Host cells engineered to bypass the CD28 co-stimulation pathway and uses thereof for inducing durable immune responses under non-inflammatory conditions

Abstract

The CD28 costimulatory role becomes particularly manifest under the poorly inflammatory conditions often encountered in tumor draining lymph nodes and the tumor microenvironment. Anti-tumoral T cell responses have thus a great reliance on CD28 signalling. The possibility of inducing high potential antitumor CAR-T cell responses independently of CD28 pathway engagement by CD80 and CD86 represents thus an appealing approach. Now, the inventors surprisingly show that the mere presence in normal T cells of one gain of function mutation at position 538 in the mouse ortholog of the human isoform 3 of Carmil2 does not drive the development of T-cell cancers. The inventors also show that this mutation switches Carmil2 into an activated state which mimics the activated Carmil2 state resulting from physiological CD28 engagement. Therefore, the gain of function mutation can substitute for CD28 engagement and primes the NF-kB signalling pathway for cooperating with the TCR signalling pathway in normal T cells responding in vivo to antigenic stimuli including tumor antigens. Once and only once the TCR has been engaged by an antigen, including a tumor antigen, it permits full T cell activation in absence of co-receptor CD28 engagement. Therefore, rewriting the CD28 costimulatory pathway of CAR-T cells via the introduction of said gain of function mutation in Carmil2 can induce long-lasting anti-tumor T responses in absence of inflammatory cues.

Description

HOST CELLS ENGINEERED TO BYPASS THE CD28 CO-STIMULATION PATHWAY AND USES THEREOF FOR INDUCING DURABLE IMMUNE RESPONSES UNDER NON-INFLAMMATORY CONDITIONS 5 FIELD OF THE INVENTION

[0001] The present invention is in the field of medicine, in particular immunology and oncology. BACKGROUND OF INVENTION 10

[0002] Targeted immunotherapies rely on the use of immune cells or molecules (e.g., immune checkpoint inhibitors) to treat a variety of diseases, including cancer, infectious and autoimmune disorders. Recently, the genetic modification of T cells to express chimeric antigen receptors (CARs) that target tumor antigens has dramatically expanded the success rate of cancer immunotherapy, especially in CD19-expressing 15 blood cancers. However, most attempts to eradicate solid tumors using CAR T cells have led to therapeutic failures. Given that solid tumors account for approximately 90% of cancer-related deaths, numerous studies are being conducted worldwide to address this challenge by increasing the proliferation, functional efficacy, durability, and resistance to functional exhaustion of CAR-T cells while making them less aggressive to normal 20 tissues.

[0003] First-generation CAR design contained an extracellular antibody-derived antigen- binding domain (scFv) and a single intracellular CD3ζ signaling domain connected via an extracellular spacer and transmembrane domain. Due to limited efficacy in clinical trials, one (second generation) or two (third generation) co-stimulatory signaling domains 25 were incorporated in the CAR to improve CAR T cell persistence and, hence, effector functions. For instance, it was shown that CARs that incorporate a CD28 costimulatory domain drive high T-cell activation. In particular, it was demonstrated that CARscontaining said CD28 transmembrane domain (TMD) can heterodimerize with the endogenous CD28 receptor leading to a stronger signal transduction, and thus facilitating CAR T-cell activation in the context of low levels of CAR antigen. This illustrates the importance of CD28-induced signals for the efficiency of CAR-T cell therapy but also 5 highlights that the lack or poor engagement of CD28 with its cognate ligands (CD80 and CD86) can thus limit the potential of CAR-T cell therapy. For instance, in the absence of inflammatory conditions - which is the case of some so-called “cold” tumor microenvironments – dendritic cells express CD80 and CD86 molecules in a reduced manner and induce suboptimal CD4+ and CD8+ T cell responses (Bonaventura, Paola, 10 et al. "Cold tumors: a therapeutic challenge for immunotherapy." Frontiers in immunology 10 (2019): 168; Duong, Ellen, et al. "Type I interferon activates MHC class I-dressed CD11b+ conventional dendritic cells to promote protective anti-tumor CD8+ T cell immunity." Immunity 55.2 (2022): 308-323). It should also be noted that the presence of Foxp3+ regulatory T cells in the tumor microenvironment may also contribute 15 to reduce the expression of CD80 and CD86 molecules on the surface of DCs and abolish their capacity to activate anti-tumor T cells (Binnewies, Mikhail, et al. "Unleashing type- 2 dendritic cells to drive protective antitumor CD4+ T cell immunity." Cell 177.3 (2019): 556-571). The possibility of inducing high potential antitumor CAR-T cell responses independently of CD28 signaling pathway engagement by CD80 and CD86 ligands in 20 tumor-draining lymph nodes and the tumor microenvironment is therefore a particularly promising approach.

[0004] The Carmil2 cytosolic protein, also known as Rltpr, is essential for CD28 co- stimulation (Liang, Yinming, et al. "The lymphoid lineage–specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory 25 T cells." Nature immunology 14.8 (2013): 858-866 ; Roncagalli, Romain, et al. "The scaffolding function of the RLTPR protein explains its essential role for CD28 co- stimulation in mouse and human T cells." Journal of Experimental Medicine 213.11 (2016): 2437-2457). In particular, Carmil2 acts as a scaffold, bridging CD28 to the CARD11 / CARMA1 cytosolic adaptor and thereby induces the NF-κB signaling pathway 30 (Roncagalli, Romain, et al. "The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells." Journal ofExperimental Medicine 213.11 (2016): 2437-2457). In human, loss of function mutations in RLPTR lead to a combined immunodeficiency (CID) affecting at least the CD28- responsive pathway in T cells (Wang, Yi et al. Dual T cell- and B cell-intrinsic deficiency in humans with biallelic RLTPR mutations. “Journal of Experimental Medicine 213.11 5 (2016): 2413-2435). Recently, the p.Q575E mutation in Carmil2 was identified as a potential oncogenic driver mutation in human cutaneous T cell lymphoma (Park, Joonhee, et al. "Genomic analysis of 220 CTCLs identifies a novel recurrent gain-of- function alteration in RLTPR (p. Q575E)." Blood, The Journal of the American Society of Hematology 130.12 (2017): 1430-1440). More specifically, upon overexpression in a 10 transformed T cell line (Jurkat) and stimulation via a T cell antigen receptor (TCR) pharmacologic mimics composed of PMA and ionomycin, the p.Q575E mutation increases binding of Carmil2 to the CARD11 / CARMA1 cytosolic adaptor and selectively upregulates the NF-κB pathway and the production of interleukin 2 transcripts by 34-fold. However, the interest of Carmil2 gain of function mutations in the context of antigenic 15 activation of normal T cells and of CAR-T cells in in vivo conditions has never been investigated. SUMMARY

[0005] The present invention is defined by the claims. In particular, the present invention 20 relates to host cells engineered to bypass the CD28 co-stimulation pathway and uses thereof for inducing durable immune responses under non-inflammatory conditions.

[0006] The present invention relates to a host cell that is engineered to express a Carmil2 polypeptide that comprises i) an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:1 wherein the glutamine residue (Q) 25 at position 539 is mutated or ii) an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:2 wherein the glutamine residue (Q) at position 538 is mutated.

[0007] The glutamine residue (Q) may be substituted by a negatively charged amino acid residue. The glutamine residue (Q) may be substituted by a glutamic acid residue (E). The glutamine residue (Q) may be substituted by an aspartic acid residue (D).

[0008] The host cell may be a murine or a human cell. The host cell may be a T cell that 5 is a CD4+ T cell or a CD8+ T cell. The host cell may be a tumor infiltrating lymphocyte. The host cell may be a pluripotent stem cell (PSC) or a hematopoietic stem cell.

[0009] The host cell may express an endogenous TCR. The host cell may express an exogenous TCR. The host cell may be engineered for expressing a CAR.

[0010] The CAR may comprise one more polypeptide(s) having an extracellular domain 10 and an intracellular domain joined by a transmembrane domain.

[0011] The CAR may be a monomeric molecule that consists of one polypeptide having an extracellular domain and an intracellular domain joined by a transmembrane domain.

[0012] The CAR may be a heterodimeric molecule that consists of two polypeptides, both having an extracellular domain and an intracellular domain joined by a transmembrane 15 domain and wherein the two polypeptides are capable of dimerizing via their respective extracellular domain.

[0013] The CAR may derive from a TCR heterodimer wherein the immunoglobulin (Ig)- variable (V) domains are substituted by a variable domain of an antibody.

[0014] The CAR may derive from a TCR heterodimer wherein the immunoglobulin (Ig)- 20 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.25

[0015] The CAR may derive 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 VHdomain 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.

[0016] The CAR may be capable of associating with a CD3 complex to form the T-cell co-receptor. 5

[0017] The CAR may replace a native and / or an endogenous TCR in the CD3 / TCR complex of the host cell.

[0018] The host cell may express a TCR or CAR that is specific for a tumor antigen or for a pathogen antigen.

[0019] The present invention further relates to a method of preparing the host cell as 10 described herein, comprising the step consisting of introducing into a host cell a polynucleotide that encodes for the Carmil2 polypeptide and optionally one or more polynucleotide(s) that encodes for the CAR or TCR of interest.

[0020] The present invention further relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a 15 population of the host cells.

[0021] The method may be suitable for the treatment of cancer or infectious diseases. The method may be suitable for the treatment of cold tumors.

[0022] The present invention further relates to a pharmaceutical composition comprising a population of the host cells, and a pharmaceutically acceptable carrier. 20 DEFINITIONS

[0023] In the present invention, the following terms have the following meanings:

[0024] 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 25 encompass an amino acid polymer that has been modified; for example, disulfide bondformation, 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. 5

[0025] As used herein, the term "negatively charged amino acid" includes any naturally occurring or unnatural amino acid having a negatively charged side chain under normal physiological conditions. Examples of negatively charged naturally occurring amino acids are aspartic acid (D) and glutamic acid (E).

[0026] As used herein, the term “polynucleotide” refers to a polymeric form of 10 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 15 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.

[0027] As used herein, the term “endogenous” or “native” refers to a polypeptide or 20 polynucleotide that is normally present in a host cell.

[0028] 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). 25

[0029] As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. Within the specification, the mutation are references according to the standard mutation nomenclature.

[0030] 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 5 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 10 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 15 “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 20 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 25 the second amino acid sequence.

[0031] 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 30 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 aminoacid 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 5 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 10 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 15 glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gln, 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, 20 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). 25

[0032] 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, 5′ cap formation, and / or 3′ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or 30 protein.

[0033] 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 5 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 10 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 15 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).

[0034] As used herein, the term “Carmil2” has its general meaning in the art an refers to the Capping protein, Arp2 / 3 and myosin-I linker protein 2 encoded by the CARMIL220 gene. The term is also known as Rltpr or LRRC16C. Carmil2 is required for CD28- mediated stimulation of NF-kappa-B signalling (Liang, Yinming, et al. "The lymphoid lineage–specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells." Nature immunology 14.8 (2013): 858-866 ; Roncagalli, Romain, et al. "The scaffolding function of the RLTPR protein explains its 25 essential role for CD28 co-stimulation in mouse and human T cells." Journal of Experimental Medicine 213.11 (2016): 2437-2457). The mouse Carmil2 gene (ENSMUSG00000050357) gives rise to several transcripts. In the publication Liang, Yinming, et al. "The lymphoid lineage–specific actin-uncapping protein Rltpr is essential for costimulation via CD28 and the development of regulatory T cells." Nature 30 immunology 14.8 (2013): 858-866, the inventors described a full-length mouseCarmil2 / Rltpr cDNA sequence that was deposited at https: / / www.ebi.ac.uk / ena / browser / view / HF678090 under the accession code HF678090. It rectified the incorrect cDNA sequence that was publicly available at the time of the publication. The Carmil2 protein sequence coded by the cDNA described under accession HF678090 was shown to give rise to a functional Carmil2 protein (Roncagalli, Romain, et al. "The scaffolding function of the RLTPR protein explains its essential role for CD28 co-stimulation in mouse and human T cells." Journal of Experimental Medicine 213.11 (2016): 2437-2457). It corresponds to transcript: ENSMUST00000213019.2 Carmil2-203 described in the latest release of http: / / www.ensembl.org / Mus_musculus. The human homologue of the mouse HF678090 / ENSMUST00000213019.2 Carmil2-203 transcript is known as “isoform 3”. Human isoform 3 differs from isoform 1 coded transcript ENST00000696176.1 Carmil2- 216 (http: / / www.ensembl.org / Homo_sapiens;) by the lack of an in-frame insertion in exon 14 and by the inclusion of exon 36. In contrast to isoform 3, isoform 1 is non- functional (Lévy, Romain et al “Human Carmil2 deficiency underlies a broader immunological and clinical phenotype than CD28 deficiency. Journal of Experimental Medicine 2023 Feb 6;220(2):e20220275). An exemplary amino acid sequence of the human and mouse functional Carmil2 isoforms 3 is shown as SEQ ID NO:1 or SEQ ID NO:2. SEQ ID NO:1 > human Carmil2 amino acid sequence The glutamine residue (Q) at position 539 is in bold and underlined. MAQTPDGISCELRGEITRFLWPKEVELLLKTWLPGEGAVQNHVLALLRWRAYLLHTTCLPLRVD CTFSYLEVQAMALQETPPQVTFELESLRELVLEFPGVAALEQLAQHVAAAIKKVFPRSTLGKLF RRPTPASMLARLERSSPSESTDPCSPCGGFLETYEALCDYNGFPFREEIQWDVDTIYHRQGCRH FSLGDFSHLGSRDLALSVAALSYNLWFRCLSCVDMKLSLEVSEQILHMMSQSSHLEELVLETCS LRGDFVRRLAQALAGHSSSGLRELSLAGNLLDDRGMTALSRHLERCPGALRRLSLAQTGLTPRG MRALGRALATNAAFDSTLTHLDLSGNPGALGASEDSGGLYSFLSRPNVLSFLNLAGTDTALDTL FAAVSRGCCTSLTHLDASRNVFSRTKSRAAPAALQLFLSRARTLRHLGLAGCKLPPDALRALLD GLALNTHLRDLHLDLSACELRSAGAQVIQDLVCDAGAVSSLDLADNGFGSDMVTLVLAIGRSRS LRHVALGRNFNVRCKETLDDVLHRIVQLMQDDDCPLQSLSVAESRLKLGASVLLRALATNPNLT ALDISGNAMGDAGAKLLAKALRVNSRLRSVVWDRNHTSALGLLDVAQALEQNHSLKAMPLPLND VAQAQRSRPELTARAVHQIQACLLRNNRADPASSDHTTRLQPLGLVSDPSEQEVNELCQSVQEH VELLGCGAGPQGEAAVRQAEDAIQNANFSLSILPILYEAGSSPSHHWQLGQKLEGLLRQVGEVC RQDIQDFTQATLDTARSLCPQMLQGSSWREQLEGVLAGSRGLPELLPEQLLQDAFTRLRDMRLS ITGTLAESIVAQALAGLSAARDQLVESLAQQATVTMPPALPAPDGGEPSLLEPGELEGLFFPEE KEEEKEKDDSPPQKWPELSHGLHLVPFIHSAAEEAEPEPELAAPGEDAEPQAGPSARGSPSPAA PGPPAGPLPRMDLPLAGQPLRHPTRARPRPRRQHHHRPPPGGPQVPPALPQEGNGLSARVDEGV EEFFSKRLIQQDRLWAPEEDPATEGGATPVPRTLRKKLGTLFAFKKPRSTRGPRTDLETSPGAAPRTRKTTFGDLLRPPTRPSRGEELGGAEGDTSSPDPAGRSRPRYTRDSKAYSMILLPAEEEATL GARPDKRRPLERGETELAPSFEQRVQVMLQRIGVSRGSGGAEGKRKQSKDGEIKKAGSDGDIMD SSTEAPPISIKSRTHSVSADPSCRPGPGSQGPESATWKTLGQQLNAELRSRGWGQQDGPGPPSP GQSPSPCRTSPSPDSLGLPEDPCLGPRNEDGQLRPRPLSAGRRAVSVHEDQLQAPAERPLRLQR SPVLKRRPKLEAPPSPSLGSGLGTEPLPPQPTEPSSPERSPPSPATDQRGGGPNP SEQ ID NO:2 > murine Carmil2 amino acid sequence The glutamine residue (Q) at position 538 is in bold and underlined. MAQTPDDISCELRGEITRFLWPKEAELLLKTWLPQEGAEQSHILALLRWRAYLLHTCLPLRVDC TFSYLEVQAMALQETPPRVTFELESLPELVLEFPCVAALEQLAQHVAAAIKKVFPRSTLGKLFR KPTPSSLLARLERSHPLESTIPSSPCGGFLETYEALCDYNGFPFREEIQWDVDTIYHRQGCRHF CLGDFSHFGSRDLALSVAALSYNLWFRRLSCEDMKLSLEVSEQILHMTSQSSYLEELVLEACGL RGDFVRRLAQALAGHFNSGLRELSLSGNLLDDRGMAALSRHLEHCPGALRRLSLAQTGLTPRGM RALGRALATNATFDSTLTHLDLSGNPGALGPSQDSGGLYTFLSRPNVLAYLNLAGTDATLGTLF TALAGGCCSSLTHLEASRNIFSRMKSQAAPAALQRFLGGTRMLRHLGLAGCKLPPEALRALLEG LALNTQIHDLHLDLSACELRSVGAQVIQDLVCDAGALSSLDLSDNGFGSDMVTLVLAIGRSRSL KHVALGRNFNVRCKETLDDVLHRIAQLMQDDDCPLQSLSVAESRLKQGASILIRALGTNPKLTA LDISGNAIGDAGAKMLAKALRVNTRLRSVIWDRNNTSALGLLDVAQALEQNHSLKSMPLPLNDV TQAHRSRPELTTRAVHQIQACLWRNNQVDSTSDLKPCLQPLGLISDHSEQEVNELCQSVQEHME LLGCGAGPQGEVAVHQAEDAIQNANFSLSILPILYEAGRSPSHHWQLQQKLESLLGQVGEICRQ DIQDFTQTTLDTTRSLCPQMLQTPGWRKQLEGVLVGSGGLPELLPEHLLQDAFSRLRDMRLSIT GTLAESIVAQALAGLHAARDRLVERLTQQAPVTMAPAVPPLGGNELSPLETGGLEELFFPTEKE EEREKDESSSWKWLEPSNCFHLVSSLHGAAEEAERDPELAAPGEDAEPQAGPSARGSPSPAAPG PPAGPLPRMDLPPAGQPLRHPTRARPRPRRQHHHRPPPGGPQVPPALLQEGNGLTARVDEGVEE FFSKRLIQQDHFWAPEEDPATEGGATPVPRTLRKKLGTLFAFKKPRSTRGPRPDLETSPGAAAR ARKSTLGDLLRPPARPGRGEEPGGAEGGTSSPDPARRNRPRYTRESKAYSMILLPAEEEAAVGT RPDKRRPLERGDTELAPSFEQRVQVMLQRIGVSRASGGAESKRKQSKDGEIKKAGSDGDIMDSS TETPPISIKSRTHSVSADPSCRPGPGGQGPESATWKTLGQQLNAELRGRGWGQQDGPGPPSPCP SPSPRRTSPAPDILSLPEDPCLGPRNEDGQLRPRPLSAGRRAVSVHEDQLQAPAERPLRLQRSP VLKRRPKLEAPPSPSLGSGLGSKPLPPYPTEPSSPERSPPSPATDQRGGGPNP

[0035] As used herein, the term “gain of function mutation” or “GOF” refers to a mutation present in the Carmil2 polypeptide that switches the protein into a state which mimics its activated state that results from the physiological CD28 engagement. Therefore, the gain of function mutation can substitute for CD28 engagement and primes the NF-kB signalling pathway for cooperating with the TCR signalling pathway in normal T cells responding to antigenic stimuli (e.g., tumor antigens). Once and only once the TCR has been engaged by an antigen, the gain-of-function mutation allows the full T cell activation in absence of co-receptor CD28 engagement. According to the present invention the gain-of-function mutation is located at position 539 in the human isoform 3 of Carmil2 (SEQ ID NO:1) and at position 538 in the mouse ortholog of the human isoform 3 of Carmil2 (SEQ ID NO:2).

[0036] As used herein, the term “Carmil2 polypeptide” refers to any polypeptide that derives from the isoform 3 of Carmil2 and that comprises the gain of function mutation of the present invention. In particular, the term “Carmil2 polypeptide” refers to i) a polypeptide that comprises an amino acid sequence having at least 90% of identity with 5 the amino acid sequence as set forth in SEQ ID NO:1 wherein the amino acid residue (Q) at position 539 is mutated or ii) a 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:2 wherein the amino acid residue (Q) at position 538 is mutated.

[0037] As used herein, the term “CD28” has its general meaning in the art and refers to 10 CD28 (Cluster of Differentiation 28) that is one of the proteins expressed on T cells. CD28 provides co-stimulatory signals required for T cell activation and survival. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.

[0038] 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 15 stably integrated, in its genome. In particular, the term “host cell” refers to a host cell that functions in an immune response or a progenitor, or progeny thereof. According to the present invention, the host cell is not an immortalized immune cell. As used herein, the term “immortalization” is defined as the acquisition of an indefinite proliferative capacity. For instance, immortalization may be induced in primary cultured cells and 20 finite cell lines by tranfection with telomerase, oncogenes, or the large T antigen of the SV40, or by infection with SV40. Immortalization is not necessarily a malignant transformation, though it may be a component of malignant transformation. Thus, the use of immortalized cell lines is thus excluded from the scope of the present invention. In particular, the host cell is not a Jurkat cell or a derivative thereof (e.g., progeny). As used 25 herein, the term “Jurkat cells” or “Jurkat cell line” refers to an immortalized acute T cell leukemia cell line which has been developed by Dr. Arthur Weiss of the University of California at San Francisco (Abraham, Robert T., and Arthur Weiss. "Jurkat T cells and development of the T-cell receptor signalling paradigm." Nature reviews immunology 4.4 (2004): 301-308). For instance, the Jurkat cells used herein may be 30 obtained from the American Type Culture Collection (ATCC; e.g., ATCC TIB-152).

[0039] 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 5 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 10 “TIL” to T cells that are present in a solid tumor.

[0040] 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. 15 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 20 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 (γ, δ, and ε) in mammals and the ζ-chain. These accessory molecules have negatively charged transmembrane regions and 25 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 30 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 essentialmolecules (e.g., LCK) inside the cell involved in the signaling of the activated T lymphocyte.

[0041] As used herein, the term “exogenous T cell receptor” or “exogenous TCR” refers to a recombinant TCR which is expressed in a host cell by introduction of 5 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. 10

[0042] 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 15 perforins induced by peptide, or degranulation.

[0043] 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 20 (e.g., a population of human host 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).

[0044] 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” 25 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 notpresent 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 5 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 10 cell to which the manipulation was applied and also any progeny of such cell.

[0045] 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 reactivity15 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.

[0046] As used herein the term "CAR-T cell" refers to a T lymphocyte that has been 20 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.

[0047] As used herein, the term “antigen” has its general meaning in the art and generally 25 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.

[0048] 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 5 receptor (e.g., CD19, MUC-16) or capable of suppressing an immune response via receptor-ligand binding (e.g., CD47, PD-L1 / L2, B7.1 / 2).

[0049] 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 10 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 15 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 (^^^^^^^) to five (^^^^) domains, a variable 20 domain (VH) and three to four constant domains (CH1, 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 25 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 30 determining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) can participate to the antibody binding site or influence theoverall 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- 5 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 10 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 15 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 heavy20 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. 25

[0050] 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 30 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 aprimary 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 5 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 10 using standard methods.

[0051] 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 15 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. 20

[0052] As used herein, the term “specificity” refers to the ability of an antibody to detectably bind target molecule (e.g., an epitope presented on an antigen) while having relatively little detectable reactivity with other target molecules. Specificity can be relatively determined by binding or competitive binding assays, using, e.g., Biacore instruments, as described elsewhere herein. Specificity can be exhibited by, e.g., an about 25 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater ratio of affinity / avidity in binding to the specific antigen versus nonspecific binding to other irrelevant molecules.

[0053] The term “affinity”, as used herein, means the strength of the binding of an antibody to an antigen (e.g., an epitope). The affinity of a binding protein is given by the dissociation constant Kd. For an antibody said Kd is defined as [Ab] x [Ag] / [Ab-Ag], 30 where [Ab-Ag] is the molar concentration of the antibody-antigen complex, [Ab] is themolar concentration of the unbound antibody and [Ag] is the molar concentration of the unbound antigen. The affinity constant Ka is defined by 1 / Kd. Preferred methods for determining the affinity of a binding protein can be found in Harlow, et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 5 1988), Coligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol.92:589-601 (1983), which references are entirely incorporated herein by reference. One preferred and standard method well known in the art for determining the affinity of binding protein is the use of Biacore instruments. 10

[0054] The term “binding” as used herein refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, and ionic and / or hydrogen-bond interactions, including interactions such as salt bridges and water bridges. In particular, as used herein, the term "binding'' in the context of the binding of an antibody to a predetermined target molecule (e.g., an antigen or epitope) typically is a15 binding with an affinity corresponding to a KD of about 10-7M or less, such as about 10-8M or less, such as about 10-9M or less, about 10-10M or less, or about 10-11M or even less.

[0055] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including 20 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 25 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 30 to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initialtreatment 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, 5 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 10 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.]).

[0056] As used herein, the expression “therapeutically effective amount” is an amount 15 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- 20 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 cells administered. 25

[0057] 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. The term “pharmaceutical composition” refers also to a composition comprising an active principle in association 30 with a pharmaceutically acceptable vehicle or excipient. A pharmaceutical compositionis for therapeutic use, and relates to health. Especially, a pharmaceutical composition may be indicated for treating or preventing a disease. According to the invention, the term “treating a disease” refers to reducing or alleviating at least one adverse effect or symptom of a disease, disorder or condition associated with a deficiency in an organ, 5 tissue or cell function. The expression “Preventing a disease” or “Inhibiting the development of a disease” refers to preventing or avoiding the occurrence of symptom.

[0058] 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 10 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. The term “pharmaceutically acceptable carrier” that may also be refereed to the term 15 “pharmaceutically acceptable excipient”, refers also to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety 20 and purity standards as required by regulatory offices, such as, for example, FDA Office or EMA.

[0059] As used herein, the term “subject” refers to a mammal, preferably a human. In some embodiments, a subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who / which is awaiting the receipt of, or is receiving medical care or 25 was / is / will be the object of a medical procedure, or is monitored for the development of a disease. In some embodiments, the subject is an adult (for example a subject above the age of 18). In some embodiments, the subject is a child (for example a subject below the age of 18). In some embodiments, the subject is a male. In some embodiments, the subject is a female. In some embodiments, the subject is an immunosuppressed subject. In some 30 embodiments, the subject is under any treatment (e.g., immunosuppressors).DETAILED DESCRIPTION

[0060] The CD28 costimulatory role becomes particularly manifest under the poorly inflammatory conditions often encountered in tumor draining lymph nodes and the tumor microenvironment. Anti-tumoral T cell responses have thus a great reliance on CD28 5 signalling. The possibility of inducing high potential antitumor CAR-T cell responses independently of CD28 pathway engagement by CD80 and CD86 represents thus an appealing approach. Now, the inventors surprisingly show that the mere presence in normal T cells of one gain of function mutation at position 538 in the mouse ortholog of the human isoform 3 of Carmil2 does not drive the development of T-cell cancers. The 10 inventors also show that this mutation switches Carmil2 into an activated state which mimics the activated Carmil2 state resulting from physiological CD28 engagement. Therefore, the gain of function mutation can substitute for CD28 engagement and primes the NF-kB signalling pathway for cooperating with the TCR signalling pathway in normal T cells responding in vivo to antigenic stimuli including tumor antigens. Once and only 15 once the TCR has been engaged by an antigen, including a tumor antigen, the documented Carmil2 gain of function mutation permits full T cell activation in absence of co-receptor CD28 engagement. Therefore, rewriting the CD28 costimulatory pathway of CAR-T cells via the introduction of said gain of function mutation in Carmil2 can induce long-lasting anti-tumor T responses in absence of inflammatory cues. 20

[0061] The first object of the present invention relates to a host cell that is engineered to express a Carmil2 polypeptide.

[0062] In some embodiments, the Carmil2 polypeptide is a constitutively activated Carmil2 polypeptide. In some embodiments, the Carmil2 polypeptide comprises a gain of function mutation. In some embodiments, the Carmil2 polypeptide is a constitutively 25 activated Carmil2 polypeptide comprising a gain of function mutation.

[0063] In some embodiments, the Carmil2 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 wherein the glutamine residue (Q) at position 539 is mutated.

[0064] In some embodiments, the Carmil2 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:2 wherein the glutamine residue (Q) at position 538 is mutated.

[0065] In some embodiments, the Carmil2 polypeptide as described herein comprises or 5 consists in an amino acid sequence as set forth in SEQ ID NO: 1 wherein the glutamine residue (Q) at position 539 is mutated.

[0066] In some embodiments, the Carmil2 polypeptide as described herein comprises or consists an amino acid sequence as set forth in SEQ ID NO: 2 wherein the glutamine residue (Q) at position 538 is mutated. 10

[0067] In some embodiments, the glutamine residue (Q) is substituted by a negatively charged amino acid residue. In some embodiments, the glutamine residue (Q) is substituted by a glutamic acid residue (E). In some embodiments, the glutamine residue (Q) is substituted by an aspartic acid residue (D). In some embodiments, the glutamine residue (Q) is substituted by a glutamic acid residue (E) or an aspartic acid residue (D). 15

[0068] In some embodiments, the host cell of the present invention expresses an endogenous TCR.

[0069] In some embodiments, the host 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 α-and β- 20 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.

[0070] In some embodiments, the host cell expresses an endogenous TCR or an 25 exogenous TCR. In some embodiments, the host cell expresses an antigen receptor.

[0071] In some embodiments, the host cell of the present invention expresses or is engineered for expressing an antigen receptor. Non-limiting examples of antigen receptors include endogenous TCR, exogenous TCR, CAR, and TCR-like chimericreceptors (including T cell Receptor Fusion Construct (TRuC), antibody-TCR (AbTCR), HLA-independent T cell receptor (HIT receptor), synthetic T cell receptor and antigen receptor (STAR), and T cell antigen coupler (TAC)). In some embodiments, the host cell of the present invention expresses or is engineered for expressing an antigen receptor and 5 displays the antigen receptor at the cell surface.

[0072] In some embodiments, the host cell of the present invention is engineered for expressing a CAR. In some embodiments the host cell of the present invention is engineered for expressing a CAR and displays the CAR at the cell surface.

[0073] In some embodiments, the host cell of the present invention is engineered for 10 expressing an HLA-independent T cell receptor (HIT receptor) or synthetic T cell receptor and antigen receptor (STAR). In some embodiments, the host cell of the present invention is engineered for expressing an HLA-independent T cell receptor (HIT receptor) or synthetic T cell receptor and antigen receptor (STAR), and displays the HIT or STAR at the cell surface. 15

[0074] In some embodiments, the host cell of the present invention is engineered for expressing an HLA-independent T cell receptor (HIT receptor). In some embodiments, the host cell of the present invention is engineered for expressing an HLA-independent T cell receptor (HIT receptor) and displays the HIT at the cell surface.

[0075] In some embodiments, the host cell of the present invention is engineered for 20 expressing a synthetic T cell receptor and antigen receptor (STAR). In some embodiments, the host cell of the present invention is engineered for expressing a synthetic T cell receptor and antigen receptor (STAR), and displays the STAR at the cell surface.

[0076] In some embodiments, the host cell is engineered for expressing a CAR, an HLA- 25 independent T cell receptor (HIT receptor) or synthetic T cell receptor and antigen receptor (STAR). In some embodiments, the host cell is engineered for expressing a CAR, an HLA-independent T cell receptor (HIT receptor) or synthetic T cell receptor and antigen receptor (STAR) and displays the CAR, HIT or STAR at the cell surface.

[0077] In some embodiments, the host cell is a murine or a human cell.

[0078] In some embodiments, the host cell is an allogeneic cell or autologous cell. In some embodiments, the host cell is an allogeneic cell. In some embodiments, the host cell is an autologous cell. 5

[0079] As used herein “autologous cell” refers to a cell derived from the same individual to whom it is later to be re-introduced.

[0080] As used herein “allogeneic cell” refers to a cell derived from a different individual of the same specie as the individual to whom the cell is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci 10 are not identical.

[0081] 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. 15

[0082] 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 20 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 γδ T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of 25 inducing the death of infected somatic or tumor cells.

[0083] In some embodiments, the host 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. In some embodiments, the T cell is a CD4+ T cell or a CD8+T cell. In some embodiments, the T cell is a CD4+ T cell, a CD8+ T cell, a double positive T cell, a double negative T cell, a γδ T cell, a memory T cells a regulatory CD4+ T cell, a mucosal associated invariant T (MAIT) cell, an innate-like T cell, or an invariant Natural Killer T (iNKT) cell. 5

[0084] In some embodiments, the host cells of the present invention are thus CAR-T cells.

[0085] In some embodiments, the host 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). iPSCs10 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 15 cultures by defined factors". Cell 126 (4): 663-76. In the present invention, the obtention of human embryonic stem cells does not comprise a step of destruction of human embryos.

[0086] In some embodiments, the host cell is a hematopoietic stem cell. As used herein, the term “hematopoietic stem cell” or “HSC” refers to blood cells that have the capacity 20 to self-renew 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- CD34+CD38+IL-3aloCD45RA−, and Lin-CD34+CD38+CD10+(Daley et al., Focus 25 18:62-67, 1996; Pimentel, E., Ed., Handbook of Growth Factors Vol. III: 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 30 are isolated form peripheral blood cells.

[0087] The present invention further relates to a population of host cells as described herein.

[0088] Any CARs that are suitable for engineering host cells for use in adoptive immunotherapy therapy can be used in the present invention. CARs that can be used in 5 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.

[0089] The CAR of the present invention typically comprises one more polypeptides 10 having an extracellular domain and an intracellular domain joined by a transmembrane domain. The CAR of the present invention typically comprises one or more polypeptides, wherein each polypeptide comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain and the intracellular domain are joined by the transmembrane domain. 15

[0090] 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. In some embodiments, the CAR is a monomeric molecule consisting of one polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain and the 20 intracellular domain are joined by the transmembrane domain.

[0091] 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. In some embodiments, the CAR is a heterodimeric 25 molecule consisting of two polypeptides, wherein each polypeptide comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain and the intracellular domain of each polypeptide are joined by the transmembrane domain, and wherein the two polypeptides are capable of dimerizing via their respective extracellular domain.

[0092] The extracellular domain of the CAR, expressed on the surface of the host cell, comprises an antigen binding domain.

[0093] In some embodiments, the antigen binding domain is derived from an antibody, preferably a humanized or a human antibody. In some embodiments, the antigen binding 5 domain of the CAR comprises an antibody fragment, preferably of a humanized antibody or human antibody.

[0094] In some embodiments, the antigen binding domain is an antibody fragment selected from fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody 10 fragments, single chain variable fragments (scFv), single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments, diabodies, and multi-specific antibodies formed from antibody fragments.

[0095] 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. 15 In some embodiments the antigen binding domain is selected from a Fab and a scFv. In some embodiments, the antigen binding domain is a scFv. In some embodiments, when the antigen binding domain is a scFv, 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 20 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 and25 comprises one or more repeats of the amino acid sequence Gly-Gly-Gly-Ser or Gly-Gly- Gly-Gly-Ser.

[0096] 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 bindingdomain 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 5 is designed to be sufficiently flexible to allow the antigen binding domain to orient in a manner that allows antigen recognition.

[0097] The hinge may be derived from or include at least a portion of an immunoglobulin Fc region, for example, an IgG1 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. The hinge may 10 comprise at least a portion of an immunoglobulin Fc region, for example, an IgG1 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.

[0098] In some embodiments, the hinge domain includes at least a portion of an IgG1, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls 15 within its CH2 and CH3 domains. In some embodiments, the hinge domain comprises at least a portion of an IgG1, an IgG2, an IgG3, an IgG4, an IgE, an IgM, or an IgA immunoglobulin Fc region that falls within its CH2 and CH3 domains.

[0099] Exemplary hinges include, but are not limited to, a CD8a hinge, a CD28 hinge, IgG1 / IgG4 (hinge-Fc part) sequences, IgG4 hinge alone, IgG4 hinge linked to CH2 and 20 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 WO2014031687, 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 25 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 sequenceis derived from a CD8 alpha molecule or a CD28 molecule. In some embodiments, the hinge sequence comprises a portion of a CD8 alpha molecule or a CD28 molecule.

[0100] 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 5 spacer and intracellular sequences.

[0101] In some embodiments, the transmembrane domain is derived either from a natural or from a synthetic source. In some embodiments, the transmembrane domain of the CAR comprises a portion of a membrane-bound protein or a transmembrane protein, or comprises a synthetic amino acid sequence. 10

[0102] Where the source is natural, the domain in some embodiments is derived from any membrane -bound or transmembrane protein. In some embodiments, the transmembrane domain comprises a portion of a membrane-bound protein or a 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, 15 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.

[0103] Alternatively, the transmembrane domain in some embodiments is synthetic. In some embodiments, the synthetic transmembrane domain comprises predominantly 20 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, 25 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.

[0104] The role of the intracellular domain of the CAR is to produce an activation signal to the host cell (e.g., T cell) as soon as the extracellular domain has recognized theantigen. In particular, the intracellular domain of the CAR triggers or elicits activation of at least one of the normal effector functions of the host 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 5 protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, 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 CD3ζ. The intracellular domain of the CAR can be designed to comprise a signalling domain (such as the CD3ζ signalling 10 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), OX40 (CD134), CD30, CD40, CD244 (2B4), ICOS, lymphocyte function- associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that 15 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), OX4020 (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.

[0105] 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 25 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 CD3ζ chain as a single signalling domain. 30 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 CD3ζ and CD28 signalling domains, while third-generation CAR may contain CD3ζ, CD28 and either OX40 (CD134) or 4-1BB (CD137). “TRUCKs” represent the recently developed “fourth- generation” CARs. TRUCKs (T cells redirected for universal cytokine killing) are CAR- 5 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 10 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.

[0106] In some embodiments, the CAR of the present invention consists in a TCR mimic. 15 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” 20 as described in Liu, Yue, et al. "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 WO2020029774 that are incorporated by 25 reference or “HLA-independent T cell receptor” or “HIT receptor” as described in Mansilla-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, 30 the most sensitive design to date, can provide. The STAR and HIT architecture cannot incorporate a CD28 transmembrane domain. Moreover, steric hindrance limits thebenefits 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 5 immunes cells armed with STAR and HIT receptors will be particularly prone to benefit of the costimulatory signals provided by co-expressed the Carmil2 polypeptide of the present invention.

[0107] In some embodiments, the CAR of the present invention comprises or consists in a STAR or HIT receptor, wherein the STAR or HIT receptor is a TCR heterodimer. In 10 some embodiments, the STAR or HIT receptor is a TCR heterodimer.

[0108] In some embodiments, the STAR or HIT receptor is a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domains are substituted by a variable domain of an antibody as described herein. In some embodiments, the STAR or HIT receptor comprises a variable domain of an antibody as described herein. In some embodiments, the STAR 15 or HIT receptor comprises the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of an antibody specific for a given antigen.

[0109] 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. 20

[0110] In some embodiments, the CAR comprises a TCR heterodimer, wherein the immunoglobulin (Ig)-variable (V) domains are substituted by a variable domain of an antibody.

[0111] 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 25 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.

[0112] In some embodiments, the STAR or HIT receptor is 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 5 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.

[0113] In some embodiments, the CAR comprises a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is substituted by a first10 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. 15

[0114] 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) 20 that binds to the antigen of interest.

[0115] In some embodiments, the STAR or HIT receptor is 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 25 dimerizing with the VH domain to form a fragment variable (Fv) that binds to the antigen of interest.

[0116] In some embodiments, the CAR comprises 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 chainis 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.

[0117] According to the present embodiments, the CAR is thus capable of associating 5 with a CD3 complex to form the T-cell co-receptor. In some embodiments, the CD3 complex comprises a CD3γ chain, a CD3δ chain, and two CD3ε 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 10 TCR in the CD3 / TCR complex of host cell.

[0118] In some embodiments, the host cell expresses a TCR or CAR that is specific for a tumor antigen. Non-limiting examples of tumor antigens include carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD8, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CLL1, CD34, CD38, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, 15 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-α, Ganglioside G2 (GD2), Ganglioside G3 (GD3), human Epidermal Growth 20 Factor Receptor 2 (HER-2), human telomerase reverse transcriptase (hTERT), Interleukin-13 receptor subunit alpha-2 (IL-13Ra2), κ-light chain, kinase insert domain receptor (KDR), Lewis Y (LeY), L1 cell adhesion molecule (L1CAM), melanoma antigen family A, 1 (MAGE-A1), Mucin 16 (MUC16), Mucin 1 (MUC1), Mesothelin (MSLN), ERBB2, MAGEA3, p53, MART1, GP100, Proteinase3 (PR1), Tyrosinase, Survivin, 25 hTERT, EphA2, NKG2D ligands, cancer-testis antigen NY-ESO-1, oncofetal antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), ROR1, 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.

[0119] In some embodiments, the host 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.

[0120] The host cell of the present invention is preparing by any conventional method 5 well known in the art. Typically, the host cells are engineered for expressing the Carmil2 polypeptide of the present invention and optionally for expressing a TCR or CAR of interest.

[0121] Thus, a further object of the present invention relates to a method of preparing a host cell of the present invention, comprising the step consisting of introducing into a host 10 cell a polynucleotide that encodes for the Carmil2 polypeptide of the present invention and optionally one or more polynucleotide(s) that encodes for the CAR or TCR of interest.

[0122] In some embodiments, the method of preparing a host cell of the present invention is an in vitro method.

[0123] In some embodiments, the in vitro method of preparing a host cell as described 15 herein, comprises delivering to the host cell a polynucleotide that encodes for the Carmil2 polypeptide.

[0124] It is contemplated that a polynucleotide can be introduced into the host cells as naked DNA or in a suitable vector.

[0125] Naked DNA generally refers to the DNA contained in a plasmid expression vector 20 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 25 emulsions, micelles, mixed micelles, and liposomes.

[0126] In some embodiments, the polynucleotide is introduced into the host 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. Insome 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 5 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 10 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. 15 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 20 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 25 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 30 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 aviral 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. 5

[0127] 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 10 be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

[0128] 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 15 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 20 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), β-actin, ubiquitin, and a simian virus 40 (SV40) / CD43 composite promoter, elongation factor (EF)-1a and 25 the spleen focus-forming virus (SFFV) promoter.

[0129] 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 30 identical polypeptide to be encoded by a variety of nucleotide sequences. A variety ofcodon 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.

[0130] Use of polycistronic expression cassettes that can both express the Carmil2 polypeptide and the TCR or CAR are preferably used. Typically the polycistronic 5 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-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picornavirus IRES, poliovirus IRES and encephalomyocarditis virus IRES) and / or cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A and F2A 10 peptides). Thus use of polycistronic polynucleotides or vectors encoding for both the Carmil2 variant and the TCR or CAR are particularly suitable for preparing the host cells of the present invention.

[0131] In some embodiments, the polynucleotides that respectively encode for the Carmil2 polypeptide and the TCR or CAR are expressed by an host cell through a 15 modified genomic locus. In some embodiments, an expression cassette, preferably a polycistronic expression cassette that encodes for the Carmil2 polypeptide and the TCR or CAR, is integrated into a targeted genomic locus of an host cell through targeted genome editing methods. In some embodiments, the targeted genomic locus can be CD3δ, CD3ε, CD247, B2M, TRAC, TRBC1, TRBC2, TRGC1 and / or TRGC2 loci. In some 20 embodiments, the CAR or TCR of the present invention and the Carmil2 polypeptide are expressed by an host 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 25 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 Carmil2 polypeptide are expressed from an expression cassette placed in an endogenous TRAC locus and / or a TRBC locus of an host cell. In some embodiments, the 30 placement of expression cassette disrupts or abolishes the endogenous expression of aTCR comprising a native TCR α chain and / or a native TCR β chain in the host cell. In some embodiments, the placement of the expression cassette prevents or eliminates mispairing between the recombinant CAR or TCR and a native TCR α chain and / or a native TCR β chain in the host cell. 5

[0132] In some embodiments, the TCR, CAR, HIT or STAR is expressed under control of an endogenous promoter. In some embodiments, the TCR, CAR, HIT or STAR is expressed under control of an endogenous promoter of T cell receptor (TCR). In some embodiments, the TCR, CAR, HIT or STAR is expressed under control of an endogenous promoter of T cell receptor (TCR), such as for example TCRa promoter, TCRb promoter, 10 CD3d promoter, CD3g promoter, CD3e promoter, and CD3z promoter. In some embodiments, the TCR, CAR, HIT or STAR is expressed under control of an endogenous promoter of T cell receptor (TCR), selected from TCRa promoter, TCRb promoter, CD3d promoter, CD3g promoter, CD3e promoter, and CD3z promoter.

[0133]

[0094] In some embodiments, the TCR, CAR, HIT or STAR is not expressed 15 under control of an endogenous promoter.

[0134] In some embodiments, the TCR, CAR, HIT or STAR is expressed under control of an exogenous promoter. Non-limiting examples of exogenous promoter include, the cytomegalovirus (CMV) promoter, the ubiquitin promoter, the simian virus 40 (SV40), the spleen focus-forming virus (SFFV) promoter, the myeloproliferative sarcoma virus 20 enhancer negative control region (MND) promoter, the human elongation factor 1 alpha (EFla) promoter, the gamma retroviral long terminal repeats (LTR) promoter, the phosphoglycerate kinase (PGK) promoter, the actin promoter, the CD11a promoter, the CD25 promoter, the CD27 promoter, the CD28 promoter, the CD38 promoter, the CD45RA promoter, the CD45RO promoter, the CD57 promoter, the CD58 promoter, the 25 CD62L promoter, the CD69 promoter, the CD95 promoter, the CD99 promoter, the CD103 promoter, the CD122 promoter, the CD127 promoter, the CD130 promoter, the CD132 promoter, the CD161 promoter, the KLRG-1 promoter, the HLA-DR promoter, the Ki-67 promoter, the CCR4 promoter, the CCR5 promoter, the CCR6 promoter, the CCR7 promoter, the CCR9 promoter, the CCR10 promoter, the CXCR3 promoter, the 30 CXCR4 promoter, the CLA promoter, the Granzyme A promoter, the Granzyme Bpromoter, the Perforin promoter, the T-bet promoter, the IFNgamma promoter, the TIM3 promoter, the GATA3 promoter, the IL1 promoter, the IL2 promoter, the IL4 promoter, the IL5 promoter, the IL6 promoter, the IL7 promoter, the IL10 promoter, the IL13 promoter, the IL15 promoter, the IL17A promoter, the IL-18Ra promoter, the IL21 5 promoter, the IL23R promoter, the FoxP3 promoter, the CTLA4 promoter, the programmed death 1 (PD-1) promoter, the c-Kit promoter, the nuclear factor of activated T cells (NFAT) promoter, the T cell immunoglobulin mucin-3 (TIM-3) promoter, the cytotoxic T lymphocyte antigen-4 (CTLA4) promoter, the lymphocyte-activation protein 3 (LAG-3) promoter, the tumor necrosis factor (TNF)-related apoptosis-inducing ligand 10 (TRAIL) promoter, the B- and T-lymphocyte attenuator (BTLA) promoter, the Fas ligand (FasL) promoter, the TIGIT promoter, the TGF-beta promoter, the Eomes promoter, the 2B4 promoter, the Type I interferon (IFN) alpha, the Type I IFN beta promoter, the IFN gamma promoter, the TNF-alpha promoter, the IRF3 promoter, the IRF7 promoter, the NFkB promoter, the AP-1 promoter, the NR4A1 promoter, the NR4A2, the NR4A3 15 promoter, and a truncated version of the PGK promoter (PGK 100).

[0135] 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 guide20 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 25 cell receptor loci in the cell are modified to eliminate the endogenous expression of the endogenous TCR chain.

[0136] 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 α-and β-chains with preservation of near-physiological T-cell function." Nature 30 biomedical engineering 3.12 (2019): 974-984 and Rohaan, M. W., et al. "MART-1 TCRgene-modified peripheral blood T cells for the treatment of metastatic melanoma: a phase I / IIa clinical trial." Immuno-Oncology and Technology 15 (2022): 100089.

[0137] Once the population of host cells is obtained, functionality of the cells may be evaluated according to any standard method which typically include a suppressive assay. 5 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 10 blotting, FACS, or Radioimmunoassays may be applicable to the present invention.

[0138] In some embodiments, the host 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. 15

[0139] The present invention further relates to a composition comprising, consisting essentially of, or consisting of the host cell or the population of host cells as described herein.

[0140] The composition as described herein may further comprise at least one pharmaceutically acceptable excipient. 20

[0141] Thus, a further object of the present invention relates to a pharmaceutical composition comprising the population of host cells of the present invention and a pharmaceutically acceptable carrier.

[0142] The pharmaceutical composition may comprise, consist essentially of or consist of the host cell or the population of host cells of the present invention, and a 25 pharmaceutically acceptable carrier.

[0143] 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 withhuman serum albumin. A treatment-effective amounts of cells in the composition is dependent on the relative representation of the host 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 5 approximately 103 / kg, preferably 5x103 / 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 10 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.

[0144] Pharmaceutically acceptable carriers or excipients that may be used in the pharmaceutical composition of the invention include, but are not limited to, ion 15 exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as, for example, human serum albumin, buffer substances such as, for example, phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as, for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, 20 colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (for example sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene- polyoxypropylene- block polymers, polyethylene glycol and wool fat.

[0145] In some embodiments, the pharmaceutical composition of the present invention 25 is, or is for use as, a medicament. Consequently, the present invention further relates to a medicament.

[0146] In some embodiments, the medicament comprises, consists essentially of or consists of the host cell or the population of host cells of the present invention.

[0147] As used herein, the term “consisting essentially of”, with reference to a composition, pharmaceutical composition or medicament, means that the host cell, or the population of host cells of the invention are the only one therapeutic agents or agents with a biologic activity within said composition, pharmaceutical composition or medicament. 5

[0148] In some embodiments, the host cell, the population of host cells, the composition, the pharmaceutical composition, or the medicament as described herein, is / are formulated for administration to a subject in need thereof.

[0149] In some embodiments, the host cell, the population of host cells, the composition, the pharmaceutical composition or the medicament of the invention is administered (or is 10 to be administered or is for administration) by intravesical administration, intravaginal administration, intraosseous administration, intraperitoneal administration, intrauterine administration, intraocular administration, intradermal administration, intraarterial administration, intracerebral administration, intranasal administration, enteral administration, buccal administration, intranasal administration, oral administration, 15 rectal administration, or by inhalation.

[0150] In some embodiments, the host cell, the population of host cells, the composition, the pharmaceutical composition or the medicament as described herein, is / are to be administered to a subject in need thereof in a therapeutically effective amount.

[0151] It will be however understood that the total daily usage of the host cell, the 20 population host cells, the composition, the pharmaceutical composition, or the medicament, as defined hereinabove, will be decided by the attending physician within the scope of sound medical judgment.

[0152] In particular, the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disease being treated and the 25 severity of the disease; activity of the host cell, the population host cells, the composition, the pharmaceutical composition, or the medicament, as defined hereinabove, employed; the age, body weight, general health, gender and diet of the subject; the time of administration, route of administration, and rate of excretion of the host cell, the population host cells, the composition, the pharmaceutical composition, or themedicament, as defined hereinabove, employed; the duration of the treatment; drugs used in combination or coincidental with the host cell, the population host cells, the composition, the pharmaceutical composition, or the medicament, as defined hereinabove, employed; and like factors well-known in the medical arts. The total dose 5 required for each treatment may be administered by multiple doses or in a single dose.

[0153] 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 cells of the present invention.

[0154] Another object of the present invention relates to a method of therapy in a subject 10 in need thereof comprising administering to the subject a therapeutically effective amount of the host cell, population of host cells, composition, pharmaceutical composition, or medicament as described herein.

[0155] The population of host cells prepared as described above can be thus utilized in methods and compositions for adoptive immunotherapy in accordance with known 15 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.

[0156] Currently, most adoptive immunotherapies are autolymphocyte therapies (ALT) directed to treatments using the patient's own immune cells. These therapies involve 20 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 biologics and drugs to convey them to a T cell profile. Once the T cells are engineered to express the Carmil2 polypeptide and optionally the TCR or CAR of interest, these ex vivo cells are reinfused into the patient 25 to induce an immune response against the antigen of interest. Thus, in some embodiments, the host cell is isolated from a subject to whom the engineered host cells are to be adoptively transferred. In some embodiments, a population of host 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 Carmil2polynucleotide 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 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- 5 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 Carmil2 polypeptide as well as a TCR / CAR of interest.

[0157] In particular, the method of the present invention is particularly suitable for the 10 treatment of cancer or infectious diseases.

[0158] Thus, the present invention also relates to a method for treating cancer or infectious diseases in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the host cell, population of host cells, composition, pharmaceutical composition, or medicament as described herein. 15

[0159] 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 20 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; 25 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; 30 adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solidcarcinoma; 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; 5 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 10 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, 15 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; 20 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, 25 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; 30 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 5 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 10 leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.

[0160] Advantageously, the host cell of the present invention is particularly suitable for the treatment of cold tumors. In some embodiments, the host cell, population of host cells, composition, pharmaceutical composition, or medicament as described herein is 15 particularly suitable for the treatment of cold tumors. In some embodiments, the host cell, population of host cells, composition, pharmaceutical composition, or medicament as described herein is particularly suitable for the treatment of immunogenic tumors. In some embodiments, the host cell, population of host cells, composition, pharmaceutical composition, or medicament as described herein is particularly suitable for the treatment 20 of cold tumors or immunogenic tumors.

[0161] As used herein, the term “cold tumor” has its general meaning in the art and describes a tumor that is not likely to trigger a strong immune response. Cold tumors tend to be surrounded by cells that are able to suppress the immune response and keep T cells (a type of immune cell) from attacking the tumor cells and killing them. Cold tumors 25 usually do not respond to immunotherapy. Most cancers of the breast, ovary, prostate, pancreas, and brain (glioblastoma) are considered cold tumors. More particularly, the host cells of the present invention are engineered to bypass the CD28 co-stimulation pathway and thus are particularly suitable for inducing durable anti-tumor responses under non- inflammatory conditions.

[0162] As used herein, the term “immunogenic tumor” has its general meaning in the art and describes a tumor that is likely to trigger a strong immune response. Immunogenic tumors are also termed “hot tumors”. Immunogenic tumors often have many molecules on their surface, which allow cytotoxic T cells to attack and kill the tumors cells. 5 Immunogenic tumors tend to response to immunotherapy. Melanoma, non-small cell lung cancer, bladder cancer, head and neck cancer, kidney cancer, and liver cancer are usually considered immunogenic tumors.

[0163] As used herein, the term "infectious diseases" has its general meaning in the art and includes, but is not limited to disorders caused by for example bacteria, viruses, fungi 10 or parasites.

[0164] Advantageously, the host 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 since the CD28-Rltpr / Carmil2 signalling axis enable signalling threshold to be overcame when TCR occupancy is low. In some embodiments, the host cells 15 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 20 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 25 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 30 between about 4,000 molecules per cell and about 2,000 molecules per cell, betweenabout 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.

[0165] The quantity of host 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 1×108, about 2×108, about 3×108, about 4×108, or about 5×108of the host cells are administered to the subject. The precise determination of what would be considered a 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.

[0166] Another object of the present invention is a host cell, a population of host cells, a composition, a pharmaceutical composition, or a medicament as described herein for use in therapy. In some embodiments, a therapeutically effective amount of the host cell, the population of host cells, the composition, the pharmaceutical composition or the medicament as described herein is for use in therapy.

[0167] Another object of the present invention is a host cell, a population of host cells, a composition, a pharmaceutical composition, or a medicament as described herein for use as a medicament. In some embodiments, a therapeutically effective amount of the host cell, the population of host cells, the composition, the pharmaceutical composition or the medicament as described herein is for use as a medicament.

[0168] The present invention relates also to a host cell, a population of host cells, a composition, a pharmaceutical composition, or a medicament according to the invention or a therapeutically effective amount of the host cell, the population of host cells, thecomposition, the pharmaceutical composition or the medicament as described herein, for use in the treatment of cancer or infectious diseases.

[0169] Another object of the present invention is the use of a host cell, a population of host cells, a composition, a pharmaceutical composition, or a medicament as described 5 herein or a therapeutically effective amount of the population of the host cell, the host cells, the composition, the pharmaceutical composition or the medicament as described herein, for the manufacture of a medicament particularly suitable for the treatment of cancer or infectious diseases.

[0170] Another object of the present invention is a host cell, a population of host cells, a 10 composition, a pharmaceutical composition, or a medicament as described herein or a therapeutically effective amount of the population of the host cell, the host cells, the composition, the pharmaceutical composition or the medicament as described herein for treating cancer or infectious diseases.

[0171] In some embodiments, the subject is a human. In some embodiments, the subject 15 is affected, preferably diagnosed with a cancer. In some embodiments, the subject is affected, preferably is diagnosed, with an infectious disease.

[0172] 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. 20 BRIEF DESCRIPTION OF THE DRAWINGS

[0173] Figures 1A-1B. Structure of the two mutated regions present in the mouse Carmil2Q538E-NterOSTgene. (Fig. 1A) The CAG codon found in exon 20 of the mouse Carmil2 gene 25 (ENSMUST00000213019.2 Carmil2-203) and coding for the glutamine residue found at position 538 of the CARMIL2 protein was mutated into a GAG codon coding for a glutamic acid. The resulting mutant allele coding for a CARMIL2 p.Q538E mutation isdenoted as Carmil2Q538E. Knock-in mice homozygous (Carmil2Q538E) or heterozygous (Carmil2Q538E / +) for the Carmil2Q538Eallele were established. (Fig. 1B) A sequence coding for a Twin-Strep-tag (SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO:10); (Junttila et al., 2005)) and a GlySerGly linker was inserted between the first (ATG) and the second (GCA) codons of the first exon of the mouse Carmil2Q538Emutant allele to give rise to a Carmil2Q538E-NterOSTallele. Knock-in mice homozygous for the Carmil2Q538E-NterOSTallele were established. In parallel experiments, knock-in mice expressing a Twin-Strep-tag at the amino terminus of wild-type (WT) CARMIL2 protein were also established and denoted as Carmil2NterOST. Sequences of Figure 1 are represented by SEQ ID NO:13 to 18. In particular, SEQ ID NO:14 and SEQ ID NO:17 are the complementary sequences of SEQ ID NO:13 and SEQ ID NO:16 in the sense 5’->3’.

[0174] Figures 2A-2C. Phenotype of T cells in wild-type, Cd28– / –, Carmil2Q538E, and Carmil2Q538ECd28– / –mice. (Fig.2A) Spleen of wild-type (WT), Cd28– / –, Carmil2Q538E, and Carmil2Q538ECd28– / –mice were analyzed by flow cytometry for expression of CD3 and TCR^. Numbers indicate the percentage of cells in the specified quadrants. (Fig.2B) T cells from the spleen of WT, Cd28– / –, Carmil2Q538E, and Carmil2Q538ECd28– / –mice were analyzed by flow cytometry for expression of CD4 and CD8. Numbers indicate the percentage of cells in the specified quadrants. (Fig. 2C) CD4+T cells from the spleen of WT,Carmil2Q538E, and Carmil2Q538ECd28– / –mice were analyzed by flow cytometry for expression of CD25 and Foxp3. Numbers indicate the percentage of Foxp3+CD25+ regulatory CD4+ T cells. Data are representative of at least three independent experiments, with two to three mice per genotype.

[0175] Figure 3. Prior to activation the primary CD4+T cells of Carmil2Q538E-NterOSTmouse show levels of Carmil2Q538E-NterOST-Card11 interaction stoichiometry that are comparable to those observed following activation of CD4+T cells from Carmil2NterOSTmice and the stoichiometry of the constitutive Carmil2Q538E-NterOST- Card11 interaction is further increased 10-fold following TCR activation.Dot plot showing the interaction stoichiometry of Carmil2NterOSTand Carmil2Q538E-NterOSTproteins with Card11 proteins in CD4+T cells that were isolated from Carmil2NterOSTand Carmil2Q538E-NterOSTmice, respectively, and analyzed before (NS = not stimulated) and 30 s, 2 min, 5 min,10 min, 15 min and 30 min after stimulation with pervanadate, a TCR pharmacologic mimics. Left part of the Figure. Prior to activation, minute numbers of Carmil2 proteins are found associated with Card11 proteins in Carmil2NterOSTCD4+T cells. Following TCR activation, the Carmil2NterOST-Card11 interaction stoichiometry increased 10-fold and this increase is sustained for at least 30 min after TCR activation. Right part of the Figure. Prior to activation, the Carmil2Q538E-NterOST-Card11 interaction stoichiometry already reaches levels similar to the maximal Carmil2NterOST-Card11 interaction stoichiometry reached in TCR-activated Carmil2NterOSTCD4+T cells. Upon TCR activation, the stoichiometry of the constitutive Carmil2Q538E-NterOST-Card11 interaction stoichiometry further increases 10-fold. Data are representative of at least three independent experiments.

[0176] Figures 4A-4B. Physiological levels of Carmil2Q538Eprotein fully substitute for CD28-CD80 / CD86 engagement during antigen-induced proliferation and cytokine production of OT-1 T cells in response to antigen-laden antigen presenting cells. Mice expressing the OT-I TCR specific for the N4 ovalbumin peptide (Barnden et al, 1998) were crossed onto Carmil2Q538Emice to give rise to OT-I Carmil2Q538Emice. Naïve CD8+T cells were purified from the spleen of either OT-I or OT-I Carmil2Q538Emice by immunomagnetic negative selection. Purified T cells were stimulated with antigen presenting cells (APC) corresponding to irradiated H-2 Kb-positive spleen cells isolated from T cell-deficient mice expressing (Cd3e^5 / ^5WT APC) or lacking CD80 and CD86 (Cd3eΔ5 / Δ5Cd80− / −Cd86− / −APC). APC were pulsed for 2 h with the N4 agonist OVA peptide (Hogquist et al., 1994) and used to stimulate OT-I or OT-I Carmil2Q538ET cells. After 48 h of culture, T-cell proliferation (Fig.4A) and IL-2 production (Fig.4B) were assessed. Data are representative of two independent experiments.

[0177] Figures 5A-5B. Physiological levels of Carmil2Q538Eprotein fully substitute for CD28-CD80 / CD86 engagement during anti-tumor responses.(Fig.5A) Cohort (n = 5) of 10 weeks-old, wild-type (WT),Carmil2Q538E, and Cd28– / –Carmil2Q538ECd28– / –C57BL / 6 mice were injected subcutaneously into the flank with 106cells of the C57BL / 6-derived mouse melanoma tumor BRAFV600EPtgs– / –and monitored for tumor growth. (Fig.5B) Consistent with the view that BRAFV600EPtgs– / –tumor cells are immunogenic and form spontaneously regressing tumor in WT mice, all the mice belonging to the WT cohort rejected the BRAFV600EPtgs– / –tumor cells. The BRAFV600EPtgs– / –tumor has been used to establish that cells belonging to innate (NK cells, dendritic cells) and adaptive (T cells) immunity contribute to its eradication (Bottcher et al., 2018; Zelenay et al., 2015). Consistent with the view that the activation of the CD28 costimulatory pathway plays an essential role in the establishment of anti- tumor T-cell responses, the results depicted formice in the present Figure show that the lack of CD28 permit BRAFV600EPtgs– / –tumor formation. Mice homozygous for the Carmil2Q538Emutation rejected the BRAFV600EPtgs– / –tumor as efficiently as WT mice. Importantly, analysis of Carmil2Q538ECd28– / –mice shows that the sole expression of Carmil2Q538Emolecules is capable of fully substituting for CD28-CD80 / CD86 engagement during anti-tumor responses. Data are representative of three independent experiments.

[0178] Figures 6A-6B. Mice expressing a single copy of the Carmil2Q538Eallele show increased numbers of Foxp3+regulatory T cells and a single copy of Carmil2Q538Eallele suffices to substitute for CD28-CD80 / CD86 engagement for Foxp3+regulatory T cell generation. (Fig.6A) Spleenmice were analyzed by flow cytometry for their cellularity. (Fig. 6B) Spleen of WT,Carmil2Q538E / +, and Carmil2Q538E / +Cd28– / –mice were analyzed by flow cytometry for the numbers of Foxp3+ CD4+ regulatory T cells (T reg). Numbers indicate the percentage of cells in the specified quadrants. Data are representative of at least three independent experiments, with three mice per genotype. Mean and SD are shown. **P ≤ 0.001, ***P ≤ 0.001; multiple unpaired T-test; E two-way ANOVA test.

[0179] Figures 7A-7B. Upon stimulation with suboptimal amounts of anti-CD3, naïve T cells from mice expressing a single copy of the Carmil2Q538Eallele are capableof producing levels of IL-2 and IFN^ comparable to those of wild-type, naive T cells stimulated with a combination of anti-CD3 plus anti-CD28 antibodies. Naïve CD4+T cells isolated from the spleen of WT and Carmil2Q538E / +mice were left non activated (na) or activated with suboptimal amount of plate-bound anti-CD3 in absence (a-CD3) or presence (a-CD3 plus anti-CD28) of soluble anti-CD28. Cells were also stimulated with PMA and ionomycin (PI). After 48 h of culture, (Fig.7A) IFN^^and (Fig.7B) IL-2 were measured in the culture supernatant. Data are representative of three independent experiments, with three mice per genotype. Mean and SD are shown. **P ≤ 0.001, ***P ≤ 0.001; multiple unpaired T-test; E two-way ANOVA test. ns, non significant.

[0180] Figures 8A-8D: In vivo efficacy of a cell therapy protocol utilizing T-cells engineered with the gene coding for Carmil2Q538Ein the context of solid cancers. MC38-Ova cancer cells were administered to immunocompetent mice in order to observe subcutaneous tumor development. Mice were injected intravenously 7 days after tumor inoculation either with an isotype control antibody (Fig.8A), an anti-PD1 antibody (Fig. 8B), naive CD8+ T cells from OT-1 mice (Fig.8C) or naive CD8+ T cells from OT-1 Carmil2Q538Emice (Fig. 8D). Efficacy of a cell therapy protocol is demonstrated by measuring the tumor volume (mm3) over time.

[0181] Figures 9A-9D: Phenotype of Carmil2Q539Egene expressing CAR-T cells. Two batches of human CAR-T cells expressing the Carmil2Q539Egene were generated from healthy donor’s peripheral blood T cells and evaluated for their phenotype at the end of the production period. Fig.9A shows the expression of CD19 CAR, tNGFR, CD4, CD8 and OST tag. Fig.9B and FIG.9C show respectively IFN-γ and IL-2 secretion of CAR- T cell populations in coculture during 48h with CD19-positive Nalm6 tumors cells. Fig. 9D shows the Cytotoxicity of CAR T-cell populations against CD19-positive Nalm6 tumors cells. * p< 0.05, ** p<0.01 Unpaired T-test.EXAMPLES Introduction The CD28 costimulatory pathway plays an essential role in the establishment of anti- tumor T-cell responses 5

[0182] The Cancer-Immunity cycle is initiated when dendritic cells (DC) infiltrating the tumor microenvironment (TME) detect tumor-triggered danger signals and capture tumor antigens. The resulting activated and immunogenic DC migrate to the tumor-draining lymph nodes (DLN) where they activate naive T lymphocytes specific for the tumor antigens displayed at the DC surface. The Cancer-Immunity cycle continues with the 10 migration of the activated tumor-specific effector T cells out of the tumor DLN and ends up with their infiltration into the TME where they kill the tumor cells. In contrast to DC egressing from inflamed TME, those originating from poorly inflamed TME express reduced levels of CD80 and CD86 molecules. As a result, they do not trigger CD28 – the major T cell co-stimulatory molecule – and induce suboptimal CD4+and CD8+T 15 lymphocyte anti-tumor responses, which ultimately fail eradicating the tumor. It should be noted that the presence of Foxp3+regulatory T cells within the TME further reduces via trogocytosis the expression of CD80 and CD86 molecules on the surface of DCs and contribute to diminish their ability to induce potent anti-tumor effector T lymphocytes (Binnewies et al., 2019; Kidani et al., 2022). Accordingly, the possibility of inducing 20 efficient anti-tumor T responses independently of the activation of the CD28 pathway by CD80 and CD86 ligands constitutes a particularly promising approach since it should relieve naïve and effector T cells from the need of CD28 / CD80-CD86 engagement and render them immune to Foxp3+regulatory T cell-mediated inhibition. The present patent application describes an experimental approach that permit to readily reach such 25 beneficial possibility in anti-tumor immunity conditions. As demonstrated in a mouse tumor model, the approach permits to elicit productive anti-tumor T cell responses independently of the CD28 costimulatory molecule. Therefore, our invention overcomes the need for the expression of the CD80 and CD86 ligands on the surface of the DC found in tumor DLN and TME and should allow adoptively transferred autologous T cellsexpressing native TCR, HLA-independent TCR, CAR-T cells, and CAR-NK cells reactive to tumor antigen to appropriately function in poorly inflamed or cold tumor. The CARMIL2 molecule is essential to the function of the CD28 co-stimulatory pathway in mice and humans 5

[0183] After engaging the CD80 or CD86 ligands that are optimally expressed on the DCs having matured in an inflammatory environment, the CD28 molecules expressed at the T cell surface trigger the association of the cytosolic adaptor CARD11 (also known under the name of CARMA1) with the BCL10 and MALT1 molecules. As a result, the CARD11-BCL10-MALT1 complex induces the activation of the NF-κB signaling 10 pathway, a vital component of full-blown T cell activation (Juilland and Thome, 2016). The identification of a loss-of-function mutation in the mouse Carmil2 gene (also known as Rltpr) enabled the inventors to demonstrate for the first time that Carmil2 is essential for CD28 co-stimulation (Liang et al., 2013). This quickly led to the identification of the first humans with Carmil2 deficiency, confirming the essential role of Carmil2 in CD28 15 co-stimulation in both mice and humans (Roncagalli et al., 2016; Wang et al., 2016).

[0184] Carmil2 is a 1399 amino acid protein that includes a non-canonical pleckstrin homology domain at its amino-terminus, a domain containing leucine-rich repeats (LRR), a domain of homodimerization, a motif that interacts with proteins involved in actin filament polymerization, and a carboxy-terminus that contains a proline-rich region 20 (Edwards et al., 2014; Zwolak et al., 2013). Affinity purification coupled to mass spectrometry (AP-MS) studies conducted by the inventors showed that the active form of Carmil2 (resulting from the engagement of CD28 by its ligands CD80 and CD86) initiates the NF-κB signaling pathway via its direct interaction with CARD11 (Roncagalli et al., 2016). Following their engagement with their respective ligands, the TCR and CD28 form 25 microclusters that move towards the center of the T-cell immune synapse and form a central supramolecular activating complex (cSMAC) (Yokosuka and Saito, 2010). At the cSMAC, the CD28 and TCR microclusters separate into a zone called CD28lowCD3highwhich seems inert in terms of signaling, and a zone called CD28highCD3lowwhich continues to deliver CD28 co-stimulatory signals via Carmil2, PKC^ and CARD11. By30 blocking the incorporation of CARD11 into CD28 microclusters, the CARMIL2 loss-of-function mutation identified by the inventors prevents the triggering of the NF-kB signaling pathway and the generation of effector T cells with high functional potential (Liang et al., 2013). Example 1 5 Material & methods Mice

[0185] Mice were on a C57BL / 6 background and 8-10 week. They were maintained under specific pathogen-free conditions at Centre d’Immunophénomique (accreditation B1301407) or Centre d’Immunologie de Marseille-Luminy (accreditation F13005). OT- 10 I mice (Hogquist et al., 1994). Cd28– / –(Shahinian et al., 1993), Cd3e^5 / ^5(Malissen et al.,(Borriello et al., 1997), and Cd3eΔ5 / Δ5Cd80− / −Cd86− / −(Chevrier et al., 2012) mice have been described. Animal experimental guidelines

[0186] Mice were handled in accordance with national and European laws for laboratory 15 animal welfare and experimentation (European Economic Community Council Directive 2010 / 63 / EU, September 2010) and protocols approved by the Marseille Ethical Committee for Animal Experimentation and with Chinese (Xinxiang Medical University) guidelines for animal care. Generation and validation of knock-in mouse expressing a Carmil2NterOSTallele (related 20 to Figure 1)

[0187] A targeting vector was assembled to introduce a nucleotide sequence coding for a Twin-Strep-tag (ASWSHPQFEKGGGSGGGSGGGSWSHPQFEK (SEQ ID NO:3); (Junttila et al., 2005)) and a GGA spacer between the first (ATG) and the second (GCA) codon of the Carmil2 gene (ENSMUST00000213019.2 Carmil2-203). A self-excising 25 CAN cassette (Bunting et al., 1999) was introduced in the intron located between exons 1 and 2. Finally, a cassette permitting expression of a diphtheria toxin fragment was abutted to the targeting construct. JM8.F6 C57BL / 6N ES cells (Pettitt et al., 2009) wereelectroporated with the targeting vector. After selection in G418, ES cell clones were screened for proper homologous recombination by Southern blot and PCR analysis. A probe specific for the neorcassette was also used to ensure that adventitious nonhomologous recombination events had not occurred in the selected clones. Mutant ES cells were injected into FVB blastocysts. Screening for proper auto-deletion of the CAN cassette and for the presence of the sequence coding for the Twin-Strep-tag (abbreviated below as OST) was performed by PCR and sequencing. The resulting mutant mice are denoted as Carmil2NterOSTmice and also known as B6-Rltprtm3Mal. Genotyping of the Carmil2NterOSTallele was performed by PCR using two pair of primers. The first pair (sense 5′-CTGGCTTCCTGTGTACGCTC-3′ (SEQ ID NO:4) and antisense 5′- ACCTGGTGATCTCGCCTGTG-3′ (SEQ ID NO:5)) amplified a 369-bp band in the case of the WT allele, whereas the second pair (sense 5′- AGATCTCGAGCTCGCGAAAG-3′ (SEQ ID NO:6) and antisense 5′- ACCTGGTGATCTCGCCTGTG -3′ (SEQ ID NO:7)) amplified a 233-bp band in the case of the Carmil2NterOSTallele. Generation and validation of knock-in mouse expressing a Carmil2Q538Eallele (related to Figure 1)

[0188] A single guide RNA (sgRNA) targeting exon 20 of the Carmil2 gene and a double- stranded DNA (dsDNA) homology-directed repair (HDR) template intended to convert the CAG codon found in exon 20 of the Carmil2 gene and coding for the glutamine residue present at position 538 of CARMIL2 into a GAG codon coding for a glutamic acid were designed. Fertilized eggs from C57BL / 6 female were microinjected with Cas9 mRNA and the designed sgRNA and HDR template as described (Voisinne et al., 2019). Tail genomic DNA was isolated from the resulting F0 mice and the region encompassing exon 20 amplified and then sequenced. An F0 mouse expressing the intended Q538E mutation was used to establish mice homozygous for the mutation. These mice are denoted as Carmil2Q538Emice and also known as B6-Rltprtm4Mal. Carmil2Q538Emice were genotyped by sequencing a 509-bp DNA fragment that was amplified using the following pair of PCR primers: sense 5’-GACATGGTGACACTGGTGCT-3’ (SEQ ID NO:8) and antisense 5’-GAGCCTTGGCTAGCATCTTG-3’ (SEQ ID NO:9).Generation and validation of knock-in mouse expressing a Carmil2Q538E-NterOSTallele (related to Figure 1)

[0189] A sgRNA targeting exon 1 of the Carmil2Q538Egene and a dsDNA HDR template intended to insert a sequence coding for an improved Twin-Strep-tag (SAWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO:10); (Junttila et al., 2005)) and a GSG spacer between the first (ATG) and the second (GCA) codon of the first exon of the Carmil2Q538Egene were designed. Fertilized eggs from C57BL / 6 female mice homozygous for the Carmil2Q538Eallele were microinjected with Cas9 mRNA, and the designed sgRNA and HDR template as described (Voisinne et al., 2019). Tail genomic DNA was isolated from the resulting F0 mice and the region encompassing exon 1 amplified and then sequenced. An F0 mouse expressing the intended Twin-Strep-Tag insertion was used to establish homozygous mutant mice. These mice are denoted as Carmil2Q538E-NterOSTmice and also known as B6-Carmil2tm5Mal. Carmil2Q538E-NterOSTmice were genotyped by using the following pair of PCR primers: sense 5’- ATTCGACCATCCTCCCACAAC-3’ (SEQ ID NO:11) and antisense 5’- GAGAGGTCTGGTTTGGAGTCAG-3’ (SEQ ID NO:12). They amplified a 413-bp band in the case of the Carmil2Q538E-NterOSTallele and a 314-bp in the case of a WT Carmil2 allele. Flow cytometry (related to Figures 2 and 6)

[0190] Stained cells were analyzed using a LSR II system and FAC SDiva 8.1 software (BD). Cell viability was evaluated using SYT OX Blue (Life Technologies) or Aqua Dead (Molecular Probes). The following antibodies were used: anti-CD3 (145-2C11), anti-CD4 (RMA-5), anti-CD5 (53–7.3), anti- CD8 (53–6.7), anti-CD11b (MI / 70), anti-CD28 (37.51), anti-CD44 (IM7), anti-CD45R (RA3-6B2), anti-CD62L (MEL-14), anti-CD138 (281–2), anti-Ly-6G (1A8), and anti-TCRβ (H57-597), all purchased from BD; anti- CD24 (M1 / 69), anti-CD25 (PC61.5), anti-CD27 (LG3A10), anti-CD19 (6D5), anti- CD161 (PK136), anti-SiglecH (551), all obtained from BioLegend; and anti-CD317 (927), anti-Foxp3 (FJK-16s), and anti-TCRδ (GL-3), all purchased from eBioscience. Stained cell samples were analysed on a LSR Fortessa or a FACSymphony Flow Cytometer equipped with FACSDiva software (BD Biosciences), and both instrumentswere validated prior to data acquisition using Flow Cytometry Calibration Particles (Spherotech, RQC-30-5A). Photomultiplier tube voltages were also adjusted to minimize fluorescence spillover. Single-stain controls were prepared with UltraComp eBeads (Thermo Fisher Scientific) following the manufacturer’s instructions, and were used to 5 calculate a compensation matrix. To be able to compare our different experiments, all our data acquisitions were standardized and application settings were recorded. Data were analyzed with either BD FACSDiva™ V9 Software or FlowJo™ V10.7 Software (BD Biosciences). Mouse T cell proliferation and cytokine production (related to Figure 5) 10

[0191] Naïve T cells were purified by immunomagnetic negative selection using the EasySepMouse T Cell Isolation Kit and then stimulated with plate-bound anti-CD3 (145- 2C11; Exbio) and soluble anti- CD28 (37–51; Exbio) antibodies or with phorbol 12- myristate 13-acetate (PMA) and ionomycin. After 48 h of culture, T cell proliferation was assessed with CellTiter-Glo Luminescent Cell Viability Assay (Promega). The resulting 15 luminescence, which is proportional to the ATP content of the culture, was measured with a Victor 2 luminometer (Wallac; Perkin Elmer Life Science). For CTV-dilution assay, T cells were stained with 5 μM CTV (Molecular Probes) and analyzed by FACS 72 h after stimulation. IL-2 and IFN^ production was measured using Cytometric Bead Array (BD).20

[0192] Naïve CD8+T cells were purified from the spleen of either OT-I or OT-I Carmil2Q538Emice by immunomagnetic negative selection using Dynabeads Untouched TM Mouse CD8 cells, (Invitrogen). Purified T cells were stimulated with irradiated H-2 Kb-positive spleen cells isolated from T cell- deficient mice and pulsed for 2 h with the N4 agonist OVA peptide (Hogquist et al., 1994). After 48 h of culture, T-cell proliferation 25 was assessed with CellTiter-Glo Luminescent Cell Viability Assay (Promega). Tumor cells and tumor cell inoculation (related to Figure 5)

[0193] A BRAFV600Emouse melanoma tumor and its Ptgs1 / Ptgs2– / –(cyclooxygenase- deficient) variant (Zelenay et al., 2015) were provided by C. Reis e Sousa (Francis CrickInstitute, London, UK). The tumor cell lines were cultured under standard conditions prior to injection into mice.

[0194] Tumor cells were harvested by trypsinization, washed three times with PBS, and 106tumor cells were injected subcutaneously into the flank of recipient mice in 100 μl of endotoxin-free PBS. Tumor size was quantified as the mean of the longest diameter and its perpendicular. CD4+T cell isolation from Carmil2Q538E-NterOSTand Carmil2NterOSTmice and short-term expansion prior to AP-MS analysis (related to Figure 3)

[0195] CD4+T cells were purified (> 95%) from pooled lymph nodes and spleens from Carmil2Q538E-NterOSTand Carmil2NterOSTmice with Dynabeads Untouched Mouse CD4+T Cell Kits (Life Technologies). CD4+T cells were activated with plate-bound anti-CD3 (145-2C11, 5 μg / ml) and soluble anti-CD28 (37-51; 1 μg / ml) antibodies, both from Exbio Praha. After 48 h of culture, CD4+T cells were harvested and grown in the presence of IL-2 (10 U / ml) for 48 h before AP-MS analysis. Wild-type CD4+T cells were subjected to the same expansion protocol and used as controls. Stimulation and lysis of short-term and long-term expanded mouse CD4+T cells prior to affinity purification coupled to mass spectrometry (AP-MS) analysis (related to Figure 3)

[0196] Short-term expanded CD4+T cells (100 × 106) from WT, Carmil2Q538E-NterOSTand Carmil2NterOSTmice mice were left unstimulated or stimulated at 37°C with pervanadate for 30, 120, 300, and 600 s. Pervanadate stock solution was made by mixing 7.6 volumes of water with 1.9 volumes of hydrogen peroxide (10 mM final concentration), and with 0.5 volume of sodium orthovanadate (100 μM final concentration), and left for 15 min at 20°C before addition to T cells. Stimulation was stopped by the addition of a twice- concentrated lysis buffer (100 mM Tris, pH 7.5, 270 mM NaCl, 1 mM EDTA, 20% glycerol, 0.4% n-dodecyl-β-D-maltoside) supplemented with protease and phosphatase inhibitors. After 10 min of incubation on ice, cell lysates were centrifuged at 21,000 g for 5 min at 4 °C. Postnuclear lysates were then used for affinity purification.Affinity purification of OST-tagged protein complexes (related to Figure 3)

[0197] Equal amounts of postnuclear lysates were incubated with Strep-Tactin Sepharose beads (IBA GmbH) for 1.5 h at 4° C on a rotary wheel. Beads were then washed five times with 1 ml of lysis buffer in the absence of detergent and of protease and phosphatase 5 inhibitors. Proteins were eluted from the Strep-Tactin Sepharose beads with 2.5 mM D- biotin, a ligand that binds to Strep-Tactin with a higher affinity than the OST sequence does. Tandem MS analysis (related to Figure 3)

[0198] Following affinity purification, protein samples were air dried in a Speed-Vac 10 concentrator, and either reconstituted in Laemmli buffer and processed by SDS-PAGE and trypsin in-gel digestion as previously described (Voisinne et al., 2019), or reconstituted in 5% SDS - 50mM ammonium bicarbonate and processed for trypsin digestion using a S-trap micro device (Protifi) according to the manufacturer instructions. Tryptic peptides were resuspended in 17 µl of 2% acetonitrile and 0.05% trifluoroacetic 15 acid and analyzed by nano-liquid chromatography (LC) coupled to tandem MS, using an UltiMate 3000 system (NCS-3500RS Nano / Cap System; Thermo Fisher Scientific) coupled to an Orbitrap Q Exactive mass spectrometer (model Q Exactive Plus or HFX, Thermo Fisher Scientific). Five microliters of each sample were loaded on a C18 precolumn (300 µm inner diameter × 5 mm, Thermo Fisher Scientific) in a solvent made 20 of 2% acetonitrile and 0.05% trifluoroacetic acid, at a flow rate of 20 µl / min. After 5 min of desalting, the precolumn was switched online with the analytical C18 column (75 µm inner diameter × 50 cm, Acclaim PepMap C18, 2 µM, Thermo Fisher Scientific, or in- house packed with 3µm Reprosil C18) equilibrated in 95% solvent A (5% acetonitrile, 0.2% formic acid) and 5% solvent B (80% acetonitrile, 0.2% formic acid). Peptides were 25 eluted using a 10%-45% gradient of solvent B over 60 min at a flow rate of 350 nl / min. The mass spectrometer was operated in data-dependent acquisition mode with an Xcalibur software. On the Q Exactive HFX or Q Exactive Plus mass spectrometer, MS survey scans were acquired with a resolution of respectively 60,000 or 70,000 and an AGC target of 3e6. The 12 or 10 most intense ions respectively were selected for 30 fragmentation by high energy collision induced dissociation, and the resulting fragmentswere analyzed at a resolution of 15000 or 17500, respectively, using an AGC target of 1e5 and a maximum fill time of 22 ms or 50 ms, respectively. Dynamic exclusion was used within 30 s to prevent repetitive selection of the same peptide. Protein identification and quantification for interaction proteomics (related to Figure 3) 5

[0199] Raw MS files were processed with MaxQuant software (version 1.5.2.8) for database search with the Andromeda search engine and quantitative analysis. Data were searched against Mus musculus entries of the UniProt KB protein database (release UniProtKB / Swiss-Prot+TrEMBL 2017_01, 89297 entries including isoforms), plus the One-Strep-tag peptide sequence, and the set of common contaminants provided by 10 MaxQuant. Carbamidomethylation of cysteines was set as a fixed modification, whereas oxidation of methionine, protein N-terminal acetylation, and phosphorylation of serine, threonine, and tyrosine were set as variable modifications. Specificity of trypsin digestion was set for cleavage after K or R, and two missed trypsin cleavage sites were allowed. The precursor mass tolerance was set to 20 ppm for the first search and 4.5 ppm for the 15 main Andromeda database search. The mass tolerance in tandem MS mode was set to 0.5 Da. Minimum peptide length was set to 7 amino acids, and minimum number of unique or razor peptides was set to 1 for validation. The I=L option of MaxQuant was enabled to avoid erroneous assignation of undistinguishable peptides belonging to very homologous proteins. Andromeda results were validated by the target decoy approach using a reverse 20 database, with a false discovery rate set at 1% at both PSM (peptide sequence match) and protein level. For label-free relative quantification of the samples, the match between runs option of MaxQuant was enabled with a match time window of 1 min, to allow cross- assignment of MS features detected in the different runs, after alignment of the runs with a time window of 20 min. Protein quantification was based on unique and razor peptides. 25 The minimum ratio count was set to 1 for LFQ calculation, and computation of the iBAQ metric was also enabled. Data processing and identification of specific interactors (related to Figure 3)

[0200] From the "proteinGroups.txt" files generated by MaxQuant with the options described above, proteins groups with negative identification scores were filtered as wellas proteins identified as contaminants. In situations were protein groups corresponded to the same gene name, protein intensities in a given sample were summed over the redundant protein groups. Protein intensities were normalized across all samples by the median intensity. Normalized intensities corresponding to different technical replicates 5 were averaged (geometric mean) and missing values were replaced after estimating background binding from WT intensities. For each bait and each condition of stimulation, we used a two-tailed Welch t-test to compare normalized log-transformed protein intensities detected in OST-tagged samples across all biological replicates to WT intensities pooled from all conditions of stimulation. To avoid spurious identification of 10 interactors due to missing value imputation, we repeated this process (missing value imputation followed by a two-tailed Welch t-test) 10 times and estimated fold-changes and p-values as their respective average (geometric mean) across all 10 tests. Specific interactors were identified as preys showing a greater than 10-fold enrichment with a p- value below 0.005 in at least one condition of stimulation. 15 Calculation of interaction stoichiometries (related to Figure 3)

[0201] For a given condition of stimulation (represented by the time of stimulation t, with t=0s corresponding to the non-stimulated condition), the stoichiometry of the interaction between a prey x and a given bait (denoted bait<x ) was computed using : Sbait<x(t) = <IOST,x(t)> / <IOST,bait(t)> * Npep,bait / Npep,x 20 <IOST,x> corresponds to the normalized intensity of protein x in OST-tagged samples and stimulation time t averaged (geometric mean) across all biological replicates, and Npep corresponds to the number of tryptic peptides theoretically observables as estimated from iBAQ values. We also computed stoichiometries independently for each biological replicate and used those values to quantify the regulation of bait-prey association 25 following TCR engagement. For a given condition of stimulation, log-transformed stoichiometries were compared to that of the non-stimulated condition using a two-tailed Welch t-test. We selected preys whose interaction stoichiometry changed at least 2-fold with a p-value below 0.05 in at least one condition of stimulation as compared to the non- stimulated condition.Results

[0202] Analysis of a panel of human cutaneous T cell lymphoma showed that a recurrent p.Q575E mutation affects the Carmil2 gene and may constitute a potential oncogenic driver mutation (Park et al., 2017). Upon overexpression in a transformed T cell line (Jurkat) and stimulation via a T cell antigen receptor (TCR) pharmacologic mimics composed of PMA and ionomycin, the p.Q575E mutation increased the binding of Carmil2 to the CARD11 / CARMA1 cytosolic adaptor and selectively upregulated the NF- κB pathway and the production of interleukin 2 transcripts. Considering that Jurkat T cells lack key signaling proteins (Astoul et al., 2001), it is difficult to generalize the conclusions of this study to normal T cells stimulated under physiological conditions. Therefore, the inventors developed and analyzed the immunophenotype of knock-in mice that express a Carmil2 pQ538E mutation and are denoted as Carmil2Q538Emice (Figures 1A-B). Based on the amino-acid numbering corresponding to isoform 3 of the mouse and human Carmil2 genes, the mouse Q538E mutation corresponds to the ortholog of the human Q539E mutation. As described below, Carmil2Q538Emice allowed the inventors to define the translational interest of Carmil2 gain-of-function mutations in the context of physiological antigenic activation of normal T cells, including tumor-specific T cells.

[0203] As expected for a gain-of-function mutation, analysis of T cell development in mice homozygous for the Carmil2Q538Emutation revealed functional effects that are opposite to those resulting from a Carmil2 deficiency (Roncagalli et al., 2016). For instance, whereas the development of Foxp3+CD4+regulatory T cells (T reg cells) is greatly diminished in mice deprived of CD28 (Cd28– / –, see Figure 2C) or expressing a Carmil2 null mutation (Liang et al., 2013; Roncagalli et al., 2016), mice expressing the Carmil2Q538Emutation expressed increased numbers of T reg cells (see Figure 2C). In contrast to T reg cells, the development of other T cell subsets was identical in wild-type Carmil2 and mutant Carmil2Q538Emice (Figures 2A-C).

[0204] Knock-in mice expressing wild-type Carmil2 or Carmil2Q538Eproteins tagged at their amino terminus with an affinity Twin-Strep-tag (OST) were also developed by the inventors and denoted as Carmil2NterOSTor Carmil2Q538E-NterOSTmice, respectively(Figures 1A-B). Note that the introduction of the OST tag does not change the levels of expression of the tagged proteins and had no adventitious developmental effect since Carmil2NterOSTor Carmil2Q538E-NterOSTmice show the same T cell phenotype as Carmil2 and Carmil2Q538Emice, respectively. Due to the expression of the OST tag, the Carmil2NterOSTand Carmil2Q538E-NterOSTproteins expressed in freshly isolated CD4+T cells are amenable to quantitative interactomics via affinity purification coupled to mass spectrometry (AP-MS) (Voisinne et al., 2019)). It demonstrated that the Carmil2Q538E-NterOSTmolecules of ex vivo primary CD4+T cells are constitutively bound to Card11, whereas the association between wild-type Carmil2NterOSTmolecules and Card11 required prior activation of the TCR signaling pathway (Figure 3). AP-MS analysis also showed that the presence of Carmil2Q538Emolecules has no impact on the composition and dynamics of assembly of the Carmil2Q538Einteractome beyond the induction of constitutive Carmil2Q538E-Card11 association.

[0205] Functional analysis showed that the expression of Carmil2Q538Eincreased the proliferation and interleukin-2 (IL-2) production of T cells expressing the OT-I TCR in response to antigen-laden antigen presenting cells (APC, Figures 4A-B). Particularly relevant for the present invention, the expression of Carmil2Q538Emolecules also relieve OT-I T cells from the need of CD28 costimulatory signals as demonstrated using OT-I Carmil2Q538ET cells responding to antigen-laden APC lacking both CD80 and CD86 (Figure 4A). Likewise, expression of Carmil2Q538Emolecules was capable of fully substituting for the need of CD28-CD80 / CD86 engagement during the generation of Foxp3+regulatory T cells (Figure 2C).

[0206] The peripheral T cells present in Carmil2Q538Emice kept under specific pathogen free conditions and in absence of intended TCR stimulation show no detectable sign of activation. Furthermore, macroscopic and flow cytometry analysis of the primary and secondary lymphoid organs and of the blood of a cohort of 10 months old Carmil2Q538Emice showed no detectable sign of T cell lymphoma or hematological malignancies. Therefore, the sole expression of the Carmil2Q538Emutation has by itself no detectable oncogenic property.

[0207] The above results demonstrate that the constitutive association of Carmil2Q538Emolecules with Card11 (1) enhances cytokine production and proliferation of primary T cells stimulated under physiological antigenic conditions, (2) enhances T reg cell development, and (3) permits Carmil2Q538Eproteins to fully substitute for CD28 engagement in terms of proliferation and cytokine production in responses to antigen- laden APC. Therefore, the Carmil2Q538Emutation behaves as a gain-of-function mutation in that it is capable of fully substituting for CD28-CD80 / CD86 engagement during TCR- triggered responses. AP-MS studies (Figures 2A-C) further suggest that Carmil2Q538Emolecules constitutively mimics the active form of wild-type Carmil2, a form that normally results from the engagement of CD28 by its CD80 and CD86 ligands and allows the recruitment of Card11. Moreover, analysis of Carmil2Q538Emice shows that the Carmil2Q538Emutation has no detectable phenotypic effect without TCR signals. The Carmil2Q538Emutation manifests its gain-of-function effects solely in the presence of coincident TCR signals.

[0208] Considering that the activation of the CD28 costimulatory pathway plays an essential role in the establishment of anti-tumor T-cell responses, the inventors assessed next whether physiological levels of Carmil2Q538Eprotein can fully substitute for CD28- CD80 / CD86 engagement during anti-tumor responses. Cohort (n = 5) of 10 weeks-old wild-typemice were injected subcutaneously into the flank with 106cells of the C57BL / 6-derived mouse melanoma tumor BRAFV600EPtgs– / –and then monitored for tumor growth (Figure 5A). The BRAFV600EPtgs– / –tumor has been previously used to establish that cells belonging to both the innate (NK cells, dendritic cells) and adaptive (T cells) immune systems contribute to its eradication (Bottcher et al., 2018; Zelenay et al., 2015). Consistent with the view that BRAFV600EPtgs– / –tumor cells are immunogenic and form spontaneously regressing tumor in WT C57BL / 6 mice, all the mice belonging to the WT cohort rejected the BRAFV600EPtgs– / –tumor cells (Figure 5B). Analysis of the Cd28– / –cohort showed that in this tumor model the engagement of the costimulatory molecule CD28 is essential for the anti-tumoral effector function of T cells in that the lack of CD28 permitted BRAFV600EPtgs– / –tumor formation. A result consistent with the view that the activation of the CD28 costimulatory pathway plays an essential role in the establishment of anti-tumor T-cell responses (Duraiswamy et al., 2021). Mice homozygous for the Carmil2Q538Emutation rejected the BRAFV600EPtgs– / –tumor as efficiently as WT mice. Importantly, analysisCd28– / –mice shows that the sole expression of Carmil2Q538Emolecules is capable of fully substituting for CD28-CD80 / CD86 engagement in a CD28-dependent anti-tumor response model. This constitutes the central tenet of the invention.

[0209] Finally, and as part of the invention, the Carmil2Q538Egain-of-function mutation was shown to manifest its phenotypic effects even if expressed as a single copy. Analysis of mice with a single copy of the Carmil2Q538Eallele shows that they contained increased numbers of Foxp3+regulatory T cells and that a single copy of Carmil2Q538Eallele sufficed to substitute for CD28-CD80 / CD86 engagement in terms Foxp3+regulatory T cells generation (Figures 6A-B). Likewise, upon stimulation with suboptimal amounts of anti-CD3, naïve T cells from mice with a single copy of the Carmil2Q538Eallele were capable of producing levels of IL-2 and IFN^ in amounts comparable to those of wild- type, naive T cells stimulated with a combination of anti-CD3 plus anti-CD28 antibodies (Figures 7A-B). Therefore, the dominant property of the Carmil2Q538Egain-of-function mutation permits to exploit its beneficial immunogenic properties in cells expressing wild-type Carmil2 molecules, a condition that will happen when Carmil2Q538Ewill be expressed via lentivirus or Crispr-Cas9 editing in autologous T cells expressing native TCR, HLA-independent TCR, CAR-T cells, and CAR-NK cells reactive to tumor antigen.Example 2 Material & methods

[0210] 106MC38-Ova cancer cells (provided by J. P. Böttcher from Technical University of Munich, Germany) were administered subcutaneously to C57Bl / 6 mice. After 7 days, groups of 10 mice were treated with isotype control antibodies (Fig.8A), anti-PD1 antibodies (Fig.8B) or 106CD8+ T cells from OT-1 mice (Fig.8C) or OT-1 Carmil2Q538Emice (Fig.8D). Naive CD8+ T cells were purified from the spleen of either OT-I mice or OT-I Carmil2Q538Emice by immunomagnetic negative selection using Dynabeads Untouched TM Mouse CD8 cells (Invitrogen) and were left unstimulated prior to inoculation. Results

[0211] Figure 8A-D show proof-of-concept results of the in vivo efficacy of a cell therapy protocol utilizing T-cells engineered with the gene coding for Carmil2Q538Ein the context of solid cancers. A colon carcinoma model was used via administration of the MC38-Ova mouse cancer line to immunocompetent mice. The MC38-Ova cell line expresses ovalbumin-specific antigens that can be recognized by specific CD8+ T lymphocytes from OT1 mice.

[0212] This cell line grows rapidly in mice in the absence of treatment, leaving no surviving mice after around 30 days (Fig.8A).

[0213] Treatment mice with antibodies specific to the PD1 molecule induced complete responses, with 100% of mice cured within 25 days (Fig. 8B). These initial results demonstrate that the MC38-Ova tumor inhibits the immune system including endogenous CD8+ T lymphocytes of mice via the PDL1-PD1 axis.

[0214] Moreover, administration of non-genetically modified naive CD8+ OT1 cells had little effect on tumor growth (Fig.8C), with only 2 out of 10 mice surviving after 30 days of follow-up.

[0215] In contrast, when CD8+ cells administered to MC38-Ova inoculated mice expressed the Carmil2Q538Egene, tumor regression was rapid and complete in all mice (Fig.8D). These results demonstrate that expression of the Carmil2Q538Egene in T cells triggers an extremely strong anti-tumor activity even in the presence of PDL1-type inhibitory signals in a solid cancer model. Example 3 Material & methods

[0216] CAR-T cells were produced using PBMC from healthy donors. Briefly, PBMC were separated by ficoll gradient centrifugation and activated via CD3 and CD28 using the TransActTMPolymer Matrix system from Miltenyi during 48 hours in RPMI media supplemented with 10% FCS (Gibco).

[0217] Activated cells were then electroporated with the Lonza S Nucleofector 4D Kit, in order to introduce the Carmil2Q539Egene tagged using an OST sequence together with the CD19-CAR (composed of a human CD19 specific scFv, a CD8 transmembrane domain, 4-1BB and CD3^^) and the selection marker tNGFR via insertion of a nanoplasmid (Aldevron) into the TCRα locus using Crips / Cas9 and specific RNA guides (IDT).

[0218] After electroporation, cells were amplified using IL-15 and IL-7 (Miltenyi) during 7 days in Serum Free Prime-XV media (Irvine Scientific). Transfected cells were positively selected based on tNGFR surface expression using the EasysepTMpositive selection kit from StemCell technologies. tNGFR positive cells were then cultured again during 5 days in IL-15 and IL-7.

[0219] At Day 14 of the production process, CAR-T cells were assessed for the expression of the CAR using the CD19 CAR detection reagent from Miltenyi, and for the expression of CD4 and CD8, as well as tNGFR and the OST tag (Fig.9A). CAR-T cells were also cultured in the presence of CD19-positive Nalm6 lymphoma cells during 18 hours. IL-2 (Fig.9C) and IFN-γ concentrations (Fig.9B) were then evaluated in the co-culture supernatants by ELISA (R&D systems) and cytotoxicity was assessed by evaluating the number of Nalm6 lysed cells in the coculture wells (Fig.9D). Control cell populations were generated in parallel and constituted of CAR-T cells not expressing the Carmil2Q539Egene and non-CAR engineered T cells. Results

[0220] Two batches of human CAR-T cells expressing the Carmil2Q539Egene were generated from healthy donor’s peripheral blood T cells and evaluated for their phenotype at the end of the production period as described in the Material and Methods section.

[0221] CAR-T cell batches strongly and homogenously expressed the chimeric receptor targeting CD19, as well as the tNGFR selection molecule and the Carmil2 gene- associated OST tag (Fig.9A). In addition, the CD4 / CD8 ratio was evaluated and was seen consistent between the two batches (Fig.9A).

[0222] Cytokine secretion assays on the two produced batches show a statistically significant increase of pro-inflammatory cytokine production (IFN-γ and IL-2, Fig.9B and 9C, respectively) by the CAR-T cells expressing Carmil2Q539Egene, as compared to control cells either expressing the same CAR molecule, or non-CAR engineered T cells.

[0223] Cytotoxicity assays demonstrated a strong CD19 positive Nalm6 tumors cell lysis with both control CAR-T cells and Carmil2Q539Egene expressing CAR-T cells, but not with non-CAR engineered T cells (Fig.9D). References - Astoul, E., Edmunds, C., Cantrell, D.A., and Ward, S.G. (2001). PI 3-K and T-cell activation: limitations of T-leukemic cell lines as signaling models. Trends Immunol 22, 490-496. - Binnewies, M., Mujal, A.M., Pollack, J.L., Combes, A.J., Hardison, E.A., Barry, K.C., Tsui, J., Ruhland, M.K., Kersten, K., Abushawish, M.A., et al. (2019). UnleashingType-2 Dendritic Cells to Drive Protective Antitumor CD4(+) T Cell Immunity. Cell 177, 556-571 e516.10.1016 / j.cell.2019.02.005. - Borriello, F., Sethna, M.P., Boyd, S.D., Schweitzer, A.N., Tivol, E.A., Jacoby, D., Strom, T.B., Simpson, E.M., Freeman, G.J., and Sharpe, A.H. (1997). B7-1 and B7-2 5 have overlapping, critical roles in immunoglobulin class switching and germinal center formation. 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[0224] 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 cell that is engineered to express a Carmil2 polypeptide that comprises i) an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:1 wherein the glutamine residue (Q) at position 539 is 5 mutated or ii) an amino acid sequence having at least 90% of identity with the amino acid sequence as set forth in SEQ ID NO:2 wherein the glutamine residue (Q) at position 538 is mutated.

2. The host cell of claim 1, wherein the glutamine residue (Q) is substituted by a negatively charged amino acid residue, preferably by a glutamic acid residue (E) 10 or an aspartic acid residue (D).

3. The host cell according to claim 1 or 2, wherein the host cell is a murine or a human cell, preferably a T cell that is a CD4+ T cell or a CD8+ T cell, more preferably a tumor infiltrating lymphocyte.

4. The host cells according to any one of claims 1 to 3, wherein the host cell is a 15 pluripotent stem cell (PSC) or a hematopoietic stem cell.

5. The host cell according to any one of claims 1 to 4, wherein the host cell expresses an endogenous TCR or an exogenous TCR.

6. The host cell according to any one of claim 1 to 4, wherein the host cell is engineered for expressing a CAR. 20 7. The host cell of claim 6, wherein the CAR comprises one more polypeptides having an extracellular domain and an intracellular domain joined by a transmembrane domain, preferably wherein (i) the CAR is a monomeric molecule that consists of one polypeptide having an extracellular domain and an intracellular domain joined by a transmembrane domain, or (ii) the CAR is a heterodimeric molecule that 25 consists of two polypeptides, both 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.

8. The host cell of claim 6, wherein the CAR derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domains are substituted by a variable domain of an antibody, preferably wherein the CAR derives from a TCR heterodimer wherein the immunoglobulin (Ig)-variable (V) domain of the alpha chain is 5 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, more preferably 10 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 15 antigen of interest.

9. The host cell according to claim 8, wherein the CAR is capable of associating with a CD3 complex to form the T-cell co-receptor.

10. The host cell according to claim 8 or 9, wherein the CAR replaces a native and / or an endogenous TCR in the CD3 / TCR complex of host cell. 20 11. The host cell according to any one of claims 1 to 10, wherein the host cell expresses a TCR or CAR that is specific for a tumor antigen or for a pathogen antigen.

12. A method of preparing the host cell according to any one of claims 1 to 11, comprising the step consisting of introducing into a host cell a polynucleotide that encodes for the Carmil2 polypeptide and optionally one or more polynucleotide(s) 25 that encodes for the CAR or TCR of interest.

13. 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 11.

14. The method of claim 13, wherein the method is suitable for the treatment of cancer or infectious diseases.

15. A pharmaceutical composition comprising the population of the host cells according to any one of claims 1 to 11, and a pharmaceutically acceptable carrier.

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