WO2021071431A1 - Genetically modified t cells and uses thereof - Google Patents

Abstract

Disclosed herein include a T cell genetically modified to express a recombinant chimeric antigen receptor (CAR) and a recombinant chimeric co-stimulatory receptor (CCR), a pharmaceutical composition comprising the T cell, methods of preparing the T cell, and method of treating cancer or tumor using the T cell. The CAR may comprise an extracellular domain comprising the extracellular domain of an NKG2D receptor, a transmembrane domain, and an intracellular signalling domain, and does not comprise a co-stimulatory signalling domain. The CCR may comprise an extracellular domain comprising a PD-L1 specific single-chain variable fragment, a transmembrane domain, a co -stimulatory signalling domain, and does not comprise an intracellular signalling domain.

Description

GENETICALLY MODIFIED T CELLS AND USES THEREOF FIELD OF THE INVENTION [0001] The present invention relates generally to the field of biotechnology and cell therapy. In particular, the present invention relates to genetically modified T cells for cancer immunotherapy, their preparation method and use in patients in need thereof. BACKGROUND OF THE INVENTION [0002] Adoptive transfer of T lymphocytes bearing a chimeric antigen receptor (CAR) is highly effective in eliminating substantial burdens of certain types of blood cancers and has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of B-cell leukemia. CAR constructs combine the specificity and high-affinity binding function of monoclonal antibodies secreted by B-cells with the intracellular signaling domain of the T cell receptor (TCR) and the cytotoxic potency of T cells to re-direct T cells towards cancer cells and enable effective killing of target cells. T cell activation by a CAR is independent of the interaction between TCR and the major histocompatibility complex (MHC)-peptide complexes, thus overcoming the immune evasion mechanisms often used by tumors cells, such as the downregulation of MHC class I molecules and development of defective antigen processing. [0003] The first-generation CARs consist of an extracellular tumor-specific domain, such as a single-chain variable fragment (scFv) from a monoclonal antibody, a transmembrane domain, and an intracellular signaling moiety for T cell activation. Despite great efforts, the studies with first-generation CAR-T cells did not achieve the desired clinical outcomes, mainly because of inadequate proliferation of the CAR-T cells, resulting in a short life span in vivo and insufficient tumor killing. [0004] The full activation of T-cells requires two signals. The first signal is antigen- specific, provided through the T cell receptor (TCR) that interacts with the antigenic peptide- MHC complex on the surface of antigen-presenting cells. The second signal is antigen nonspecific and delivered by the interaction between co-stimulatory molecules expressed on the antigen-presenting cells and co-stimulatory receptors on the T cell. One such example is the interaction of B7 ligands with the co-stimulatory receptor CD28; the interaction between 4-1BB Ligand (CD137L) and 4-1BB (CD137) receptor is another example. The co- stimulatory signal promotes the synthesis and secretion of cytokines that are either produced by the antigen-presenting cell and/or by the activated T cell itself. The second signal is critical for appropriate T cell activation without anergy, promoting long-term proliferation of activated T cells. Given the important role of dual signaling for T cell activation, the second- generation CARs include intracellular costimulatory moieties, such as those from CD28 and 4-1BB receptors, and significantly improve the functionality of the modified CAR-T cells, especially the in vivo proliferation and persistence of CAR-T cells, making these CAR-T cells “true living drugs” in the patient’s body. [0005] While adoptive transfer of CAR-T cells constitutes a highly promising strategy for leukemia therapy, the translation of the approach to non-haematological malignancies is, however, challenging due to the special pathophysiological characteristics of solid tumors. Major limitations of clinical CAR-T cell technology against solid tumors include factors affecting target antigen selection (for example tumor heterogeneity and antigen loss variants), intrinsic negative regulatory mechanisms of tumor microenvironment (TME) such as the PD- L1/PD-1 signaling pathway, and on-target, off-tumor toxicities. [0006] The programed cell death 1 (PD-1) receptor (CD279) is an inhibitory immune receptor, which plays important roles as an immune checkpoint, especially in T cell co- inhibition and exhaustion. The interaction between PD-1 and its cognate ligand PD-L1 is one of the major processes used by many tumors for immune suppression and evasion. PD-1 is expressed on activated lymphocytes and has been shown to negatively regulate antigen receptor signaling upon engagement of PD-L1 expressed on tumor cells, leading to impaired immune cell effector functions such as reduced cell proliferation, cytokine secretion, and tumor cell lysis. Using PD-L1 detection antibodies and immunohistochemistry staining, high PD-L1 expression in tumor cells has been found to correlate with poor prognosis in terms of overall survival in various human cancers. Therefore, PD-L1 expression has been introduced into clinical practice as a biomarker to select responders to PD-1/PD-L1 antibody treatments. Modulating the PD-1/PD-L1 interaction using monoclonal antibodies (mAbs), an approach termed “immune checkpoint blockade”, has yielded good clinical responses and improved overall survival in patients with melanoma, renal cell cancer, non-small cell lung cancer, and other tumors. However, PD-1 mAb treatment can lead to immune-related adverse events (irAEs), which could be serious and even fatal. The most severe adverse event with PD-1 mAb treatment is pneumonitis, leading to 3 deaths in an early-phase study. . [0007] The current invention addresses some of the shortcomings as described above in the area of chimeric antigen receptor (CAR)-modified T lymphocyte therapy against cancer, in particular solid tumors. SUMMARY OF THE INVENTION [0008] In one aspect, the present invention discloses a T cell genetically modified to express: i) a recombinant chimeric antigen receptor (CAR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a co- stimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of an NKG2D receptor; and ii) a recombinant chimeric co-stimulatory receptor (CCR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) a co-stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv). [0009] In another aspect, the present invention discloses a pharmaceutical composition comprising a pharmaceutically effective amount of the T cell of the present invention, and a pharmaceutically acceptable excipient. [0010] In another aspect, the present invention discloses a method of preparing the T cell of the present invention, the method comprises: a) obtaining or providing T cell; b) providing i) a recombinant nucleic acid encoding the recombinant CAR; and ii) a recombinant nucleic acid encoding the CCR; and c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the CCR into the T cell. [0011] In yet another aspect, the present invention discloses a method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the T cell or the pharmaceutical composition of the present invention. [0012] In a further aspect, the present invention discloses a method of treating cancer or tumor in a subject in need thereof, the method comprises: a) obtaining T cells from the subject, or from a donor which is different from the subject to be treated; b) providing i) a recombinant nucleic acid encoding a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a co-stimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of an NKG2D receptor; and ii) a recombinant nucleic acid encoding a recombinant chimeric co- stimulatory receptor (CCR), wherein the recombinant CCR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) a co- stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-1L1 specific single-chain variable fragment (scFv); c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR into the T cell to obtain genetically modified T cells; and d) administering to the subject a pharmaceutically effective amount of the T cells obtained from c). BRIEF DESCRIPTION OF THE DRAWINGS [0013] The invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, in which: [0014] Fig. 1 shows schematic representations of chimeric receptors described in this application. Fig. 1A shows that the 1st generation NKG2D CAR is generated by fusing the extracellular domain of NKG2D receptor with an activation domain. The activation domain is derived from either CD3 zeta or DAP12. Fig. 1B shows that αPD-L1 CCR is generated by fusing a PD-L1-specific scFv to a single co-stimulatory signaling domain. Fig. 1C shows that the PD-1 chimeric switch receptor (CSR) is generated by fusing the extracellular domain of PD-1 inhibitory receptor to a single co-stimulatory signaling domain. In Fig. 1B and 1C, the co-stimulatory signaling domain is derived from either 4-1BB or CD28. Fig. 1D shows that the two NKG2D CAR-based combinations described in this application are generated by pairing the 1st generation NKG2D CAR with either the αPD-L1 CCR (CAR-T1) or the PD-1 CSR (CAR-T2). CAR, chimeric antigen receptor; CCR, chimeric costimulatory receptor; CSR, chimeric switch receptor. [0015] Fig. 2 shows bar chart results of cytotoxicity assay showing that CAR-T1 and CAR-T2-modified T cells exhibit dose-dependent cytotoxicity against a panel of cancer cell lines. HepG2 (NKG2DL+), CAOV3 (NKG2DL+, PD-L1+) and Detroit-562 (NKG2DL+, PD-L1+, PD-L2+) cancer cells were used as target cells in a standard 2-hour Delfia time- resolved cytotoxicity assay. CAR-T1 and CAR-T2-modified T cells were seeded with target cells at various E:T ratios: 20:1 and 10:1. Both CAR-T1 and CAR-T2 demonstrated a dose- dependent increase in cytotoxicity against target cells. No significant difference was observed between CAR-T1 and CAR-T2 in their cytotoxicity against all three cell lines (NS, p>0.05). E:T, effector to target. Data shown are mean ± SD of triplicates, representative of three independent experiments. Statistical analysis was performed by 2-way ANOVA, followed by Tukey’s multiple comparisons. [0016] Fig. 3 Antigen-dependent expansion of NKG2D CAR-T cells is further enhanced by αPD-L1 CCR. CAR-T1 and CAR-T2 cells were expanded with K562 myelogenous leukemia cells that express both NKG2D and PD-1 ligands. A fresh batch of γ-irradiated K562 feeder cells was used every 10 days at an E:T ratio of 2:1. Data shown are cell numbers obtained from Trypan blue exclusion assay on day 17, 27 and 37 post DNA electroporation from one donor, representative of three independent experiments. [0017] Fig. 4 Inclusion of αPD-L1 CCR enhanced the development of CCR7-/CD45RA- effector memory T cells. Memory T development was characterized for both CAR-T1 and CAR-T2 groups through detection of both CCR7 and CD45RA antigens. (A) Proportion of CCR7- CD45RA+ effector T cells in CAR-T1 and CAR-T2 in the middle and at the end of expansion phase. (B) Proportion of CCR7- CD45RA- effector memory cells in CAR-T1 and CAR-T2 in the middle and at the end of expansion phase. Data shown are single measurements of one donor, representative of three independent experiments. [0018] Fig. 5 Inclusion of αPD-L1 CCR alleviated the expression of T cell exhaustion markers. (A) Single end-point measurements from three donors: the expression levels of PD- 1, TIGIT, TIM3 and LAG3 were assessed on CAR-T1 and CAR-T2 cells at the end of the expansion phase described in Fig. 3. (B) Pooled average of expression levels from each exhaustion marker categorized according to CAR experimental group. [0019] Fig. 6 CAR-T cell treatment eliminates established tumors in a mouse xenograft model. Four groups of mice (5 mine per group) received i.p. injection of 2×10⁶ HCT116-Luc human colorectal cancer cells (day 0) followed by i.p. injection of PBS, control CAR-T cells, T cells modified with NKG2D CAR + PD-1 CSR or T cells modified with NKG2D CAR+ αPD-L1 CCR on day 7 and day 32 (1×10⁷ CAR-T cells per mouse per injection). Growth of HCT116 was monitored by bioluminescent imaging on the indicated days. Bioluminescent images are shown. [0020] Fig. 7 CAR-T cell treatment significantly prolongs the survival of mice inoculated with human cancer cells. In the mouse xenograft model described in Fig. 6, animal survival was monitored up to 150 days post tumor inoculation and was analyzed by the Kaplan-Meier method. [0021] Fig. 8 shows schematic overview of the design concepts for constructs useful for modifying the T cells as described in the present disclosure. DEFINITIONS [0022] A “genetically modified cell” means any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA molecule, or protein or polypeptide. [0023] A “T cell” is a type of lymphocyte which develops in the thymus gland and plays a central role in the immune response. T cells can be distinguished from other lymphocytes by the presence of a T-cell receptor on the cell surface. These immune cells originate as precursor cells, derived from bone marrow, and develop into several distinct types of T cells once they have migrated in to the thymus gland. T cells were initially grouped into a series of subsets based on their function, but also have been grouped into subsets based on associated gene or protein expression patterns. The T-cell receptor (TCR) is a molecule found on the surface of T cells, and is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR is composed of two different protein chains (i.e., it is a heterodimer). In humans, in the majority of the T cells the TCR consists of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), thus being referred to as αβ T cells. In a small portion of the T cells the TCR consists of a gamma (γ) and a delta (δ) chain (encoded by TRG and TRD, respectively), thus being referred to as γδ T cells. For different classes of T cells, the capacity for differentiation and proliferation increases in the following order: effector T cells, effector memory T cells, central memory T cells, stem cell memory T cells. Conversely, their effector functions, for example, cell-mediated cytotoxicity and cytokine release, decrease in the same order. The clinical efficacy of CAR-T cells is directly correlated with the capacity to proliferate and persist in the body. Thus, a higher memory order allows CAR-T cells to persist in the patient’s body longer and thus exert longer-lasting anti-tumor activity. [0024] The term "chimeric antigen receptor" or the short form “CAR” as used herein refers to artificial receptor proteins, or chimeric immunoreceptors, and encompass engineered receptors that graft an artificial specificity onto a particular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. CARs typically comprise an intracellular activation domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. CARs can combine antibody-based specificity for a desired antigen with a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific cellular immune activity such as anti-tumor cellular immune activity. In some cases, molecules can be co- expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the NK cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. [0025] The term “chimeric co-stimulatory receptor” or the short form “CCR” as used herein refers to artificial receptor proteins, or chimeric immunoreceptors, which assists the full activation of a particular immune effector cell, such as T cell. For example, T cells require two signals to become fully activated, with a first, antigen-specific signal provided by the T cell receptor (TCR), and a second, antigen nonspecific co-stimulatory signal provided through the interaction between co-stimulatory molecules and co-stimulatory receptors. CCRs typically comprise an extracellular domain comprising an antigen binding region, a transmembrane domain, a co-stimulatory signaling domain, but do not comprise an intracellular activation domain. As such, the use of CCR alone does not result in the full activation and generation of the immune effector cells. CCRs typically have to be used together with another recombinant receptor that comprises an intracellular activation domain (such as CAR) in order to fully activate the immune effector cells. [0026] As used herein, the term "antigen" is a molecule capable of being bound by an antibody or cell surface receptor. An antigen may generally be used to induce a humoral immune response and/or a cellular immune response leading to the production of lymphocytes. [0027] Natural-killer group 2, member D, also known as Klrk1 (NKG2D), is a C-type lectin-like receptor, which was firstly identified in NK cells as an activating immune receptor. NKG2D is a type II transmembrane glycoprotein, which does not contain any known signaling elements in the intracellular domain. Resembling many activating receptors, NKG2D depends on an adaptor molecule to initiate signaling transduction and cellular activation. In human, NKG2D is not only expressed by all NK cells, but is also expressed by all CD8⁺ T cells, and subsets of γδ⁺ T cells as a co-stimulatory receptor. NKG2D expression and signaling can be regulated by cytokines and tumor-derived factors. Cytokines, such as IL-2, IL-7, IL-12, IL-15, and type I interferons (IFNs) increase cell surface expression of NKG2D. Cytokines such as IL-21, IFN-γ, and TGF-β have been shown to decrease NKG2D expression. IL-21 has been reported to reduce expressions of NKG2D in human CD8⁺ T cells and NK cells. In mice, IL-21 stimulation of NK cells is dependent on regulating the NKG2D expression in mouse model of breast carcinoma. [0028] Programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a protein that in humans is encoded by the CD274 gene. PD-L1 is a 40kDa type 1 transmembrane protein that acts as a ligand of PD-1. Engagement of PD-L1 with its receptor PD-1 on T cells delivers a signal that inhibits TCR- mediated activation of IL-2 production and T cell proliferation. The binding of PD-L1 to the inhibitory checkpoint molecule PD-1 transmits an inhibitory signal based on interaction with phosphatases (SHP-1 or SHP-2) via Immunoreceptor Tyrosine-Based Switch Motif (ITSM) motif. This reduces the proliferation of antigen-specific T-cells in lymph nodes, while simultaneously reducing apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells) - further mediated by a lower regulation of the gene Bcl-2. Upregulation of PD-L1 may allow cancers to evade the host immune system. [0029] Programmed cell death protein 1, also known as PD-1 and CD279 (cluster of differentiation 279), in humans is encoded by the PDCD1 gene. PD-1 is a cell surface receptor that belongs to the immunoglobulin superfamily and is expressed on T cells and pro- B cells. PD-1 binds two ligands, PD-L1 and PD-L2. PD-1 has a role in regulating the immune system's response to the cells of the human body by down-regulating the immune system and promoting self-tolerance by suppressing T cell inflammatory activity. PD-1 is an immune checkpoint and guards against autoimmunity through two mechanisms. First, it promotes apoptosis (programmed cell death) of antigen-specific T-cells in lymph nodes. Second, it reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells). An exemplary sequence of the extracellular domain of PD-1 is (from the N-terminal to the C- terminal): PGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTD KLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQI KESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 1), encoded by the following exemplary nucleotide sequence:

[0030] The term “single-chain variable fragment” or “scFv” as used herein refers to a fusion protein of the variable regions of the heavy (V_H) and light chains (V_L) of immunoglobulins, connected with a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V_H with the C-terminus of the V_L, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. [0031] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogues thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogues. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labelling component. The term also refers to both double- and single- stranded molecules. Unless otherwise specified or required, a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. [0032] The term “recombinant nucleic acid” as used herein refers to nucleic acid formed by laboratory methods of genetic recombination (such as molecular cloning) to bring together genetic material from multiple sources. The nucleic acid sequences used in the construction of recombinant nucleic acid molecules can originate from any species. For example, human nucleic acid may be joined with bacterial nucleic acid. In addition, nucleic acid sequences that do not occur anywhere in nature may be created by the chemical synthesis of nucleic acid, and incorporated into recombinant molecules. Proteins that can result from the expression of recombinant nucleic acid within living cells are termed recombinant proteins. When recombinant nucleic acid encoding a protein is introduced into a host organism, the recombinant protein is not necessarily produced. Expression of foreign proteins requires the use of specialized expression vectors and often necessitates significant restructuring by foreign coding sequences. [0033] As used herein, the term "vector" refers to a non-chromosomal nucleic acid comprising an intact replicon such that the vector may be replicated when placed within a permissive cell, for example by a process of transformation. A vector may replicate in one cell type, such as bacteria, but have limited ability to replicate in another cell, such as mammalian cells. Vectors may be viral or non-viral. Exemplary non-viral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DNA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defined-length oligopeptides, and polyethylene imine, in some cases contained in liposomes; and the use of ternary complexes comprising a virus and polylysine-DNA. [0034] The terms “transfer” or “transfect” as used herein refer to the general process by which exogenous nucleic acid is introduced into the host cell, said process can be mechanical transfection (including electroporation), chemical transfection or viral transduction. A “transfected” cell is one which has been transfected with exogenous nucleic acid using any of the above mentioned methods. The cell includes the primary subject cell and its progeny. [0035] As used herein, the term “autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual. [0036] As used herein, the term “allogeneic” refers to any material derived from an individual other than the individual to which it is later to be introduced into. [0037] As used herein, "percent identity" refers to sequence identity between two peptides or between two nucleic acid molecules. Percent identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are identical at that position. [0038] As used herein, the terms “peptide”, “polypeptide”, and “protein” are used interchangeably, and refer to a compound having amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can include a protein's or peptide's sequence. Polypeptides include any peptide or protein having two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides, and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof. [0039] As used herein, the phrase "homologous" or "variant" nucleotide sequence, or "homologous" or "variant" amino acid sequence refers to sequences characterized by identity, at the nucleotide level or amino acid level, of at least a specified percentage. Homologous nucleotide sequences include those sequences coding for naturally occurring allelic variants and mutations of the nucleotide sequences set forth herein. Homologous nucleotide sequences include nucleotide sequences encoding for a protein of a mammalian species other than humans. Homologous amino acid sequences include those amino acid sequences which contain conservative amino acid substitutions and which polypeptides have the same binding and/or activity. In some examples, a homologous nucleotide or amino acid sequence has at least 60% or greater, for example at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99%, with a comparator sequence. In some examples, a homologous nucleotide or amino acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a comparator sequence. In some examples, a homologous amino acid sequence has no more than 15, or no more than 10, or no more than 5 or no more than 3 conservative amino acid substitutions. Percent identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman. In some examples, the recombinant nucleic acid molecules used to modify the T cells according to the present disclosure are homologous to the exemplary nucleotide sequences disclosed herein, such as the sequences provided in any of SEQ ID NO: 24, 26, 28 and 30. In some other examples, the chimeric antigen receptor (CAR) and/or chimeric co- stimulatory receptor (CCR) expressed in the modified T cells are homologous to the exemplary amino acid sequences disclosed herein, such as the sequences provided in any of SEQ ID NO: 23, 25, 27 and 29. [0040] The term “express” refers to the production of a gene product in a cell. [0041] The term "transient" when referred to expression means a polynucleotide is not incorporated into the genome of the cell. In contrast, the term “stable” when referred to expression means a polynucleotide is incorporated into the genome of the cell. Transient expression can occur from introduced constructs which contain expression signals functional in the host cell, but which constructs do not replicate and rarely integrate in the host cell, or where the host cell is not proliferating. Transient expression also can be accomplished by inducing the activity of a regulatable promoter operably linked to the gene of interest, although such inducible systems frequently exhibit a low basal level of expression. Stable expression can be achieved by introduction of a nucleic acid construct that can integrate into the host genome or that autonomously replicates in the host cell. Stable expression of the gene of interest can be selected for through the use of a selectable marker located on or transfected with the expression construct, followed by selection for cells expressing the marker. When stable expression results from integration, integration of constructs can occur randomly within the host genome or can be targeted through the use of constructs containing regions of homology with the host genome sufficient to target recombination with the host locus. Where constructs are targeted to an endogenous locus, all or some of the transcriptional and translational regulatory regions can be provided by the endogenous locus. To achieve expression in a host cell, the transformed nucleic acid is operably associated with transcriptional and translational initiation and termination regulatory regions that are functional in the host cell. [0042] The term “tumor” as used herein refers to a swelling of a part of the body, generally without inflammation, caused by an abnormal growth of tissue. Tumors can be benign or malignant (i.e cancerous). A benign tumor does not invade nearby tissue or spread to other parts of the body. Common types of benign tumors include adenomas, fibromas (or fibroids), hemangiomas, lipomas, meningiomas, myomas, neuromas, and osteochondromas. Adenomas are benign tumors starting in the epithelial tissue of a gland or gland-like structure. A common type of adenoma is a polyp in the colon. Adenomas might also grow in the liver or the adrenal, pituitary, or thyroid gland. Fibromas (or fibroids) are tumors of fibrous or connective tissue that can grow in any organ. Hemangiomas are a buildup of blood vessel cells in the skin or internal organs. Lipomas grow from fat cells. They are the most common benign tumor in adults, often found in the neck, shoulders, back, or arms. Meningiomas are tumors that develop from the membrane surrounding the brain and spinal cord. Myomas are tumors that grow from muscle. Neuromas are tumors that develop from the nerves. Osteochondromas are tumors that develop from the bones. [0043] The term “cancer” as used herein refers to an unregulated proliferation of cells due to loss or normal controls, resulting in unregulated growth, lack of differentiation, local tissue invasion, and, often, metastasis. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord. DETAILED DESCRIPTION OF THE PRESENT INVENTION [0044] The inventors of the present application have found that after genetically modifying T cells to express a recombinant CAR comprising the extracellular domain of an NKG2D receptor, and a recombinant CCR specifically targeting PD-L1, the genetically modified T cells facilitate more precise cancer targeting by displaying specificity towards cancer cells that express both PD-L1 and NKG2D ligand(s), thus minimizing the on-target off-tumor risk associated with CAR-T cell therapy. The inventors have also found that such genetically modified T cells can evade a major immunosuppressive mechanism within tumor microenvironment (TME). [0045] Thus, in one example, the present invention refers to a T cell genetically modified to express: i) a recombinant chimeric antigen receptor (CAR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a co- stimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of an NKG2D receptor; and ii) a recombinant chimeric co-stimulatory receptor (CCR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) a co-stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv). [0046] In some examples, the extracellular domain of the recombinant CAR is the extracellular domain of an NKG2D receptor. In one example, the extracellular domain of the NKG2D receptor has the following amino acid sequence (from the N-terminal to the C- terminal):

, encoded by the following exemplary nucleotide sequence:

NKG2D ligands are structural

homologs of MHC class I molecules. NKG2D ligands are absent or rarely expressed in normal tissues, but are extensively expressed in various malignancies and viral-infected tissues. Examples of human NKG2D ligands include class-I-related chains-related molecules A and B (MICA and MICB) proteins and retinoic acid early transcripts-1 (RAET1), also known as UL-16 binding proteins. Examples of mouse NKG2D ligands include five different RAET1 isoforms (RAET1α, RAET1 β, RAET1 γ, RAET1 δ, and RAET1 ε), three different H60 isoforms (H60a, b, and c), and UL16 binding protein 1 (encoded by MULT1 gene). Although NKG2D ligands are structural homologs of MHC class I molecules, they do not present antigen to T cells or bind β2-microglobulin. In some examples, the NKG2D ligands for which the extracellular domain of the chimeric antigen receptor binds to are membrane- bound ligands. [0047] In some examples, the extracellular domain of the recombinant CCR is a PD-L1 specific scFv, referred to as αPD-L1 scFv. In one specific example, the PD-L1 scFv has the following amino acid sequence (from the N-terminal to the C-terminal):

NWPRTFGQGTKVEIKAS (SEQ ID NO: 5), encoded by the following exemplary nucleotide sequence:

. In one specific example, the heavy chain

variable domain of the PD-L1 scFv has the following amino acid sequence (from the N- terminal to the C-terminal):

. In one specific example, the light chain variable domain of the PD-L1 scFv has the following amino acid sequence (from the N-terminal to the C-terminal):

9), encoded by the following exemplary nucleotide sequence:

( Q NO: 10). The heavy chain variable domain and the light chain variable domain of the PD-L1 scFv can be linked by a linker peptide. In one specific example, the heavy chain variable domain and the light chain variable domain of the PD-L1 scFv are linked by a (G4S)₃ linker peptide having the following amino acid sequence (from the N-terminal to the C-terminal): GGGGSGGGGSGGGGS (SEQ ID NO: 11), encoded by the following exemplary nucleotide sequence:

(SEQ ID NO: 12). [0048] The extracellular domain of a recombinant chimeric antigen receptor (CAR) and/or a recombinant chimeric co-stimulatory receptor (CCR) can also comprise a hinge region. The hinge region is a sequence positioned between for example, the antigen binding region, and the transmembrane domain. The sequence of the hinge region can be obtained from, for example, any suitable sequence from any genus, including human or a part thereof. In some examples, the hinge region includes the hinge region of a human protein including CD3, CD-8 alpha, CD28, 4-1BB, OX-40, T cell receptor α or β chain, a CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof. In some specific examples, the hinge region is the hinge region of CD3, CD8 or CD28. In some examples, the hinge region of the recombinant CAR and the hinge region of the recombinant CCR are the same. In some other examples, the hinge region of the recombinant CAR and the hinge region of the recombinant CCR are different. In some examples, the hinge region can be one selected from, but is not limited to, immunoglobulin (e.g. IgG1, IgG2, IgG3, IgG4, and IgD). [0049] It is understood that the antigen binding region may include some variability within its sequence and still be selective for the targets disclosed herein. Therefore, it is contemplated that the polypeptide of the antigen binding region may be at least 95%, at least 90%, at least 80%, or at least 70% identical to the antigen binding region polypeptide sequences disclosed herein and still be selective for the targets described herein and be within the scope of the disclosure. [0050] The transmembrane domain of a recombinant CAR and/or a recombinant CCR includes a hydrophobic polypeptide that spans the cellular membrane. In particular, the transmembrane domain spans from one side of a cell membrane (extracellular) through to the other side of the cell membrane (intracellular or cytoplasmic). [0051] In some examples, the transmembrane domain is artificially designed so that more than 25%, more than 50% or more than 75% of the amino acid residues of the domain are hydrophobic residues such as leucine and valine. [0052] The transmembrane domain may be in the form of an alpha helix or a beta barrel, or combinations thereof. The transmembrane domain may include a polytopic protein, which has many transmembrane segments, each alpha-helical, beta sheets, or combinations thereof. [0053] In one example, the transmembrane domain that is naturally associated with one of the domains in the CAR or CCR is used. In another example, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. [0054] For example, a transmembrane domain includes a transmembrane domain of a T- cell receptor α or β chain, a CD3ζ chain, CD3ε, CD8, CD45, CD4, CD5, CD7, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD68, CD134, CD137, ICOS, CD41, CD154, functional derivatives thereof, and combinations thereof. In some examples, the transmembrane domain of the recombinant CAR and the transmembrane domain of the recombinant CCR are the same. In some other examples, the transmembrane domain of the recombinant CAR and the transmembrane domain of the recombinant CCR are different. In one specific example, the transmembrane domain of the recombinant CAR is a CD28 transmembrane domain. In one specific example, the transmembrane domain of the recombinant CAR has the following amino acid sequence (from the N-terminal to the C- terminal): ESKYGPPCPPCPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 13), encoded by the following exemplary nucleotide sequence:

In one specific example, the transmembrane domain of the

recombinant CCR is a CD8 transmembrane domain. In one specific example, the transmembrane domain of the recombinant CCR has the following amino acid sequence (from the N-terminal to the C-terminal):

encoded by the following exemplary nucleotide sequence:

[0055] The intracellular signaling domain of a recombinant CAR is responsible for activation of at least one of the normal effector functions of the immune cell in which the recombinant CAR has been placed. The term "effector function" refers to a specialized function of a differentiated cell, such as a T cell. The intracellular signaling domain generally includes at least one immunoreceptor tyrosine based activation motif (ITAM)-containing domain. Each ITAM possesses two repeats of the consensus sequence Tyr-X-X-Leu/Ile (X being any amino acid) spaced by six to eight amino acids. The tyrosine residues within ITAM become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signaling pathways of the cell. In some examples, the intracellular signaling domain of the recombinant CAR comprises only one ITAM, or not more than one ITAM. In some examples, the T cells that are genetically modified to express a recombinant CAR containing only one ITAM in the intracellular signaling domain release lower levels of cytokines as compared to T cells expressing a recombinant CAR containing more than one ITAM in the intracellular signaling domain. In some examples, intracellular signaling domains in the CAR include part or all of CD3ζ, or DAP molecules such as DAP 12, or a combination thereof. In one specific example, the intracellular signaling domain used is CD3ζ. In one specific example, the intracellular signaling domain used is DAP12. In one specific example, the intracellular signaling domain of the CAR has the following amino acid sequence (from the N-terminal to the C-terminal): YFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK (SEQ ID NO: 17), encoded by the following exemplary nucleotide sequence:

NO: 8). [0056] In some examples, the recombinant CAR disclosed herein does not comprise a co- stimulatory signaling domain, such as OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; Natural killer Group 2 member C (NKG2C); Natural killer Group 2 member D (NKG2D), B7-H3; a ligand that binds to at least one of CD83, ICAM-1, LFA-1 (CDl la/CD18), ICOS, and 4-1BB (CD137); CDS; ICAM-1; LFA-1 (CD1a/CD18); CD40; CD27; CD7; B7-H3; NKG2C; PD-1; ICOS; active fragments thereof; functional derivatives thereof; and combinations thereof. [0057] In some examples, between the extracellular domain and the transmembrane domain of the recombinant CAR and/or the recombinant CCR, or between the intracellular signaling domain and the transmembrane domain of the recombinant CAR, there is incorporated a spacer domain. As used herein, the term "spacer domain" generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the intracellular domain in the polypeptide chain, A spacer domain can comprise up to about 300 amino acids, or about 10 to about 100 amino acids, or about 25 to about 50 amino acids. [0058] In one specific example, the desired recombinant CAR in the genetically modified T cell comprises NKG2D receptor, CD28 transmembrane domain, and DAP12 intracellular signaling domain. In one specific example, the desired recombinant CCR in the genetically modified T cell comprises αPD-L1 scFv, CD8 transmembrane domain, and 4-1BB co- stimulatory signaling domain. [0059] The terms “CD3ζ”, “CD3zeta”, “CD3z” are used interchangeably herein to refer to the same T-cell surface glycoprotein CD3 zeta chain. [0060] The co-stimulatory signaling domain of a recombinant CCR can enhance the proliferation, survival and/or development of the T cells. Examples of co-stimulatory signaling domains include but are not limited to: 4-1BB, CD27, CD28, OX-40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, functional derivatives thereof, and combinations thereof. In one specific example, the co-stimulatory signaling domain is 4-1BB. 4-1BB, also known as CD137, is a member of the tumor necrosis factor (TNF) receptor family. In one specific example, the co- stimulatory signaling domain of the CCR has the following amino acid sequence (from the N- terminal to the C-terminal):

Q Q Q ( Q 19), encoded by the following exemplary nucleotide sequence:

In another specific example, the co-stimulatory signaling domain is CD28. [0061] In some examples, in order to generate the recombinant CAR and/or the recombinant CCR, a signal peptide is added to the 5’ end of the recombinant CAR/CCR construct. In one specific example the signal peptide is a GM-CSF signal peptide, having the sequence (from the N-terminal to the C-terminal): MLLLVTSLLLCELPHPAFLLIPGAHA (SEQ ID NO: 21), encoded by the exemplary nucleotide sequence

( Q ) [0062] In one example, the recombinant CAR without the signal peptide has the following sequence (from the N-terminal to the C-terminal):

CCG C C CCC CGGCC C C (S Q NO: ). In one example, the recombinant CAR with the signal peptide has the following sequence (from the N- terminal to the C-terminal):

nucleotide sequence

[0063] In one example, the recombinant CCR without the signal peptide has the following sequence (from the N-terminal to the C-terminal):

KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 27), encoded by the exemplary nucleotide sequence

In one example, the recombinant CCR with the signal peptide has the following sequence (from the N-terminal to the C-terminal):

(SEQ ID NO: 29), encoded by the exemplary nucleotide sequence

[0064] In some examples, in order to measure the surface expression level of the recombinant CAR, a peptide tag is added to the recombinant CAR construct. In one example, the peptide tag is added between the extra-cellular domain and the transmembrane domain. In one specific example, the peptide tag is a ST2 tag, having the sequence (from the N-terminal to the C-terminal): NWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGGGGSNWSHPQFEKGGGGSGG GGS (SEQ ID NO: 31), encoded by the exemplary nucleotide sequence

[0065] Expression of the recombinant CAR and/or the recombinant CCR in the genetically modified T cells can be transient or stable. In some examples, the constructs in each of Fig. 1A and 1B are transiently or stably expressed in the genetically modified T cells. [0066] In some examples, the genetically modified T cells as disclosed herein target cancer or tumor cells that express both NKG2D ligand(s) and PD-L1. The binding of the NKG2D ligand(s) to the extracellular domain of the recombinant CAR activates the genetically modified T cells, which then proceed to proliferate, synthesize and secrete cytokines. The binding of PD-L1 expressed on the tumor cells to the extracellular domain of the recombinant CCR activates the co-stimulatory signaling domain of the recombinant CCR, thus enhancing the proliferation, survival and/or development of the T cells, and promoting the synthesis and secretion of cytokines. Upon binding to both PD-L1 and NKG2D ligand(s) expressed by the target tumor cells, the genetically modified T cells become fully activated and functional. [0067] The genetically modified T cells as described herein can be provided as a composition or a pharmaceutical composition. Thus, in one example, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of the T cell as disclosed herein and a pharmaceutically acceptable excipient. The compositions as described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired. Administration may be topical, pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal) or systemic such as oral, and/or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. In some examples, the route of administration may be selected from the group consisting of systemic administration, oral administration, intravenous administration and parenteral administration. [0068] A composition or pharmaceutical composition as described herein can be provided in unit dosage form wherein each dosage unit, e.g., an injection, contains a predetermined amount of the T cells as disclosed herein, alone or in appropriate combination with other active agents. The term “unit dosage form” as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the T cells as disclosed herein, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically acceptable diluent, carrier, or vehicle, where appropriate. The specifications for the unit dosage forms depend on the particular pharmacodynamics associated with the pharmaceutical composition in the particular subject. Unit dosage forms can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid or semi- liquid carriers. [0069] The compositions as described herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritic, astringents, local anaesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as buffer, dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers or combination thereof appropriate for use with the pharmacologically active agent that may be added to solution in any concentration suitable for use in eye drops. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colourings, flavourings and/or aromatic substances and the like which do not deleteriously interact with the T cells of the formulation. [0070] In another example, there is provided a method of preparing the T cell genetically modified to express a recombinant CAR and a recombinant CCR as disclosed herein, the method comprises: a) obtaining or providing T cell; b) providing i) a recombinant nucleic acid encoding the recombinant CAR; and ii) a recombinant nucleic acid encoding the CCR; and c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the CCR into the T cell. In some examples, the method further comprises, after step a), culturing the T cells to expand the number of T cells. T cell expansion methods known in the art can be used. In some examples, the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR are transferred into the T cells simultaneously. In some examples, the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR are transferred into the T cells simultaneously, the recombinant nucleic acids encoding the recombinant CAR and the recombinant CCR are cloned into the same vector. In some other examples, the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR are transferred into the T cells sequentially. [0071] A recombinant nucleic acid encoding the recombinant CAR and/or the recombinant CCR can be created using methods known in the art. A base sequence encoding an amino acid sequence can be obtained from the NCBI RefSeq IDs or accession numbers of GenBenk for an amino acid sequence of each domain, and the nucleic acid as disclosed herein can be prepared using a standard molecular biological and/or chemical procedure. For example, based on the base sequence, a polynucleotide can be synthesized, and the polynucleotide of the present disclosure can be prepared by combining DNA fragments which are obtained from a cDNA library using a polymerase chain reaction (PCR). The sequence of the open reading frame encoding the CAR and/or the CCR can be obtained from a genomic DNA source, a cDNA source, or can be synthesized (e.g. via PCR), or combinations thereof. Depending upon the size of the genomic DNA and the number of introns, it may be desirable to use cDNA or a combination thereof as it is found that introns stabilize the mRNA. Also, it may be further advantageous to use endogenous or exogenous non-coding regions to stabilize the mRNA. [0072] The nucleotide sequence encoding for the recombinant CAR and/or the recombinant CCR can be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence. A variety of host-vector systems may be utilized to express the protein- coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.), insect cell systems infected with virus (e.g., baculovirus), microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA, transgenic plants or transgenic non-human animals. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used. [0073] Any of the known methods for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric nucleotide sequence consisting of appropriate transcriptional/translational control signals and the protein coding sequences. Exemplary methods include in vitro recombinant DNA and synthetic techniques. Expression of a recombinant nucleic acid sequence encoding the recombinant CAR and/or the recombinant CCR may be regulated by a second nucleic acid sequence so that the recombinant CAR and/or the recombinant CCR is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the recombinant CAR and/or the recombinant CCR may be controlled by any promoter/enhancer element known in the art. [0074] In some examples, the basic backbone of the recombinant expression vector is a commercially available vector into which each of the above elements is inserted. Exemplary expression vectors included but are not limited to pFastbac1, pALTER-Ex1, pALTER-Ex2, pCal-n, pCal-n-EK, pCal-c, pCal-Kc, pcDNA 2.1, pDUAL, pET-3a-c, pET-9a-d, pET-11a-d, pET-12a-c, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b(+),pET-21a-d(+), pET-22b(+), pET-23a-d(+), pET-24a-d(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28a- c(+), pET-29a-c(+), pET-30a-c(+), pET-31b(+), pET-32a-c(+), pET-33b(+), pET-34b(+), pET-35b(+), pET-36b(+), pET-37b(+), pET-38b(+), pET-39b(+), pET-40b(+), pET-41a-c(+), pET-42a-c(+), pET-43a-c(+), pETBlue-1, pETBlue-2, pETBlue-3, pGEMEX-1, pGEMEX-2, pRSET, pTriEx-1, and pTriEx-2 expression vectors. [0075] Methods generally known in the field can be used for the transcription of mRNA from the recombinant expression vectors described above. For example, PCR can be performed using the recombinant expression vectors as the DNA template to generate the linear DNA template. In vitro transcription of the linear DNA template generated can then be used to generate the recombinant CAR and/or the recombinant CCR mRNA. Optionally, the linear DNA template is purified before in vitro transcription is carried out. [0076] The nucleic acid encoding the recombinant CAR and/or the recombinant CCR can be cloned into the expression vector using molecular cloning techniques commonly known in the art. [0077] The expression vector(s) comprising the recombinant nucleic acid encoding the recombinant CAR and/or the recombinant CCR can be transferred into the T cell using any techniques known in the art, such as electroporation, non-viral chemical transfection, and viral transduction. In one particular example, the T cells are genetically modified to express the recombinant CAR and recombinant CCR by electroporation. [0078] The genetically modified T cells of the present invention provide improved T cell proliferation, reduced expression of T cell exhaustion markers, and enhanced in vivo tumor killing, as compared to T cells genetically modified to express recombinant NKG2D CAR and recombinant PD-1 chimeric switch receptor (CSR), as evidenced by the data presented in the Experimental Section. In some examples, the T cells genetically modified to express the recombinant CAR and the recombinant CCR are able to replicate in vivo resulting in long- term persistence that can lead to sustained tumor control. [0079] In some examples, a medical disease or disorder can be treated by administering a population of the T cell as disclosed herein. In some examples, the medical disease or disorder is cancer or tumor. In another example, there is provided a method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the T cell as disclosed herein, or the pharmaceutical composition as disclosed herein. In another example, there is provided use of a pharmaceutically effective amount of the T cell as disclosed herein in the manufacture of a medicament for treating cancer or tumor. In yet another example, there is provided the T cells or the pharmaceutical composition as disclosed herein for use in treating cancer or tumor. In some examples, the genetically modified T cells used to treat cancer or tumor are prepared during the course of treatment. Thus, in one example, there is provided a method of treating cancer or tumor in a subject in need thereof, the method comprises: a) obtaining T cells from the subject, or from a donor which is different from the subject to be treated; b) providing i) a recombinant nucleic acid encoding a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, , wherein the recombinant CAR does not comprise a co-stimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of an NKG2D receptor; and ii) a recombinant nucleic acid encoding a recombinant chimeric co- stimulatory receptor (CCR), wherein the recombinant CCR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) a co- stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv); c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR into the T cell to obtain genetically modified T cells; and d) administering to the subject a pharmaceutically effective amount of the T cells obtained from c). In some other examples, the method further comprises, after step a), culturing the T cells to expand the number of T cells. T cell expansion methods are known in the art and exemplified in the Experimental Section of the present application. [0080] The term “pharmaceutically effective amount” as used herein includes within its meaning a sufficient but non-toxic amount of the T cells as described herein to provide the desired treatment effect. Desirably an effective amount or sufficient number of the T cells as described herein is present in the composition and introduced into the subject such that long- term, specific, anti-tumor responses are established to reduce the size of a tumor or eliminate tumor growth or regrowth than would otherwise result in the absence of such treatment. Desirably, the amount of T cells introduced into the subject causes at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 100% decrease in tumor size when compared to otherwise same conditions wherein the T cells are not present. The exact amount of T cells required will vary from subject to subject depending on factors such as the species being treated, the age and general condition of the subject, the severity of the condition being treated (e.g. the stage and /or size of the tumor), the mode of administration, and so forth. In general, the concentration of T cells desirably should be sufficient to provide in the subject being treated at least from about 1×10⁶ to about 1×10⁹ T cells, even more desirably, from about 1×10⁷ to about 5×10⁸ T cells, although any suitable amount can be utilized either above, e.g., 5×10⁸ cells, or below, e.g., 1×10⁷ cells. The dosing schedule can be based on well-established cell-based therapies, or an alternate continuous infusion strategy can be employed. These values provide general guidance of the range of T cells to be utilized by the practitioner upon optimizing the method of treatment as disclosed herein. The recitation herein of such ranges by no means precludes the use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule can vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on inter-individual differences in pharmacokinetics, drug disposition, and metabolism. In any given case, an appropriate “effective amount” may be determined by one of ordinary skill in the art using only routine experimentation. [0081] Cancers or tumors for which the present treatment methods are useful include any malignant cell type, such as those found in a solid tumor or a hematological tumor. In some specific examples, the malignancy is a solid tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon, cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast. In some specific examples, the malignancy is a hematological tumor. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma. The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra-mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia. In some examples, the cancer/tumor that can be treated using the genetically modified T cells provided herein include colorectal cancer, ovarian cancer, head and neck cancer, liver cancer, breast cancer, cervical cancer and glioma. In one specific example, the cancer/tumor that can be treated is colorectal cancer. [0082] In some examples, the cancer/tumor expresses both NKG2D ligand and PD-L1. In some other examples, the cancer/tumor expresses NKG2D ligand and PD-L1 after treatment with another drug, radiation or biological agent. [0083] The terms “treat”, “treatment”, and grammatical variants thereof, refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder or disease or obtain beneficial or desired clinical results. Such beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of extent of condition, disorder or disease; stabilized (i.e. not worsening) state of condition, disorder or disease; delay or slowing of condition, disorder or disease progression; amelioration of the condition, disorder or disease state, remission (whether partial or total), whether detectable or undetectable; or enhancement or improvement of condition, disorder or disease. Treatment includes eliciting a cellular response that is clinically significant, without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment. [0084] The terms "decrease", "reduced", "reduction", "decrease", “removal” or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, "reduced", "reduction" or "decrease", "removal", or "inhibit" means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level (e.g., in the absence of a treatment as described herein). [0085] In some examples, the subject or patient to be treated is an animal, mammal, human, including, without limitation, animals classed as bovine, porcine, equine, canine, lupine, feline, murine, ovine, avian, piscine, caprine, corvine, acrine, or delphine. In one example, the patient is a human. [0086] The source of T cells that can be used for treating a medical disease or disorder may be of any kind, but in specific examples the cells are obtained from a bank of umbilical cord blood, peripheral blood, human embryonic stem cells, or induced pluripotent stem cells. [0087] In some examples, the T cells that can be used for treating a medical disease or disorder are autologous, i.e. obtained from the same individual to which the treatment is to be administered. For example, autologous source of T cells can be collected from a patient in need of treatment and T cells are activated and modified using the methods described herein and known in the art and then infused back into the patient. Some autologous source of T cells include PBMCs, umbilical cord blood obtained when the patient was born and subsequently preserved, and induced pluripotent stem cells derived from cells obtained from the patient. [0088] In some other examples, the T cells that can be used for treating a medical disease or disorder are allogeneic, i.e. derived from a different individual of the same species as the patient, such as a T cell donor. In some other examples, the T cells that can be used for treating a medical disease or disorder are xenogeneic, i.e. derived from an animal of a different species as the patient. Genetically modified T cells derived from allogeneic xenogeneic sources can provide an off-the-shelf product. [0089] Allogeneic or autologous T cells induce a rapid immune response but disappear relatively rapidly from the circulation due to their limited lifespan. Thus, there is reduced concern of persisting side effects using the treatment methods as disclosed herein. [0090] In certain examples, the T cells as described herein are administered in combination with a second therapeutic agent. For example, the second therapeutic agent may comprise an immunomodulatory agent, a monoclonal antibody, or a chemotherapeutic agent. In non- limiting examples, the immunomodulatory agent is lenolidomide, the monoclonal antibody is alemtuzumab, rituxumab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, adotranstuzumab, blinatunomab, daratumumab or elotuzumab, and the chemotherapeutic agent is fludarabine or cyclophosphamide. [0091] Following administration of the genetically modified T cells as disclosed herein for treating or preventing a cancer/tumor, the efficacy of the treatment can be assessed in various ways well known to the skilled practitioner. For instance, one of ordinary skill in the art will understand that a therapeutic genetically modified cell delivered in conjunction with the chemo-adjuvant is efficacious in treating or inhibiting a cancer in a patient by observing that the therapeutic genetically modified cell reduces the cancer cell load or prevents a further increase in cancer cell load. Cancer cell loads can be measured by methods that are known in the art, for example, using polymerase chain reaction assays to detect the presence of certain cancer cell nucleic acids or identification of certain cancer cell markers in the blood using, for example, an antibody assay to detect the presence of the markers in a sample (e.g., but not limited to, blood) from a subject or patient, or by measuring the level of circulating cancer cell antibody levels in the patient. [0092] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [0093] As used in this application, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a genetic marker” includes a plurality of genetic markers, including mixtures and combinations thereof. [0094] As used herein, the term “about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value. [0095] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [0096] Certain embodiments may also be described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the disclosure. This includes the generic description of the embodiments with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [0097] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein. [0098] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. EXPERIMENTAL SECTION [0099] Methods and Materials [00100] Cell lines and culture conditions [00101] Human K562 myelogenous leukaemia feeder cells engineered to express CD64, CD86 and CD137L were maintained in IMDM. HepG2 and Detroit-562 were maintained in DMEM. CAOV3 was maintained in RPMI. All types of medium were supplemented with 10% FBS (Gibco). [00102] CAR-T cell preparation [00103] PBMCs of healthy donors were isolated by gradient centrifugation from buffy coats obtained from Health Sciences Authority (HSA, Singapore) with approved IRB. PBMCs were seeded at 5×10⁶ cells/ml in T cell media (AIM-V + 5% AB serum) and activated for 2-3 days with soluble OKT3 (Ebioscience) at 1 µg/ml or TransAct (Miltenyi Biotec, Germany), 100 µl of TransAct was added for every 1×10⁷ PBMCs. Recombinant IL-2 (300 IU/ml; Peprotech) was at the start of the culture and replenished every other day. [00104] On day 2 or 3, activated T cells were harvested and transduced with a non-viral electroporation method. Modified T cells were cultured in AIM-V supplemented with either 5% AB serum or 1% human plasma and 300IU/ml IL-2 for a further five days. On day 8, genetically modified T Cells were numerated and co-cultured with γ-irradiated K562 or K562#6 cells at 1:2 ratio in AIM-V medium supplemented with 300 IU/ml of IL-2. Re- stimulation of CAR T cells was done every 10 days with addition of new γ-irradiated K562 cells at 1:2 ratio. For propagation of non-modified T cells, T cells were co-cultured with γ- irradiated K562#6 at a 1:50 ratio supplemented with 60 ng/ml of OKT3 at the start of the co- culture. 300 IU/ml of IL-2 was added at the start of the culture and replenished every other day. [00105] CD56+ cells were removed from day 17 CAR-T cell cultures based on manufacturer’s protocol (Miltenyi Biotec, Germany). Briefly, up to 1×10⁷ cells were suspended in 80 µl of autoMACS running buffer and incubated with 20µl of CD56 microbeads at 4°C in the dark for 15 min. Cells were centrifuged at 300g for 10min and resuspended in 500µl of autoMACS running buffer. LS columns were placed in a MACS MultiStand and equilibrated by rinsing twice with 1ml of autoMACS running buffer. Cell suspensions were introduced into the LS columns to capture tagged CD56+ cells. CD56- flow-through fraction was collected at the bottom of the column in a 15ml conical centrifuge tube (BD Biosciences, USA). [00106] Flow cytometry [00107] For phenotypic analysis of T cells, the following conjugated anti-human antibodies were used: CD3 (clone: OKT3; eBioscience), CD45RO (clone: UCHL1; BD Biosciences), CCR7 (clone: 3D12; eBioscience), PD-1 (clone: MIH4; BD Pharmingen), TIGIT (clone: MBSA43; eBioscience), TIM3 (clone: F38-2E2; eBioscience) and LAG3 (clone: 3DS223H; eBioscience). Appropriate isotype controls were used to validate gating. The expression of NKG2D CAR (which included a Strep Tag) on T cell was determined using THETM NWSHPQFEK Tag Antibody (Genescript) at day 5 post electroporation and day 12 after one week of co-culture with γ-irradiated K562#6. Flow cytometry analysis was performed and analyzed using BD Accuri™ C6 (BD Biosciences). [00108] In vitro cytotoxicity assay [00109] The cytolytic activity of CAR-modified T cells was examined with the DELFIA EuTDA Cytotoxicity Reagents kit (PerkinElmer). The effector to target (E:T) ratios used ranged from 20:1 to 1:1. Control groups were set up to measure spontaneous release (only target cells added), maximum release (target cells added with 10 µl lysis buffer), and medium background (no cell added). Killing efficacy was calculated by using the following formula: [00110] % Specific release = (Experimental release (counts) -Spontaneous release (counts))/(Maximum release (counts) -Spontaneous release (counts))×100 [00111] In vivo experiments [00112] Animal experiments were performed according to protocols reviewed and approved by Institutional Animal Care and Use Committee (IACUC), the Biological Resource Centre (BRC), the Agency for Science, Technology and Research (A*STAR), Singapore (Permit number BRC IACUC#181324). Non-obese diabetic/severe combined immuno- deficiency/IL-2Rγcnull (NSG) mice (8-10 weeks old, male, The Jackson Laboratory) were maintained and used in the current study. All luminescent signals and images were acquired and analyzed with the Xenogen living imaging software v3.2. [00113] To establish a mouse colorectal cancer (CRC) xenograft model, mice were injected i.p. with 2×10⁶ HCT116-luc human colorectal cancer cells on day 0. On day 7 post-tumor inoculation, tumor engraftment was confirmed by live bioluminescence imaging (BLI) monitored using an IVIS Spectrum Imaging platform with Living Image software (PerkinElmer). Mice with similar BLI signal intensity randomly divided into 4 different treatment groups containing 5 mice per group. On day 7, mice were treated via i.p. injection of 100 µl of PBS or 100 µl cell suspension containing 1×10⁷ T cells of three different group: (1) Control T cell, (2) NKG2D CAR+ PD-1 CSR dual CAR-modified T cells or (3) NKG2D CAR+ αPD-L1 CCR dual CAR-modified T cells. A second injection at the same dose (1×10⁷ T cells/mice) was given at day 32 post tumor inoculation. Tumor progression was monitored by BLI every week. Mice were monitored closely and humanely euthanized after observing the development of moribund condition characterized by obvious abdominal bloating due to ascites, palpable hypothermia, inability to walk, and/or lack of overt response to manipulation. [00114] Statistics [00115] For in vitro and in vivo experiments, we used unpaired Student’s t test to evaluate continuous variable of 2 groups, and 1-way ANOVA with post-test Bonferroni to evaluate continuous variables of more than 2 groups. Survival was analyzed by the Kaplan-Meier method and the log-rank (Mantel-Cox) test to compare pairs of groups. Statistics were computed using GraphPad Prism 7.0 (GraphPad Software). Differences were considered significant when the P value was less than 0.05. [00116] Results [00117] To improve the in vivo persistence of tumor-reactive T cells, the use of a PD-1- based CSR has been investigated and validated in several studies, either in combination with a tumor-reactive TCR or CAR. However, PD-1 naturally recognizes both PD-L1 and PD-L2, of which the expression pattern of PD-L2 is mostly restricted to cells of the myeloid lineage while PD-L1 is more commonly found to be upregulated on various tumors. As such, there exists a need to further improve the tumor-specificity of this approach. In the present invention, a first generation CAR was constructed by fusing the extracellular domain of NKG2D receptor with an activation domain derived from DAP12 (Fig. 1A). To specifically target PD-L1, an αPD-L1 CCR that only recognizes PD-L1 but not PD-L2 was generated (Fig. 1B). In parallel, a PD-1-based CSR for comparative studies was also generated (Fig. 1C). In both constructs, the antigen-targeting moiety was expressed in tandem with a single co-stimulatory domain derived from 4-1BB. Hereinafter, the combination of NKG2D CAR and αPD-L1 CCR will be referred to as CAR-T1, whereas the combination of NKG2D CAR and PD-1 CSR will be referred to as CAR-T2. [00118] To evaluate the functionality of CAR-T1 and CAR-T2 cells, beads-activated T cells were co-electroporated with plasmids coding for piggyBac transposase, NKG2D CAR and either αPD-L1 CCR or PD-1 CSR. To expand and enrich for NKG2D CAR-expressing T cells, these T cells were co-cultured with the human K562 myelogenous leukaemia cell line at an effector to target (E:T) ratio of 1:2. Expanded CAR-T1 and CAR-T2 cells were then used as effector cells in a 2-hour cytotoxicity assay to assess their cytotoxicity against cancer cells bearing NKG2D and PD-1 ligands. Three different cancer cell lines with various expression levels of PD-1 ligands were used: HepG2 expresses only NKG2DL but neither of the PD-1 ligands; CAOV3 expresses NGK2DL and PD-L1; Detroit-562 expresses NKG2DL and both PD-1 ligands. As shown in Fig. 2, there was no observable statistical difference in the cytotoxicity exerted by CAR-T1 and CAR-T2 cells against each of these three target cell lines. [00119] To evaluate the potential for large-scale manufacturing, CAR-T1 and CAR-T2 cells were serially expanded with a fresh batch of γ-irradiated K562 feeder cells every 10 days in the presence of IL-2 alone. The increase in cell numbers were enumerated across a period of 30 days and observed robust expansion of these CAR-T cells (Fig. 3). While there was no observable difference in the cytotoxicity between NKG2D CAR-T cells engrafted with either αPD-L1 CCR or PD-1 CSR, there was an improvement in the total cell yield for CAR-T1 as compared to CAR-T2 when co-cultured with K562 feeder cells (Fig. 3). This trend of improvement was consistent across all three donors tested. [00120] Next, it was evaluated how the incorporation of αPD-L1 CCR could further improve the development of memory T cell subsets. Using CCR7 and CD45RA as markers for classifying the various memory subsets, the co-expression of these two markers on CAR- T1 and CAR-T2 cells in the middle and at the end of the expansion phase were evaluated. As seen in Fig. 4, development of memory T cells differed in the two CAR-T groups. Comparing the compositions of T cells in the middle of expansion against the end of expansion, the proportion of effector T cells in CAR-T2 increased slightly from 33.2% to 34.5% whereas in CAR-T1, this proportion decreased from 26.6% to 15.2%. Conversely, the percentage of effector memory T cells decreased from 66.2% to 64.9% in CAR-T2 but increased from 73.3% to 84.6% in CAR-T1. While there were no detectable levels of either central memory T or stem cell memory T cells, Fig. 4 shows a clear divergence in effector memory T development between αPD-L1 CCR and PD-1 CSR. [00121] Chronic antigenic activation typically results in the elevated expression of canonical T cell exhaustion markers. To investigate how αPD-L1 CCR could relieve the expression of T cell exhaustion markers and possibly reverse the fate of T cells in an immunosuppressive environment, the expression of PD-1, TIGIT, LAG3 and TIM3 on CAR- T1 and CAR-T2 cells were measured at the end of the expansion phase. In congruence with the results seen in Figs. 3 and 4, CAR-T1 cells exhibited lowered expression levels of these exhaustion markers compared to CAR-T2 cells (Fig. 5). The experimental setup does not discern between endogenous inhibitory PD-1 receptors and the exogenous PD-1 CSR and hence, the higher levels of PD-1 on CAR-T2 may not be indicative of an elevated PD-1 inhibition. However, the expression levels for TIGIT, TIM3 and LAG3 were consistently higher on CAR-T2 cells across all three donors tested. Single end-point measurements from individual donors (Fig. 5A) and pooled average values (Fig. 5B) collectively showed that incorporation of αPD-L1 CCR relieved the T cell exhaustion to a larger extent as compared to PD-1 CSR. [00122] It was further investigated whether there would be a difference in the in vivo tumor killing effects between CAR-T1 (NKG2D CAR + αPD-L1 CCR) and CAR-T2 (NKG2D CAR + PD-1 CSR). We established a mouse colorectal model in NSG mice by intraperitoneal (i.p.) injection of human HCT116-luc CRC cells. Tumor progression was monitored by whole body bioluminescence imaging (Fig. 6). On day 7, when all mice had established tumors in the peritoneal cavity, animals were randomly divided into 4 groups for treatment: group 1 was subjected to two i.p. injections of PBS, group 2 to two i.p. injections of 10⁷ control CAR-T cells, group 3 and group 4 received two i.p. injections of 10⁷ CAR-T1 cells and CAR-T2 cells, respectively. As shown in Fig. 6, 4 groups of mice displayed a similar tumor burden on day 7, before treatment, as demonstrated by similar bioluminescence intensities. The established tumors continued to grow in PBS and control CAR-T cell treated mice and all mice died by day 28. With dual CAR-T treatment, the established tumors were totally eliminated and their bioluminescence signals became undetectable on day 14. Significant reduction of the disease by the treatment maintained for >8 weeks and by day 62, 4 out of 10 treated mice showed tumor regrowth, 3 in the CAR-T2 group and 1 in the CAR- T1 group. By day 150, while three mice died and two survived in the CAR-T2 group, all mice still survived well in the CAR-T1 group: 4 of them were tumor free and one mice displayed stable disease. The Kaplan–Meier curves and results of the log rank tests are shown in Fig. 7, indicating that there is a significant difference between the survival curves of the two groups. Overall, this animal experiment demonstrated that while both CAR-T1 and CAR-T2 were able to effectively eradicate established solid tumors in vivo, the tumor-killing efficacy was further enhanced significantly by replacing PD-1 CSR with αPD-L1 CCR for dual CAR construction. [00123] Conclusions [00124] Because of their special biological properties, such as clonal heterogeneity and inhibitory tumor microenvironments (TME) hostile to T cells, the eradication of large solid tumors for most cancer patients remains challenging. [00125] PD-1 recognizes both PD-L1 and PD-L2. PD-L1 is overexpressed on tumor cells and expressed at low levels on a wide range of non-haematopoietic cells. PD-L2 has a more restricted expression pattern compared with PD-L1 and its expression is primarily restricted to antigen presenting cells such as dendritic cells, macrophages, B1 B cells and mast cells. Thus, of the two PD-1 ligands, PD-L1 is considered to be the primary mediator of cancer immune evasion and PD-L2 does not seem to be a dominant mediator of T cell inhibition at the tumor site, although PD-L2 may play a role in certain cases. Another difference is their affinity to PD-1: PD-L2 exhibits 2 to 6-fold higher affinity to PD-1 than PD-L1 and shows different association/dissociation kinetics compared to the interaction between PD-1 and PD- L1. These differential binding properties of PD-L2 and PD-L1 may be responsible for differential contributions of these two PD-1 ligands to immune responses. [00126] Previous studies have utilized PD-1 receptor-based CSRs to protect the transduced T cells from PD-L1–induced T cell inhibition and to turn an inhibitory signal into the required co-stimulation signal for optimal T cell function. When used for bi-specific dual CAR construction, the higher binding properties of PD-L2 to PD-1 may guide PD-1-based dual CAR-T cells to be activated more easily by PD-L2 positive cells. To improve cancer specificity and reduce side effects on antigen presenting cells and adverse events associated with PD-1 blockade, a PD-L1-specific CSR should be used, which is unfortunately difficult to construct by using the extracellular structure of the PD-1 receptor. To circumvent the problem, the current invention has utilized a scFv fragment specific to PD-L1 to generate chimeric costimulatory receptor (CCR). When used together with 1st generation NKG2D CAR, it has been demonstrated that the use of this anti-PD-L1 CCR based bi-specific dual CAR provides an improved T cell proliferation, reduced expression of T cell exhaustion markers and most importantly, enhanced in vivo tumor killing. [00127] As CAR-T cell therapy based clinical trials continue to contend with the struggles of on-target off-tumor effects, immune evasion and immune suppression, new strategies are needed to complement the on-going clinical efforts. As a concluding remark, the bi-specific dual CAR developed here presents attractive opportunities imaginable in different therapy scenarios targeting PD-L1- and NKG2D ligand-positive cancer types. When a tumor type presents selective overexpression of PD-L1, the usage of the presently disclosed dual CAR enables T cells to overcome the immunosuppressive TME, while activating T cell cytotoxicity against NKG2G ligand-expressing cancer cells. Importantly, such a bi-specific dual CAR is theoretically safe and would not pose any conceivable threat to patients, thereby paving the way for smoother translational application.

Claims

CLAIMS 1. A T cell genetically modified to express: i) a recombinant chimeric antigen receptor (CAR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a co-stimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of an NKG2D receptor; and ii) a recombinant chimeric co-stimulatory receptor (CCR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) a co-stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-L1 specific single-chain variable fragment (scFv).

2. The T cell of claim 1, wherein the extracellular domain of the recombinant CAR is the extracellular domain of an NKG2D receptor, and/or wherein the extracellular domain of the recombinant CCR is a PD-L1 specific scFv.

3. The T cell of claim 1 or 2, wherein the intracellular signaling domain of the recombinant CAR is CD3ζ or DAP12.

4. The T cell of any one of claims 1 to 3, wherein the co-stimulatory signaling domain of the recombinant CCR is 4-1BB or CD28.

5. The T cell of any one of claims 1 to 4, wherein the transmembrane domain of the recombinant CAR and the transmembrane domain of the CCR are different.

6. A pharmaceutical composition comprising a pharmaceutically effective amount of the T cell of any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

7. A method of preparing the T cell of any one of claims 1 to 5, the method comprises: a) obtaining or providing T cell; b) providing i) a recombinant nucleic acid encoding the recombinant CAR; and ii) a recombinant nucleic acid encoding the CCR; and c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the CCR into the T cell.

8. A method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the T cell of any one of claims 1 to 5, or the pharmaceutical composition of claim 6.

9. The method of claim 8, wherein the T cells are derived from allogeneic cells or autologous cells.

10. A method of treating cancer or tumor in a subject in need thereof, the method comprises: a) obtaining T cells from the subject, or from a donor which is different from the subject to be treated; b) providing i) a recombinant nucleic acid encoding a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the recombinant CAR does not comprise a co-stimulatory signaling domain, and wherein the extracellular domain of the recombinant CAR comprises the extracellular domain of an NKG2D receptor; and ii) a recombinant nucleic acid encoding a recombinant chimeric co-stimulatory receptor (CCR), wherein the recombinant CCR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, and (c) a co-stimulatory signaling domain, wherein the recombinant CCR does not comprise an intracellular signaling domain, and wherein the extracellular domain of the recombinant CCR comprises a PD-1L1 specific single-chain variable fragment (scFv); c) transferring the recombinant nucleic acid encoding the recombinant CAR and the recombinant nucleic acid encoding the recombinant CCR into the T cell to obtain genetically modified T cells; and d) administering to the subject a pharmaceutically effective amount of the T cells obtained from c).

11. The method of any one of claims 8 to 10, wherein the cancer or tumor is a solid tumor.

12. The method of any one of claims 8 to 11, wherein the cancer or tumor expresses both NKG2D ligand(s) and PD-L1.

13. The method of any one of claims 8 to 12, wherein the cancer or tumor is colorectal cancer or tumor.

14. The method of any one of claims 7 and 10-13, wherein transferring the recombinant nucleic acids in step c) is carried out using electroporation.