence:

[0017] ATGGTGAATCCGGGCAGCAGCTCGCAGCCGCCCCCGGTGACGGCCGGCTCCCTCTC CTGGAAGCGCTGCGCAGGCTGCGGGGGCAAGATCGCGGACCGCTTTCTGCTCTATG CCATGGACAGCTACTGGCATAGCCGCTGCCTCAAGTGCTCCTGCTGCCAGGCGCAG CTGGGCGACATTGGCACGTCCTGTTACACCAAGAGCGGCATGATCCTTTGCAGAAA TGACTACATTAGGTTATTTGGGAATAGCGGTGCTTGCAGTGCCTGTGGACAGTCGA TTCCTGCAAGTGAGCTCGTCATGAGGGCCCAAGGCAACGTGTATCATCTCAAGTGT TTCACATGTTCTACCTGCCGGAATCGCCTGGTCCCGGGAGATCGGTTTCACTACAT CAATGGCAGTTTATTTTGTGAACATGATAGACCCACAGCCCTCATCAATGGCCATT TGAATTCACTTCAGAGCAATCCACTACTGCCAGACCAGAAGGTCTGCGC ( SEQ ID No . 2 )

[0018] As will be appreciated, any nucleic acid sequence encoding for a functional LMO4 polypeptide may be used within the spirit of the inventions. This includes codon-optimized versions of the nucleic acids sequence of SEQ ID No. 2, as well as truncated, optimized or otherwise modified nucleic acid sequences encoding a functional LM04 polypeptide.

[0019] The term "cell" as used herein refers to a single cell or a plurality of cells.

[0020] The term "host cell" as used herein refers to a cell comprising a nucleic acid and / or a vector. In the context of the present disclosure, the term host cell refers to a cell comprising a nucleic acid and / or a vector encoding a functional LM04 polypeptide. Such host cell will express the functional LM04 polypeptide and is suitable to be used in medicine. Preferred host cells of the present invention are eukaryotic host cells, such as immune cells. Particularly preferred host cells are T cells, more particularly CD8-positive T cells.

[0021] The terms "immune cell" as used herein refers to any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., an autoantigen). In various embodiments, an immune cell is, e.g., a T cell, a B cell, a dendritic cell, a monocyte, a natural killer cell, a macrophage, Langerhan's cells, or Kupffer cells. Preferred immune cells are T cells, such as CD8-positive T cells.

[0022] The term "T cell" as used herein refers to a type of lymphocyte that plays a central role in cell- mediated immunity. T cells, also referred to as T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor (TCR) on the cell surface. There are several subsets of T cells with distinct functions, including but not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells and natural killer T cells. In certain embodiments, the T cell is an engineered T cell. In other embodiments, the T cell is a CD8-positive T cell. In yet other embodiments, the T cell is a CAR-T cell.

[0023] The terms "CD8" or "cluster of differentiation 8" as used herein refer to a transmembrane glycoprotein (UniProt: P01732) that is present on certain T cells and serves as a co-receptor for the T- cell receptor (TCR). Along with the TCR, the CD8 co-receptor plays a role in T cell signaling and aiding with cytotoxic T cell-antigen interactions. T cells expressing CD8 are referred to as "CD8-positive T cells" or "CD8+ T cells".

[0024] The term "CAR" or "chimeric antigen receptor" as used herein refers to an artificial cell surface receptor that is designed to bind to certain proteins on cells, for example cancer cells. When certain CARs are expressed by a T cell, binding of the CAR extracellular binding moiety with a target antigen can activate the T cell. CARs are also known as chimeric T cell receptors or chimeric immunoreceptors. Typical CARs comprise (i) an extracellular domain that includes a moiety that binds a target antigen; (ii) a transmembrane domain; and (ill) an intracellular signaling domain that sends activating signals when the CAR is stimulated by binding of the extracellular binding moiety with a target antigen.

[0025] The term "CAR-T cell" as used herein refers to a T cell that has been engineered to express a CAR.

[0026] The terms "T cell receptor" or "TCR" as used herein refer to a complex of integral membrane proteins that participates in the activation of T cells in response to the binding of an antigen. The TCR is a d isu If ide-l i n ked membrane-anchored heterodimer normally consisting of the highly variable alpha and beta chains expressed as the part of a complex with the invariant CD3 (cluster of differentiation 3) chain molecules.

[0027] The terms "polynucleotide" and / or "nucleic acid sequence" and / or "nucleic acid" as used herein refer to a sequence of nucleoside or nucleotide monomers consisting of bases, sugars and intersugar (backbone) linkages. The term includes DNA and RNA and can be either double stranded or single stranded and represents the sense or antisense strand. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil, and xanthine and hypoxanthine. The nucleic acids of the present disclosure may be isolated from biological organisms, formed by laboratory methods of genetic recombination or obtained by chemical synthesis or other known protocols for creating nucleic acids.

[0028] The terms "isolated polynucleotide" or "isolated nucleic acid sequence" as used herein refer to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.

[0029] The terms "recombinant nucleic acid" or "engineered nucleic acid" as used herein refer to a nucleic acid or polynucleotide that is not found in a biological organism. For example, recombinant nucleic acids may be formed by laboratory methods of genetic recombination (such as molecular cloning) to create sequences that would not otherwise be found in nature. Recombinant nucleic acids may also be created by chemical synthesis or other known protocols for creating nucleic acids. Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present application herein described for which they are suitable as would be understood by a person skilled in the art.

[0030] The term "polypeptide" or "protein" as used herein describes a chain of amino acids. A polypeptide or protein of this disclosure can be a peptide, which usually describes a chain of amino acids of from two to about 30 amino acids. The term protein as used herein also describes a chain of amino acids having more than 30 amino acids and can be a fragment or domain of a protein or a full-length protein. Furthermore, as used herein, the term protein can refer to a linear chain of amino acids or it can refer to a chain of amino acids that has been processed and folded into a functional protein. It is understood, however, that 30 is an arbitrary number with regard to distinguishing peptides and proteins and the terms can be used interchangeably for a chain of amino acids. The proteins of the present disclosure can be obtained by isolation and purification of the proteins from cells where they are produced naturally, by enzymatic (e.g., proteolytic) cleavage, and / or recombinantly by expression of nucleic acid encoding the proteins or fragments of this disclosure. The proteins and / or fragments of this disclosure can also be obtained by chemical synthesis or other known protocols for producing proteins and fragments.

[0031] The term "isolated polypeptide" refers to a polypeptide substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.

[0032] The term "vector" as used herein refers to a polynucleotide that can be used to deliver a nucleic acid to the inside of a cell. In one embodiment, a vector is an expression vector comprising expression control sequences (for example, a promoter) operatively linked to a nucleic acid to be expressed in a cell. Vectors known in the art include, but are not limited to, plasmids, phages, cosmids, and viruses. The terms "ectopic expression", "ectopically expressed" or similar refers to a nucleic acid that is or can be expressed in a cell or a cell type other than the cell or the cell type in which it normally is expressed, or at a time other than the time at which it normally is expressed.

[0033] The terms "recipient", "individual", "subject", "host", and "patient", are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.

[0034] As used herein, the terms "treatment," "treating," and the like, in some embodiments, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of affecting a partial or complete cure for a disease and / or symptoms of the disease. The terms include treatment of a disease or disorder (e.g. cancer) in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease); (b) inhibiting the disease (i.e., arresting its development); and (c) relieving the disease (i.e., causing regression of the disease). The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with diseases (e.g., inflammation).

[0035] The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0036] The terms "signaling domain" or "intracellular signaling domain" in the context of a CAR-T cell refers to the intracellular domain of the CAR which transduced the activation signal. The signaling domain may be an effector domain that can directly or indirectly promote a biological or physiological response in a cell when receiving the appropriate signal. An effector domain may directly promote a cellular response. An effector domain may also indirectly promote a cellular response by associating with one or more other proteins that promote a cellular response, such as co-stimulatory domains. Effector domains can provide for activation of at least one function of a modified cell upon binding to the cellular marker expressed by a cancer cell. Activation of the modified cell can include one or more of differentiation, proliferation and / or activation or other effector functions. In particular embodiments, an effector domain can include an intracellular signaling component including a T cell receptor and a co-stimulatory domain which can include the cytoplasmic sequence from co-receptor or co- stimulatory molecule. An effector domain can include receptor signaling domains, intracellular signaling components (e.g., cytoplasmic signaling sequences), co-stimulatory domains, or combinations thereof. Exemplary effector domains include signaling and stimulatory domains selected from: 4-1BB (CD137), CARD11, CD3y, CD35, CD3s, CD3^, CD27, CD28, CD79A, CD79B, DAP10, FcRa, FcRp (FcsRIb), FcRy, Fyn, HVEM (LIGHTR), ICOS, LAG 3, LAT, Lek, LRP, NKG2D, NOTCH1, pToc, PTCH2, 0X40, ROR2, Ryk, SLAMF1, Slp76, TCRoc, TCRp, TRIM, Wnt, Zap70, or any combination thereof. Exemplary effector domains include signaling and co-stimulatory domains selected from: CD86, FcyRlla, DAP12, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8oc, CD8P, IL2RP, I L2Ry, IL7Roc, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, ITGAM, CDllb, ITGAX, CDllc, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB- A, Lyl08), SLAM (CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, PAG / Cbp, NKp44, NKp30, or NKp46.

[0037] The terms "is", "are", "is derived from" and "are derived from" in the context of a polypeptide or domain of a polypeptide refers to the amino acid sequence of said polypeptide or domain of a polypeptide and indicates that the amino acid sequence is either identical to the native version of said polypeptide or domain of a polypeptide, or a variant of said polypeptide or domain of a polypeptide which is functionally indistinguishable form from the native version of said polypeptide or domain of a polypeptide.

[0038] The terms "recombinant" as used herein refers to molecules or cells that are prepared, generated or created by recombinant means, such as genetic engineering or molecular biological technologies. Recombinant molecules do not occur naturally in nature. For example, a recombinant polypeptide or a recombinant nucleic acid refer to a polypeptide (or nucleic acid) which has been modified or which has been put into another context (e.g. by cloning it behind certain regulatory elements, such as a promoter) as compared to the respective wild-type molecule. A recombinant immune cell, such as a recombinant T cell, comprises a recombinant polypeptide and / or a recombinant nucleic acid.

[0039] The term "cancer" as used herein it its broadest sense refers to diseases in which abnormal cells divide without control.

[0040] The term "hematological cancer" as used herein refers to a cancer of the blood, and includes leukemia, lymphoma and myeloma among others. "Leukemia" refers to a cancer of the blood in which too many white blood cells that are ineffective in fighting infection are made, thus crowding out the other parts that make up the blood, such as platelets and red blood cells. It is understood that cases of leukemia are classified as acute or chronic. Certain forms of leukemia include, by way of non-limiting examples, acute lymphocytic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenous leukemia (CML); Myeloproliferative disorder / neoplasm (MPDS); and myelodysplasia syndrome. "Lymphoma" may refer to a Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell), among others. Myeloma may refer to multiple myeloma (MM), giant cell myeloma, heavychain myeloma, and light chain or Bence-Jones myeloma.

[0041] The term "solid tumor" or "solid cancer" as used herein refers to tumors that usually do not contain cysts or liquid areas. Solid tumors as used herein include sarcomas and carcinomas, such as e.g. breast tumors, ovarian tumors, gastric tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, thyroid tumors non-small-cell lung cancer (NSCLC) and kidney tumors.

[0042] The terms "pharmaceutical carrier" or "pharmaceutically acceptable carrier", as used herein refer to an excipient, carrier, or adjuvant that can be administered to a subject, together with at least one therapeutic agent, and that is generally safe, non-toxic, and has no effect on the pharmacological activity of the therapeutic agent.

[0043] Figure legends

[0044] Figure 1 shows a Volcano plot displaying median sgRNA log2-fold change (I FN-yhl / l° sorting bin counts) for each gene tested in the genome-wide CRISPRa screen. Genes included in the analysis were exclusively transcription factors or transcriptional regulators.

[0045] Figure 2 shows a heatmap depicting the expression levels (assessed by RNA-seq) of the top negative hits identified in the CRISPRa screen in CD8-positive T cells cultured for 4 d under different conditions: No Cytokine (NC), IL-2, IL-2 + LDHi, IL-21, IL-21 + LDHi.

[0046] Figure 3 shows that priming naive CD8-positive T cells in the absence of TWS119 profoundly downregulates LM04, whereas LM04 levels are mildly increased when the GSK-3 inhibitor is added to the culture medium. q-PCR of Lmo4 mRNA in CD8-positive T cells cultured for 72 h with or without TWS119.

[0047] Figure 4 shows the experimental design to assess the impact of LM04 overexpression on stem-like CD8-positiveT cell formation. Figure 5 shows an immunoblot of LMO4 in Thyl.l and Lmo4-Thyl.l overexpressing T cells. ACTB served as control.

[0048] Figures 6 and 7 show the flow cytometry analysis (Figure 6) and quantification (Figure 7) of splenic pmel-1 CD8-positiveT cells following transfer of either 1 x 105pmel-1 Ly5.1+Thyl.l+or Lmo4-Thyl.l+CD8-positive T cells into wild-type mice infected with gplOO-vv. Assessment was conducted at various time points from 3 to 30 days post-transfer, with three mice per group for each time point. Data are from one representative of 3 experiments.

[0049] Figures 8 and 9 show the flow cytometry analysis (Figure 8) and percentages (Figure 9) of CD62L KLRG1+splenic pmel-1 T cells 5 d after transfer.

[0050] Figures 10 and 11 show the flow cytometry analysis (Figure 10) and percentages (Figure 11) of CD62L KLRG1+splenic pmel-1 T cells 30 d after transfer.

[0051] Figure 12. Panel A shows a UMAP plot of concatenated Thyl.l+and Lmo4-Thyl.l+pmel-1 CD8+T cells isolated from spleens 5 d after treatment, showing the distribution of clusters (Cl) identified by FlowSOM. Panel B shows a "Parts of whole" plot of concatenated Thyl.l* and Lmo4-Thyl.l* pmel-1 CD8-positive T cells isolated from spleens 5 d after treatment highlighting more precisely the distribution of clusters assessed by FlowSOM. *P < 0.05, **P < 0.01, (unpaired two-tailed Student's t- test).

[0052] Figure 13 shows the experimental design investigating the impact of LMO4 overexpression on CD8- positive T cell secondary responses.

[0053] Figure 14 shows the flow cytometry analysis of splenic pmel-1 CD8-positive T cells following transfer of either 1 x 105pmel-1 Ly5.1+Thyl.l* or Lmo4-Thyl.l* CD8+T cells into wild-type mice infected with gplOO-vv (primary infection) and gplOO-Adv (secondary infection). Assessment was conducted at various time points: d30 after primary infection and d5 or d30 after secondary infection (recall); with seven mice per group for d30 and three mice per group for d5 recall and d30 recall.

[0054] Figure 15 shows of the percentage analysis of the flow cytometry data shown in Figure 14. Panel A: d30 after primary infection, Panel B: d5 after secondary infection (recall), Panel C: d30 after secondary infection (recall).

[0055] Figure 16 shows the flow cytometry analysis of pmel-1 T cells 5 d after transfer as described in Example 4.

[0056] Figure 17 shows of the percentage analysis of the flow cytometry data shown in Figure 16. Panel A: CD62L KLRG1+splenic pmel-1 T cells, Panel B: CD62L+KLRG1 splenic pmel-1 T cells. Figure 18. Panel A shows a UMAP plot of concatenated Thyl.l+and Lmo4-Thyl.l+pmel-1 CD8+T cells isolated from spleens 30 d after secondary transfer showing the distribution of clusters (Cl) identified by FlowSOM. Panel B shows a "Parts of whole" plot of concatenated Thyl.l+and Lmo4-Thyl.l+pmel- 1 CD8+T cells isolated from spleens 5 d after treatment highlighting more precisely the distribution of clusters assessed by FlowSOM. *P < 0.05, **P < 0.01, (unpaired two-tailed Student's t-test).

[0057] Figure 19 shows the experimental design investigating the effect of Lmo4 overexpression on the antitumor immune response of CD8-positive T cells.

[0058] Figures 20 and 21 show the tumor size (Figure 20) and survival curve (Figure 21) of B16KVP tumorbearing wild-type mice after transfer of either 3.5 x 105pmel-1 Thyl.l+or Lmo4-Thyl.l+CD8+T cells into wild-type mice treated with gplOO-vv and IL-2. *P< 0.05 (Figure 20: Wilcoxon rank sum test; Figure 21: log-rank (Mantel-Cox) test).

[0059] Figure 22 shows a Volcano plot displaying changes in gene expression between pmel-1 Thyl.l+and Lmo4-Thyl.1+CD8+T cells. Gene expression was evaluated by RNA-seq of pmel-1 CD62L KLRG1 T cells collected 5 d after transfer of 1 x 105pmel-1 Thyl.l+and Lmo4-Thyl.l+CD8+T cells into wild-type mice infected with gplOO-vv (five mice per group).

[0060] Figure 23 shows the results of Gene Set Enrichment Analysis (GSEA). Genes upregulated in response to IL-21 in CD4-positive T cells are shown in the left panel and genes displaying one or more STAT3 binding motifs (GSEA C3:STAT3_02) on the right panel.

[0061] Figure 24 shows an immunoblot of pSTAT3 and STAT3 (control) in Thyl.l and LMO4-Thyl.l overexpressing T cells at 0, 0.5 and 2h after adding IL-6 (left panel), IL-10 (middle panel) or IL-21 (right panel) to the cell culture.

[0062] Figure 25 shows results of a q-PCR of Tcf7 (left panel), Zfp36 (middle left panel), Socs3 (middle right panel) and Junb (right panel) mRNA in Lmo4-Thyl.l relative to Thyl.l overexpressing T cells that were cultured for 16 h with IL-6 or IL-21.

[0063] Figure 26 shows the experimental design to assess the impact of Stat3 versus Thyl.2 (control) deletion on LMO4-induced stem-like T cell formation.

[0064] Figure 27 shows flow cytometry histograms showing Thyl.2 (left panel) and STAT3 (right panel) to assess knockout efficiency compared to controls in pmel-1 CD8-positive T cells.

[0065] Figure 28 shows a flow cytometry analysis (of splenic pmel-1 CD8-positive T cells 5 d after transfer of either 1 x 105pmel-1 Ly5.1+ Thyl.l+Thyl.2KO, Thyl.l+StatSKO, Lmo4-Thyl.l+Thyl.2KO or Lmo4- Thyl.l+Stat3KO CD8-positive T cells into wild-type mice infected with gplOO-vv (five mice per group). Figure 29 shows the analysis of the flow cytometry data shown in Figure 28 (unpaired two-tailed Student's t-test).

[0066] Figure 30 shows a flow cytometry analysis of CD62L+CD44+ splenic pmel-1 T cells 30 d after transfer.

[0067] Figure 31 shows the percentage analysis of the flow cytometry data shown in Figure 30 (unpaired two- tailed Student's t-test).

[0068] Figures 32 and 33 show the tumor size (Figure 32) and survival curve (Figure 33) of B16KVP tumorbearing wild-type mice after transfer of either 3.5 x 105pmel-1 Thyl.l+Thyl.2KO, Thyl.l+Stat3KO, Lmo4-Thyl.l+Thyl.2KO or Lmo4-Thyl.l+Stat3KO CD8+ T cells into wild-type mice treated with gplOO- vv and IL-2. *P < 0.05, **P < 0.01, (Figure 32: Wilcoxon rank sum test; Figure 33: log-rank (Mantel-Cox) test).

[0069] Figure 34 shows that LMO4 is poorly expressed in human CD8-positive T cells. Violin plots depicting the expression levels of LMO4, TCF7, and PRDM1 in (a) healthy donors (10X genomic database) or (b) HNSCC patients.

[0070] Figure 35 shows the experimental design investigating the effect of LMO4 overexpression on the antitumor immune response of OT-1 CD8- positive T cells.

[0071] Figure 36 and 37 shows the tumor size (Figure 36) and survival (Figure 37) of LLCl-OVA-bearing mice, after transfer of 106-Thyl.l or Thyl.l transduced OT-1 T cells into lymphodepleted mice and subsequent vaccination with OVA peptide-loaded dendritic cells (n = 9-10 mice / group).

[0072] Figure 38 shows the percentage of TSCM cells in LMO4-Thyl.l and Thyl.l transduced CD8+ T cells after activation by TransAct and subsequent culture in IL-7 and IL-21 for 7 days (n = 5).

[0073] Figure 39 shows the experimental design investigating the effect of LMO4 overexpression on the antitumor immune response of human CD19-CAR-modifiedCD8+ T cells (n = 5 to 7 mice / group).

[0074] Figure 40 shows the percentage of human CD8+ T cells in the peripheral blood of NXG mice bearing NALM6-GL leukemia 7 days after adoptive transfer of LMO4- Thyl.l or Thyl.l CD19-CAR CD8+ T cells in conjunction with recombinant human IL-15.

[0075] Figure 41 shows survival of NALM6-GL-bearing NXG mice treated as indicated in Figure 40 (*P < 0.05 g, unpaired one-tailed Student's t-test, i, P = 0.0597 log-rank (Mantel-Cox test).

[0076] Figure 42 shows Venn diagram illustrating the overlap between pathways significantly upregulated in LMO4-Thyl.l+ and down-regulated inMybfl / flpmel-1 CD8-positive+ T cells, generated under identical experimental conditions. Embodiments of the invention

[0077] Strategies to enhance the sternness and persistence of antitumor T cells are actively pursued given the central role of stem-like cells in eliciting productive antitumor responses. Several pathways and relevant players influencing the formation of these cells have been characterized in preclinical tumor models offering exploitable targets for potentiating T-cell therapy. A CRISPRa gain-of-function screen was used to broaden actionable targets to augment CD8-positive T-cell sternness beyond normal physiology. As shown herein, this led to the identification of LM04 as a potent modulator of CD8- positive T-cell sternness and memory formation when ectopically expressed. LM04 boosted CD8- positive T-cell responses while preserving a substantial pool of stem-like memory T cells even after repetitive antigenic exposure.

[0078] Mechanistically, it was found that LM04, rather than directly modulating gene transcription, boosted STAT3 signaling in response to IL-21, activating a complex GRN that comprised the upregulation of several STAT3 target molecules known to shape CD8+T-cell differentiation and sternness. Interestingly, many of the upregulated genes in this GRN, including JUN B, JUN, FOS, FOSB, and ZFP36, were recently found to be enriched in a subset of highly functional T cells isolated from human head and neck squamous cell carcinoma (bioRxiv (2023). https: / / doi.org:10.1101 / 2023.09.26.559470), underscoring the potential role of STAT3 signaling in enhancing the antitumor effectiveness of LMO4-overexpressing T cells.

[0079] The findings disclosed herein have significant implications for the development of more effective T cell-based immunotherapies. Current strategies promoting the generation of stem-like T cells have the drawback of restricting cell proliferation, thus limiting the cell yield available for infusion. LMO4 overexpression offers the advantage of enabling robust T-cell expansion in vivo without adversely impacting the longevity of stem-like memory T cells, even when T cells are produced in the presence of IL-2, a potent driver of terminal effector differentiation. Indeed, this synthetic-engineering strategy led to the generation of highly effective T cells capable of achieving curative responses in the majority of tumor-bearing animals. The compact genetic footprint of LMO4, spanning less than 500 nucleotides, offers the additional advantage of easy integration in CAR and TCR vectors currently employed in the clinic. Lastly, the capacity to elicit curative responses against solid tumors without the need for lymphodepletion preconditioning enhances the attractiveness of this technology for broad adoption. Ectopic expression of LM04

[0080] The present invention is based on the finding that the ectopic overexpression of LM04 in T cells has a beneficial effect in cancer therapy. Said T cell expresses LM04 at higher levels compared to a wild type T cell.

[0081] In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a functional LM04 polypeptide for use in medicine. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a polypeptide comprising an amino acid sequence of SEQ ID No. 1 or a functional variant thereof.

[0082] In certain embodiments, the present disclosure relates to a recombinant T cell overexpressing a recombinant nucleic acid encoding a functional LM04 polypeptide compared to a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant T cell expressing a recombinant nucleic acid encoding a functional LM04 polypeptide at higher levels than a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant T cell ectopically overexpressing a recombinant nucleic acid encoding a functional LM04 polypeptide compared to a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant T cell transgenically overexpressing a recombinant nucleic acid encoding a functional LM04 polypeptide compared to a wild type T cell for use in the treatment of a cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant T cell transgenically expressing a recombinant nucleic acid encoding a functional LM04 polypeptide at higher levels than a wild type T cell for use in the treatment of a cancer or a tumor.

[0083] In certain embodiments, the present disclosure relates to a recombinant nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine.

[0084] The nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1, or a functional variant thereof, is ectopically expressed in a host cell, such as a T cell, preferably a CD8- positive T cell. Said host cell may comprise an additional, genomic copy of LM04. The ectopically expressed LM04 polypeptide leads to a beneficial effect in cancer therapy. The ectopically expressed nucleic acid encoding said polypeptide comprising an amino acid sequence of SEQ ID No. 1, or a functional variant thereof, may be operably linked to a promoter. This promoter is responsible for the overexpression of LM04 in the host cell. Any suitable promoter may be used for this purpose. The promoter may be an inducible promoter, i.e. a promoter which triggers expression of the target gene (LM04) upon the presence of a stimuli or inducer. The promoter may also be a constitutive promoter that directs high level expression of LM04.

[0085] Therefore, in certain embodiments, the present disclosure relates to a recombinant nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said nucleic acid is operably linked to a promoter. In certain embodiments, the present disclosure relates to a recombinant nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said nucleic acid is operably linked to a promoter. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine, wherein said nucleic acid is operably linked to a promoter. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine, wherein said nucleic acid is operably linked to a promoter. In certain embodiments, said promoter is an inducible promoter. In other embodiments, said promoter is a constitutive promoter.

[0086] In the broadest sense, the present invention may also be practiced by introducing regulatory elements into the host cell which lead to an overexpression of LM04 from its natural genomic locus. This may be achieved by introducing the respective regulatory elements into the appropriate genomic locus of the host cell, for example via genome editing technologies, such as Crispr-Cas9, base editing or prime editing.

[0087] The LM04 polypeptide overexpressed within the spirit of the present disclosure may be encoded on any commonly used expression vector or and / or may be delivered utilizing any appropriate vehicle. Preferred expression vectors are viral vectors and plasmid.

[0088] Therefore, in certain embodiments the present disclosure relates to a recombinant nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said nucleic acid is encoded on an expression vector. In certain embodiments, the present disclosure relates to a recombinant nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said nucleic acid is encoded on an expression vector. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine, wherein said nucleic acid is encoded on an expression vector. In certain embodiments, the present disclosure relates to a recombinant nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine, wherein said nucleic acid is encoded on an expression vector. Preferably, said expression vector is a viral vector or a plasmid.

[0089] T cells expressing LM04

[0090] To achieve the therapeutic effect in the treatment of cancer or a tumor, the nucleic acid encoding the functional LM04 polypeptide is expressed in a host cell. Preferably, the host cell is an immune cell. More preferably, the immune cell is a T cell. Even more preferably, the T cell is a CD8-positive T cell.

[0091] Therefore, in certain embodiments the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine.

[0092] In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising a codon- optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine.

[0093] In certain embodiments, the present disclosure relates to a recombinant CD8-positive T cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant CD8-positive T cell comprising a nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant CD8-positive T cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine. In certain embodiments, the present disclosure relates to a recombinant CD8-positive T cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine.

[0094] Said recombinant host cell may additionally comprise a nucleic acid encoding a chimeric antigen receptor (CAR) or a T cell receptor (TCR): Typically, the CAR or TCR is specific for a cancer antigen.

[0095] Therefore, in certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising a codon- optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. Preferably, said CAR or TCR is specific for a cancer antigen.

[0096] In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said T cell additionally expresses a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in medicine, wherein said T cell additionally expresses a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in medicine, wherein said T cell additionally expresses a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in medicine, wherein said T cell additionally expresses a CAR or a TCR. Preferably, said CAR or TCR is specific for a cancer antigen.

[0097] In certain embodiments, said CD8-positive T cell is a tumor infiltrating lymphocyte (TIL). In other embodiments, said CD8-positive T cell is a peripheral blood lymphocyte ( PBL) isolated from a patient afflicted with cancer. In certain embodiments, said T cell is an isolated T cell, preferably an isolated CD8-positive T cell, even more preferably an isolated CD8-positive human T cell.

[0098] In certain embodiments, said T cell is a purified T cell, preferably a purified CD8-positive T cell, even more preferably a purified CD8-positive human T cell.

[0099] In certain embodiments, the present disclosure relates to a population of cells comprising at least one recombinant T cell described herein above.

[0100] Therapeutic use

[0101] As described herein, overexpression of LM04 in T cells is particularly useful in the treatment of cancer or a tumor.

[0102] Therefore, in certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in the treatment of cancer or a tumor. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in the treatment of cancer or a tumor.

[0103] Various types of cancer may be treated with the inventive concept disclosed herein, such as hematological cancer or solid cancer. Therefore, in certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of a hematological cancer. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising a codon- optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of a hematological cancer. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in the treatment of a hematological cancer. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in the treatment of a hematological cancer. Said hematological cancer may be, among others, leukemia, lymphoma or myeloma. Said leukemia may be acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), Myeloproliferative disorder / neoplasm (MPDS), or and myelodysplasia syndrome. Said lymphoma may be Hodgkin's lymphoma, both indolent and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell), among others. Myeloma may be multiple myeloma (MM), giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jones myeloma.

[0104] In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of a solid cancer. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of a solid cancer. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in the treatment of a solid cancer. In certain embodiments, the present disclosure relates to a recombinant immune cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in the treatment of a solid cancer. Said solid tumor may be, among others, a sarcoma or a carcinoma, including breast tumors, ovarian tumors, gastric tumors, lung tumors, pancreatic tumors, prostate tumors, melanoma tumors, colorectal tumors, lung tumors, head and neck tumors, bladder tumors, esophageal tumors, liver tumors, thyroid tumors non-small-cell lung cancer (NSCLC) and kidney tumors.

[0105] In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of cancer or a tumor, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid comprising a codon-optimized sequence of the nucleic acid sequence of SEQ ID No. 2 for use in the treatment of cancer or a tumor, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a polypeptide comprising the amino acid sequence of SEQ ID No. 1 for use in the treatment of cancer or a tumor, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a recombinant T cell comprising a nucleic acid encoding a functional variant of the polypeptide comprising the amino acid sequence of SEQ ID No.l for use in the treatment of cancer or a tumor, wherein said T cell additionally comprises a nucleic acid encoding a CAR or a TCR. In certain embodiments, the present disclosure relates to a method of increasing CD8-positive T cell mediated immunity in a subject comprising administering to said subject a recombinant T cell overexpressing LMO4 as described herein.

[0106] In certain embodiments, the disclosure relates to a method to augment the sternness of CD8-positive T cells comprising administering to said subject a recombinant T cell overexpressing LMO4 as described herein.

[0107] In certain embodiments, the disclosure relates to a method to promote adoptive transfer-mediated antitumor activity comprising administering to said subject a recombinant T cell overexpressing LMO4 as described herein.

[0108] In certain embodiments, the disclosure relates to boost CD8-positive T-cell antitumor immunity comprising administering to said subject a recombinant T cell overexpressing LMO4 as described herein.

[0109] In certain embodiments, the disclosure relates to a method of reducing the size of a tumor in a subject comprising administering to said subject a recombinant T cell overexpressing LMO4 as described herein.

[0110] In certain embodiments, the present disclosure relates to a method of increasing CD8-positive T cell mediated immunity in a subject comprising administering to said subject a recombinant T cell overexpressing LMO4 as described herein, wherein said method comprises: isolating a population of the subject's T cells; introducing into said T cells a nucleic acid molecule leading to the overexpression of LMO4, and reintroducing said T cells into said subject.

[0111] In certain embodiments, said nucleic acid is introduced into the immune cells by transduction or transfection. In certain embodiments, said immune cell is autologous to the mammal. In certain embodiments, said immune cells are isolated from the blood of the subject.

[0112] Examples

[0113] Example 1: Materials and methods

[0114] Ethics statement All mouse experiments were performed with the approval of the National Cancer Institute, the National Heart, Lung, and Blood Institute Animal Care and Use Committees, and the Government of Lower Franconia. Experiments performed at NIH were done according to NIH guidelines for research using mice.

[0115] Mice

[0116] C57BL / 6NCr and B6-Ly5.1 / Cr mice were obtained from Charles River Frederick or Sulzfeld; pmel-1 (B6. Cg-Thyla / Cy Tg (TcraTcrb)8Rest / J) mice were obtained from the Jackson Laboratory; Cre-ERT2(B6- Gt(ROSA)26Sortm9(cre / Esrl|Arte) mice from Taconic. Lmo4fl / flmice5 were obtained from Bogi Andersen and were back-crossed with C57BL / 6NCr mice for more than 30 generations. Pmel-1 mice were crossed with Lmo4fl / fl mice for the formation of pme-1 Lmo4fl / flmice and were further crossed with Cre-ERT2mice for the generation of pmel-1 Cre-ERT2Lmo4fl / flmice. Immunodeficient NCG and NXG mice ages 6- 8 weeks were obtained from Janvier Labs. All mouse experiments were performed with the approval of the National Cancer Institute, the National Heart, Lung, and Blood Institute Animal Care and Use Committees, the Institutional Animal Care and Use Committee programme (IACUC n° 1178 and 1208) and the Government of Lower Franconia. Experiments performed at NIH were done according to NIH guidelines for research using mice.

[0117] Cell lines

[0118] Platinum-E cells were from Cell Biolabs, being authenticated and validated mycoplasma free. B16 melanoma expressing human gplOO (B16KVP); JCI Insight 4 (2019)) were obtained from K.-l. Hanada, National Cancer Institute, Bethesda, Lewis Lung Carcinoma (LLC1) cells from ATCC, NALM6-GL cells were obtained from T. Fry (National Cancer Institute, Bethesda). All cell lines were validated by PCR- assay to be mycoplasma free.

[0119] Antibodies, flow cytometry and cell sorting

[0120] Anti-LY5.1 (A20), Anti-LY5.2 (104), anti-THYl.l (OX-7), anti-CD62L (MEL-14), anti-IFNy (XMG1.2), anti- TNFa (MP6-XT2), anti-TNFa (MP6-XT2), anti-CD8 (HIT8a), anti-THYl.l (HIS51), anti-CCR7 (2-L1-A), anti- CD45RA (5H9), anti-CD45R0 (UCHL1) were bought from BD Biosciences; anti-CD8a (53-6.7), anti- THY1.2 (30-H12), anti-KLRG-1 (2F1), anti-CD44 (IM7), anti-IL-2 (JE56-5H4), anti-STAT3 (4G4B45), anti- IL-21R (4A9), anti-CD95 (DX2) and anti-CD62L (DREG-56) were from Biolegend. For intracellular staining of STAT3, cells were fixed and permeabilized (eBioscience, 00-5524). Leukocyte Activation Cocktail containing phorbol myristate acetate and ionomycin (BD Biosciences) was employed to activate CD8- positive T cells for intercellular cytokine staining. A Fixation / Permeabilization Solution Kit (BD Biosciences) was used to fix and permeabilize the cells. Flow Cytometry Acquisition was done on an LSR II or BDFortessa (BD Biosciences) flow cytometer. The raw data were analyzed by using FlowJo software (TreeStar). For naive CD8-positive T-cell enrichment, the Naive CD8-positive T-cell isolation kit from Stem Cell Technology was used. All other T-cell isolations were done on FACSAria (BD Biosciences).

[0121] Quantitative PCR

[0122] The isolation of RNA was performed using the RNeasy Mini Kit (Qiagen). Complementary DNA (Applied Biosystems) was obtained by PCR. qPCR was done using primers from Applied Biosystems and a Quant Studio 3 (Applied Biosystems) using Powerllp SYBR Green Master Mix (Applied Biosystems). Findings are shown relative to Actb or Rpll3 expression.

[0123] List of primers:

[0124] ActbF, ActbR: Primers from Applied Biosystems were used.

[0125] Rpll3F: CGAGGCATGCTGCCCCACAA (SEQ ID No. 3)

[0126] Rpll3R: AGCAGGGACCACCATCCGCT (SEQ ID No. 4)

[0127] Tcf7F: AGCTTTCTCCACTCTACGAACA (SEQ ID No. 5)

[0128] Tcf7R: AATCCAGAGAGATCGGGGGTC (SEQ ID No. 6)

[0129] Socs3F: ATGGTCACCCACAGCAAGTTT (SEQ ID No. 7)

[0130] Socs3R TCCAGTAGAATCCGCTCTCCT (SEQ ID No. 8)

[0131] JunbF: TCACGACGACTCTTACGCAG (SEQ ID No. 9)

[0132] JunbR: CCTTGAGACCCCGATAGGGA (SEQ ID No. 10)

[0133] Zfp36F CCACCTCCTCTCGATACAAGA (SEQ ID No. 11)

[0134] Zfp36R GCTTGGCGAAGTTCACCCA (SEQ ID No. 12)

[0135] Immunoblot analysis

[0136] 11 Proteins were segregated by 4-12% SDS-PAGE, then standard immunoblot analysis was performed with anti-LMO4 (Cell Signaling Technology, clone D6V4Z), anti-ACTB (Cell Signaling, clone 8H10D10), anti-STAT3 (Cell Signaling Technology, clone 124H6), anti-pSTAT3(Tyr705) (Cell Signaling Technology, clone M9C6), horseradish peroxidase-conjugated goat anti-mouse IgG (sc-2031; Santa Cruz Biotechnology) and horseradish peroxidase-conjugated anti-rabbit IgG (sc-2030; Santa Cruz Biotechnology).

[0137] Immunoprecipitation of JAK1 was performed using anti-JAKl antibody (Cell Signaling Technology, clone D1T6W). lgG2a was used as negative control (Cell Signaling Technology, clone E5Y6Q). In brief, the cells were washed twice with PBS and lysed in in ice-cold lysis buffer (50 mM Tris-CI (pH 7.5), 150 mM NaCI, and 1% NP-40) with protease and phosphatase inhibitors (Cell Signaling). The cell lysates containing 1 to 3 mg of protein were pre-cleared with 30 pl protein A / G-Sepharose beads (Thermo Scientific) for 1 h at 4C° and thereafter incubated with the appropriate antibody overnight at 4° C, following another incubation with 30 pl of protein A / G-Sepharose beads for additional 2 h at 4°C. The immunoprecipitates were washed three times with washing buffer (20 mM Tris-CI (pH7.5), 150 mM NaCI, and 0,1% NP- 40), separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) after being boiled in Laemmli buffer, and transferred to a 0.2 pm nitrocellulose membrane. The membrane was blocked with PBS containing 0.1% Tween-20 and 1% bovine serum albumin before it was incubated with the appropriate primary and secondary antibodies. The bound antibodies were visualized using Pierce ECL WB Substrate (Thermo Fisher).

[0138] Retroviral vector generation and virus synthesis

[0139] Lmo4 was cloned into the MSGV-l-Thyl.l vector as formerly reported. Platinum-E (mouse setting) or 293GP (human setting) cell lines were employed for gamma-retroviral vector generation by transfection with DNA plasmids using Lipofectamine 2000 (Invitrogen) and collecting the virus 40 h after transfection.

[0140] Mouse CD8-positive T-cell in vitro stimulation and transduction

[0141] Naive CD8-positive T cells were stimulated in 24-well tissue culture plates coated with anti-CD3e (2 pg ml1; 145-2C11; BD Biosciences) and soluble anti-CD28 (1 pg ml-1; 37.51; BD Biosciences) in culture medium including recombinant human (rh) IL-2 (10 ng ml-1; Prometheus Laboratories). The virus was 'spin-inoculated' at 2,000 g for 2 h at 32°C on non-tissue culture plates coated with RetroNectin (Takara). CD8-positive T cells stimulated for 24 h were spun on these plates after aspiration of the viral supernatant. The transduction efficiency was measured after 48 h. For experiments evaluating STAT3 phosphorylation and target gene expression, CD8-positive T cells were washed from rhlL-2 supplemented medium 3 days after stimulation and incubated 0 / N at 37°C. CD8-positive T cells were then exposed for 30 min to 20 ng ml-1 of mouse IL-6, IL-10 and IL-21 (all Miltenyi Biotec). LLC1 cells were transduced with virus-containing supernatant and selected for ALNGFr expression by magnetic beads (Dynabeads M-450, Dynal) coated with the LNGFr-specific mAb 20.4 (ATCC).

[0142] Human CD8+ T cell in vitro stimulation and transduction

[0143] PBMCs (NIH, US and University Hospital Regensburg, Germany) were enriched for naive CD8+ T cells using naive CD8+ T cell isolation kit (Stem Cell Technologies) before freezing. To generate TSCM- enriched cells, naive CD8+ T cells were thawed and activated with GMP grade T Cell TransAct (Miltenyi Biotec) in TexMACS GMP-grade medium (Miltenyi Biotec), 0.5% human AB serum (ZKT Tubingen, FSM / TV 276), supplemented with 1% Pen-Strep (Gibco) in the presence of 5 ng / mL IL-7 and 30 ng / mL IL-21 (both Miltenyi Biotec). Cells were transduced on days 2 and 3 and expanded for 5 more days in media containing IL-7 and IL-21. To generate TSCM-enriched cells modified with CD19-CAR, naive CD8+ T cells were thawed and stimulated using anti-CD3 / CD28 beads (Dynabeads Human T-Expander CD3 / CD28; Thermo Fisher Scientific) at a 1:1 bead-to-cell ratio in AIM-V (Gibco), supplemented with 5% human AB serum 2 mM GMAX (Gibco), 5 ng / mL IL-7 and 30 ng / mL IL-21. Transduction with the PG13 expressing CD19-CAR (FMC63-28-?)) retrovirus was performed on days 2 and 3, followed by expansion in media containing IL-7 and IL-21 for an additional 5 days after the removal of activating beads on day 4.

[0144] Generation of dendritic cell vaccine

[0145] Dendritic cells (DCs) were generated from bone marrow (BM) of femurs and tibias. BM was flushed out with RPMI, and a single cell suspension was prepared by passing the BM solution through a 19- gauge needle. BM cells re-suspended at 5 x 105 cells / ml in 6-well plates were cultured in IMDM 10 % FBS, GM-CSF and IL-4 (PeproTech), both at 20 ng / ml. At day 2, cells were split 1:2 by adding fresh medium and cytokines. At day 5, we removed and discarded 2 ml of supernatant and added 2 ml of fresh medium with GM-CSF and IL-4 (both 40 ng / ml). At day 7, DCs were activated with LPS (1 mg / ml) for 16 hrs. The day after DCs were collected and pulsed with 5 pg / ml of the OVA-derived peptide SIINFEKL, for 1 h at 37 °C. Peptide-pulsed DCs were then washed and resuspended at 5 x 106 cells / ml. We injected 100 01 of PBS containing peptide pulsed DCs / mice s.c. the day after ACT. Tamoxifen injections, adoptive cell transfer, virus infection and tumor inoculation

[0146] Cre-ERT2activity was prompted by intraperitoneal injection of 2 mg tamoxifen (Sigma-Aldrich) dissolved in corn oil (Sigma-Aldrich) on 4 successive days. The adoptive transfer of pmel-1 CD8-positive T cells (1.5 x 105to 10 x 105cells) into 6- to 8-week-old C57BL / 6 mice was performed together with the vaccination of 2 x 107PFU recombinant vaccinia virus expressing human gplOO (gplOO-VV). Experiments to evaluate the recall response were conducted 35 d after primary infection with gplOO- VV by secondary transfer of normalized numbers of pmel-1 memory T cells into C57BL / 6 mice infected with 108PFU recombinant adenovirus type 2 expressing human gplOO. In tumor experiments, 2 x 105B16KVP cells were inoculated subcutaneously into 6- to 8-week-old C57BL / 6 mice. After 10 days mice were adoptively transferred with 3.5 x 105pmel-1 CD8-positive T cells and vaccinated intravenously with 2 x 107PFU gplOO-VV. Recombinant human IL-2 (2.4e5 IU per dose) was injected intraperitoneally twice a day for a total of six doses. To prevent potential rejection in conditional knockout experiments investigating the late contraction phase of the immune response and in secondary treatment experiments, we used recipient mice carrying the Lmo4flallele. In these experiments, as wild-type controls, we used pmel-1 CD8-positive T cells isolated from tamoxifen-injected littermates carrying the Lmo4flallele but not Cre-ERT2.

[0147] Murine syngeneic lung carcinoma model:

[0148] Mice were injected subcutaneously with LLC1 cells (5 x 105) expressing ovalbumin (LLC1-OVA). Seven days later, mice were injected intraperitoneally with a single dose of cyclophosphamide (CTX 3 mg / mouse). After 24 hours, mice were randomized and adoptively transferred with OT-1 T cells transduced with Thyl.l or LMO4-Thyl.l ( 106cells). The day after, mice were subcutaneously injected with dendritic cells (5 x 105) loaded with the SIINFEKL peptide. Tumor volume was measured every 2 days.

[0149] Xenograft acute lymphoblastic leukemia model:

[0150] NXG host mice received intravenous injections of NALM6-GL (8 x 10s) cells, followed by the administration of 1.25 x 105CD19-CAR+ CD8+ T cells after a 3-day interval. Recombinant human IL-15 (NCI) was administered intraperitoneally every other day at a dosage of 1 pg per mouse. Tumor burden was assessed using the IVIS Lumina III In Vivo Imaging System (PerkinElmer). After 7 days, blood samples were collected from the mice to estimate the engraftment of CD8+ T cells

[0151] Quantification of adoptively transferred cells After processing the spleen, cells were counted by trypan blue exclusion of dead cells. The percentage of transferred CD8-positive T cells was measured by analyzing the expression of CD8 and Thyl.l / Ly5.1 or Ly5.2 by flow cytometry. The absolute quantity of transferred cells was calculated by multiplying the total cell count by the frequency of CD8-positive Thyl.l+Ly5.1+or CD8+Ly5.2+cells.

[0152] RNA-seq

[0153] Total cellular RNA was isolated from CD8-positive T-cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. The concentration and quality of the purified RNA was analyzed using the RNA ScreenTape Kit (Agilent). Generation of dsDNA libraries for Illumina sequencing from total cellular RNA was carried out using the TruSeq Stranded Total RNA Kit (Illumina) according to the manufacturer's instructions. The quality of dsDNA libraries was analyzed using the High Sensitivity D1000 ScreenTape Kit (Agilent) and concentrations were assessed with the Qubit dsDNA HS Kit (Thermo Fisher Scientific). Sequencing was performed using an Illumina NextSeq550 sequencer. Sequenced reads were processed and aligned to the mmlO genome using splice-aware aligner TopHat 2.1.1. Then, raw mapped read counts were processed in R with edgeR (Bioinformatics 26:139-40 (2010)) to generate normalized read counts and determine differentially expressed genes with Iog2 FC > 0.3 and P < 0.05. Gene set enrichment analysis was performed with ClusterProfiler package and volcano plot was generated using EnhancedVolcano package. Volcano plot shows differentially expressed genes (P < 0.05, Iog2 FC > 0.3 and < -0.3) in pmel-1 Lmo4-Thyl.l+compared to Thyl.l+CD8+T cells.

[0154] CRISPRa screen analysis

[0155] Genes that are deemed to be transcription factors or transcription regulators were filtered from the list of genes targeted in a genome-wide CRISPRa screen analysis. Filtering was done based on the Transcription Factor dataset available on Transcription Factor checkpoint 2.0 database (https: / / www.tfcheckpoint.org / index.php), which is a resource for Human, Mouse, and Rat Transcription Factors. The filtered gene list was used to generate a volcano plot with EnhancedVolcano package. Volcano plot shows median sgRNA Iog2-fold change (IFN-Yhl / I° sorting bin counts) for each gene (P < 0.05, Iog2 FC > 0.52 and < 0.531).

[0156] Cas9 / RNP nucleofection pmel-1 CD8-positive T cells isolated from Cas9+ C57BL / 6 mice were stimulated 24 h before transfection. To prepare the crRNA-tracrRNA duplex each Alt-R crRNA and Alt-R tracrRNA (IDT) was reconstituted to 100 pM with nuclease-Free Duplex Buffer (IDT). After mixing the oligos at equimolar concentrations [0.75 pl Alt-R crRNA and 0.75 pl Alt-R tracrRNA per guide with 1.5 pl Buffer (Mirus Ingenio Electroporation)] in a sterile PCR tube the mix was annealed by heating at 95°C for 5 min in PCR thermocycler and then slowly cooled down to room temperature. For CAS9 / RNP precomplexing, resulted 3 pl crRNA-tracrRNA duplex and 1.2 pl TrueCut CAS9 Protein v2 (Thermo Fisher) were gently mixed and incubated at room temperature for 20 min. After prewarming 200 pl complete T-cell media containing rhlL-2 (10 ng ml1) per well of a 96-well plate, 1 million CD8-positive T cells were resuspended in 20 pl primary cell nucleofection solution (P4 Primary Cell 4D-Nucleofector X kit; 32 RCT; V4XP-4032, Lonza). T cells were then mixed and incubated with 4.2 pl RNP and 15.8 ul Buffer (Mirus) for a total volume of 20 pl at room temperature for 2 min in round bottom 96-well plate. Cell / RNP mix was transferred to Nucleofection cuvette strips (4D-Nucleofector X kit S; Lonza) and electroporated using a 4D nucleofector (4D-Nucleofector X Unit: AAF-1002X, Lonza) and a CM137 pulse. Following nucleofection, transfected cells were transferred to 96-well plates containing 200 pl prewarmed complete T-cell media per well and incubated at 37°C for 6 hours, preceding retroviral transduction and adoptive transfer into C57BL / 6 mice.

[0157] List of crRNAs used:

[0158] Thyl.2#l:

[0159] / AltRI / rCrCrU rUrGrG rUrGrU rUrArU rUrCrU rCrArU rGrGrG rUrUrU rUrArG rArGrC rUrArU rGrCrU / AltR2 / (SEQ ID No. 13)

[0160] Thyl.2#2:

[0161] / AltRI / rGrArG rCrArG rGrArG rArGrC rGrArC rGrCrU rGrArG rUrUrU rUrArG rArGrC rUrArU rGrCrU / AltR2 / (SEQ. ID No. 14)

[0162] Stat3#l:

[0163] / AITRl / rCrArArCrArUrCrUrGrCrCrUrGrGrArCrCrGrUrCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU / AI tR2 / (SEQ ID No. 15)

[0164] Stat3#2:

[0165] / AITRl / rArGrUrUrGrArArArUrCrArArArGrUrCrGrUrCrCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU / AI tR2 / (SEQ ID No. 16) IL-21RW1:

[0166] / AltRl / rGrUrCrArArUrGrUrGrArCrGrGrArCrCrArGrUrCrGrUrUrUrUrArGrArGrCrUrArUrGrCrU / AI tR2 / (SEQ ID No. 17)

[0167] IL-21R3W2:

[0168] / AltRl / rCrCrCrUrCrCrArArCrUrArCrGrUrGrCrUrGrArGrGrUrUrUrUrArGrArGrCrUrArUrGrCrU / Alt R2 / (SEQ. ID No. 18)

[0169] Gene-set enrichment and STRING network analyses

[0170] Mouse gene symbols were initially mapped to the orthologous human genes using the homology data from the MGI website (ftp: / / ftp.informatics.jax.org / pub / reports / HMD_HGNC_Accession.rpt) and were ranked by the fold changes of the gene expression as profiled by RNA-seq. Subsequently, geneset enrichment analyses were performed using ClusterProfiler R package (Innovation (Camb) 2:100141 (2021)). C7 immunologic signature gene set from MSigDB (https: / / www.gsea- msigdb.org / gsea / msigdb / ), was employed for GSEA of naive / memory and effector signatures. Gene sets of interest were visualized on a bubble plot, generated using the ggplot2 R package. Pathway analysis was performed using Panther DB (https: / / www.pantherdb.org / ). Significant pathways with p- value <0.05 were selected for downstream analysis. Pathways of interest showing a reverse relationship between Lmo4- Thyl.l and Myb-deficient T cells were visualized on bidirectional bar plot. The Stat3 GRN was created using Cytoscape software and STRING database together with our RNA-seq dataset.

[0171] Statistical analyses

[0172] Two-tailed Student's t-test was performed with Graphpad Prism 9 software for comparison of data such as gene expression levels, cell proliferation and functionality (numbers and percentages). Comparisons between multiple groups was done with Kruskal-Wallis test for multiple comparisons. Tumor growth graphs were analyzed using Wilcoxon Rank Sum test on growth slopes. For comparison of survival curves, a log-rank (Mantel-Cox) test was used. Fisher's exact test was used to calculate the significance of the overlapping pathways in the Venn diagram. GSEA's significance was assessed using the Kolmogorov-Smirnov test. Example 2: Identification of LMO4 as a candidate gene to enhance CD8-positive T-cell sternness

[0173] To identify transcriptional regulators that could be harnessed to augment stem-like behavior in CD8- positive T cells beyond physiological function, we analyzed a recently published genome-wide CRISPRa screen dataset (Science 375, 513 (2022)). Interferon-y (IFN-y) production was used as a proxy for terminal differentiation for the selection of the most potent negative regulators of effector programs.

[0174] Filtering was done based on the Transcription Factor dataset available on Transcription Factor checkpoint 2.0 database (https: / / www.tfcheckpoint.org / index.php), which is a resource for Human, Mouse, and Rat Transcription Factors. The filtered gene list was used to generate a volcano plot with EnhancedVolcano package. Volcano plot (Figure 1) shows median sgRNA Iog2-fold change (IFN-yhl / l° sorting bin counts) for each gene (P < 0.05, Iog2 FC > 0.52 and < -0.531).

[0175] Top positive hits included well-established transcription factors that orchestrate effector differentiation such as EOMES, TBX21, PRDM1, and IRF4. MYB and BACH2, which are known regulators of CD8-positive T-cell sternness (Nat Immunol 20, 337-349 (2019); Nat Immunol 17, 851-860 (2016)) were among the negative hits, validating the robustness of the assay. Notably, with the exception of IKZF1, which has recently been demonstrated to restrain effector differentiation (Journal of Immunology 192, 5118-5129 (2014)), the most significant negative hits have unknown functions in CD8-positive T-cell memory formation.

[0176] To narrow down the list of candidate genes, the expression of the top 14 transcriptional regulators was analyzed in culture conditions favoring the formation of stem-like T cells. It was previously shown that stem-like T cells can be efficiently induced by activating CD8-positive T cells in the presence of IL- 21 and an inhibitor of lactate dehydrogenase (LDHi; Proc Natl Acad Sci U S A 117, 6047-6055 (2020)). Several of the candidates, such as Foxfl, Foxf2, Foxl2, Cebpb and Gata6 were not expressed in CD8- positive T cells. Strikingly, only LMO4 and Ikzfl were upregulated in IL-21 + LDHi cultured cells compared to cells cultured with no cytokine (NC), IL-2, IL-2 + LDHi, or IL-21 (Figure 2).

[0177] Given the known function of LMO4 protein in cell differentiation, and the as-yet-undetermined role of LMO4 in mature CD8-positive+ T cells, this molecule was selected for further investigation. To determine whether the LMO4 induction represents a common trait of cells undergoing stem-like T-cell differentiation, the abundance of Lmo4 transcripts in CD8-positive T cells activated in the presence of TWS119 was investigated. TWS119 is a potent inhibitor of glycogen synthase kinase-3 (GSK-3 ), which was shown to drive the generation of both human and mouse stem-like T cells (Nat Med 15, 808-813 (2009); Blood 128, 519-528 (2016)), and that is used in a clinical trial evaluating CD19 CAR-modified stem-like T cells (NCT01087294). Priming naive CD8-positive T cells in the absence of TWS119 profoundly downregulated LMO4, whereas LMO4 levels were mildly increased when the GSK-3 inhibitor was added to the culture medium (Figure 3).

[0178] Altogether, these results reveal the maintenance of LM04 as a common signature of stem-like T cells, indicating that modulating its expression might regulate CD8-positive T-cell sternness.

[0179] Example 3: Ectopic LMO4 expression promotes the generation of stem-like T cells while restraining terminal differentiation

[0180] LMO4 is very poorly expressed at physiological conditions in both, healthy persons and tumor patients (see Figure 34). To test if CD8-positive T-cell sternness could be enhanced by increasing LMO4 expression at supraphysiological levels, a synthetic biology approach to overexpress LMO4 in CD8- positive T cells was employed in which the ability to generate long-lived memory cells was measured. pmel-1 Ly5.1+T cells (which recognize the shared melanoma-melanocyte differentiation antigen gplOO) were transduced with Lmo4-Thyl.l or Thyl.l alone and transferred into wild-type mice infected with gplOO-VV (Figure 4). The LMO4 polypeptide overexpressed in this and the following experiments has the amino acid sequence of SEQ. ID No. 1 and is encoded by a nucleic acid sequence of SEQ ID No. 2.

[0181] Overexpression of LMO4 protein was confirmed by western blotting before adoptive T-cell transfer (Figure 5). It was found that enforced expression of LMO4 promoted the expansion of pmel-1 T cells, which accumulated in the spleen at almost three-fold the frequency of control Thyl.l cells during the expansion phase (day 3) and at the peak (day 5) of the immune response. Similar findings were observed in lymph nodes and lungs, indicating that LMO4 overexpression enhances the accumulation of antigen-specific T cells in both lymphoid and peripheral tissues. These differences were amplified in the memory phase of the immune response as manifested by a five-fold increase in pmel-1 Lmo4- Thyl.l T-cell frequencies and numbers 30 days after infection (Figures 6 and 7). Likewise, a notable increase in pmel-1 T Lmo4-Thyl.l cells on day 30 in the lungs was observed, and an even more pronounced accumulation of these cells in the lymph nodes, which are known to serve as a niche for stem-like T cells (Sci Immunol 6, eabg7836 (2021)).

[0182] Next, it was investigated whether LMO4 overexpression could also qualitatively affect the immune response by altering the frequencies of CD8-positive T-cell effector and memory subsets. It was found that enforced expression of LMO4 restrained terminal effector differentiation, resulting in lower frequencies of CD62L KLRG1+short-lived effectors compared to control Thyl.l cells both at the peak of expansion and in the late memory phase (Figures 8-11).

[0183] Conversely, knocking out Lmo4 did not have a significant impact on pmel-1 T cell accumulation but resulted in higher frequencies of CD62L KLRG1+short-lived effectors and a decline in CD62L+KLRG1- memory precursor compared to pmel-1 Lmo4fl / flT cells. These findings underscore that LM04 exerts influence on the control of T-cell differentiation, even when expressed at physiological levels. As T cells undergo differentiation into terminal effectors, they progressively lose the capacity to produce various types of cytokines, ultimately transitioning into monofunctional IFN-y producers.

[0184] To determine if LM04 overexpression also impacted CD8-positive T-cell function, IL-2, TNF-a and IFN- y production in memory T cells was measure by intracellular cytokine staining (ICS). Results are shown in Figure 12. It was found that LMO4-overexpressing cells not only exhibited increased frequencies of cytokine-producing cells but that most of these cells displayed stronger polyfunctionality (i.e. produced 2 or more cytokines). Notably, the ability of LMO4-transduced CD8+ T cells to release IL-2 could serve as a potential mechanism for sustaining their proliferative advantage through autocrine or paracrine signals.

[0185] Taken together, these findings demonstrate that enhancing LM04 expression in CD8-positive T cells is an effective strategy to enhance the generation of polyfunctional stem-like memory T cells.

[0186] Example 4: LM04 overexpression enhances CD8-positive T-cell recall responses

[0187] Having established LM04 as a key player in the regulation of CD8-positive T-cell differentiation and formation of stem-like memory T cells, it was next investigated if LMO4-overexpression could promote more robust recall responses after secondary infection. Thirty days after primary infection with gplOO- vv, pmel-1 T cells overexpressing Lmo4-Thyl.l or Thyl.l were isolated and transferred in equal numbers into new wild-type hosts together with a gplOO-encoding adenovirus type 2 (gplOO-Adv) (Figure 13).

[0188] Strikingly, as in primary infection, an increased expansion of LMO4-overexpressingT cells at the peak of the recall response (day 5) was observed, and an even greater accumulation at secondary memory phase (day 30) in all organs examined (Figures 14 and 15). Similar to primary infection, a more marked differences in T-cell accumulation in lymph node reservoirs was detected. Repetitive antigen stimulation is known to drive CD8-positive T cells toward terminal differentiation, while progressively reducing the pool of stem-like T cells (Immunity 33, 128-140 (2010)). Herein it was found that enforced expression of LMO4 reduced the percentage of terminal effectors, whereas it doubled the frequencies of stem-like T cells 30 days after secondary infection with gplOO-adV (Figures 16 and 17). Again, no significant defects in pmel-1 T-cell expansion were observed when Lmo4 was knocked out, but a consistent greater differentiation into KLRG1+ effectors in pmel-1 Lmo4A / AT cells was observed compared to controls. Similar to primary infection, it was also observed that overexpression of LM04 boosted secondary memory CD8-positive T-cell polyfunctionality (Figure 18).

[0189] Taken together, these results show that elevated levels of LM04 enhance CD8-positive T-cell recall responses and facilitate the maintenance of a robust pool of stem-like T cells.

[0190] Example 5: Enforced expression of LM04 promotes curative CD8-positive T-cell antitumor responses

[0191] Given the ability of LM04 overexpression to enhance stem-like T-cell formation, it was determined whether LM04 overexpression could potentiate the therapeutic efficacy of pmel-1 T cells in a model of neoantigen-targeted adoptive immunotherapy. Therefore, pmel-1 T cells overexpressing LM04- Thyl.l or Thyl.l control were adoptively transferred into wild-type mice bearing subcutaneous B16 melanomas carrying the mutated gplOO epitope (KVP; JCI Insight 4 (2019)). To enhance the expansion and function of transferred T cells, a gplOO-VV vaccine was co-delivered intravenously, and high doses of IL-2 were administered intraperitoneally (Figure 19).

[0192] It was found that pmel-1 T cells overexpressing LMO4 were much more effective at controlling tumor growth than control cells (Figure 20). This robust antitumor effect led to curative responses in eight out of nine mice. In contrast, Thyl.l control cells were able to cure only a small fraction of the animals (Figure 21).

[0193] Of note, these strong responses were achieved without the need for lymphodepletion preconditioning, a standard clinical practice utilized to enhance the engraftment and functionality of adoptively transferred T cells (J Exp Med 202, 907-912 (2005); Sci Transl Med 8, 355rall6 (2016)). These findings underscore the potential of LMO4 overexpression to enhance T-cell therapy against challenging solid tumors.

[0194] Next, the investigation was extended by examining the effects of LMO4 overexpression in an additional challenging solid tumor model, which relies on the targeting of LLC1-OVA lung carcinomas by OVA-specific OT-1 T cells (Figure 35). Again, enhanced tumor control was observed in mice administered with Lmo4-Thyl.l T cells (Figures 36 and 37).

[0195] To gauge the relevance of this approach in humans, it was sought to determine whether ectopic expression of LMO4 could be employed to enhance the generation of human stem-like T cells. Naive CD8+ T cells were activated in the presence of IL-21 and IL-7, a combination of cytokines commonly used in the production of clinical-grade CAR stem-like T cells and subsequently transduced with either LMO4-Thyl.l orThyl.l control. Strikingly, LM04 overexpression significantly increased the generation of stem cell memory T cells (TSCM) across several donors (Figure 38).

[0196] Next it was evaluated whether the LM04 technology could be adopted to potentiate the efficacy of human CAR T cells in a humanized model of acute lymphoblasts leukemia (NALM6-GL) 5 (Fig 39). Overexpression of LM04 promoted the expansion of CD8+ T cells in the circulation of NALM6-bearing NXG mice seven days after adoptive transfer (Figure 40). This enhanced engraftment was associated with improved tumor control and extended survival of the animals (Figure 41). These findings highlight the potential of LMO4 overexpression to enhance T-cell therapy, demonstrating efficacy not only in syngeneic tumor models but also in human xenograft settings.

[0197] Example 6: LMO4 overexpression boosts IL-21-mediated STAT3 signaling to orchestrate a memorypromoting gene regulatory network

[0198] To elucidate the molecular mechanisms by which LMO4 influences CD8-positive T-cell memory differentiation and antitumor immunity, RNA-seq analysis was performed on LMO4-overexpressing pmel-1 T cells and control pmel-1 T cells harvested 5 d after adoptive transfer into mice infected with gplOO-VV. To avoid potential misinterpretation arising from transcriptional variations influenced by the skewed composition of T-cell subsets in control and LMO4-overexpressing T cells, a homogeneous population of CD62L-KLRG1- cells was sorted with purities exceeding 99%. 889 genes were found to be upregulated and 776 downregulated (p <0.05) comparing LMO4-overexpressing T cells and Thyl.l. Even after phenotypic subset normalization, LMO4-overexpressing cells were enriched for genes known to regulate memory T-cell differentiation (Figure 22). In particular, Tcf7, a pivotal regulator of T-cell sternness and longevity (Proc Natl Acad Sci U S A 107, 9777-9782 (2010)), was highly expressed in LMO4-overexpressing T cells compared to control cells. Likewise, Socs3, a suppressor of cytokine signaling that has been shown to prevent terminal differentiation by shielding developing memory CD8-positive T cells from inflammatory cytokines (Immunity 35, 792-805), was elevated in LMO4- overexpressing T cells. LMO4-overexpressing T cells also displayed increased mRNA levels of ZFP36, an RNA-binding protein that was recently shown to dampen CD8-positive T-cell effector responses, facilitating the formation of memory precursors (Elife 7 (2018)). The AP-1 transcription factor subunit, Junb, was upregulated in CD8-positive T cells expressing heightened levels of LMO4. AP-1 has been mostly linked to effector differentiation (Nat Immunol 17, 851-860 (2016)), but recent evidence has demonstrated the role of its subunits (e.g., JUNB and JUN) for T-cell survival and sternness (Front Immunol 13, 901030 (2022); Nature 576, 293-300 (2019)). These seemingly opposing effects might be dictated by the presence of STAT3 signaling, as AP-1 has been shown to cooperatively work with STAT3 to activate specific genes promoting cell survival (Mol Cell 7, 517-528 (2001)).

[0199] Among the pathways influenced by both LMO4 and c-Myb manipulation, we also observed a highly significant enrichment of the Reactome pathway R-MMU-1280215, which relates to cytokine signaling (P < 0.001) and includes several molecules involved in STAT signaling. Interestingly, Tcf7, Zfp36, Socs3, and Junb are all known targets of STAT341, implying a potential involvement of the STAT3 pathway in the actions of LMO4. To substantiate this hypothesis, the RNA-seq results were validated by q-PCR, confirming the upregulation of these pro-memory factors in LMO4-overexpressing cells. Then a GSEA was conducted utilizing STAT3 datasets. It was found that Lmo4-Thyl.l T cells exhibited an enrichment of STAT3-dependent genes that are upregulated in CD4+ T cells in response to IL- 2142, whereas Myb- deficient cells were negatively enriched. Moreover, LMO4-overexpressing T cells were enriched with genes displaying one or more STAT3 binding motifs (GSEA C3:STAT3_02). Notably, LMO4, Tcf7, Junb, Socs3, and Zfp36 were interconnected in a complex gene regulatory network that interfaces with Stat3 overexpressing CD8+ T cells.

[0200] Conversely, control cells displayed heightened expression of effector transcripts, such as Prdml, Zeb2, and Klrgl. Accordingly, Gene Set Enrichment Analysis (GSEA) revealed that LMO4-overexpressing cells exhibited enrichment for gene sets linked to naive and memory CD8-positive T cells or IL-7Rhlmemory precursors. By contrast, gene signatures associated with effectors as well as TCFl-deficient memory T cells, were negatively enriched. Remarkably, ectopic expression of LMO4 upregulated genes that are universally characteristic of stem-like T cells across various diseases, including acute and chronic viral infections as well as tumors.

[0201] Recently it was identified that c-Myb is a key transcription factor promoting CD8+ T cell sternness. To assess the degree to which LMO4 and c-Myb regulate overlapping transcriptional programs associated with sternness, a pathway analysis on LMO4-overexpressing cells was performed. Results were then compared with those obtained from pmel-1 CD8-positive MybA / AT cells, which were previously generated under identical experimental conditions. Out of the 174 pathways significantly enriched in Lmo4-Thyl.l, 74 (42.5%) displayed opposite enrichment patterns in Myb-deficient cells (P < 0.00001). Strikingly, many of the upregulated pathways in LMO4-overexpressing cells were associated with sternness, including the regulation of WNT10, Hedgehog35, and NRF2 (also known as NFE2L2) signaling36. Additionally, pathways related to mitochondrial metabolism, vital for the fitness of stemlike memory cells37, were also prominently upregulated (Figure 42). Inhibition of the MAPK pathway has similarly been associated with the development of stemlike T cells. Notably, ectopic expression of LM04 led to the upregulation of gene sets associated with the negative regulation of this signaling pathway Among the negatively enriched pathways in Lmo4-Thyl.l cells, only one was oppositely regulated in MybA / AT cells. This pathway relates to the ATR response to replication stress, suggesting that LM04 overexpression promoted cellular quiescence during the peak of the immune response, which likely facilitates the commitment of cells to enter the memory pool. Taken together, these findings highlight the capacity of LMO4to orchestrate several transcriptional programs associated with sternness and cellular fitness.

[0202] Tcf7, Junb, Socs3, and Zfp36 are all known targets of STAT3 (Cell Rep 19, 1888-1901 (2017)), implying a potential involvement of the STAT3 pathway in the actions of LMO4. To substantiate this hypothesis, a Gene Set Enrichment Analysis (GSEA) was performed utilizing STAT3 datasets. It was found that Lmo4-Thyl.l T cells exhibited an enrichment of STAT3-dependent genes that are upregulated in CD4- positive T cells in response to IL-21 (Proc Natl Acad Sci U S A 112, 9394-9399 (2015)) (Figure 23). Moreover, LMO4-overexpressing T cells were enriched with genes displaying one or more STAT3 binding motifs (GSEA C3:STAT3_02). Notably, LMO4, Tcf7, Junb, Socs3, and Zfp36 were interconnected in a complex gene regulatory network (GRN) that interfaces with Stat3.

[0203] Taken together, these analyses further corroborate an enhancement of STAT3 signaling in LMO4- overexpressing CD8-positive T cells. LMO4 has been previously demonstrated to enhance STAT3 signaling in Jurkat cells, by associating with and stabilizing the IL-6-gpl30 complex (J Biol Chem 280, 12747-12757 (2005)). Furthermore, LMO4 has been shown to enhance the activity of the STAT3 pathway in various cell types and in response to diverse STAT3-signaling molecules (Cell Mol Life Sci 67, 949-957 (2010); J Invest Dermatol 138, 1078-1087 (2018); Proc Natl Acad Sci U S A 104, 17305- 17310 (2007)). IL-6 (J Immunol 178, 778-787 (2007)), IL-10 (Immunity 35, 792-805 (2011)), and IL-21 (Nat Rev Drug Discov 13, 379-395 (2014); Blood 111, 5326-5333 (2008)) are well-established STAT3- signaling cytokines that promote CD8-positive T-cell memory. To determine which of these cytokines had a predominant effect in our system, CD8-positive T cells transduced with Lmo4-Thyl.l or Thyl.l controls were stimulated with IL-6, IL-10, or IL-21 and the level of phosphorylated STAT3 (pSTAT3) was measured at different time points. IL-6 and especially IL-21 induced a more pronounced STAT3 phosphorylation in LMO4-overexpressing T cells compared to controls (Figure 24). IL-10, on the other hand, induced pSTAT3 without noticeable differences between the two experimental groups, indicating that this cytokine does not play a major role in the pro-memory function of LMO4.

[0204] To further dissect the potential involvement of IL-6 and IL-21, the impact of these two cytokines on the expression of Tcf7, Zfp36, Socs3, and Junb in LMO4-overexpressing T cells and controls was investigated. Strikingly, IL-21, but not IL-6, induced the expression of all these molecules, though with distinct kinetics (Figure 25). IL-6 was only able to trigger a rapid but transient induction of Junb (Figure

[0205] 25, right panel).

[0206] Collectively, these findings confirm that LM04 enhances T-cell memory by boosting IL-21-mediated STAT3 signaling and its downstream pro-memory factors.

[0207] To dissect the potential involvement of IL-6 and IL-21, the impact of these two cytokines on the expression of Tcf7, Zfp36, Socs3, and Junb in LMO4-overexpressing T cells and controls was evaluated. Strikingly, IL-21, but not IL-6, induced the expression of all these molecules, though with distinct kinetics. IL-6 was only able to trigger a rapid but transient induction of Junb .

[0208] To substantiate the involvement of IL-21 in LM04-mediated sternness, the impact of deleting Il21r on the generation of CD62L+ memory T cell precursors in response to gplOO-vv was assessed. Using CRISPR / Cas9, a profound downregulated IL21R expression on CD8+ T cell surface was observed. Five days after pmel-1 T cell transfer, knocking out Il21r reduced the frequencies of splenic CD62L+KLRG1' T cells in Lmo4-Thyl.l T cells relative to Thyl.2 knockout controls. Conversely, the percentage of CD62L+KLRGl terminal effectors was increased upon deletion of the IL-21 receptor). Notably knocking out 1121 r did not have a significant impact on CD8+ T cells transduced with Thyl.l. The negative impact of Il21r deletion on the generation of CD62L+ memory T cell precursors abrogated the typical accumulation of Lmo4-Thyl.l T cells in lymph nodes, whereas it had no effects on Thy.1.1 controls. While the involvement of other STAT3-signaling cytokines cannot be excluded, the findings highlight the pivotal role of IL-21 signaling in the setting of LM04 overexpression. This supports a model by which LM04 enhances T-cell sternness by boosting IL21-STAT3 signaling and its downstream promemory factors.

[0209] Example 7: LM04 overexpression increases CD8-positive T-cell sternness and antitumor immunity in a STAT3-dependent manner

[0210] To demonstrate that STAT3 signaling is a crucial mediator of the memory phenotype induced by LM04, the effects of knocking out Stat3 on the expansion and memory differentiation of pmel-1 Lmo4- Thyl.l T cells was evaluated (Figure 26). CRISPR / Cas9 technology was employed to delete Stat3 or Thyl.2 control with knock-out efficiencies of 90% and 78%, respectively (Figure 27).

[0211] Next, pmel-1 Lmo4-Thyl.l and pmel-1 Thyl.l T cells either deficient for Stat3 or Thyl.2 were adoptively transferred into wild-type mice infected with gplOO-VV, and antigen-specific CD8-positive T-cell expansion as well as memory formation was measured over time. It was found that the deletion of Stat3 abrogated the beneficial effects of LMO4 overexpression on T-cell expansion (Figures 28 and 29). Of note no major impact of Stat3 removal on Thyl.l control cells was observed, highlighting the specific role of STAT3 in the context of LM04 overexpression. Similarly, Stat3 deficiency impaired the augmented formation of stem-like T cells observed in the setting of LM04 overexpression (Figure 30 and 31). These results demonstrate the central role of the STAT3 signaling pathway in LMO4-induced regulation of CD8-positive T-cell responses and their development of stem-like cells.

[0212] To further determine if STAT3 signaling is also essential for the antitumor effects of LMO4- overexpressing T cells, pmel-lCas9 Lmo4-Thyl.l and Thyl.l T cells nucleofected with sgRNAs specific for Stat3 or Thyl.2 were adoptively transferred into mice bearing subcutaneous B16KVP melanomas in conjunction with gplOO-VV and IL-2. Consistent with the findings in the infection model, it was found that the enhanced antitumor responses promoted by the heightened levels of LMO4 also depend on STAT3 (Figures 32 and 33). Altogether, these findings highlight the pivotal role of STAT3 in mediating the effects of LMO4 in enhancing CD8-positive T-cell sternness and antitumor immunity.

Claims

Claims1. A recombinant CD8-positive T cell overexpressing a recombinant nucleic acid encoding a functionalLM04 polypeptide for use in the treatment of a cancer or a tumor.

2. The recombinant CD8-positive T cell for use according to claim 1, wherein said recombinant nucleic acid encoding said functional LM04 polypeptide is ectopically expressed.

3. The recombinant CD8-positive T cell for use according to claim 1 or 2, wherein said recombinant nucleic acid is operably linked to a promoter.

4. The recombinant CD8-positive T cell for use according to any one of the preceding claims, wherein said recombinant nucleic acid is encoded on an expression vector, preferably a viral vector or a plasmid.

5. The recombinant CD8-positive T cell for use according to claim 4, wherein said expression vectors triggers the overexpression of said recombinant nucleic acid encoding said functional LM04 polypeptide.

6. The recombinant CD8-positive T cell for use according to any one of the preceding claims, wherein said recombinant nucleic acid encoding said functional LM04 polypeptide is different from a genomic copy of LM04 in said host cell.

7. The recombinant CD8-positive T cell for use according to any one of the preceding claims, wherein said functional LM04 polypeptide comprises the amino acid sequence of SEQ ID No. 1.

8. The recombinant CD8-positive T cell for use according to any one of the preceding claims, wherein said recombinant nucleic acid encoding the functional LM04 polypeptide comprises the nucleic acid sequence of SEQ ID No. 2.

9. The recombinant CD8-positive T cell for use according to any one of the preceding claims, wherein said T cell further comprises a nucleic acid encoding a chimeric antigen receptor or a T cell receptor.

10. The recombinant CD8-positive T cell for use according to claim 9, wherein said chimeric antigen receptor or said T cell receptor is specific for a cancer antigen.

11. The recombinant CD8-positive T cell for use according to any one of the preceding claims, wherein said cancer or tumor is a solid cancer or a hematological cancer.

12. The recombinant T cell for use according to any one of the preceding claims, wherein said T cell is an isolated or purified T cell, preferably an isolated or purified human T cell.

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