Lat-based nk cars and their use

Abstract

The invention relates to chimeric antigen receptor constructs and their use for treatments and therapies. Further disclosed are methods of treating and / or preventing a disease, such as e.g. a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

Description

[0001] LAT-based NK CARs and their use

[0002] FIELD OF THE INVENTION

[0003] The invention relates to chimeric antigen receptor constructs and their use for treatments and therapies. Further disclosed are methods of treating and / or preventing a disease, such as e.g. a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0004] BACKGROUND OF THE INVENTION

[0005] Chimeric Antigen Receptor (CAR) T cells have shown to be an attractive cellular therapy. It is a promising approach as it can potentially cure malignancies that are refractory to current treatments such as chemotherapy, radiotherapy or surgery.

[0006] CARs are synthetic receptors made of a scFv or another ligand binding protein which is linked to co-stimulatory and activating domains through a transmembrane domain. The scFv allows target cell recognition in a MHC independent manner. The activating domain gives the signal to the effector cell that will initiate the elimination of a target cell. It was shown that the addition of costimulatory domain(s) in between the transmembrane domain and the activating domain increases survival of the effector cells and their antitumor efficacy in vivo.

[0007] CAR engineered immune cells obtain supra-physiological properties enabling them to eliminate any potential target cell expressing the targeted membrane protein. CAR T cells therapy has shown remarkable outcomes in the clinic against certain subsets of B cell malignancies.

[0008] CAR T cell therapy requires a complex workforce to isolate and engineer the patient’s own T cells due to HLA matching restrictions. Also, the treatment efficacy depends on the patient’s T cell quality which is not always optimal. Moreover, its application to solid cancer remains challenging since CAR T cells often show limited tumor infiltration and restricted trafficking. Finally, T cells can induce cytokine release syndrome (CRS), a potentially lifethreatening toxicity characterized by elevated circulating cytokines such as IL6 and IFNy.

[0009] Recent advances in natural killer (NK) cell-based immunotherapy underlined their high potential as a novel therapeutic. Unlike T cells, NK cells can be established as an off-the-shelf therapy. Also, NK cells often show superior tumor infiltration than T cells suggesting their high potential to control solid cancer growth. Clinical trials have shown that NK cells show reduced adverse effects including CRS or graft-versus-host disease (GVHD).

[0010] CAR NK can be engineered from various NK sources such as iPSC-derived NK cells, NK cells isolated from blood (PB-NK), NK isolated from the umbilical cord blood or NK cell lines such as NK92. However, current clinical trials were restricted to PB-NK or NK92 (NCT00995137, NCT01974479, NCT02839954, NCT02892695, NCT02742727, and NCT02944162).

[0011] Until now, several studies have tried to develop CAR designs for NK cells but restricted their activation signaling domain to ITAM-containing domains such as the CD3z and more rarely FceRlg or DAP12 intracellular domains. Even though CAR NK cells are a promising therapeutic in the clinic, CAR designs currently used in NK cells were initially developed in T cells and might not be optimal for NK cells.

[0012] Therefore, there is still a need for optimized CAR T and NK cells showing an increased cytotoxicity towards their targets and mitigated exhaustion.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention provides a chimeric antigen receptor polypeptide comprising an extracellular domain that binds to a cell surface marker, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a recognition domain for a SH2 domaincontaining protein selected from the recruited proteins comprising PIK3R1, PI3K, p65 alpha subunit of PI3K, p85 subunit of PI3K, p50 subunit of PI3K, GRB2, GRAP, GRAP2, PLCG1, PLCG2, SHB, SKAP2, CLNK, and FCGR1 A, a variant, an isoform or a combination of one or more thereof.

[0015] Further provided is an isolated polynucleotide encoding the chimeric antigen receptor (CAR) of the invention. Also provided is a vector comprising the isolated polynucleotide of the invention.

[0016] Also provided is an immune cell comprising i) an isolated polynucleotide, and / or a vector of the invention, or ii) expressing a chimeric antigen receptor according to the invention.

[0017] Also provided is an immune cell according to the invention for use in treating a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0018] Also provided is a pharmaceutical composition comprising i) an isolated polynucleotide of the invention, ii) a vector of the invention, or iii) an immune cell of invention, and pharmaceutically acceptable carrier, diluent and / or excipient.

[0019] Further provided is a method of treating a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease comprising administering i) an isolated polynucleotide of the invention, ii) a vector of the invention, iii) an immune cell of the invention, or iv) a pharmaceutical composition of the invention, to a subject in need thereof.

[0020] FIGURES

[0021] Figure 1: LAT-based CAR expression and activity in NK cells in vitro. A) Schematic of the 19LAT and the two common (1928z and 19BBz) CAR constructs used. The shaded grey area depicts the transmembrane domain. B) Expression of the different CAR constructs in NK92 measured by Flow cytometry. An HA tag is used to detect the CAR construct at the membrane, and GFP serves as a marker for transduction. C) In vitro killing capacity of NK92 stably expressing different CAR constructs after 4h co-culture with Nalm6 at effectortarget (E:T) ratios of 3 : 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the first challenge (target cells only added once). D) Rechallenge killing assay layout. New target cells were added to the coculture every 24 hours and the killing of the last population added was measured after 4 hours of co-culture. E) In vitro killing capacity of NK92 stably expressing different CAR constructs after 4h co-culture with Nalm6 at effectortarget (E:T) ratio of 3: 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the third and fourth challenges.

[0022] Figure 2: The 19LAT CAR is able to induce solid cancer growth control by engineered NK92. A) In vitro killing capacity of NK92 stably expressing different CAR constructs after 4h co-culture with A1847-CD19 at effectortarget (E:T) ratio of 10: 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the fourth challenge. B) Experimental layout for in vivo killing assay. Mice were injected i.p. with 5xlOA4 A1847 ovarian cancer cell line stably expressing Luciferase and CD19. Five and seven days after tumor injection, mice were treated with 5 million transduced NK92 cells expressing the different CAR constructs. Tumor burden was measured by IVIS at indicated time points. C) Comparison of in vivo efficacy of LAT in NK92 against A1847 ovarian cancer cell line stably expressing Luciferase and CD 19. Tumor burden of mice measured by bioluminescence at indicated time points. Arrows symbolize the injection of NK92. Data depicts total flux (p / s) mean + s.e. (n=4-6).

[0023] Figure 3: 19LAT CAR construct tuning and optimization. A) Schematic of the 19LAT(Y134F) compared to the 19LAT CAR construct. The shaded grey area depicts the transmembrane domain. B) In vitro killing capacity of NK92 stably expressing the 19LAT(Y134F) compared to the 19LAT construct after 4h co-culture with Nalm6 at effectortarget (E:T) ratio of 1 :2. Cytotoxicity is measured by annexin V staining within the target population. C) Schematic of the kinetic 19LAT mutants. The 19LAT(G133D), 19LAT(P132D) and the 19LAT(DD) constructs are represented compared to the 19LAT CAR construct. The shaded grey area depicts the transmembrane domain. D) In vitro killing capacity of NK92 stably expressing kinetic 19LAT mutants compared to the 19LAT construct after 4h co-culture with Nalm6 at effectortarget (E:T) ratio of 3: 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the first and the fourth challenge. E) Schematic of the PLCg site mutants. The 19LAT(VEGFR2) and the 19LAT(Hybrid) constructs are represented compared to the 19LAT CAR. The shaded grey area depicts the transmembrane domain. F) In vitro killing capacity of NK92 stably expressing PLCg site 19LAT mutants compared to the 19LAT construct after 4h co-culture with Nalm6 at effectortarget (E:T) ratio of 1 :2. Cytotoxicity is measured by annexin V staining within the target population. G) Schematic of the 19LAT stabilized mutant. The 19LAT(RR) and the 19LAT CARs constructs are represented. The shaded grey area depicts the transmembrane domain. H) In vitro killing capacity of NK92 stably expressing 19LAT stabilized mutants compared to the 19LAT construct after 4h co-culture with Nalm6 ateffectortarget (E:T) ratio of 3: 1. Cytotoxicity is measured by annexin V staining within the target population. I) Schematic of the 19LAT monomeric hinge mutant. The 19(CS)LAT and the 19LAT CARs constructs are represented. The shaded grey area depicts the transmembrane domain. J) In vitro killing capacity of NK92 stably expressing 19(CS)LAT compared to the 19LAT construct after 4h co-culture with Nalm6 and A1847-CD19 at the indicated effectortarget (E:T) ratios. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the fourth challenge. K) Long-term killing assay experiment layout. Effector and target cells are co-cultured for 7 days. Following this co-culture, fresh target cells are added and the functional killing is measured by flow cytometry by PI and annexinV staining. L) In vitro killing capacity of NK92 stably expressing 19(CS)LAT compared to the 1928z and 19BBz CAR constructs during a long-term killing assay at effector Target (E:T) ratio of 1 : 10. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the functional killing. M) Comparison of in vivo efficacy of 19(CS)LAT in NK92 against Al 847 ovarian cancer cell line stably expressing Luciferase and CD 19. Tumor burden of mice measured by bioluminescence at indicated time points. Arrows symbolize the injection of NK92. Data depicts total flux (p / s) mean + s.e. (n=5-6).

[0024] Figure 4: 19LAT can be optimized for use in T cells. A) Expression level of CAR in T cells measured by flow cytometry. The HA tag in the CAR construct is 5abelled by antibody staining. B) In vitro killing capacity of T cells stably expressing 19LAT compared to the 1928z and the 19BBz CAR constructs after 16h co-culture with Nalm6 at effectortarget (E:T) ratio 2: 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the first and the third challenge. C) Schematic of the 19LAT variants optimized for T cells. The 1928LAT, 19BBLAT, 19LAT28 and the 19LAT(G133D)28 CARs constructs are represented. The shaded grey area depicts the transmembrane domain. D) Expression level of 1928LAT, 19BBLAT compared to the 1928z and 19BBz CARs in T cells is measured by flow cytometry. The HA tag in the CAR construct is labelled by antibody staining. E) In vitro killing capacity of T cells stably expressing 1928LAT and 19BBLAT compared to the 1928z and the 19BBz CAR constructs after 16h co-culture with Nalm6 at effectortarget (E:T) ratio of 2: 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the first and the third challenge. F) Comparison of in vivo efficacy of 19BBLAT compared to the 19BBz and the 1928z CARs in T cells against Nalm6 cell line stably expressing Luciferase. Tumor burden of mice measured by bioluminescence at indicated time points. Data depicts total flux (p / s) mean + s.d. (n=5-6). G) Expression level of CAR in T cells is measured by flow cytometry. The HA tag in the CAR construct is labelled by antibody staining. H) In vitro killing capacity of T cells stably expressing 19LAT28(G133D) compared to the 19LAT28, 1928z and the 19BBz CAR constructs after 16h co-culture with Nalm6 at effectortarget (E:T) ratio of 2: 1. Cytotoxicity is measured by annexin V staining within the target population. The figure shows the first and the third challenge. I) Comparison of in vivo efficacy of 19LAT28(G133D) and 19LAT28 compared to the 1928z CAR in T cells against Nalm6 cell line stably expressing Luciferase. Tumor burden of mice measured by bioluminescence at indicated time points. Arrows symbolize the injection of T cells. Data depicts total flux (p / s) mean + s.e. (n=5-6).

[0025] Figure 5: 19LAT can induce engineered immune cell functionality upon antigen recognition. A) Proliferation assay layout for PBMCs in co-culture with regulatory T cells. CAR T reg are first activated on a CD 19 coated surface for 16 hours. Then PBMCs are added to the culture. PBMCs proliferation is assessed by CFSE measurement by flow cytometry after three days. B) Expression level of CAR expressed by regulatory T cells purified from blood is measured by flow cytometry. The HA tag in the CAR construct is labeled by antibody staining. C) Division assay to compare the regulatory function of the CAR Tregs. The division of activated conventional T cells (Tconv) is measured via the dilution of CFSE. Mean fluorescent intensity (MFI) of CFSE is measured after 3 days of co-culture. D) Phagocytosis assay layout. Phagocytosis is measured by GFP positive Nalm6 target cells were plated with CAR THP1 stained with cell trace violet. Following a 24 hours incubation, the phagocytosis rate is extracted from the flow cytometry data. E) Expression level of CAR expressed by THP-1 cells measured by flow cytometry. The HA tag in the CAR construct is labeled by antibody staining. F) The rate of phagocytosis in non differentiated THP1 is measured by flow cytometry as the percentage of GFP cells within the THP1 population after 24h co-culture at indicated effector to target ratios.

[0026] Figure 6: LAT-based CAR optimization for increased functional cytotoxicity induction in NK cells. A) Schematic of the 19LAT variants optimized for NK cells. The controls A and B, as well as 19LAT variants such as the 19LAT(ITTc), 19LAT(ITTi) and the 19LAT(G133D, ITTi)4-lBB CARs constructs are represented. The shaded grey area depicts the transmembrane domain. B) Expression level of different CAR constructs in NK92 measured by flow cytometry with HA staining. C, D, E, F) In vitro re-challenge killing assay of NK cells stably expressing variants of the 19LAT CAR constructs at an effectortarget (E:T) ratio of 0.3: 1. Plots shows the cytotoxicity of NK92 cells toward C) A1847-CD19 after three challenges, D) A1847-CD19 after four challenges, E) Nalm6 after three challenges, and F) Nalm6 after four challenges.

[0027] DESCRIPTION OF THE INVENTION

[0028] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0029] In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

[0030] The term "comprise / comprising" is generally used in the sense of "include / including", that is to say permitting the presence of one or more features or components. This term also encompasses the more restricted term "consist / consisting".

[0031] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

[0032] The term "amino acid" includes all of the naturally occurring amino acids as well as modified amino acids.

[0033] As used herein, "at least one" means "one or more", "two or more", "three or more", etc.

[0034] The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus ten (10) percent “Homology” refers to the percent identity between two polynucleotide or two polypeptide moieties. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% or at least about 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence. Alternatively, homology can be determined by readily available computer programs or by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0035] In general, “identity” refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. In some aspects, a nucleotide or amino acid sequence of the invention, or a portion thereof, is at least 80%, namely, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a corresponding nucleotide or amino acid sequence (SEQ ID NO identifier).

[0036] As used herein, the terms "peptide", "protein", "polypeptide", "polypeptide chain", "polypeptidic" and "peptidic" are used interchangeably to designate a series of amino acid residues connected to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.

[0037] The present invention discloses a chimeric antigen receptor (CAR) polypeptide comprising an extracellular domain that binds to a cell surface marker, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain derives from, comprises, or consists of a recognition domain that recognizes a SH2 domain-containing protein selected from the recruited proteins comprising PIK3R1, PI3K, p65 alpha subunit of PI3K, p85 subunit of PI3K, p50 subunit of PI3K, GRB2, GRAP, GRAP2, PLCG1, PLCG2, SHB, SKAP2, CLNK, and FCGR1A, a variant, an isoform, or a combination of one or more thereof.

[0038] In a preferred aspect, the transmembrane domain derives from, comprises, or consists of a linker for activation of T cells transmembrane domain (LAT-tm), a fragment or a variant thereof.

[0039] The recruitment of PLCG1, GRB2, GRAP2 and PIK3R1 onto LAT enables immune cell activation (including T cells and NK cells). Conventional CARs mainly rely on ZAP70 recruitment, through their CD3z chain, which initiates an amplification of signal in order to initiate the signaling cascade in T cells.

[0040] Surprisingly, the Inventors have shown that point mutations, such as e.g. affecting tyrosines in the LAT sequence, alter the LAT -based CAR efficacy by either increasing the LAT capacity to recruit PLCG1, GRB2, GRAP2, PI3K, p65 alpha subunit of PI3K, p85 subunit of PI3K, p50 subunit of PI3K, and PIK3R1 and / or increasing the stability of said CAR.

[0041] In one aspect, the extracellular domain that binds to a cell surface marker is selected from the group comprising an antigen-binding polypeptide, a receptor, or a natural ligand for a target cell antigen or receptor.

[0042] Preferably, the antigen-binding polypeptide is an antibody or antibody fragment, selected from the group comprising murine antibodies, rabbit antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, single domain antibody variable domains, nanobodies (VHHs), and camelized antibody variable domains. Preferably also, the single-chain Fragment variant (scFv), is derived from an antibody or a ligand or a receptor. In some instances, the extracellular domain comprises a hinge portion (also called linker or spacer). A variety of hinges or linkers can be employed in accordance with the invention as described infra.

[0043] In one aspect, the antigen recognized by the antigen-binding polypeptide is usually selected from the group comprising a cancer cell associated antigen, an infection-associated antigen and an auto-antigen.

[0044] Preferably, the antigen recognized by the antigen-binding polypeptide is selected from the group comprising CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, fibroblast activation protein (FAP), CA125, MUC-1, PSMA, PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3 -antigen, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (EUF-I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF -I), KC4-antigen, KS-1 -antigen, KS1-4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleophosmin, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, antigen specific for PAM- 4 antibody, placental growth factor, p53, prostatic acid phosphatase, RS5, S1OO, TAC, TAG- 72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, NeuGcGM3, N-glycolyl GM3 ganglioside, Neu5Gc, GM3 -Ganglioside, GD3, GM2, carbohydrate antigens, ganglioside antigens, Lewis Y, Lewis B, MOG, MBP, aB-crystallin, PLP, GlialCAM, p-synuclein, HLA-A2, TNP, CD123 or Kappa chain of immunoglobulin, and a combination of one or more thereof.

[0045] Where a linker region is interposed between the extracellular region and the transmembrane domain, it will be selected from the group comprising i) an immunoglobulin hinge region or a linker region derived from CD8, CD8a, or CD28, or any variant thereof, and ii) a (GnS)m linker, wherein G is glycine, S is serine, n is an integer between 2-12, preferably between 3- 10, more preferably between 3-5, even more preferably n=3 and m is an integer between 2-12, preferably between 3-10, more preferably between 3-5, even more preferably m=3.

[0046] In one aspect, the transmembrane domain and hinge is usually fused to the extracellular domain of the CAR. It can similarly be fused to the intracellular signaling domain of the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (comprise, or correspond to) CD28, CD28T, OX-40, 4- 1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), CDl-la / CD18, CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof. Preferably, the transmembrane domain derives from, comprises, or consists of a linker for activation of T cells transmembrane domain (LAT-tm), a fragment or a variant thereof. In one aspect, the LAT transmembrane domain increases the activity of the CAR.

[0047] In one aspect, the LAT transmembrane domain (e.g. human LAT transmembrane domain) comprises the amino acid sequence of SEQ ID No. 9 (ILVPCVLGLLLLPILAMLMALCV), a fragment or a variant thereof.

[0048] Optionally, short linkers may form linkages between any or some of the extracellular, transmembrane, and intracellular domains of the CAR.

[0049] One of ordinary skills in the field will appreciate that any s hinge can be used in this invention. In a preferred aspect, the hinge in the CAR of the invention is a CD8 (e.g. CD8a). In one aspect, the CD8 transmembrane domain (e.g. CD8a transmembrane domain) and hinge (e.g. CD8a hinge or spacer domain) comprises the transmembrane portion and hinge of the amino acid sequence of SEQ ID No. 11, a fragment or a variant thereof.

[0050] Examples of variants of the CD8a hinge comprise one or more mutations at positions 27 and / or 44, such as .e.g. in SEQ ID No. 13.

[0051] In one aspect, the LAT transmembrane domain (e.g. human LAT transmembrane domain) comprises the amino acid sequence of SEQ ID No. 9, a fragment or a variant thereof.

[0052] The intracellular signaling domain of the CAR of the invention comprises one or more recognition domain(s) for a SH2 domain-containing protein.

[0053] As used herein, a "recognition domain" refers to a domain comprising one or more tyrosine that are phosphorylated upon activation to allow the recruitment of one or more SH2 containing-protein. The recruitment can be direct or indirect, i.e. via a first SH-2 domaincontaining protein (e.g. GRB2 or GRAP2). In one aspect, the one or more SH2 domain-containing proteins directly recruited by the recognition domain are selected from the non-limiting recruiting proteins comprising Phosphoinositide-3 -Kinase Regulatory Subunit 1 (PIK3R1), Phosphoinositide-3 -Kinase (PI3K), p65 alpha subunit of PI3K, p85 subunit of PI3K, p50 subunit of PI3K, Growth factor receptor-bound protein 2 (GRB2), GRB2 Related Adaptor Protein (GRAP), GRB2 Related Adaptor Protein 2 (GRAP2), Phospholipase C Gamma 1 (PLCG1), Phospholipase C Gamma 2 (PLCG2), SHB, Src Kinase Associated Phosphoprotein 2 (SKAP2), Cytokine Dependent Hematopoietic Cell Linker (CLNK), Fc Gamma Receptor la (FCGR1 A), a variant, an isoform or a combination of one or more thereof.

[0054] In one aspect, the one or more SH2 domain-containing proteins indirectly recruited by the recognition domain are selected from the non-limiting recruiting proteins comprising Casitas B-lineage lymphoma (CBL), SOS Ras / Rac Guanine Nucleotide Exchange Factor 1 (Sosl), VAV, Lymphocyte cytosolic protein 2 (LCP2, also known as SLP76), Thymocyte-expressed molecule involved in selection protein (THEMIS), Thymocyte-expressed molecule involved in selection protein 2 (THEMIS2), SH2-containing protein tyrosine phosphatase-1 (SHP-1), SH2-containing protein tyrosine phosphatase-1 (SHP-2), Interleukin-2 -inducible T-cell kinase (ITK), GRB2-associated-binding protein 2 (GAB-2), a variant, an isoform or a combination of one or more thereof.

[0055] For example, GRAP2 once activated and recruited will recruit SLP76 and / or ITK .

[0056] In one aspect, the recognition domain is selected from the group comprising a linker for activation of T cells (LAT), a fragment or a variant thereof.

[0057] Preferably, the LAT of the invention is selected from SEQ ID No. 1, a fragment or a variant thereof.

[0058] As used herein, a “fragment” of a LAT sequence of the invention, preferably of a human LAT sequence, refers to a sequence containing less nucleotides in length than the respective nucleic acid sequence or less nucleotides in length than the respective polypeptide sequence . Preferably, this sequence contains less than 90%, preferably less than 60%, in particular less than 30% nucleotides in length than the respective polypeptide sequence or nucleic acid sequence (e.g. SEQ ID No. 1).

[0059] The term “variant”, when it refers to a LAT sequence (whether a nucleotide or a peptide sequence) of the invention, means one or more biologically active derivatives of a LAT, preferably of a human LAT sequence of the invention. In general, the term “variant” refers to molecules having a native sequence with one or more additions, substitutions (such as e.g. one or more mutations) and / or deletions, relative to the native molecule, and which are “substantially homologous” to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). In general, the sequences of such variants will have a high degree of sequence homology or identity to the reference sequence, e.g., sequence homology or identity of more than 25%, generally more than 50% to 70%, even more particularly 80%, or 85% or more, such as at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 99%, when the two sequences are aligned.

[0060] It is understood that the term variant also encompasses human isoforms of a LAT sequence.

[0061] In one aspect, the LAT variant comprises one or more additions, substitutions (such as e.g. one or more mutations) and / or deletions within SEQ ID No. 1.

[0062] In one aspect, said one or more mutations is / are independently selected from the group comprising aspartic acid at position 132, aspartic acid at position 133, aspartic acid at position 171, aspartic acid at position 172, aspartic acid at position 191, aspartic acid at position 192, aspartic acid at position 226, aspartic acid at position 227, glycine at position 132, glycine at position 133, glycine at position 171, glycine at position 172, glycine at position 191, glycine at position 192, glycine at position 226, glycine at position 227, glutamic acid at position 132, glutamic acid at position 133, glutamic acid at position 171, glutamic acid at position 172, glutamic acid at position 191, glutamic acid at position 192, glutamic acid at position 226, glutamic acid at position 227, methionine at position 137, methionine at position 176, methionine at position 196, methionine at position 231, methionine at position 135, methionine at position 174, methionine at position 194, methionine at position 229, asparagine at position 136, asparagine at position 175, asparagine at position 195, asparagine at position 230, phenylalanine at position 136, phenylalanine at position 175, phenylalanine at position 195, phenylalanine at position 230, serine at position 130, serine at position 169, serine at position 189, serine at position 224, isoleucine at position 131, isoleucine at position 170, isoleucine at position 190, isoleucine at position 225. In one aspect, said one or more mutations is / are within positions 130 to 138 (SEQ ID NO. 3: HNPGYLVVL) and / or positions 224 to 231 (SEQ ID No. 26: GAPDYENL).

[0063] In one aspect, the LAT variant comprises, from i) positions 130 to 138, a sequence selected from the group comprising SEQ ID No. 4 (DGKDYIVLP), SEQ ID No. 5 (DGKDYIPIN), SEQ ID No. 6 (ADSGYIIPL), and SEQ ID No. 7 (TSFGYDKPH), or a combination thereof.

[0064] In one aspect, the LAT variant comprises, from ii) positions 224 to 231, a sequence selected from the group comprising SEQ ID No. 27 (SIDDYMNM), SEQ ID No. 38 (SIDDYMFM) or any ITT-like motif, preferably comprising the consensus sequence YxxM, with x being any amino acid.

[0065] It is also understood that the LAT variant can comprise one or more mutations within SEQ ID No. 1 and an insertion from positions 130 to 138, as described supra; one or more mutations within SEQ ID No. 1 and an insertion from positions 224 to 231 ; or one or more mutations within SEQ ID No. 1, an insertion from positions 130 to 138 and an insertion from positions 224 to 231, as described supra.

[0066] In one aspect, the CAR of the invention further comprises one or more intracellular costimulatory domain(s). The co-stimulatory domain can be placed between the transmembrane domain and the LAT i.e., on the N-terminal end of the LAT (TM domain>Co-Stim Domain>LAT) or after i.e., on the C-terminal end of the LAT (TM domain >LAT>Co-Stim Domain) or on both the N-terminal end and C-terminal end of the LAT.

[0067] Preferably, the intracellular co-stimulatory domain is selected from the non-limiting group comprising CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen- 1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP- 10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, a variant thereof or any combination thereof.

[0068] In one aspect, the intracellular co-stimulatory domain is as set forth in SEQ ID No. 15, a fragment or a variant thereof.

[0069] In one aspect, the CAR of the invention can further comprise a signal sequence (e.g. a signal peptide such as SEQ ID No. 18, a fragment or a variant thereof) that is preferably added to its N-term. Once the CAR is incorporated into the cell membrane, the sequence is then cleaved.

[0070] In some instances, the extracellular domain comprises a CD8a hinge known to dimerize through the trans-disulphide bridge formation of two cysteines in the CD8a hinge. One may thus understand that, where necessary, the invention provides that these two cysteines are mutated to, e.g. serine to abolish the dimerization of the constructs (Fig. 31).

[0071] The chimeric antigen receptor polypeptides of the invention may thus exist in a monomeric state or in a dimeric state.

[0072] In one aspect of the invention, the chimeric antigen receptor is selected from the group comprising SEQ ID No. 21, SEQ ID No. 25, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43, SEQ ID No. 45, SEQ ID No. 47, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 53, SEQ ID No. 55, SEQ ID No. 57, SEQ ID No. 59, SEQ ID No. 61, and SEQ ID No. 63, a fragment or a variant thereof.

[0073] The invention further contemplates an isolated polynucleotide encoding the chimeric antigen receptor (CAR) of the invention, as well as a fragment or a variant thereof, including a domain or region disclosed herein.

[0074] In one aspect of the invention, the isolated polynucleotide encoding the chimeric antigen receptor (CAR) of the invention is selected from the group comprising SEQ ID No. 20, SEQ ID No. 22, SEQ ID No. 24, SEQ ID No. 28, SEQ ID No. 30, SEQ ID No. 32, SEQ ID No. 34, SEQ ID No. 36, SEQ ID No. 38, SEQ ID No. 40, SEQ ID No. 42, SEQ ID No. 44, SEQ ID No. 46, SEQ ID No. 48, SEQ ID No. 50, SEQ ID No. 52, SEQ ID No. 54, SEQ ID No. 56, SEQ ID No. 58, SEQ ID No.60, and SEQ ID No. 62, a fragment or a variant thereof.

[0075] The terms "nucleic acid", "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any kind of deoxyribonucleotide (e.g. DNA, cDNA, ...) or ribonucleotide (e.g. RNA, mRNA, ...) polymer or a combination of deoxyribonucleotide and ribonucleotide (e.g. DNA / RNA) polymer, in linear or circular conformation, and in either single - or double - stranded form. These terms are not to be construed as limiting with respect to the length of a polymer and can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and / or phosphate moieties (e.g. phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity, i.e., an analogue of A will base-pair with T.

[0076] Further contemplated is a vector comprising an isolated polynucleotide of the invention. The term "vector", as used herein, refers to any vector known in the art that can be suitable for the present invention. In some aspects, the vector refers to a viral vector or to a nucleic acid (DNA or RNA) molecule such as a plasmid or other vehicle, which contains one or more heterologous nucleic acid sequence(s) of the invention and, preferably, is designed for transfer between different host cells and / or for amplification purposes. The terms "expression vector", “gene delivery vector” and "gene therapy vector" refer to any vector that is effective to incorporate and express one or more nucleic acid(s) of the invention, in a cell, preferably under the regulation of a promoter. A cloning or expression vector may comprise additional elements, for example, regulatory and / or post-transcriptional regulatory elements in addition to a promoter.

[0077] In one aspect, the vector is selected from the non-limiting group comprising a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, an RNA (e.g. mRNA) targeted lipid nanoparticles (LNPs), a liposome or any combination thereof.

[0078] The present invention further provides a host cell comprising an isolated nucleic acid of the invention, an expression vector of the invention or a CAR of the invention.

[0079] In one aspect, the cell is a mammalian cell, whether an autologous or an allogeneic cell. Preferably, the cell is selected from the group comprising a cytotoxic cell, an immune cell, a stem cell, a progenitor cell, a cell line and a cell derived from a stem cell or a progenitor cell.

[0080] Where the cell is an immune cell, said immune cell will be selected from the non-limiting group comprising T cell, tumor infiltrating lymphocyte (TIL), NK cell, regulatory T cell (Treg cell), macrophage, TCR-expressing cell, eosinophil, basophil, neutrophil, myeloid cells, B cell, plasma cell, regulatory B cell (Breg), innate lymphoid cell 1 (ICL1), ILC2, ICL3, dendritic cell, or NK-T cell. There are a variety of available techniques know in the art for isolation and enrichment of T cells, such as e.g. PBMC obtained from peripheral blood samples.

[0081] Where the immune cell is a T-cell or NK cell, it will be either an autologous or an allogeneic T cell or NK cell. In some aspects, the immune cell is a T-cell line or NK cell line. Any cell line derived from any primary cell can be used in the present invention. Non-limiting examples of an NK cell line comprise NK92, YTS or KHYG1 cell lines.

[0082] Where the cell is derived from a stem cell, it will be either a natural or an induced stem cell (e.g., an iPSC-derived cell).

[0083] As shown in the examples, the LAT -based CAR constructs of the invention are not only able to induce cytotoxicity in NK and T cells but can also be used in other cell types to functionally activate them. Non-limiting examples of other cell types that can be functionally activated comprise regulatory T cells and macrophages. In one aspect, the LAT -based CAR constructs of the invention activate phagocytosis of the target cell (macrophages), or induce an immunosuppressive environment around their target (Tregs).

[0084] The results of example 2 show that the LAT -based CAR can be optimized for use in NK cells to take advantage of their full cytotoxic capacity. The engineering of specific tyrosine surrounding site enabling the increased of phosphorylation kinetics or the recruitment of additional signalling proteins is possible due to the highly modulable structure of LAT signalling domain. The modularity of YxxM motifs used here to modify an existing ITT motif can further be expanded to take advantage of other properties such as inserting the YxxM motif YMFM to also recruit the p85 and p50a subunits of PI3K. On the opposite way, the insertion of the YxxM motif YVKM would enable the recruitment of the p85 subunit or PI3K and SHP2 which can help to dampen down the CAR signalling strength.

[0085] The present invention further contemplates a CAR, a host cell, e.g. an immune cell or a pharmaceutical composition, according to the present invention for use in treating a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0086] Where the disease to be treated is an inflammatory or inflammation-induced disease, it is selected from the non-limiting group comprising fibrosis (lung or cardiac fibrosis), chronic obstructive pulmonary disease, cardiovascular diseases, diabetes, asthma, fatty liver disease, , gout, and scleroderma.

[0087] Where the disease to be treated is a chronic disease, it is selected from the non-limiting group comprising fibrosis, Alzheimer disease, lupus erythematosus, and chronic kidney disease.

[0088] Where the disease to be treated is an infectious disease, it is selected from the non-limiting group comprising HIV, Hepatitis C and Human Cytomegalovirus.

[0089] Where the disease to be treated is cancer, it is selected from a solid cancer or a liquid cancer.

[0090] Where the cancer to be treated is solid, it is selected from the non-limiting group comprising lung cancer, breast cancer, ovarian cancer, cervical cancer, uterus cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal carcinoma, kidney cancer, prostate cancer, gastric cancer, bronchus cancer, pancreatic cancer, urinary bladder cancer, hepatic cancer and brain cancer and skin cancer, in particular melanoma, or a combination of one or more thereof.

[0091] Where the cancer to be treated is liquid it refers to cancer cells that are present in body fluids, such as blood, lymph and bone marrow. Liquid cancer is selected from the non-limiting group comprising leukemia, myeloma, myelodysplastic syndrome (MDS), and liquid lymphomas. For example, liquid cancer can be acute myeloid leukemia (AML). Liquid lymphomas include lymphomas that contain cysts or liquid areas.

[0092] Where the disease to be treated is an autoimmune disease, it is selected from the non-limiting group comprising rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, inflammatory bowel disease (IBD), psoriasis, and psoriatic arthritis, or a combination of one or more thereof.

[0093] The present invention provides compositions comprising i) an isolated polynucleotide of the invention, ii) a vector of the invention, or iii) a host cell, such as e.g. an immune cell expressing a chimeric antigen receptor according to the invention.

[0094] The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of i) an isolated polynucleotide of the invention, ii) a vector of the invention, or iii) a host cell, such as e.g. an immune cell expressing a chimeric antigen receptor according to the invention, and pharmaceutically acceptable carrier, diluent and / or excipient.

[0095] The present invention further provides the use of pharmaceutical compositions comprising a therapeutically effective amount of i) an isolated polynucleotide of the invention, ii) a vector of the invention, or iii) a host cell, such as e.g. an immune cell expressing a chimeric antigen receptor according to the invention, and pharmaceutically acceptable carrier, diluent and / or excipient in the manufacture of a medicament for treating a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0096] The term "therapeutically effective amount" as used herein means an amount of an immune cell, nucleic acid, plasmid or vector, high enough to significantly positively modify the symptoms and / or condition to be treated, but low enough to avoid serious side effects (at a reasonable risk / benefit ratio), within the scope of sound medical judgment.

[0097] The therapeutically effective amount of a host cell, e.g. an immune cell, nucleic acid, plasmid or vector as described herein is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient or subject; the severity of the condition or disease (e.g. cancer, infection or autoimmune disease ) to be treated; the route of administration; the renal and hepatic function of the patient or subject. A physician of ordinary skill in the art can readily determine and prescribe the effective amount of the immune cell, nucleic acid, plasmid of vector required to prevent, counter or arrest the progress of the disease, such as e.g. a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0098] “Pharmaceutically acceptable carrier or diluent” means a carrier or diluent that is useful in preparing pharmaceutical compositions that is generally safe, non-toxic, and desirable, and includes carriers or diluents that are acceptable for human pharmaceutical use.

[0099] Such pharmaceutical compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants e.g., aluminum hydroxide); and preservatives.

[0100] Any of the compositions provided herein can be provided in any appropriate pharmaceutical composition and be administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, the inhalation, intra-arterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes. Pharmaceutical compositions of the present invention are preferably formulated for intravenous administration.

[0101] The pharmaceutical compositions (solutions, suspensions or the like), may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0102] The pharmaceutical compositions of the invention can further comprise at least one additional therapeutic agent or therapy. A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein.

[0103] In one aspect, said at least one additional therapeutic agent or therapy is an anticancer agent or anticancer therapy, useful to treat a cancer, preferably a solid cancer. Preferably, the one or more anti-cancer therapy will be selected from the group comprising radiotherapy, chemotherapy, immune checkpoint inhibitor, immunotherapy and hormone therapy, or a combination of one of more thereof.

[0104] Preferably, the immune checkpoint inhibitor is selected from the group comprising a PD-1 inhibitor, a PD-L1 inhibitor, and a CTLA-4 inhibitor, or a combination of one of more thereof.

[0105] For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0106] For example, potentially useful additional therapeutic agents include PD-L1 inhibitors such as atezolizumab, avelumab, AMP -224, MEDI-0680, RG-7446, GX-P2, durvalumab, KY-1003, KD-033, MSB-0010718C, TSR-042, ALN-PDL, STI-A1014, CX-072, and BMS-936559.

[0107] Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy) (also known as BMS-734016, MDX-010, MDX- 101 ) and tremelimumab (formerly ticilimumab, CP-675,206).

[0108] A chemotherapy of the present invention can concern agents that damage DNA and / or prevent cells from multiplying, such as genotoxins. Genotoxins can be selected from the group comprising alkylating agents, antimetabolites, DNA cutters, DNA binders, topoisomerase poisons and spindle poisons. Examples of alkylating agents are lomustine, carmustine, streptozocin, mechlorethamine, melphalan, uracil nitrogen mustard, chlorambucil, cyclosphamide, iphosphamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazin, procarbazine, hexamefhyl melamine, triethylene melamine, busulfan, pipobroman, mitotane and other platine derivatives.

[0109] An example of DNA cutters is bleomycin.

[0110] Topoisomerases poisons can be selected from the group comprising topotecan, irinotecan, camptothecin sodium salt, daorubicin, doxorubicin, idarubicin, mitoxantrone teniposide, adriamycin and etoposide.

[0111] Examples of DNA binders are dactinomycin and mithramycin whereas spindle poisons can be selected among the group comprising vinblastin, vincristin, navelbin, paclitaxel and docetaxel.

[0112] A chemotherapy of the present invention can concern antimetabolites selected among the following coumpounds: methotrexate, trimetrexate, pentostatin, cytarabin, ara-CMP, fludarabine phosphate, hydroxyurea, fluorouracyl, fioxuridine, chlorodeoxyadenosine, gemcitabine, thioguanine and 6-mercaptopurine.

[0113] Radiotherapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, without limitation, external radiation therapy and internal radiation therapy (also called brachytherapy).

[0114] External radiation therapy is most common and typically involves directing a beam of direct or indirect ionizing radiation to a tumor or cancer site. While the beams of radiation, the photons, the Cobalt or the particule therapy are focused to the tumor or cancer site, it is nearly impossible to avoid exposure of normal, healthy tissue. Energy source for external radiation therapy is selected from the group comprising direct or indirect ionizing radiation (for example: x-rays, gamma rays and particle beams or combination thereof).

[0115] Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, etc., inside the body, at, or near to the tumor site. Energy source for internal radiation therapy is selected from the group of radioactive isotopes comprising: iodine (iodinel25 or iodinel31), strontium89, radioisotopes of phosphorous, palladium, cesium, indium, phosphate, or cobalt, and combination thereof. Such implants can be removed following treatment, or left in the body inactive. Types of internal radiation therapy include, but are not limited to, interstitial, and intracavity brachytherapy (high dose rate, low dose rate, pulsed dose rate).

[0116] A currently less common form of internal radiation therapy involves biological carriers of radioisotopes, such as with radio-immunotherapy wherein tumor-specific antibodies bound to radioactive material are administered to a patient or subject. The antibodies bind tumor antigens, thereby effectively administering a dose of radiation to the relevant tissue.

[0117] Methods of administering radiation therapy are well known to those of skill in the art.

[0118] A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein.

[0119] Additional therapeutic agents suitable for use in combination with the invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin11), trastuzumab (Herceptin11), trastuzumab emtansine (KADCYLA11), imatinib (Gleevec11), cetuximab (Erbitux11), panitumumab (Vectibix11), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, masitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, cabozantinib, imatinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as Everolimus and Temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib, CDK inhibitors such as CDK inhibitor (palbociclib).

[0120] In additional aspects, the additional therapeutic agent can be an anti-inflammatory agent. Antiinflammatory agents or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), nonsteroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF medications, cyclophosphamide and my cophenolate. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox- 2 inhibitors, and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol of proporxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors, such as the TNF antagonists, (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®) and infliximab (REMICADE®), chemokine inhibitors and adhesion molecule inhibitors. The biological response modifiers include monoclonal antibodies as well as recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofin) and intramuscular) and minocycline.

[0121] The present invention further contemplates methods of treating and / or preventing a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease comprising administering i) an isolated polynucleotide of the invention, ii) a vector of the invention, iii) a host cell, e.g. an immune cell of the invention, or a pharmaceutical composition of the invention, to a subject in need thereof.

[0122] The term "treatment" or "treating" means any administration of a composition, pharmaceutical composition, therapeutic agent, compound, etc. . . of the disclosure to a subject for the purpose of:

[0123] (i) inhibiting the disease, that is, arresting the development of clinical symptoms; and / or

[0124] (ii) relieving the disease, that is, causing the regression of clinical symptoms.

[0125] As used herein, the term “prevention” or “preventing” means any administration of a composition, pharmaceutical composition, therapeutic agent, compound, etc... of the disclosure to a subject for the purpose of preventing the disease, that is, causing the clinical symptoms of the disease not to develop. In the context of the present invention, the disease is a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease .

[0126] As used herein the terms "subject" / " subject in need thereof, or "patient" / "patient in need thereof " are well-recognized in the art, and, are used interchangeably herein to refer to a mammal, including dog, cat, rat, mouse, monkey, cow, horse, goat, sheep, pig, camel, and, most preferably, a human. In some cases, the subject is a subject in need of treatment or a subject with a disease or disorder. However, in other aspects, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. Preferably, the subject is a human, most preferably a human that suffer or might be at risk of suffering from a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

[0127] In one aspect, the method of treatment and / or prevention of a cancer, an infection or an autoimmune disease in a patient or subject comprises (i) removing and isolating immune cells, preferably immune cells, more preferably native T cells or NK cells, from said patient or subject, (ii) genetically engineering said T cells or NK cells with one recombinant construct (e.g. vector, plasmid or polynucleotide) encoding a recombinant construct encoding a chimeric antigen receptor (CAR) or the invention, (iii) expanding ex vivo into a larger population of engineered immune cells, e.g. T cells or NK cells, and (iv) reintroducing said engineered immune cells, e.g. T cells, into the patient or subject. After the immune cells, e.g. T cells or NK cells are reintroduced into the patient or subject, they exert their function, which varies depending on the immune cell type used. Examples of functions are triggering cell death on the target cells (T and NK), phagocytosis of the target cell (macrophages), or induce an immunosuppressive environment around their target (Tregs).

[0128] Alternatively, the method of treatment and / or prevention of a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease in a patient or subject comprises (i) providing genetically engineered immune cells such as e.g.T cells or NK cells with one recombinant construct (e.g. vector, plasmid or polynucleotide of the invention) encoding a chimeric antigen receptor (CAR) or the invention, (ii) expanding ex vivo into a larger population of engineered immune cells, e.g. T cells or NK cells, and (iii) reintroducing said engineered immune cells, e.g. T cells, into the patient or subject. In one aspect, the NK cells is a cell line, preferably NK-92 cells.

[0129] NK cells can, for example, be engineered from various NK sources such as iPSC-derived NK cells, NK cells isolated from blood (PB-NK), NK isolated from the umbilical cord blood or NK cell lines comprising NK92, YTS or KHYG1 cell lines.

[0130] Examples of clinical trials using NK cells comprise NCT00995137, NCT01974479, NCT02839954, NCT02892695, NCT02742727, and NCT02944162

[0131] (https: / / www.clinicaltrials.gov / ).

[0132] The polypeptides disclosed herein, or nucleic acids encoding such, may be introduced into the host cells using transfection and / or transduction techniques known in the art. The nucleic acid may be integrated into the host cell DNA or may be maintained extrachromosomally. The nucleic acid may be maintained transiently or may be a stable introduction. Transfection may be accomplished by a variety of means known in the art including but not limited to calcium phosphate-DNA co-precipitation, DEAE- dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of a gene(s) using a viral or retroviral vector by means of viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vectors into virions prior to contact with a cell. For example, a nucleic acid encoding a transmembrane polypeptide carried by a retroviral vector can be transduced into a cell through infection and pro virus integration.

[0133] In certain aspects, the nucleic acid or vector, e.g. viral vector, is transferred via ex vivo transformation. Methods for transfecting cells and tissues removed from an organism in an ex vivo setting are known to those of skill in the art. Thus, it is contemplated that cells (or tissues) may be removed and transfected ex vivo using the polynucleotides presented herein. In particular aspects, the transplanted cells or tissues may be placed into an organism. Thus, it is well within the knowledge of one skilled in the art to isolate antigen-presenting cells ( e.g., T- cells or NK cells) from an animal ( e.g., human), transfect the cells with the expression vector and then administer the transfected or transformed cells back to the animal (e.g. human).

[0134] In certain aspects, the nucleic acid or vector is transferred via injection. In certain aspects, a polynucleotide is introduced into an organelle, a cell, a tissue or an organism via electroporation. In certain aspects, a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol.

[0135] In one aspect the method of treatment and / or prevention of a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease in a subject comprises administering a pharmaceutical composition of the invention to a subject in need thereof.

[0136] In one aspect, the methods of treatment and / or prevention described above, can further comprise administrating at least one additional therapeutic agent or therapy.

[0137] Preferably, the at least one additional therapeutic agent or therapy will be an anticancer agent or anticancer therapy, more preferably a therapeutically effective amount or dose of an anticancer agent or anticancer therapy. Said one or more anti-cancer agent or therapy will be selected among the non-limiting group comprising radiotherapy, chemotherapy, immune checkpoint inhibitor, immunotherapy and hormone therapy, or a combination of one of more thereof, as described herein.

[0138] The invention also contemplates kits for the treatment and / or prevention of a disease of the invention. In one aspect of the invention, the kit comprises a pharmaceutical composition of the invention.

[0139] The kits of the invention may also comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating the disease of disorder of the invention and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Alternatively, or additionally, the kits may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution) and / or at least one additional therapeutic agent (such as e.g. a ligand of the invention) . It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0140] The label or package insert may comprise instructions for use thereof. Instructions included may be affixed to packaging material or may be included as a package insert. While the instructions are typically written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure.

[0141] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991); Carey and Sundberg Advanced Organic Chemistry 3. sup. rd Ed. (Plenum Press) Vols A and B (1992).

[0142] The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

[0143] Table 1 recites the sequences referred to herein.

[0144] Table 1

[0145] Human LAT wild type intracellular (SEQ ID NO. 1)

[0146] HCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQ PLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPP

[0147] EPACEDADEDEDDYHNPGYLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDY VNVPESGESAEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYEN LQELN

[0148] Human LAT wild type intracellular (SEQ ID NO. 2) cattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttcaagagacctcatacagt ggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattccaagatctcctcagcctc tgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacgagaatgaaggagcctc tggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcctgccacctgaaccagc atgcgaagatgccgatgaggatgaggatgactaccacaacccaggctatctggtggtgctgcctgattctacaccagccacatctaca gccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacgactacgtgaatgtgcct gagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgcatcctggagctgccaaa acagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaatctgcaggagctgaac

[0149] Human LAT wild type site surrounding tyrosine 134 (from histidine 130 to leucine 138 (SEQ ID NO. 3))

[0150] HNPGYLVVL

[0151] Human LAT intracellular variant 1 of SEQ ID NO. 3 (SEQ ID NO. 4)

[0152] DGKDYIVLP

[0153] Human LAT intracellular variant 2 of SEQ ID NO. 3 (SEQ ID NO. 5)

[0154] DGKD YIPIN

[0155] Human LAT intracellular variant 3 of SEQ ID NO. 3 (SEQ ID NO. 6)

[0156] ADSGYIIPL

[0157] Human LAT intracellular variant 4 of SEQ ID NO. 3 (SEQ ID NO. 7) TSFGYDKPH

[0158] Human LAT transmembrane (SEQ ID NO. 8) atcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgctgatggccctgtgtgtg

[0159] Human LAT transmembrane (SEQ ID NO. 9)

[0160] ILVPCVLGLLLLPILAMLMALCV

[0161] CD19scFv (IgG2a mouse monoclonal antibody specific for human CD19) (SEQ ID NO. 10)

[0162] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGV

[0163] PSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGG

[0164] SGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVI

[0165] WGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAM

[0166] DYWGQGTSVTVSS

[0167] Human CD8a (hinge) (SEQ ID NO. 11)

[0168] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

[0169] Human CD8a (hinge) (SEQ ID NO. 12) acaacaacacctgctccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggc ggcgctgtgcacactaggggcctggattttgcctgcgat

[0170] CD8a variant (hinge) (SEQ ID NO. 13)

[0171] TTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASD

[0172] Human CD28 intracellular (SEQ ID NO. 14) cgctccaaaagatccagactcctccattccgattatatgaatatgacacctagacggcctggccctacacgcaaacattatcaaccttat gcgcccccccgtgattttgcggcgtatcgttcc

[0173] Human CD28 intracellular (SEQ ID NO. 15)

[0174] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS Influenza hemagglutinin HA tag (SEQ ID NO. 16) tacccttacgacgtgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgcc

[0175] Influenza hemagglutinin HA tag (SEQ ID NO. 17)

[0176] YPYDVPDYAGAPGYPYDVPDYA

[0177] Signal peptide (SEQ ID NO. 18) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgcc

[0178] Signal peptide (SEQ ID NO. 19)

[0179] MRCFAQLLGLLVLWIPGSTA

[0180] Example of a LAT -based CAR optimized for T cells (SEQ ID NO. 20) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccaggctatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaaccgctccaaaagatccagactcctccattccgattatatgaatatgacacctagacggcctggccctacacgca aacattatcaaccttatgcgcccccccgtgattttgcggcgtatcgttcctaa Example of a LAT -based CAR optimized for T cells (SEQ ID NO. 21)

[0181] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ

[0182] QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY

[0183] TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

[0184] YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD

[0185] DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP

[0186] DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL

[0187] VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA

[0188] YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ

[0189] AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTPATSTAAPS

[0190] APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP

[0191] AALSSQEAEEVEEEGAPDYENLQELNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY

[0192] APPRDFAAYRS

[0193] Human Anti-FAP (SEQ ID NO. 22) caggtgcagctcaagcagtccggggccgagctggtcaagcccggcgccagcgtgaaactgagctgcaagacaagcggctacaca ttcacagaaaacatcatccactgggtgaagcagcggtccggccagggcctggagtggattggctggtttcaccccggctctgggagc atcaagtacaacgaaaagaaggataaggccaccctgactgccgacaaatcatcttccaccgtgtacatggagctgtccagactgacct ctgaggactccgctgtgtacttctgtgccaggcacgggggaacagggaggggagctatggactattgggggcagggaactagcgtg accgtgagctccggcggagggggaagtggaggcggcggctctggaggaggaggatctggcggcggagatatcctgatgacaca gtctcccgccagctctgtggtgtccctgagcggacaacgggccactattagctgcagggcctccaagagcgtgtccacatctgcctat agctatatgcattggtaccagcagaaaccaggacagccccccaaactcctgatctacctggcgtcaaatctggaaagcggagtgcca ccccgctttagcggcagcggctctggcacagatttcactctgaacattcaccccgtggaagaggaggatgctgccacctattattgcca gcacagcagggagctgccctacacattcggcggcggcaccaaactggaaattaag

[0194] Human Anti-FAP (SEQ ID NO. 23)

[0195] QVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWVKQRSGQGLEWIGWFHPGSG SIKYNEKKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHGGTGRGAMDYWGQ GTSVTVSSGGGGSGGGGSGGGGSGGGDILMTQSPASSVVSLSGQRATISCRASKSVS TSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGSGSGTDFTLNIHPVEEEDA ATYYCQHSRELPYTFGGGTKLEIK

[0196] Example of a LAT -based CAR optimized for NK cells - 19LAT (SEQ ID NO. 24) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgcccaggtgcagctcaagcagtccggggccg agctggtcaagcccggcgccagcgtgaaactgagctgcaagacaagcggctacacattcacagaaaacatcatccactgggtgaag cagcggtccggccagggcctggagtggattggctggtttcaccccggctctgggagcatcaagtacaacgaaaagaaggataaggc caccctgactgccgacaaatcatcttccaccgtgtacatggagctgtccagactgacctctgaggactccgctgtgtacttctgtgccag gcacgggggaacagggaggggagctatggactattgggggcagggaactagcgtgaccgtgagctccggcggagggggaagtg gaggcggcggctctggaggaggaggatctggcggcggagatatcctgatgacacagtctcccgccagctctgtggtgtccctgagc ggacaacgggccactattagctgcagggcctccaagagcgtgtccacatctgcctatagctatatgcattggtaccagcagaaaccag gacagccccccaaactcctgatctacctggcgtcaaatctggaaagcggagtgccaccccgctttagcggcagcggctctggcacag atttcactctgaacattcaccccgtggaagaggaggatgctgccacctattattgccagcacagcagggagctgccctacacattcggc ggcggcaccaaactggaaattaagtccatggccggctacccttacgacgtgcctgactacgccggagcacccggctacccttacgac gtgcctgactacgccggagcacccgggagcagtacaacaacacctgctccaagacctcctacacctgctcctaccattgctagtcagc ctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtgcacactaggggcctggattttgcctgcgatcctagggcc atcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgctgatggccctgtgtgtgcattgtcacagactgcctggatc ctatgatagcaccagctctgacagcctgtacccaagaggcatccagttcaagagacctcatacagtggccccttggcctcctgcttatc ctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattccaagatctcctcagcctctgggaggaagccacagaaca ccaagcagcagaagagactctgatggagccaattctgtggccagctacgagaatgaaggagcctctggaattagaggcgctcaggc tggatggggagtttggggaccttcttggacaagactgacacctgtgagcctgccacctgaaccagcatgcgaagatgccgatgagga tgaggatgactaccacaacccaggctatctggtggtgctgcctgattctacaccagccacatctacagccgccccttctgctcctgctct gtctacacctggcatcagagattctgccttcagcatggagagcatcgacgactacgtgaatgtgcctgagagcggagaatctgctgag gcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgcatcctggagctgccaaaacagaacctgctgccctgtcttc tcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaatctgcaggagctgaac

[0197] Example of a LAT -based CAR optimized for NK cells - 19LAT (SEQ ID NO. 25)

[0198] MRCFAQLLGLLVLWIPGSTAQVQLKQSGAELVKPGASVKLSCKTSGYTFTENIIHWV KQRSGQGLEWIGWFHPGSGSIKYNEKKDKATLTADKSSSTVYMELSRLTSEDSAVYF CARHGGTGRGAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSGGGDILMTQSPASSV VSLSGQRATISCRASKSVSTSAYSYMHWYQQKPGQPPKLLIYLASNLESGVPPRFSGS GSGTDFTLNIHPVEEEDAATYYCQHSRELPYTFGGGTKLEIKSMAGYPYDVPDYAGA PGYPYDVPDYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD FACDPRAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPH TVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEG ASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTP ATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELH PGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN

[0199] Human LAT wild type site surrounding tyrosine 228 (from glycine 224 to leucine 231) (SEQ ID NO. 26)

[0200] GAPDYENL

[0201] Human LAT intracellular variant 4 of SEQ ID NO. 26 ( enhanced ITT site to integrate a YxxM motif where x represents any amino-acids (SEQ ID NO. 27))

[0202] SIDDYMNM

[0203] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTi) (SEQ

[0204] ID NO. 28) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaagcatcgacgactatatgaat atgcaggagctgaac

[0205] Example of a LAT-based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTi) (SEQ ID NO. 29)

[0206] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEESIDDYMNMQELN

[0207] Example of a LAT-based CAR optimized for NK cells - 19LAT(G133D, ITTc) (SEQ ID NO. 30) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaacagcatcgacgactatatgaatatgactccacgccgg

[0208] Example of a LAT -based CAR optimized for NK cells - 19LAT(G133D, ITTc) (SEQ ID NO. 31)

[0209] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELNSIDDYMNMTPRR

[0210] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTi)41BB (SEQ ID NO. 32) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaagcatcgacgactatatgaat atgcaggagctgaacaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagagg aagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg

[0211] Example of a LAT-based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTi)41BB (SEQ ID NO. 33)

[0212] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS

[0213] APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP

[0214] AALSSQEAEEVEEESIDDYMNMQELNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCR

[0215] FPEEEEGGCEL

[0216] Example of a LAT-based CAR optimized for NK cells - 19LAT(Y134F) (SEQ ID NO. 34) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggcaaaccaatccca aatcctctgctgggcctggatagcactggaggtggcggcagcggtggcggcggcagcggtaagcctattcctaacccactcctcggt ctcgactctaccggcggtggtggttctggtggtggtggtcccgggagcagtacaacaacacctgctccaagacctcctacacctgctc ctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtgcacactaggggcctggattttg ccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgctgatggccctgtgtgtgcattgt cacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttcaagagacctcatacagtggccc cttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattccaagatctcctcagcctctggga ggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacgagaatgaaggagcctctggaat tagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcctgccacctgaaccagcatgcga agatgccgatgaggatgaggatgactaccacaacccaggcttcctggtggtgctgcctgattctacaccagccacatctacagccgcc ccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacgactacgtgaatgtgcctgagagc ggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgcatcctggagctgccaaaacagaa cctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaatctgcaggagctgaac

[0217] Example of a LAT-based CAR optimized for NK cells - 19LAT(Y134F) (SEQ ID NO. 35) MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGKPIPNPLLGLDSTGGGGSGG

[0218] GGSGKPIPNPLLGLDSTGGGGSGGGGPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPA AGGAVHTRGLDFASDPRAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDS LYPRGIQFKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSD GANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYH

[0219] NPGFLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDG SREYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN

[0220] Example of a LAT -based CAR optimized for NK cells - 19LAT(G133D) (SEQ ID NO. 36) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaac

[0221] Example of a LAT-based CAR optimized for NK cells - 19LAT(G133D) (SEQ ID NO. 37)

[0222] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELN

[0223] Example of a LAT-based CAR optimized for NK cells - 19LAT(P132D) (SEQ ID NO. 38) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacgacggctatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaac

[0224] Example of a LAT-based CAR optimized for NK cells - 19LAT(P132D) (SEQ ID NO. 39)

[0225] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP

[0226] DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ

[0227] AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNDGYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELN

[0228] Example of a LAT-based CAR optimized for NK cells - 19LAT(DD) (SEQ ID NO. 40) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacgacgactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaac

[0229] Example of a LAT -based CAR optimized for NK cells - 19LAT(DD) (SEQ ID NO. 41)

[0230] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNDDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELN

[0231] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT (SEQ ID NO. 42) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccaggctatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaac

[0232] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT (SEQ ID NO. 43)

[0233] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELN Example of a LAT-based CAR optimized for NK cells - 19LATCVEGFR2) (SEQ ID NO.

[0234] 44) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggcaaaccaatccca aatcctctgctgggcctggatagcactggaggtggcggcagcggtggcggcggcagcggtaagcctattcctaacccactcctcggt ctcgactctaccggcggtggtggttctggtggtggtggtcccgggagcagtacaacaacacctgctccaagacctcctacacctgctc ctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtgcacactaggggcctggattttg ccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgctgatggccctgtgtgtgcattgt cacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttcaagagacctcatacagtggccc cttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattccaagatctcctcagcctctggga ggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacgagaatgaaggagcctctggaat tagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcctgccacctgaaccagcatgcga agatgccgatgaggatgaggatgactacgatggcaaagactacatcgtgctgcctatttctgaaacaccagccacatctacagccgcc ccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacgactacgtgaatgtgcctgagagc ggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgcatcctggagctgccaaaacagaa cctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaatctgcaggagctgaac

[0235] Example of a LAT-based CAR optimized for NK cells - 19LAT(VEGFR2) (SEQ ID NO.

[0236] 45)

[0237] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGKPIPNPLLGLDSTGGGGSGG GGSGKPIPNPLLGLDSTGGGGSGGGGPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPA AGGAVHTRGLDFASDPRAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDS LYPRGIQFKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSD GANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYD

[0238] GKDYIVLPISETPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGS REYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN

[0239] Example of a LAT -based CAR optimized for NK cells - 19LAT(Hybrid) (SEQ ID NO. 46) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggcaaaccaatccca aatcctctgctgggcctggatagcactggaggtggcggcagcggtggcggcggcagcggtaagcctattcctaacccactcctcggt ctcgactctaccggcggtggtggttctggtggtggtggtcccgggagcagtacaacaacacctgctccaagacctcctacacctgctc ctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtgcacactaggggcctggattttg ccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgctgatggccctgtgtgtgcattgt cacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttcaagagacctcatacagtggccc cttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattccaagatctcctcagcctctggga ggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacgagaatgaaggagcctctggaat tagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcctgccacctgaaccagcatgcga agatgccgatgaggatgaggatgactaccacaacccaggctatatcgtgctgcctatttctgaaacaccagccacatctacagccgcc ccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacgactacgtgaatgtgcctgagagc ggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgcatcctggagctgccaaaacagaa cctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaatctgcaggagctgaac

[0240] Example of a LAT -based CAR optimized for NK cells - 19LAT(Hybrid) (SEQ ID NO. 47) MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGKPIPNPLLGLDSTGGGGSGG

[0241] GGSGKPIPNPLLGLDSTGGGGSGGGGPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPA AGGAVHTRGLDFASDPRAILVPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDS LYPRGIQFKRPHTVAPWPPAYPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSD GANSVASYENEGASGIRGAQAGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYH NPGYIVLPISETPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGS REYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN

[0242] Example of a LAT -based CAR optimized for NK cells - 19LAT(RR) (SEQ ID NO. 48) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca gaagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccaggctatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccagaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaac

[0243] Example of a LAT -based CAR optimized for NK cells - 19LAT(RR) (SEQ ID NO. 49)

[0244] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFRRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAARTEP AALSSQEAEEVEEEGAPDYENLQELN

[0245] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(ITTc) (SEQ ID NO.

[0246] 50) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccaggctatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaacagcatcgacgactatatgaatatgactccacgccgg

[0247] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(ITTc) (SEQ ID NO. 51)

[0248] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA

[0249] YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELNSIDDYMNMTPRR

[0250] Example of a LAT -based CAR optimized for NK cells - 19LAT(ITTi) (SEQ ID NO. 52) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccaggctatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaagcatcgacgactatatgaat atgcaggagctgaac

[0251] Example of a LAT -based CAR optimized for NK cells - 19LAT(ITTi) (SEQ ID NO. 53)

[0252] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPGYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEESIDDYMNMQELN

[0253] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTi) (SEQ

[0254] ID NO. 54) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaagcatcgacgactatatgaat atgcaggagctgaac

[0255] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTi) (SEQ

[0256] ID NO. 55)

[0257] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEESIDDYMNMQELN

[0258] Example of a LAT -based CAR optimized for NK cells - 19LAT(G133D, ITTc) (SEQ ID NO. 56) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaacagcatcgacgactatatgaatatgactccacgccgg

[0259] Example of a LAT -based CAR optimized for NK cells - 19LAT(G133D, ITTc) (SEQ ID NO. 57) MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELNSIDDYMNMTPRR

[0260] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTc) (SEQ ID NO. 58) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaacagcatcgacgactatatgaatatgactccacgccgg

[0261] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D, ITTc) (SEQ

[0262] ID NO. 59)

[0263] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ

[0264] AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELNSIDDYMNMTPRR

[0265] Example of a LAT -based CAR optimized for NK cells - 19LAT(G133D, ITTi) (SEQ ID

[0266] NO. 60) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctgcagaccagccgctggcggcgctgtg cacactaggggcctggattttgcctgcgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaagcatcgacgactatatgaat atgcaggagctgaac

[0267] Example of a LAT -based CAR optimized for NK cells - 19LAT(G133D, ITTi) (SEQ ID

[0268] NO. 61)

[0269] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA

[0270] YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEESIDDYMNMQELN

[0271] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D) (SEQ ID NO. 62) atgaggtgctttgctcagctcctggggctgctggtgctgtggattcctggctccaccgccgatatacagatgacgcagacaacgtcaag tctttccgccagcttgggagaccgagtgactatatcttgtagagcaagccaggatatttctaagtatcttaactggtaccaacaaaagccc gatggaacggttaagctgcttatataccataccagtagactccactccggcgtaccatcacggttttctggcagtggctccgggaccga ctattctttgacgatctctaatctcgaacaagaggatattgcaacatacttttgtcagcaaggcaataccttgccatatacgtttgggggcg ggacaaaacttgagataaccggcggcggtggttcaggcggtggcggttccggtggtgggggatcagaggttaagcttcaggaatcc ggaccaggtttggttgcccccagccaatctctcagcgttacatgcacggtttcaggcgtcagtctccccgattacggtgtaagttggattc ggcaacctccgcgaaagggtctggaatggctgggggttatttgggggagtgagacaacttattacaactctgcacttaagagtcggctt accatcatcaaggataattcaaaatcacaagtattcctgaagatgaactcattgcaaacagatgatacagctatatactattgtgccaagc attactattatggtggttcttatgcaatggattactgggggcaaggcacgtcagtgacagtgagctccatggccggctacccttacgacg tgcctgactacgccggagcacccggctacccttacgacgtgcctgactacgccggagcacccgggagcagtacaacaacacctgct ccaagacctcctacacctgctcctaccattgctagtcagcctttgtctcttagacctgaagcctctaggccagccgctggcggcgctgtg cacactaggggcctggattttgccagtgatcctagggccatcctggtgccttgtgtgctgggactgctgctgctgcctattctggctatgc tgatggccctgtgtgtgcattgtcacagactgcctggatcctatgatagcaccagctctgacagcctgtacccaagaggcatccagttca agagacctcatacagtggccccttggcctcctgcttatcctcctgtgaccagctatcctcctctgagccagcctgatctgctgcctattcc aagatctcctcagcctctgggaggaagccacagaacaccaagcagcagaagagactctgatggagccaattctgtggccagctacg agaatgaaggagcctctggaattagaggcgctcaggctggatggggagtttggggaccttcttggacaagactgacacctgtgagcc tgccacctgaaccagcatgcgaagatgccgatgaggatgaggatgactaccacaacccagactatctggtggtgctgcctgattctac accagccacatctacagccgccccttctgctcctgctctgtctacacctggcatcagagattctgccttcagcatggagagcatcgacg actacgtgaatgtgcctgagagcggagaatctgctgaggcctctctggatggcagcagagagtatgtgaatgtgtctcaggagctgca tcctggagctgccaaaacagaacctgctgccctgtcttctcaggaagctgaggaagtggaagaagaaggagcccctgactatgaaaa tctgcaggagctgaac

[0272] Example of a LAT -based CAR optimized for NK cells - 19(CS)LAT(G133D) (SEQ ID NO. 63)

[0273] MRCFAQLLGLLVLWIPGSTADIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQ QKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPY TFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTD DTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSMAGYPYDVPDYAGAPGYPYDVP DYAGAPGSSTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFASDPRAIL VPCVLGLLLLPILAMLMALCVHCHRLPGSYDSTSSDSLYPRGIQFKRPHTVAPWPPA YPPVTSYPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASYENEGASGIRGAQ AGWGVWGPSWTRLTPVSLPPEPACEDADEDEDDYHNPDYLVVLPDSTPATSTAAPS APALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEP AALSSQEAEEVEEEGAPDYENLQELN EXAMPLES

[0274] Material & Methods

[0275] Experimental model and subject details

[0276] All experiments with mice were authorized by the Canton of Vaud (license VD3747) and were performed according to accepted guidelines for animal handling.

[0277] Mice

[0278] All mice were kept on a 12-hour light / dark cycle in individually ventilated cages. We used RAG- / - IL2Rg- / - for all in vivo tumor killing assays. All mice were bred and maintained in pathogen-free barrier animal facilities in accord with Swiss regulations for the care and use of mice in experimental research

[0279] Healthy 8-10 weeks old female mice were used in the study. Mice used in this study had no previous history of drug administration, surgery, or behavioural testing.

[0280] Cell culture

[0281] Nalm6 and Al 847 were cultured in RPMI media (61870010, Thermo Fisher Scientific) supplemented with 10% FBS (F7524, Sigma-Aldrich) and lx pen strep (15140-122, Thermo Fisher Scientific). NK92 cells were cultured in MEM Alpha media (1-23S50-I, BioConcept) supplemented with 25% FBS (F7524, Sigma-Aldrich) and lx pen strep and human IL-2 (lOOU / ml, Peprotech).

[0282] Al 847 cells were passaged using Trypsin-EDTA (25300-054, Thermo Fisher Scientific) every 2-3 days or when they reached 90% confluence. All cells were maintained in a 37C, 5% CO2 atmosphere.

[0283] Plasmid construction and lentivirus preparations

[0284] Genes constructs were designed by us. Lentiviral vectors were then constructed and packaged by VectorBuilder (vectorbuilder.com).

[0285] Transduction by electroporation of NK92 or THP1 cell lines

[0286] NK92 or THP1 cell lines were co-transfected with the expression plasmid and the retrotransposase by electroporation, in an exponential growth phase, using the Elepo21 electroporator from NepaGene with the following parameters: Poring Pulse (125V, Length 5ms, Interval 50ms, No 2, Polarity +) and Transfer Pulse (20V, Length 50ms, Interval 50ms, No. 5, Polarity + / -). Electroporation was performed in Opti-MEM media (31985070, Thermo Fisher Scientific) and electroporated cells were immediately cultured in their respective culture media. Following 4 days of recovery and expansion, NK92 or THP1 cells were selected with Puromycin (final concentration of 2 ng / ml, cat. number: ant-pr-1, InvivoGen).

[0287] Human blood samples

[0288] All human blood samples (prepared as buffy coats) were purchased from Interregional Blood Transfusion SRC Ltd. with informed consent of anonymous healthy donors.

[0289] Transduction and expansion of human T cells

[0290] Human PBMCs were purified from anonymous healthy donor buffy coats by density gradient centrifugation against Ficoll-Paque PLUS. Cells were then cultured at a density of 0.5-1 *106cells / ml in complete RPMI medium, which contained RPML1640 (61870010, Thermo Fisher Scientific), FBS (10% v / v), HEPES (pH 7.2-7.5, 20mM), penicillin / streptomycin (1% v / v), supplemented with human IL-7(10 ng / ml, Peprotech) and human IL-15 (10 ng / ml, Peprotech). hPBMCs were activated with CD3 and CD28 magnetic beads at a 1 : 1 ratio for 48h before transduction. Cells were transduced in minimal media during 5 hours at an MOI of 3.

[0291] Flow cytometry antibody staining

[0292] Cells are stained at 4°C for 20 minutes in PBS with the desired antibody diluted according to the manufacturer information. Cells are then washed twice with PBS and resuspended at a concentration of 107cells / ml. For Annexin V staining, Annexin buffer was used instead of PBS, for staining and further processing of the cells.

[0293] In vitro killing assay measured by FACS

[0294] On the day of the co-culture, the target cells were stained using Cell Trace Violet, Cell Trace Far-Red or Cell Trace CFSE (C34557, C34564, or C34554, Thermo Fisher Scientific) following manufacturer instructions.

[0295] Effector cells were added at the correct ratios, and after a determined amount of time, the killing of the target population was extracted from the double negative population of Annexin V and PI staining.

[0296] For the serial and long killing assays, target cells were added at the indicated time points before the measurement of the killing. For the quantification of the killing, the target cells were gated in the correct Cell Trace channel, and the living fraction (Annexin V and PI negative) was extracted. Then the killing was calculated relative to the viability of the control condition with no effector cells, using the following formula: Killing = (% Viability Control - % Viability sample) / %Viability Control *100

[0297] Regulatory T cells isolation and transduction

[0298] Regulatory T cells were purified from PBMCs using the EasySep™ Human CD4+CD1271owCD25+ Regulatory T Cell Isolation Kit (Stemcell technologies). Cells were then cultured at a density of 0.5-1 *106cells / ml in complete RPMI medium, which contained RPMI-1640 (Thermo Fisher Scientific), FBS (10% v / v), HEPES (pH 7.2~7.5, 20mM), penicillin / streptomycin (1% v / v), supplemented with human IL-2 ( lOOU / mL in normal conditions, and 300U / mL when we want to encourage growth (e.g. after sort), Peprotech).

[0299] Division assay

[0300] PBMCs were preactivated with CD28 and CD3 magnetic beads for 48 hours before the assay. Then beads were removed, and the cells were stained with CFSE (Thermo Fisher Scientific) following manufacturer instructions. Tregs were stimulated 24 hours prior to the assay in a CD19 coated plate. CFSE pre activated PBMCs were then co-cultured with stimulated or unstimulated Tregs during 3 to 5 days in the CD19 coated plate. Then the division was measured by flow cytometry as a dilution of CFSE.

[0301] Phagocytosis assay

[0302] THP1 cells electroporated or not to express the CAR construct were stained with Cell Trace Violet (Thermo Fisher Scientific) following manufacturer instructions. Then they were cocultured with target cells expressing GFP at different ratios for 24h. Then the percentage of THP1 cells acquiring GFP was measured by Flow Cytometry.

[0303] Ovarian Cancer Xenografts with NK cell treatment

[0304] A1847 CD19 GFP-Luc (5*105cells per mouse) were suspended in lOOuL of PBS and were injected into RAG- / - IL2Rg- / - female mice. 4 days after tumor engraftment mice were randomised and assigned to the different treatment groups, and l-5*106NK92 cells per mouse were injected i.p.. Tumor progression was monitored twice per week by bioluminescence imaging using the IVIS Lumina II in vivo imaging system (PerkinElmer). Mice were euthanized when the signal exceeded a threshold before any signs of discomfort were detected.

[0305] Leukaemia Xenograft with T cell treatment

[0306] Nalm6 GFP-Luc (5*106cells per mouse) were suspended in lOOuL of PBS and were i.v. injected into RAG- / - IL2Rg- / - female mice. 4 days after tumor engraftment mice were randomised and assigned to the different treatment groups, and 5*106transduced T cells per mouse were injected i.v.. Tumor progression was monitored twice per week by bioluminescence imaging using the IVIS Lumina II in vivo imaging system (PerkinElmer). Mice were euthanized when the signal exceeded a threshold before any signs of discomfort were detected.

[0307] Example 1

[0308] Results

[0309] LAT-based CAR expression and activity in NK cells in vitro.

[0310] The 19LAT activating domain is made of the intracellular portion of the linker for activation of T cells (LAT). The extracellular portion of the 19LAT is made of a scFv and the extracellular spacer region (i.e. linker or hinge) of CD8a. The inventors compared our new CAR construct to two well documented CARs (1928z and 19BBz) used as control CARs in our study. Their signalling domain is made of the commonly used CD28 or 4-1BB costimulatory domains respectively and the CD3z activating domain (Fig. 1 A).

[0311] Expression level of CARs in NK92, as assessed by flow cytometry, was similar among all CAR constructs (Fig. IB).

[0312] In vitro activity of thel9LAT was compared to the activity of the 1928z and 19BBz CARs in NK92 cells in an in vitro killing assay (Fig. 1C). The cytotoxicity was similar among all constructs showing the potential of our new CAR design.

[0313] To better define the efficacy of our new CAR construct, the inventors assessed for different constructs various parameters in NK92 in in vitro killing assays. Since we are comparing, the length of the stimulation phase by the CAR may affect the NK cytotoxic capacity. The inventors performed a re-challenge killing assay, which mimics chronic inflammation in vitro (Fig. ID). In this assay, new target cells were added to the coculture every 24 hours and the killing of the last population added was measured after 4 hours of co-culture. This assay reveals the ability of the engineered NK cells to retain cytotoxicity while being continuously surrounded by target cells. The inventors hypothesize that the continuous CAR signalling to the NK cell might affect the cytotoxicity in the long term. In these settings, all CARs were equivalent in terms of cytotoxicity after three and four rechallenges (Fig. IE).

[0314] Altogether, these results show that the 19LAT can induce NK cytotoxicity at a similar level than commonly used 1928z and 19BBz CAR constructs in NK cells.

[0315] The 19LAT CAR is able to induce solid cancer growth control by engineered NK92.

[0316] One advantage of NK cells over T cells is their increased ability to penetrate solid cancers. The inventors next compared the efficacy of the 19LAT to the 19BBz and 1928z CARs in NK to eliminate the ovarian human cancer cell line Al 847 engineered to express CD 19 as a cancerspecific marker.

[0317] The inventors first assessed the cytotoxicity of the CAR NK cells against the Al 847 ovarian cells expressing CD 19 in vitro. No differences were observed following the four hours coculture between the three CARs. However, the 19LAT shows a better activity compared to the 1928z CAR in the serial killing assay after four challenges (Fig. 2A). This data shows the superior function of the 19LAT against solid tumours in vitro.

[0318] The inventors then compared the efficacy of the 19LAT to the 1928z CAR in vivo. On day 0, A1847 were injected in immunodeficient mice. CAR NK were injected intraperitoneally once the tumour was established, on day 3 (Fig. 2B). Interestingly, the 19LAT and 1928z CARs have a similar anti-tumor efficacy in vivo (Fig. 2C).

[0319] Altogether, these data show that NK92 engineered with the 19LAT have a strong antitumor induction capacity against solid cancer cells in vitro and in vivo.

[0320] 19LAT CAR construct tuning and optimization.

[0321] The kinetic of PLCy recruitment to LAT is known to modulate T cell activation thresholds. The inventors hypothesized that modulating the PLCy recruitment kinetic would allow to finetune the 19LAT activity.

[0322] The inventors first made sure that the recruitment of PLCy on the tyrosine 134 was not only required but also necessary for the 19LAT. This was done by making a Y134F CAR mutant, namely 19LAT(Y134F) (Fig. 3 A). This mutation was shown in the literature to abolish PLCy recruitment on LAT and the inventors observed that the CAR mutant was unable to induce cytotoxicity (Fig. 3B).

[0323] The tyrosine 134 phosphorylation kinetic on LAT by ZAP70 can be increased by introducing an acidic residue on the -1 and -2 positions relative to the tyrosine. Corresponding 19LAT mutants were generated (Fig. 3C). The cytotoxicity of the mutants was assessed in vitro but no differences were observed compared to the 19LAT construct after 4 hours co-culture and neither after four challenges (Fig. 3D).

[0324] The inventors then wanted to see if PLCy can be recruited not only with the original PLCy recruiting site of LAT but also by the PLCy recruiting site from another protein known to bind PLCy. The inventors chose to use VEGFR, which contains a protein sequence known to recruit PLCy. It has been shown that within these PLCy docking sites, the tyrosine is phosphorylated by a kinase which is recruited by the aminoacid on the N-terminal side of the tyrosine. Whereas the amino acid located on the C-terminal side of the tyrosine confers the specificity for the recruited protein (PLCy in our case) on the phospho-tyrosine. Since the tyrosine needs to be phosphorylated by ZAP-70 / Syk the inventors generated a hybrid LAT / VEGFR docking site for PLCy where the tyrosine can be phosphorylated by ZAP-70 (the amino acids on the N- terminal side of the tyrosine are from the LAT sequence) and recruit PLCy by the VEGFR recruiting site (the amino acids on the C-terminal side of the tyrosine are from the VEGFR sequence) (Fig. 3E).

[0325] The in vitro functional assay shows that the 19L AT (Hybrid) was functional to a similar activity level as the initial 19LAT CAR construct. However, a 19LAT(VEGFR2) construct lacking the N-terminal amino acid residue required for ZAP70 / Syk recruitment does not show any cytotoxicity (Fig. 4F). These data shows that the recruitment of PLCy can be mediated by other docking sites known to recruit PLCy thereby extending our invention to any protein sequence which allows kinase-inducible PLCy recruitment. Since in the case of a CAR, the tyrosine has to be phosphorylated by the tyrosine kinase ZAP70 / Syk known to be expressed by NK cells, such docking sites need to be adapted (as shown above) to allow phosphorylation by this kinase family.

[0326] The stability and surface expression of LAT is known to be controlled by the ubiquitination of two lysine residues in LAT. The inventors therefore mutated these residues to Arginine - namely 19LAT(RR) - in order to see the effect of the stabilisation of the 19LAT construct on membrane expression (Fig. 4G). The inventors assessed the cytotoxicity of those mutants and the inventors saw that they were showing similar activity compared to the 19LAT construct both after 4 hours co-culture and after four challenges (Fig. 4H). More investigation needs to be done to see if the stabilisation of the CAR could confer any advantages to the NK cell.

[0327] LAT is endogenously present in the membrane as a monomeric protein. The CD8a hinge is known to dimerize through the trans-disulphide bridge formation of two cysteines in the CD8a hinge. The inventors therefore generated a mutant of the 19LAT CAR named 19(CS)LAT where these two cysteines have been mutated to serine to abolish the dimerization of the constructs (Fig. 41). The functionality of the 19(CS)LAT CAR was assessed in vitro and was measured to be similar to the 1928z and 19BBz CAR constructs after 4 hours co-culture. Interestingly it shows superior tumor elimination capacity after four challenges both against Nalm6 and Al 847 cell lines (Fig. 4J). The inventors then performed a long-term killing assay experiment which mimics an acute inflammation in vitro (Fig. 4K). This long-term killing assay assesses functional killing by CAR constructs following a co-culture of effector and target cells over seven days. Functional killing / cytotoxicity toward target cells is measured following this long co-culture after an exposure of fresh target cells to CAR NK cells for four hours. This assay was designed in order to assess the activated / resting state reversibility of the engineered NK cells. In these settings, NK92 engineered with the 19(CS)LAT showed a superior functional cytotoxicity compared to NK92 with the 1928z or the 19BBz CARs (Fig. 4L). Interestingly, the 19(CS)LAT construct showed better tumour control than the 1928z CAR which was not the case of the 19LAT demonstrating that the monomeric 19(CS)LAT CAR is superior in vivo (Fig. 4M).

[0328] Altogether, these results show that our LAT -based CAR is highly engineerable to modulate the threshold of activation or the stability of the receptors at the membrane. Moreover, with the 19(CS)LAT superior in vitro and in vivo capacity were achieved compared to the commonly used 1928z CAR.

[0329] 19LAT can be optimised for use in T cells.

[0330] The inventors showed that our new 19LAT exhibits a great advantage in NK cells. The inventors were wondering if those features were the same in T cells.

[0331] T cells were able to express the 19LAT (Fig. 4A) but were showing reduced cytotoxicity against target cells in vitro compared to 1928z and 19BBz after three challenges (Fig. 4B). For the first generation of CARs using the CD3z activating domain, it was shown that the introduction of a co-stimulatory domain such as CD28 or 4-1BB was increasing the cytotoxicity of CARs.

[0332] The inventors generated a 1928LAT and 19BBLAT CAR where CD28 or 4-1BB were introduced in between the transmembrane and the intracellular domain of LAT respectively (Fig. 4C). These constructs were successfully expressed in T cells (Fig. 4D) and the cytotoxicity of the CAR T cells could be assessed (Fig. 4E). However, the 19BBLAT CAR had reduced tumor control capacity compared to the 1928z and 19BBz CARs in vivo (Fig. 4F). The inventors hypothesized that LAT might require membrane proximity in order to efficiently recruit signalling proteins such as PLCy. Therefore, the inventors generated a new CAR, named 19LAT28, where CD28 was placed on the C-terminal side of the LAT signalling domain. This allows the LAT signalling domain to be in a membrane proximal position while taking advantage of the CD28 co-stimulatory signalling. In addition, to further boost the activity of the 19LAT28, the inventors included the previously described mutation that increases the phosphorylation kinetic by ZAP70 / Syk of the PLCy docking site on the CAR, namely 19LAT28(G133D) (Fig. 4C). These constructs could be expressed in T cells (Fig. 4G). Their cytotoxicity could be measured and was similar to the conventional CAR construct 1928z (Fig. 4H). The 19LAT28 construct showed mild tumour control in vivo, whereas the 19LAT28(G133D) showed improved tumour control comparable to the 1928z (Fig. 41). Altogether, this study shows that our 19LAT can be adapted to T cells by introducing the CD28 co-stimulatory domain on the C-terminal side of the LAT signalling domain, and its functionality can be boosted by increasing the PLCy docking site phosphorylation kinetic by ZAP70 / Syk.

[0333] 19LAT can induce engineered immune cell functionality upon antigen recognition.

[0334] CAR therapy is not restricted to cancer treatment and recent findings have shown its potential to treat other diseases. In the future, CAR therapy might not be restricted to T or NK cells but extended to other immune cell types such as regulatory T cells or macrophages.

[0335] To see if our new CAR construct can also trigger functional activity in regulatory T cells the inventors performed a proliferation assay for PBMCs in co-culture with regulatory CAR T cells (Fig. 5 A). Regulatory T cells were purified from donor’s blood and transduced to express the CAR (Fig. 5B). CD28 signalling has been shown to be beneficial to regulatory T cells whereas 4-1BB signalling is known to negatively affect regulatory T cell phenotype stability. This is why the inventors only focus our experiment on constructs based on the CD28 signalling, namely 19LAT28 and 1928z CAR constructs. The proliferation assay shows that the 19LAT28 and 19LAT(G133D)28 CARs were able to activate regulatory T cells to inhibit proliferation of PBMCs in a comparable manner as the 1928z CAR (Fig. 5C).

[0336] To assess if the 19LAT was able to induce phagocytosis in macrophages, the inventors used the human leukemia monocytic THP-1 cell line to perform a phagocytosis assay (Fig. 5D). First the inventors transduced THP-1 cells with the 19LAT CAR construct and the expression was validated by flow cytometry (Fig. 5E). The 19LAT was able to induce phagocytosis specifically of CD 19 expressing target cells, in a more efficient manner than the non transduced parental THP-1 cells (Fig. 5F).

[0337] Altogether, these data shows that the new LAT -based CAR constructs of the invention are not only able to induce cytotoxicity in NK and T cells but can also be used in other cell types such as regulatory T cells or macrophages to functionally activate them.

[0338] Example 2

[0339] 19LAT CAR signalling domain optimization to increase NK cell cytotoxicity.

[0340] The inventors showed that the 19LAT was able to induce a high level of cytotoxicity in NK cells. However, critical tyrosine within the LAT signalling domain were untouched in the 19LAT design. The inventors hypothesize that the engineering of these sites could further enhance the functionality of the LAT -based CAR.

[0341] The inventors produced the 19LAT(G133D) and 19(CS)LAT(G133D) CAR constructs. The G133D mutant was earlier described in the context of T cells. The inventors are now exploring the effect of this mutation in NK cells (Fig. 6A).

[0342] In addition, the inventors generated 19LAT mutant in the site surrounding the tyrosine 228 initially known to recruit GRB2 and other adaptors. Since this tyrosine is known to recruit similar adaptor proteins than other tyrosine including tyrosine 173 and 193, the inventors thought that engineering of the tyrosine 228 site will not negatively affect LAT signalling. To promote the recruitment of PI3K through the generation of a YxxM motif withing the existing ITT motif, the inventors mutated the site initially GAPDYENL to SIDDYMNM. Mutation on the C-terminus side of the tyrosine (from GAPD to SIDD) was made to promote the phosphorylation kinetics of the tyrosine 228. Mutation of the N-terminus side of the tyrosine (ENL to MNM) was made to create a YxxM motif within the existing ITT motif, possibly recruiting PI3K. This strategy where an existing ITT motif (xYxN) is mutated in order to include a YxxM motif enables to add the function associated with the YxxM motif, such as recruiting PI3K, while keeping the initial function of the ITT motif, such as recruiting GRB2 / GADS. This “enhanced ITT mutant” combine properties of both ITT and YxxM motifs. These mutants are referred as 19LAT(ITTi) or 19(CS)LAT(ITTi). To see if the G133D and the enhanced ITT mutations synergize together the inventors combined both mutations on the same CAR construct called 19LAT(G133D, ITTi) or 19(CS)LAT(G133D, ITTi) (Fig. 6 A).

[0343] Since the mutation of site surrounding the tyrosine 228 could negatively affect the functionality of the LAT signalling domain, the inventors also generated 19LAT variants that contains the enhanced ITT motif on the C-terminus end of the LAT domain. These CAR constructs are named 19LAT(ITTc) and 19(CS)LAT(ITTc). This enhanced ITT motif addition was also combined with the G133D mutant, generating the 19LAT(G133D, ITTc) and the 19(CS)LAT(G133D, ITTc) (Fig. 6A).

[0344] Finally, to fully promote the NK cell functionality, the inventors introduced the 4-1BB signalling domain on the C-terminus side of the 19LAT variants. As an example, Fig. 6A depicts the 19(CS)LAT(G133D, ITTi) with the addition of the 41BB domain, generating the 19(CS)LAT(G133D, ITTi)41BB (Fig. 6A). CAR expressions were confirmed by flow cytometry (Fig. 6B).

[0345] In order to distinguish functional difference between all CAR constructs the Inventors plated a serial killing assay at low NK ratio (0.3 NK cells for 1 target) (Fig. 6C-F).

[0346] Results

[0347] Interestingly, the 19LAT is more cytotoxic compared to the previously reported CAR constructs made of the LAT signalling domain fused to the CD28 transmembrane domain (see control 6 A). Also, single mutants namely 19LAT(G133D), 19(CS)LAT(G133D), 19LAT(ITTi), 19(CS)LAT(ITTi), 19LAT(ITTc), and 19LAT(ITTc) display increased cytotoxicity compared to the 19LAT or 19(CS)LAT CAR constructs. This suggest that the mutations enhance the recruitment of signalling molecules resulting in increased cytotoxicity. Moreover, the combination G133D and enhanced ITT motif generation mutants, namely 19LAT(G133D, ITTi), 19(CS)LAT(G133D, ITTi), 19LAT(G133D, ITTc), and 19(CS)LAT(G133D, ITTc), shows a stronger cytotoxicity compared to both unmutated and single mutated LAT -based CARs. This synergistic effect was unexpected. Finally, in order to generate a CAR that takes advantage of the full cytotoxic capacity of NK cells, the inventors generated a CAR construct made to activate a broad variety of activating signalling pathways required for optimal NK cells activation. The addition of the 4-1BB signalling domain to the 19(CS)LAT(G133D, ITTi), namely 19(CS)LAT(G133D, ITTi)41BB resulted to an increased cytotoxicity (Fig. 6C-F).

[0348] The following table 2 summarizes the different CAR constructs described above.

[0349] Table 2

[0350] Altogether, these results show that the LAT -based CAR can be optimized for use in NK cells to take advantage of their full cytotoxic capacity. The engineering of specific tyrosine surrounding site enabling the increased of phosphorylation kinetics or the recruitment of additional signalling proteins is possible due to the highly modulable structure of LAT signalling domain. The modularity of YxxM motifs used here to modify an existing ITT motif can further be expanded to take advantage of other properties such as inserting the YxxM motif YMFM to also recruit the p85 and p50a subunits of PI3K. On the opposite way, the insertion of the YxxM motif YVKM would enable the recruitment of the p85 subunit or PI3K and SHP2 which can help to dampen down the CAR signalling strength.

Claims

CLAIMS1. A chimeric antigen receptor polypeptide comprising an extracellular domain that binds to a cell surface marker, a transmembrane domain, and an intracellular signaling domain, wherein the transmembrane domain derives from, comprises, or consists of a linker for activation of T cells transmembrane domain (LAT-tm), a fragment or a variant thereof, and wherein the intracellular signaling domain derives from, comprises, or consists of a recognition domain for a SH2 domain-containing protein selected from the recruited proteins comprising PIK3R1, PI3K, p65 alpha subunit of PI3K, GRB2, GRAP, GRAP2, PLCG1, PLCG2, SHB, SKAP2, CLNK, and FCGR1 A, a variant, an isoform or a combination of one or more thereof.

2. The chimeric antigen receptor according to claim 1, wherein the recognition domain comprises a sequence selected from the group comprising a linker for activation of T cells (LAT), a fragment or a variant thereof.

3. The chimeric antigen receptor according to claim 2, wherein the LAT is selected from SEQ ID No. 1, a fragment or a variant thereof.

4. The chimeric antigen receptor according to claim 3, wherein the LAT variant comprises one or more additions, substitutions (such as e.g. one or more mutations) and / or deletions within SEQ ID No. 1.

5. The chimeric antigen receptor according to claim 3, wherein the substitutions consist of one or more mutations.

6. The chimeric antigen receptor according to claim 5, wherein the one or more mutations within SEQ ID No. 1 is selected from the group comprising aspartic acid at position 132, aspartic acid at position 133, aspartic acid at position 171, aspartic acid atposition 172, aspartic acid at position 191, aspartic acid at position 192, aspartic acid at position 226, aspartic acid at position 227, glycine at position 132, glycine at position 133, glycine at position 171, glycine at position 172, glycine at position 191, glycine at position 192, glycine at position 226, glycine at position 227, glutamic acid at position 132, glutamic acid at position 133, glutamic acid at position 171, glutamic acid at position 172, glutamic acid at position 191, glutamic acid at position 192, glutamic acid at position 226, glutamic acid at position 227, methionine at position 137, methionine at position 176, methionine at position 196, methionine at position 231, methionine at position 135, methionine at position 174, methionine at position 194, methionine at position 229, asparagine at position 136, asparagine at position 175, asparagine at position 195, asparagine at position 230, phenylalanine at position 136, phenylalanine at position 175, phenylalanine at position 195, phenylalanine at position 230, serine at position 130, serine at position 169, serine at position 189, serine at position 224, isoleucine at position 131, isoleucine at position 170, isoleucine at position 190, isoleucine at position 225.

7. The chimeric antigen receptor according to any one of the preceding claims, wherein the LAT variant comprises,- from positions 130 to 138, a sequence selected from the group comprising, SEQ ID No. 4 (DGKDYIVLP), SEQ ID No.5 (DGKDYIPIN), SEQ ID No.6 (ADSGYIIPL), SEQ ID No.7 (TSFGYDKPH), SEQ ID No. 35 (HNPDYLVV), SEQ ID No. 36 (HNDGYLVV), and SEQ ID No. 37 (HNDDYLVV), and / or- from positions 224 to 231, a sequence selected from the group comprising SEQ ID No. 27 (SIDDYMNM), and SEQ ID No. 38 (SIDDYMFM).

8. The chimeric antigen receptor according to any one of the preceding claims, further comprising one or more intracellular co-stimulatory domain.

9. The chimeric antigen receptor according to claim 8, wherein the intracellular costimulatory domain is selected from the group comprising CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD1 la / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class I molecule, TNF receptor proteins, an Immunoglobulin protein, cytokine receptor, integrins, Signaling Lymphocytic Activation Molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD1 lb, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, GADS, SLP-76, PAG / Cbp, CD 19a, a ligand that specifically binds with CD83, a variant thereof or any combination thereof.

10. The chimeric antigen receptor according to any one of the preceding claims, further comprising a linker region interposed between the extracellular region and the transmembrane domain.

11. The chimeric antigen receptor according to claim 10, wherein the linker region is selected from the group comprising i) an immunoglobulin hinge region or a linker region derived from CD8, CD8a, or CD28 and ii) a GnS linker, wherein G is glycine, S is serine and n is an integer between 2-12.

12. The chimeric antigen receptor according to any one of the preceding claims, wherein the extracellular domain that binds to a cell surface marker is an antigen-binding polypeptide, a receptor, or a natural ligand for a target cell antigen or receptor.

13. The chimeric antigen receptor according to claim 12, wherein the antigen-binding polypeptide is an antibody or antibody fragment, selected from the group comprising murine antibodies, rabbit antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), camelid antibody variable domains and humanized versions, shark antibodyvariable domains and humanized versions, single domain antibody variable domains, nanobodies (VHHs), and camelized antibody variable domains.

14. The chimeric antigen receptor according to claim 12, wherein the antigen recognized by the antigen-binding polypeptide is selected from a cancer cell associated antigen, an infection-associated antigen and an auto-antigen.

15. The chimeric antigen receptor according to any one of claims 12 to 14, wherein the antigen recognized by the antigen-binding polypeptide is CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, fibroblast activation protein (FAP), CA125, MUC-1, PSMA, PSA, CD44 surface adhesion molecule, mesothelin, carcinoembryonic antigen (CEA), CEACAM5, CEACAM6, epidermal growth factor receptor (EGFR), EGFRvIII, vascular endothelial growth factor receptor-2 (VEGFR2), high molecular weight-melanoma associated antigen (HMW- MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CD1, CDIa, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, FIt-I, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia inducible factor (HIF-I), la, IL-2, IL-6, IL-8, insulin growth factor- 1 (IGF -I), KC4-antigen, KS-1 -antigen, KS1- 4, Le-Y, macrophage inhibition factor (MIF), MAGE, MUC1, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, tyrosinase, PRAME, EBNA, KLK3, HPV E7, LMP2, NY-ESO-1, PAP, reverse transcriptase, nucleophosmin, PRTN3 / ELANE, CT83 / KKLC1, MUC16, DNTT, antigen specific for PAM-4 antibody, placental growth factor, p53, prostatic acid phosphatase, RS5, SI 00, TAC, TAG-72, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin, 17-lA-antigen, NeuGcGM3, N- glycolyl GM3 ganglioside, Neu5Gc, GM3 -Ganglioside, GD3, GM2, carbohydrate antigens, ganglioside antigens, Lewis Y, Lewis B, MOG, MBP, aB-crystallin, PLP, GlialCAM, P- synuclein, HLA-A2, TNP, CD123 or Kappa chain of immunoglobulin.

16. The chimeric antigen receptor according to any one of the preceding claims, wherein the LAT-tm is selected from SEQ ID No. 9, a fragment or a variant thereof.

17. The chimeric antigen receptor according to any one of the preceding claims, wherein the chimeric antigen receptor is selected from the group comprising SEQ ID No. 21, SEQ ID No. 25, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No. 33, SEQ ID No. 35, SEQ ID No. 37, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 43, SEQ ID No. 45, SEQ ID No. 47, SEQ ID No. 49, SEQ ID No. 51, SEQ ID No. 53, SEQ ID No. 55, SEQ ID No. 57, SEQ ID No. 59, SEQ ID No. 61, and SEQ ID No. 63, a fragment or a variant thereof.

18. An isolated polynucleotide encoding a chimeric antigen receptor of any one of claims 1 to 17, a fragment or a variant thereof.

19. A vector comprising an isolated polynucleotide of claim 18.

20. The vector of claim 19, which is selected from the group comprising a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus associated vector, a lentiviral vector, an RNA (e.g. mRNA) targeted lipid nanoparticles (LNPs), a liposome or any combination thereof.

21. A host cell comprising an isolated nucleic acid of claim 18 , an expression vector of claim 19 or 20 or expressing a chimeric antigen receptor of any one of claims 1 to 17, a fragment or a variant thereof .

22. The host cell of claim 21, wherein the host cell is an autologous or an allogeneic mammalian cell.

23. The host cell of claim 21 or 22, wherein the host cell is selected from the group comprising a cytotoxic cell, an immune cell, a stem cell, a progenitor cell, a cell line and a cell derived from a stem cell or from a progenitor cell.

24. The host cell of claim 23, wherein the immune cell is selected from the group comprising T cell, tumor infiltrating lymphocyte (TIL), NK cell, regulatory T cell (Treg cell), macrophage, TCR-expressing cell, eosinophil, basophil, neutrophil, myeloid cells, B cell, plasma cell, regulatory B cell (Breg), innate lymphoid cell 1 (ICL1), ILC2, ICL3, dendritic cell, or NK-T cell.

25. The host cell of claim 24, wherein the immune cell is a T-cell line or NK cell line.

26. The host cell of claim 25, wherein the NK cell line is selected from the group comprising NK92, YTS and KHYG1 cell lines.

27. A pharmaceutical composition comprising i) an isolated polynucleotide of claim 18, ii) a vector of claim 19 or 20, or iii) a host cell of any one of claims 21 to 26, and pharmaceutically acceptable carrier, diluent and / or excipient.

28. The pharmaceutical composition of claim 27 for use in treating a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease.

29. A method of treating a cancer, an infectious disease, an inflammatory or inflammation- induced disease, a chronic disease or an autoimmune disease comprising administering i) an isolated polynucleotide of claim 18, ii) a vector of claim 19 or 20, iii) a host cell of any one of claims 21 to 26, or iv) a pharmaceutical composition of claim 27 or 28, to a subject in need thereof.

30. A method of treatment and / or prevention of a cancer, an infection or an auto-immune disease in a patient or subject comprising (i) removing and isolating host cells, preferably immune cells, more preferably native T cells or NK cells, from said patient or subject, (ii) genetically engineering said T cells or NK cells with one recombinant construct (e.g. vector, plasmid or polynucleotide) encoding a recombinant construct encoding a chimeric antigen receptor (CAR) of any one of claims 1 to 17, (iii) expanding ex vivo into a larger population of engineered immune cells, e.g. T cells or NK cells, and (iv) reintroducing said engineered immune cells, e.g. T cells, into the patient or subject.

31. A method of treatment and / or prevention of a cancer, an infectious disease, an inflammatory or inflammation-induced disease, a chronic disease or an autoimmune disease in a patient or subject comprising (i) providing genetically engineered immune cells such as e.g. T cells or NK cells with one recombinant construct (e.g. vector, plasmid or polynucleotide of the invention) encoding a chimeric antigen receptor (CAR) of any one of claims 1 to 17, (ii) expanding ex vivo into a larger population of engineered immune cells, e.g. T cells or NK cells, and (iii) reintroducing said engineered immune cells, e.g. T cells, into the patient or subject.

32. The method of claim 31, wherein the NK cell is a cell line, preferably a NK-92 cell.

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