Host immune cells engineered to overexpress a FOXK1 polypeptide

Abstract

T lymphocytes play a key role in the immune response and their functions are intimately linked to metabolic programs. During immune responses, T cells undergo a metabolic reprogramming notably characterized by an increased aerobic glycolysis. Using a quantitative phosphoproteomic approach, the inventors have identified a new transcription factor called Foxk1 as being highly phosphorylated in T cells upon T Cell Receptor (TCR) engagement. The results also indicate that Foxk1 phosphorylation and nuclear translocation is dependent of the AKT-mTOR kinase activities. Using T-cell specific Foxk1 deficient mice (Foxk1- / -), we demonstrated that Foxk1 is required for full T cell activation. Foxk1-deficient T cells exhibited reduced proliferation and cytokine secretion following TCR stimulation. Furthermore, T cells from Foxk1- / - mice were less prone to acquire an effector like phenotype than wild-type cells when challenged in vivo. Conversely, Foxk1 overexpression in T cells enhanced their effector functions in a TCR-dependent manner. In CD8+ T cells, this effect also results into enhanced cancer cell killing capacity in vitro, and improved tumor rejection in vivo. Altogether, these results indicated that Foxk1 is a major regulator of T cell metabolism and thus, of T cell effector functions. Thus, the present invention relates to host immune cells engineered to overexpress a Foxk1 polypeptide and their use of the treatment of cancer.

Description

[0001] HOST IMMUNE CELLS ENGINEERED TO OVEREXPRESS A FOXK1 POLYPEPTIDE

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular immunology.

[0004] BACKGROUND OF THE INVENTION:

[0005] T lymphocytes are essential for the recognition and elimination of cancer cells, which often evade the immune system by expressing or suppressing molecules that interfere with T cell activation. Therefore, enhancing the metabolic fitness and function of T cells is a promising strategy to improve cancer immunotherapy. One of the most successful approaches in this field is the use of chimeric antigen receptor (CAR) T cells, which are genetically modified to express a synthetic receptor that recognizes a specific antigen on tumor cells. CAR T cells can bypass the conventional mechanisms of T cell activation and directly target and kill cancer cells. However, CAR T cells also face metabolic challenges in the tumor microenvironment, such as nutrient deprivation, hypoxia, and immunosuppression. Thus, modulating the metabolic pathways of T cells and in particular CAR T cells could enhance their efficacy and persistence in cancer treatment. During immune responses, T cells undergo a metabolic reprogramming characterized by increased aerobic glycolysis, nutrient uptake and macromolecules synthesis. These processes are crucial for cell growth, differentiation, and acquisition of effector functions. In this regard, the AKT-mTOR signalling pathway has been shown to be an essential hub for regulating T cell metabolism and, consequently cell fate.

[0006] Among the metabolic regulators of T cell function, the forkhead box (Fox) family of transcription factors has emerged as a key player. Fox proteins modulate the expression of genes involved in cell cycle, apoptosis, differentiation, and metabolism. In particular, FoxOl and FoxO3 have been extensively studied in T cells and shown to control their quiescence, activation, and memory formation. However, less is known about other members of the Fox family, such as Foxkl, which belongs to the subfamily K of Fox proteins. Foxkl is highly expressed in skeletal muscle and regulates its growth and regeneration. Recently, it has been reported that FoxKl is also expressed in lymphocytes and could play a role in their functions (Sukonina, Valentina, et al. "F0XK1 and F0XK2 regulate aerobic glycolysis. " Nature 566. 7743 (2019): 279-283). SUMMARY OF THE INVENTION:

[0007] The present invention is defined by the claims. In particular, the present invention relates to a host immune cell that is engineered to overexpress a Foxkl polypeptide.

[0008] DETAILED DESCRIPTION OF THE INVENTION:

[0009] Main definitions:

[0010] As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

[0011] As used herein, the term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0012] As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).

[0013] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention, a first amino acid sequence having at least 90% of identity with a second amino acid sequence means that the first sequence has 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence.

[0014] As used herein, the term “expression” of a polynucleotide sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5Zcap formation, and / or 3Zend formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0015] As used herein, the term "overexpression" refers to the production of a protein or RNA at a level that is higher than its normal or physiological level in a cell or organism. Overexpression can be achieved by various methods, such as introducing additional copies of a gene or cDNA encoding the protein or RNA into a cell, using strong promoters or enhancers to drive the expression of the gene or cDNA, deleting or inhibiting negative regulators of the gene or cDNA, or activating or enhancing positive regulators of the gene or cDNA. Overexpression can be measured by comparing the level of the protein or RNA in a cell or organism with a reference level, such as the level in a control cell or organism, or the average level in a population of cells or organisms.

[0016] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase “polynucleotide sequence that encodes a protein or a RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0017] As used herein, the term "Foxkl" refers to a member of the forkhead family of transcription factors and refers to the Forkhead Box Protein KI that is encoded by the Foxkl gene (Gene 221937). Foxkl is also known as Mnf. Foxkl induces aerobic glycolysis by upregulating the enzymatic machinery required for this (for example, hexokinase-2, phosphofructokinase, pyruvate kinase, and lactate dehydrogenase), while at the same time suppressing further oxidation of pyruvate in the mitochondria by increasing the activity of pyruvate dehydrogenase kinases 1 and 4. Together with suppression of the catalytic subunit of pyruvate dehydrogenase phosphatase 1 this leads to increased phosphorylation of the El a regulatory subunit of the pyruvate dehydrogenase complex, which in turn inhibits further oxidation of pyruvate in the mitochondria — instead, pyruvate is reduced to lactate. Suppression of Foxkl induces the opposite phenotype. An exemplary amino acid sequence of Foxkl is shown as SEQ ID NO:1. SEQ ID NO : 1 >sp | P85037 | FOXK1_HUMAN Forkhead box protein KI OS=Homo sapiens OX=9606 GN=FOXK1 PE=1 SV=1 MAEVGEDSGARALLALRSAPCSPVLCAAAAAAAFPAAAPPPAPAQPQPPPGPPPPPPPPL PPGAIAGAGSSGGSSGVSGDSAVAGAAPALVAAAAASVRQSPGPALARLEGREFEFLMRQ PSVTIGRNSSQGSVDLSMGLSSFI SRRHLQLSFQEPHFYLRCLGKNGVFVDGAFQRRGAP ALQLPKQCTFRFPSTAIKIQFTSLYHKEEAPASPLRPLYPQI SPLKIHI PEPDLRSMVSP VPSPTGTI SVPNSCPASPRGAGSSSYRFVQNVTSDLQLAAEFAAKAASEQQADTSGGDSP KDESKPPFSYAQLIVQAI SSAQDRQLTLSGIYAHITKHYPYYRTADKGWQNSIRHNLSLN RYFIKVPRSQEEPGKGSFWRIDPASEAKLVEQAFRKRRQRGVSCFRTPFGPLSSRSAPAS PTHPGLMSPRSGGLQTPECLSREGSPI PHDPEFGSKLASVPEYRYSQSAPGSPVSAQPVI MAVP P RP S S L VAK P VAYMP AS I VT S QQ P AGHAI H WQQAP T VTMVRWT T SAN SAN GY I L TSQGAAGGSHDAAGAAVLDLGSEARGLEEKPTIAFATI PAAGGVIQTVASQMAPGVPGHT VTILQPATPVTLGQHHLPVRAVTQNGKHAVPTNSLAGNAYALTSPLQLLATQASSSAPW VT RVC E VG P KE P AAAVAAT AT T T PAT AT TASASASSTGEP E VKRS RVE EPS GAVT T P AGV IAAAGPQGPGTGE

[0018] As used herein, the term “Foxkl polypeptide” refers to any polypeptide that comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

[0019] A used herein, the term "host cell" or "recipient cell" refers to a cell that was genetically engineered, i.e. harboring an exogenous nucleotide sequence, preferably stably integrated, in its genome.

[0020] As used herein, the term “host immune cell” refers to a host cell that functions in an immune response or a progenitor, or progeny thereof that may express a native or an exogenous TCR on its surface, depending on the context and the purpose of the invention. As used herein, the term “host immune cell” refers to a host cell that functions in an immune response or a progenitor, or progeny thereof. A host immune cell may also be a precursor cell that can differentiate into a T cell, such as a hematopoietic stem cell or a common lymphoid progenitor. A progeny of a host immune cell may be a cell that is derived from a host immune cell by cell division, genetic modification, or fusion.

[0021] As used herein, the term “TCR” has its general meaning in the art and refers to the molecule found on the surface of T cells that is responsible for recognizing antigens bound to MHC molecules. The TCR heterodimer consists of an alpha and beta chain in 95% of T cells, whereas 5% of T cells have TCRs consisting of gamma and delta chains. Engagement of the TCR with antigen and MHC results in activation of its T lymphocyte through a series of biochemical events mediated by associated enzymes, co-receptors, and specialized accessory molecules. Each chain of the TCR is a member of the immunoglobulin superfamily and possesses one N- terminal “immunoglobulin (Ig)-variable (V) domain”, one “Ig-constant (C) domain”, a “transmembrane region”, and a short “cytoplasmic tail” at the C-terminal end. The constant domain of the TCR consists of short connecting sequences in which a cysteine residue forms a disulfide bond, making a link between the two chains. The structure allows the TCR to associate with other molecules like CD3 which possess three distinct chains (y, 5, and a) in mammals and the ^-chain. These accessory molecules have negatively charged transmembrane regions and are vital to propagating the signal from the TCR into the cell. The CD3 chains, together with the TCR, form what is known as the TCR complex. The signal from the TCR complex is enhanced by simultaneous binding of the MHC molecules by a specific co-receptor. On helper T cells, this co-receptor is CD4 (specific for class II MHC); whereas on cytotoxic T cells, this co-receptor is CD8 (specific for class I MHC). The co-receptor not only ensures the specificity of the TCR for an antigen, but also allows prolonged engagement between the antigen presenting cell and the T cell and recruits essential molecules (e.g., LCK) inside the cell involved in the signaling of the activated T lymphocyte.

[0022] As used herein, the term “T cell” has its general meaning in the art and represent an important component of the immune system that plays a central role in cell-mediated immunity. T cells are known as conventional lymphocytes as they recognize the antigen with their TCR (T cell receptor for the antigen) with presentation or restriction by molecules of the complex major histocompatibility. There are several subsets of T cells each having a distinct function such as CD8+ T cells, CD4+ T cells, and gamma delta T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. The term also encompasses tumor infiltrating lymphocytes. As used herein, the term “tumor infiltrating lymphocyte” or “TIL” to T cells that are present in a solid tumor.

[0023] As used herein, the term “T-cell response” means the specific proliferation and activation of effector functions induced by an antigen in vitro or in vivo. For instance, MHC class I restricted cytotoxic T-cells, effector functions may be lysis of antigen-presenting target-cells, secretion of cytokines, preferably Interferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion of effector molecules, preferably granzymes or perforins induced by peptide, or degranulation.

[0024] As used herein, the term "population" refers to a population of cells, wherein the majority (e.g., at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80%) of the total number of cells have the specified characteristics of the cells of interest and express the markers of interest (e.g. a population of human host immune cells comprises at least about 50%, preferably at least about 60%, more preferably at least about 70%, and even more preferably at least about 80% of cells which have the highly immune functions).

[0025] As used herein, the term “engineered” refers to an aspect of having been manipulated and altered by the hand of man. In particular, the term “engineered cell” refers to a cell that has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation. In some embodiments, a genetic manipulation is or comprises one or more of (i) introduction of a polynucleotide not present in the cell prior to the manipulation (i.e., of a heterologous polynucleotide); (ii) removal of a polynucleotide, or portion thereof, present in the cell prior to the manipulation; and / or (iii) alteration (e.g., by sequence substitution) of a polynucleotide, or portion thereof, present in the cell prior to the manipulation. In some embodiments, a an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a polynucleotide, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. Those of ordinary skill in the art will appreciate that reference to an “engineered cell” herein may, in some embodiments, encompass both the particular cell to which the manipulation was applied and also any progeny of such cell.

[0026] As used herein, the term “chimeric antigen receptor” or “CAR” has its general meaning in the art and comprises one or more artificially constructed hybrid polypeptides containing an antigen binding domain linked to one or more T- cell signalling domains. Characteristics of CARs include their ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC -restricted manner, exploiting e.g. the antigen-binding properties of monoclonal antibodies. The chimeric antigen receptor of the present invention typically comprises one more polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain.

[0027] As used herein the term "CAR-T cell" refers to a T lymphocyte that has been genetically engineered to express a CAR. The T lymphocytes that are genetically modified may be "derived" or "obtained" from the patient who will receive the treatment using the genetically modified T cells or they may be "derived" or "obtained" from a different patient.

[0028] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0029] As used herein, the expression “therapeutically effective amount” is an amount sufficient to effect a beneficial or desired clinical result upon treatment. An effective amount can be administered to a subject in one or more doses. In terms of treatment, an effective amount is an amount that is sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease. The effective amount is generally determined by the physician on a case-by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the subject, the condition being treated, the severity of the condition and the form and effective concentration of the host immune cells administered.

[0030] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0031] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0032] Engineered host immune cells of the present invention:

[0033] The first object of the present invention related to a host immune cell that is engineered to overexpress a Foxkl polypeptide.

[0034] Foxkl polypeptide:

[0035] In some embodiments, the Foxkl polypeptide of the present invention comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

[0036] In some embodiments, the Foxkl polypeptide of the present invention comprises the amino acid sequence h as set forth in SEQ ID NO: 1 that comprises one or more conservative modification, more particularly one or more conservative substitutions. As used herein, the term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the biologic function of the protein containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into a protein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0037] Host immune cells:

[0038] In some embodiments, the host immune cell of the present invention expresses an endogenous TCR. As used herein, the term “endogenous” or “native” refers to a polypeptide or polynucleotide that is normally present in a host cell.

[0039] In some embodiments, the host immune cell of the present invention expresses an exogenous TCR (i.e. the host cell is engineered for expressing a TCR of interest) as described in Schober, Kilian, et al. "Orthotopic replacement of T-cell receptor a-and f-chains with preservation of near-physiological T-cell function. "Nature biomedical engineering 3.12 (2019): 974-984 and Rohaan, M. W., et al. "MART-1 TCR gene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I / IIa clinical trial. " Immuno-Oncology and Technology 15 (2022): 100089.

[0040] As used herein, the term “exogenous T cell receptor” or “exogenous TCR” refers to a recombinant TCR which is expressed in a host immune cell by introduction of exogenous polynucleotides encoding for a TCR, i.e. one polynucleotide encoding for the alpha chain and one polynucleotide encoding for the beta chain. In particular, the exogenous TCR may be expressed in a cell in which the TCR is either not natively expressed or is expressed at levels that are insufficient to induce a response by the cell or a responder cell upon TCR ligand binding.

[0041] In some embodiments, the host immune cell is a murine or a human cell.

[0042] In some embodiments, the host cell is a hematopoietic cell from the lymphoid lineage that comprises peripheral blood mononuclear cells (PBMC), and other blood cell subsets such as, but not limited to, T-cells such as tumor infiltrating lymphocytes (TILS), CD4+ T-cells or CD8+ T-cells. Non-limiting examples of host cells of the lymphoid lineage include T cells, and precursors thereof including embryonic stem cells, and pluripotent stem cells (e.g., those from which lymphoid cells may be differentiated). T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cells, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, Regulatory T cells (also known as suppressor T cells), and y5 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells.

[0043] In some embodiments, the host immune cell is a T cell. The T cell can be a CD4+ T cell or a CD8+ T cell. In some embodiments, the T cell is a CD4+ T cell. In some embodiments, the T cell is a CD8+ T cell.

[0044] In some embodiments, the host immune cell is a pluripotent stem cell (PSC). PSCs can be indeed be modified by a CAR and then can be used for deriving T cells (e.g. WO 2017100403). PSCs include embryonic stem cell (ESCs) and induced pluripotent stem cell (iPSCs). iPSCs can be generated directly from adult cells (e.g., somatic cells). iPSCs can be typically derived or generated by introducing a specific set of pluripotency-associated genes, or "reprogramming factors", into a given cell type. Reprogramming factors include, but are not limited to, OCT4 (also known as "POU5FL"), SOX2, cMYC, and KLF4, which are also known as Yamanaka factors. See Takahashi, K; Yamanaka, S (2006). "Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors". Cell 126 (4): 663-76.

[0045] In some embodiments, the host immune cell is a hematopoietic stem cell. As used herein, the term “hematopoietic stem cell” or “HSC” refers to blood cells that have the capacity to selfrenew and to differentiate into precursors of blood cells. These precursor cells are immature blood cells that cannot self-renew and must differentiate into mature blood cells. Hematopoietic stem progenitor cells display a number of phenotypes, such as Lin- CD34+CD38-CD90+CD45RA-, Lin-CD34+CD38-CD90-CD45RA-, Lin-

[0046] CD34+CD38+IL-3aloCD45RA-, and Lin-CD34+CD38+CD 10+(Daley et al., Focus 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. Ill: Hematopoietic Growth Factors and Cytokines, pp. 1-2, CRC Press, Boca Raton, Fla., 1994). Within the bone marrow microenvironment, the stem cells self-renew and maintain continuous production of hematopoietic stem cells that give rise to all mature blood cells throughout life. In some embodiments, the hematopoietic progenitor cells or hematopoietic stem cells are isolated form peripheral blood cells.

[0047] In some embodiments, the host immune cell of the present invention is engineered for expression a CAR. In some embodiments, the host immune cells of the present invention are CAR-T cells.

[0048] Chimeric antigen receptor (CAR):

[0049] Any CARs that are suitable for engineering host immune cells for use in adoptive immunotherapy therapy can be used in the present invention. CARs that can be used in the present invention include those described in Sadelain, et al., “The Basic Principles of Chimeric Antigen Receptor Design.” Cancer Discovery, OF1-11, (2013), Chicaybam, et al., (2011), Brentjens et al. Nature Medicine 9:279-286 (2003), and U.S. Pat. No. 7,446,190, which are herein incorporated by reference in their entireties.

[0050] The CAR of the present invention typically comprises one more polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain.

[0051] In some embodiments, the CAR is a monomeric molecule that consists of one polypeptide having an extracellular domain and an intracellular domain joined by a transmembrane domain.

[0052] In some embodiments, the CAR is a heterodimeric molecule that consists of two polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain and wherein the two polypeptides are capable of dimerizing via their respective extracellular domain.

[0053] Extracellular domain:

[0054] The extracellular domain of the CAR, expressed on the surface of the host immune cell, comprises an antigen binding domain.

[0055] In some embodiments, the antigen binding domain is derived from an antibody, preferably a humanized or a human antibody.

[0056] As used herein the term "antibody" and "immunoglobulin" have the same meaning, and will be used equally in the present invention. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody fragments as well as variants (including derivatives) of antibodies and antibody fragments. In natural antibodies, two heavy chains are linked to each other by disulfide bonds and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chain, lambda (1) and kappa (k). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE. Each chain contains distinct sequence domains. The light chain includes two domains, a variable domain (VL) and a constant domain (CL). The heavy chain includes three (a, 5, y) to five (p, s) domains, a variable domain (VH) and three to four constant domains (CHI, CH2, CH3 and CH4 collectively referred to as CH). The variable regions of both light (VL) and heavy (VH) chains determine binding recognition and specificity to the antigen. The constant region domains of the light (CL) and heavy (CH) chains confer important biological properties such as antibody chain association, secretion, trans-placental mobility, complement binding, and binding to Fc receptors (FcR). The Fv fragment is the N-terminal part of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of the antibody resides in the structural complementarity between the antibody combining site and the antigenic determinant. Antibody combining sites are made up of residues that are primarily from the hypervariable or complementarity determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence the overall domain structure and hence the combining site. CDRs refer to amino acid sequences which together define the binding affinity and specificity of the natural Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H- CDR1, H-CDR2, H-CDR3, respectively. An antigen-binding site, therefore, typically includes six CDRs, comprising the CDR set from each of a heavy and a light chain V region. Framework Regions (FRs) refer to amino acid sequences interposed between CDRs. The residues in antibody variable domains are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1987, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereafter “Kabat et al ”). This numbering system is used in the present specification. The Kabat residue designations do not always correspond directly with the linear numbering of the amino acid residues in SEQ ID sequences. The actual linear amino acid sequence may contain fewer or additional amino acids than in the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, whether framework or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues may be determined for a given antibody by alignment of residues of homology in the sequence of the antibody with a “standard” Kabat numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31-35B (H-CDR1), residues 50-65 (H-CDR2) and residues 95-102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24-34 (L-CDR1), residues 50-56 (L-CDR2) and residues 89-97 (L-CDR3) according to the Kabat numbering system.

[0057] In some embodiments, the antigen binding domain is an antibody fragment.

[0058] As used herein, the term "antibody fragment" refers to at least one portion of an intact antibody, preferably the antigen binding region or variable region of the intact antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. “Fragments” comprise a portion of the intact antibody, generally the antigen binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2, and Fv fragments; diabodies; any antibody fragment that is a polypeptide having a primary structure consisting of one uninterrupted sequence of contiguous amino acid residues (referred to herein as a “single-chain antibody fragment” or “single chain polypeptide”), including without limitation (1) single - chain Fv molecules (2) single chain polypeptides containing only one light chain variable domain, or a fragment thereof that contains the three CDRs of the light chain variable domain, without an associated heavy chain moiety and (3) single chain polypeptides containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region, without an associated light chain moiety; and multispecific antibodies formed from antibody fragments. Fragments of the present antibodies can be obtained using standard methods.

[0059] In some embodiments, the antigen binding domain is selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments. In some embodiments, the antigen binding domain is a single-chain antibody fragment (comprising a variable heavy chain region and / or a variable light chain region. In some embodiments the antigen binding domain is selected from a Fab and a scFv. In some embodiments, when the antigen binding domain is a scFv.

[0060] As used herein, the term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0061] In some embodiments, the scFv can be derived from the variable heavy chain (VH) and variable light chain (VL) regions of an antigen-specific mAb linked by a flexible linker. The scFv retains the same specificity and a similar affinity as the full antibody from which it is derived. The peptide linker connecting scFv VH and VL domains joins the carboxyl terminus of one variable region domain to the amino terminus of the other variable domain without compromising the fidelity of the VH-VL paring and antigen-binding sites. Peptide linkers can vary from 10 to 30 amino acids in length. In some embodiments, the scFv peptide linker is a Gly / Ser linker and comprises one or more repeats of the amino acid sequence Gly-Gly-Gly-Ser or Gly-Gly-Gly-Gly-Ser.

[0062] In some embodiments, the extracellular domain optionally comprises a spacer or hinge domain linking the antigen binding domain to the transmembrane domain. In some embodiments, the CAR thus comprises a hinge sequence between the antigen binding domain and the transmembrane domain and / or between the transmembrane domain and the cytoplasmic domain. One ordinarily skilled in the art will appreciate that a hinge sequence is a short sequence of amino acids that facilitates flexibility. In particular, the spacer or hinge domain linking the antigen binding domain to the transmembrane domain is designed to be sufficiently flexible to allow the antigen binding domain to orient in a manner that allows antigen recognition. The hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgGl Fc region, an IgG2 Fc region, an IgG3 Fc region, an IgG4 Fc region, an IgE Fc region, an IgM Fc region, or an IgA Fc region. In some embodiments, the hinge domain includes at least a portion of an IgGl, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains. Exemplary hinges include, but are not limited to, a CD8a hinge, a CD28 hinge, IgGl / IgG4 (hinge-Fc part) sequences, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, international patent application publication number W02014031687, U.S. Pat. No. 8,822,647 or published app. No. US2014 / 0271635. As hinge domain, the invention relates to all or a part of residues 118 to 178 of CD8a (GenBank Accession No. NP_001759.3), residues 135 to 195 of CD8 (GenBank Accession No. AAA35664), residues 315 to 396 of CD4 (GenBank Accession No. NP_000607.1), or residues 137 to 152 of CD28 (GenBank Accession No. NP 006130.1) can be used. Also, as the spacer domain, a part of a constant region of an antibody H chain or L chain (CHI region or CL region) can be used. Further, the spacer domain may be an artificially synthesized sequence. In some embodiments, for example, the hinge sequence is derived from a CD8 alpha molecule or a CD28 molecule.

[0063] Transmembrane domain

[0064] The transmembrane domain of the CAR functions to anchor the receptor on the cell surface. The choice of the transmembrane domain may depend on the neighbouring spacer and intracellular sequences.

[0065] In some embodiments, the transmembrane domain is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some embodiments is derived from any membrane -bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T- cell receptor, CD28, CD3 zeta, CD3 epsilon, CD3 gamma, CD3 delta, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS / CD278, GITR / CD357, NKG2D, and DAP molecules. Alternatively, the transmembrane domain in some embodiments is synthetic. In some embodiments, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. A transmembrane domain is thermodynamically stable in a membrane. It may be a single alpha helix, a transmembrane beta barrel, a beta-helix of gramicidin A, or any other structure. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular signalling domain(s) of the CAR. A glycine-serine doublet may provide a suitable linker.

[0066] Intracellular domain

[0067] The role of the intracellular domain of the CAR is to produce an activation signal to the host immune cell (e.g. T cell) as soon as the extracellular domain has recognized the antigen. In particular, the intracellular domain of the CAR triggers or elicits activation of at least one of the normal effector functions of the host immune cell. Examples of intracellular domain sequences that are of particular use in the invention include those derived from an intracellular signalling domain of a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3< FcRy, FcRp, CD3y, CD35, CD3s, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRI, DAP10, and DAP12. It is particularly preferred that the intracellular domain in the CAR comprises a cytoplasmic signalling sequence derived from CD3(^. The intracellular domain of the CAR can be designed to comprise a signalling domain (such as the CD3(^ signalling domain) by itself or combined with costimulatory domain(s). A costimulatory molecule can be defined as a cell surface molecule that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4- 1BB (CD137), 0X40 (CD134), CD30, CD40, CD244 (2B4), ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D. The intracellular signalling portion of the above recited co-stimulatory domains can be used alone or in combination with other co-stimulatory domains. In particular, the CAR can comprise any combination of two or more co-stimulatory domains from the group consisting of CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, CD244 (2B4), ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, CD8, CD4, b2c, CD80, CD86, DAP10, DAP12, MyD88, BTNL3, and NKG2D.

[0068] The CAR of the invention may be a first generation, a second generation, or a third generation CAR as described hereabove. Preferably, the CAR is a second or third generation CAR. Typically, “first-generation CARs” contain a single signalling domain. CARs containing a signalling domain together with one additional costimulatory domain are termed “second generation” while those containing a signalling domain together with two additional costimulatory domains are listed as “third generation”. For example, first-generation CARs contain solely the CD3(^ chain as a single signalling domain. Second- and third-generation CARs consist of one or two additional costimulatory signalling domains, respectively, such as CD28, CD27, OX-40 (CD134) and 4-1BB (CD137). For example, second-generation CAR may contain CD3(^ and CD28 signalling domains, while third-generation CAR may contain CD3(^, CD28 and either 0X40 (CD134) or 4-1BB (CD137). “TRUCKs” represent the recently developed “fourth-generation” CARs. TRUCKs (T cells redirected for universal cytokine killing) are CAR-redirected T cells used as vehicles to produce and release a transgenic product that accumulates in the targeted tissue. The product, for example a pro-inflammatory cytokine, may be constitutively produced or induced once the T cell is activated by the CAR. Other substances such as enzymes or immunomodulatory molecules may be produced in the same way and deposited by CAR-redirected T cells in the targeted lesion. This strategy involves two separate transgenes expressing for example (i) the CAR and (ii) a cell activation responsive promoter linked to a cytokine such as IL-12. Consequently, immune stimulatory cytokine such as IL-12 is secreted upon CAR engagement. In a particular embodiment, the CAR is a CAR of fourth generation as defined above.

[0069] HLA-independent T cell receptor (HIT receptor) or synthetic T cell receptor (TCR) and antigen receptors (STAR):

[0070] In some embodiments, the CAR of the present invention consists in a TCR mimic. TCR mimics were recently developed in which the Va and Vb domains of native TCR were replaced by the VH and VL domains of an antibody specific for a given antigen. They confer HLA-independent recognition of antigen and coincidently permit to benefit of the high antigen sensitivity supplied by the structure of the native TCR-CD3 architecture. They are denoted as “Synthetic TCR and Antigen Receptor” or “STAR” as described in / . / > / , Yue, etal. "Chimeric STAR receptors using TCR machinery mediate robust responses against solid tumors. " Science Translational Medicine 13.586 (2021): eabb5191.,' Wang, Jiasheng, et al. "A Novel Adoptive Synthetic TCR and Antigen Receptor (STAR) T - Cell Therapy for B - Cell Acute Lymphoblastic Leukemia. " American Journal of Hematology (2022) and in W02020029774 that are incorporated by reference or “HLA-independent T cell receptor” or “HIT receptor” as described mMansilla- Soto, J., Eyquem, J., Haubner, S. et al. HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 28, 345- -352 (2022), and in WO2019157454 that are incorporated by reference. Upon expression in human T cells, STARs and HIT receptors mediate tumor recognition beyond what CD28-based CARs, the most sensitive design to date, can provide. The STAR and HIT architecture cannot incorporate a CD28 transmembrane domain. Moreover, steric hindrance limits the benefits expected from the incorporation of the CD28 intracytoplasmic segment at the carboxy-terminus of the TCR a or b chains (Wang, Jiasheng, et al. "A Novel Adoptive Synthetic TCR and Antigen Receptor (STAR) T - Cell Therapy for B - Cell Acute Lymphoblastic Leukemia. " American Journal of Hematology (2022)). Therefore, host immunes cells armed with STAR and HIT receptors will be particularly prone to benefit of the costimulatory signals provided by co-expressed the Foxkl polypeptide of the present invention.

[0071] In some embodiments, the CAR of the present invention thus derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domains are substituted by a variable domain of an antibody.

[0072] In some embodiments, the CAR of the present invention derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a first variable domain of an antibody (e.g. a VL or VH domain) and the immunoglobulin Invariable (V) domain of the beta chain is substituted by a second variable domain of an antibody (e.g. a VL or VH domain) wherein the first variable domain is capable of dimerizing with the second variable domain to form a fragment variable (Fv) that binds to the antigen of interest.

[0073] In some embodiments, the CAR of the present invention derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a VL domain of an antibody and the immunoglobulin (Ig)-variable (V) domain of the beta chain is substituted by a VH domain of an antibody wherein the VL domain is capable of dimerizing with the VH domain to form a fragment variable (Fv) that binds to the antigen of interest.

[0074] According to the present embodiments, the CAR is thus capable of associating with a CD3 complex to form the T-cell co-receptor. In some embodiments, the CD3 complex comprises a CD3y chain, a CD35 chain, and two CD3s chains. In some embodiments, the CAR of the present invention and the CD3 complex form an antigen recognizing receptor complex similar to a native TCR / CD3 complex. In some embodiments, the CAR of the present invention replaces a native and / or an endogenous TCR in the CD3 / TCR complex of host immune cell.

[0075] Antigens:

[0076] In some embodiments, the host immune cell expresses a TCR or CAR that is specific for an antigen.

[0077] As used herein, the term “antigen” has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized and selectively bound by an antibody or by a T cell antigen receptor, resulting in induction of an immune response. Antigens according to the invention are typically, although not exclusively, peptides and proteins. Antigens may be natural or synthetic and generally induce an immune response that is specific for that antigen.

[0078] In some embodiments, the host immune cell expresses a TCR or CAR that is specific for a tumor antigen.

[0079] As used herein, the term “tumor antigen” refers to an antigen (e.g., a polypeptide) that is uniquely or differentially expressed on a tumor cell compared to a normal cell. In some embodiments, a tumor antigen includes any polypeptide expressed by a tumor that is capable of activating or inducing an immune response via an antigen recognizing receptor (e.g., CD 19, MUC-16) or capable of suppressing an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0080] Non-limiting examples of tumor antigens include carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD 10, CD 19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, CD123, CD44V6, an antigen of a cytomegalovirus (CMV) infected cell (e.g., a cell surface antigen), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine-protein kinases erb-B2,3,4 (erb-B2,3,4), folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2), K-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), LI cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MARTI, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), R0R1, tumor-associated glycoprotein 72 (TAG-72), vascular endothelial growth factor R2 (VEGF-R2), and Wilms tumor protein (WT-1), BCMA, NKCS1, EGF1R, EGFR-VIII, CD99, CD70, ADGRE2, CCR1, LILRB2, LILRB4, PRAME and ERBB.

[0081] In some embodiments, the host immune cell expresses a TCR or CAR that is specific for a pathogen antigen. Non-limiting examples of pathogen includes a virus, bacteria, fungi, parasite and protozoa capable of causing an infectious disease.

[0082] Methods for preparing the host immune cells of the present invention

[0083] The host immune cell of the present invention is preparing by any conventional method well known in the art. Typically, the host immune cells are engineered for expressing the Foxkl polypeptide of the present invention and optionally for expressing a TCR or CAR of interest.

[0084] Thus, a further object of the present invention relates to a method of preparing a host immune cell of the present invention, comprising the step consisting of introducing into a host immune cell a polynucleotide that encodes for the Foxkl polypeptide of the present invention and optionally one or more polynucleotide(s) that encodes for the CAR or TCR of interest.

[0085] The method herein disclosed can be carried out ex vivo or in vivo. As used herein, the term “e vivo" refers to a process that occurs outside of a living organism, typically in a controlled laboratory environment, where cells, tissues, or organs are manipulated or maintained. This approach is often used to engineer immune cells or introduce specific genetic modifications before they are reintroduced into the host organism. As used herein, the term vivo" refers to a process or experiment conducted within a living organism. This typically involves introducing or manipulating cells, tissues, or genetic material directly in the organism.

[0086] It is contemplated that the polynucleotide can be introduced into the host immune cells as naked or in a suitable vector. The polynucleotide may be DNA, circRNA or mRNA. In some embodiments, naked DNA or messenger RNA (mRNA) may be used.

[0087] In some embodiments, the polynucleotide is a naked DNA. Naked DNA generally refers to the DNA contained in a plasmid expression vector in proper orientation for expression. Physical methods for introducing a polynucleotide construct into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, nucleofection, and the like. Other means can be used including colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0088] In some embodiments, the polynucleotide is associated with one or more lipid components. In some embodiments, the one or more lipid components can comprise a cationic lipid. In some embodiments, the one or more lipid components may comprise a non-cationic lipid. In some embodiments, the one or more lipid components may comprise a polar lipid, and / or a non-polar lipid. In some embodiments, the one or more lipid components may comprise a neutral lipid. In some embodiments, the one or more lipid components may comprise a conjugated lipid. In some embodiments, the one or more lipid components may comprise a liposome around the polynucleotide. In some embodiments, the one or more lipid components may comprise a lipid nanoparticle encapsulating the polynucleotide. In some embodiments, the polynucleotide is introduced into the host immune cell by lipid nanoparticle (LNP) encapsulation. Typically, in said embodiments, the polynucleotide is a mRNA or a DNA. As used herein, the term “lipid nanoparticle” or “LNP” refers to a nanoscale particle consisting of lipids, which are typically used to encapsulate and deliver therapeutic agents such as nucleic acids (e.g., DNA or RNA) to specific cells or tissues. These particles are designed to improve the stability, bioavailability, and targeted delivery of the encapsulated material. The LNP encapsulated DNA or RNA can thus be used for transfecting host immune cells ex vivo, or can be administered to a subject (in vivo). The LNP may be designed for targeted delivery for uptake by a host immune cell when delivered locally or systemically to a subject. In some embodiments, the LNP may comprise one or more targeting moieties, such as an antibody or a ligand, or a biomolecule or part thereof that binds to a surface element of a host immune cell and facilitates uptake of the mRNA encapsulated LNP by host immune cells. In some embodiments, the LNP can be targeted to a tissue by one or more antibodies, ligands, binders, aptamers that me be associated or incorporated into the LNP. In some embodiments, the LNP comprises one or more polymers. In some embodiments, the LNP comprises a synthetic polymer. In some embodiments, the LNP comprises a cationic lipid. In some embodiments, the LNP comprises a non-cationic lipid. In some embodiments the LNP comprises a neutral lipid. In some embodiments, the LNP comprises one or more PEGylated lipids. In some embodiments, the LNP is about 100 nm to about 200 nm in diameter. In some embodiments, the LNP is about 150 nm in diameter, such as 80-120 nm, 100-140 nm, 100-130 nm, 70-140 nm, 80-150 nm, or 90-180 nm in diameter. In some embodiments, the LNP is less than 100 nm, such as less than 90 nm, or less than 80 nm in diameter. Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid. The lipid can be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin- MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In some embodiments, the lipid is a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid can be the lipids described in and / or made by the methods described in US Patent Publication No. US20130150625. As a non-limiting example, the cationic lipid can be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2- {[(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-l-yloxy]-2-{[(9Z)-octadec-9-en-l- yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca- 9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]-2-{ [(9Z, 12Z)-octadeca-9, 12- dien-l-yloxy]methyl}propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. Nanoparticle formulations of the present disclosure can be coated with a surfactant or polymer in order to improve the delivery of the particle. In some embodiments, the nanoparticle is coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge. The hydrophilic coatings can help to deliver nanoparticles with larger payloads such as, but not limited to, polynucleotides within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in US Patent Publication No. US20130183244.

[0089] In some embodiments, the polynucleotide is introduced into the host immune cell by a viral vector that is an adeno-associated virus (AAV), a retrovirus, lentivirus, bovine papilloma virus, an adenovirus vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is a retroviral. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, the polynucleotide of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and / or env genes but without the LTR and / or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1, HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign polynucleotide, for selection and for transfer of the polynucleotide into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a polynucleotide encoding a viral gag and a pol gene and another vector that can provide a polynucleotide encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the vector of the present invention includes "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

[0090] Another polynucleotide sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”. To increase the expression, polynucleotides of the present invention may be operably linked to strong promoters, such as retroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murine stem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), P-actin, ubiquitin, and a simian virus 40 (SV40) / CD43 composite promoter, elongation factor (EF)-la and the spleen focus-forming virus (SFFV) promoter.

[0091] In some embodiments, the sequence of the polynucleotides is codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e., codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least U.S. Pat. Nos. 5,786,464 and 6,114,148.

[0092] Use of polycistronic expression cassettes that can both express the Foxkl polypeptide and the TCR or CAR are preferably used. Typically the polycistronic expression cassettes comprise various viral and non-viral Internal Ribosome Entry Sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-KB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picomavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and / or cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A and F2A peptides). Thus use of polycistronic polynucleotides or vectors encoding for both the Foxkl variant and the TCR or CAR are particularly suitable for preparing the host immune cells of the present invention.

[0093] In some embodiments, the polynucleotides that respectively encode for the Foxkl polypeptide and the TCR or CAR are expressed by an host immune cell through a modified genomic locus. In some embodiments, an expression cassette, preferably a polycistronic expression cassette that encodes for the Foxkl polypeptide and the TCR or CAR, is integrated into a targeted genomic locus of an host immune cell through targeted genome editing methods. In some embodiments, the targeted genomic locus can be CD35, CD3s, CD247, B2M, TRAC, TRBC1, TRBC2, TRGC1 and / or TRGC2 loci. In some embodiments, the CAR or TCR of the present invention and the Foxkl polypeptide are expressed by an host immune cell through a modified endogenous T cell receptor locus. In some embodiments, the expression cassette is integrated at an endogenous T cell receptor locus. In some embodiments, the expression cassette is integrated within the T cell receptor alpha locus (TRA, GenBank ID: 6955). In some embodiments, the expression cassette is integrated within the T cell receptor beta locus (TRB, GenBank ID: 6957). In some embodiments, the expression cassette is integrated within the T cell receptor gamma locus (TRG, GenBank ID: 6965). Thus, in some embodiments, the recombinant CAR (or TCR) and Foxkl polypeptide are expressed from an expression cassette placed in an endogenous TRAC locus and / or a TRBC locus of an host immune cell. In some embodiments, the placement of expression cassette disrupts or abolishes the endogenous expression of a TCR comprising a native TCR a chain and / or a native TCR P chain in the host immune cell. In some embodiments, the placement of the expression cassette prevents or eliminates mispairing between the recombinant CAR or TCR and a native TCR a chain and / or a native TCR P chain in the host immune cell.

[0094] Any suitable genetic editing methods and systems can be used to modify an endogenous T cell receptor locus. In some embodiments, a CRISPR system is used to modify T cell receptor locus. In some embodiments, the CRISPR system targets exon 1 of a human TRAC locus. In some embodiments, the CRISPR system comprises a guide RNA (gRNA) that targets exon 1 of a human TRAC locus. In some embodiments, a zinc-finger nuclease is used to modify an endogenous T cell receptor locus. In some embodiments, a TALEN system is used to modify an endogenous T cell receptor locus. In some embodiments, when one endogenous T cell receptor locus in a cell is modified to express the CAR or TCR of the present invention, one or more other endogenous T cell receptor loci in the cell are modified to eliminate the endogenous expression of the endogenous TCR chain.

[0095] Methods for expressing an exogenous TCR are well known in the art and includes gene editing as described in Schober, Kilian, et al. "Orthotopic replacement of T-cell receptor a-and flchains with preservation of near-physiological T-cell function. "Nature biomedical engineering 3.12 (2019): 974-984 and Rohaan, M. W., et al. "MART-1 TCR gene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I / IIa clinical trial. " ImmunoOncology and Technology 15 (2022): 100089.

[0096] Once the population of host immune cells is obtained, functionality of the cells may be evaluated according to any standard method which typically include a suppressive assay. Cell surface phenotype of the cells with the appropriate binding partners can also be confirmed. Quantifying the secretion of various cytokines may also be performed. Methods for quantifying secretion of a cytokine in a sample are well known in the art. For example, any immunological method such as but not limited to ELISA, multiplex strategies, ELISPOT, immunochromatography techniques, proteomic methods, Western blotting, FACS, or Radioimmunoassays may be applicable to the present invention.

[0097] Methods of therapy:

[0098] A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of host immune cells of the present invention.

[0099] The population of host immune cells prepared as described above can be thus utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure. See, e.g., US Patent Application Publication No. 2003 / 0170238 to Gruenberg et al; see also US Patent No. 4,690,915 to Rosenberg. Currently, most adoptive immunotherapies are autolymphocyte therapies (ALT) directed to treatments using the patient's own immune cells. These therapies involve processing the patient's own lymphocytes to enhance the response towards specific antigens. Typically, the treatments are accomplished by removing the patient's lymphocytes and exposing these cells in vitro to biologies and drugs to convey them to a T cell profile. Once the T cells are engineered to express the Foxkl polypeptide and optionally the TCR or CAR of interest, these ex vivo cells are reinfused into the patient to induce an immune response against the antigen of interest. Thus, in some embodiments, the host immune cell is isolated from a subject to whom the engineered host immune cells are to be adoptively transferred. In some embodiments, a population of host immune cells of the present invention are obtained by isolating a population of T-cells from a subject, optionally expanding said population of T cells, and by subsequently proceeding with the Foxkl polynucleotide and optionally TCR / CAR polynucleodie transfer ex vivo and subsequent immunotherapy of the subject by adoptive transfer of the transduced T cells. Alternatively the population of host immune cells is, or is derived from, a population of stem cells, such as a haemopoietic stem cells (HSC). An advantage of this approach is that the gene- modified stem cells are a continuous source of mature T-cells with the desired functionality and antigen specificity. The cell may therefore be a gene-modified stem cell, which, upon differentiation, produces a T-cell expressing the Foxkl polypeptide as well as a TCR / CAR of interest.

[0100] A further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a polynucleotide that encodes for the Foxkl polypeptide of the present invention and optionally one or more polynucleotide(s) that encodes for the CAR or TCR of interest.

[0101] This method of therapy also extends to in vivo approaches where polynucleotides encoding the Foxkl polypeptide, along with the CAR or TCR, are directly introduced into the patient's system. The polynucleotides can be inserted into a vector engineered for high efficiency of delivery and stability, or they can be encapsulated within delivery vehicles such as lipid nanoparticles, which facilitate targeted transport to the desired cells and enhance transfection efficacy. These strategies ensure the therapeutic polynucleotides reach their intended destination while maintaining cellular integrity, thereby enabling precise and effective modification of host immune cells within the patient’s body. In particular, the methods of therapy herein disclosed are particularly suitable for the treatment of cancer or infectious diseases.

[0102] As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood borne tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the present invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malig melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0103] Advantageously, the host immune cells of the present invention exhibit enhanced effector functions in a TCR-dependent manner. In particular, the host immune cells of the present invention enhanced cancer cell killing capacity. Advantageously, the host immune cell of the present invention is also capable of inducing an immune response when binding to an antigen that has a low density on the surface of a tumor cell. In some embodiments, the host immune cells comprising the CAR of the present invention can be used to treat a subject having tumor cells with a low expression level of a surface antigen, e.g., from a relapse of a disease, wherein the subject received treatment which leads to residual tumor cells. In some embodiments, the tumor cells have a low density of an antigen on the surface of the tumor cells. In some embodiments, an antigen having a low density on the cell surface has a density of less than about 5,000 molecules per cell, less than about 4,000 molecules per cell, less than about 3,000 molecules per cell, less than about 2,000 molecules per cell, less than about 1,500 molecules per cell, less than about 1,000 molecules per cell, less than about 500 molecules per cell, less than about 200 molecules per cell, or less than about 100 molecules per cell. In some embodiments, an antigen having a low density on the cell surface has a density of less than about 2,000 molecules per cell. In some embodiments, an antigen having a low density on the cell surface has a density of less than about 1,500 molecules per cell. In some embodiments, an antigen having a low density on the cell surface has a density of less than about 1,000 molecules per cell. In some embodiments, an antigen having a low density on the cell surface has a density of between about 4,000 molecules per cell and about 2,000 molecules per cell, between about 2,000 molecules per cell and about 1,000 molecules per cell, between about 1,500 molecules per cell and about 1,000 molecules per cell, between about 2,000 molecules per cell and about 500 molecules per cell, between about 1,000 molecules per cell and about 200 molecules per cell, or between about 1,000 molecules per cell and about 100 molecules per cell.

[0104] The quantity of host immune cells to be administered will vary for the subject being treated. In some embodiments, between about 104and about 1010, between about 105and about 109, or between about 106and about 108of the hors immune cells are administered to the subject. More effective cells may be administered in even smaller numbers. In some embodiments, at least about l * 108, about 2* 108, about 3* 108, about 4* 108, or about 5* 108of the host immune cells are administered to the subject. The precise determination of what would be considered an therapeutically effective amount may be based on factors individual to each subject, including their size, age, sex, weight, and condition of the particular subject. Dosages can be readily ascertained by those skilled in the art from this disclosure and the knowledge in the art.

[0105] Pharmaceutical compositions:

[0106] In some embodiments, the host immune cells of the present invention are formulated by first harvesting them from their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (i.e. a pharmaceutically acceptable carrier) in a treatment-effective amount. Thus a further object of the present invention relates to a pharmaceutical composition comprising the population of host immune cells of the present invention and a pharmaceutically acceptable carrier.

[0107] Suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), but also 5% dextrose in water or Ringer's lactate can be utilized. The infusion medium can be supplemented with human serum albumin. A treatment-effective amounts of cells in the composition is dependent on the relative representation of the host immune cells with the desired specificity, on the age and weight of the recipient, on the severity of the targeted condition and on the immunogenicity of the targeted Ags. These amounts of cells can be as low as approximately 103 / kg, preferably 5xl03 / kg; and as high as 107 / kg, preferably 108 / kg. The number of cells will depend upon the ultimate use for which the composition is intended, as will the type of cells included therein. For example, if cells that are specific for a particular Ag are desired, then the population will contain greater than 70%, generally greater than 80%, 85% and 90-95% of such cells. For uses provided herein, the cells are generally in a volume of a liter or less, can be 500 ml or less, even 250 ml or 100 ml or less. The clinically relevant number of immune cells can be apportioned into multiple infusions that cumulatively equal or exceed the desired total amount of cells.

[0108] In some embodiments, a pharmaceutical composition is provided comprising a polynucleotide encoding the Foxkl polypeptide, along with one or more polynucleotides encoding chimeric antigen receptors (CAR) and / or T cell receptors (TCR). These polynucleotides may be formulated with advanced delivery vehicles such as liposomes or lipid nanoparticles, which enhance their stability, targeting efficiency, and cellular uptake. The lipid-based nanoparticles serve as carriers that protect the genetic material during delivery, facilitate its transport to the target cells, and ensure effective transfection within the patient's body. This formulation is designed to optimize the therapeutic efficacy of the composition by enabling precise modification of host immune cells, which enhances their effector functions and cancer cellkilling capacity in a TCR-dependent manner. The pharmaceutical composition is suitable for therapeutic applications, including the treatment of cancer and other diseases requiring enhanced immune response. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0109] FIGURES:

[0110] Figure 1: Impact of Foxkl overexpression in mouse T cells. (A) Analysis of Foxkl protein expression in primary T cells retro-infected with Foxkl or control virus. (B) IFN-g secretion of CD4+T cells stimulated with anti-CD3 / CD28 antibodies and retro-infected with Foxkl or control virus. The percentage of CD98 positive cells were assessed in the same conditions (C) IFN-g secretion and cytotoxicity activity of OT-I CD8+T cells retro-infected with Foxkl or control virus and co-incubated with tumor cells expressing chicken ovalbumin protein.

[0111] Figure 2: Analysis of Foxkl deficient T cells. (A) Purified CD4+ T cells from Foxkl fl / fl and Foxkl- / - mice were stimulated for 48h with anti-CD3e plus anti-CD28 antibodies or maintained quiescent with IL-7, and concentration of IL-2 and IFN-y were measured by ELISA. (B) Purified CD8+ T cells from OT-I Foxklfl / fl and Foxkl- / - mice were stimulated for 72 h with antigen presenting cells pulsed with OVA peptides (T4; 10-8M or N4; 10-10M) and concentration of IFN-y were measured by ELISA. (C,D) Purified CTV-labelled naive CD4+ T cells from Foxklfl / fl or Foxkl- / - mice, were injected into CD3s- / - mice. After 6 days, spleens and lymph nodes were harvested and CD4+ T cells were analyzed by flow cytometry for CTV dilution and CD62L and CD44 expression. (E,F) Purified naive CD4+ T cells from Foxklfl / fl or Foxkl- / - mice were injected into CD3s- / - mice. Spleens and lymph nodes were harvested 21 days later and CD4+ T cells were analyzed by flow cytometry for CD62L, CD44, KLRG1 and CD127 expression. Naive, Central Memory (CM) and Effector memory (EM) cells were identified using CD62L and CD44 markers. Short Live Effector Cells (SLECs) and Memory Precursor Effector Cells (MPECs) were identified as KLRGlhighCD1271ow and KLRGllowCD127high cells respectively. Data are representative of two or three independent experiments and are presented as the mean ± SD (3-4 mice / group). The statistical analysis was performed using Welch’s t-tests *p < 0.05, **p < 0.01, ***p < 0.001.

[0112] Figure 3: Impact of Foxkl inactivation on T cell metabolic processes. (A) Purified CD4+ T cells from Foxklfl / fl and Foxkl- / - mice were left untreated (non-stimulated; NS) or were stimulated for 24h with anti- CD3s plus anti-CD28 antibodies or with phorbol myristate plus ionomycin (PI) or maintained quiescent with IL-7, and analyzed for CD71 and CD98 expression by flow cytometry. PI was used as positive control and IL-7 was used as negative control. (B) Analysis of oxygen consumption rate (OCR) on addition of oligomycin (Oligo), fluorocarbonyl cyanide phenylhydrazone (FCCP) and rotenone plus antimycin A (Rot / AA) in CD4+T cells from Foxklfl / fl and Foxkl- / - mice. OCR at maximal respiration is shown on the right. (C,D) Analysis of extracellular acidification rate (ECAR) on addition of glucose (GLC), oligomycin (Oligo) and 2-deoxyglucose (2-DG) C or following CD3 s / CD28 stimulation D in CD4+T cells from Foxklfl / fl and Foxkl- / - mice. (E) Glucose uptake of purified CD4+ T cells from Foxklfl / fl and Foxkl- / - mice, left untreated (non-stimulated; NS) or stimulated for 48h with anti-CD3 antibody in absence or presence of anti-CD28 antibody, or with IL-7. (F) Lactate secretion of similar cells as in (g). Data are representative of two independent experiments and are presented as the mean ± SD (3-4 mice / group). The statistical analysis was performed using Welch’s t-tests *p < 0.05, **p < 0.01, ***p < 0.001.

[0113] Figure 4: Identification of Foxkl targeted genes in T cells. qChIP analysis of Foxkl at the promoters of autophagy genes of unstimulated (Oh) and 4h stimulated CD4+ T cells.

[0114] Figure 5: Role of Foxkl in anti-tumor T cell responses. (A) Purified OT-I CD8+ T cells from Foxklfl / fl and Foxkl- / - mice were co-incubated with A20-SCT cells for 48h and IFN-y+ cells were detected by flow cytometry. A20 cells were used as negative control. (B) Supernatants of the conditions described in A were harvested to perform ELISA to measure IL-2 and IFN-y secretion. (C,D) Specific lysis of A20-SCT and N4 pulsed EL4 cells co-cultured with OT-I CD8+ T cells from Foxklfl / fl and Foxkl- / - mice at the indicated E:T ratios. (E) Foxklfl / fl or Foxkl- / - mice were injected (s.c) in the flank with MC38 colon carcinoma cells and monitored for tumor growth at the indicated time. 6 days post tumor injection mice were treated with anti- PD-1 or Isotype control (ISO) every 3 days. (F) Foxklfl / fl or Foxkl- / - mice were injected with 105 BRAFV600E ptgsl / ptgs2- / - melanoma cells and monitored for tumor growth at the indicated time. Data are representative of two (B, D) or three (A, C) independent experiments and are presented as the mean ± SD with (3-4 mice / group). Data from n=5-9 (E) n=21-24 (F) mice per group. Data are presented as mean ± SEM. The statistical analysis was performed by multiple unpaired t-tests (A-D) and by two way-anova (E-F). *p < 0.05, **p < 0.01, ***p < 0.001.

[0115] Figure 6: Impact of Foxkl overexpression in anti-tumor T cell responses. (A) Control and transduced Foxkl CD4+T cells were activated with anti-CD3 in absence or presence of anti- CD28 for 48h and IFN-y+ cells were detected by flow cytometry. (B) Control and transduced Foxkl OT-I CD8+T cells were co-incubated with A20 and A20-SCT cells for 48h and IFN-y+ cells were detected by flow cytometry. (C,D) Specific lysis of A20-SCT and N4 pulsed EL4 cells co-cultured with control and transduced Foxkl OT-I CD8+T cells at the indicated E:T ratios. (E,F) C57B6 / J mice were injected with 1x106 B16-0VA melanoma cells e, or 5x105 MC38-OVA carcinoma cells F. Day 8 post tumor injection, IM of control or transduced Foxkl OT-I CD8+T cells were adoptively transferred in mice bearing tumor and tumor growth was monitored at the indicated time. Data are representative of two (B, C, E, F) or three (A, D) independent experiments and are presented as the mean ± SD (A-D) or the mean ± SEM with 5-10 mice per group (E, F). The statistical analysis was performed by multiple unpaired t-tests (a-d) and by two way-anova (e-f). *p < 0.05, **p < 0.01, ***p < 0.001.

[0116] Figure 7: Molecular control of Foxkl nuclear localization. Molecular organization of Foxkl and the engineered Foxkl mutants. Wild-type or Foxkl- / - CD4+ T cells were transduced with an empty vector (control), vectors containing wild-type Foxkl (Foxkl) or Foxkl mutants (Ml, Ml, M2, M3, M4) and were left unstimulated (NS-) or stimulated with anti-CD3 plus anti- CD28 for 24h and ZFN-y+ cells, CD62L, CD44, CD27 expression were detected by flow cytometry.

[0117] EXAMPLE:

[0118] EXAMPLE 1

[0119] T lymphocytes play a key role in the immune response and their functions are intimately linked to metabolic programs. During immune responses, T cells undergo a metabolic reprogramming characterized by increased aerobic glycolysis, nutrient uptake and macromolecules synthesis. These processes are crucial for cell growth, differentiation, and acquisition of effector functions. In this regard, the AKT-mTOR signalling pathway has been shown to be an essential hub for regulating T cell metabolism and, consequently cell fate.

[0120] Using a quantitative phosphoproteomic approach, we have identified a new transcription factor called Foxkl as being highly phosphorylated in T cells upon T Cell Receptor (TCR) engagement. Our results also indicate that Foxkl phosphorylation and nuclear translocation is dependent of the AKTmTOR kinase activities. Using T-cell specific Foxkl deficient mice (Foxkl- / -), we demonstrated that Foxkl is required for full T cell activation. Foxkl -deficient T cells exhibited reduced proliferation and cytokine secretion following TCR stimulation. Furthermore, T cells from Foxkl- / - mice were less prone to acquire an effector like phenotype than wild-type cells when challenged in vivo. Conversely, Foxkl overexpression in T cells enhanced their effector functions in a TCR- dependent manner. In CD8+ T cells, this effect also results into enhanced cancer cell killing capacity in vitro, and improved tumor rejection in vivo.

[0121] Global proteomic analysis of TCR-stimulated CD4+ and CD8+ T cells from Foxkl deficient mice revealed defective expression of critical proteins involved in metabolism, and in particular of effectors and enzymes of the glycolysis pathway. Accordingly, Foxkl deficient T cells had reduced maximal mitochondrial respiratory capacity compared to wild-type and exhibited an impaired ability to enhance aerobic glycolysis upon TCR stimulation or glucose supplementation.

[0122] Altogether, these results indicated that Foxkl is a major regulator of T cell metabolism and thus, of T cell effector functions. Its molecular targeting can be envisioned as a part of T-cell reprogramming to fight cancer.

[0123] EXAMPLE 2

[0124] Results

[0125] TCR stimulation triggers Foxkl nuclear translocation in an Akt-mTOR dependent manner

[0126] Foxkl and Foxk2 are present in all mouse tissues, mRNA expression databases indicate high expression of Foxkl in T lymphocytes (biogps.org). Our previous quantitative phosphoproteomic analysis of primary CD4+ and CD8+ T cells also identified Foxkl but not Foxk2 as phosphorylated at multiple serine (Ser) and threonine (Thr) residues following TCR engagement12, 13. In addition, immunoblot analysis of cellular extracts from mouse lymphoid organs showed low expression of Foxkl in the thymus, but a substantial abundance of Foxkl proteins in purified mature CD4+ and CD8+ T cells, while Foxk2 was undetectable in these conditions (Data not shown). Protein abundance of Foxkl was also higher in naive T cells compared with activated T cells (Data not shown). Analysis of the cellular localization of Foxkl in resting CD4+ and OT-I CD8+ T cells showed detection of the TF mainly in the cytoplasm (Data not shown). Of interest, TCR stimulation triggers nuclear accumulation of Foxkl, starting after 2 min and peaking at 10 min. To better characterize the upstream signalling pathways promoting Foxkl nuclear translocation, we treated T cells with specific inhibitors of mTOR (Ku63794), Aktl / 2 (AKTi VIII), or Gsk3 (ARA014418) and reassessed the transcription factor’s cellular localization. Akt or mTOR inhibition fully prevented Foxkl nuclear translocation following TCR stimulation. In contrast, Gsk3 inhibition enhanced Foxkl accumulation in the nucleus even in the absence of TCR engagement (Data not shown). Therefore, these results indicated that Foxkl is mainly localized in cytoplasm in resting T cells and translocates into the nucleus after TCR stimulation in an Akt-mTOR signaling pathwaydependent manner.

[0127] Foxkl is required for full activation of T cells

[0128] To evaluate the role of Foxkl in T cells, we crossed the Foxklfl / flmouse11with a CD4-Cre transgenic mouse in order to obtain mice whose T cells lack Foxkl proteins (hereafter Foxkl- / -) (Data not shown). Analysis of thymi from Foxkl- / - mice showed that their T cells developed normally, with similar cell numbers (Data not shown). The periphery was populated with T cells with a normal phenotype, although we observed a slight reduction of the numbers of CD4+ T cells, CD8+ T cells and Treg cells in spleens and lymph nodes (Data not shown). To evaluate the importance of Foxkl during T cell activation, we purified CD4+ T cells from Foxkl- / - and control mice and analysed their phenotypes after TCR stimulation with anti-CD3s in the presence or absence of anti-CD28. Foxkl -deficient cells showed a normal upregulation of the activation markers CD69 and CD44, but much less proliferation and secretion of IL-2 and IFN- y as compared with controls (Fig. 2A). In addition, when naive T cells were cultured in vitro under skewing conditions promoting T helper (Th) cell differentiation, less IFN-g+ and IL-4+ producing cells were detected in Foxkl- / - T cells than in control cells under Thl- and Th2- polarizing conditions respectively (Data not shown). In contrast, no major differences were observed under Treg and Thl7 skewing conditions.

[0129] To examine the functions of Foxkl in CD8+ T cells, Foxkl- / - mice were crossed with OT-I transgenic mice expressing a TCR specific for the ovalbumin (OVA). Purified OT-I CD8+ T cells were co-incubated with irradiated splenocytes from CD3s- / - mice (acting as antigen- presenting cells), pulsed with increasing doses of the ovalbumin-derived peptide (N4), or its variant of lower affinity (T4). When stimulated with N4 or T4 peptides, the absence of Foxkl resulted in much less production of IFN-y and reduced proliferation of OT-I CD8+ T cells compared with control cells, despite their similar capacity to integrate certain signals, as reflected by increased CD69 and CD44 expression levels compared with the unstimulated condition (Fig. 2B).

[0130] To evaluate the proliferative capacity of Foxkl -deficient T cells in vivo, naive CD4+ T cells were labelled with Cell Trace Violet (CTV) and injected into CD3s- / - mice, which are devoid of T cells. Because of the lymphopenic environment of the hosts, injected T cells proliferate in a manner dependent on TCR-self antigen interactions14, 15. Flow cytometry analysis after 6 days showed that a lower proportion of Foxkl -deficient T cells had completely diluted the CTV, indicative of slower rates of proliferation (Fig. 2C). Moreover, staining of injected cells with CD62L and CD44 markers, revealed reduced ability of Foxkl- / - T cells to differentiate into effector memory T (Tern) cells (Fig. 2D). To explore this defect further, we performed a similar analysis 21 days following injection. Although Foxkl -deficient T cells eventually succeed in differentiating into Tern cells, the relative proportions of naive cells, central memory cells (Tcm) and Tern cells indicate that they lag behind control cells in their ability to differentiate into effector / memory cell populations (Fig. 2E). In addition, analysis of memory phenotypes showed that transferred Foxkl- / - T cells were more prone to differentiate into KLRGllowCD127hlghmemory precursor effector cells (MPECs) than into KLRGllhlghCD127lowshort-lived effector cells (SLECs), compared with their control counterparts (Fig. 2F). Consistently, we also found increased percentages of the PD-llowCD27hlghpopulation with injected Foxkl -deficient T cells, indicative of the persistence of low-activated cells (Data not shown). Taken together, these results demonstrate that Foxkl is required to trigger T cell activation and regulate effector T cell differentiation.

[0131] Foxkl regulates metabolic pathways of T cells

[0132] To investigate the molecular mechanisms by which Foxkl regulates T cell activation, we performed transcriptomic analysis on purified naive CD4+ T cells from Foxkl- / - and control mice that were left resting or activated with anti-CD3 plus anti-CD28 for 6 and 24 hours. The absence of Foxkl during T activation led to a decreased expression of 355 genes and an increased expression of 618 other genes (Data not shown). Gene set enrichment analysis (GSEA) using Hallmark gene sets indicated that the absence of Foxkl decreased the expression of target genes of c-Myc and E2F, of genes involved in the Akt-mTOR signaling pathway, and in processes of oxidative phosphorylation (OXPHOS) and glycolysis (Data not shown). For the latter, transcripts with reduced expression included the glucose receptor (Slc2al) and glycolytic enzymes (Hk2, Pfkl, Pgkl, Pgaml, Tpil, Ldhd) (Data not shown). Given the importance of metabolic changes on cellular function and T-cell differentiation, we then performed a GSEA analysis with immune-specific gene signatures. This analysis indicated that activated Foxkl- deficient CD4+ T cells failed to switch to an effector / memory cell state as evidenced by their inability to upregulate Tbx21. Irf8. Ifng. 112. Pomes. while remaining positive for some naive markers such as Ccr7 and Bach2 (Data not shown). As observed previously in Figure 2, we noted that this differentiation defect is partial, since the expression of some markers characterizing the transition of naive to effector / memory cells (Cd44, Sell, Il7 ) is regulated similarly to that of controls. (Data not shown).

[0133] To further characterize the molecular pathways controlled by Foxkl, we determined by quantitative mass spectrometry (MS) analysis, the proteomes of control and Foxkl- / - CD4+ T cells activated with CD3 / CD28 for 24 and 48 hours. After normalization and filtering, more than 8000 proteins were identified and quantified across all conditions. Cell stimulation of Foxkl- / - CD4+ T cells resulted in a change in the abundance of 1376 proteins compared with control cells, with no major impact on the increase in total protein mass associated with cell activation (Data not shown). GSEA with Hallmark terms on proteomic data showed enrichment of similar terms with those identified by transcriptomic analysis (Data not shown). Thus, gene signatures associated with Myc targets and mTor signaling were significantly repressed in Foxkl -deficient cells. Although induction of Myc expression itself was unaffected, Foxkl- deficient T cells exhibits reduced expression of some of its targets such as the heterodimer SLC7A5 / SLC3A2(also CD98) and the Transferrin receptor TFRC (also CD71) required for the uptake of essential amino-acids and iron, respectively (Fig 3A). Concomitant with transcriptomic data, Foxkl is also required to induce the high-level protein abundance of the glucose receptor SLC2A1 and crucial components of glycolysis pathways (HK2, PFKL, ALDOA, PGK1, PGAM1, SLC16A3) that is reached in control cells following TCR stimulation (Data not shown).

[0134] In order to functionally assess the metabolic processes affected by protein expression changes, we analyzed aerobic glycolysis and OXPHOS of TCR-activated CD4+ T cells. We observed that Oxygen consumption rate (OCR) at maximal capacity and extracellular acidification rate (ECAR) in response to glucose and to CD3 D / CD28 stimulation were significantly decreased in Foxkl- / - T cells compared with their control counterparts (Fig. 3B-D). Consistently, in the absence of Foxkl, TCR stimulation failed to increase glucose uptake and production of lactate to levels comparable to those in control T cells (Fig. 3E & 3F). Overall, these analyses revealed that Foxkl is required to induce mTor-mediated metabolic processes enabling effector T cell differentiation.

[0135] Foxkl represses autophagy transcriptional programs

[0136] Analysis of transcriptomic data from the first 6h of cell activation showed a predominant increase in transcripts in Foxkl- / - T cells compared with controls, which is not observed at 24h (Data not shown). This suggested that Foxkl could exert a direct repressive function on the transcription of certain genes shortly after TCR stimulation. In myoblasts, Foxkl has been shown to repress autophagy and atrophy genes9. At 6h following activation, GSEA analysis revealed a significant enrichment for the KEGG autophagy pathway driven by core components of the autophagy machinery such as Autophagy-related protein family members (AtglOl, Atgl2, Atgl3, Atgl4, Alg2a), Wdr45, Ulkl, Taxlbpl and Mapllc3a / b (Data not shown). To further explore these findings, we sought to identify direct genomic targets of Foxkl in T cells. First, we performed chromatin immunoprecipitation coupled with sequencing (ChlP-seq) in the BI-141 T cell line expressing a tagged version of Foxkl (Foxkl-Flag) (Data not shown). ChlP- seq analysis with anti -Flag or anti-Foxkl antibodies led to the identification of 296 shared peaks mainly localized in gene promoters (Data not shown). The sequences of these peaks were enriched for a Foxkl consensus binding motif previously identified in non-immune cells (Data not shown). Interestingly we found, that this set of genes with Foxkl bound promoters was enriched for genes in the KEGG autophagy pathway (Data not shown). Consistently, genes associated with these enriched terms were also up-regulated transcripts in 6h-activated Foxkl- deficient T cells (Data not shown). Furthermore, ChlP-seq analysis revealed additional autophagy genes (Beas 3, Bnip3l, Ccpgl and Calcocol) not listed in the KEGG pathway, as well as the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32') involved in muscle cell atrophy, all upregulated in Foxkl- / - T cells16(Data not shown). We next sought to validate our findings in primary CD4+ T cells for a subset of these gene promoters using qPCR-based ChIP quantification. In these conditions, we confirmed the DNA binding of Foxkl on promoters of Calcocol, Wipi2, Wdr45, Taxlbpl and Fbxo32, and its increase following T cell activation (Fig. 4). Importantly, comparative analysis of the protein abundance of these target genes when detected in the proteome between 24h-activated Foxkl -deficient and control CD4+T cells, indicated that the transcriptional repression mediated by Foxkl is also echoed at the protein level (Data not shown). Collectively, these data demonstrate that Foxkl represses autophagic programs during the first hours of T cell activation, thereby enabling the triggering and sustaining of expression of proteins involved in energetic processes.

[0137] T cell-specific deletion of Foxkl impairs anti-tumor immunity

[0138] Given the importance of T-cell metabolism in anti-tumor responses, we postulated that Foxkl could be an important mediator of T-cell functions in a such context. To test this hypothesis, we co-incubated OT-I CD8+ T cells from either WT or Foxkl-deficient mice with a mouse A20 lymphoma B-cell line engineered to express a single chain trimer (SCT) fusion protein, consisting of the N4-peptide fused with b2-microglobulin and H-2Kb MHC-I17(Data not shown). As expected, expression of SCT in A20 (A20-SCT+) promoted IFN-y production in OT-I CD8+T cells as compared with the parental A20 (A20-SCT-) cell line (Fig. 5A & 5B). Importantly, Foxkl -deficient T cells showed a lower capacity to produce IFN-y compared with control cells. Co-incubation with increasing numbers of A20-SCT+, also demonstrates a reduced ability of Foxkl- / - CD8+ T cells to produce IL-2 (Fig 5B). We next assessed T cell- mediated cytotoxicity using A20-SCT+ cells or the EL-4 T cell lymphoma cell line that was pulsed with the N4 peptide. As for cytokine secretion, Foxkl- / - OT-I CD8+ T cells showed compromised responses toward both types of target cells as compared with Foxkl-sufficient T cells (Fig 5C & 5D) To explore the role of Foxkl in controlling tumor growth in vivo, we subcutaneously engrafted Foxkl- / - or control mice with carcinoma MC38 cells and injected either blocking anti-PD-1 or isotype control antibodies. In the absence of effective immunotherapy, our data indicated that MC38 grew similarly in control and Foxkl- / - mice (Fig 5E). In contrast, anti-PD-1 treatment showed impaired ability of Foxkl- / - mice to delay tumor growth as compared with control mice (Fig 5E). Next, we took advantage of the melanoma model BrafV600E in which the genes coding for cyclooxygenase (COX)-l and -2 have been inactivated (PtgsllPtgs2-l- BrafV600E)18. As previously described, Ptgsl / Ptgs2- / - BrafV600Ecells subcutaneously injected into immunocompetent control mice were rejected and tumor failed to grow throughout the duration of the analysis (Fig 5F). In a striking contrast, mice whose T cells lacked Foxkl had larger size tumors, indicating a defective function of anti-tumor T cell responses. Taken together, these data support the view that Foxkl is involved in the direct cytotoxic response of T cells against cancer cells, as well as in the longer-term control of tumor growth.

[0139] Enforced Foxkl expression enhances effector functions of T cells

[0140] Observations made with Foxkl -deficient T cells prompted us to investigate the extent to which Foxkl could potentiate T cell effector functions. For this purpose, mouse CD4+ T cells were retro-infected with a mouse Foxkl cDNA or control construct. Immunoblot analysis of lysates from EGFP+FACS-sorted cells showed a strong increase in Foxkl protein expression in Foxkl transduced-cells as compared with control cells (Data not shown). When stimulated with anti- CD3s in the absence or presence of anti-CD28, antigen-experienced Foxkl-overexpressing T cells showed a greater capacity to produce IFN-y than control cells (Fig. 6A). The Foxkl- overexpression effect was also evaluated in OT-I CD8+ T cells. After retro-infection, EGFP+ OT-I cytotoxic T lymphocytes (CTLs) were sorted and co-incubated with A20-SCT- or A20- SCT+ cells. In contrast to A20-SCT- cells, the addition of increasing numbers of A20-SCT+ cells resulted an increased percentage of IFN-y+cells when Foxkl was overexpressed, and at a higher level than in control cells (Fig. 6B). Furthermore, using A20-SCT+ cells and N4-pulsed EL-4 cells, we found that Foxkl-overexpressing CTLs exhibited higher cytolytic activity against target cells than control cells (Fig. 6C & 6D). We next sought to evaluate whether enforcing Foxkl expression could also enhance anti-tumor immunosurveillance in vivo. To do so, C57BL / 6 wild-type mice were subcutaneously injected the B16F10 melanoma or MC38 carcinoma cell lines expressing the OVA protein (B16F10-OVA, MC38-OVA). Control or Foxkl-overexpressing OT-I CD8+ T cells were then adoptively transferred and tumor growth was analyzed. Mice that received OT-I CD8+ T cells overexpressing Foxkl were more effective in controlling tumor progression than control cells, as evidenced by the reduction in measured tumor size (Fig. 6E & 6F) Overall, these results indicate that enforcing Foxkl expression induces T cell reprogramming towards more effective anti-tumor effector functions.

[0141] Molecular control of Foxkl nuclear localization enhances T cell effector functions

[0142] Since our data showed that Foxkl nuclear translocation in T cells was dependent on the kinases mTOR, AKT, and GSK3, we sought to investigate whether various Ser / Thr motifs present in the Foxkl sequence could control its subcellular localization. Given the large number of potential phosphorylation sites in addition to those already identified12, 19, 20(phosphosite.org), and the difficulty of associating specific kinase activity with motifs, we opted for a systemic Ser-to-Ala and Thr-to-Val mutation approach to assess their impact on Foxkl roles. We constructed multiple Foxkl-tagged mutants, including one (Ml) targeting a series of Ser and Thr residues between the Forkhead-associated (FAH) domain and the Forkhead (FH) domain (SI 99 to S243), a second mutant (M2, S402 to S485) targeting a Ser-rich region located downstream of the FH domain, and a third mutant (M3) that combines all the mutations from Ml and M2 (Data not shown). As most of these Ser / Thr are part of Ser / Thr-Pro-containing motifs, they could be potential target of the kinases mTOR and GSK321, 22. Transduction of each Foxkl form into wild-type CD4+T cells by retro-infection, followed by an analysis of Foxkl subcellular localization, revealed that the tagged wildtype form was predominantly cytoplasmic, and like the endogenous form, translocated to the nucleus upon TCR stimulation (Data not shown). The situation was different with all mutants, which appeared already localized into the nucleus, even in the absence of TCR stimulation. This effect was particularly pronounced with the M3 mutant, which exhibited a much higher nuclear-to-cytoplasmic ratio of Foxkl localization compared to the other mutants (Data not shown). We also noticed that expression of the M3 mutant resulted in a marked increase in mobility likely related to loss of phosphorylation. To evaluate the impact of this Foxkl-subcellular re-localization on T cell effector functions, we transduced each Foxkl mutant into Foxkl -deficient CD4+ T cells and stimulated them with anti-CD3 plus anti-CD28 antibodies for 24h. Interestingly, analysis of the transduced cells prior to TCR stimulation showed a graded downregulation of CD62L and CD27 expression depending on the mutant expressed, with the strongest effect observed for M3 (Fig. 7). TCR stimulation led to a marked downregulation of CD62L in all cell types, as well as CD27, except in Foxkl -deficient CD4+T cells transduced with the control vector, where CD27 expression remained high. As expected, IFN-y secretion in response to TCR stimulation was reduced in Foxkl" / " CD4+T cells but was restored by overexpression of either the wildtype or mutant Foxkl forms (Fig. 7). Notably, although modest, the M3 mutant induced a higher frequency of IFN-y+cells compared to wild-type Foxkl overexpression. These results support the idea that enforcing Foxkl nuclear localization through phosphorylation-dependent regulations license T cells prior to stimulation and enhances their effector function under activating conditions.

[0143] Discussion

[0144] The present study reports the critical role of Foxkl in T cell effector functions and differentiation. We found that, shortly after TCR stimulation, Foxkl translocates into the nucleus in an AKT-mTOR axis-dependent manner and binds to the promoters of autophagy genes, leading to their repression. This finding reveals Foxkl as an important molecular player acting downstream of mTOR and involved in the transient repression of the autophagy flux operating during the initiation of T cell responses23. Such a mechanism is essential to trigger the subsequent metabolic reprogramming of T cells to a level allowing full differentiation into effector cells24Consistent with T cell phenotypes reported following inhibition of mTOR signals by rapamycin 3 or loss of RAS homolog enriched in brain (RHEB)25, Foxkl -deficient T cells showed a reduced propensity to differentiate into effector cells and reduced effector functions but remained potent to acquire a long-lived memory-like phenotype. The observed reduction in effector functions resulted in a lower capacity to kill tumor cells and control tumor growth. Conversely, enforced expression of Foxkl in CD4+ and CD8+ T cells is sufficient to enhance effector cell differentiation and functions.

[0145] In naive T cells, metabolic processes are under the control of a high autophagy flux23‘24This flux is imposed by the low expression level of nutrient transporters that characterize naive T cells, and enable constant recycling of the intracellular metabolites necessary for survival. Indeed, autophagy induced by amino acid starvation in T cells selectively targets nutrient transporters for degradation, leading to reduced glycolysis. The autophagy genes repressed by Foxkl could be the core mediator of autophagy flux in naive T cells. Thus, to trigger the rapid differentiation into effector cells, one of the first events induced by TCR stimulation must lead to the termination of this flux, even before translation of nutrient transporters and glycolytic enzymes is initiated. A short lag of time between the termination of the autophagy flux and the induction of protein expression is probably sufficient to prevent degradation of the reservoir of proteins involved in metabolic pathways, enabling their accumulation and promoting a positive feedback process2. Given the final impact of the mTOR axis, and Foxkl in particular, on T cell fate, it would be valuable to investigate how much the variation in the expression of metabolic players in these initial steps of activation, accounts for the efficiency of differentiation into effector or memory cells.

[0146] The characterization of Foxkl clearly draws a parallel with the Foxos TF, also involved in T cell differentiation. Although Foxos and Foxkl are both regulated by AKT, the operating mechanism differs significantly. Foxos are constantly active and localized in the nucleus of quiescent T cells. Upon TCR stimulation, activation of AKT induces their phosphorylation on Ser and Thr residues which promote their nuclear exclusion. Phosphorylation of Foxos provides docking sites for the 14-3-3 proteins, which, along with the masking of the Nuclear Localization Signal (NLS), results in their sequestration in the cytoplasm compartment, thereby suspending their transcriptional activity26, 27. In contrast, Foxkl has a subcellular localization symmetrically inverted to that of Foxos. Our data suggests that sequestration of Foxkl in the cytoplasm in resting T cells is operated by GSK3. Indeed, GSK3 is constitutively active in resting T cells and its inhibition by AKT following TCR stimulation could release the constraint exerted by GSK3 on Foxkl28, 29. Our kinase inhibition assays also indicated that additional events of phosphorylation mediated by mTOR on Foxkl could be necessary to enhance Foxkl nuclear shuttling. The mutational approach targeting Ser / Thr motif in Foxkl, showed that some phosphorylation sites might be more effective in regulating Foxkl subcellular localization. The impact of combined mutations suggests that GSK3 might have a prominent role in maintaining Foxkl in the cytoplasm of resting T cells. Although early studies in other cell types have observed phosphorylation-dependent localization of Foxkl, there is no clear consensus on the exact phosphorylation sites and associated kinases responsible for each modification10, 19, 20. The challenge arises from the numerous potential phosphorylation sites, their close proximity, and their ability to substitute for one another when mutated. Nevertheless, it appears that enforcing Foxkl nuclear localization is sufficient to enhance T cell effector functions presumably through its transcriptional repression activity. Further investigation will be required to clarify how phosphorylation regulations modify Foxkl localization and functional activities. Finally, noting that the Foxkl binding motif and its targets differ from those of Foxos, it is tempting to speculate that the mTOR-AKT axis mediates a synergistic cooperation of these transcription factors in order to induce full T cell effector differentiation.

[0147] Overall, our results provide a basis for further investigation into how Foxkl control T cell differentiation and function in diverse physiological or pathological contexts. Since the AKT- mTOR axis integrates signals from contextual receptors (co-stimulatory and co-inhibitory receptors), cytokine receptors and nutrient sensors, it will be important to identify Foxkl- dependent and -independent pathways in these different contexts. Furthermore, these findings identify Foxkl as a potential novel target for modulating T cell effector functions in immunotherapeutic applications, such as those using engineered tumor-infiltrating lymphocytes and CAR-T cells.

[0148] REFERENCES: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A host immune cell that is engineered to overexpress a Foxkl polypeptide.

2. The host immune cell according to claim 2 wherein the Foxkl polypeptide comprises an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO: 1.

3. The host immune cell according to claim 2 wherein the Foxkl polypeptide comprises the amino acid sequence h as set forth in SEQ ID NO: 1 that comprises one or more conservative modification, more particularly one or more conservative substitutions.

4. The host cell according to any one of claims 1 to 3 that is a murine or a human cell.

5. The the host cell according to any one of claims 1 to45 that is a T cell that is a CD4+ T cell or a CD8+ T cell.

6. The host cell of claim 5 that a tumor infiltrating lymphocyte.

7. The host cells according to any one of claims 1 to 6 that is a pluripotent stem cell (PSC) or a hematopoietic stem cell.

8. The host cell according to any one of claims 1 to 7 that expresses an endogenous TCR.

9. The host cell according to any one of claims 1 to 7 that expresses an exogenous TCR.

10. The host cell according to any one of claim 1 to 7 that is engineered for expression of a CAR.

11. The host cell of claim 10 wherein the CAR comprises one more polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain.

12. The host cell of claim 10 wherein the CAR is a monomeric molecule that consists of one polypeptide having an extracellular domain and an intracellular domain joined by a transmembrane domain.

13. The host cell of claim 10 wherein the CAR is a heterodimeric molecule that consists of two polypeptides, both having an extracellular domain and an intracellular domainjoined by a transmembrane domain and wherein the two polypeptides are capable of dimerizing via their respective extracellular domain.

14. The host cell of claim 10 wherein the CAR derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domains are substituted by a variable domain of an antibody.

15. The host cell of claim 14 wherein the CAR derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a first variable domain of an antibody (e.g. a VL or VH domain) and the immunoglobulin (Ig)- variable (V) domain of the beta chain is substituted by a second variable domain of an antibody (e.g. a VL or VH domain) wherein the first variable domain is capable of dimerizing with the second variable domain to form a fragment variable (Fv) that binds to the antigen of interest.

16. The host cell of claim 15 wherein the CAR derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a VL domain of an antibody and the immunoglobulin (Ig)-variable (V) domain of the beta chain is substituted by a VH domain of an antibody wherein the VL domain is capable of dimerizing with the VH domain to form a fragment variable (Fv) that binds to the antigen of interest.

17. The host cell according to any one of claims 14 to 16 wherein the CAR is capable of associating with a CD3 complex to form the T-cell co-receptor.

18. The host cell according to any one of claims 14 to 17 wherein the CAR replaces a native and / or an endogenous TCR in the CD3 / TCR complex of host cell.

19. The host cell according to any one of claims 1 to 18 that expresses a TCR or CAR that is specific for a tumor antigen.

20. A method of preparing the host cell according to any one of claims 1 to 19, comprising the step consisting of introducing into a host cell a polynucleotide that encodes for the Foxkl polypeptide and optionally one or more polynucleotide(s) that encodes for the CAR or TCR of interest.

21. A method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the population of host cells according to any one of claims 1 to 18.

22. The method of claim 21 that is suitable for the treatment of cancer.

23. A pharmaceutical composition comprising the population of the host cells according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

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