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WO2021045693A1 - T cell modified with a synthetic receptor containing a single itam signaling motif - Google Patents

T cell modified with a synthetic receptor containing a single itam signaling motif Download PDF

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Publication number
WO2021045693A1
WO2021045693A1 PCT/SG2020/050520 SG2020050520W WO2021045693A1 WO 2021045693 A1 WO2021045693 A1 WO 2021045693A1 SG 2020050520 W SG2020050520 W SG 2020050520W WO 2021045693 A1 WO2021045693 A1 WO 2021045693A1
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car
cells
cell
domain
recombinant
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Shu Wang
Yu Yang NG
Zhendong Li
Chin Kang Johan TAY
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National University of Singapore
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National University of Singapore
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/4224—Molecules with a "CD" designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/7056—Lectin superfamily, e.g. CD23, CD72
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/59—Reproductive system, e.g. uterus, ovaries, cervix or testes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates generally to the field of biotechnology and cell therapy.
  • the present invention relates to recombinant chimeric antigen receptors, genetically modified ab T cell for cancer immunotherapy, their preparation method and use in patients in need thereof.
  • CARs chimeric antigen receptors
  • scFv single-chain variable fragment
  • T cells white blood cells that play a central role in immune responses that do not involve antibodies, otherwise known as the adaptive immune system.
  • the adaptive immune system uses a combination of a diverse set of specialized receptors expressed on the surface of T-cells to provide protection against different pathogens.
  • ab T cells are a type of T cell that express the alpha-beta T-cell receptor (TCR), and forms the majority of human T cells.
  • ab T cells have the capacity for immunologic memory as they have the ability to adapt and recognize processed antigenic peptide presented on the multihistocompatability complex (MHC).
  • MHC multihistocompatability complex
  • This immunologic memory arises from genetic recombination of the a and b chains of thymocytes in the thymus, thereby giving rise to a diverse repertoire of ab T cells and their ability to distinguish a vast array of peptides. Therefore, adoptive cell transfer therapy with ab T cells is one of the most promising immunotherapeutic modalities for cancer patients.
  • CRS cytokine release syndrome
  • ITAM immunoreceptor tyrosine- based activation motif
  • a pharmaceutical composition comprising a pharmaceutically effective amount of the ab T cell of the present invention and a pharmaceutically acceptable excipient.
  • a method of treating cancer or tumor in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of the ab T cell of the present invention, or the pharmaceutical composition of the present invention.
  • a method of treating cancer or tumor in a subject in need thereof comprises: (i) obtaining ab T cells from the subject, or from a ab T cell donor which is different from the subject to be treated; (ii) providing 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, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM); (iii) transferring the recombinant nucleic acid encoding the recombinant CAR into the ab T cell to obtain genetically modified ab T cells; and
  • ITAM immunoreceptor tyrosine-based activ
  • a method of preparing the ab T cell of the present invention comprises: (i) obtaining or providing ab T cell; (ii) providing a recombinant nucleic acid encoding the recombinant chimeric antigen receptor (CAR); and (iii) transferring the recombinant nucleic acid encoding the recombinant CAR into the ab T cell.
  • CAR chimeric antigen receptor
  • a recombinant chimeric antigen receptor comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • Fig.2 is a collection of Fig.2A – Fig.2F showing the characterization of NKG2D CAR-T cells bearing a DAP12 activating domain.
  • Fig.2A is a set of schematic representation of the overview of the design concepts for the CAR constructs as described in the present disclosure.
  • NKG2Dp – first generation CAR construct with a granulocyte-macrophage colony- stimulating factor signal peptide (GM-CSF SP) domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 transmembrane (CD28 TM) domain and a DAP12 activating domain
  • GM-CSF SP granulocyte-macrophage colony- stimulating factor signal peptide
  • CD28 TM CD28 transmembrane
  • DAP12 activating domain (middle) NKG2Dbp – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 TM domain, a 4-1BB co-stimulatory signaling domain and a DAP12 activating domain
  • bottom control (Ctrl) CAR – second generation anti-CD22 CAR construct with a GM-CSF SP domain, an aCD22 s
  • Fig. 2C is a schematic representation of the pZTS4-Ef1a-NKG2D-IgG4H-CD28TM-DAP12 vector map.
  • Fig. 2D is a line graph showing the antigen-dependent expansion of NKG2Dp CAR-T cells and NKG2Dbp CAR-T cells. The expansion of the two groups of CAR-T cells are performed with g-irradiated parental K562 feeder cells that was driven by DAP12 activation and enhanced by 4-1BB co-stimulation. 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.
  • Fig.2E is an image of two histogram diagrams that show the intensity of CAR expression and proportion of NKG2Dp CAR-T cells (left) and NKG2Dbp CAR-T cells (right).
  • CAR expression intensity mean fluorescence intensity (MFI) values
  • proportion of CAR-expressing cells % were analyzed in the middle (area under the curve is colored black) and end (area under the curve is colored grey) of the CAR-T cell expansion phase.
  • Unstained sample (black line with no color under the curve) is used as a background control.
  • the histogram diagrams from middle and end of expansion phase were superimposed to illustrate the enrichment of CAR-expressing T cells.
  • the results show that the intensity of CAR expression and proportion of NKG2Dp CAR-T cells and NKG2Dbp CAR-T cells increased consecutively upon repeated cultures with K562 feeder cells.
  • Fig. 2F is a column chart that shows the percentages of effector T cells, effector memory T cells, central memory T cells and stem cell memory T cells in the middle and at the end of the expansion phase.
  • Memory T cell development was characterized for each NKG2D CAR group through detection of both CCR7 and CD45RA antigens.
  • Effector T cell is CCR7-CD45RA + ; effector memory T cell is CCR7-CD45RA-; central memory T cell is CCR7 + CD45RA-; and stem cell memory T cell is CCR7 + CD45RA + .
  • composition of memory T subsets was analyzed both in the middle and at the end of each expansion phase and arranged side-by-side to illustrate the divergence in memory T subset development. The results show that the inclusion of 4-1BB co-stimulatory signaling domain in NKG2Dbp CAR-T cells enhanced the development of CC7-/CD45RA- effector memory T cells.
  • Fig. 3 is a collection of Fig. 3A – Fig.
  • FIG. 3C showing the effects of the NKG2Dp CAR-T cell and NKG2Dpb CAR-T cell in a mouse model of HCT116 human colorectal (CRC) cells.
  • Fig. 3A shows photos of bioluminescent images of four groups of mice. The mice received intraperitoneal (i.p.) injection of 2 ⁇ 10 6 HCT116 cells (day 0) followed by i.p. injection of PBS, control CAR-T cells, NKG2Dp CAR-T cells, NKG2Dbp CAR-T cells on day 7 and day 30 (2 ⁇ 10 6 cells per mouse). Growth of HCT116 was monitored by bioluminescent imaging on the indicated days. Bioluminescent images of 5 mice per group are shown.
  • FIG. 3B is a line graph that shows the bioluminescence flux values from each mouse of respective groups from Fig.3A to monitor growth of tumor.
  • Fig.3C is a line graph that shows the survival plot of the mice from Fig. 3A.
  • the data was analyzed by the Kaplan-Meier method. The differences in survival were then compared using the log-rank test. ****P ⁇ 0.0001.
  • the graph shows that all the mice treated with NKG2Dbp CAR-T cells continued to survive up to Day 90 of treatment.
  • Fig. 4 is a collection of Fig. 4A – Fig.
  • FIG.4A is a set of schematic representation of three different CAR constructs: (top) NKG2Dbz – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 TM domain, a 4- 1BB co-stimulatory signaling domain and a full length CD3zeta activating domain; (middle) NKG2Dbz1 – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 TM domain, a 4-1BB co-stimulatory signaling domain and a CD3zeta activating domain consisting the first ITAM only; (bottom) NKG2Dbp – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4
  • FIG. 4B is an image of four scatter plots that represent flow cytometry plots showing the expression of ST2 (NKG2Dbz, NKG2Dbz1 or NKG2Dbp CAR-T cells) 7 days after electroporation in T cells.
  • the flow cytometry plots show the efficiency of T cell electroporated with NKG2D CARs using a Piggybac transposon system. Efficiency ranged about 50-80%.
  • FIG. 4C is an image of four scatter plots that represent flow cytometry plots showing the expression of ST2 in electroporated T-cells after co-culture with K562#6 artificial antigen presenting cells (aAPC) at day 28. The results show that the expression of CARs in T cells can be enriched upon co-culture with K562#6.
  • Fig.5 is a collection of Fig.5A – Fig.5B showing T cell memory subsets in CAR- T cells electroporated with the three different NKG2D CAR constructs shown in Fig. 4A.
  • Fig. 5A is an image of three scatter plots that represent flow cytometry plots showing the expression of CD45RO (x-axis) and CCR7 (y-axis).
  • Fig. 5B depicts bar charts that show the percentage of each T cell memory subtypes from three different donors modified with either NKG2Dbz, NKG2Dbz1 or NKG2Dbp Majority of the T cells showed an effector memory subtype.
  • Fig. 6 is a collection of Fig. 6A – Fig. 6C showing the function of CAR-T cells electroporated with NKG2Dbz, NKG2Dbz1 or NKG2Dbp CAR constructs, measured in terms of cytolytic activity, cytokine release and cell expansion.
  • Fig. 6A shows the cytolytic activity of the modified CAR-T cells against HCT116 colorectal cancer cell lines.
  • HCT116 was labelled with DELFIA BATDA reagent (DELFIA® EuTDA Cytotoxicity Reagents, Perkin Elmer) followed by co-culture with CAR-T cells at indicated effector to target ratios. Cytotoxicity assay was carried out over 4 hours (left) and 16 hours (right), % cytotoxicity is then calculated by measuring Europium release signal from the target tumor cells using a plate reader. The results shown are based on one representative experiment out of two independent experiments with 2 different donors. The results show that no difference in killing was observed in T-cell modified with three different NKG2D CAR constructs.
  • Fig.6B shows the cytokine release of CAR-T cells after co-culture with HCT116.
  • Fig. 6C shows the expansion profile of the CAR-T cells enumerated at two different time points: (top) from Day 14 to Day 21; and (bottom) from Day 21 to Day 28.
  • the CAR-T cells were expanded with K562C6 cells in the presence or absence of exogenous IL-2.
  • Fig. 7 is a collection of Fig. 7A and Fig.
  • FIG. 7B showing the function of CAR-T cells electroporated with NKG2Dbz or NKG2Dbp CAR constructs, measured in terms of cytolytic activity and IFN-g production.
  • Fig. 7A shows collection of four line graphs showing the percentage cytotoxicity of control, NKG2Dbz or NKG2Dbp CAR-T cells against solid tumor cell lines SKOV3, HCT116, U87 or FaDu.
  • SKOV3, HCT116, U87 or FaDu were labelled with BATDA dye (DELFIA® EuTDA Cytotoxicity Reagents, Perkin Elmer) followed by co- culture with CAR-T cells at indicated effector to target ratio. Cytotoxicity was calculated after 4 hours by measurement of Europium release signal using a plate reader.
  • Fig. 7B shows one photo and one column chart that shows interferon-gamma (IFN-g) release from NKG2Dbz or NKG2Dbp CAR-T cells against HCT116 human CRC cell line in each well.
  • the photo shows IFN-g production by control T cells (left column), NKG2Dbz CAR-T cells (middle column) and NKG2Dbp CAR-T cells (right column). IFN-g production is shown by the number of spots per well.
  • NKG2Dbz CAR- T cells or NKG2Dbp CAR-T cells were co-cultured with HCT116 at 1:1 ratio overnight to assess IFN-g production using human IFN-g Elispot kit (Mabtech). Number of spots per well were acquired by a plate reader and number of spots per well were plotted in a column chart. ****: P ⁇ 0.0001. The results indicate that NKG2Dbz CAR-T cells showed higher IFN-g release compared to NK2Dbp CAR-T cells.
  • Fig. 8 shows a collection of 10 column charts showing the levels of different cytokines released when control, NKG2Dbz or NKG2Dbp CAR-T cells were used against HCT116 human CRC cell lines.
  • cytokines measured are granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-g), tumor necrosis factor alpha (TNF-a), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10) and interleukin 12 (IL-12).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IFN-g interferon-gamma
  • TNF-a tumor necrosis factor alpha
  • IL-2 interleukin 2
  • IL-4 interleukin 4
  • IL-5 interleukin 5
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • IL-12 interleukin 10
  • IL-12 interleukin 12
  • cytokines Concentration of cytokines were measured in 16 hour supernatants by LUNARISTM Human 11-Plex Cytokine Kit (Ayoxxa Biosystems). Shown in column charts are the mean ⁇ standard deviation (SD) for 10 out of 11 cytokines tested from three different peripheral blood mononuclear cells (PBMC) donors. Statistical difference * (P ⁇ 0.05 denoted by asterisk) only for IFN-g, TNF-a and IL-2 analysis between NKG2Dbz CAR-T cells vs NKG2Dbp CAR-T cells.
  • Fig. 9 is a collection of Fig. 9A – Fig. 9C showing the effects of NKG2Dbp or NKG2Dbz CAR-T cells in a mouse model of HCT116 human CRC Cells.
  • Fig. 9A is an image showing the timeline and experimental outline for the investigation of NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells anti-tumor effect in an i.p HCT116 xenograft model. Four groups of mice received i.p.
  • HCT116 HCT116 cells
  • day 0 day 0
  • PBS control CAR-T cells
  • NKG2Dbz CAR-T cells NKG2Dbp CAR-T cells
  • day 7 1 ⁇ 10 7 cells per mouse
  • Growth of HCT116 was monitored by bioluminescent imaging on the indicated days.
  • Fig. 9B shows photos of bioluminescent images of four groups of mice from the experiment as described in Fig.9A. The bioluminescent images of 5 mice per group are shown. Based on the images taken on Day 14, 21 and 28, the tumor sizes are smaller in NKG2Dbz CAR-T cells and NKG2Dbp CAR-T cells treated mice as compared to PBS and control T-cell treated mice.
  • Fig. 9B shows photos of bioluminescent images of four groups of mice from the experiment as described in Fig.9A. The bioluminescent images of 5 mice per group are shown. Based on the images taken on Day 14, 21 and 28, the tumor sizes are smaller in NKG2Dbz CAR-
  • FIG. 10 is a collection of Fig. 10A – Fig. 10E showing the effects of NKG2Dbp and NKG2Dbz human CAR-T cells on mice and death caused by graft versus host disease (GvHD).
  • Fig. 10 is a collection of Fig. 10A – Fig. 10E showing the effects of NKG2Dbp and NKG2Dbz human CAR-T cells on mice and death caused by graft versus host disease (GvHD).
  • FIG. 10A shows photos of bioluminescent images of two groups of mice treated with NKG2Dbp and NKG2Dbz CAR-T cells in a mouse model of HCT116 human colorectal (CRC) cells.
  • the growth of HCT116 was monitored by bioluminescent imaging on day 7 up to day 70.
  • Mice from NKG2Dbz CAR-T cells group were euthanized starting from day 35 onwards due to evidence of xenogeneic GvHD despite negligible tumor burden based on bioluminescent imaging.
  • a cross (“X”) represents death or termination of the mouse by euthanasia. (Criteria of xenogeneic GvHD included >15% weight loss, hunched posture, ruffled fur, reduced mobility).
  • Fig. 10B is a line graph that shows the survival plot of the mice that are monitored up to 150 days post tumor inoculation. The data was analysed by the Kaplan-Meier method.
  • Fig.10C shows the serum cytokine concentration obtained from mice treated with either NKG2Dbp or NKG2Dbz CAR-T cells. It was observed that treatment with NKG2Dbp CAR-T cells triggered significantly lower level secretions of the cytokines IL-2, TNF-a and IFN-g.
  • Fig.10D shows the hematoxylin-eosin (H&E) staining of liver collected at euthanization.
  • H&E hematoxylin-eosin
  • Fig. 10E shows that in mice treated with NKG2Dbz CAR-T cells, alopecia (which is one of the symptoms of GvHD) can be observed. In contrast, there were no signs of alopecia in mice treated with NKG2Dbp CAR-T cells.
  • FIG. 11 is an image of three scatter plots that represent flow cytometry plots showing consecutive gating strategy (from left to right): based on cell morphology (left), CD3 and abTCR expression levels (middle) and ST2 and NKG2D levels (right).
  • the dots in the area marked up by dotted lines represent the cells that are selected for gating. 51.5% of the cells were gated based on cell morphology, wherein 94.9% of the gated cells T cells are CD3+ and express abTCR.93.2% of the gated cells expressing CD3 and abTCR also express exogenous NKG2D CAR (ST2 and NKG2D double positive cells). Endogenous T cells (2.9%) express NKG2D and are not ST2 positive.
  • Fig. 12 is a collection of Fig. 12A – Fig. 12C showing the effects of NKG2Dbp CAR-T cells in a mouse model of SKOV3 human ovarian cancer cells.
  • Fig. 12A is an image showing the timeline and experimental outline for the investigation of the anti-tumor effect of NKG2Dbp CAR-T cells in an i.p SKOV3 xenograft model.
  • Three groups of mice received i.p. injection of 1 ⁇ 10 7 SKOV3 cells (day 0) followed by i.p. injection of PBS, control CAR-T cells, or NKG2Dbp CAR-T cells on day 7 and day 14 (1 ⁇ 10 7 cells per injection per mouse).
  • Fig. 12B shows photos of bioluminescent images of three groups of mice from the experiment as described in Fig. 12A. The bioluminescent images of 5 mice per group are shown. Based on the images taken on Day 14, 28, 35 and 42, the SKOV3 tumor sizes are negligible in NKG2Dbp CAR-T cells treated mice as compared to PBS and control T-cell treated mice. A cross (“X”) represents death or termination of the mouse by euthanasia.
  • Fig. 12C is a line graph that shows the survival plot of the mice from Fig. 12B. The data was analyzed by the Kaplan-Meier method. The differences in survival were then compared using the log-rank test.
  • 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.
  • 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.
  • MHC major histocompatibility complex
  • the TCR is composed of two different protein chains (i.e., it is a heterodimer).
  • the TCR In humans, in the majority of the T cells the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), thus being referred to as ab T cells.
  • the TCR In a small portion of the T cells the TCR consists of a gamma (g) and a delta (d) chain (encoded by TRG and TRD, respectively), thus being referred to as gd T cells.
  • 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.
  • molecules can be co- expressed with the CAR, including co-stimulatory molecules, reporter genes for imaging, gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.
  • 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.
  • 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 recognizes eight stress-induced ligands belonging to two families: two MHC class I chain-related proteins MICA and MICB and six HCMV UL16-binding proteins (ULBP1-6).
  • NKG2D ligands are not usually present on the cell surface of most healthy tissues, but can be up-regulated upon DNA damage, infection and transformation of cells, thus being commonly detected on hematopoietic tumors and carcinomas.
  • 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 gd + 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- g, and TGF-b 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.
  • polynucleotide refers 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.
  • 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.
  • 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.
  • recombinant nucleic acid 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.
  • 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.
  • 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.
  • 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.
  • transfection refers to the general process by which exogenous nucleic acid is transferred or 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.
  • 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.
  • allogeneic refers to any material derived from an individual other than the individual to which it is later to be introduced into.
  • 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.
  • peptide 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.
  • polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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: 1.
  • the chimeric antigen receptor (CAR) 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: 16.
  • the term “express” or “expression” refers to the production of a gene product in a cell.
  • the term “transient” when referred to expression means a polynucleotide is not incorporated into the genome of the cell.
  • 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.
  • constructs 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.
  • the transformed nucleic acid is operably associated with transcriptional and translational initiation and termination regulatory regions that are functional in the host cell.
  • Tumor 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.
  • 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.
  • cancers 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. [0044]
  • 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.
  • T cells in particular ab T cells
  • a chimeric antigen receptor comprising at least one co-stimulatory signaling domain and an intracellular signaling domain comprising only one immunoreceptor tyrosine-based activation motif (ITAM)
  • ITAM immunoreceptor tyrosine-based activation motif
  • the present invention refers to a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co- stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NK cell activating receptor or a scFv fragment of a monoclonal antibody of an NK cell activating receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • the intracellular signaling domain of the recombinant CAR comprises only one ITAM, or not more than one ITAM.
  • the ITAM is a DAP12 ITAM.
  • the antigen recognition domain includes a polypeptide that is selective for or targets an antigen, receptor, peptide ligand, or protein ligand of the target; or a polypeptide of the target.
  • the antigen recognition domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction.
  • the antigen recognition domain may include a portion of Ig heavy chain linked with a portion of Ig light chain, constituting a single chain fragment variable (scFv) that binds specifically to a target antigen.
  • the antibody may be monoclonal or polyclonal antibody or may be of any type that binds specifically to the target antigen.
  • the antigen recognition domain can be a receptor or ligand.
  • the choice of the antigen binding region depends upon the type and number of ligands that define the surface of a target cell.
  • the antigen binding region may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands for the antigen binding region in the CARs include those associated with or specific to cancer and/or tumor cells, autoimmune diseases, and viral, bacterial and parasitic infections.
  • the antigen binding region of the extracellular domain binds to a tumor associated antigen, a tumor specific antigen, or a pathogen-specific antigen.
  • Cells comprising such CARs can then be redirected to the tumor associated antigen, tumor specific antigen or a pathogen-specific antigen based on the antigen binding specificity.
  • a tumor specific antigen is unique to tumor cells and does not occur on other cells in the body.
  • a tumor associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen.
  • the expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen.
  • Tumor associated antigens can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.
  • Non- limiting examples of tumor associated or tumor specific antigens include the following: NKG2D, differentiation antigens such as MART-l/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A- PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7.
  • NKG2D differentiation antigens
  • MART-1
  • the CAR recognizes cell-surface tumor associated antigen or tumor specific antigen independent of human leukocyte antigen (HLA) and employs one or more signaling molecules to activate the genetically modified T cells for killing, proliferation, and/or cytokine production.
  • HLA human leukocyte antigen
  • the antigen binding region of the extracellular domain of the CARs target an antigen that includes but is not limited to CD19, CD20, CD22, RORl , Mesothelin, CD33/lL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like.
  • the tumor associated antigen is a ligand of NKG2D, and therefore the extracellular domain of the chimeric antigen receptor comprises NKG2D or an antigen binding fragment thereof.
  • the antigen binding fragment of NKG2D is or comprises the extracellular domain of NKG2D having the following amino acid sequence (from the N- terminal to the C-terminal): Q Q Q (SEQ ID NO: 6) (UniProt P26718, amino acid residues 83-216), encoded by the following exemplary nucleotide sequence: 5’- (SEQ ID NO: 5).
  • 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.
  • 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.
  • mouse NKG2D ligands include five different RAET1 isoforms (RAET1a, RAET1 b, RAET1 g, RAET1 d, and RAET1 e), three different H60 isoforms (H60a, b, and c), and UL16 binding protein 1 (encoded by MULT1 gene).
  • the extracellular domain of a chimeric antigen receptor generally also comprises 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.
  • the hinge region includes the hinge region of a human protein including CD-8 alpha, CD28, 4-1BB, OX-40, T cell receptor a or b chain, a CD3z chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof.
  • the hinge region includes the CD-8 alpha hinge region.
  • the CD-8 alpha hinge has the following amino acid sequence (from the N-terminal to the C-terminal): (SEQ ID NO: 21), encoded by the following exemplary nucleotide sequence: 5’- NO: 22).
  • the hinge region can be one selected from, but is not limited to, immunoglobulin (e.g. IgG1, IgG2, IgG3, IgG4, and IgD).
  • the hinge region is an IgG4 hinge region.
  • the IgG4 hinge region has the following amino acid sequence (from the N-terminal to the C-terminal): ESKYGPPCPSCP (SEQ ID NO: 8) (UniProtKB - P01861 hinge domain, amino acid residues 99-110), encoded by the following exemplary nucleotide sequence: 5’- [0055]
  • ESKYGPPCPSCP SEQ ID NO: 8
  • 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.
  • the transmembrane domain of a CAR includes a hydrophobic polypeptide that spans the cellular membrane.
  • the transmembrane domain spans from one side of a cell membrane (extracellular) through to the other side of the cell membrane (intracellular or cytoplasmic).
  • 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.
  • 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. [0059] In one example, the transmembrane domain that is naturally associated with one of the domains in the CAR 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.
  • a transmembrane domain includes a transmembrane domain of a T- cell receptor a or b chain, a CD3z chain, CD3e, 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.
  • the transmembrane domain is a CD28 transmembrane domain.
  • the transmembrane domain of the CAR has the following amino acid sequence (from the N- terminal to the C-terminal): (SEQ ID NO: 10), encoded by the following exemplary nucleotide sequence: 5’- [0061]
  • the intracellular signaling domain of a chimeric antigen receptor is responsible for activation of at least one of the normal effector functions of the immune cell in which the chimeric antigen receptor has been placed.
  • effector function refers to a specialized function of a differentiated cell, such as a T cell, or more specifically a ab T cell.
  • the intracellular signaling domain known in the art generally includes multiple immunoreceptor tyrosine based activation motifs (ITAMs).
  • the chimeric antigen receptors (CARs) of the present invention only contain one ITAM.
  • T cells in particular ab T cells, that are genetically modified to express CARs containing only one ITAM in the intracellular signaling domain release lower levels of cytokines as compared to cells expressing CARs containing more than one ITAM in the intracellular signaling domain.
  • Such genetically modified T cells or ab T cells in particular results in lower risk of xenogeneic graft versus host disease (GvHD) as compared to T cells or ab T cells expressing CARs containing more than one ITAM in the intracellular signaling domain.
  • GvHD xenogeneic graft versus host disease
  • 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.
  • Examples of intracellular signaling domains containing only one ITAM include but are not limited to, DAP12, the cytoplasmic domain of CLEC2/CLEC1B and FcRg.
  • DAP12 also known as KARAP (killer cell activating receptor-associated protein), is a short 12 kDa transmembrane protein of 113 amino acids expressed on the cells surface.
  • the protein consists of a 27 amino- acid leader, 14 amino-acid extracellular domain containing a cysteine residue, 24 amino-acid transmembrane segment with a negatively charged aspartic acid residue, and 48 amino-acid cytoplasmic domain containing a single ITAM.
  • the peptide sequence of the ITAM of DAP12 is which has less than 25% homology with the ITAM motifs identified in human CD3zeta chain and FceRI-g chain.
  • the intracellular receptor signaling domain of the CAR has the following amino acid sequence (from the N-terminal to the C-terminal): (SEQ ID NO: 14).
  • CD3 cluster of differentiation 3 is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+ naive T cells).
  • the complex contains a CD3g chain, a CD3d chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the z-chain (zeta-chain) to generate an activation signal in T lymphocytes.
  • TCR T-cell receptor
  • zeta-chain z-chain
  • Exemplary sequences of the ITAMs in CD3 zeta chain include but are not limited to: CD3 zeta ITAM1 having the following amino acid sequence (from the N-terminal to the C-terminal): encoded by the following exemplary nucleotide sequence: 5’- (SEQ ID NO: 18); CD3 zeta ITAM2 having the following amino acid sequence (from the N-terminal to the C-terminal): (SEQ ID NO: 23), encoded by the following exemplary nucleotide sequence: 5’- (SEQ ID NO: 24); CD3 zeta ITAM3 having the following amino acid sequence (from the N-terminal to the C-terminal): (SEQ ID NO: 25), encoded by the following exemplary nucleotide sequence: 5’- (SEQ ID NO: 26).
  • the only ITAM comprised in the intracellular signaling domain of the recombinant CAR as disclosed herein is CD3zeta ITAM-1.
  • C-type lectin-like receptor 2 (CLEC-2), also known as C-Type Lectin Domain Family 1 Member B (CLEC1B), is a 32 kDa, type II transmembrane glycoprotein and member of the C-type lectin-like family of receptors.
  • CLEC-2 consists of a 33 amino acid cytoplasmic domain, a 21 aa transmembrane region, and a 175 aa extracellular domain (SwissProt #Q9P126).
  • the cytoplasmic domain contains multiple threonine and serine residues which are sites of potential phosphorylation, and an ITAM through which CLEC-2 does its signaling.
  • Ligand binding and cross-linking of CLEC-2 induces Src kinase-dependent tyrosine phosphorylation of the ITAM, inducing activation of the tyrosine kinase Syk and initiation of a signaling pathway that culminates in activation of phospholipase C gamma 2.
  • the extracellular domain contains three potential sites of N-linked glycosylation, and a single carbohydrate recognition domain (CRD) which shows conservation of six cysteine residues.
  • Human CLEC-2 shares 63% aa sequence identity with mouse CLEC-2.
  • CLEC-2 is expressed preferentially in liver, and is also detected in myeloid cells (monocytes, dendritic cells, and granulocytes), platelets, and megakaryocytes.
  • Fc receptor common g chain (FcRg) is an adaptor bearing an ITAM that transduces activation signals from various immunoreceptors.
  • the co-stimulatory signaling domain of a chimeric antigen receptor can enhance the proliferation, survival and/or development of the T cells.
  • 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.
  • LFA-1 lymphocyte function-associated antigen-1
  • CD2, CD7, LIGHT, NKG2C, B7-H3, functional derivatives thereof, and combinations thereof the chimeric antigen receptor as described herein does not comprise more than one co- stimulatory signaling domain.
  • 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.
  • TNF tumor necrosis factor
  • the at least one co-stimulatory signaling domain and intracellular signaling domain may be collectively referred to as the intracellular domain of the chimeric antigen receptor.
  • the hinge region and the antigen binding region may be collectively referred to as the extracellular domain of the chimeric antigen receptor.
  • there is incorporated a spacer domain In some examples, between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the CAR, there is incorporated a spacer domain.
  • 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.
  • the desired CAR comprises the extracellular domain of an NKG2D receptor, IgG4 hinge, CD28 transmembrane domain, co-stimulatory signaling domain 4-1BB, and the intracellular signaling domain of DAP12.
  • the desired CAR comprises the extracellular domain of an NKG2D receptor, IgG4 hinge, CD28 transmembrane domain, co-stimulatory signaling domain 4-1BB, and an intracellular signaling domain comprising CD3zeta ITAM-1.
  • each of the components of a chimeric antigen receptor including but not limited to the extracellular domain, the hinge domain, the transmembrane domain, the co- stimulatory signaling domain, and the intracellular signaling domain, is covalently bonded to the chimeric antigen receptor component adjacent to it.
  • the co-stimulatory signaling domain of the recombinant CAR is covalently bonded to the intracellular signaling domain of the same recombinant CAR, and so forth.
  • a ab T cell genetically modified to express the recombinant chimeric antigen receptor (CAR) as disclosed herein. Expression of CARs in the ab T cells can be transient or stable.
  • a method of preparing the ab T cell genetically modified to express a recombinant chimeric antigen receptor (CAR) as disclosed herein comprises: (i) obtaining or providing ab T cell; (ii) providing a recombinant nucleic acid encoding the recombinant CAR; and (iii) transfecting the recombinant nucleic acid encoding the recombinant CAR into the ab T cell.
  • the method further comprises, after step (i), culturing the ab T cells to expand the number of ab T cells.
  • T cell expansion methods particularly ab T cell expansion methods, known in the art can be used.
  • a recombinant nucleic acid encoding the recombinant CAR 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.
  • 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).
  • PCR polymerase chain reaction
  • the sequence of the open reading frame encoding the CAR 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. [0069]
  • the nucleotide sequence coding for the recombinant CAR 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.
  • 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
  • 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 may be regulated by a second nucleic acid sequence so that the recombinant CAR is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the recombinant CAR may be controlled by any promoter/enhancer element known in the art.
  • the recombinant CAR construct containing the 5’TR, the core insulator 1, Ef1a1178, the nucleotide sequence encoding the recombinant CAR, the Sv40L, the core insulator 2, the 3’TR, and the pUC has the nucleotide sequence of SEQ ID NO: 1.
  • the basic backbone of the recombinant expression vector is a commercially available vector into which each of the above elements is inserted.
  • the nucleic acid encoding the recombinant CAR can be cloned into the expression vector using molecular cloning techniques commonly known in the art.
  • the expression vector comprising the recombinant nucleic acid encoding the recombinant CAR can be transferred into the ab T cell using any techniques known in the art, such as electroporation, non-viral chemical transfection, and viral transduction.
  • the ab T cells are genetically modified to express the CAR by electroporation.
  • the ab T cells genetically modified to express the recombinant CAR are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • the ab T cells as described herein can be provided as a composition or a pharmaceutical composition.
  • 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.
  • parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
  • the route of administration may be selected from the group consisting of systemic administration, oral administration, intravenous administration and parenteral administration.
  • 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 ab T cells as disclosed herein, alone or in appropriate combination with other active agents.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the ab 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.
  • 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. [0078]
  • the compositions as described herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions.
  • 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.
  • 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.
  • 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 stabilizer
  • a medical disease or disorder can be treated by administering a population of the ab T cell as disclosed herein.
  • the medical disease or disorder is cancer or tumor.
  • a method of treating cancer or tumor in a subject in need thereof comprising administering to the subject a pharmaceutically effective amount of the ab T cell as disclosed herein, or the pharmaceutical composition as disclosed herein.
  • a pharmaceutically effective amount of the ab T cell as disclosed herein in the manufacture of a medicament for treating cancer or tumor.
  • the ab T cells or the pharmaceutical composition as disclosed herein for use in treating cancer or tumor.
  • the genetically modified ab T cells used to treat cancer or tumor are prepared during the course of treatment.
  • a method of treating cancer or tumor in a subject in need thereof comprises: (i) obtaining ab T cells from the subject, or from a ab T cell donor which is different from the subject to be treated; (ii) providing a recombinant nucleic acid encoding the recombinant chimeric antigen receptor (CAR) as disclosed herein; (iii) transfecting the recombinant nucleic acid encoding the recombinant CAR into the ab T cell to obtain genetically modified ab T cells; and (iv) administering to the subject a pharmaceutically effective amount of the ab T cells obtained from (iii).
  • CAR chimeric antigen receptor
  • the method further comprises, after step (i), culturing the ab T cells to expand the number of ab T cells.
  • ab T cell expansion methods are known in the art and exemplified in the Experimental Section of the present application.
  • the term “pharmaceutically effective amount” as used herein includes within its meaning a sufficient but non-toxic amount of the ab T cells as described herein to provide the desired treatment effect. Desirably an effective amount or sufficient number of the ab 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.
  • the amount of ab 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 ab T cells are not present.
  • the exact amount of ab 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.
  • the concentration of ab T cells desirably should be sufficient to provide in the subject being treated at least from about 1 ⁇ 10 6 to about 1 ⁇ 10 9 ab Tcells, even more desirably, from about 1 ⁇ 10 7 to about 5 ⁇ 10 8 ab T cells, although any suitable amount can be utilized either above, e.g., 5 ⁇ 10 8 cells, or below, e.g., 1 ⁇ 10 7 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 ab T cells to be utilized by the practitioner upon optimizing the method of treatment as disclosed herein.
  • 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.
  • 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.
  • 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.
  • 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
  • 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
  • the cancer/tumor that can be treated using the genetically modified ab T cells provided herein include colorectal cancer, ovarian cancer, head and neck cancer, liver cancer, breast cancer, cervical cancer and glioma.
  • the cancer/tumor that can be treated is colorectal cancer/tumor or ovarian cancer/tumor.
  • the cancer/tumor expresses NKG2D ligand. In some other examples, the cancer/tumor expresses NKG2D ligand after treatment with another drug, radiation or biological agent.
  • 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.
  • the terms “decrease” , “reduced”, “reduction” , “decrease”, “removal” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
  • “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).
  • 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.
  • the patient is a human.
  • the source of ab 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.
  • the ab 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.
  • autologous source of ab T cells can be collected from a patient in need of treatment and ab 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 ab 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.
  • the ab T cells that can be used for treating a medical disease or disorder are allogeneic, i.e.
  • the ab 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 ab T cells derived from allogeneic xenogeneic sources can provide an off-the-shelf product. [0089] Allogeneic or autologous ab 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 ab T cells as described herein are administered in combination with a second therapeutic agent.
  • the second therapeutic agent may comprise T cells other than ab T cells, an immunomodulatory agent, a monoclonal antibody, or a chemotherapeutic agent.
  • the immunomodulatory agent is lenolidomide
  • the monoclonal antibody is alemtuzumab, rituxumab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, adotranstuzumab, blinatunomab, daratumumab or elotuzumab
  • the chemotherapeutic agent is fludarabine or cyclophosphamide.
  • 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.
  • a sample e.g., but not limited to, blood
  • 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.
  • a genetic marker includes a plurality of genetic markers, including mixtures and combinations thereof.
  • 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.
  • certain embodiments may be disclosed in a range format.
  • 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.
  • PBMCs Peripheral blood mononuclear cells
  • HSA Health Sciences Authority
  • IRB Institutional Review Board
  • PBMCs were seeded at 5x10 6 cells/ml in T cell media (AIM-V + 5% AB serum) and activated for 2-3 days with soluble OKT3 (Ebioscience) at 1 ⁇ g/ml or TransAct (Miltenyi Biotec, Germany), 100 ⁇ l of TransAct was added for every 1x10 7 PBMCs.
  • Recombinant IL-2 (300 IU/ml; Peprotech) was at the start of the culture and replenished every other day.
  • activated T cells were harvested and transduced with a non-viral electroporation method.
  • Modified T cells were cultured in AIM-V medium supplemented with either 5% AB serum or 1% human plasma and 300IU/ml IL-2 for a further five days.
  • genetically modified T cells were numerated and co-cultured with g-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 7 days with addition of new g-irradiated K562 cells at 1:2 ratio.
  • T cells were co-cultured with g- 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.
  • CD56+ cells were removed from day 17 CAR-T cell cultures based on manufacturer’s protocol (Miltenyi Biotec, Germany).
  • CD3 clone: OKT3; eBioscience
  • CD45RO clone: UCHL1; BD Biosciences
  • CD45RA clone HI100; eBioscience
  • CCR7 clone: 3D12; eBioscience
  • abTCR clone IP26, BD Biosciences
  • NKG2D clone 1D11, BD Biosciences
  • NKG2D CAR which included a Strep Tag
  • ST2 THETM NWSHPQFEK Tag
  • Genescript Antibody (Genescript) at day 5 post electroporation and day 12 after one week of co-culture with g-irradiated K562#6.
  • Flow cytometry analysis was performed and analysed using BD AccuriTM C6 (BD Biosciences).
  • In vitro cytotoxicity assay [00104] 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 40:1 to 1:1. Control groups were set up to measure spontaneous release (only target cells added), maximum release (target cells added with 10ml lysis buffer), and medium background (no cell added). Killing efficacy was calculated by using the following formula:
  • Non-modified or CAR T cells were stimulated for 18 hours with HCT116 at a 1:1 effector to target (E:T) ratio.
  • the cells were lysed, and the supernatants were collected and analysed with LUNARIS TM Human 11-Plex Cytokine kit (Ayoxxa Biosystems) or BD TM Cytometric Bead Array Human Thl/Th2 Cytokine Kit (BD Biosciences)
  • Non-obese diabetic/severe combined immuno-deficiency/IL-2Rgcnull (NSG) mice (The Jackson Laboratory) were maintained and used in the current study. All luminescent signals and images were acquired and analysed with the Xenogen living imaging software v3.2.
  • NKG2D 1st Generation (NKG2Dp) CAR- T cell and NKG2D 2nd Generation (NKG2Dbp) CAR-T cells 8-10 weeks old male NSG mice were used. Male mice were subjected to intraperitoneal (i.p.) injection with 2 x 10 6 HCT116-luc cells on day 0 to establish a human colorectal cancer (CRC) xenograft model. 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).
  • BBI live bioluminescence imaging
  • mice with similar BLI signal intensity randomly divided into 4 different treatment groups containing 5 mice per group.
  • a second intraperitoneal (i.p.) injection at the same dose (2x10 6 T cells/mice) was given at day 30 post tumor inoculation. Tumor progression was monitored by bioluminescence imaging (BLI) every week.
  • mice For the study to determine the effects of NKG2D-41BB-3z CAR (NKG2Dbz) and NKG2D-41BB-Dapl2 CAR (NKG2Dpb), 8-10 weeks old female NSG mice were used. Female mice were injected intraperitoneally (i.p.) with 2x10 6 HCT116-luc cells on day 0 to establish a human CRC xenograft model. On day 7 post-tumor inoculation, tumor engraftment was confirmed by live imaging and mice with similar BLI signal intensity randomly divided into 4 different treatment groups containing 5 mice per group.
  • mice were monitored closely and humanely euthanized after showing signs of xenogeneic graft versus host disease (GvHD) characterized by >15% loss in weight, ruffled fur, hunched posture and/or hind leg paralysis.
  • GvHD xenogeneic graft versus host disease
  • tumor engraftment was confirmed by live imaging and mice with similar BLI signal intensity randomly divided into 3 different treatment groups containing 5 mice per group.
  • a second injection was given at day 14 post tumor inoculation with the same dose of 1x10 7 T cells/mice. Tumor progression was monitored by BLI every week.
  • mice were monitored closely till day 80 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.
  • Statistics [00112] For in vitro and in vivo experiments, unpaired Student’s t test was used 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 analysed 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.
  • Interferon-gamma (IFN-g) release assay [00113] To detect IFNg secretion and up-regulation by CAR T cell after co-culture with HCT116, 10 5 T cells were incubated with 10 5 HCT116 cells for 16 hours using the IFNg ELISPOT assays according to the provided protocols of ELISPOT kits (Mabtech, Nacka Strand, Sweden). The plates were analyzed by an ELISPOT scanner (CTL, Ltd., of. Cleveland, OH).
  • CTL ELISPOT scanner
  • the 1st-generation DAP12 CAR construct was generated by cloning the type II NKG2D ectodomain sequence (amino acids 82 – 216) downstream of the GM-CSF signal peptide, followed by the hinge region and transmembrane domain of the CD8 protein.
  • the single ITAM-containing DAP12 coding sequence was cloned downstream of the transmembrane. Accordingly, the 2nd-generation CAR construct incorporated an additional 4-1BB co-stimulatory signaling domain upstream of the DAP12 activating domain (Fig.2B).
  • NKG2D CAR-T cells were generated by electroporating day 2 beads-activated T cells with NKG2D CAR-encoding and piggyBac transposase-encoding plasmids, and cultured for a further five days in IL-2 alone.
  • day 7 CAR-T cells (5 days post-electroporation) were then co-cultured with the human K562 myelogenous leukaemia cell line at an effector to target (E:T) ratio of 1:2.
  • E:T effector to target
  • NK cells As the use of K562 feeder cells would inevitably cause NK cells to increase in numbers because NK cells naturally lyse K562 feeder cells, this leads to the competition of NK cells with CAR-T cells for the recognition of K562 feeder cells. Therefore, a CD56 depletion step was incorporated on day 17 prior to further co-culture procedures, wherein the depletion of CD56-expressing NK cells would allow for selective enrichment of NKG2D CAR-T cells. [00116] Concomitantly, CAR expression intensity and proportion of CAR-expressing T cells increased as antigen-dependent proliferation took place (Fig.2E). By the end of CAR-T cell expansion, nearly 100% of T cells in both NKG2Dp and NKG2Dbp groups were CAR- positive.
  • NKG2Dbp mean fluorescence intensity (MFI) value: 19037
  • NKG2Dp mean fluorescence intensity (MFI) value: 28263).
  • the incorporation of 4-1BB co-stimulatory signaling domain also increased the formation of memory T cell subsets.
  • the memory T cell subsets can be determined by the combination of expression of the cell surface markers. For example, effector T cell is CCR7- CD45RA + ; effector memory T cell is CCR7-CD45RA-; central memory T cell is CCR7 + CD45RA-; and stem cell memory T cell is CCR7 + CD45RA + . As seen in Fig.
  • NKG2Dbp CAR-T cells showed increased development of CCR7-CD45RA- effector memory T cells than NKG2Dp CAR-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.
  • 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.
  • composition of memory T subsets was comparable in the middle of expansion phase (Effector memory T cell: 67.1% for NKG2Dp vs 76.4% for NKG2Dbp; effector T cell: 32.7% for NKG2Dp vs 23.4% for NKG2Dbp), there was a clear divergence in memory T cell development by the end of the expansion phase (Effector memory T cell: 39.6% for NKG2Dp vs 87.5% for NKG2Dbp; effector T cell: 60.3% for NKG2Dp vs 12.4% for NKG2Dbp).
  • NKG2Dbp NKG2D 1st generation CAR
  • NKG2Dbp T cells stably expressing NKG2D 2nd generation CAR
  • mice in both PBS and control CAR T cell groups were all euthanized prior to day 28 due to developing moribund condition.
  • Mice that received NKG2D 1st generation CAR-T cells or 2nd generation CAR-T cells showed reduction in tumor burden on day 14 after receiving a single dose T cell injection (Fig.3A and 3B).
  • NKG2D 2nd generation CAR (NKG2Dbp) had impressive in vitro and in vivo tumor killing effects
  • NKG2Dbp CAR construct was compared against two NKG2D CAR constructs that use CD3z (instead of Dap12) as the activation and signaling domain.
  • CD3z instead of Dap12
  • NKG2Dbz the CAR construct that consists of NKG2D-41BB-CD3z
  • ITAM1 the CAR construct that consists of NKG2D- 41BB-CD3z
  • NKG2D-41BB-Dap12 construct was termed NKG2Dbp
  • ST2 A 3x streptomycin – tag II (ST2) was included in the CAR construct to detect CAR expression in T cells. Using the Piggybac transposon system, about 50-70% efficiency five was achieved days after electroporation (Fig. 4B).
  • CAR on modified T cells can be further enriched upon co-culture with K562#6 that is engineered to express CD64, CD86 and CD137L, as shown in Fig. 4C. This is because the presence of the CD86 and CD137L expressed on K562#6 allows the CAR T cell to proliferate better and therefore improve the CAR expression on total T cells.
  • the calibration of CAR activation potential determines alternative T cell fate and therapeutic potency. Therefore, the generation of T cell memory subsets amongst the three different groups of CAR-T cells was determined.
  • NKG2Dbz with three ITAM motifs the two CARs with a single ITAM, NKG2Dbz1 and NKG2Dbp, increased the fraction of effector memory CAR T cells and reduced the proportion of effector cells in response to in vitro stimulation with K562C6 cells (Fig.5A and 5B).
  • NKG2Dbp CAR-T cells, NKG2Dbz1 CAR-T cells, and NKG2Dbz CAR-T cells were then tested for their anti-tumor cytotoxicity using HCT116-luc (colorectal cancer) in a 4 hour and 16 hour cytotoxicity assays.
  • NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells displayed high cell lysis activity towards a panel of solid tumour cancer cells, including SKOV3 luc (ovarian cancer), HCT116-luc (colorectal cancer), FaDu (head and neck cancer) and U87 (glioblastoma).
  • SKOV3 luc ovarian cancer
  • HCT116-luc colonrectal cancer
  • FaDu head and neck cancer
  • U87 glioblastoma
  • NKG2Dbz CAR-T cells in general promoted higher amounts of IL-2, IFN-g, and TNF-a production as compared to NKG2Dbz1 and NKG2Dbp CAR-T cells, although the statistical differences were significant (p ⁇ 0.05) only between NKG2Dbz CAR-T cells and NKG2Dbz1 CAR-T cells for IL-2, and between NKG2Dbz CAR-T cells and NKG2Dbp CAR-T cells for all three examined pro-inflammatory cytokines (Fig.6B).
  • NKG2Dbz CAR-T cells showed higher level of IFN-g production compared to NKG2Dbp CAR-T cells after 18 hours co-culture with HCT116.
  • the production of other pro-inflammatory cytokines was also assayed in the supernatants collected from NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells.
  • NKG2Dbz CAR-T cells produced significantly higher amounts of IFN-g, TNF-a and IL-2 as compared to NKG2Dbp CAR-T cells (p ⁇ 0.05).
  • GM-CSF granulocyte-macrophage colony- stimulating factor
  • the in vivo tumor killing effects between NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells are determined using a mouse colorectal model.
  • the mouse colorectal model is established in (NOD scid gamma) NSG mice by intraperitoneal (i.p.) injection of HCT116- luc cells. Tumor progression was monitored by whole body bioluminescence imaging (Fig. 9).
  • mice 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 one intraperitoneal (i.p.) injection of PBS, group 2 was subjected to one intraperitoneal (i.p.) injection of 1x10 7 control T cells; and groups 3 and 4 received one intraperitoneal (i.p.) injection of 1x10 7 NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells respectively.
  • Bioluminescence intensities which are indicative of tumor burdens, are used for analysis. As shown in Fig.9B, the bioluminescence intensities taken in the PBS group remain high during the treatment period from day 7 to 28.
  • mice that received control CAR T cell groups were slower compared to PBS group. This finding is represented in a bar graph as shown in Fig.9C.
  • the groups of mice that received NKG2Dbp CAR-T cells or NKG2Dbz CAR-T cells showed reduction in tumor burden after receiving a single dose T cell injection (Fig. 9B).
  • tumor burden remained undetectable (Fig. 9B and 9C) in most of the mice in the two groups. Bioluminescence signals were detected only at the site of injection for two mice, suggesting tumor relapse (Fig. 9B).
  • mice that had received NKG2Dbz CAR-T cells injection began to show signs of xenogeneic graft versus host disease (x-GvHD) as evidenced by > 15% loss of weight, ruffled fur and hunched posture from day 35 onwards. Mice that showed signs of xenogeneic graft versus host disease (x-GvHD) were euthanized according to IACUC protocol.
  • Xenogeneic graft versus host disease was not observed in mice that had received NKG2Dbp CAR-T cells.
  • the animal study was terminated at day 150 post tumor inoculation, where all mice that received NKG2Dbp CAR-T cells remained healthy. Only one mouse from the NKG2Dbz CAR-T cell treatment group survived. Therefore, treatment with NKG2Dbp CAR-T cells is less susceptible to xenogeneic graft versus host disease (x-GvHD). This could be explained by the lower production of pro-inflammatory cytokines present in the mice serum compared to treatment with NKG2Dbz CAR-T cells (Fig. 10C).
  • NKG2Dbp CAR can control the proliferation of CAR ab T cells, thereby reducing the occurrence of x-GvHD.
  • a mouse model of SKOV3 human ovarian cancer cells in NSG mice was established (Fig. 12).
  • Growth of SKOV3 was monitored by bioluminescent imaging on the indicated days (Fig. 12A).
  • Fig. 12B shows photos of bioluminescent images of three groups of mice from the experiment as described in Fig. 12A.
  • NKG2Dbp CAR-T cell has only one ITAM motif per CAR molecule, which can stimulate relatively lower levels of cytokine release upon interaction with target cells, in comparison to T cells expressing NKG2Dbz that contains three ITAM motifs per CAR molecule. This suggests that cytokine release upon CAR activation could be controlled by simply decreasing the ITAM density of the CAR construct.
  • the reduction in cytokine release by NKG2Dbp is primarily attributable to the use of the single ITAM configuration, which can affect downstream functional output through the mitogen-activated protein kinase pathway.
  • NKG2Dbp CAR-T would possibly provide a potential clinical advantage in reducing the risk of CRS and is the most important finding of this study.
  • both NKG2Dbp and NKG2Dbz CAR-T products result in similar in vivo anti-tumor activities.
  • the single ITAM configuration favours the persistence of highly functional CARs, balancing the replicative capacity of long-lived memory cells and the acquisition of effective antitumor function, thereby yielding CAR designs with enhanced therapeutic profiles.
  • the most common toxicities observed after CAR T-cell therapy are cytokine release syndrome (CRS) and cerebral edema/neurotoxicity. These involve the release of excess amounts of cytokines, and could be potentially life-threatening.
  • CRS cytokine release syndrome
  • the NKG2Dbp construct comprises a CD8a hinge and a transmembrane domain.
  • the CAR construct was designed to comprise only one ITAM motif so as to reduce the ITAM density of NKG2Dbp. This is to reduce the cytokine release that is commonly seen in present CAR-T therapies, and yet retain the tumor killing capacity of NKG2Dbp CAR T cells.
  • a single-chain CAR design targeting NKG2D ligand has been developed by fusing the NKG2D extracellular domain to the DAP12 internal signaling domain containing a single ITAM of the amino acid sequence ESPYQELQGQRSDVYSDL (NKG2D-DAP12).
  • a functional synthetic NKG2D-DAP12 protein has also been demonstrated in human T cells.
  • T cells genetically modified with NKG2D-DAP12 stimulate a lower level of cytokine release during tumor cell lysis, and can eliminate established tumor xenografts in mice.

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Abstract

The present invention refers to a αβ T cell genetically modified to express a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) a co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM); the recombinant CAR used to modify the αβ T cell; a pharmaceutical composition comprising the genetically modified αβ T cell; method of treating cancer or tumor using the genetically modified αβΤ cell; and method of preparing the genetically modified αβ T cell. In one embodiment, the genetically modified αβΤ cell comprises a CAR comprising the extracellular domain of NKG2D receptor, an lgG4 hinge region, a CD28 transmembrane domain, a 4-1 BB co-stimulatory signaling domain and a single ITAM-containing DAP 12 domain.

Description

T CELL MODIFIED WITH A SYNTHETIC RECEPTOR CONTAINING A SINGLE ITAM SIGNALING MOTIF CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority of the Singapore application No. 10201908256R, filed on 6 September 2019, the contents of it being hereby incorporated by reference in its entirety for all purposes. FIELD OF THE INVENTION [0002] The present invention relates generally to the field of biotechnology and cell therapy. In particular, the present invention relates to recombinant chimeric antigen receptors, genetically modified ab T cell for cancer immunotherapy, their preparation method and use in patients in need thereof. BACKGROUND OF THE INVENTION [0003] Recent years have seen tremendous progress in utilizing immune effector cells for cancer therapy. Cancer immunotherapy employing genetically engineered T lymphocytes that express chimeric antigen receptors (CARs) is an effective approach in treating several haematological cancers. CARs are composed of an extracellular antigen-binding domain, typically the single-chain variable fragment (scFv) derived from a monoclonal antibody, and an intracellular signaling domain. T cells are white blood cells that play a central role in immune responses that do not involve antibodies, otherwise known as the adaptive immune system. The adaptive immune system uses a combination of a diverse set of specialized receptors expressed on the surface of T-cells to provide protection against different pathogens. ab T cells are a type of T cell that express the alpha-beta T-cell receptor (TCR), and forms the majority of human T cells. ab T cells have the capacity for immunologic memory as they have the ability to adapt and recognize processed antigenic peptide presented on the multihistocompatability complex (MHC). This immunologic memory arises from genetic recombination of the a and b chains of thymocytes in the thymus, thereby giving rise to a diverse repertoire of ab T cells and their ability to distinguish a vast array of peptides. Therefore, adoptive cell transfer therapy with ab T cells is one of the most promising immunotherapeutic modalities for cancer patients. [0004] However, the approach has not been rather successful due to the risk of cytokine release syndrome (CRS). Despite recent success in clinical trials, several major limitations are associated with CAR-modified T cells. For example, CAR-T cell therapy holds an inherent risk of cytokine release syndrome (CRS), which is a systemic inflammatory response caused by the large, rapid release of cytokines when CAR-T cells are activated. Cytokine release syndrome (CRS) can result in various symptoms that can range from mild symptoms such as fever, nausea or rash, to being potentially life-threatening. [0005] Furthermore, the translation of ab T cells therapy to non-haematological malignancies is challenging due to the special pathophysiological characteristics of solid tumors, including target antigen heterogeneity, obstacles to CAR immune cell trafficking, and intrinsic negative regulatory mechanisms of tumor microenvironment. [0006] In addition, the need to generate an autologous CAR-T cell product for each individual patient is logistically demanding and restrictive for a wider adoption in the medical practice. Furthermore, it is not always possible to collect enough lymphocytes from heavily pretreated patients to generate sufficient quantities of CAR-T cells. An allogeneic “off-the- shelf” product could overcome these challenges, but allogeneic T cells pose significant risk of graft-versus-host disease (GvHD). [0007] Therefore, it is an objective of the present invention to provide improved recombinant chimeric antigen receptors and improved ab T cells for cancer immunotherapy, which address some or all of the above mentioned problems. SUMMARY [0008] In one aspect, there is provided a ab T cell genetically modified to express a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine- based activation motif (ITAM). [0009] In another aspect, there is provided a pharmaceutical composition comprising a pharmaceutically effective amount of the ab T cell of the present invention and a pharmaceutically acceptable excipient. [0010] In a further aspect, 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 ab T cell of the present invention, or the pharmaceutical composition of the present invention. [0011] In yet a further aspect, there is provided a method of treating cancer or tumor in a subject in need thereof, the method comprises: (i) obtaining ab T cells from the subject, or from a ab T cell donor which is different from the subject to be treated; (ii) providing 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, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM); (iii) transferring the recombinant nucleic acid encoding the recombinant CAR into the ab T cell to obtain genetically modified ab T cells; and (iv) administering to the subject a pharmaceutically effective amount of the ab T cells obtained from (iii). [0012] In yet a further aspect, there is provided a method of preparing the ab T cell of the present invention, the method comprises: (i) obtaining or providing ab T cell; (ii) providing a recombinant nucleic acid encoding the recombinant chimeric antigen receptor (CAR); and (iii) transferring the recombinant nucleic acid encoding the recombinant CAR into the ab T cell. [0013] In yet a further aspect, there is provided a recombinant chimeric antigen receptor (CAR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM). BRIEF DESCRIPTION OF THE DRAWINGS [0014] 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: [0015] Fig. 1 is an image showing the overall schematic of the different CAR-T construct and the effects on T cell activity and cytokine release. The different domains that make up the CAR-T constructs are provided in the legend. [0016] Fig.2 is a collection of Fig.2A – Fig.2F showing the characterization of NKG2D CAR-T cells bearing a DAP12 activating domain. Fig.2A is a set of schematic representation of the overview of the design concepts for the CAR constructs as described in the present disclosure. Fig. 2B is a set of schematic representation of three different CAR constructs: (top) NKG2Dp – first generation CAR construct with a granulocyte-macrophage colony- stimulating factor signal peptide (GM-CSF SP) domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 transmembrane (CD28 TM) domain and a DAP12 activating domain; (middle) NKG2Dbp – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 TM domain, a 4-1BB co-stimulatory signaling domain and a DAP12 activating domain; (bottom) control (Ctrl) CAR – second generation anti-CD22 CAR construct with a GM-CSF SP domain, an aCD22 scFv ectodomain, IgG4 hinge domain, a CD28 TM domain, a single 4-1BB co-stimulatory signaling domain and a single DAP12 activating domain. Fig. 2C is a schematic representation of the pZTS4-Ef1a-NKG2D-IgG4H-CD28TM-DAP12 vector map. Fig. 2D is a line graph showing the antigen-dependent expansion of NKG2Dp CAR-T cells and NKG2Dbp CAR-T cells. The expansion of the two groups of CAR-T cells are performed with g-irradiated parental K562 feeder cells that was driven by DAP12 activation and enhanced by 4-1BB co-stimulation. 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. The results show that both NKG2Dp and NKG2Dbp CAR-T cells underwent robust expansion in response to antigenic stimulation from K562 cells, with NKG2Dbp CAR-T cells showing higher fold of expansion after day 27. Fig.2E is an image of two histogram diagrams that show the intensity of CAR expression and proportion of NKG2Dp CAR-T cells (left) and NKG2Dbp CAR-T cells (right). Within each CAR group, CAR expression intensity (mean fluorescence intensity (MFI) values) and proportion of CAR-expressing cells (%) were analyzed in the middle (area under the curve is colored black) and end (area under the curve is colored grey) of the CAR-T cell expansion phase. Unstained sample (black line with no color under the curve) is used as a background control. Within each CAR group, the histogram diagrams from middle and end of expansion phase were superimposed to illustrate the enrichment of CAR-expressing T cells. The results show that the intensity of CAR expression and proportion of NKG2Dp CAR-T cells and NKG2Dbp CAR-T cells increased consecutively upon repeated cultures with K562 feeder cells. Fig. 2F is a column chart that shows the percentages of effector T cells, effector memory T cells, central memory T cells and stem cell memory T cells in the middle and at the end of the expansion phase. Memory T cell development was characterized for each NKG2D CAR group through detection of both CCR7 and CD45RA antigens. Effector T cell is CCR7-CD45RA+; effector memory T cell is CCR7-CD45RA-; central memory T cell is CCR7+CD45RA-; and stem cell memory T cell is CCR7+CD45RA+. Within each CAR group, composition of memory T subsets was analyzed both in the middle and at the end of each expansion phase and arranged side-by-side to illustrate the divergence in memory T subset development. The results show that the inclusion of 4-1BB co-stimulatory signaling domain in NKG2Dbp CAR-T cells enhanced the development of CC7-/CD45RA- effector memory T cells. [0017] Fig. 3 is a collection of Fig. 3A – Fig. 3C showing the effects of the NKG2Dp CAR-T cell and NKG2Dpb CAR-T cell in a mouse model of HCT116 human colorectal (CRC) cells. Fig. 3A shows photos of bioluminescent images of four groups of mice. The mice received intraperitoneal (i.p.) injection of 2×106 HCT116 cells (day 0) followed by i.p. injection of PBS, control CAR-T cells, NKG2Dp CAR-T cells, NKG2Dbp CAR-T cells on day 7 and day 30 (2×106 cells per mouse). Growth of HCT116 was monitored by bioluminescent imaging on the indicated days. Bioluminescent images of 5 mice per group are shown. A cross represents death or termination of the mouse by euthanasia. Fig. 3B is a line graph that shows the bioluminescence flux values from each mouse of respective groups from Fig.3A to monitor growth of tumor. Fig.3C is a line graph that shows the survival plot of the mice from Fig. 3A. The data was analyzed by the Kaplan-Meier method. The differences in survival were then compared using the log-rank test. ****P < 0.0001. The graph shows that all the mice treated with NKG2Dbp CAR-T cells continued to survive up to Day 90 of treatment. The results demonstrate that mice that received NKG2Dp CAR-T cells or NKG2Dbp CAR-T cells showed reduction in tumor burden on day 14 after receiving a single dose T cell injection. However, from day 21 onwards, tumor burden for mice that received NKG2Dp CAR-T cells increased progressively, and although a second CAR T cell injection was given at day 30, the tumor burden did not decrease. Mice that received NKG2Dp CAR-T cells injection were all euthanized by day 62. In contrast, tumor burden for the group of mice that received NKG2Dbp CAR-T cells showed progressive reduction and became undetectable up to day 62. [0018] Fig. 4 is a collection of Fig. 4A – Fig. 4C showing the characterisation of T-cell modified with different NKG2D CAR constructs. Fig.4A is a set of schematic representation of three different CAR constructs: (top) NKG2Dbz – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 TM domain, a 4- 1BB co-stimulatory signaling domain and a full length CD3zeta activating domain; (middle) NKG2Dbz1 – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 TM domain, a 4-1BB co-stimulatory signaling domain and a CD3zeta activating domain consisting the first ITAM only; (bottom) NKG2Dbp – second generation CAR construct with a GM-CSF SP domain, a NKG2D ectodomain, IgG4 hinge domain, a CD28 transmembrane (CD28 TM), a 4-1BB co- stimulatory signaling domain and a DAP12 activating domain. A ST2 (streptomycin tag II) tag was included to detect CAR expression for flow cytometry analysis. Fig. 4B is an image of four scatter plots that represent flow cytometry plots showing the expression of ST2 (NKG2Dbz, NKG2Dbz1 or NKG2Dbp CAR-T cells) 7 days after electroporation in T cells.The flow cytometry plots show the efficiency of T cell electroporated with NKG2D CARs using a Piggybac transposon system. Efficiency ranged about 50-80%. Fig. 4C is an image of four scatter plots that represent flow cytometry plots showing the expression of ST2 in electroporated T-cells after co-culture with K562#6 artificial antigen presenting cells (aAPC) at day 28. The results show that the expression of CARs in T cells can be enriched upon co-culture with K562#6. [0019] Fig.5 is a collection of Fig.5A – Fig.5B showing T cell memory subsets in CAR- T cells electroporated with the three different NKG2D CAR constructs shown in Fig. 4A. Fig. 5A is an image of three scatter plots that represent flow cytometry plots showing the expression of CD45RO (x-axis) and CCR7 (y-axis). Staining of CD45RO and CCR7 showed that relative to NKG2Dbz with three ITAM motifs, the two CARs with a single ITAM (i.e. NKG2Dbz1 and NKG2Dbp CAR constructs), increased the fraction of effector memory CAR T cells and reduced the proportion of effector cells in response to in vitro stimulation with K562C6. T cells electroporated with NKG2Dbz, NKG2Dbz1 or NKG2Dbp CAR were subjected to three rounds of K562#6 artificial antigen presenting cells (aAPC) stimulation before determining the percentage of T cell memory subsets based on CD45RO and CCR7 staining. Representative FACS staining shown for one PBMC donor. Fig. 5B depicts bar charts that show the percentage of each T cell memory subtypes from three different donors modified with either NKG2Dbz, NKG2Dbz1 or NKG2Dbp Majority of the T cells showed an effector memory subtype. [0020] Fig. 6 is a collection of Fig. 6A – Fig. 6C showing the function of CAR-T cells electroporated with NKG2Dbz, NKG2Dbz1 or NKG2Dbp CAR constructs, measured in terms of cytolytic activity, cytokine release and cell expansion. Fig. 6A shows the cytolytic activity of the modified CAR-T cells against HCT116 colorectal cancer cell lines. HCT116 was labelled with DELFIA BATDA reagent (DELFIA® EuTDA Cytotoxicity Reagents, Perkin Elmer) followed by co-culture with CAR-T cells at indicated effector to target ratios. Cytotoxicity assay was carried out over 4 hours (left) and 16 hours (right), % cytotoxicity is then calculated by measuring Europium release signal from the target tumor cells using a plate reader. The results shown are based on one representative experiment out of two independent experiments with 2 different donors. The results show that no difference in killing was observed in T-cell modified with three different NKG2D CAR constructs. Fig.6B shows the cytokine release of CAR-T cells after co-culture with HCT116. Supernatants were collected to determine cytokine production by the CBA (Cytometric Bead Array) Human Th1/Th2 Cytokine Kit (BD Biosciences). Data shown in bar graph are mean ± SD for 3 cytokines in supernatants tested with CAR-T cells from 3 different PBMC donors. The results show that NKG2Dbz CAR-T cells in general promoted higher amounts of IL-2, IFN-g, and TNF-a production, as compared to NKG2Dbz1 and NKG2Dbp CAR-T cells, although the statistical differences were significant (p < 0.05) only between NKG2Dbz-T and NKG2Dbz1- T for IL-2. The results also show that between NKG2Dbz1-T cells and NKG2Dbp-T cells, the latter stimulated the production of IL-2 and IFN-g at lower levels (p < 0.05). Fig. 6C shows the expansion profile of the CAR-T cells enumerated at two different time points: (top) from Day 14 to Day 21; and (bottom) from Day 21 to Day 28. The CAR-T cells were expanded with K562C6 cells in the presence or absence of exogenous IL-2. It was observed that in the absence of exogenous IL-2, cell numbers were consistently lower in the CAR-T samples modified with NKG2Dbz1 and NKG2Dbp, as compared with those modified with NKG2Dbz, indicating that NKG2Dbz1 and NKG2Dbp CAR-T cells proliferated less than NKG2Dbz CAR-T without the support of exogenous IL-2. Nevertheless, in the presence of exogenous IL-2 (300 IU/ml), there were no obvious differences in the antigen-stimulated CAR-T cell proliferation between the three types of CAR-T cells. [0021] Fig. 7 is a collection of Fig. 7A and Fig. 7B showing the function of CAR-T cells electroporated with NKG2Dbz or NKG2Dbp CAR constructs, measured in terms of cytolytic activity and IFN-g production. Fig. 7A shows collection of four line graphs showing the percentage cytotoxicity of control, NKG2Dbz or NKG2Dbp CAR-T cells against solid tumor cell lines SKOV3, HCT116, U87 or FaDu. SKOV3, HCT116, U87 or FaDu were labelled with BATDA dye (DELFIA® EuTDA Cytotoxicity Reagents, Perkin Elmer) followed by co- culture with CAR-T cells at indicated effector to target ratio. Cytotoxicity was calculated after 4 hours by measurement of Europium release signal using a plate reader. The results show that there were no observable differences in killing efficiency between NKG2Dbz and NKG2Dbp CAR-T cell groups. Fig. 7B shows one photo and one column chart that shows interferon-gamma (IFN-g) release from NKG2Dbz or NKG2Dbp CAR-T cells against HCT116 human CRC cell line in each well. The photo shows IFN-g production by control T cells (left column), NKG2Dbz CAR-T cells (middle column) and NKG2Dbp CAR-T cells (right column). IFN-g production is shown by the number of spots per well. NKG2Dbz CAR- T cells or NKG2Dbp CAR-T cells were co-cultured with HCT116 at 1:1 ratio overnight to assess IFN-g production using human IFN-g Elispot kit (Mabtech). Number of spots per well were acquired by a plate reader and number of spots per well were plotted in a column chart. ****: P < 0.0001. The results indicate that NKG2Dbz CAR-T cells showed higher IFN-g release compared to NK2Dbp CAR-T cells. [0022] Fig. 8 shows a collection of 10 column charts showing the levels of different cytokines released when control, NKG2Dbz or NKG2Dbp CAR-T cells were used against HCT116 human CRC cell lines. The cytokines measured are granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-g), tumor necrosis factor alpha (TNF-a), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 10 (IL-10) and interleukin 12 (IL-12). NKG2Dbz or NKG2Dbp CAR-T cells were co-cultured with HCT116 at 1:1 ratio overnight to access a panel of cytokine production. Concentration of cytokines were measured in 16 hour supernatants by LUNARIS™ Human 11-Plex Cytokine Kit (Ayoxxa Biosystems). Shown in column charts are the mean ± standard deviation (SD) for 10 out of 11 cytokines tested from three different peripheral blood mononuclear cells (PBMC) donors. Statistical difference * (P<0.05 denoted by asterisk) only for IFN-g, TNF-a and IL-2 analysis between NKG2Dbz CAR-T cells vs NKG2Dbp CAR-T cells. This shows that HCT116 treated NKG2Dbp CAR-T cells has less IFN-g, TNF-a and IL-2 release as compared to HCT116 treated with NKG2Dbz CAR-T cells. [0023] Fig. 9 is a collection of Fig. 9A – Fig. 9C showing the effects of NKG2Dbp or NKG2Dbz CAR-T cells in a mouse model of HCT116 human CRC Cells. Fig. 9A is an image showing the timeline and experimental outline for the investigation of NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells anti-tumor effect in an i.p HCT116 xenograft model. Four groups of mice received i.p. injection of 2×106 HCT116 cells (day 0) followed by i.p. injection of PBS, control CAR-T cells, NKG2Dbz CAR-T cells or NKG2Dbp CAR-T cells on day 7 (1×107 cells per mouse). Growth of HCT116 was monitored by bioluminescent imaging on the indicated days. Fig. 9B shows photos of bioluminescent images of four groups of mice from the experiment as described in Fig.9A. The bioluminescent images of 5 mice per group are shown. Based on the images taken on Day 14, 21 and 28, the tumor sizes are smaller in NKG2Dbz CAR-T cells and NKG2Dbp CAR-T cells treated mice as compared to PBS and control T-cell treated mice. Fig. 9C is a line graph that shows the bioluminescence flux values on Day 7 and Day 28 from each mice of respective groups from Fig. 9B to monitor growth of tumor. Taken together, the results show that NKG2Dbp and NKG2Dbz CAR-T cells can reduce the size of the peritoneal tumor. [0024] Fig. 10 is a collection of Fig. 10A – Fig. 10E showing the effects of NKG2Dbp and NKG2Dbz human CAR-T cells on mice and death caused by graft versus host disease (GvHD). Fig. 10A shows photos of bioluminescent images of two groups of mice treated with NKG2Dbp and NKG2Dbz CAR-T cells in a mouse model of HCT116 human colorectal (CRC) cells. The growth of HCT116 was monitored by bioluminescent imaging on day 7 up to day 70. Mice from NKG2Dbz CAR-T cells group were euthanized starting from day 35 onwards due to evidence of xenogeneic GvHD despite negligible tumor burden based on bioluminescent imaging. A cross (“X”) represents death or termination of the mouse by euthanasia. (Criteria of xenogeneic GvHD included >15% weight loss, hunched posture, ruffled fur, reduced mobility). Fig. 10B is a line graph that shows the survival plot of the mice that are monitored up to 150 days post tumor inoculation. The data was analysed by the Kaplan-Meier method. Fig.10C shows the serum cytokine concentration obtained from mice treated with either NKG2Dbp or NKG2Dbz CAR-T cells. It was observed that treatment with NKG2Dbp CAR-T cells triggered significantly lower level secretions of the cytokines IL-2, TNF-a and IFN-g. Fig.10D shows the hematoxylin-eosin (H&E) staining of liver collected at euthanization. Multiple irregular patchy areas of hepatic coagulative necrosis were observed in liver sections from NKG2Dbz-T treated mice but not in those from NKG2Dbp-T treated mice. The pathological necrosis was characterized by hepatocellular loss and absence of nuclei, being accompanied with the infiltration of mononuclear inflammatory cells and bile duct hyperplasia. Fig. 10E shows that in mice treated with NKG2Dbz CAR-T cells, alopecia (which is one of the symptoms of GvHD) can be observed. In contrast, there were no signs of alopecia in mice treated with NKG2Dbp CAR-T cells. [0025] Fig. 11 is an image of three scatter plots that represent flow cytometry plots showing consecutive gating strategy (from left to right): based on cell morphology (left), CD3 and abTCR expression levels (middle) and ST2 and NKG2D levels (right). The dots in the area marked up by dotted lines represent the cells that are selected for gating. 51.5% of the cells were gated based on cell morphology, wherein 94.9% of the gated cells T cells are CD3+ and express abTCR.93.2% of the gated cells expressing CD3 and abTCR also express exogenous NKG2D CAR (ST2 and NKG2D double positive cells). Endogenous T cells (2.9%) express NKG2D and are not ST2 positive. [0026] Fig. 12 is a collection of Fig. 12A – Fig. 12C showing the effects of NKG2Dbp CAR-T cells in a mouse model of SKOV3 human ovarian cancer cells. Fig. 12A is an image showing the timeline and experimental outline for the investigation of the anti-tumor effect of NKG2Dbp CAR-T cells in an i.p SKOV3 xenograft model. Three groups of mice received i.p. injection of 1×107 SKOV3 cells (day 0) followed by i.p. injection of PBS, control CAR-T cells, or NKG2Dbp CAR-T cells on day 7 and day 14 (1×107 cells per injection per mouse). Growth of SKOV3 was monitored by bioluminescent imaging on the indicated days. Fig. 12B shows photos of bioluminescent images of three groups of mice from the experiment as described in Fig. 12A. The bioluminescent images of 5 mice per group are shown. Based on the images taken on Day 14, 28, 35 and 42, the SKOV3 tumor sizes are negligible in NKG2Dbp CAR-T cells treated mice as compared to PBS and control T-cell treated mice. A cross (“X”) represents death or termination of the mouse by euthanasia. Fig. 12C is a line graph that shows the survival plot of the mice from Fig. 12B. The data was analyzed by the Kaplan-Meier method. The differences in survival were then compared using the log-rank test. P < 0.01. The graph shows that all the mice treated with NKG2Dbp CAR-T cells continued to survive up to Day 80 of treatment. DEFINITIONS [0027] 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. [0028] 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 (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), thus being referred to as ab T cells. In a small portion of the T cells the TCR consists of a gamma (g) and a delta (d) chain (encoded by TRG and TRD, respectively), thus being referred to as gd T cells. [0029] 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 T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors. [0030] 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. [0031] 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 recognizes eight stress-induced ligands belonging to two families: two MHC class I chain-related proteins MICA and MICB and six HCMV UL16-binding proteins (ULBP1-6). These NKG2D ligands are not usually present on the cell surface of most healthy tissues, but can be up-regulated upon DNA damage, infection and transformation of cells, thus being commonly detected on hematopoietic tumors and carcinomas. 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 gd+ 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- g, and TGF-b 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. [0032] 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. [0033] 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. [0034] 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. [0035] The term “transfection” as used herein refers to the general process by which exogenous nucleic acid is transferred or 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. [0036] 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. [0037] 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. [0038] 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. [0039] 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. [0040] 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: 1. In some other examples, the chimeric antigen receptor (CAR) 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: 16. [0041] The term “express” or "expression" refers to the production of a gene product in a cell. [0042] 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. [0043] 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. [0044] 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 [0045] The inventors of the present application have found that after genetically modifying T cells, in particular ab T cells, to express a chimeric antigen receptor (CAR) comprising at least one co-stimulatory signaling domain and an intracellular signaling domain comprising only one immunoreceptor tyrosine-based activation motif (ITAM), the genetically modified T cells display enhanced cell expansion, reduced cytokine release, as well as reduced xenogeneic graft versus host disease (GvHD). [0046] Thus, in one example, the present invention refers to a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co- stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NK cell activating receptor or a scFv fragment of a monoclonal antibody of an NK cell activating receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM). 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 ITAM is a DAP12 ITAM. [0047] The antigen recognition domain includes a polypeptide that is selective for or targets an antigen, receptor, peptide ligand, or protein ligand of the target; or a polypeptide of the target. The antigen recognition domain may be obtained from any of the wide variety of extracellular domains or secreted proteins associated with ligand binding and/or signal transduction. The antigen recognition domain may include a portion of Ig heavy chain linked with a portion of Ig light chain, constituting a single chain fragment variable (scFv) that binds specifically to a target antigen. The antibody may be monoclonal or polyclonal antibody or may be of any type that binds specifically to the target antigen. In another embodiment, the antigen recognition domain can be a receptor or ligand. [0048] The choice of the antigen binding region depends upon the type and number of ligands that define the surface of a target cell. For example, the antigen binding region may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Examples of cell surface markers that can act as ligands for the antigen binding region in the CARs include those associated with or specific to cancer and/or tumor cells, autoimmune diseases, and viral, bacterial and parasitic infections. [0049] In some examples of CARs, the antigen binding region of the extracellular domain binds to a tumor associated antigen, a tumor specific antigen, or a pathogen-specific antigen. Cells comprising such CARs can then be redirected to the tumor associated antigen, tumor specific antigen or a pathogen-specific antigen based on the antigen binding specificity. A tumor specific antigen is unique to tumor cells and does not occur on other cells in the body. A tumor associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. Tumor associated antigens can be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. Non- limiting examples of tumor associated or tumor specific antigens include the following: NKG2D, differentiation antigens such as MART-l/MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A- PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE- 6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1 , p15, p16, 43-9F, 5T4, 791Tgp72, alpha- fetoprotem, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733VEpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein, Acyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. [0050] In some examples, the CAR recognizes cell-surface tumor associated antigen or tumor specific antigen independent of human leukocyte antigen (HLA) and employs one or more signaling molecules to activate the genetically modified T cells for killing, proliferation, and/or cytokine production. [0051] In some examples, the antigen binding region of the extracellular domain of the CARs target an antigen that includes but is not limited to CD19, CD20, CD22, RORl , Mesothelin, CD33/lL3Ra, c-Met, PSMA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 TCR, MAGE A3 TCR, and the like. [0052] In some specific examples, the tumor associated antigen is a ligand of NKG2D, and therefore the extracellular domain of the chimeric antigen receptor comprises NKG2D or an antigen binding fragment thereof. [0053] In one example, the antigen binding fragment of NKG2D is or comprises the extracellular domain of NKG2D having the following amino acid sequence (from the N- terminal to the C-terminal):
Figure imgf000021_0001
Q Q Q
Figure imgf000021_0002
(SEQ ID NO: 6) (UniProt P26718, amino acid residues 83-216), encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000021_0003
Figure imgf000021_0004
(SEQ ID NO: 5). 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 (RAET1a, RAET1 b, RAET1 g, RAET1 d, and RAET1 e), 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 b2-microglobulin. In some examples, the NKG2D ligands for which the extracellular domain of the chimeric antigen receptor binds to are membrane- bound ligands. [0054] The extracellular domain of a chimeric antigen receptor generally also comprises 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 CD-8 alpha, CD28, 4-1BB, OX-40, T cell receptor a or b chain, a CD3z chain, CD28, CD3e, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, and combinations thereof. In one specific example, the hinge region includes the CD-8 alpha hinge region. In one specific example, the CD-8 alpha hinge has the following amino acid sequence (from the N-terminal to the C-terminal):
Figure imgf000022_0001
(SEQ ID NO: 21), encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000022_0002
NO: 22). 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). In one example, the hinge region is an IgG4 hinge region. In one specific example, the IgG4 hinge region has the following amino acid sequence (from the N-terminal to the C-terminal): ESKYGPPCPSCP (SEQ ID NO: 8) (UniProtKB - P01861 hinge domain, amino acid residues 99-110), encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000022_0003
[0055] 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. [0056] The transmembrane domain of a CAR 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). [0057] 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. [0058] 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. [0059] In one example, the transmembrane domain that is naturally associated with one of the domains in the CAR 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. [0060] For example, a transmembrane domain includes a transmembrane domain of a T- cell receptor a or b chain, a CD3z chain, CD3e, 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 one specific example, the transmembrane domain is a CD28 transmembrane domain. In one specific example, the transmembrane domain of the CAR has the following amino acid sequence (from the N- terminal to the C-terminal):
Figure imgf000023_0001
(SEQ ID NO: 10), encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000023_0002
[0061] The intracellular signaling domain of a chimeric antigen receptor is responsible for activation of at least one of the normal effector functions of the immune cell in which the chimeric antigen receptor has been placed. The term "effector function" refers to a specialized function of a differentiated cell, such as a T cell, or more specifically a ab T cell. The intracellular signaling domain known in the art generally includes multiple immunoreceptor tyrosine based activation motifs (ITAMs). However, the chimeric antigen receptors (CARs) of the present invention only contain one ITAM. The inventors found that T cells, in particular ab T cells, that are genetically modified to express CARs containing only one ITAM in the intracellular signaling domain release lower levels of cytokines as compared to cells expressing CARs containing more than one ITAM in the intracellular signaling domain. Such genetically modified T cells or ab T cells in particular results in lower risk of xenogeneic graft versus host disease (GvHD) as compared to T cells or ab T cells expressing CARs containing more than one ITAM in the intracellular signaling 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. Examples of intracellular signaling domains containing only one ITAM include but are not limited to, DAP12, the cytoplasmic domain of CLEC2/CLEC1B and FcRg. DAP12, also known as KARAP (killer cell activating receptor-associated protein), is a short 12 kDa transmembrane protein of 113 amino acids expressed on the cells surface. The protein consists of a 27 amino- acid leader, 14 amino-acid extracellular domain containing a cysteine residue, 24 amino-acid transmembrane segment with a negatively charged aspartic acid residue, and 48 amino-acid cytoplasmic domain containing a single ITAM. In one example, the peptide sequence of the ITAM of DAP12 is
Figure imgf000024_0001
which has less than 25% homology with the ITAM motifs identified in human CD3zeta chain and FceRI-g chain. Ligation of a DAP12-associated receptor leads to activation of SRC-family kinases, phosphorylation of paired tyrosine residues in the ITAM of DAP12, and the subsequent recruitment of the cytoplasmic kinase ZAP-70, converting the ligation event into downstream signaling. In one specific example, the intracellular receptor signaling domain of the CAR has the following amino acid sequence (from the N-terminal to the C-terminal):
Figure imgf000024_0002
(SEQ ID NO: 14). CD3 (cluster of differentiation 3) is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cell (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). It is composed of four distinct chains. In mammals, the complex contains a CD3g chain, a CD3d chain, and two CD3e chains. These chains associate with the T-cell receptor (TCR) and the z-chain (zeta-chain) to generate an activation signal in T lymphocytes. Each CD3 zeta chain contains three distinct ITAMs. Exemplary sequences of the ITAMs in CD3 zeta chain include but are not limited to: CD3 zeta ITAM1 having the following amino acid sequence (from the N-terminal to the C-terminal):
Figure imgf000025_0001
encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000025_0002
Figure imgf000025_0003
(SEQ ID NO: 18); CD3 zeta ITAM2 having the following amino acid sequence (from the N-terminal to the C-terminal):
Figure imgf000025_0004
(SEQ ID NO: 23), encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000025_0005
Figure imgf000025_0006
(SEQ ID NO: 24); CD3 zeta ITAM3 having the following amino acid sequence (from the N-terminal to the C-terminal):
Figure imgf000025_0007
(SEQ ID NO: 25), encoded by the following exemplary nucleotide sequence: 5’-
Figure imgf000025_0008
Figure imgf000025_0009
(SEQ ID NO: 26). In one specific example, the only ITAM comprised in the intracellular signaling domain of the recombinant CAR as disclosed herein is CD3zeta ITAM-1. C-type lectin-like receptor 2 (CLEC-2), also known as C-Type Lectin Domain Family 1 Member B (CLEC1B), is a 32 kDa, type II transmembrane glycoprotein and member of the C-type lectin-like family of receptors. CLEC-2 consists of a 33 amino acid cytoplasmic domain, a 21 aa transmembrane region, and a 175 aa extracellular domain (SwissProt #Q9P126). The cytoplasmic domain contains multiple threonine and serine residues which are sites of potential phosphorylation, and an ITAM through which CLEC-2 does its signaling. Ligand binding and cross-linking of CLEC-2 induces Src kinase-dependent tyrosine phosphorylation of the ITAM, inducing activation of the tyrosine kinase Syk and initiation of a signaling pathway that culminates in activation of phospholipase C gamma 2. The extracellular domain contains three potential sites of N-linked glycosylation, and a single carbohydrate recognition domain (CRD) which shows conservation of six cysteine residues. Human CLEC-2 shares 63% aa sequence identity with mouse CLEC-2. CLEC-2 is expressed preferentially in liver, and is also detected in myeloid cells (monocytes, dendritic cells, and granulocytes), platelets, and megakaryocytes. Fc receptor common g chain (FcRg) is an adaptor bearing an ITAM that transduces activation signals from various immunoreceptors. [0062] The co-stimulatory signaling domain of a chimeric antigen receptor 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 example, the chimeric antigen receptor as described herein does not comprise more than one co- stimulatory signaling domain. 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. [0063] As used herein, the at least one co-stimulatory signaling domain and intracellular signaling domain may be collectively referred to as the intracellular domain of the chimeric antigen receptor. As used herein, the hinge region and the antigen binding region may be collectively referred to as the extracellular domain of the chimeric antigen receptor. [0064] In some examples, between the extracellular domain and the transmembrane domain of the CAR, or between the intracellular domain and the transmembrane domain of the 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. [0065] In one specific example, the desired CAR comprises the extracellular domain of an NKG2D receptor, IgG4 hinge, CD28 transmembrane domain, co-stimulatory signaling domain 4-1BB, and the intracellular signaling domain of DAP12. In another specific example, the desired CAR comprises the extracellular domain of an NKG2D receptor, IgG4 hinge, CD28 transmembrane domain, co-stimulatory signaling domain 4-1BB, and an intracellular signaling domain comprising CD3zeta ITAM-1. Unless otherwise specified, it is understood that each of the components of a chimeric antigen receptor, including but not limited to the extracellular domain, the hinge domain, the transmembrane domain, the co- stimulatory signaling domain, and the intracellular signaling domain, is covalently bonded to the chimeric antigen receptor component adjacent to it. For example, the co-stimulatory signaling domain of the recombinant CAR is covalently bonded to the intracellular signaling domain of the same recombinant CAR, and so forth. [0066] In another example, there is provided a ab T cell genetically modified to express the recombinant chimeric antigen receptor (CAR) as disclosed herein. Expression of CARs in the ab T cells can be transient or stable. [0067] In another example, there is provided a method of preparing the ab T cell genetically modified to express a recombinant chimeric antigen receptor (CAR) as disclosed herein, the method comprises: (i) obtaining or providing ab T cell; (ii) providing a recombinant nucleic acid encoding the recombinant CAR; and (iii) transfecting the recombinant nucleic acid encoding the recombinant CAR into the ab T cell. In some examples, the method further comprises, after step (i), culturing the ab T cells to expand the number of ab T cells. T cell expansion methods, particularly ab T cell expansion methods, known in the art can be used. [0068] A recombinant nucleic acid encoding the recombinant CAR 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 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. [0069] The nucleotide sequence coding for the recombinant CAR 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. [0070] 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 may be regulated by a second nucleic acid sequence so that the recombinant CAR is expressed in a host transformed with the recombinant DNA molecule. For example, expression of the recombinant CAR may be controlled by any promoter/enhancer element known in the art. [0071] In one specific example, the recombinant CAR construct containing the 5’TR, the core insulator 1, Ef1a1178, the nucleotide sequence encoding the recombinant CAR, the Sv40L, the core insulator 2, the 3’TR, and the pUC, has the nucleotide sequence of SEQ ID NO: 1. [0072] 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. [0073] The nucleic acid encoding the recombinant CAR can be cloned into the expression vector using molecular cloning techniques commonly known in the art. [0074] The expression vector comprising the recombinant nucleic acid encoding the recombinant CAR can be transferred into the ab T cell using any techniques known in the art, such as electroporation, non-viral chemical transfection, and viral transduction. In one particular example, the ab T cells are genetically modified to express the CAR by electroporation. [0075] In some examples, the ab T cells genetically modified to express the recombinant CAR are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. [0076] The ab 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 ab 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. [0077] 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 ab 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 ab 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. [0078] 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 ab T cells of the formulation. [0079] In some examples, a medical disease or disorder can be treated by administering a population of the ab 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 ab 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 ab T cell as disclosed herein in the manufacture of a medicament for treating cancer or tumor. In yet another example, there is provided the ab T cells or the pharmaceutical composition as disclosed herein for use in treating cancer or tumor. In some examples, the genetically modified ab 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: (i) obtaining ab T cells from the subject, or from a ab T cell donor which is different from the subject to be treated; (ii) providing a recombinant nucleic acid encoding the recombinant chimeric antigen receptor (CAR) as disclosed herein; (iii) transfecting the recombinant nucleic acid encoding the recombinant CAR into the ab T cell to obtain genetically modified ab T cells; and (iv) administering to the subject a pharmaceutically effective amount of the ab T cells obtained from (iii). In some other examples, the method further comprises, after step (i), culturing the ab T cells to expand the number of ab T cells. ab 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 ab T cells as described herein to provide the desired treatment effect. Desirably an effective amount or sufficient number of the ab 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 ab 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 ab T cells are not present. The exact amount of ab 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 ab T cells desirably should be sufficient to provide in the subject being treated at least from about 1×106 to about 1×109 ab Tcells, even more desirably, from about 1×107 to about 5×108 ab 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 ab 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 ab 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/tumor or ovarian cancer/tumor. [0082] In some examples, the cancer/tumor expresses NKG2D ligand. In some other examples, the cancer/tumor expresses NKG2D ligand 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 ab 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 ab 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 ab T cells can be collected from a patient in need of treatment and ab 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 ab 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 ab 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 an ab T cell donor. In some other examples, the ab 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 ab T cells derived from allogeneic xenogeneic sources can provide an off-the-shelf product. [0089] Allogeneic or autologous ab 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 ab T cells as described herein are administered in combination with a second therapeutic agent. For example, the second therapeutic agent may comprise T cells other than ab T cells, 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 ab 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 Materials and Methods Cell lines and culture conditions [0099] Human K562 myelogenous leukaemia feeder cells or K562#6 engineered to express CD64, CD86 and CD137L was maintained in Iscove's Modified Dulbecco's Media (IMDM). SKOV3-luc and HCT116-luc cells were maintained in McCoy’s 5A medium, whereas FaDu and U87 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM). All types of medium were supplemented with 10% Fetal Bovine Serum (FBS) (Gibco). NKG2D CAR-T cell preparation [00100] Peripheral blood mononuclear cells (PBMCs) of healthy donors were isolated by gradient centrifugation from buffy coats obtained from Health Sciences Authority (HSA, Singapore) with ethics approved by the Institutional Review Board (IRB). PBMCs were seeded at 5x106 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 1x107 PBMCs. Recombinant IL-2 (300 IU/ml; Peprotech) was at the start of the culture and replenished every other day. [00101] 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 medium 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 g-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 7 days with addition of new g-irradiated K562 cells at 1:2 ratio. For propagation of non-modified T cells, T cells were co-cultured with g- 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. [00102] CD56+ cells were removed from day 17 CAR-T cell cultures based on manufacturer’s protocol (Miltenyi Biotec, Germany). Briefly, up to 1x107 cells were suspended in 80ml of autoMACS running buffer and incubated with 20ml of CD56 microbeads at 4°C in the dark for 15 minutes. Cells were centrifuged at 300g for 10 minutes and resuspended in 500ml 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). Flow cytometry [00103] For phenotypic analysis of T cells, the following conjugated anti-human antibodies were used: CD3 (clone: OKT3; eBioscience), CD45RO (clone: UCHL1; BD Biosciences), CD45RA (clone HI100; eBioscience), CCR7 (clone: 3D12; eBioscience), abTCR (clone IP26, BD Biosciences), NKG2D (clone 1D11, BD Biosciences) 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 (ST2) Antibody (Genescript) at day 5 post electroporation and day 12 after one week of co-culture with g-irradiated K562#6. Flow cytometry analysis was performed and analysed using BD Accuri™ C6 (BD Biosciences). In vitro cytotoxicity assay [00104] 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 40:1 to 1:1. Control groups were set up to measure spontaneous release (only target cells added), maximum release (target cells added with 10ml lysis buffer), and medium background (no cell added). Killing efficacy was calculated by using the following formula:
Figure imgf000039_0001
Multiplex cytokine quantification assay
[00105] Non-modified or CAR T cells were stimulated for 18 hours with HCT116 at a 1:1 effector to target (E:T) ratio. The cells were lysed, and the supernatants were collected and analysed with LUNARIS TM Human 11-Plex Cytokine kit (Ayoxxa Biosystems) or BD TM Cytometric Bead Array Human Thl/Th2 Cytokine Kit (BD Biosciences)
In vivo experiments
[00106] 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).
[00107] Non-obese diabetic/severe combined immuno-deficiency/IL-2Rgcnull (NSG) mice (The Jackson Laboratory) were maintained and used in the current study. All luminescent signals and images were acquired and analysed with the Xenogen living imaging software v3.2.
[00108] For the study to determine the effects of NKG2D 1st Generation (NKG2Dp) CAR- T cell and NKG2D 2nd Generation (NKG2Dbp) CAR-T cells, 8-10 weeks old male NSG mice were used. Male mice were subjected to intraperitoneal (i.p.) injection with 2 x 106 HCT116-luc cells on day 0 to establish a human colorectal cancer (CRC) xenograft model. 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, 100mI of PBS or 100mI cell suspension containing 2x106 T cells of three different groups: (1) Control T cell, (2) NKG2D 1st Generation (NKG2Dp) CAR-T cells or (3) NKG2D 2nd Generation (NKG2Dbp) CAR-T cells were injected intraperitoneally (i.p.). A second intraperitoneal (i.p.) injection at the same dose (2x106 T cells/mice) was given at day 30 post tumor inoculation. Tumor progression was monitored by bioluminescence imaging (BLI) every week.
[00109] For the study to determine the effects of NKG2D-41BB-3z CAR (NKG2Dbz) and NKG2D-41BB-Dapl2 CAR (NKG2Dpb), 8-10 weeks old female NSG mice were used. Female mice were injected intraperitoneally (i.p.) with 2x106 HCT116-luc cells on day 0 to establish a human CRC xenograft model. On day 7 post-tumor inoculation, tumor engraftment was confirmed by live imaging and mice with similar BLI signal intensity randomly divided into 4 different treatment groups containing 5 mice per group. On day 7, 100ml of PBS or 100ml cell suspension containing 1x107 T cells of three different group: (1) Control T cell (2) NKG2D-41BB-3z CAR T Cell or (3) NKG2D-41BB-Dap12 CAR T cell was injected i.p. vein . A second injection was given at day 32 at a reduced dose of 2x106 T cells/mice post tumor inoculation. Tumor progression was monitored by BLI every week. [00110] 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. Similarly, mice were monitored closely and humanely euthanized after showing signs of xenogeneic graft versus host disease (GvHD) characterized by >15% loss in weight, ruffled fur, hunched posture and/or hind leg paralysis. [00111] For the study to determine the effects of NKG2D-41BB-Dap12 CAR (NKG2Dpb) in a human SKOV3 ovarian cancer xenograft model, 8-10 weeks old female NSG mice were used. Female mice were injected intraperitoneally (i.p.) with 1x107 SKOV3-luc cells on day 0 to establish a human ovarian cancer xenograft model. On day 7 post-tumor inoculation, tumor engraftment was confirmed by live imaging and mice with similar BLI signal intensity randomly divided into 3 different treatment groups containing 5 mice per group. On day 7, 100ml of PBS or 100ml cell suspension containing 1x107 T cells of two different group: (1) Control T cell or (2) NKG2D-41BB-Dap12 CAR T cell was injected i.p.. A second injection was given at day 14 post tumor inoculation with the same dose of 1x107T cells/mice. Tumor progression was monitored by BLI every week. Mice were monitored closely till day 80 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. Statistics [00112] For in vitro and in vivo experiments, unpaired Student’s t test was used 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 analysed 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. Interferon-gamma (IFN-g) release assay [00113] To detect IFNg secretion and up-regulation by CAR T cell after co-culture with HCT116, 105 T cells were incubated with 105 HCT116 cells for 16 hours using the IFNg ELISPOT assays according to the provided protocols of ELISPOT kits (Mabtech, Nacka Strand, Sweden). The plates were analyzed by an ELISPOT scanner (CTL, Ltd., of. Cleveland, OH). Results [00114] The 1st-generation DAP12 CAR construct was generated by cloning the type II NKG2D ectodomain sequence (amino acids 82 – 216) downstream of the GM-CSF signal peptide, followed by the hinge region and transmembrane domain of the CD8 protein. The single ITAM-containing DAP12 coding sequence was cloned downstream of the transmembrane. Accordingly, the 2nd-generation CAR construct incorporated an additional 4-1BB co-stimulatory signaling domain upstream of the DAP12 activating domain (Fig.2B). [00115] NKG2D CAR-T cells were generated by electroporating day 2 beads-activated T cells with NKG2D CAR-encoding and piggyBac transposase-encoding plasmids, and cultured for a further five days in IL-2 alone. For large-scale expansion and longitudinal study of memory T development, day 7 CAR-T cells (5 days post-electroporation) were then co-cultured with the human K562 myelogenous leukaemia cell line at an effector to target (E:T) ratio of 1:2. Both NKG2Dp and NKG2Dbp CAR-T cells underwent robust proliferation in response to antigenic stimulation from K562 cells (Fig. 2D). As the use of K562 feeder cells would inevitably cause NK cells to increase in numbers because NK cells naturally lyse K562 feeder cells, this leads to the competition of NK cells with CAR-T cells for the recognition of K562 feeder cells. Therefore, a CD56 depletion step was incorporated on day 17 prior to further co-culture procedures, wherein the depletion of CD56-expressing NK cells would allow for selective enrichment of NKG2D CAR-T cells. [00116] Concomitantly, CAR expression intensity and proportion of CAR-expressing T cells increased as antigen-dependent proliferation took place (Fig.2E). By the end of CAR-T cell expansion, nearly 100% of T cells in both NKG2Dp and NKG2Dbp groups were CAR- positive. While proportions were comparable, the expression intensity of NKG2Dbp (mean fluorescence intensity (MFI) value: 19037) was lower compared to NKG2Dp (mean fluorescence intensity (MFI) value: 28263). [00117] The incorporation of 4-1BB co-stimulatory signaling domain also increased the formation of memory T cell subsets. The memory T cell subsets can be determined by the combination of expression of the cell surface markers. For example, effector T cell is CCR7- CD45RA+; effector memory T cell is CCR7-CD45RA-; central memory T cell is CCR7+CD45RA-; and stem cell memory T cell is CCR7+CD45RA+. As seen in Fig. 2F, NKG2Dbp CAR-T cells showed increased development of CCR7-CD45RA- effector memory T cells than NKG2Dp CAR-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. While composition of memory T subsets was comparable in the middle of expansion phase (Effector memory T cell: 67.1% for NKG2Dp vs 76.4% for NKG2Dbp; effector T cell: 32.7% for NKG2Dp vs 23.4% for NKG2Dbp), there was a clear divergence in memory T cell development by the end of the expansion phase (Effector memory T cell: 39.6% for NKG2Dp vs 87.5% for NKG2Dbp; effector T cell: 60.3% for NKG2Dp vs 12.4% for NKG2Dbp). While the expansion folds from both groups were comparable in the initial phases of co-culture with K562, a higher proportion of effector memory T cells in NKG2Dbp group resulted in a more persistent expansion of T cells driven by 4-1BB signaling. [00118] The in vivo tumor killing effects between T cells stably expressing NKG2D 1st generation CAR (NKG2Dp) and T cells stably expressing NKG2D 2nd generation CAR (NKG2Dbp) were analysed. A mouse colorectal model was established in NOD scid gamma (NSG) mice by intraperitoneal (i.p.) injection of HCT116-luc cells. Tumor progression was monitored by whole body bioluminescence imaging (Fig. 3A). As shown in Fig. 3A and 3B, the bioluminescence intensities, which is indicative of tumor burdens, in the PBS group and the control CAR T cell groups increased rapidly. Due to the aggressiveness of the model, mice in both PBS and control CAR T cell groups were all euthanized prior to day 28 due to developing moribund condition. [00119] Mice that received NKG2D 1st generation CAR-T cells or 2nd generation CAR-T cells showed reduction in tumor burden on day 14 after receiving a single dose T cell injection (Fig.3A and 3B). However, from day 21 onwards (Fig.3B), tumor burden for mice that received NKG2D 1st generation CAR-T cells increased progressively and although a second CAR T cell injection was given at day 30, the tumor burden did not decrease (Fig. 3B). Mice that received NKG2D 1st generation CAR-T cells injection were all euthanized by day 62. In contrast, tumor burden for the group of mice that received NKG2D 2nd generation CAR-T cells showed progressive reduction and became undetectable up to day 62. Mice continued to survive up to day 90 at the termination of the animal study (Fig.3C). [00120] After showing that NKG2D 2nd generation CAR (NKG2Dbp) had impressive in vitro and in vivo tumor killing effects, the NKG2Dbp CAR construct was compared against two NKG2D CAR constructs that use CD3z (instead of Dap12) as the activation and signaling domain. As a comparison, the full length sequence of CD3z, and CD3z consisting only one single ITAM, were used. As shown in Fig. 4, the CAR construct that consists of NKG2D-41BB-CD3z was termed NKG2Dbz, the CAR construct that consists of NKG2D- 41BB-CD3z (ITAM1) was termed NKG2Dbz1, while NKG2D-41BB-Dap12 construct was termed NKG2Dbp. A 3x streptomycin – tag II (ST2) was included in the CAR construct to detect CAR expression in T cells. Using the Piggybac transposon system, about 50-70% efficiency five was achieved days after electroporation (Fig. 4B). The expression of CAR on modified T cells can be further enriched upon co-culture with K562#6 that is engineered to express CD64, CD86 and CD137L, as shown in Fig. 4C. This is because the presence of the CD86 and CD137L expressed on K562#6 allows the CAR T cell to proliferate better and therefore improve the CAR expression on total T cells. [00121] The calibration of CAR activation potential determines alternative T cell fate and therapeutic potency. Therefore, the generation of T cell memory subsets amongst the three different groups of CAR-T cells was determined. Staining of CD45RO and CCR7 showed that relative to NKG2Dbz with three ITAM motifs, the two CARs with a single ITAM, NKG2Dbz1 and NKG2Dbp, increased the fraction of effector memory CAR T cells and reduced the proportion of effector cells in response to in vitro stimulation with K562C6 cells (Fig.5A and 5B). [00122] NKG2Dbp CAR-T cells, NKG2Dbz1 CAR-T cells, and NKG2Dbz CAR-T cells were then tested for their anti-tumor cytotoxicity using HCT116-luc (colorectal cancer) in a 4 hour and 16 hour cytotoxicity assays. It was found that the three CAR-T types showed indistinguishable tumor cell lysis activities, while all of them were highly effective in killing the investigated tumor cell lines (Fig 6A). Both NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells displayed high cell lysis activity towards a panel of solid tumour cancer cells, including SKOV3 luc (ovarian cancer), HCT116-luc (colorectal cancer), FaDu (head and neck cancer) and U87 (glioblastoma). However, there were no significant differences in cytotoxicity between the two NKG2D CAR-T cell groups (Fig.7A). [00123] To further distinguish function of CAR-T cells electroporated with NKG2Dbz, NKG2Dbz1 or NKG2Dbp CAR constructs, cytokine release and proliferation were investigated. NKG2Dbz CAR-T cells in general promoted higher amounts of IL-2, IFN-g, and TNF-a production as compared to NKG2Dbz1 and NKG2Dbp CAR-T cells, although the statistical differences were significant (p < 0.05) only between NKG2Dbz CAR-T cells and NKG2Dbz1 CAR-T cells for IL-2, and between NKG2Dbz CAR-T cells and NKG2Dbp CAR-T cells for all three examined pro-inflammatory cytokines (Fig.6B). ELISPOT assay as shown in Fig. 7B also confirmed that NKG2Dbz CAR-T cells showed higher level of IFN-g production compared to NKG2Dbp CAR-T cells after 18 hours co-culture with HCT116. [00124] The production of other pro-inflammatory cytokines was also assayed in the supernatants collected from NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells. As shown in Fig. 8, NKG2Dbz CAR-T cells produced significantly higher amounts of IFN-g, TNF-a and IL-2 as compared to NKG2Dbp CAR-T cells (p <0.05). Notably, it was observed that NKG2Dbz CAR-T cells produced higher levels of granulocyte-macrophage colony- stimulating factor (GM-CSF) as compared to NKG2Dbp CAR-T cells although it did not reach statistically significant level (p = 0.0519). [00125] The in vivo tumor killing effects between NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells are determined using a mouse colorectal model. The mouse colorectal model is established in (NOD scid gamma) NSG mice by intraperitoneal (i.p.) injection of HCT116- luc cells. Tumor progression was monitored by whole body bioluminescence imaging (Fig. 9). 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 one intraperitoneal (i.p.) injection of PBS, group 2 was subjected to one intraperitoneal (i.p.) injection of 1x107 control T cells; and groups 3 and 4 received one intraperitoneal (i.p.) injection of 1x107 NKG2Dbp CAR-T cells and NKG2Dbz CAR-T cells respectively. Bioluminescence intensities, which are indicative of tumor burdens, are used for analysis. As shown in Fig.9B, the bioluminescence intensities taken in the PBS group remain high during the treatment period from day 7 to 28. In addition, tumor progression for mice that received control CAR T cell groups was slower compared to PBS group. This finding is represented in a bar graph as shown in Fig.9C. [00126] In comparison, the groups of mice that received NKG2Dbp CAR-T cells or NKG2Dbz CAR-T cells showed reduction in tumor burden after receiving a single dose T cell injection (Fig. 9B). At Day 28, tumor burden remained undetectable (Fig. 9B and 9C) in most of the mice in the two groups. Bioluminescence signals were detected only at the site of injection for two mice, suggesting tumor relapse (Fig. 9B). A second T cell injection was given at a lower dose (2x106 cells/mice) in an attempt to eradicate the relapsed tumor and was successful (Fig.10). [00127] The group of mice that had received NKG2Dbz CAR-T cells injection began to show signs of xenogeneic graft versus host disease (x-GvHD) as evidenced by > 15% loss of weight, ruffled fur and hunched posture from day 35 onwards. Mice that showed signs of xenogeneic graft versus host disease (x-GvHD) were euthanized according to IACUC protocol. Xenogeneic graft versus host disease (x-GvHD) was not observed in mice that had received NKG2Dbp CAR-T cells. The animal study was terminated at day 150 post tumor inoculation, where all mice that received NKG2Dbp CAR-T cells remained healthy. Only one mouse from the NKG2Dbz CAR-T cell treatment group survived. Therefore, treatment with NKG2Dbp CAR-T cells is less susceptible to xenogeneic graft versus host disease (x-GvHD). This could be explained by the lower production of pro-inflammatory cytokines present in the mice serum compared to treatment with NKG2Dbz CAR-T cells (Fig. 10C). One of the causes of x-GvHD is excess cytokine-release after T-cell activation, which triggers a high level of proliferation of T cells and observed only in ab T cells, not other immune cells such as gamma delta T cells and natural killer (NK) cells. When NKG2Dbp CAR is used, reduced cytokine release was observed, as compared to NKG2Dbz CAR. Therefore, NKG2Dbp CAR can control the proliferation of CAR ab T cells, thereby reducing the occurrence of x-GvHD. [00128] To demonstrate that NKG2Dbp CAR-T cells exhibit broad anti-tumor effects beyond colorectal cancer, a mouse model of SKOV3 human ovarian cancer cells in NSG mice was established (Fig. 12). The mice, 5 per group, received i.p. injection of 1×107 SKOV3 cells (day 0), followed by i.p. injection of PBS, control CAR-T cells, or NKG2Dbp CAR-T cells on day 7 and day 14 (1×107 cells per injection per mouse). Growth of SKOV3 was monitored by bioluminescent imaging on the indicated days (Fig. 12A). Fig. 12B shows photos of bioluminescent images of three groups of mice from the experiment as described in Fig. 12A. The bioluminescent images of 5 mice per group demonstrated that the SKOV3 tumors in mice receiving NKG2Dbp CAR-T cells were eliminated from Day 14 onwards. Consequently, the CAR-T treatment significantly prolonged the survival of the mice in this group over the two controls (Fig. 12C) (P < 0.01). While all mice died in the two control groups by day 42, the mice treated with NKG2Dbp CAR-T cells survived for at least 80 days. Together with the results from the above CRC model, the data demonstrates that NKG2Dbp CAR-T cells are able to reduce and/or eradicate established solid tumors, thus prolonging the survival of tumor-bearing mice. CAR triggering lower cytokine release [00129] The presently disclosed NKG2Dbp CAR-T cell has only one ITAM motif per CAR molecule, which can stimulate relatively lower levels of cytokine release upon interaction with target cells, in comparison to T cells expressing NKG2Dbz that contains three ITAM motifs per CAR molecule. This suggests that cytokine release upon CAR activation could be controlled by simply decreasing the ITAM density of the CAR construct. We hypothesize that the reduction in cytokine release by NKG2Dbp is primarily attributable to the use of the single ITAM configuration, which can affect downstream functional output through the mitogen-activated protein kinase pathway. This effect provided by NKG2Dbp CAR-T would possibly provide a potential clinical advantage in reducing the risk of CRS and is the most important finding of this study. In addition, both NKG2Dbp and NKG2Dbz CAR-T products result in similar in vivo anti-tumor activities. This demonstrates that a single functional ITAM is sufficient for potent in vivo antitumor efficacy and superior to that afforded by the triple- ITAM-containing wild-type CD3z chain. Moreover, the single ITAM configuration favours the persistence of highly functional CARs, balancing the replicative capacity of long-lived memory cells and the acquisition of effective antitumor function, thereby yielding CAR designs with enhanced therapeutic profiles. [00130] The most common toxicities observed after CAR T-cell therapy are cytokine release syndrome (CRS) and cerebral edema/neurotoxicity. These involve the release of excess amounts of cytokines, and could be potentially life-threatening. Several factors including, for example, high tumor burden, high CAR-T cell dose, and lymphodepletion using cyclophosphamide and fludarabine, have been identified as being associated with a higher risk of cytokine release syndrome (CRS) and/or neurotoxicity among patients receiving CD19-targeted CAR T cell therapies. [00131] The NKG2Dbp construct comprises a CD8a hinge and a transmembrane domain. Furthermore, the CAR construct was designed to comprise only one ITAM motif so as to reduce the ITAM density of NKG2Dbp. This is to reduce the cytokine release that is commonly seen in present CAR-T therapies, and yet retain the tumor killing capacity of NKG2Dbp CAR T cells. Conclusion [00132] A single-chain CAR design targeting NKG2D ligand has been developed by fusing the NKG2D extracellular domain to the DAP12 internal signaling domain containing a single ITAM of the amino acid sequence ESPYQELQGQRSDVYSDL (NKG2D-DAP12). A functional synthetic NKG2D-DAP12 protein has also been demonstrated in human T cells. The T cells genetically modified with NKG2D-DAP12 stimulate a lower level of cytokine release during tumor cell lysis, and can eliminate established tumor xenografts in mice. Tables [00133] Table 1: Table of sequences used in the construction of recombinant CARs
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001

Claims

CLAIMS: 1. A ab T cell genetically modified to express a recombinant chimeric antigen receptor (CAR), wherein the recombinant CAR comprises (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM).
2. The ab T cell of claim 1, wherein the ITAM is a DAP12 ITAM.
3. The ab T cell of claim 2, wherein the DAP12 ITAM is ESPYQELQGQRSDVYSDL (SEQ ID NO: 15).
4. The ab T cell of claim 1, wherein the ITAM is CD3zeta ITAM-1.
5. The ab T cell of claim 4, wherein the CD3zeta ITAM-1 is APAYQQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 19).
6. The ab T cell of any one of claims 1 to 3, wherein the intracellular signaling domain is the intracellular signaling domain of DAP12.
7. The ab T cell of claim 4 or 5, wherein the intracellular signaling domain comprises APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL (SEQ ID NO: 27).
8. The ab T cell of any one of claims 1 to 7, wherein the transmembrane domain is CD28 transmembrane domain.
9. The ab T cell of any one of claims 1 to 8, wherein the co-stimulatory signaling domain is selected from the group consisting of 4-1BB, CD28, CD2 and OX-40.
10. The ab T cell of any one of claims 1 to 9, wherein the recombinant CAR further comprises, between the antigen binding region and the transmembrane region, a hinge region.
11. The ab T cell of claim 10, wherein the hinge region is an IgG4 hinge region.
12. A method of preparing the ab T cell of any one of claims 1 to 11, the method comprises: (i) obtaining or providing ab T cell; (ii) providing a recombinant nucleic acid encoding the recombinant chimeric antigen receptor (CAR); and (iii) transferring the recombinant nucleic acid encoding the recombinant CAR into the ab T cell.
13. A pharmaceutical composition comprising a pharmaceutically effective amount of the ab T cell of any one of claims 1 to 11 and a pharmaceutically acceptable excipient.
14. A method of treating cancer or tumor in a subject in need thereof, comprising administering to the subject a pharmaceutically effective amount of the ab T cell of any one of claims 1 to 11, or the pharmaceutical composition of claim 13.
15. The method of claim 14, wherein the ab T cells are derived from allogeneic or autologous ab T cells.
16. A method of treating cancer or tumor in a subject in need thereof, the method comprises: (i) obtaining ab T cells from the subject, or from a ab T cell donor which is different from the subject to be treated; (ii) providing 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, (c) at least one co-stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM); (iii) transferring the recombinant nucleic acid encoding the recombinant CAR into the ab T cell to obtain genetically modified ab T cells; and (iv) administering to the subject a pharmaceutically effective amount of the ab T cells obtained from (iii).
17. The method of any one of claims 14 to 16, wherein the cancer or tumor expresses NKG2D ligand(s).
18. The method of any one of claims 14 to 17, wherein the cancer or tumor is a solid cancer or tumor.
19. The method of any one of claims 14 to 18, wherein the cancer or tumor is colorectal cancer or colorectal tumor, or ovarian cancer or ovarian tumor.
20. The method of any one of claims 12 and 16-19, wherein transferring the recombinant nucleic acid in step (iii) is carried out using electroporation.
21. A recombinant chimeric antigen receptor (CAR) comprising (a) an extracellular domain comprising an antigen binding region, (b) a transmembrane domain, (c) at least one co- stimulatory signaling domain, and (d) an intracellular signaling domain, wherein the extracellular domain comprises the extracellular domain of an NKG2D receptor, and wherein the intracellular signaling domain comprises one immunoreceptor tyrosine-based activation motif (ITAM).
22. The recombinant CAR of claim 21, wherein the ITAM is a DAP12 ITAM.
23. The recombinant CAR of claim 22, wherein the DAP12 ITAM is ESPYQELQGQRSDVYSDL (SEQ ID NO: 15).
24. The recombinant CAR of any one of claims 21 to 23, wherein the intracellular signaling domain is the intracellular signaling domain of DAP12.
25. The recombinant CAR of any one of claims 21 to 24, wherein the transmembrane domain is CD28 transmembrane domain.
26. The recombinant CAR of any one of claims 21 to 25, wherein the at least one co- stimulatory signaling domain is selected from the group consisting of 4-1BB, CD28, CD2 and OX-40.
27. The recombinant CAR of any one of claims 21 to 26, wherein the recombinant CAR further comprises, between the antigen binding region and the transmembrane region, a hinge region.
28. The recombinant CAR of claim 27, wherein the hinge region is an IgG4 hinge region.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022251120A3 (en) * 2021-05-24 2023-02-16 Kite Pharma, Inc. Nkg2d-based chimeric antgen receptor
JP2024525807A (en) * 2021-07-14 2024-07-12 スジョウ イミュノフォコ バイオテクノロジー カンパニー リミテッド Novel chimeric receptor compositions, recombinant vectors, cells and their applications
WO2024167875A1 (en) * 2023-02-07 2024-08-15 Xyphos Biosciences Inc. Chimeric antigen receptor and antibody-nkg2d ligand domain fusion protein

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115725001A (en) * 2022-12-08 2023-03-03 湖南光琇高新生命科技有限公司 CD 19-targeted chimeric antigen receptor, nucleic acid, recombinant vector and CAR-T cell

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018140725A1 (en) * 2017-01-26 2018-08-02 Novartis Ag Cd28 compositions and methods for chimeric antigen receptor therapy
WO2018147805A1 (en) * 2017-02-08 2018-08-16 Agency For Science, Technology And Research Gamma delta t cells and a method of augmenting the tumoricidal activity of the same
WO2019133969A2 (en) * 2017-12-29 2019-07-04 Memorial Sloan-Kettering Cancer Center Enhanced chimeric antigen receptors and uses thereof

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2961636A1 (en) * 2014-09-17 2016-03-24 Boris ENGELS Targeting cytotoxic cells with chimeric receptors for adoptive immunotherapy
AU2018335266A1 (en) * 2017-09-22 2020-02-06 H. Lee Moffitt Cancer Center And Research Institute, Inc. Chimeric antigen receptors with enhanced NFκB signaling
CN109971713B (en) * 2017-12-28 2023-06-20 上海细胞治疗研究院 Muc 1-specific CAR-T cells stably expressing PD-1 antibodies and uses thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018140725A1 (en) * 2017-01-26 2018-08-02 Novartis Ag Cd28 compositions and methods for chimeric antigen receptor therapy
WO2018147805A1 (en) * 2017-02-08 2018-08-16 Agency For Science, Technology And Research Gamma delta t cells and a method of augmenting the tumoricidal activity of the same
WO2019133969A2 (en) * 2017-12-29 2019-07-04 Memorial Sloan-Kettering Cancer Center Enhanced chimeric antigen receptors and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LANIER L.L. ET AL.: "NKG2D Receptor and Its Ligands in Host Defense", CANCER IMMUNOL RES., vol. 3, no. 6, 2015, pages 575 - 582, XP055486221, DOI: 10.1158/2326-6066.CIR-15-0098> *
LUCÍA FERNÁNDEZ, JEAN-YVES METAIS, ADELA ESCUDERO, MARÍA VELA, JAIME VALENTÍN, ISABEL VALLCORBA, ALEJANDRA LEIVAS, JUAN TORRES, AN: "Memory T Cells Expressing an NKG2D-CAR Efficiently Target Osteosarcoma Cells", CLIN CANCER RES., vol. 23, no. 19, 28 June 2017 (2017-06-28), pages 5824 - 5835, XP055802284, DOI: 10.1158/1078-0432.CCR-17-0075> *
NG Y.Y. ET AL.: "T Cells Expressing NKG2D CAR with a DAP12 Signaling Domain Stimulate Lower Cytokine Production While Effective in Tumor Eradication", MOL THER., 5 September 2020 (2020-09-05), DOI: 10.1016/J.YMTHE.2020.08.016> *
YANG DONG, SUN BIN, DAI HONGJIU, LI WENXUAN, SHI LAN, ZHANG PEIXIAN, LI SHIRONG, ZHAO XUDONG: "T cells expressing NKG2D chimeric antigen receptors efficiently eliminate glioblastoma and cancer stem cells", J IMMUNOTHER CANCER., vol. 7, 9 July 2019 (2019-07-09), pages 171, XP055802285, DOI: 10.1186/S40425-019-0642-9> *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022251120A3 (en) * 2021-05-24 2023-02-16 Kite Pharma, Inc. Nkg2d-based chimeric antgen receptor
US12331315B2 (en) 2021-05-24 2025-06-17 Kite Pharma, Inc. Chimeric antigen receptor
AU2022282266B2 (en) * 2021-05-24 2025-09-18 Kite Pharma, Inc. NKG2D-based chimeric antigen receptor
JP2024525807A (en) * 2021-07-14 2024-07-12 スジョウ イミュノフォコ バイオテクノロジー カンパニー リミテッド Novel chimeric receptor compositions, recombinant vectors, cells and their applications
EP4372010A4 (en) * 2021-07-14 2025-04-23 Suzhou Immunofoco Biotechnology Co., Ltd. NOVEL CHIMERIC RECEPTOR COMPOSITION, RECOMBINANT VECTOR, CELL AND APPLICATION THEREOF
WO2024167875A1 (en) * 2023-02-07 2024-08-15 Xyphos Biosciences Inc. Chimeric antigen receptor and antibody-nkg2d ligand domain fusion protein

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