LAT-based NK CAR and its use

Abstract

The present invention relates to chimeric antigen receptor constructs and their use for treatment and therapy. Additionally, methods for treating and / or preventing diseases, such as cancer, infectious diseases, inflammatory or pro-inflammatory diseases, chronic diseases, or autoimmune diseases, are disclosed.

Description

[Technical Field]

[0001] The present invention relates to chimeric antigen receptor constructs and their use for treatment and therapy. Additionally, methods for treating and / or preventing diseases, such as cancer, infectious diseases, inflammatory or pro-inflammatory diseases, chronic diseases, or autoimmune diseases, are disclosed. [Background technology]

[0002] Chimeric antigen receptor (CAR) T cells have been shown to be an attractive cell therapy, a promising approach due to their potential to cure malignancies that are refractory to current treatments such as chemotherapy, radiation therapy, or surgery.

[0003] CAR is a synthetic receptor made from scFv or another ligand-binding protein linked to a costimulatory domain and an activation domain via a transmembrane domain. The scFv allows target cell recognition in an MHC-independent manner. The activation domain provides a signal to effector cells to initiate target cell elimination. The addition of a costimulatory domain between the transmembrane domain and the activation domain has been shown to increase the survival of effector cells and their anti-tumor efficacy in vivo.

[0004] CAR-engineered immune cells acquire supraphysiological properties that enable them to eliminate any potential target cells that express the targeted membrane protein. CAR T-cell therapy has demonstrated remarkable clinical outcomes for specific subsets of B-cell malignancies.

[0005] CAR T cell therapy requires complex efforts to isolate and engineer a patient's own T cells due to HLA-matching restrictions. Furthermore, therapeutic efficacy depends on the quality of the patient's T cells, which is not always optimal. Furthermore, CAR T cells often exhibit limited tumor penetration and trafficking, making their application to solid tumors challenging. Finally, T cells can induce cytokine release syndrome (CRS), a potentially life-threatening toxicity characterized by elevated levels of circulating cytokines such as IL-6 and IFN-γ.

[0006] Recent advances in natural killer (NK) cell-based immunotherapy have highlighted their high potential as novel therapeutic agents. Unlike T cells, NK cells can be established as off-the-shelf (easily available) therapies. NK cells also often exhibit superior tumor infiltration compared with T cells, suggesting their potential for controlling solid tumor growth. Clinical trials have shown that NK cells exhibit reduced adverse effects, such as CRS or graft-versus-host disease (GVHD).

[0007] 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 umbilical cord blood, or NK cell lines such as NK92. However, current clinical trials have been limited to PB-NK or NK92 (NCT00995137, NCT01974479, NCT02839954, NCT02892695, NCT02742727, and NCT02944162).

[0008] To date, several studies have attempted to develop CAR designs for NK cells, but have limited their activation signaling domains to ITAM-containing domains such as CD3z and, more rarely, FceR1g or DAP12 intracellular domains. Although CAR NK cells are promising therapeutics in the clinic, the CAR designs currently used in NK cells were first developed in T cells and may not be optimal for NK cells. Summary of the Invention [Problem to be solved by the invention]

[0009] Therefore, there remains a need for optimized CAR T and NK cells that exhibit increased cytotoxicity against targets and reduce attrition. [Means for solving the problem]

[0010] The present invention provides a chimeric antigen receptor polypeptide, comprising: an extracellular domain that binds to a cell surface marker; a transmembrane domain; Intracellular signaling domains and wherein the intracellular signaling domain comprises a recognition domain for an SH2 domain-containing protein selected from recruited proteins including PIK3R1, PI3K, the p65 α subunit of PI3K, the p85 subunit of PI3K, the p50 subunit of PI3K, GRB2, GRAP, GRAP2, PLCG1, PLCG2, SHB, SKAP2, CLNK, and FCGR1A, one or more variants, isoforms, or combinations thereof. Chimeric antigen receptor polypeptides are provided.

[0011] Additionally provided is an isolated polynucleotide encoding the chimeric antigen receptor (CAR) of the present invention.

[0012] Also provided are vectors comprising the isolated polynucleotides of the invention.

[0013] Also provided are immune cells that i) comprise the isolated polynucleotide and / or vector of the invention, or ii) express the chimeric antigen receptor of the invention.

[0014] There is also provided an immune cell according to the invention for use in the treatment of cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease or an autoimmune disease.

[0015] 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 the invention, and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0016] Further provided is a method for treating cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease, comprising the step of administering to a subject in need thereof 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. [Brief explanation of the drawings]

[0017] [Figure 1A] Expression and activity of LAT-based CARs in NK cells in vitro. A) Schematic diagram of the 19LAT and two common (1928z and 19BBz) CAR constructs used. The gray shaded area indicates the transmembrane domain. [Figure 1B] Expression and activity of LAT-based CARs in NK cells in vitro. B) Expression of different CAR constructs in NK92 measured by flow cytometry. The HA tag was used to detect the CAR constructs at the membrane, and GFP served as a marker for transduction. [Figure 1C]Expression and activity of LAT-based CARs in NK cells in vitro. C) In vitro killing capacity of NK92 stably expressing different CAR constructs after 4 hours of co-culture with Nalm6 at a 3:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the first challenge (target cells are added only once). [Figure 1D] Expression and activity of LAT-based CARs in NK cells in vitro. D) Reload killing assay layout. Fresh target cells were added to the co-culture every 24 hours, and killing of the last added population was measured after 4 hours of co-culture. [Figure 1E] Expression and activity of LAT-based CARs in NK cells in vitro. E) In vitro killing capacity of NK92 stably expressing different CAR constructs after 4 hours of co-culture with Nalm6 at a 3:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the third and fourth loadings. [Figure 2A] 19LAT CAR can induce solid tumor growth control through engineered NK92. A) In vitro killing capacity of NK92 stably expressing different CAR constructs after 4 hours of co-culture with A1847-CD19 at an effector:target (E:T) ratio of 10:1. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the fourth loading. [Figure 2B] The 19LAT CAR can induce solid tumor growth control through engineered NK92. B) Experimental layout for in vivo killing assay. Mice were intraperitoneally injected with 5x104 A1847 ovarian cancer cell lines stably expressing luciferase and CD19. Five and seven days after tumor injection, mice were treated with 5 million transduced NK92 cells expressing different CAR constructs. Tumor burden was measured by IVIS at the indicated time points. [Figure 2C]The 19LAT CAR can induce solid tumor growth control through engineered NK92. C) Comparison of the in vivo efficacy of LAT on NK92 against the A1847 ovarian cancer cell line stably expressing luciferase and CD19. Tumor burden in mice was measured by bioluminescence at the indicated time points. Arrows indicate NK92 injection. Data show the mean total flux (p / s) + SE (n = 4-6). [Figure 3A] Tuning and optimization of the 19LAT CAR construct. A) Schematic of 19LAT(Y134F) compared to the 19LAT CAR construct. The grey shaded area indicates the transmembrane domain. [Figure 3B] Tuning and optimization of the 19LAT CAR construct. B) In vitro killing capacity of NK92 stably expressing 19LAT(Y134F) compared to the 19LAT construct after 4 hours of co-culture with Nalm6 at an effector:target (E:T) ratio of 1:2. Cytotoxicity is measured by Annexin V staining within the target population. [Figure 3C] Tuning and optimization of the 19LAT CAR construct. C) Schematic of kinetic 19LAT mutants. The 19LAT(G133D), 19LAT(P132D), and 19LAT(DD) constructs are depicted relative to the 19LAT CAR construct. The grey shaded area indicates the transmembrane domain. [Figure 3D] Tuning and optimization of the 19LAT CAR construct. D) In ​​vitro killing capacity of NK92 stably expressing the kinetic 19LAT variant compared to the 19LAT construct after 4 hours of co-culture with Nalm6 at a 3:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the first and fourth loadings. [Figure 3E] Tuning and optimization of the 19LAT CAR construct. E) Schematic of PLCg site mutants. 19LAT(VEGFR2) and 19LAT(hybrid) constructs are depicted relative to the 19LAT CAR. The grey shaded area indicates the transmembrane domain. [Figure 3F]Tuning and optimization of the 19LAT CAR construct. F) In vitro killing capacity of NK92 stably expressing the PLCg site 19LAT mutant compared to the 19LAT construct after 4 hours of co-culture with Nalm6 at an effector:target (E:T) ratio of 1:2. Cytotoxicity is measured by Annexin V staining within the target population. [Figure 3G] Tuning and optimization of the 19LAT CAR construct. G) Schematic diagram of the 19LAT stabilization mutants. The 19LAT(RR) and 19LAT CAR constructs are shown. The grey shaded area indicates the transmembrane domain. [Figure 3H] Tuning and optimization of the 19LAT CAR construct. H) In vitro killing capacity of NK92 stably expressing the 19LAT stabilized mutant compared to the 19LAT construct after 4 hours of co-culture with Nalm6 at a 3:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. [Figure 3I] Tuning and optimization of the 19LAT CAR construct. I) Schematic of the 19LAT monomer hinge mutant. The 19(CS)LAT and 19LAT CAR constructs are shown. The grey shaded area indicates the transmembrane domain. [Figure 3J] Tuning and optimization of the 19LAT CAR construct. J) In vitro killing capacity of NK92 stably expressing 19(CS)LAT compared to the 19LAT construct after 4 hours of co-culture with Nalm6 and A1847-CD19 at the indicated effector:target (E:T) ratios. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the fourth loading. [Figure 3K] Tuning and optimization of the 19LAT CAR construct. K) Experimental layout for the long-term killing assay. Effector and target cells are co-cultured for 7 days. After this co-culture, fresh target cells are added and functional killing is measured by flow cytometry with PI and Annexin V staining. [Figure 3L]Tuning and optimization of the 19LAT CAR construct. L) In vitro killing capacity of NK92 stably expressing 19(CS)LAT compared to 1928z CAR and 19BBz CAR constructs in a long-term killing assay at an effector:target (E:T) ratio of 1:10. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows functional killing. [Figure 3M] Tuning and optimization of the 19LAT CAR construct. M) Comparison of the in vivo efficacy of 19(CS)LAT in NK92 versus A1847 ovarian cancer cell lines stably expressing luciferase and CD19. Tumor burden in mice was measured by bioluminescence at the indicated time points. Arrows indicate injection of NK92. Data represent total flux (p / s) mean + SE (n = 5-6). [Figure 4A] 19LAT can be optimized for use in T cells. A) CAR expression levels in T cells measured by flow cytometry. The HA tag in the CAR construct was labeled by antibody staining. [Figure 4B] 19LAT can be optimized for use in T cells. B) In vitro killing capacity of T cells stably expressing 19LAT compared to 1928z CAR and 19BBz CAR constructs after 16 hours of co-culture with Nalm6 at a 2:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the first and third loadings. [Figure 4C] 19LAT can be optimized for use in T cells. C) Schematic of 19LAT variants optimized for T cells. The 1928LAT CAR, 19BBLAT CAR, 19LAT28 CAR, and 19LAT(G133D)28 CAR constructs are shown. The gray shaded area indicates the transmembrane domain. [Figure 4D]19LAT can be optimized for use in T cells. D) Expression levels of 1928LAT, 19BBLAT compared to 1928z CAR and 19BBz CAR in T cells are measured by flow cytometry. The HA tag in the CAR construct is labeled by antibody staining. [Figure 4E] 19LAT can be optimized for use in T cells. E) In vitro killing capacity of T cells stably expressing 1928LAT and 19BBLAT compared to 1928z CAR and 19BBz CAR constructs after 16 hours of co-culture with Nalm6 at a 2:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the first and third loadings. [Figure 4F] 19LAT can be optimized for use in T cells. F) Comparison of in vivo efficacy of 19BBLAT compared to 19BBz CAR and 1928z CAR in T cells against a Nalm6 cell line stably expressing luciferase. Tumor burden in mice was measured by bioluminescence at the indicated time points. Data represent total flux (p / s) mean + SD (n = 5-6). [Figure 4G] 19LAT can be optimized for use in T cells. G) CAR expression levels in T cells are measured by flow cytometry. The HA tag in the CAR construct is labeled by antibody staining. [Figure 4H] 19LAT can be optimized for use in T cells. H) In vitro killing capacity of T cells stably expressing 19LAT28(G133D) compared to 19LAT28 CAR, 1928z CAR, and 19BBz CAR constructs after 16 hours of co-culture with Nalm6 at a 2:1 effector:target (E:T) ratio. Cytotoxicity is measured by Annexin V staining within the target population. The figure shows the first and third loadings. [Figure 4I]19LAT can be optimized for use in T cells. I) Comparison of the in vivo efficacy of 19LAT28(G133D) and 19LAT28 compared to the 1928z CAR in T cells against a Nalm6 cell line stably expressing luciferase. Tumor burden in mice was measured by bioluminescence at the indicated time points. Arrows indicate T cell injection. Data show the total flux (p / s) mean + SE (n = 5-6). [Figure 5A] 19LAT can induce engineered immune cell functionality upon antigen recognition. A) Proliferation assay layout of PBMCs in co-culture with regulatory T cells. CAR Tregs are first activated on a CD19-coated surface for 16 hours. PBMCs are then added to the culture. PBMC proliferation is assessed after 3 days by CFSE measurement by flow cytometry. [Figure 5B] 19LAT can induce engineered immune cell functionality upon antigen recognition. B) The 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. [Figure 5C] 19LAT can induce engineered immune cell functionality upon antigen recognition. C) Division assay to compare the regulatory function of CAR Tregs. Division of activated conventional T cells (Tconv) is measured by CFSE dilution. The mean fluorescence intensity (MFI) of CFSE is measured after 3 days of co-culture. [Figure 5D] 19LAT can induce engineered immune cell functionality upon antigen recognition. D) Phagocytosis assay layout. Phagocytosis is measured by GFP-positive Nalm6 target cells plated with CAR THP1 stained with cell trace violet. After 24 hours of incubation, the phagocytosis rate is extracted from the flow cytometry data. [Figure 5E]19LAT can induce engineered immune cell functionality upon antigen recognition. E) Expression levels of CAR expressed by THP-1 cells measured by flow cytometry. The HA tag in the CAR construct was labeled by antibody staining. [Figure 5F] 19LAT can induce engineered immune cell functionality upon antigen recognition. F) The rate of phagocytosis in undifferentiated THP1 cells was measured by flow cytometry as the percentage of GFP cells within the THP1 population after 24 hours of co-culture at the indicated effector-to-target ratios. [Figure 6A] Optimization of LAT-based CARs for increased induction of functional cytotoxicity in NK cells. A) Schematic of 19LAT variants optimized for NK cells. Controls A and B, as well as 19LAT variants such as 19LAT(ITTc), 19LAT(ITTi), and 19LAT(G133D,ITTi)4-1BB CAR constructs, are shown. The gray shaded area indicates the transmembrane domain. [Figure 6B] Optimization of LAT-based CARs for increased induction of functional cytotoxicity in NK cells. B) Expression levels of different CAR constructs in NK92 measured by flow cytometry using HA staining. [Figure 6C] Optimization of LAT-based CARs for increased induction of functional cytotoxicity in NK cells. C) In vitro reload killing assay of NK cells stably expressing variants of the 19LAT CAR construct at an effector:target (E:T) ratio of 0.3:1. The plot shows the cytotoxicity of NK92 cells against A1847-CD19 after three rounds of loading. [Figure 6D] Optimization of LAT-based CARs for increased induction of functional cytotoxicity in NK cells. D) In ​​vitro reload killing assay of NK cells stably expressing variants of the 19LAT CAR constructs at an effector:target (E:T) ratio of 0.3:1. The plot shows the cytotoxicity of NK92 cells against A1847-CD19 after four rounds of loading. [Figure 6E]Optimization of LAT-based CARs for increased induction of functional cytotoxicity in NK cells. E) In vitro re-challenge killing assay of NK cells stably expressing variants of the 19LAT CAR construct at an effector:target (E:T) ratio of 0.3:1. The plot shows the cytotoxicity of NK92 cells against Nalm6 after three rounds of challenge. [Figure 6F] Optimization of LAT-based CARs for increased induction of functional cytotoxicity in NK cells. F) In vitro re-challenge killing assay of NK cells stably expressing variants of the 19LAT CAR construct at an effector:target (E:T) ratio of 0.3:1. Plots show F) cytotoxicity of NK92 cells against Nalm6 after 4 rounds of challenge. DETAILED DESCRIPTION OF THE INVENTION

[0018] 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 should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Additionally, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0019] In case of conflict, the present specification, including definitions, will control. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. As used herein, the following definitions are provided to facilitate understanding of the present invention.

[0020] The term "comprise / comprising" is generally used in the sense of include / including, i.e., allowing for the presence of one or more features or components. This term also encompasses the more specific term "consist / consisting."

[0021] As used in this specification and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0022] The term "amino acid" includes all naturally occurring and modified amino acids.

[0023] As used herein, "at least one (one type)" means "one (one type) or more," "two (two types) or more," "three (three types) or more," etc.

[0024] The term "about," particularly with respect to a given quantity, is meant to encompass a deviation of ±10%.

[0025] "Homology" refers to the percent identity between two polynucleotides or two polypeptide moieties. Two nucleic acid sequences or two polypeptide sequences are "substantially homologous" to one another if they 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 molecule. As used herein, "substantially homologous" also refers to sequences that exhibit complete identity to a specific sequence. Alternatively, homology can be determined by readily available computer programs or by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with a single-strand-specific nuclease and sizing of the digested fragments. Substantially homologous DNA sequences can be identified, for example, in a Southern hybridization experiment under stringent conditions defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.

[0026] Generally, "identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotide or polypeptide sequences, respectively. Percent identity can be determined by direct comparison of the sequence information between the 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 embodiments, a nucleotide or amino acid sequence of the invention, or a portion thereof, is at least 80%, i.e., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the corresponding nucleotide or amino acid sequence (SEQ ID NO: identifier).

[0027] As used herein, the terms "peptide," "protein," "polypeptide," "polypeptide chain," "polypeptidic," and "peptidic" are used interchangeably to refer to a series of amino acid residues connected to one another by peptide bonds between the α-amino and carboxy groups of adjacent residues.

[0028] The present invention provides a chimeric antigen receptor (CAR) polypeptide comprising: an extracellular domain that binds to a cell surface marker; a transmembrane domain; Intracellular signaling domains and wherein the intracellular signaling domain is derived from, comprises, or consists of a recognition domain that recognizes an SH2 domain-containing protein selected from recruitment proteins including PIK3R1, PI3K, the p65 alpha subunit of PI3K, the p85 subunit of PI3K, the p50 subunit of PI3K, GRB2, GRAP, GRAP2, PLCG1, PLCG2, SHB, SKAP2, CLNK, and FCGR1A, and one or more variants, isoforms, or combinations thereof. Chimeric antigen receptor (CAR) polypeptides are disclosed.

[0029] In a preferred embodiment, the transmembrane domain is derived from, comprises or consists of a linker for activation of T cells transmembrane domain (LAT-tm), a fragment or variant thereof.

[0030] Recruitment of PLCG1, GRB2, GRAP2, and PIK3R1 onto LAT enables immune cell activation (including T cells and NK cells). Conventional CARs primarily rely on ZAP70 recruitment via their CD3z chain, which initiates signal amplification to initiate a signaling cascade in T cells.

[0031] Surprisingly, the inventors have shown that point mutations, such as those affecting tyrosines in the LAT sequence, alter the potency of LAT-based CARs by increasing the ability of LAT to recruit PLCG1, GRB2, GRAP2, PI3K, the p65 α subunit of PI3K, the p85 subunit of PI3K, the p50 subunit of PI3K, and PIK3R1, and / or by increasing the stability of this CAR.

[0032] In one embodiment, 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 an antigen or receptor on the target cell.

[0033] 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 (VHH), and camelized antibody variable domains.

[0034] Preferably, the single-chain fragment variants (scFv) are also derived from antibodies or ligands or receptors. In some instances, the extracellular domain comprises a hinge portion (also called a linker or spacer). A variety of hinges or linkers may be used in accordance with the present invention, as described below.

[0035] In one embodiment, the antigen recognized by the antigen-binding polypeptide is generally selected from the group comprising cancer cell-associated antigens, infection-associated antigens and self-antigens.

[0036] Preferably, 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 cell 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-MEA), or a combination thereof. antigen, HMW-MAA), MAGE-A1, IL-13R-a2, GD2, carbonic anhydrase EX, alpha-fetoprotein, A3, A33 antibody-specific antigen, Ba733, BrE3-antigen, CD1, CD1a, CD3, CD5, CD15, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (colon-specific antigen-p, CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia-inducible factor (HIF-I), Ia, 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, HPVE7, 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, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A-antigen, NeuGcGM3, N-glycolyl GM3 ganglioside, Neu5Gc, GM3-ganglioside, GD3, GM2, carbohydrate antigen, ganglioside antigen, Lewis Y, Lewis B, MOG, MBP, aB-crystallin, PLP, GlialCAM, β-synuclein, HLA-A2, TNP, CD123 or the kappa chain of immunoglobulin, and combinations of one or more thereof.

[0037] When a linker region is inserted between the extracellular region and the transmembrane domain, the linker region is selected from the group consisting of: i) an immunoglobulin hinge region or a linker region derived from CD8, CD8α, or CD28, or any variant thereof; and ii) a (GnS)m linker, wherein G is glycine, S is serine, n is an integer of 2 to 12, preferably 3 to 10, more preferably 3 to 5, even more preferably n=3, and m is an integer of 2 to 12, preferably 3 to 10, more preferably 3 to 5, even more preferably m=3.

[0038] In one embodiment, the transmembrane domain and hinge are usually fused to the extracellular domain of the CAR. The transmembrane domain and hinge may also be fused to the intracellular signaling domain of the CAR. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or different surface membrane protein, minimizing interaction with other members of the receptor complex. The transmembrane domain can be derived from either natural or synthetic sources. If the source is natural, this domain can be derived from any membrane-bound or transmembrane protein.Transmembrane regions that are particularly useful in the present invention include CD28, CD28T, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 death-1, PD-1), inducible T cell costimulatory molecule (ICOS), CD11a / CD18, CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class 1 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA 1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11 a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The ligand may be derived from (or may include or correspond to) a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83, or any combination thereof.

[0039] Preferably, the transmembrane domain is derived from, comprises, or consists of a linker transmembrane domain of T cell activation (LAT-tm), a fragment or variant thereof, hi one embodiment, the LAT transmembrane domain increases the activity of the CAR.

[0040] In one embodiment, the LAT transmembrane domain (eg, a human LAT transmembrane domain) comprises the amino acid sequence of SEQ ID NO: 9 (ILVPCVLGLLLLPILAMLMALCV), a fragment or variant thereof.

[0041] Optionally, a short linker may form the link between any or some of the extracellular domain, transmembrane domain, and intracellular domain of the CAR.

[0042] Those skilled in the art will understand that any hinge can be used in the present invention. In a preferred embodiment, the hinge in the CAR of the present invention is CD8 (e.g., CD8a). In one embodiment, the CD8 transmembrane domain (e.g., CD8a transmembrane domain) and hinge (e.g., CD8a hinge or spacer domain) comprise the transmembrane portion and hinge of the amino acid sequence of SEQ ID NO: 11, a fragment or variant thereof.

[0043] Examples of variants of the CD8a hinge include one or more mutations at positions 27 and / or 44, such as those in SEQ ID NO:13.

[0044] In one embodiment, the LAT transmembrane domain (eg, a human LAT transmembrane domain) comprises the amino acid sequence of SEQ ID NO: 9, a fragment or variant thereof.

[0045] The intracellular signaling domain of the CAR of the present invention comprises one or more recognition domains for SH2 domain-containing proteins.

[0046] As used herein, "recognition domain" refers to a domain containing one or more tyrosines that are phosphorylated upon activation to allow recruitment of one or more SH2-containing proteins. This recruitment can be direct or indirect, i.e., via a first SH-2 domain-containing protein (e.g., GRB2 or GRAP2).

[0047] In one embodiment, the one or more SH2 domain-containing proteins directly recruited by the recognition domain are phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1), phosphoinositide-3-kinase (PI3K), p65 α 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 (SRC), or a combination thereof. 2 (SKAP2), Cytokine Dependent Hematopoietic Cell Linker (CLNK), Fc Gamma Receptor Ia (FCGR1A), and variants, isoforms, or combinations of one or more thereof.

[0048] In one embodiment, the one or more SH2 domain-containing proteins indirectly recruited by the recognition domain are selected from the group consisting of Casitas B-lineage lymphoma, SOS Ras / Rac Guanine Nucleotide Exchange Factor 1 (Sos1), 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), and / or IL-1. The recruitment proteins are selected from, but are not limited to, interleukin-1 (ITK), GRB2-associated-binding protein 2 (GAB-2), and variants, isoforms, or combinations of one or more thereof.

[0049] For example, when activated and recruited, GRAP2 recruits SLP76 and / or ITK.

[0050] In one embodiment, the recognition domain is selected from the group comprising linker for activation of T cells (LAT), a fragment or variant thereof.

[0051] Preferably, the LAT of the present invention is selected from SEQ ID NO: 1, a fragment or a variant thereof.

[0052] As used herein, a "fragment" of an LAT sequence of the invention, preferably a human LAT sequence, refers to a sequence containing a shorter nucleotide length than the respective nucleic acid sequence or a shorter nucleotide length than the respective polypeptide sequence. Preferably, the sequence contains less than 90%, preferably less than 60%, and especially less than 30% of the nucleotides in length of the respective polypeptide or nucleic acid sequence (e.g., SEQ ID NO: 1).

[0053] The term "variant," when referring to an LAT sequence of the invention (whether a nucleotide sequence or a peptide sequence), refers to one or more biologically active derivatives of LAT, preferably a human LAT sequence of the invention. Generally, the term "variant" refers to a molecule having a native sequence that has one or more additions, substitutions (e.g., one or more mutations), and / or deletions compared to the native molecule, and that is "substantially homologous" to the reference molecule (Gorby et al., Sci. Signal. 13, eabc0653, 2020; Saxton et al., Science 371, eabc8433, 2021). Generally, the sequence of such a variant will have a high degree of sequence homology or sequence identity to a reference sequence, for example, greater than 25%, generally greater than 50% to 70%, even more particularly 80%, or 85% or more, for example 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% sequence homology or sequence identity when the two sequences are aligned.

[0054] It is understood that the term variant also encompasses human isoforms of the LAT sequence.

[0055] In one embodiment, a variant of LAT comprises one or more additions, substitutions (eg, one or more mutations), and / or deletions within SEQ ID NO:1.

[0056] In one embodiment, the one or more mutations are independently selected from the group consisting of 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, and glutamic acid at position 227. glutamic acid 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, and isoleucine at position 225.

[0057] In one embodiment, the one or more mutations are located within positions 130 to 138 (SEQ ID NO: 3: HNPGYLVVL) and / or positions 224 to 231 (SEQ ID NO: 26: GAPDYENL).

[0058] In one aspect, the LAT variant comprises: i) 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), and SEQ ID NO: 7 (TSFGYDKPH), or a combination thereof.

[0059] In one embodiment, the LAT variant comprises ii) a sequence selected from the group comprising any ITT-like motif from positions 224 to 231, including SEQ ID NO: 27 (SIDDYMNM), SEQ ID NO: 38 (SIDDYMFM) or preferably the consensus sequence YxxM (where x is any amino acid).

[0060] The LAT variant is one or more mutations in SEQ ID NO: 1 and the above insertions at positions 130 to 138, one or more mutations and insertions from positions 224 to 231 in SEQ ID NO: 1, or One or more mutations in SEQ ID NO: 1, the insertions at positions 130 to 138 and the insertions at positions 224 to 231 described above It is also understood that the present invention may include:

[0061] In one embodiment, the CAR of the invention further comprises one or more intracellular costimulatory domains, which can be located between the transmembrane domain and LAT, i.e., at the N-terminus of LAT (TM domain > costimulatory domain > LAT), or after LAT, i.e., at the C-terminus of LAT (TM domain > LAT > costimulatory domain), or at both the N- and C-termini of LAT.

[0062] Preferably, the intracellular costimulatory domain is selected from the group consisting of CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), lymphocyte function-associated antigen-1, and the like. antigen-1, LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1) , NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49 a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LF A-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The ligands are selected from the non-limiting group including AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​GADS, SLP-76, PAG / Cbp, CD19a, ligands that specifically bind to CD83, variants thereof, or any combination thereof.

[0063] In one embodiment, the intracellular costimulatory domain is as set forth in SEQ ID NO: 15, a fragment or variant thereof.

[0064] In one embodiment, the CAR of the present invention can further comprise a signal sequence (e.g., a signal peptide such as SEQ ID NO: 18, a fragment or variant thereof) preferably attached to its N-terminus. This sequence is then cleaved when the CAR is integrated into the cell membrane.

[0065] In some instances, the extracellular domain comprises a CD8a hinge, which is known to dimerize via trans-disulfide bridge formation of two cysteines in the CD8a hinge. Thus, it can be appreciated that, if necessary, the invention provides for mutating these two cysteines to, for example, serine to abolish dimerization of the construct (FIG. 3I).

[0066] Thus, the chimeric antigen receptor polypeptides of the present invention may exist in a monomeric or dimeric state.

[0067] In one embodiment 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 variant thereof.

[0068] The present invention further contemplates isolated polynucleotides encoding the chimeric antigen receptors (CARs) of the present invention, as well as fragments or variants of said polynucleotides comprising the domains or regions disclosed herein.

[0069] 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 variant thereof.

[0070] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to any type of deoxyribonucleotide (e.g., DNA, cDNA, etc.) or ribonucleotide (e.g., RNA, mRNA, etc.) polymer or combination of deoxyribonucleotides and ribonucleotides (e.g., DNA / RNA) polymer, in linear or circular structure and in either single- or double-stranded form. These terms should not be construed as limiting with respect to the length of the polymer and can encompass known analogues of natural nucleotides as well as nucleotides modified in the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones). In general, analogues of a particular nucleotide have the same base-pairing specificity, i.e., an analogue of A will base pair with T.

[0071] Vectors comprising the isolated polynucleotides of the present invention are further contemplated. As used herein, the term "vector" refers to any vector known in the art that may be suitable for the present invention. In some embodiments, a vector refers to a viral vector, or a nucleic acid (DNA or RNA) molecule, such as a plasmid or other vehicle, that contains one or more heterologous nucleic acid sequences of the present invention, preferably designed for transfer and / or amplification between different host cells. The terms "expression vector," "gene delivery vector," and "gene therapy vector" refer to any vector effective for incorporating and expressing one or more nucleic acids of the present invention in a cell, preferably under the control of a promoter. A cloning vector or expression vector may contain additional elements in addition to a promoter, such as regulatory elements and / or post-transcriptional regulatory elements.

[0072] In one embodiment, the vector is selected from the non-limiting group including a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, an RNA (e.g., mRNA)-targeted lipid nanoparticle (LNP), a liposome, or any combination thereof.

[0073] 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.

[0074] In one embodiment, the cells are mammalian cells, whether autologous or allogeneic. Preferably, the cells are selected from the group comprising cytotoxic cells, immune cells, stem cells, progenitor cells, cell lines, and cells derived from stem or progenitor cells.

[0075] When the cell is an immune cell, the immune cell is selected from the non-limiting group including T cells, tumor infiltrating lymphocytes (TILs), NK cells, regulatory T cells (Treg cells), macrophages, TCR-expressing cells, eosinophils, basophils, neutrophils, myeloid cells, B cells, plasma cells, regulatory B cells (Bregs), innate lymphoid cell 1 (ICL1), ILC2, ICL3, dendritic cells, or NK-T cells. There are a variety of available techniques known in the art for the isolation and enrichment of T cells, for example, PBMCs obtained from peripheral blood samples.

[0076] When the immune cells are T cells or NK cells, they can be either autologous or allogeneic T cells or NK cells. In some embodiments, the immune cells are T cell lines or NK cell lines. Any cell line derived from any primary cell can be used in the present invention. Non-limiting examples of NK cell lines include NK92, YTS, or KHYG1 cell lines.

[0077] When the cells are derived from stem cells, they are either naturally occurring stem cells or artificial stem cells (eg, iPSC-derived cells).

[0078] As shown in the Examples, the LAT-based CAR constructs of the present invention are not only capable of inducing cytotoxicity in NK cells 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 include regulatory T cells and macrophages.

[0079] In one embodiment, the LAT-based CAR constructs of the present invention activate phagocytosis of target cells (macrophages) or induce an immunosuppressive environment around their targets (Tregs).

[0080] The results of Example 2 demonstrate that LAT-based CARs can be optimized for use in NK cells to utilize their full cytotoxic potential. The highly modular (regulatable) structure of the LAT signaling domain allows for the manipulation of specific tyrosine-enriched sites to increase phosphorylation kinetics or recruit additional signaling proteins. The modularity of the YxxM motif used in the present invention to modify existing ITT motifs can be further expanded to utilize other properties, such as inserting the YxxM motif YMFM to also recruit the p85 and p50a subunits of PI3K. Conversely, inserting the YxxM motif YVKM allows for the recruitment of the p85 subunit or PI3K and SHP2, which may help attenuate CAR signaling strength.

[0081] The present invention further contemplates a CAR, a host cell, e.g., an immune cell, or a pharmaceutical composition according to the invention for use in the treatment of cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease.

[0082] When the disease to be treated is an inflammatory or pro-inflammatory disease, the disease is selected from the non-limiting group including fibrosis (pulmonary fibrosis or cardiac fibrosis), chronic obstructive pulmonary disease, cardiovascular disease, diabetes, asthma, fatty liver disease, gout, and scleroderma.

[0083] When the disease being treated is a chronic disease, the chronic disease is selected from the non-limiting group including fibrosis, Alzheimer's disease, lupus erythematosus, and chronic kidney disease.

[0084] When the disease being treated is an infectious disease, the infectious disease is selected from the non-limiting group including HIV, hepatitis C, and human cytomegalovirus.

[0085] When the disease to be treated is cancer, the cancer is selected from solid tumors or liquid tumors.

[0086] If the cancer to be treated is solid, it is selected from the non-limiting group including lung cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, head and neck cancer, glioblastoma, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, kidney cancer, prostate cancer, stomach cancer, bronchial cancer, pancreatic cancer, bladder cancer, liver cancer and brain cancer and skin cancer, particularly melanoma, or a combination of one or more thereof.

[0087] When the cancer to be treated is liquid-based, it refers to cancer cells present in body fluids, such as blood, lymph, and bone marrow.Liquid-based cancers are selected from the non-limiting group including leukemia, myeloma, myelodysplastic syndrome (MDS), and liquid lymphoma.For example, liquid-based cancers may be acute myeloid leukemia (AML).Liquid lymphomas include lymphomas containing cysts or liquid areas.

[0088] When the disease to be treated is an autoimmune disease, the autoimmune disease is selected from the non-limiting group including rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, inflammatory bowel disease (IBD), psoriasis, and psoriatic arthritis, or a combination of one or more thereof.

[0089] The present invention provides a composition comprising i) an isolated polynucleotide of the present invention, ii) a vector of the present invention, or iii) a host cell, such as an immune cell, that expresses a chimeric antigen receptor of the present invention.

[0090] The present invention also provides pharmaceutical compositions comprising a therapeutically effective amount of i) an isolated polynucleotide of the present invention, ii) a vector of the present invention, or iii) a host cell, such as an immune cell, expressing a chimeric antigen receptor of the present invention, and a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0091] The present invention further provides use of a pharmaceutical composition comprising a therapeutically effective amount of i) an isolated polynucleotide of the present invention, ii) a vector of the present invention, or iii) a host cell, such as an immune cell, expressing a chimeric antigen receptor of the present invention, and a pharmaceutically acceptable carrier, diluent, and / or excipient, in the manufacture of a medicament for treating cancer, an infectious disease, an inflammatory disease or a pro-inflammatory disease, a chronic disease, or an autoimmune disease.

[0092] As used herein, the term "therapeutically effective amount" means, within the scope of sound medical judgment, an amount of immune cells, nucleic acid, plasmid, or vector that is high enough to result in a significant desired modification of the symptoms and / or condition being treated, yet low enough (at a reasonable risk / benefit ratio) to avoid serious side effects.

[0093] A therapeutically effective amount of the host cells, e.g., immune cells, nucleic acids, plasmids, or vectors described herein is selected according to various factors, including the type, species, age, weight, sex, and medical condition of the patient or subject; the severity of the condition or disease (e.g., cancer, infectious disease, or autoimmune disease) being treated; the route of administration; and the renal and hepatic function of the patient or subject. A physician skilled in the art can readily determine and prescribe the effective amount of immune cells, nucleic acids, plasmids, or vectors required to prevent, counter, or halt the progression of a disease, such as cancer, infectious disease, inflammatory or pro-inflammatory disease, chronic disease, or autoimmune disease.

[0094] "Pharmaceutically acceptable carrier or diluent" means a carrier or diluent that is generally safe, non-toxic, and useful for preparing a desired pharmaceutical composition, and includes a carrier or diluent that is acceptable for human pharmaceutical use.

[0095] Such pharmaceutical compositions may include buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, e.g., glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, e.g., glycine; antioxidants; chelating agents, e.g., EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0096] Any of the compositions provided herein can be provided in any suitable pharmaceutical composition and administered by any suitable route of administration. Suitable routes of administration include, but are not limited to, inhalation, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes. The pharmaceutical compositions of the present invention are preferably formulated for intravenous administration.

[0097] The pharmaceutical compositions (solutions, suspensions, etc.) may contain one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, etc.; fixed oils such as synthetic monoglycerides or diglycerides that may function as solvents or suspending media, polyethylene glycol, glycerin, propylene glycol, or other solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes, or multiple-dose vials. Pharmaceutical compositions for injection are preferably sterile.

[0098] The pharmaceutical compositions of the present 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.

[0099] In one embodiment, the at least one additional therapeutic agent or therapy is an anti-cancer agent or therapy useful for treating cancer, preferably a solid cancer. Preferably, the one or more anti-cancer therapies will be selected from the group comprising radiation therapy, chemotherapy, immune checkpoint inhibitors, immunotherapy, and hormonal therapy, or a combination of one or more thereof.

[0100] 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 or more thereof.

[0101] For example, potentially useful additional therapeutic agents include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0102] 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.

[0103] 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).

[0104] Chemotherapy of the present invention can involve agents that damage DNA and / or prevent cells from proliferating, such as genotoxins.

[0105] The genotoxin can be selected from the group consisting of 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, cyclosulfamide, ifosfamide, cisplatin, carboplatin, mitomycin, thiotepa, dacarbazine, procarbazine, hexamethylmelamine, triethylenemelamine, busulfan, pipobroman, mitotane and other platin derivatives.

[0106] An example of a DNA cutter is bleomycin.

[0107] The topoisomerase poison may be selected from the group including topotecan, irinotecan, camptothecin sodium salt, daunorubicin, doxorubicin, idarubicin, mitoxantrone, teniposide, adriamycin and etoposide.

[0108] Examples of DNA binders are dactinomycin and mithramycin, while spindle poisons can be selected from the group including vinblastine, vincristine, navelbine, paclitaxel and docetaxel.

[0109] The chemotherapy of the present invention may involve an antimetabolite selected from the following compounds: methotrexate, trimetrexate, pentostatin, cytarabine, ara-CMP, fludarabine phosphate, hydroxyurea, fluorouracil, floxuric acid, chlorodeoxyadenosine, gemcitabine, thioguanine, and 6-mercaptopurine.

[0110] Radiation therapy refers to the use of high-energy radiation to shrink tumors and kill cancer cells. Examples of radiation therapy include, but are not limited to, external radiation therapy and internal radiation therapy (also called brachytherapy).

[0111] External radiation therapy is the most common and typically involves directing a beam of direct or indirect ionizing radiation to the tumor or cancer site. The radiation beam, photon, cobalt, or particle therapy, is focused on the tumor or cancer site, but it is almost impossible to avoid exposing normal, healthy tissue. The energy source for external radiation therapy is selected from the group including direct or indirect ionizing radiation (e.g., x-rays, gamma rays, and particle beams, or a combination thereof).

[0112] Internal radiation therapy involves implanting a radiation-emitting source, such as a bead, wire, pellet, or capsule, inside the body at or near the tumor site. The energy source for internal radiation therapy is selected from the group of radioisotopes, including iodine (iodine-125 or iodine-131), strontium-89, phosphorus, palladium, cesium, indium, phosphate, or cobalt radioisotopes, and combinations thereof. Such implants can be removed after treatment or left inactive in the body. Types of internal radiation therapy include, but are not limited to, interstitial and intracavitary brachytherapy (high-dose rate, low-dose rate, and pulsed-dose rate).

[0113] Currently less common forms of internal radiation therapy involve biological carriers of radioisotopes, such as in radioimmunotherapy, where tumor-specific antibodies conjugated to radioactive material are administered to the patient or subject. The antibodies bind to tumor antigens, thereby effectively delivering a dose of radiation to the relevant tissue.

[0114] Methods for administering radiation therapy are well known to those skilled in the art.

[0115] A variety of other additional therapeutic agents may be used in conjunction with the compositions described herein.

[0116] Additional therapeutic agents suitable for use in combination with the present invention include ibrutinib (Imbruvica®), ofatumumab (Arzerra®), rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®), and rituximab (Rituxan®). (Kadcyla®), imatinib (Gleevec®), cetuximab (Erbitux®), panitumumab (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axinib Tinib, 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 These include, but are not limited to, tinib, 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 the CDK inhibitor (palbociclib).

[0117] In a further embodiment, the additional therapeutic agent may be an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDS) including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolic acid. Exemplary NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors, and sialylate. Exemplary analgesics include acetaminophen, oxycodone, tramadol, or propoxyphene 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 TNF antagonists (e.g., etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE®)), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies and recombinant forms of molecules. Exemplary DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold (oral (auranofin) and intramuscular), and minocycline.

[0118] The present invention further contemplates a method for treating and / or preventing cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease, comprising the step of administering to a subject in need thereof 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.

[0119] The term "treatment" or "treating" means (i) inhibiting the disease, i.e., arresting the development of clinical symptoms, and / or (ii) relieving the disease, i.e., causing regression of clinical symptoms; "Administration" refers to the administration of any of the compositions, pharmaceutical compositions, therapeutic agents, compounds, etc. of the present disclosure to a subject for the purpose of:

[0120] As used herein, the terms "prevention" or "preventing" refer to the administration of any of the disclosed compositions, pharmaceutical compositions, therapeutic agents, compounds, etc. to a subject for the purpose of preventing disease, i.e., not developing clinical symptoms of disease.

[0121] In the context of the present invention, the disease is cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease or an autoimmune disease.

[0122] As used herein, the terms "subject" / "subject in need" or "patient" / "patient in need" are well-recognized in the art and are used interchangeably herein to refer to mammals, including dogs, cats, rats, mice, monkeys, cows, horses, goats, sheep, pigs, camels, and most preferably humans. In some cases, the subject is a subject in need of treatment or a subject suffering from a disease or disorder. However, in other embodiments, the subject can be a normal subject. The term does not denote a particular age or sex. Thus, it is intended to encompass adult and newborn subjects, whether male or female. Preferably, the subject is a human, most preferably a human who may be suffering from or at risk of suffering from cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease.

[0123] In one embodiment, a method for treating and / or preventing cancer, infectious disease, or autoimmune disease in a patient or subject includes the steps of (i) removing and isolating immune cells, preferably immune cells, more preferably natural T cells or NK cells, from the patient or subject; (ii) genetically engineering the T cells or NK cells with a recombinant construct (e.g., a vector, plasmid, or polynucleotide) encoding a recombinant construct encoding a chimeric antigen receptor (CAR) of the present invention; (iii) ex vivo expansion (expansion) of the engineered immune cells, e.g., T cells or NK cells, into a larger population; and (iv) reintroducing the 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 functions, which vary depending on the type of immune cell used. Examples of functions include inducing cell death of target cells (T and NK), phagocytosis of target cells (macrophages), or induction of an immunosuppressive environment around the target (Tregs).

[0124] Alternatively, a method for treating and / or preventing cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease in a patient or subject comprises the steps of: (i) providing immune cells, e.g., T cells or NK cells, that have been genetically engineered with a recombinant construct (e.g., a vector, plasmid, or polynucleotide of the invention) encoding a chimeric antigen receptor (CAR) of the invention; (ii) expanding ex vivo into a larger population of engineered immune cells, e.g., T cells or NK cells; and (iii) reintroducing the engineered immune cells, e.g., T cells, into the patient or subject. In one embodiment, the NK cells are a cell line, preferably NK-92 cells.

[0125] NK cells can be engineered from a variety of NK sources, such as iPSC-derived NK cells, NK cells isolated from blood (PB-NK), NK cells isolated from umbilical cord blood, or NK cell lines, including the NK92, YTS, or KHYG1 cell lines.

[0126] Examples of clinical trials using NK cells include NCT00995137, NCT01974479, NCT02839954, NCT02892695, NCT02742727, and NCT02944162 (https: / / www.clinicaltrials.gov / ).

[0127] The polypeptides disclosed herein or nucleic acids encoding them may be introduced into host cells using transfection and / or transduction techniques known in the art. The nucleic acids may be integrated into host cell DNA or maintained extrachromosomally. The nucleic acids may be transiently maintained or stably introduced. Transfection may be achieved by various means known in the art, including, but not limited to, calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics. Transduction refers to the delivery of genes using viral or retroviral vectors by viral infection rather than by transfection. In certain embodiments, retroviral vectors are transduced by packaging the vector into virions before contacting cells. For example, nucleic acids encoding transmembrane polypeptides carried by retroviral vectors can be transduced into cells by infection and proviral integration.

[0128] In certain embodiments, the nucleic acid or vector, e.g., a 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 with the polynucleotides presented herein. In certain embodiments, transplanted cells or tissues may be placed into an organism. Thus, it is well within the knowledge of one of skill in the art to isolate antigen-presenting cells (e.g., T cells or NK cells) from an animal (e.g., a human), transfect the cells with an expression vector, and then administer the transfected or transformed cells back to the animal (e.g., a human).

[0129] In certain embodiments, the nucleic acid or vector is transferred by injection. In certain embodiments, the polynucleotide is introduced into an organelle, cell, tissue, or organism via electroporation. In certain embodiments, the polynucleotide is delivered to cells using DEAE-dextran followed by polyethylene glycol.

[0130] In one aspect, a method for treating and / or preventing cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease in a subject comprises administering to a subject in need thereof a pharmaceutical composition of the present invention.

[0131] In one embodiment, the above methods of treatment and / or prevention can further comprise administering at least one additional therapeutic agent or therapy.

[0132] Preferably, the at least one additional therapeutic agent or therapy is an anti-cancer agent or therapy, more preferably a therapeutically effective amount or dose of an anti-cancer agent or therapy, wherein the one or more anti-cancer agents or therapies are selected from the non-limiting group including radiation therapy, chemotherapy, immune checkpoint inhibitors, immunotherapy, and hormonal therapy as described herein, or one or more combinations thereof.

[0133] The present invention also contemplates kits for the treatment and / or prevention of the diseases of the present invention. In one embodiment of the present invention, the kit comprises a pharmaceutical composition of the present invention.

[0134] 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, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating a disease or disorder of the invention and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). Alternatively, or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer (e.g., bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution) and / or at least one additional therapeutic agent (e.g., a ligand of the invention). The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0135] The label or package insert may include instructions for use of the kit. The included instructions may be affixed to the packaging material or included as a package insert. The instructions are typically written or printed, but are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by the present disclosure.

[0136] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, 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., latest edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ, 1991); Carey and Sundberg Advanced Organic Chemistry 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0137] The present disclosure is therefore to be considered in all respects as illustrative and not restrictive, the scope of the present invention being indicated by the appended claims, and all changes that come within the meaning and range of equivalents are intended to be embraced therein.

[0138] Table 1 lists the sequences referred to herein.

[0139] [Table 1(1)] [Table 1(2)] [Table 1(3)] [Table 1(4)]

Table 1(5)

Table 1(8)

Table 1(9)

Table 1(10)

Table 1(11)

Table 1(12)

Table 1(13)

Table 1(14)

Table 1(15)

Table 1(16)

Table 1(18)

Table 1(19)

Table 1(20)

[0140] Materials and Methods Experimental model and subject details All experiments with mice were authorized by the Canton of Vaud (license VD3747) and were carried out in accordance with accepted guidelines for animal care.

[0141] mouse All mice were housed in individually ventilated cages with a 12-hour light / dark cycle. We used RAG- / -IL2Rg- / - mice for all in vivo tumor killing assays. All mice were bred and maintained in a pathogen-free barrier animal facility in accordance with the Swiss regulations for the care and use of mice in experimental research.

[0142] Healthy 8-10 week old female mice were used in the study. The mice used in this study had no history of drug administration, surgery, or behavioral testing.

[0143] cell culture Nalm6 and A1847 cells were cultured in RPMI medium (61870010, Thermo Fisher Scientific) supplemented with 10% FBS (F7524, Sigma-Aldrich) and 1x penicillin-streptomycin (15140-122, Thermo Fisher Scientific). NK92 cells were cultured in MEM Alpha medium (1-23S50-1, BioConcept) supplemented with 25% FBS (F7524, Sigma-Aldrich), 1x penicillin-streptomycin, and human IL-2 (100 U / ml, Peprotech).

[0144] A1847 cells were passaged every 2–3 days using trypsin-EDTA (25300-054, Thermo Fisher Scientific) or when they reached 90% confluence. All cells were maintained at 37°C in a 5% CO atmosphere.

[0145] Plasmid construction and lentivirus preparation The gene construct was designed by the present inventors, and then a lentiviral vector was constructed and packaged using VectorBuilder (vectorbuilder.com).

[0146] Transduction of NK92 or THP1 cell lines by electroporation NK92 or THP1 cell lines were cotransfected with the expression plasmid and retrotransposase by electroporation during exponential growth using a NepaGene Elepo21 electroporator with the following parameters: Poring Pulse (125 V, 5 ms duration, 50 ms interval, No. 2, positive polarity) and Transfer Pulse (20 V, 50 ms duration, 50 ms interval, No. 5, positive polarity). Electroporation was performed in Opti-MEM medium (31985070, Thermo Fisher Scientific), and electroporated cells were immediately cultured in the respective culture medium. After 4 days of recovery and expansion, NK92 or THP1 cells were selected with puromycin (final concentration 2 ng / ml, catalog number: ant-pr-1, InvivoGen).

[0147] human blood samples All human blood samples (prepared as buffy coats) were purchased from Interregional Blood Transfusion SRC Ltd. with informed consent from anonymous healthy donors.

[0148] Transduction and expansion of human T cells Human PBMCs were purified from anonymized healthy donor buffy coats by density gradient centrifugation on Ficoll-Paque PLUS. Cells were then cultured at 0.5–1 × 10 cells in complete RPMI medium containing RPMI-1640 (61870010, Thermo Fisher Scientific), FBS (10% v / v), HEPES (pH 7.2–7.5, 20 mM), penicillin / streptomycin (1% v / v), and supplemented with human IL-7 (10 ng / ml, Peprotech) and human IL-15 (10 ng / ml, Peprotech). 6hPBMCs were cultured at a density of 1000 cells / ml. hPBMCs were activated with CD3 and CD28 magnetic beads at a 1:1 ratio for 48 hours prior to transduction. Cells were transduced at an MOI of 3 in minimal medium for 5 hours.

[0149] Flow cytometry antibody staining Stain the cells with the desired antibody diluted according to the manufacturer's instructions in PBS for 20 minutes at 4°C. Then, wash the cells twice with PBS and add 10 7 Resuspend at a concentration of 1000 cells / ml. For Annexin V staining, Annexin buffer was used instead of PBS for staining and further processing of the cells.

[0150] In vitro killing assay measured by FACS On the day of co-culture, target cells were stained using Cell Trace Violet, Cell Trace Far-Red, or Cell Trace CFSE (C34557, C34564, or C34554, Thermo Fisher Scientific) according to the manufacturer's instructions.

[0151] Effector cells were added at the correct ratio and after a determined time, killing of the target population was subtracted from the double-negative population by Annexin V and PI staining.

[0152] For continuous and long-term killing assays, target cells were added at the indicated time points before measuring killing.

[0153] For quantification of killing, target cells were gated in the correct Cell Trace channel and the viable fraction (Annexin V and PI negative) was extracted. Killing was then calculated relative to the viability of the control condition without effector cells using the following formula: Killing = (% Viability Control - % Viability Sample) / % Viability Control x 100

[0154] Isolation and transduction of regulatory T cells Regulatory T cells were purified from PBMCs using the EasySep™ Human CD4+CD127lowCD25+ Regulatory T Cell Isolation Kit (Stemcell Technologies). Cells were then cultured at 0.5–1 × 10 cells in complete RPMI medium containing RPMI-1640 (Thermo Fisher Scientific), FBS (10% v / v), HEPES (pH 7.2–7.5, 20 mM), penicillin / streptomycin (1% v / v), and supplemented with human IL-2 (100 U / mL for normal conditions, 300 U / mL if we wish to promote proliferation (e.g., after selection), Peprotech). 6 The cells were cultured at a density of 1000 cells / ml.

[0155] Mitotic assay PBMCs were pre-activated with CD28 and CD3 magnetic beads for 48 hours prior to the assay. The beads were then removed, and cells were stained with CFSE (Thermo Fisher Scientific) according to the manufacturer's instructions. Tregs were stimulated in CD19-coated plates for 24 hours prior to the assay. CFSE-preactivated PBMCs were then co-cultured with stimulated or unstimulated Tregs in CD19-coated plates for 3-5 days. Cell proliferation was then measured by flow cytometry as a dilution of CFSE.

[0156] Phagocytosis assay THP1 cells, either electroporated to express the CAR construct or not, were stained with Cell Trace Violet (Thermo Fisher Scientific) according to the manufacturer's instructions. They were then co-cultured with GFP-expressing target cells at different ratios for 24 hours. The percentage of THP1 cells acquiring GFP was then measured by flow cytometry.

[0157] Ovarian cancer xenografts receiving NK cell treatment A1847 CD19 GFP-Luc (5 × 105 The cells (1–5 × 10 cells / mouse) were suspended in 100 μL of PBS and injected into RAG- / -IL2Rg- / - female mice. Four days after tumor engraftment, mice were randomized and assigned to different treatment groups, with 1–5 × 10 cells / mouse. 6 NK92 cells were injected i.p. Tumor progression was monitored twice weekly by bioluminescence imaging using an IVIS Lumina II in vivo imaging system (PerkinElmer). Mice were euthanized when the signal exceeded a threshold before signs of discomfort were detected.

[0158] T-cell-treated leukemia xenografts Nalm6 GFP-Luc (5 × 10 per mouse 6 The cells were suspended in 100 μL of PBS and injected intravenously into RAG− / −IL2Rg− / − female mice. Four days after tumor engraftment, mice were randomized and assigned to different treatment groups, with 5 × 10 cells per mouse. 6 Transduced T cells were injected intravenously. Tumor progression was monitored twice weekly by bioluminescence imaging using an IVIS Lumina II in vivo imaging system (PerkinElmer). Mice were euthanized when the signal exceeded a threshold before signs of discomfort were detected.

[0159] Example 1 result Expression and activity of LAT-based CARs in NK cells in vitro The 19LAT activation domain is composed of the intracellular portion of the linker for T cell activation (LAT). The extracellular portion of 19LAT is composed of the extracellular spacer region (i.e., linker or hinge) of the scFv and CD8a. We compared our novel CAR construct with two well-documented CARs (1928z and 19BBz) that were used as control CARs in our studies. Their signaling domains are composed of the commonly used CD28 or 4-1 BB costimulatory domain and the CD3z activation domain, respectively (Figure 1A).

[0160] The expression levels of CAR in NK92 were similar among all CAR constructs, as assessed by flow cytometry (Fig. 1B).

[0161] The in vitro activity of 19LAT was compared to that of 1928z CAR and 19BBz CAR in NK92 cells in an in vitro killing assay (Figure 1C). Cytotoxicity was similar for all constructs, demonstrating the potential of our new CAR design.

[0162] To better define the efficacy of our novel CAR constructs, we evaluated various parameters of different constructs in NK92 cells in an in vitro killing assay. Since we are comparing the length of the CAR stimulation phase, it is possible that it influences NK cytotoxicity. We performed a rechallenge killing assay, which mimics chronic inflammation in vitro (Figure 1D). In this assay, new target cells were added to the co-culture every 24 hours, and the killing of the last added population was measured 4 hours after co-culture. This assay reveals the ability of engineered NK cells to retain cytotoxicity while being continuously surrounded by target cells. We hypothesize that continuous CAR signaling to NK cells may affect cytotoxicity in the long term. In these settings, all CARs were comparable in terms of cytotoxicity after three and four rechallenges (Figure 1E).

[0163] Taken together, these results indicate that 19LAT can induce NK cytotoxicity in NK cells at a level similar to that of the commonly used 1928z and 19BBz CAR constructs.

[0164] 19LAT CAR can induce solid tumor growth control through engineered NK92 One advantage of NK cells over T cells is their increased ability to penetrate solid tumors. Next, we compared the efficacy of 19LAT in NK cells with 19BBz CAR and 1928z CAR to eliminate the ovarian human cancer cell line A1847, which had been engineered to express CD19 as a cancer-specific marker.

[0165] We first evaluated the cytotoxicity of CAR NK cells against CD19-expressing A1847 ovarian cells in vitro. After 4 hours of co-culture, no difference was observed among the three CARs. However, 19LAT showed better activity compared to 1928z CAR in the serial killing assay after four rounds of challenge (Figure 2A). This data demonstrates the superior function of 19LAT against solid tumors in vitro.

[0166] We then compared the efficacy of 19LAT with 1928z CAR in vivo. On day 0, A1847 was injected into immunodeficient mice. Once tumors were established on day 3, CAR NK was injected intraperitoneally (Figure 2B). Interestingly, 19LAT and 1928z CAR have similar antitumor efficacy in vivo (Figure 2C).

[0167] Taken together, these data indicate that 19LAT-engineered NK92 has potent anti-tumor induction potential against solid cancer cells in vitro and in vivo.

[0168] Tuning and optimization of the 19LAT CAR construct The kinetics of PLCγ recruitment to LAT is known to regulate the T cell activation threshold, and we hypothesized that modulation of PLCγ recruitment kinetics would allow fine-tuning of LAT activity.

[0169] We first confirmed that PLCγ recruitment to tyrosine 134 is not only required but also necessary for 19LAT. This was done by generating a Y134F CAR mutant, i.e., 19LAT(Y134F) (Figure 3A). This mutation has been shown in the literature to abolish PLCγ recruitment to LAT, and we observed that this CAR mutant was unable to induce cytotoxicity (Figure 3B).

[0170] The kinetics of ZAP70-induced phosphorylation of tyrosine 134 on LAT can be increased by introducing acidic residues at positions -1 and -2 relative to the tyrosine. The corresponding 19LAT mutants were generated (Figure 3C). The cytotoxicity of the mutants was assessed in vitro, and no difference was observed compared to the 19LAT construct after 4 hours of co-culture, nor after 4 rounds of challenge (Figure 3D).

[0171] We next wanted to investigate whether PLCγ could be recruited not only by the original PLCγ recruitment site of LAT, but also by a PLCγ recruitment site from another protein known to bind PLCγ. We chose to use VEGFR, which contains a protein sequence known to recruit PLCγ. Within these PLCγ docking sites, tyrosines have been shown to be phosphorylated by kinases recruited by amino acids N-terminal to the tyrosine. The amino acid C-terminal to the tyrosine confers specificity for the recruiting protein (in this case, PLCγ) on phosphotyrosine. Because tyrosine phosphorylation by ZAP-70 / Syk is required, we generated a hybrid LAT / VEGFR docking site for PLCγ in which the tyrosine can be phosphorylated by ZAP-70 (the amino acids N-terminal to the tyrosine are derived from the LAT sequence) and the VEGFR recruitment site can recruit PLCγ (the amino acids C-terminal to the tyrosine are derived from the VEGFR sequence) (Figure 3E).

[0172] In vitro functional assays show that 19LAT(hybrid) was functional to activity levels similar to the initial 19LAT CAR construct. However, the 19LAT(VEGFR2) construct, lacking the N-terminal amino acid residues required for ZAP70 / Syk recruitment, did not exhibit any cytotoxicity (Figure 4F). These data indicate that PLCγ recruitment can be mediated by other docking sites known to recruit PLCγ, thereby extending our invention to any protein sequence that allows kinase-induced PLCγ recruitment. In the case of CARs, tyrosines must be phosphorylated by the tyrosine kinase ZAP70 / Syk, known to be expressed by NK cells, so such docking sites must be adapted to allow phosphorylation by this kinase family (as shown above).

[0173] The stability and surface expression of LAT are known to be regulated by ubiquitination of two lysine residues in LAT. Therefore, to examine the effect of stabilizing the 19LAT construct on membrane expression, we mutated these residues to arginine, i.e., 19LAT(RR) (Figure 4G). We evaluated the cytotoxicity of these mutants and found that they exhibited similar activity compared to the 19LAT construct both after 4 hours of co-culture and after four rounds of challenge (Figure 4H). Further studies are needed to determine whether CAR stabilization may confer any advantage to NK cells.

[0174] LAT is endogenously present in the membrane as a monomeric protein. The CD8a hinge is known to dimerize via trans-disulfide bridge formation of two cysteines in the CD8a hinge. Therefore, we generated a mutant of the 19LAT CAR, designated 19(CS)LAT, in which these two cysteines were mutated to serine to abolish dimerization of the construct (Figure 4I). The functionality of the 19(CS)LAT CAR was evaluated in vitro and was determined to be similar to that of the 1928z CAR and 19BBz CAR constructs after 4 hours of coculture. Interestingly, this 19(CS)LAT CAR exhibited superior tumor elimination ability after four rounds of challenge in both Nalm6 and A1847 cell lines (Figure 4J). We then performed a long-term killing assay experiment to mimic acute inflammation in vitro (Figure 4K). This long-term killing assay evaluates functional killing by CAR constructs after co-culture of effector and target cells over a 7-day period. Functional killing / cytotoxicity against target cells is measured after this extended co-culture, followed by a 4-hour exposure of fresh target cells to CAR NK cells. This assay was designed to assess the reversibility of the activated / resting state of engineered NK cells. In these settings, NK92 engineered with 19(CS)LAT exhibited superior functional cytotoxicity compared to NK92 engineered with 1928z or 19BBz CARs (Figure 4L). Interestingly, these results demonstrate the superiority of the monomeric 19(CS)LAT CAR in vivo, as the 19(CS)LAT construct exhibited better tumor control than the 1928z CAR, but not the 19LAT (Figure 4M).

[0175] Taken together, these results demonstrate that our LAT-based CARs are highly manipulable to modulate the activation threshold or stability of the receptor in the membrane. Furthermore, superior in vitro and in vivo potency was achieved with 19(CS)LAT compared to the widely used 1928z CAR.

[0176] 19LAT can be optimized for use in T cells The present inventors have shown that the novel 19LAT of the present invention exhibits significant advantages in NK cells. The present inventors wondered whether these characteristics were the same in T cells.

[0177] T cells were able to express 19LAT (Figure 4A), but showed reduced cytotoxicity against target cells in vitro compared to 1928z and 19BBz after three rounds of challenge (Figure 4B). For first-generation CARs using the CD3z activation domain, the introduction of a costimulatory domain such as CD28 or 4-1 BB was shown to increase the cytotoxicity of the CAR.

[0178] We generated 1928LAT and 19BBLAT CARs, in which CD28 or 4-1 BB was inserted between the transmembrane and intracellular domains of LAT (Figure 4C). These constructs were successfully expressed in T cells (Figure 4D), and the cytotoxicity of these CAR T cells could be assessed (Figure 4E). However, the 19BBLAT CAR exhibited reduced tumor-controlling ability in vivo compared with the 1928z and 19BBz CARs (Figure 4F). We hypothesized that LAT might require membrane proximity to efficiently recruit signaling proteins such as PLCγ. Therefore, we generated a new CAR, designated 19LAT28, in which CD28 was placed C-terminal to the LAT signaling domain. This allows the LAT signaling domain to be in a membrane-proximal position while utilizing CD28 costimulatory signaling. Additionally, to further enhance the activity of 19LAT28, we included the aforementioned mutation that increases the phosphorylation kinetics of the PLCγ docking site on the CAR by ZAP70 / Syk, i.e., 19LAT28(G133D) (Figure 4C). These constructs could be expressed in T cells (Figure 4G). Their cytotoxicity could be measured and was similar to that of the conventional CAR construct 1928z (Figure 4H). While the 19LAT28 construct showed mild tumor control in vivo, 19LAT28(G133D) showed improved tumor control comparable to that of 1928z (Figure 4I). Collectively, this study demonstrates that our 19LAT can be adapted for T cells by introducing a CD28 costimulatory domain C-terminal to the LAT signaling domain, and its functionality can be enhanced by increasing the phosphorylation kinetics of the PLCγ docking site by ZAP70 / Syk.

[0179] 19LAT can induce engineered immune cell functions upon antigen recognition CAR therapy is not limited to cancer treatment, and recent findings indicate its potential to treat other diseases. In the future, CAR therapy may not be limited to T cells or NK cells, but may be extended to other immune cell types, such as regulatory T cells or macrophages.

[0180] To determine whether our new CAR constructs could also induce functional activity in regulatory T cells, we performed proliferation assays of PBMCs in coculture with regulatory CAR T cells (Figure 5A). Regulatory T cells were purified from donor blood and transduced to express the CAR (Figure 5B). CD28 signaling has been shown to be beneficial for regulatory T cells, whereas 4-1BB signaling is known to adversely affect regulatory T cell phenotypic stability. For this reason, we focused our experiments solely on constructs based on CD28 signaling, namely, the 19LAT28 and 1928z CAR constructs. Proliferation assays showed that the 19LAT28 CAR and 19LAT(G133D)28 CARs were able to activate regulatory T cells and inhibit PBMC proliferation in a manner comparable to the 1928z CAR (Figure 5C).

[0181] To evaluate whether 19LAT can induce phagocytosis in macrophages, we performed a phagocytosis assay using the human leukemia-monocyte THP-1 cell line (Figure 5D). First, we transduced THP-1 cells with the 19LAT CAR construct and verified expression by flow cytometry (Figure 5E). 19LAT was able to specifically induce phagocytosis of CD19-expressing target cells more efficiently than non-transduced parental THP-1 cells (Figure 5F).

[0182] Taken together, these data demonstrate that the novel LAT-based CAR constructs of the present invention are not only capable of inducing cytotoxicity in NK cells and T cells, but can also be used to functionally activate other cell types, such as regulatory T cells or macrophages.

[0183] Example 2 Optimization of the 19LAT CAR signaling domain to increase NK cell cytotoxicity We demonstrated that 19LAT can induce high levels of cytotoxicity in NK cells. However, key tyrosines within the LAT signaling domain were not addressed in the 19LAT design. We hypothesize that manipulation of these sites may further enhance the functionality of LAT-based CARs.

[0184] We generated the 19LAT(G133D)CAR and 19(CS)LAT(G133D)CAR constructs. The G133D mutant was previously described in the context of T cells. In this example, we investigate the effect of this mutation in NK cells (Figure 6A).

[0185] In addition, we generated 19 LAT mutants surrounding tyrosine 228, which is originally known to recruit GRB2 and other adaptors. Because this tyrosine is known to recruit similar adaptor proteins as other tyrosines, including tyrosines 173 and 193, we reasoned that manipulation of tyrosine 228 would not adversely affect LAT signaling. To promote PI3K recruitment through the creation of a YxxM motif within the existing ITT motif, we first mutated the site GAPDYENL to SIDDYMNM. The C-terminal mutation of the tyrosine (GAPD to SIDD) was performed to promote the phosphorylation dynamics of tyrosine 228. The N-terminal mutation of the tyrosine (ENL to MNM) was performed to create a YxxM motif within the existing ITT motif, presumably to recruit PI3K. This strategy of mutating the existing ITT motif (xYxN) to include a YxxM motif allows for the addition of functions associated with the YxxM motif, such as recruitment of PI3K, while maintaining the initial function of the ITT motif, such as recruitment of GRB2 / GADS. This "enhanced ITT mutant" combines the properties of both the ITT and YxxM motifs. These mutants are called 19LAT(ITTi) or 19(CS)LAT(ITTi). To determine whether the G133D and enhanced ITT mutations synergize together, we combined both mutations on the same CAR construct, called 19LAT(G133D,ITTi) or 19(CS)LAT(G133D,ITTi) (Figure 6A).

[0186] Because mutations surrounding tyrosine 228 may adversely affect the functionality of the LAT signaling domain, we also generated 19LAT variants containing an enhanced ITT motif at the C-terminus of the LAT domain. These CAR constructs are called 19LAT(ITTc) and 19(CS)LAT(ITTc). The addition of this enhanced ITT motif was also combined with the G133D mutation to generate 19LAT(G133D,ITTc) and 19(CS)LAT(G133D,ITTc) (Figure 6A).

[0187] Finally, to fully promote NK cell functionality, we introduced the 4-1BB signaling domain into the C-terminus of the 19LAT variant. As an example, Figure 6A shows the 19(CS)LAT(G133D,ITTi)41BB variant, which was generated by adding the 41BB domain to 19(CS)LAT(G133D,ITTi) (Figure 6A). CAR expression was confirmed by flow cytometry (Figure 6B).

[0188] To distinguish functional differences between all CAR constructs, we plated serial killing assays at a low NK ratio (0.3 NK cells to 1 target) (Figure 6C-F).

[0189] result Interestingly, 19LAT is more cytotoxic than the previously reported CAR construct (see Control 6A) made from the LAT signaling domain fused to the CD28 transmembrane domain. The single mutants, i.e., 19LAT(G133D), 19(CS)LAT(G133D), 19LAT(ITTi), 19(CS)LAT(ITTi), 19LAT(ITTc), and 19LAT(ITTc), also show increased cytotoxicity compared to the 19LAT CAR or 19(CS)LAT CAR constructs. This suggests that the mutations enhance the recruitment of signaling molecules, resulting in increased cytotoxicity. Furthermore, the G133D and enhanced ITT motif combination-generation mutants, i.e., 19LAT(G133D,ITTi), 19(CS)LAT(G133D,ITTi), 19LAT(G133D,ITTc), and 19(CS)LAT(G133D,ITTc), exhibited stronger cytotoxicity compared to both unmutated and single-mutated LAT-based CARs. This synergistic effect was unexpected.

[0190] Finally, to generate a CAR that harnesses the full cytotoxic potential of NK cells, we engineered a CAR construct designed to activate a broad range of signaling pathways required for optimal NK cell activation. Addition of the 4-1BB signaling domain to 19(CS)LAT(G133D,ITTi) resulted in increased cytotoxicity (Figure 6C-F).

[0191] Table 2 below summarizes the different CAR constructs mentioned above.

[0192] [Table 2]

[0193] Taken together, these results indicate that LAT-based CARs can be optimized for use in NK cells to harness their full cytotoxic potential. Engineering specific tyrosine-enriched sites to increase phosphorylation kinetics or recruit additional signaling proteins is possible due to the highly modular structure of the LAT signaling domain. The modularity of the YxxM motif used in this example to modify the existing ITT motif can be further expanded to exploit other properties, such as inserting the YxxM motif YMFM to also recruit the p85 and p50a subunits of PI3K. Conversely, inserting the YxxM motif YVKM allows recruitment of the p85 subunit or PI3K and SHP2, which can help attenuate CAR signaling strength.

Claims

1. 1. A chimeric antigen receptor polypeptide comprising: an extracellular domain that binds to a cell surface marker; a transmembrane domain; Intracellular signaling domains and wherein the transmembrane domain is derived from, comprises, or consists of a linker transmembrane domain of T-cell activation (LAT-tm), a fragment or variant thereof; 10. A chimeric antigen receptor, wherein the intracellular signaling domain is derived from, comprises, or consists of a recognition domain for an SH2 domain-containing protein selected from recruitment proteins including PIK3R1, PI3K, the p65 alpha subunit of PI3K, GRB2, GRAP, GRAP2, PLCG1, PLCG2, SHB, SKAP2, CLNK, and FCGR1A, and any variant, isoform, or combination thereof.

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

3. 3. The chimeric antigen receptor of claim 2, wherein the LAT is selected from SEQ ID NO: 1, a fragment or a variant thereof.

4. The chimeric antigen receptor of claim 3, wherein the LAT variant comprises one or more additions, substitutions (e.g., one or more mutations), and / or deletions within SEQ ID NO:

1.

5. The chimeric antigen receptor of claim 3 , wherein the substitution consists of one or more mutations.

6. The one or more mutations in SEQ ID NO: 1 are 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, 6. The chimeric antigen receptor of claim 5, wherein the amino acid residues of the chimeric antigen receptor are selected from the group consisting of methionine at position 131, 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, and isoleucine at position 225.

7. The LAT manifold is A sequence selected from the group comprising, from positions 130 to 138, 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 consisting of SEQ ID NO: 27 (SIDDYMNM) and SEQ ID NO: 38 (SIDDYMFM) The chimeric antigen receptor of any one of claims 1 to 6, comprising:

8. 8. The chimeric antigen receptor of claim 1, further comprising one or more intracellular costimulatory domains.

9. The intracellular costimulatory domain may be any of the following: CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell costimulatory molecule (ICOS), lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), LIGHT (TNFSF14), NKG2C, Igα (CD79a), DAP-10, Fcγ receptor, MHC class I molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activating NK cell receptor, BTLA, Toll ligand receptor, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAF FR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT GAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT 9. The chimeric antigen receptor of claim 8, wherein the chimeric antigen receptor is selected from the group consisting of a ligand that specifically binds to AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​GADS, SLP-76, PAG / Cbp, CD19a, CD83, a variant thereof, or any combination thereof.

10. The chimeric antigen receptor according to any one of claims 1 to 9, further comprising a linker region inserted between the extracellular region and the transmembrane domain.

11. The chimeric antigen receptor of claim 10, wherein the linker region is selected from the group consisting of: i) an immunoglobulin hinge region or a linker region derived from CD8, CD8α, or CD28; and ii) a GnS linker, wherein G is glycine, S is serine, and n is an integer from 2 to 12.

12. 12. The chimeric antigen receptor of claim 1, wherein the extracellular domain that binds to a cell surface marker is an antigen-binding polypeptide, a receptor, or a natural ligand for an antigen or receptor on a target cell.

13. 13. The chimeric antigen receptor of claim 12, wherein the antigen-binding polypeptide is an antibody or antibody fragment selected from the group comprising a murine antibody, a rabbit antibody, a human antibody, a humanized antibody, a single-chain variable fragment (scFv), a camelid antibody variable domain and humanized versions, a shark antibody variable domain and humanized versions, a single-domain antibody variable domain, a nanobody (VHH), and a camelized antibody variable domain.

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

15. The antigens recognized by the antigen-binding polypeptides include CD19, CD20, CD38, CD30, Her2 / neu, ERBB2, fibroblast activation protein (FAP), CA125, MUC-1, PSMA, PSA, CD44 cell 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, A33, Br3, Br4, Br5, Br6, Br7, Br8, Br9, Br10, Br11, Br12, Br13, Br14, Br15, Br16, Br17, Br18, Br19 ... 20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD45, CD74, CD79a, CD80, CD138, colon-specific antigen-p (CSAp), CSAp, EGP-I, EGP-2, Ep-CAM, Flt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its subunits, hypoxia Inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-I), KC4-antigen, KS-1-antigen, KS1-4, Le-Y, macrophage inhibitory 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, S100, TAC, TAG-72, tenascin, TRAIL receptor, Tn antigen, Thomson-Friedenreich antigen, tumor necrosis antigen, VEGF, ED-B fibronectin, 17-1A-antigen, NeuGcGM3, N-glycolyl GM3 ganglioside, Neu5Gc, GM3-ganglioside, GD3, GM2, carbohydrate antigen, ganglioside antigen, Lewis Y, LewisThe chimeric antigen receptor according to any one of claims 12 to 14, which is selected from the group consisting of B, MOG, MBP, aB-crystallin, PLP, GlialCAM, β-synuclein, HLA-A2, TNP, CD123, and an immunoglobulin κ chain.

16. The chimeric antigen receptor according to any one of claims 1 to 15, wherein the LAT-tm is selected from SEQ ID NO: 9, a fragment or a variant thereof.

17. 17. The chimeric antigen receptor of any one of claims 1 to 16, 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 variant thereof.

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

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

20. 20. The vector of claim 19, wherein the vector is selected from the group comprising a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, an RNA (e.g., mRNA)-targeted lipid nanoparticle (LNP), a liposome, or any combination thereof.

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

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

23. 23. The host cell of claim 21 or claim 22, selected from the group comprising cytotoxic cells, immune cells, stem cells, progenitor cells, cell lines, and cells derived from stem cells or progenitor cells.

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

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

26. 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 according to claim 18, ii) a vector according to claim 19 or claim 20, or iii) a host cell according to any one of claims 21 to 26, together with a pharmaceutically acceptable carrier, diluent and / or excipient.

28. 28. The pharmaceutical composition of claim 27 for use in the treatment of cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease.

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

30. 18. A method of treating and / or preventing cancer, an infectious disease or an autoimmune disease in a patient or subject, the method comprising the steps of: (i) removing and isolating host cells, preferably immune cells, more preferably natural T cells or NK cells, from said patient or subject; (ii) genetically engineering said T cells or NK cells with a recombinant construct (e.g., a vector, plasmid or polynucleotide) encoding a recombinant construct encoding a chimeric antigen receptor (CAR) according to 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 said patient or subject.

31. 19. A method for treating and / or preventing cancer, an infectious disease, an inflammatory or pro-inflammatory disease, a chronic disease, or an autoimmune disease in a patient or subject, the method comprising the steps of: (i) providing immune cells, such as T cells or NK cells, that have been genetically engineered with a recombinant construct (e.g., a vector, a plasmid, or a polynucleotide of the invention) encoding a chimeric antigen receptor (CAR) according to any one of claims 1 to 17; (ii) expanding ex vivo into a larger population of engineered immune cells, such as T cells or NK cells; and (iii) reintroducing the engineered immune cells, such as T cells, into the patient or subject.

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

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