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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0634—Cells from the blood or the immune system
  • C12N5/0636—T lymphocytes
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
• • C—CHEMISTRY; METALLURGY
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/20—Cytokines; Chemokines
  • C12N2501/23—Interleukins [IL]
  • C12N2501/2308—Interleukin-8 (IL-8)
• • 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
• • 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
  • C12N2740/00—Reverse transcribing RNA viruses
  • C12N2740/00011—Details
  • C12N2740/10011—Retroviridae
  • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
  • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
  • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• This invention relates to methods of making immunoresponsive cells, immunoresponsive cells thereof, pharmaceutical compositions, kits, uses thereof and methods of treatment.
• CAR chimeric antigen receptor
• NKG2D ligands comprise a group of 8 stress-induced proteins (MICA, MICB, ULBP1-6) that are aberrantly expressed on virtually all tumour cell types.
• MICA, MICB, ULBP1-6 stress-induced proteins
• NKG2D ligands are also found on tumour associated stromal elements such as endothelium, regulatory T-cells and myeloid derived suppressor cells (Parihar, R., et al., 2019, Cancer Immunol. Res. 7(3):363-375; Schmiedel & Mandelboim, 2018, Front. Immunol. (9)2040).
• NKG2D mice that are genetically deficient in NKG2D demonstrate impaired immunosurveillance for both epithelial and lymphoid malignancies.
• the NKG2D receptor is naturally expressed by natural killer (NK) and some T-cell populations. Each NKG2D homodimer associates with two homodimeric DAP10 adaptor molecules via complementary charged amino acids within the plasma membrane. This interaction is required for cell surface expression and function of NKG2D.
• DAP10 resembles CD28 in its ability to provide co-stimulation via phosphatidylinositol 3-kinase but, importantly, it lacks a p56lck binding motif that promotes the unwanted recruitment of regulatory T-cells (Kofler, et al., 2011, Mol. Ther. 19:760-767). Potency of DAP10 co- stimulation is underscored by its continued ability to signal following internalization. However, since DAP10 lacks an immunoreceptor tyrosine-based activation motif (ITAM), NKG2D engagement does not lead to full T-cell activation.
• ITAM immunoreceptor tyrosine-based activation motif
• CARs have been developed which use different methods to provide ITAM- dependent signal 1 in addition to co-stimulation (also known as signal 2), as both signal types are necessary to elicit full T-cell activation.
• the first NKG2D-targeted CAR was developed by Sentman et al. and consists of a fusion of NKG2D to CD3£ (Zhang et al., 2005, Blood 106: 1544-1551). Although nominally a first-generation CAR, it associates with endogenous DAP10 in T-cells, meaning that both signals 1 and 2 are provided. This CAR is currently undergoing clinical development by Celyad Oncology as Cyad-01. More recently, Chang et al. (2013, Cancer Res.
• Ligands of the CXCR2 receptor include chemokines of the Cysteine-X-Cysteine (CXC) family that contain an (Glu-Leu-Arg) ELR motif, namely CXCL1-3 and CXCL5-8. Production of these chemokines within tumours is not only undertaken by malignant cells, but also by stromal elements such as fibroblasts and macrophages (Thuwajit et al., 2018, Med Res Rev.
• CXC Cysteine-X-Cysteine
• tumour cells can educate stromal cells to produce these factors, thereby enabling disease progression and resistance to cytotoxic chemotherapy (Le Naour, 2020, J Mol Cell Biol. 12:202-15).
• CXCL8 also known as interleukin (IL)-8.
• IL interleukin
• levels of circulating CXCL8 are elevated in patients with certain cancers, including ovarian tumours (Zhang, et al., 2019, Oncol Lett. 17:2365-9), malignant mesothelioma (Judge, et al. 2016, Ann Surg Oncol. 23: 1496-500), pancreatic cancer (Hou, et al. 2018, J Clin Med. 7:502, breast cancer (Milovanovic, et al. 2019, Cytokine 118:93-98; Autenshlyus, et al.
• the present invention provides a method of making an immunoresponsive cell.
• the method comprises:
• the method comprises:
• activating does not comprise phorbol 12-myristate 13-acetate (PMA), phytohaemagglutinin (PHA) or beads; and
• the present invention also provides an immunoresponsive cell obtainable by the method of either of the above aspects.
• an immunoresponsive cell genetically modified to express an NKG2D polypeptide, wherein the immunoresponsive cell has been activated with anti-CD3 and anti- CD28 antibodies, or fragments thereof, conjugated to a nanomatrix.
• the present invention provides a pharmaceutical composition
• a pharmaceutical composition comprising the immunoresponsive cell of the invention and a pharmaceutically or physiologically acceptable diluent and/or carrier.
• the present invention also provides a kit comprising the immunoresponsive cell or the pharmaceutical composition of the invention.
• immunoresponsive cell or the pharmaceutical composition of the invention for use in the treatment or prevention of a disease, optionally wherein the disease is cancer.
• the invention also provides a method of treating or preventing cancer in a subject, wherein the method comprises administering to the subject the immunoresponsive cell or the pharmaceutical composition of the invention.
• FIG. 1 shows schematics of the CAR constructs N1012, N1012_CXCR2, NKG2D and CYAD- 01_10.
• CYAD-01_10 a replica of Cyad-01 has been co-expressed with additional DAP10.
• Figure 2 shows development over time of s.c. CFPac-1 pancreatic tumour xenografts in mice treated 28 days after tumour inoculation with PBS, high dose (IxlO 7 ) CAR T-cells (N1012, N1012_CXCR2, CYAD-01 replica or untransduced) or low dose (4xl0 6 ) CAR T-cells (N1012, or N1012_CXCR2).
• A shows the average tumour volume per treatment group. Survival of the mice is shown in B.
• C shows individual mouse plots for the averaged data presented in A.
• Figure 3 shows the improved ability of N1012 and N1012_CXCR2 T-cells to persist and proliferate following repeated rounds of stimulation on the triple-negative breast cancer (TNBC) cell lines MDA-MB_468 and MDA-MB_231 compared to controls.
• TNBC triple-negative breast cancer
• FIG 4 shows bioluminescence emission from firefly luciferase (ffLuc)-expressing BxPC3 tumour xenografts in mice treated with N1012_CXCR2, N1012 or untransduced T-cells activated in vitro with different activation stimuli (PHA, TransAct or immobilised anti-CD3 and anti-CD28 antibodies).
• T-cells were administered i.p. on day 12 after tumour inoculation (indicated by vertical dotted lines). Tumour development is measured as total flux (photons/sec).
• FIG 5 shows the bioluminescence results presented in Figure 4 but with graphs to directly compare the anti-tumour activity of N1012, N1012_CXCR2 and untransduced T-cells when activated using the same stimulus (PHA, TransAct or immobilised anti-CD3 and anti-CD28 antibodies).
• Figure 6 shows individual plots of the results shown in Figures 4 and 5. An additional re- challenge was undertaken on day 64 in mice that had achieved a complete response. This re-challenge is designated by the second broken vertical line.
• Figure 7 shows the CD4 to CD8 ratio (as measured by flow cytometry) of N1012, N1012_CXCR2, untransduced, CYAD-01 and CYAD-01_10 T-cells at the end of the culture period (Day 10-12) following stimulation with either 5ug/mL phytohaemagglutinin-L (PHA-L) or lOuL TransAct reagent per IxlO 6 PBMCs.
• PHA-L phytohaemagglutinin-L
• TransAct lOuL TransAct reagent per IxlO 6 PBMCs.
• Activation with TransAct shifted all T-cells to a higher CD4:CD8 ratio. This shift was most apparent for N1012 and N1012_CXCR2 T-cells.
• Figure 8 shows (A) Transduction efficacy of T-cells after activation with PHA or TransAct at the end of the ex vivo culture period (Day 10-12) as assessed by flow cytometry. Transduction was assessed as any cell that stained positive for NKG2D or CXCR2 and was assessed in CD4 + T-cells due to the lack of endogenous NKG2D expression; (B) Median fluorescence intensity (MFI) of NKG2D was assessed in CD4 + T-cells by flow cytometry at the end of the ex vivo culture period.
• MFI Median fluorescence intensity
• Figure 9 shows the fold expansion of T-cells activated with either 5ug/mL phytohaemagglutinin-L (PHA-L) or lOuL TransAct reagent per IxlO 6 PBMCs. This was calculated by dividing the number of T-cells present at the end of the ex vivo culture period by the number of T-cells initially transduced.
• PHA-L phytohaemagglutinin-L
• lOuL TransAct reagent per IxlO 6 PBMCs This was calculated by dividing the number of T-cells present at the end of the ex vivo culture period by the number of T-cells initially transduced.
• FIG 10 shows the median fluorescence intensity (MFI, A) and transduction percentage (B) of cell surface NKG2D expression in pan-y6 TCR + CD3 + cells following transduction with retroviral vectors that encode N1012 or N1012_CXCR2 and 21 days of expansion. Results for untransduced T-cells are shown for comparison.
• AML acute myeloid leukaemia
• Figure 14 shows (A) Purity of CAR + y6 T-cells following restimulation on MDA-MB-468 cells
• Figure 15 shows anti-tumour efficacy of N1012 or N1012_CXCR2 y6 T-cells in NSG mice engrafted with I.P. BxPC-3-LT tumours. Cryopreserved y6 T-cells were thawed and injected at a dose of 10 million I.P. at day 11 of tumour engraftment. Untransduced y6 T-cells and PBS were used as controls.
• Figure 16 is a survival curve of I.P. BxPC3 tumour xenograft-containing mice treated with PBS or 1 X 10 7 CAR T-cells (N1012, N1012_CXCR2, or untransduced).
• Figure 17 is a survival curve showing pooled data from in vivo experiments for CFPacl, BxPC3, Kuramochi, Ovsaho, Mesothelioma, triple-negative breast cancer or SKOV3 tumour xenograft-containing mice treated with PBS or 1 x 10 7 CAR T-cells (N1012, N1012_CXCR2, or untransduced).
• Figure 18 shows the transduction (A-B) and expansion (C) of primary human T-cells with NKG2D_CXCR2 following activation with differing concentrations of TransActTM.
• the expression of NKG2D expression was assessed in CD4 + T-cells by flow cytometry three and ten days after transduction and used as a marker of transduction efficiency.
• Untransduced (UT) CD4 + T-cells were used as a negative control for transduction.
• Figure 19 demonstrates the anti-tumour efficacy of N1012 and N1012_CXCR2 T-cells in NSG mice engrafted subcutaneously with either LS180 or SW620 metastatic colorectal carcinoma (mCRC) cells.
• the T-cells were injected intravenously at a dose of 1 x 10 7 and tumour growth monitored by caliper measurement.
• the average growth per treatment group (A-B) or the tumour growth in individual mice in the SW620 model (C) was compared with untransduced (UT) T-cells or CYAD-01 T-cells.
• the present invention provides a method of making an immunoresponsive cell.
• the method comprises:
• the present inventors have found that activation of immune cells with anti-CD3 and anti- CD28 antibodies, or fragments thereof, conjugated to a nanomatrix and genetic modification of immune cells to express an NKG2D polypeptide results in immunoresponsive cells with unexpectedly improved functionality.
• the resulting immunoresponsive cells have unexpectedly improved anti-tumour activity.
• the method comprises:
• the method may comprise:
• step (i) occurs before step (ii).
• Activation of the immune cell with anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix may be for a time period of from about 6 hours to about 504 hours.
• activation of the immune cell with anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is for a time period of at least about 6 hours, at least about 12 hours, at least about 18 hours, at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours or at least about 168 hours.
• the method comprises step (iii) comprising culturing of the immune cell in vitro. In some embodiments, the method comprises:
• Culturing the immune cell in vitro may be for a time period of from about 24 hours to about 504 hours. In some embodiments, culturing the immune cell in vitro is for a time period of at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours, at least about 120 hours, at least about 144 hours or at least about 168 hours. In some embodiments, culturing the immune cell in vitro is for a time period of no more than about 504 hours, no more than about 480 hours, no more than about 456 hours, no more than about 432 hours, no more than about 408 hours, no more than about 384 hours or no more than about 360 hours.
• culturing the immune cell in vitro is for a time period of at least about 48 hours. More preferably, culturing the immune cell in vitro is for a time period of at least about 72 hours. Most preferably, culturing the immune cell in vitro is for a time period of at least about 96 hours. Preferably, culturing the immune cell in vitro is for a time period of no more than about 336 hours. More preferably, culturing the immune cell in vitro is for a time period of no more than about 312 hours. Most preferably, culturing the immune cell in vitro is for a time period of no more than about 288 hours.
• culturing the immune cell in vitro is for a time period of about 168 hours. In other embodiments, culturing the immune cell in vitro is for a time period of about 192 hours. In some embodiments, culturing the immune cell in vitro is for a time period of about 216 hours.
• culture of the immune cell in vitro is in an appropriate medium.
• the medium may comprise RPMI-1640 medium or DMEM high glucose medium. Other suitable media will be known to the skilled person.
• the medium may be supplemented with, for example, antibiotic, IL-2, human AB serum, FBS (foetal bovine serum) or FCS (foetal calf serum) and/or amino acids.
• the medium comprises IL-2.
• the IL-2 may be recombinant.
• the IL-2 may be human. In some embodiments the IL-2 is at a concentration in the medium of about 20 U/ml.
• culturing the immune cells in vitro comprises re-activating the immune cells in vitro.
• the re-activation may comprise activation of the immune cell with anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix.
• the re-activation may comprise activation with phorbol 12-myristate 13- acetate (PMA), phytohaemagglutinin (PHA) or anti-CD3 and anti-CD28 antibodies conjugated to beads.
• beads conjugated to anti-CD3 and anti-CD28 antibodies are typically inert and of a uniform diameter which is similar to that of antigen-presenting cells. Thus, beads typically have a diameter of from about 1 to about 10 pm in size. The beads are also typically spherical in shape with a non-porous, solid surface area. Such beads have previously commonly been used in in vitro cell culture to activate and expand T-cells. Various such beads are readily commercially available, including, but not limited to Dynabeads® (ThermoFisher Scientific, UK).
• the method does not comprise phorbol 12-myristate 13-acetate (PMA), phytohaemagglutinin (PHA) or beads.
• PMA phorbol 12-myristate 13-acetate
• PHA phytohaemagglutinin
• steps (i) and (ii) may not comprise phorbol 12-myristate 13-acetate (PMA), phytohaemagglutinin (PHA) or beads.
• PMA phorbol 12-myristate 13-acetate
• PHA phytohaemagglutinin
• the polymer chains may comprise collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions.
• polymers may include polyesters, polyethers, polyanhydrides, polyalkyl cyanoacrylates, polyacrylamides, polyorthoesters, polyphosphazenes, polyvinyl acetates, block copolymers, polypropylene, polytetrafluorethylene (PTFE), or polyurethanes.
• the polymer may be lactic acid or a copolymer.
• a copolymer may comprise lactic acid and glycolic acid (PLGA).
• the nanomatrix has a diameter of from about 1 to about 500nm.
• the nanomatrix has a diameter of at least about Inm, at least about lOnm, at least about 20nm, at least about 50nm or at least about 70nm. In some embodiments, the nanomatrix has a diameter of no more than about lOOOnm, no more than about 500nm or no more than about 200nm.
• the nanomatrix has a diameter of from about 50nm to about 200nm. More preferably, the nanomatrix has a diameter of from about lOnm to about 200nm. Most preferably, the nanomatrix has a diameter of about lOOnm.
• the nanomatrix is biodegradable.
• the nanomatrix is non-toxic to living cells. By non-toxic to living cells, this will be understood to mean that the nanomatrix does not detrimentally affect the cell viability.
• the nanomatrix is soluble or colloidal.
• the term “soluble” will be understood to mean that the nanomatrix is dissolvable in a solvent.
• the solvent may be an aqueous solution, such as a media as described above.
• the term “colloidal” refers to the nanomatrix being insoluble but suspended throughout a solvent to form a mixture comprising the nanomatrix and the solvent.
• any fragment of an anti- CD3 antibody or anti-CD-28 antibody is a functional fragment, in that it retains the functional activity of the anti-CD3 or anti-CD28 antibody.
• the anti-CD3 antibody or fragment thereof is humanized. In some embodiments, the anti-CD28 antibody or fragment thereof is humanized. In some embodiments, the anti-CD3 antibody or fragment thereof and the anti-CD28 antibody or fragment thereof are humanized.
• a humanized antibody is an antibody from a non-human species wherein the amino acid sequence of the antibody has been genetically modified to increase similarity to human antibodies, thereby reducing immunogenicity. Methods to humanize antibodies are well known in the art.
• conjugation or “conjugated” this will be understood to mean that the antibody (or other agent) is linked or coupled to the nanomatrix.
• the linkage or coupling may be covalent or non-covalent.
• Covalent linkages may comprise a linkage to a carboxyl group on a polymer chain.
• Non-covalent linkages may comprise a biotin-avidin interaction.
• the nanomatrix may be conjugated to additional agents to the anti-CD3 and anti-CD28 antibodies, or fragments thereof.
• additional agents may include, but not necessarily be limited to polynucleotides and/or proteins. Proteins may include antibodies, fragments and derivatives thereof, fusion proteins and genetically modified proteins. Preferably, additional agents comprise cell co-stimulatory molecules.
• co-stimulatory molecules include, but are not necessarily limited to anti-CD5, anti-CD4, anti-CD8, anti-MHCI, anti-MHCII, anti CTLA- 4, anti-ICOS, anti-PD-1, anti-OX40, anti-CD27L (CD70), anti 4-1BBL, anti CD30L and anti- LIGHT antibodies, fragments or derivatives thereof.
• cytokines may comprise IL-2, IFN-y, IL-12, IL-17, IL-1 IL-15, IL-4, IL-10 and TNF-o.
• chemokine receptors include, but are not necessarily limited to CCR1, CCR2, CCR3, CCR4, CCR5, and CXCR3.
• an additional agent may comprise any agent capable of binding to cellular adhesion molecules on T-cells such as mAbs, fusion proteins and the corresponding ligands or fractions thereof to adhesion molecules in the following categories: cadherins, ICAM, integrins, and selectins.
• adhesion molecules on T-cells are: CD44, CD31, CD18/CD11 a (LFA-1), CD29, CD54 (ICAM-1), CD62L (L-selectin), and CD29/CD49d (VLA- 4).
• further agents may be embedded into the nanomatrix.
• further agents may be embedded into the polymer chains.
• Such further agents may comprise magnetic agents or fluorescent agents.
• metal oxide crystals may be embedded into the polymer chains.
• iron oxide crystals are embedded into the polymer chains.
• suitable magnetic and fluorescent agents are known to those skilled in the art.
• the agent being located within one or more polymer chains of the nanomatrix.
• the polymer chain comprises the further agent.
• the nanomatrix comprises iron oxide crystals embedded into a biocompatible polysaccharide matrix with a diameter of about 100 nm. In some embodiments, the nanomatrix is colloidal and comprises iron oxide crystals embedded into a biocompatible polysaccharide matrix with a diameter of about 100 nm.
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is the nanomatrix disclosed in W02014/048920A1, which is herein incorporated in its entirety.
• This nanomatrix may be referred to as the MACS GMP TransAct CD3/CD28 Kit (Miltenyi Biotec GmbH , Order no . 170 - 076 - 140).
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is at a concentration of at least about O.luL per IxlO 6 cells, at least about 0.5uL per IxlO 6 cells, at least about luL per IxlO 6 immune cells, at least about 2uL per IxlO 6 immune cells, at least about 5uL per IxlO 6 immune cells or at least about lOuL per IxlO 6 immune cells.
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is at a concentration of less than about 50uL per IxlO 6 immune cells, less than about 40uL per IxlO 6 immune cells, less than about 30uL per IxlO 6 immune cells or less than about 20uL per IxlO 6 immune cells.
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is at a concentration of about 10 uL per IxlO 6 immune cells.
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is at a concentration of about 5 uL per IxlO 6 immune cells.
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is at a concentration of about 1 uL per IxlO 6 immune cells.
• the anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix is at a concentration of about 1 to about 5 uL per IxlO 6 immune cells.
• one or more steps of the method are automated.
• automated refers to the method being automated through use of devices and/or computers and computer softwares, such that once the automated step/method begins, there is no requirement for manual intervention. In some embodiments the method is automated.
• the immune cell is genetically modified to further express a DAP10/DAP12 fusion polypeptide.
• Genetic modification to further express a DAP10/DAP12 fusion polypeptide may be simultaneous, prior or after to genetic modification to express an NKG2D polypeptide.
• the method comprises:
• the method comprises:
• the fusion polypeptide may have the formula, from N-terminus to C-terminus:
• A is an optional N-terminal sequence
• B is a DAP10 polypeptide
• C is an optional linker sequence
• D is a DAP12 polypeptide
• E is an optional C-terminal sequence.
• the fusion polypeptide does not comprise SEQ ID NO: 84. In some embodiments the fusion polypeptide does not comprise an anti-EpCAM peptide. In some embodiments the fusion polypeptide does not comprise SEQ ID NO: 85. In some embodiments the fusion polypeptide does not comprise SEQ ID NO: 86. In some embodiments, the fusion polypeptide does not comprise both SEQ ID NO: 85 and SEQ ID NO: 86.
• the DAP10 polypeptide may be mammalian, for example human. Wild-type human DAP10 is encoded by the amino acid sequence having UniProt accession no: Q9UBK5 (SEQ ID NO: 1). This is a 93 amino acid polypeptide. The first 18 amino acids are considered to be a signal/leader sequence, amino acids 19-48 the extracellular domain, amino acids 49-69 the transmembrane domain, and amino acids 70-93 the cytoplasmic/intracellular domain.
• mammal as used herein includes both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
• the DAP10 polypeptide is a functional variant of DAP10 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP10 polypeptide of SEQ ID NO: 1.
• the DAP10 polypeptide comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 1.
• percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
• sequence comparison algorithms e.g., BLASTP and BLASTN or other algorithms available to persons of skill
• the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
• sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
• test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
• percent identity and sequence similarity is performed using the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/).
• Truncated versions of a DAP10 polypeptide may also be used in fusion polypeptides of the invention.
• the DAP10 polypeptide is a functional variant of a DAP10 polypeptide which is a truncated version of the polypeptide having the amino acid sequence of SEQ ID NO: 1.
• a truncated version of DAP10 comprises only amino acids 19-93 of SEQ ID NO: 1 (i.e. lacking amino acids 1-18, the signal/leader sequence). Such a sequence is referred to as SEQ ID NO: 2 herein.
• truncated versions may comprise amino acids 19-69 of SEQ ID NO: 1, such a sequence comprising merely the extracellular and transmembrane domains of DAP10, and referred to herein as SEQ ID NO: 3.
• a further truncated version of DAP10 used in the invention may comprise amino acids 1-71 of SEQ ID NO: 1 (i.e. the signal/leader sequence, extracellular domain, transmembrane domain and 2 amino acids from the cytoplasmic/intracellular domain), referred to as SEQ ID NO: 4 herein.
• a further truncated version of DAP10 used in the invention may comprise amino acids 19-71 of SEQ ID NO: 1 (i.e.
• a further truncated version of DAP10 used in the invention may comprise amino acids 70-93 of SEQ ID NO: 1 (i.e. the intracellular domain), referred to as SEQ ID NO: 6 herein.
• a yet further truncated version of DAP10 used in the invention may comprise amino acids 49-93 of SEQ ID NO: 1 (i.e. the transmembrane and cytoplasmic/intracellular domains), referred to as SEQ ID NO: 7 herein.
• a yet further truncated version of DAP10 used in the invention may comprise amino acids 49-69 of SEQ ID NO: 1 (i.e. the transmembrane domain), referred to as SEQ ID NO: 8 herein.
• the truncated version of DAP10 comprises or consists of amino acids 19-93, 19-69, 1-71, 19-71, 19-48, 49-69, 49-93, or 70-93 of SEQ ID NO: 1
• the DAP10 polypeptide comprises or consists of any one of SEQ ID NOs: 1-8.
• a functional variant is a variant of a wild-type protein which retains the functional activity of the wild-type protein.
• this will be understood to refer to an amino acid sequence which is altered to that of the wild-type protein.
• the variant may have at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or more point mutations relative to the wild type protein sequence.
• the point mutation(s) may comprise a substitution, deletion or addition of an amino acid.
• a functional variant of a DAP10 polypeptide retains at least 10% (for example 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild type polypeptide shown in SEQ ID NO: 1.
• the activity may be measured by assessment of tyrosine phosphorylation of DAP10 and/or recruitment and activation of the p85 subunit of phosphatidylinositol 3-kinase and the downstream anti- apoptotic kinase, AKT.
• the DAP12 polypeptide may be mammalian, for example human. Wild-type human DAP12 is encoded by the amino acid sequence having UniProt accession no: 043914 (SEQ ID NO: 9). The first 21 amino acids are considered to be a signal/leader sequence, amino acids 22-40 the extracellular domain, amino acids 41-61 the transmembrane domain, and amino acids 62-113 the cytoplasmic/intracellular domain.
• the DAP12 polypeptide is a functional variant of DAP12 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP12 polypeptide having the sequence of SEQ ID NO: 9.
• the DAP12 polypeptide comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 9.
• Truncated versions of a DAP12 polypeptide may also be used in fusion polypeptides of the invention.
• the DAP12 polypeptide is a truncated version of the DAP12 polypeptide having the amino acid sequence of SEQ ID NO: 9.
• An exemplary truncated version of DAP12 comprises only amino acids 22-113 of SEQ ID NO: 9 (i.e. lacking amino acids 1-21, the signal/leader sequence). Such a sequence is referred to as SEQ ID NO: 10 herein.
• Other truncated versions may comprise amino acids 62-113 of SEQ ID NO: 9, such a sequence comprising merely the cytoplasmic/intracellular domain of DAP12 and referred to herein as SEQ ID NO: 11.
• truncated versions may comprise amino acids 41-61 of SEQ ID NO: 9 (i.e. the transmembrane domain), referred to as SEQ ID NO: 12 herein.
• Another truncated version may comprise amino acids 22-61 of SEQ ID NO: 9 (i.e., the extracellular and transmembrane domains), referred to as SEQ ID NO: 13 herein.
• the truncated version of DAP12 comprises or consists of amino acids 22-113, 62-113, 22-61, or 41-61 of SEQ ID NO: 9.
• the DAP12 polypeptide comprises or consists of any one of SEQ ID NOs: 9-13.
• a functional variant of a DAP12 polypeptide may retain at least 10% (for example 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild type polypeptide shown in SEQ ID NO: 9.
• the activity may be measured using functional assays, such as MTT and measuring cytokine secretion by ELISA.
• the NKG2D polypeptide may be a mammalian polypeptide.
• the NKG2D polypeptide is a human polypeptide.
• Wild-type human NKG2D is encoded by the amino acid sequence having UniProt accession no: P26718 (SEQ ID NO: 14).
• the wild type NKG2D polypeptide is considered to comprise a cytoplasmic domain (amino acids 1-51), a transmembrane domain (amino acids 52-72) and an extracellular domain (amino acids 73- 216).
• the NKG2D polypeptide is a functional variant of NKG2D.
• the functional variant of NKG2D may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the NKG2D polypeptide having the sequence of SEQ ID NO: 14.
• the NKG2D polypeptide comprises or consists of SEQ ID NO: 14.
• the functional variant of NKG2D may comprise or consist of a truncated version of NKG2D.
• the NKG2D polypeptide may be a truncated version of NKG2D comprising only amino acids 73-216 of SEQ ID NO: 14 (i.e., the extracellular domain. Such a sequence is referred to as SEQ ID NO: 15 herein.
• Other truncated versions may comprise amino acids 82-216 of SEQ ID NO: 14, such a sequence comprising part of the extracellular domain of NKG2D and referred to herein as SEQ ID NO: 16.
• a further truncated version comprises amino acids 52-216 of SEQ ID NO: 14 (i.e. the transmembrane and extracellular domains), referred to as SEQ ID NO: 17 herein.
• a functional variant of a NKG2D polypeptide may retain at least 10% (for example 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of the wild type polypeptide shown in SEQ ID NO: 14.
• the activity may be measured using flow cytometry to confirm continued binding to NKG2D ligands and through various cell culture assays (such as MTT and ELISA) aimed at confirming target T-cell lysis, cytokine secretion and co-stimulation.
• the functional variant of the NKG2D polypeptide is a chimeric NKG2D polypeptide.
• the chimeric NKG2D polypeptide may be a human-murine chimeric polypeptide.
• the term "murine” refers to a rodent of the subfamily Murinae.
• the term “murine” comprises rat and mouse.
• chimeric NKG2D polypeptide refers to an NKG2D receptor formed of domains from two or more different organisms. Chimeric NKG2D polypeptides are described in more detail in WO 2021/234163, the disclosure of which is herein incorporated by reference in its entirety. In some embodiments, the chimeric NKG2D polypeptide comprises from N terminus to C terminus the murine NKG2D transmembrane domain or a variant thereof and a human NKG2D extracellular domain or a variant thereof.
• the immune cell is genetically modified to express a CXCR2 polypeptide.
• Genetic modification to express a CXCR2 polypeptide may be simultaneous, prior to or after genetic modification to express an NKG2D polypeptide.
• the method comprises:
• the method comprises:
• the method comprises:
• the method comprises:
• the method comprises:
• the method comprises:
• the CXCR2 polypeptide is a mammalian polypeptide. More preferably, the CXCR2 polypeptide is a human polypeptide. Wild-type human CXCR2 is encoded by the amino acid sequence having UniProt accession no: P25025 (SEQ ID NO: 87).
• the CXCR2 polypeptide comprises a functional variant of the wildtype CXCR2 polypeptide.
• the CXCR2 polypeptide may be a functional variant of the CXCR2 polypeptide of SEQ ID NO: 87.
• the functional variant of the CXCR2 polypeptide may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the CXCR2 polypeptide having the sequence of SEQ ID NO: 87.
• the CXCR2 polypeptide comprises or consists of amino acid sequence of SEQ ID NO: 87.
• a functional variant of the CXCR2 polypeptide may retain at least 10% (for example, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99% or more) of the activity of wild-type human CXCR2 (SEQ ID NO: 87).
• Activity of the polypeptide may be measured using flow cytometry to confirm binding of CXCR2 to ligand (e.g., IL-8).
• ligand e.g., IL-8
• activity of the polypeptide is measured through cell culture assays known in the art.
• the DAP10 and DAP12 polypeptides of the fusion polypeptide described herein can be directly bonded to each other in a contiguous polypeptide chain or may be indirectly bonded to each other through a suitable linker.
• the DAP10/12 fusion polypeptide may comprise a linker sequence.
• the linker may be a peptide linker.
• Peptide linkers are commonly used in fusion polypeptides and methods for selecting or designing linkers are well-known. (See, e.g., Chen X et al., 2013, Adv. Drug Deliv. Rev. 65(10): 135701369 and Wriggers W et al., 2005, Biopolymers 80:736-746.).
• Linkers may also be used to join the fusion polypeptide of the disclosure to another polypeptide (such as the NKG2D polypeptide and/or the CXCR2 polypeptide) in a chimeric construct as described further below.
• Peptide linkers generally are categorized as i) flexible linkers, ii) helix forming linkers, and iii) cleavable linkers, and examples of each type are known in the art.
• a flexible linker is included in the fusion polypeptides described herein.
• Flexible linkers may contain a majority of amino acids that are sterica lly unhindered, such as glycine and alanine.
• the hydrophilic amino acid Ser is also conventionally used in flexible linkers.
• flexible linkers include, without limitation: polyglycines (e.g., (Gly)4 and (Gly)s), polyalanines poly(Gly-Ala), and poly(Gly-Ser) (e.g., (Gly n -Ser n ) n or (Ser n -Gly n )n, wherein each n is independently an integer equal to or greater than 1).
• polyglycines e.g., (Gly)4 and (Gly)s
• poly(Gly-Ser) e.g., (Gly n -Ser n ) n or (Ser n -Gly n )n, wherein each n is independently an integer equal to or greater than 1).
• Peptide linkers can be of a suitable length.
• the peptide linker sequence may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or more amino acid residues in length.
• a peptide linker can be from about 5 to about 50 amino acids in length; from about 10 to about 40 amino acids in length; from about 15 to about 30 amino acids in length; or from about 15 to about 20 amino acids in length. Variation in peptide linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.
• the peptide linker sequence may be comprised of naturally or non-naturally occurring amino acids, or a mixture of both naturally and non-naturally occurring amino acids.
• the linker sequence comprises or consists of the amino acids glycine and serine.
• the linker region may comprise sets of glycine repeats (GSG3) n (SEQ ID NO: 18), where n is a positive integer equal to or greater than 1 (for example 1 to about 20). More specifically, the linker sequence may be GSGGG (SEQ ID NO: 19). The linker sequence may be GSGG (SEQ ID NO: 20).
• the linker region orientation comprises sets of glycine repeats (SerGlys)n, where n is a positive integer equal to or greater than 1 (for example 1 to about 20) (SEQ ID NO: 21).
• a linker may contain glycine (G) and serine (S) in a random or a repeated pattern.
• the linker can be (GGGGS) n (SEQ ID NO: 22), wherein n is an integer ranging from 1 to 20, for example 1 to 4.
• n is 4 and the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 23).
• n is 3 and the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 24).
• a linker may contain glycine (G), serine (S) and proline (P) in a random or repeated pattern.
• the linker can be (GPPGS) n , wherein n is an integer ranging from 1 to 20, for example 1-4. In a particular example, n is 1 and the linker is GPPGS (SEQ ID NO: 25).
• linker is not immunogenic when administered in a patient, such as a human.
• linkers may be chosen such that they have low immunogenicity or are thought to have low immunogenicity.
• the linkers described herein are exemplary, and the linker can include other amino acids, such as Glu and Lys, if desired.
• the peptide linkers may include multiple repeats of, for example, (G 3 S) (SEQ ID NO: 26), (G 4 S) (SEQ ID NO: 27), (GYS) (SEQ ID NO: 28), and/or (GlySer) (SEQ ID NO: 29), if desired.
• the peptide linkers may include multiple repeats of, for example, (SG 4 ) (SEQ ID NO: 30), (SG 3 ) (SEQ ID NO: 31), (SG 2 ) (SEQ ID NO: 32), (SG) 2 (SEQ ID NO: 33) or (SerGly) (SEQ ID NO: 34).
• the peptide linkers may include combinations and multiples of repeating amino acid sequence units, such as (G 3 S)+(G 4 S)+(GlySer) (SEQ ID NO: 26 +SEQ ID NO: TJ +SEQ ID NO: 29).
• Ser can be replaced with Ala e.g., (G 4 A) (SEQ ID NO: 35) or (G 3 A) (SEQ ID NO: 36).
• the linker comprises the motif (EAAAK) n , where n is a positive integer equal to or greater than 1, for example 1 to about 20 (SEQ ID NO: 37).
• peptide linkers also include cleavable linkers.
• the linkers may comprise further domains and/or features, such as a furin cleavage site (RRKR)(SEQ ID NO: 38), a P2A ribosomal skip peptide (ATNFSLLKQAGDVEENPGP)(SEQ ID NO: 39) and/or a T2A ribosomal skip peptide (EGRGSLLTCGDVEENPGP)(SEQ ID NO: 40).
• RRKR furin cleavage site
• ATNFSLLKQAGDVEENPGP P2A ribosomal skip peptide
• EGRGSLLTCGDVEENPGP T2A ribosomal skip peptide
• linkers comprising these domains include SGSG + a P2A ribosomal skip peptide (SGSGATNFSLLKQAGDVEENPGP)(SEQ ID NO: 41), SGSG + a T2A ribosomal skip peptide (SGSGEGRGSLLTCGDVEENPGP)(SEQ ID NO: 42), and versions also including a furin cleavage site, i.e.
• furin cleavage site + SGSG + a P2A ribosomal skip peptide (RRKRSGSGATNFSLLKQAGDVEENPGP) (SEQ ID NO: 43) and furin cleavage site + SGSG + a T2A ribosomal skip peptide (RRKRSGSGEGRGSLLTCGDVEENPGP) (SEQ ID NO: 44).
• Alternative ribosomal skip peptides that may be used in the invention include F2A (VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO: 45) and E2A (QCTNYALLKLAGDVESNPGP) (SEQ ID NO: 46).
• sequences may be attached to the N- or C-terminus of the fusion polypeptide, the NKG2D polypeptide and/or the CXCR2 polypeptide disclosed herein. These sequences may be functional, such as signal peptides, purification tags/sequences, or half-life extension moieties, or may simply comprise spacer sequences. Alternatively, they may comprise another function, such as a T-cell stimulatory function.
• tags or markers may be attached to the N- or C-terminus of the fusion polypeptide, the NKG2D polypeptide and/or the CXCR2 polypeptide to assist with purification.
• a tag may comprise an affinity tag.
• affinity tags are a His-tag, a FLAG-tag, Arg-tag, T7-tag, Strep-tag, S-tag, aptamer-tag, V5 tag, AviTagTM, myc epitope tag or any combination of these tags.
• the affinity tag is a His-tag (usually comprising 5-10 histidine residues), for example a 6His tag (i.e. HHHHHH) (SEQ ID NO: 47).
• the affinity tag is a FLAG tag (i.e. DYKDDDDK) (SEQ ID NO: 48). In some embodiments, the affinity tag is an AviTagTM (i.e. GLNDIFEAQKIEWHE) (SEQ ID NO: 49). In some embodiments, the affinity tag is a V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 50) or (IPNPLLGLD) (SEQ ID NO: 51). In some embodiments, the affinity tag is a myc epitope tag recognised by the 9el0 antibody (EQKLISEEDL) (SEQ ID NO: 52). Various other tags are suitable for use and well known in the art.
• affinity tags may also be used, either comprising one or more tags at the N-terminus, one or more tags at the C-terminus, or one or more tags at each of the N- terminus and the C-terminus.
• examples of such combinations include a His tag (H) combined with an AviTag (A), or a His tag (H) combined with both an AviTag (A) and a FLAG tag (F).
• the tags may be in either orientation, thus the AviTag/His tag may have the orientation N-AH-C or N-HA-C, while the Avi/His/FLAG tag may have the orientation N-AHF- C, N-FHA-C, etc.
• a fusion polypeptide may comprise an "AHF” tag having the sequence "GLNDIFEAQKIEWHEGGHHHHHHDYKDDDDK” (SEQ ID NO: 53).
• a fusion polypeptide may comprise an "FHA” tag having the sequence "DYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE” (SEQ ID NO: 54).
• the CD8o leader sequence (amino acids 1-21 of UniProt: P01732 or a shortened derivative comprising amino acids 1-18), is a commonly used T-cell sequence, and is referred to as SEQ ID NO: 55 herein.
• the fusion polypeptide, NKG2D polypeptide and/or CXCR2 polypeptide may comprise SEQ ID NO: 55.
• the N terminus of the fusion polypeptide, NKG2D polypeptide and/or CXCR2 polypeptide comprises SEQ ID NO: 55.
• T-cell co-stimulatory activation sequences are known from previous work to engineer CAR-T-cells.
• the fusion polypeptide, NKG2D polypeptide and/or CXCR2 polypeptide may further comprise or be fused to a T-cell co-stimulatory activation sequence.
• the 4-1BB endodomain (amino acids 214-255 of UniProt: Q07011) may also be used as an N- or C-terminal sequence.
• the 4-1BB endodomain is referred to as SEQ ID NO: 56 herein.
• the 4-1BB endodomain may act as a co-stimulatory domain.
• the CD27 endodomain (amino acids 213-260 of UniProt: P26842) may also be used as an N- or C-terminal sequence.
• the CD27 endodomain is referred to as SEQ ID NO: 57 herein.
• the CD27 endodomain may act as a co-stimulatory domain.
• the human IgGl hinge (amino acids 218-229 or 218-232 of UniProt: PODOX5) may also be used as an N- or C-terminal sequence.
• the human IgGl hinge is referred to as SEQ ID NO: 58 or SEQ ID NO: 104.
• a truncated CD8o hinge (amino acids 138-182 of Uniprot: P01732) may also be used as an N- or C-terminal sequence.
• the truncated CD8a hinge is referred to as SEQ ID NO: 59.
• the NKG2D polypeptide and the DAP10/12 fusion polypeptide may be genetically encoded as part of a contiguous chimeric construct.
• the fusion polypeptide and NKG2D polypeptide may then be separated during translation (e.g. using a ribosomal skip peptide) or by post translation cleavage (e.g. using a furin cleavage site).
• the fusion polypeptide and NKG2D polypeptide may therefore be joined by an optional linker.
• Such a linker may comprise a cleavage site to facilitate cleavage.
• the NKG2D polypeptide and the CXCR2 polypeptide may be genetically encoded as part of a contiguous chimeric construct.
• the CXCR2 polypeptide and NKG2D polypeptide may then be separated during translation (e.g. using a ribosomal skip peptide) or by post translation cleavage (e.g. using a furin cleavage site).
• the CXCR2 polypeptide and NKG2D polypeptide may therefore be joined by an optional linker.
• Such a linker may comprise a cleavage site to facilitate cleavage.
• the NKG2D polypeptide, DAP10/12 fusion polypeptide and the CXCR2 polypeptide may be genetically encoded as part of a contiguous chimeric construct.
• the CXCR2 polypeptide, DAP10/12 fusion polypeptide and NKG2D polypeptide may then be separated during translation (e.g. using a ribosomal skip peptide) or by post translation cleavage (e.g. using a furin cleavage site).
• the present disclosure provides the following exemplary fusion polypeptide constructs in
• the fusion polypeptides may be expressed as a single chimeric construct with a NKG2D and optionally a CXCR2 polypeptide, for translation-associated or post-translational cleavage.
• the translated polypeptides are cleaved to create the separate polypeptides which then self-associate to form a CAR.
• a fusion polypeptide is cleaved from a NKG2D polypeptide. Examples of such constructs are shown in Table 2:
• Genetic modification will be understood to refer to the introduction of exogenous polynucleotide(s) into a cell or organism to enable the expression of the protein(s) encoded by the exogenous polynucleotide(s) in the cell.
• transfection encompasses a wide variety of techniques commonly used for the introduction of exogenous DNA into a cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
• the exogenous polynucleotide may be DNA or RNA.
• the polynucleotide may encode the polypeptide sequence of any one or more of SEQ ID NOs: 60-69, 87, 90, and 102.
• the polynucleotide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity with a polynucleotide sequence encoding any of SEQ ID NOS: 60-69, 87, 90, and 102. Sequence identity is typically measured along the full length of the reference sequence.
• the polynucleotide may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity with any one of SEQ ID NOs: 70-79, 88, 91, and 103.
• the polynucleotide comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 70-79, 88, 91, and 103.
• polynucleotide encompasses polynucleotides containing known analogues of natural nucleotides that have similar properties as the reference polynucleotide and are metabolized in a manner similar to naturally occurring nucleotides.
• analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphorates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
• a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et a/., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).
• the polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below).
• Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68: 109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22: 1859; and the solid support method of U.S. Pat. No. 4,458,066.
• PCR Technology Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1: 17.
• genetic modification comprises or consists of transduction.
• transduction involves the introduction of exogenous polynucleotide(s) into a cell by a virus or viral vector.
• genetic modification may comprise the delivery of a vector encoding the NKG2D polypeptide (and optionally other polypeptides disclosed), into the cell.
• Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345).
• non-viral vectors useful for expression of the polynucleotides and polypeptides of the invention in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1/His, pEBVHis A, B and C, (Invitrogen, San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins.
• Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV).
• retroviral, lentiviral, adenoviral or adeno- associated viral vectors are commonly used for expression in T-cells.
• retroviral, lentiviral, adenoviral or adeno- associated viral vectors are commonly used for expression in T-cells.
• examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737).
• genetic modification comprises the delivery of a viral vector encoding the NKG2D polypeptide (and optionally other polypeptides disclosed), into the cell.
• the viral vector is a lentiviral or retroviral vector.
• the lentiviral vector may comprise a self- inactivating lentiviral vector (so-called SIN vectors).
• the retroviral vector may comprise an SFG retroviral expression vector.
• the vector may comprise expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
• expression control sequences such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68)
• necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
• These vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable.
• Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the EFl alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art.
• the metallothionein promoter the constitutive adenovirus major late promoter
• the dexamethasone-inducible MMTV promoter the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter
• the tetracycline-inducible CMV promoter such as the human immediate-early CMV promoter
• the vector comprises a polynucleotide sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97% or at least about 99% sequence identity with a polynucleotide encoding any of SEQ ID NOS: 60-69, 87, 90, and 102. In some embodiments, the vector comprises a polynucleotide sequence encoding one or more of SEQ ID NOs: 60-69, 87, 90, and 102. In some embodiments, the vector comprises the polynucleotide sequence of any one of SEQ ID NOs: 70-79, 88, 91, and 103.
• an immune cell is a cell of the immune system.
• an immune cell will be understood to be a cell which can respond to infectious organisms. Any such immune cell is suitable for the present invention.
• Exemplary immune cells may include, but not necessarily be limited to neutrophils, eosinophils, basophils, mast cells, monocytes, macrophages, dendritic cells, natural killer cells, and lymphocytes.
• lymphocytes include B cells and T-cells.
• the immune cell is a neutrophil, macrophage, dendritic cell, natural killer cell or lymphocyte.
• the immune cell is a B cell, T-cell or Natural Killer cell.
• the immune cell is a T-cell or a Natural Killer (NK) cell.
• the immune cell is a T-cell.
• T-cells include CD4 T-cells, CD8 T-cells and NKT-cells.
• the T-cell is a CD4 + or CD8 + T-cell.
• the immune cell is a CD4 + T-cell. In other embodiments, the immune cell is a CD8 + T-cell.
• the immune cell is an o[3 or a y6 T-cell. In some embodiments, the immune cell is a 00 T-cell. In other embodiments, the immune cell is a y6 T-cell.
• the immune cell is a peripheral blood mononuclear cell (PBMC).
• PBMC peripheral blood mononuclear cell
• the PBMC may differentiate into another immune cell described above.
• the PBMC may have differentiated into a T-cell.
• the method is a method of making an immunoresponsive T or NK cell and comprises:
• genetically modifying the PBMC to express the NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide wherein genetic modification comprises transduction of the PBMC with a retroviral vector encoding the NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide.
• the method is a method of making an immunoresponsive T or NK cell and comprises:
• genetically modifying the T or NK cell to express the NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide wherein genetic modification comprises transduction of the T or NK cell with a retroviral vector encoding the NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide.
• the method is a method of making an immunoresponsive T or NK cell and comprises: (i) activating a PBMC with anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix such that the PBMC differentiates into a T or NK cell, wherein the nanomatrix comprises iron oxide crystals embedded into a biocompatible polysaccharide matrix with a diameter of about 100 nm; and
• genetically modifying the T or NK cell to express the NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide wherein genetic modification comprises transduction of the T or NK cell with a retroviral vector encoding the NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide.
• the method is a method of making an immunoresponsive T or NK cell and comprises:
• activating does not comprise phorbol 12-myristate 13-acetate (PMA), phytohaemagglutinin (PHA) or beads; and
• the order of activation and genetic modification may be as defined above.
• the genetic modification and immune cell may be as defined above.
• genetic modification may comprise transduction.
• the immune cell may be a PBMC.
• the immune cell is a T-cell or neutrophil.
• Genetic modification may comprise genetically modifying an immune cell to express an NKG2D polypeptide and a DAP10/DAP12 fusion polypeptide. In some embodiments genetic modification comprises genetically modifying an immune cell to express an NKG2D polypeptide and a CXCR2 polypeptide. In some embodiments genetic modification comprises genetically modifying an immune cell to express an NKG2D polypeptide, a DAP10/DAP12 fusion polypeptide and a CXCR2 polypeptide. Immunoresponsive cells
• the invention also provides an immunoresponsive cell obtainable by any of the methods of the invention.
• an immunoresponsive cell genetically modified to express an NKG2D polypeptide; wherein the immunoresponsive cell has been activated with anti-CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to a nanomatrix.
• the immunoresponsive cell is genetically modified to express an NKG2D polypeptide and a fusion polypeptide comprising (i) a DNAX-activating 10 (DAP10) polypeptide, or a functional variant thereof and (ii) a DNAX-activating protein 12 (DAP12) polypeptide, or a functional variant thereof.
• DAP10 DNAX-activating 10
• DAP12 DNAX-activating protein 12
• the NKG2D polypeptide may be as defined above.
• the fusion polypeptide may be as defined above.
• the DAP10 polypeptide is a functional variant of DAP10 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP10 polypeptide of SEQ ID NO: 1.
• the DAP10 polypeptide comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 1.
• the DAP10 polypeptide is a truncated version of DAP10 which comprises or consists of amino acids 19-93, 19-69, 1-71, 19-71, 19-48, 49-69, 49-93, or 70-93 of SEQ ID NO: 1.
• the DAP10 polypeptide comprises or consists of any one of SEQ ID NOs: 1-8.
• the DAP12 polypeptide is a functional variant of DAP12 comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the DAP12 polypeptide having the sequence of SEQ ID NO: 9.
• the DAP12 polypeptide comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 9.
• the DAP12 polypeptide is a truncated version of DAP12 which comprises or consists of amino acids 22-113, 62-113, 22-61, or 41-61 of SEQ ID NO: 9.
• the DAP12 polypeptide comprises or consists of any one of SEQ ID NOs: 9-13.
• the NKG2D polypeptide is a human polypeptide.
• the NKG2D polypeptide is a functional variant of NKG2D.
• the functional variant of NKG2D may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% sequence identity to the NKG2D polypeptide having the sequence of SEQ ID NO: 14.
• the NKG2D polypeptide comprises or consists of SEQ ID NO: 14.
• the functional variant of NKG2D may comprise or consist of a truncated version of NKG2D.
• the NKG2D polypeptide may be a truncated version of NKG2D comprising only amino acids 73-216 of SEQ ID NO: 14 (i.e., the extracellular domain. Such a sequence is referred to as SEQ ID NO: 15 herein.
• Other truncated versions may comprise amino acids 82-216 of SEQ ID NO: 14, such a sequence comprising part of the extracellular domain of NKG2D and referred to herein as SEQ ID NO: 16.
• a further truncated version comprises amino acids 52-216 of SEQ ID NO: 14 (i.e. the transmembrane and extracellular domains), referred to as SEQ ID NO: 17 herein.
• the functional variant of the NKG2D polypeptide is a chimeric NKG2D polypeptide.
• the chimeric NKG2D polypeptide may be a human-murine chimeric polypeptide.
• the chimeric NKG2D polypeptide comprises from N terminus to C terminus the murine NKG2D transmembrane domain or a variant thereof and a human NKG2D extracellular domain or a variant thereof.
• the immune cell is genetically modified to further express a CXCR2 polypeptide.
• the immunoresponsive cell is genetically modified to express an NKG2D polypeptide and a CXCR2 polypeptide.
• the immunoresponsive cell is genetically modified to express an NKG2D polypeptide, a CXCR.2 polypeptide and a DAP10/DAP12 fusion polypeptide as defined above.
• the CXCR2 polypeptide may be as defined above.
• the CXCR2 polypeptide may be a mammalian polypeptide.
• the CXCR2 polypeptide may be a human polypeptide.
• the CXCR2 polypeptide comprises a functional variant of the wildtype CXCR2 polypeptide.
• the CXCR2 polypeptide may be a functional variant of the CXCR2 polypeptide of SEQ ID NO: 87.
• the functional variant of the CXCR2 polypeptide may comprise an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the CXCR2 polypeptide having the sequence of SEQ ID NO: 87.
• the CXCR2 polypeptide comprises or consists of amino acid sequence of SEQ ID NO: 87.
• the immunoresponsive cell is an immune cell.
• the immunoresponsive cell may comprise a neutrophil, an eosinophil, a basophil, mast-cell, monocyte, macrophage, dendritic cell, natural killer cell or lymphocyte.
• the immune cell is a neutrophil, macrophage, dendritic cell, natural killer cell or lymphocyte.
• the immunoresponsive cell is a lymphocyte or natural killer (NK) cell.
• the immunoresponsive cell may be a T-cell, B-cell or NK cell.
• the immunoresponsive cell is a T-cell or a Natural Killer (NK) cell.
• the immunoresponsive cell is a T-cell.
• the T-cell is a CD4 + or CD8 + T-cell.
• the immunoresponsive cell is a CD4 + T-cell.
• the immunoresponsive cell is a CD8 + T-cell.
• the immunoresponsive cell is an o[3 or a y6 T-cell. In some embodiments, the immunoresponsive cell is a 00 T-cell. In other embodiments, the immunoresponsive cell is a y6 T-cell.
• the immunoresponsive cell is a peripheral blood mononuclear cell (PBMC).
• PBMC peripheral blood mononuclear cell
• the immunoresponsive cell may be derived from a PBMC.
• "derived from a PBMC” will be understood to refer to a cell which has differentiated from a PBMC.
• the immunoresponsive cell comprises a population of immunoresponsive cells. More preferably, the immunoresponsive cell comprises a population T or NK cells. Most preferably, the immunoresponsive cell comprises a population of T-cells.
• the population of T-cells may comprise CD4 + T-cells.
• the population of T-cells may comprise CD8 + T-cells. In some embodiments, the population of T-cells comprises CD4 + T-cells and CD8 + T-cells.
• immunoresponsive cells activated with anti- CD3 and anti-CD28 antibodies, or fragments thereof, conjugated to the nanomatrix of the invention have greater fold expansion and thus population numbers than immunoresponsive cells activated using other activation stimuli.
• the population comprises at least about 1 xlO 5 immunoresponsive cells, at least about 5 xlO 5 immunoresponsive cells, at least about IxlO 6 immunoresponsive cells, at least about 5 xlO 6 immunoresponsive cells, at least about IxlO 7 immunoresponsive cells, at least about 5xl0 7 immunoresponsive cells, at least about IxlO 8 immunoresponsive cells, at least about 5xl0 8 immunoresponsive cells, at least about IxlO 9 immunoresponsive cells, at least about 5xl0 9 immunoresponsive cells, at least about IxlO 10 immunoresponsive cells, at least about 5xlO 10 immunoresponsive cells or at least about IxlO 11 immunoresponsive cells. In some embodiments the population comprises at least about IxlO 6 immunoresponsive cells.
• the population of immunoresponsive cells has an expansion rate of at least about five-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about ten-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 20-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 30-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 40-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 50-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 60-fold every ten days.
• the population of immunoresponsive cells has an expansion rate of at least about 70-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 80-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 90-fold every ten days. In some embodiments, the population of immunoresponsive cells has an expansion rate of at least about 100-fold every ten days.
• the immunoresponsive cell comprises a population of T-cells having an expansion rate of at least about five-fold every ten days. More preferably, the immunoresponsive cell comprises a population of T-cells having an expansion rate of at least about ten-fold every ten days. Most preferably, the immunoresponsive cell comprises a population of T-cells having an expansion rate of at least about 20-fold every ten days.
• the population of T-cells comprises a CD4:CD8 ratio of at least about 1 : 1.
• the population of T-cells comprises a CD4:CD8 ratio of at least about 2: 1.
• the population of T-cells may comprise a CD4:CD8 ratio of at least about 3: 1, optionally of about 4: 1.
• composition comprising the immunoresponsive cell obtainable by either of the methods disclosed herein or the immunoresponsive cell of the above aspect and a pharmaceutically or physiologically acceptable diluent and/or carrier.
• pharmaceutical composition refers to the combination of an active ingredient with a carrier, inert or active, making the composition especially suitable for therapeutic or diagnostic use in vitro, in vivo or ex vivo.
• compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
• the carrier is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
• these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media.
• Suitable agents for inclusion in the pharmaceutical compositions include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogen-sulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and di
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically-acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included.
• Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose.
• agents to adjust tonicity of the composition for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic.
• Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present.
• the precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22nd Edition).
• a pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and/or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, for example by injection or infusion.
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intra-sternal injection and infusion.
• the pharmaceutical composition is administered intratumourally.
• the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition.
• a particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved.
• the pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake.
• the pharmaceutical composition described herein can be administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
• a non-parenteral route such as a topical, epidermal or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually or topically.
• the pharmaceutical composition is for subcutaneous administration.
• suitable formulation components and methods for subcutaneous administration of polypeptide therapeutics are known in the art, see, for example, US2011/0044977, US8465739 and US8476239.
• the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g, amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents.
• the pharmaceutical composition comprising the immunoresponsive cell is administered to the subject by intravenous infusion.
• Administration of the pharmaceutically useful composition of the present invention is preferably in a "therapeutically effective amount” or “prophylactically effective amount", this being sufficient to show benefit to the individual.
• the actual amount administered, and rate and time-course of administration will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
• a pharmaceutical composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
• administering refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., peptide) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs.
• Exemplary routes of administration to the human body can be through space under the arachnoid membrane of the brain or spinal cord (intrathecal), the eyes (ophthalmic), mouth (oral), skin (topical or transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal or lingual), ear, rectal, vaginal, by injection (e.g., intravenously, subcutaneously, intratumourally, intraperitoneally, etc.) and the like.
• injection e.g., intravenously, subcutaneously, intratumourally, intraperitoneally, etc.
• the invention also provides a kit comprising the immunoresponsive cell obtainable by the methods disclosed herein, the immunoresponsive cell of the invention or the pharmaceutical composition of the invention.
• the kit may further comprise instructions for use.
• the immunoresponsive cells and/or the pharmaceutical composition is provided in an aqueous solution in the kit, optionally buffered solution and/or at a temperature of at least -20°C.
• kits for example, any of the vectors, as well as the mammalian cells, related buffers, media, triggering agents, or other components related to cell culture and virion production can be provided, with optional components frozen and packaged as a kit, alone or along with separate containers of any of the other agents and optional instructions for use.
• the kit may comprise culture vessels, vials, tubes, or the like.
• Methods of treatment Also provided is a method of treating or preventing cancer in a subject, wherein the method comprises administering to the subject the immunoresponsive cell or the pharmaceutical composition of the invention.
• treatment means an approach to obtaining a beneficial or intended clinical result.
• the beneficial or intended clinical result can include alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of a disease or condition, steadying diseases in a non-advanced state, delaying the progress of a disease, and/or improvement or alleviation of disease conditions.
• the method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the immunoresponsive cell or the pharmaceutical composition of the invention.
• a therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease and/or abolishes one or more symptoms, such as all the symptoms, of the disease.
• the therapeutically effective amount preferably cures the disease.
• a prophylactically effective amount is an amount which prevents the onset of the disease and/or prevents the onset of one or more symptoms, such as all the symptoms, of the disease.
• the prophylactically effective amount preferably prevents the subject from developing the disease. Suitable amounts are discussed in more detail below.
• the immunoresponsive cells or pharmaceutical composition of the invention may be administered to a subject that displays symptoms of disease.
• the immunoresponsive cells or pharmaceutical composition of the invention may be administered to a subject that is asymptomatic, i.e. does not display symptoms of disease.
• the immunoresponsive cells or pharmaceutical composition of the invention may be administered when the subject's disease status is unknown or the patient is expected not to have a disease.
• the immunoresponsive cells or pharmaceutical composition of the invention may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease.
• subject broadly refers to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.).
• human and non-human animals e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.
• the subject may be a mammal.
• the subject is a human, horse, dog or cat.
• the subject is human.
• the subject may be a horse.
• the cancer may include, but not necessarily be limited to, a solid tumour cancer, a soft tissue tumour, a metastatic lesion, and a haematological cancer.
• the cancer can be liver cancer, lung cancer, breast cancer, prostate cancer, lymphoid cancer, colon cancer, renal cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non-Hodgkin's lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the cancer
• the cancer can be breast cancer, such as an estrogen receptor- positive (ER pos) breast cancer and/or a metastatic form of breast cancer.
• ER pos estrogen receptor- positive
• the cancer may be a solid tumour cancer.
• the cancer is selected from the group consisting of cancer of the head and/or neck, ovarian cancer, malignant mesothelioma, breast cancer, pancreatic cancer, colorectal cancer, lung cancer, gastric cancer, bladder cancer, prostate cancer, oesophageal cancer, endometrial cancer, hepatobiliary cancer, chronic or acute leukaemia including acute myeloid leukaemia, duodenal carcinoma, thyroid carcinoma, cancer of the central nervous system or renal cell carcinoma.
• the cancer is selected from ovarian cancer, breast cancer, optionally triple-negative breast cancer, pancreatic cancer, chronic or acute leukaemia including acute myeloid leukaemia, malignant mesothelioma, and combinations of said cancers.
• the cancer is breast cancer.
• Breast cancer includes, but is not necessarily limited to estrogen receptor-positive (ER pos) breast cancer, estrogen receptor- negative (ER neg) breast cancer, progesterone receptor-positive (PR pos) breast cancer, progesterone receptor-negative (PR neg) breast cancer, HER2-positive (HER2 pos) breast cancer, HER2-negative (HER2 neg) breast cancer, and triple negative (TNBC) breast cancer.
• TNBC breast cancer is breast cancer wherein the cancer cells test negative (i.e. express no or negligible amounts) for the estrogen receptor, progesterone receptor and HER2.
• the breast cancer comprises ER pos, ER neg, PR pos, PR neg, HER2 pos, HER2 neg, and/or combinations thereof.
• the breast cancer comprises triple negative breast cancer (TNBC).
• TNBC triple negative breast cancer
• treatment of the cancer involves targeting of non-tumour cells, such as tumour-associated stromal cells.
• non-tumour cells such as tumour-associated stromal cells.
• tumour-associated stromal cells include pancreatic stromal cells.
• Other types of non-tumour cells that may be targeted include macrophages, regulatory T-cells and myeloid-derived suppressor cells.
• the subject may have been pre-treated with a chemotherapeutic agent.
• the administration of immunoresponsive cells or pharmaceutical composition of the invention to the subject may result in a decrease in tumour size of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated tumour.
• the number of cells administered to the subject should take into account the route of administration, the disease being treated, the weight of the subject and/or the age of the subject. In general, from about 1 x 10 6 to about 1 x 10 11 immunoresponsive cells may be administered to the subject. In some embodiments, from about 1 x 10 7 to about 1 x 10 10 immunoresponsive cells, or from about 1 x 10 8 to about 1 x 10 9 immunoresponsive cells are administered to the subject.
• the invention also provides the immunoresponsive cell or pharmaceutical composition of the invention for use in any of the therapeutic methods described above.
• the immunoresponsive cell or pharmaceutical composition of the invention for use in the treatment or prevention of a disease.
• the disease is cancer. This may otherwise be referred to as for use in therapy.
• the invention provides the immunoresponsive cells or pharmaceutical composition of the invention for use in the treatment or prevention of breast cancer.
• the invention provides the immunoresponsive cells or pharmaceutical composition of the invention for use in the treatment or prevention of triple negative breast cancer
• the immunoresponsive cell or the pharmaceutical composition of the invention for the manufacture of a medicament for the treatment or prevention of a disease.
• the disease is cancer, as described above.
• the invention also provides use of the immunoresponsive cell or the pharmaceutical composition of the invention for (i) therapy or (ii) the treatment of cancer.
• the cancer is breast cancer.
• the breast cancer is triple-negative breast cancer. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
• PBMCs Peripheral blood mononuclear cells
• IxlO 6 activated PBMC were plated onto a RetroNectin-coated plate that had been pre-treated with 3mL retroviral supernatant. Each well was subsequently treated with 3mL fresh viral supernatant and lOOIU/mL IL-2. Retroviral transduction was performed with viral particles produced by stable gibbon ape leukemia virus (GALV)-pseudotyped 293TVec stable packaging cells. Thereafter, T-cells were fed with lOOIU/mL in RPMI1640 media + 5% normal human AB serum, with fresh media and IL-2 (lOOIU/mL) provided thrice weekly.
• GALV stable gibbon ape leukemia virus
• T-cells were activated using paramagnetic beads coated with anti-human CD3 and anti- human CD28 antibodies (1:2 celkbead ratio), phytohaemagglutinin (PHA) at a concentration of 5ug/mL or lOuL TransAct reagent per IxlO 6 PBMCs.
• PHA phytohaemagglutinin
• T-cell transduction and transfection of 293T-cells was assessed by flow cytometry, making comparison, where indicated, with an appropriate isotype control.
• To assess the expression of the NKG2D-based constructs cells were stained with mouse anti-human CD4-FITC, mouse anti-human NKG2D-PE and mouse anti-human CD8-APC and compensated appropriately. Due to high levels of endogenous NKG2D expression in CD8 + T-cells, transduction efficiency was compared against NKG2D expression in untransduced CD4 + T- cells. Prior to use, the transduction efficiency was normalized between constructs by spiking in the requisite proportion of untransduced T-cells. This ensured that all conditions were identical for the total number of CAR + T-cells and overall T-cell concentration.
• T-cells were removed from tumour cell co-cultures and stained with FITC-conjugated anti-human CD45RO, allophycocyanin (APC)-conjugated anti-human CD62L, PE-conjugated anti-human CCR7, FITC-conjugated anti-human CD4, APCCy7-conjugated anti-human CD8o and phycoerythrin cyanine7 (PECy7)-conjugated anti-human CD27 antibodies. After washing in 2 mL ice-cold PBS the cells were re-suspended in 0.5 mL ice-cold PBS and assessed by flow cytometry. Staining efficiency was assessed using T-cells stained with the appropriate isotype and fluorescence minus one (FMO) controls.
• FMO fluorescence minus one
• IxlO 4 tumour cells were plated per well (lOOpL) of a 96-well plate and incubated at 37°C and 5% CO2 overnight. Twenty-four hours later T-cells were added at log 2 CAR T-cell: tumour cell ratios ranging from 1: 1 to 1:64. After 72 hours, the T-cells were removed and lOOpL MTT solution (500pg/mL) added, before the plates were incubated at 37°C and 5% C0 2 for approximately 1 hour. Following removal of the MTT solution, the resulting formazan crystals were solubilized in DMSO (lOOpL/well) and the absorbance measured at 560nm. Tumour cell viability was calculated as follows: (Absorbance of monolayer with T- cells/absorbance of monolayer without T-cells)*100.
• IxlO 5 tumour cells were plated in triplicate wells of a 24-well plate and incubated for 24 hours at 37°C and 5% CO 2 . Twenty-four hours later, ImL containing IxlO 5 CAR + T-cells were added per well. After 72 hours, the T-cells were gently removed and the well was washed with ImL PBS. Following removal of the PBS, ImL of MTT (at a final concentration of 500pg/mL) was added to each well and the plate incubated at 37°C and 5% CO 2 for approximately 1 hour. Absorbance was measured in the appropriate wells at 560nm and tumour cell viability calculated as detailed in the 'dose response' section. A re-stimulation was considered successful if the tumour cell viability was measured as less than 50%.
• T-cells that had been removed from the plate were centrifuged at 400xg for 5 minutes and the supernatant removed.
• the pellet was re-suspended in 3.2mL R5 media and ImL added to each well of fresh tumour monolayer (IxlO 5 tumour cells per well of a 24-well plate) in triplicate.
• Total T-cell number was assessed by trypan blue exclusion of a small aliquot of the remaining 200pL.
• ffLUC firefly luciferase
• Tumour growth was monitored by BLI, with all data presented as total flux (photons/second) or average total flux (photons/second) per treatment. Mice were monitored closely and weighed three times per week for signs of ill health.
• mice IxlO 5 CFPac-1 or BxPC3 cells were injected subcutaneously in 50 pl of Matrigel (1 : 1 PBS) into the left flank of NSG mice. Twenty-nine days after inoculation with CFPac-1 cells or fourteen days after inoculation with BxPC3 cells, mice were treated intravenously with either PBS or IxlO 7 CAR T-cells (N1012_CXCR2, N1012, NKG2D, CYAD-01 replica, untransduced (UT)).
• Example 1 Evaluating anti-tumour efficacy of N1012 CXCR.2 T-cells in vivo in mouse models of pancreatic cancer
• T-cells expressing a CAR construct were generated by isolating peripheral blood mononuclear cells (PBMCs), activating the T-cells, and transducing the T-cells with virus containing a polynucleotide encoding the CAR ( Figure 1).
• PBMCs peripheral blood mononuclear cells
• HEK293T-cells were seeded in a 10cm 2 tissue culture dish in lOmL of IMDM media containing 10% FBS and 2mM L-glutamine (110 media) and incubated for 24 hours at 37°C at 5% CO2. The following day, transfection mix was generated for each CAR construct according to the protocol in Table 3.
• the HEK293T-cells were separately transfected with the following plasmids: N1012 (encoding DAP10/12 fusion protein and human NKG2D receptor (SEQ ID NO: 74)), N1012_CXCR2 (encoding DAP10/12 fusion protein, human NKG2D receptor, and CXCR2 (SEQ ID NO: 91)), CYAD-01 replica (encoding NKG2D receptor fused to CD3£ (SEQ ID NO: 93)), and CYAD-01_10 (encoding CYAD-01 replica and DAP10 (SEQ ID NO: 94).
• HEK293T-cells were fed with 10 mL of fresh 110 media and returned to the incubator. After an additional 24 hours, the supernatant was harvested a second time from the HEK293T- cells and combined with the supernatant harvested 48 hours after transfection. The combined supernatant was aliquoted into pre-labelled tubes, snap frozen, and stored at - 80°C.
• PBMCs were isolated using standard Ficoll Paque-mediated density centrifugation. Once re- suspended at a concentration of IxlO 6 cells/mL in RPMI + 5% normal human AB serum and 2mM L-glutamine ('R5' media), the T-cells were activated. Forty-eight hours after activation, IxlO 6 T-cells were plated onto RetroNectin-coated non-tissue culture treated plates and mixed with 3mL viral supernatant harvested from transiently transfected HEK 293T-cells. T-cells were fed with lOOIU/mL IL-2 in RPMU640 media + 5% normal human AB serum, with fresh media and IL-2 (lOOIU/mL) provided thrice weekly.
• Results shown in Figure 2A demonstrate that efficacy of treatment with a low dose of N1012_CXCR2 T-cells tracks treatment with a high dose of N1012 T-cells.
• Figure 2B shows that the probability of survival was highest for N1012_CXCR2 treated mice, particularly those administered the high dose of N1012_CXCR2 cells.
• Example 2 Anti-tumour efficacy of N1012 CXCR2 T-cells against breast cancer cells To confirm the efficacy of N1012_CXCR2 T-cells against another cancer model, the restimulation and cytotoxic capacity of N1012_CXCR2 T-cells against the triple negative breast cancer cell lines MDA-MB-468 and MDA-MB-231 was compared to N1012 T-cells.
• N1012 or N1012_CXCR2 T-cells were co-cultured with fresh monolayer twice weekly until monolayer destruction was not observed.
• IxlO 5 MDA-MB-468 or MDA- MB-231 cells were plated in triplicate wells of a 24-well plate and incubated for 24 hours at 37°C and 5% CO2.
• NKG2D + T-cells were used as a control. Twenty-four hours later, IxlO 5 CAR + T-cells were added per well at a final concentration of IxlO 5 CAR + /mL. After 72 hours, the T-cells were gently removed and centrifuged at 400xg for 5 minutes and the supernatant removed. The pellet was re-suspended in 3.2mL R5 media and ImL added to each well of fresh tumour monolayer in triplicate. Total T-cell number was assessed by trypan blue exclusion of a small aliquot of the remaining 200pL.
• N1012_CXCR2 T-cells When compared to the N1012 and NKG2D T-cells, N1012_CXCR2 T-cells underwent significantly more rounds of re-stimulation upon MDA-MB-468 and MDA-MB-231 cells ( Figure 3).
• Example 3 Anti-tumour efficacy of N1012 CXCR.2 T-cells activated using different activation stimuli
• CAR T-cells activated using different activation stimuli were then evaluated.
• CAR T-cells were activated in vitro with PHA, the clinically compliant activation stimuli TransAct (Miltenyi Biotec GmbH) or the clinically compliant immobilised anti-CD3 and anti-CD28 antibodies.
• N1012 and non-transduced (UT) T-cells were used for comparison.
• BxPC3_LT firefly luciferase-tagged BxPC3 cells
• 4xl0 6 T-cells activated as described above
• PBS PBS
• Figures 4 and 5 show that treatment efficacy was highest when CAR T-cells were activated with TransAct, especially N1012_CXCR2 CAR T-cells activated with TransAct.
• An additional rechallenge was undertaken on day 64 in mice that had achieved a complete response, as shown by the second broken vertical line in Figure 6, which shows individual mice from Figures 4 and 5.
• Example 4 N1012 CXCR2 T-cells activated using TransAct are skewed towards a CD4 + profile
• N1012_CXCR2 T-cells activated using different activation stimuli were then assessed.
• CAR T-cells were activated in vitro with PHA or the clinically compliant activation stimuli TransAct. N1012, CYAD-01 replica cells and non- transduced (UT) T-cells (also activated using the two different stimuli) were used as comparators.
• the CYAD-01 CAR consists of a fusion of NKG2D to CD3£ and represents a human version of the mouse CAR originally described by Sentman et al. (Zhang et al., 2005, Blood 106: 1544-1551).
• CYAD-01 CAR Although nominally a first-generation CAR, it associates with endogenous DAP10 in T-cells, meaning that both signals 1 and 2 are provided.
• the CYAD-01 CAR is currently undergoing clinical development by Celyad Oncology, and a replicate of this CAR is provided in these examples for the purposes of comparison only.
• CYAD-01_10 a replica of Cyad-01 has been co-expressed with additional DAP10.
• Figure 7 shows that at the end of the in vitro culture period (day 10-12), N1012_CXCR2 T- cells activated with TransAct have a higher CD4 to CD8 ratio than N1012_CXCR2 T-cells activated with PHA. Whereas N1012_CXCR2 T-cells activated with PHA had more CD8 cells than CD4 cells, N1012_CXCR2 T-cells activated with TransAct had more CD4 than CD8 cells. This was unexpected. However, the transduction efficacy and median fluorescence intensity (MFI) of N1012_CXCR2 T-cells activated using TransAct was comparable to N1012_CXCR2 cells activated using PHA ( Figure 8).
• MFI median fluorescence intensity
• T-cells activated with either 5ug/mL phytohaemagglutinin-L (PHA) or lOuL TransAct reagent per IxlO 6 PBMCs was then assessed. This was calculated by dividing the number of T-cells present at the end of the ex vivo culture period (of 10 to 12 days) by the number of T-cells initially transduced.
• PHA phytohaemagglutinin-L
• N1012 and N1012_CXCR2 constructs were reproducibly expressed at high levels at the surface of primary human y6 T- cells.
• Example 6 Assessment of N1012 and N1012 CXCR2 v5 T-cell expansion and anti-tumour cytotoxicity
• N1012 or N1012_CXCR2 expressing y6 T-cells was then assessed.
• N1012 expressing y6 T-cells or N1012_CXCR2 expressing y6 T- cells were activated as described above.
• Fold expansions as shown in Figure 11A, were calculated on the basis of % pan-y6 TCR + CD3 + cells at day 0, day 7, day 14 and day 21 in culture.
• both the % of cells and the fold expansion rate were similar in untransduced, N1012 and N1012_CXCR2 expressing y6 T-cells.
• Example 7 Anti-tumour in vivo efficacy of N1012 and N1012 CXCR.2 v5 T-cells
• N1012 CXCR2 T-cells exhibit consistently enhanced anti-tumour efficacy Survival curves from various in vivo experiments were pooled.
• survival curves from CFPacl pancreatic, two models
• BxPC3 pancreatic, two models
• Kuramochi High grade serous ovarian, one model
• Ovsaho epidermal ovarian cancer, one model
• Mesothelioma patient-derived xenograft, one model
• triple-negative breast cancer patient-derived xenograft, one model
• SKOV3 epihelial ovarian cancer, one model tumour xenograft-containing mice treated with PBS or 1 x 10 7 CAR T-cells (N1012, N1012_CXCR2, or untransduced) were pooled.
• CAR T-cells (N1012_CXCR2, CYAD-01 or PBS buffer as control) were injected into mice bearing CFPac-1 tumour cells or BxPC3 cells tumour cells.
• Example 9 Efficient transduction and expansion of N1012 CXCR.2 can be achieved across a range of TransActTM concentrations, including those substantially below the concentration recommended by the manufacturer
• Transduction efficiency as measured by CXCR.2 and/or NKG2D expression in CD4+ T-cells was assessed three and ten days post-transduction by flow cytometry, using fluorescein isothiocyanate (FITC)- conjugated anti-human CD4, allophycocyanin-cyanine? (APC-Cy7)-conjugated anti-human CD8, phycoerythrin (PE)-conjugated anti-human NKG2D and AlexaFluor647-conjugated anti-human CXCR2 antibodies, as detailed in Figure 18.
• FITC fluorescein isothiocyanate
• APC-Cy7 allophycocyanin-cyanine?
• PE phycoerythrin
• AlexaFluor647-conjugated anti-human CXCR2 antibodies as detailed in Figure 18.
• Expansion of the untransduced (UT) and N1012_CXCR2 T-cells was assessed three and ten days post-transduction by trypan blue exclusion. The cell concentration was multiplied by total culture volume to provide a definitive cell number.
• Example 10 Anti-tumour efficacy of N1012 CXCR2 T-cells in metastatic colorectal carcinoma xenografts models
• IxlO 6 LS180 or SW620 tumour cells were injected subcutaneously into the left flank of NSG mice. Tumour growth was measured using weekly caliper measurements. Twelve days post- engraftment, mice were treated with IxlO 7 CAR + T-cells or IxlO 7 untransduced T-cells.
• SEQ ID NO: 1 human DAP10 full sequence
• MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL CARPRRSPAQ EDGKVYINMP GRG
• SEQ ID NO: 3 (DAP10 aal9-69 - extracellular/transmembrane domain)
• MIHLGHILFL LLLPVAAAQT TPGERSSLPA FYPGTSGSCS GCGSLSLPLL AGLVAADAVA SLLIVGAVFL C
• SEQ ID NO: 6 (DAP10 aa70-93 - intracellular domain)
• SEQ ID NO: 7 (DAP10 aa49-93 - transmembrane and intracellular domain)
• SEQ ID NO: 8 (DAP10 aa49-69 - transmembrane domain)
• SEQ ID NO: 12 (DAP12 aa41-61 - transmembrane domain)
• SEQ ID NO: 13 (DAP12 aa22-61 - extracellular and transmembrane domains)
• SEQ ID NO: 14 human NKG2D full sequence
• VAMGIRFI IM VAIWSAVFLN SLFNQEVQI P LTESYCGPCP KNWICYKNNC YQFFDESKNW
• SEQ ID NO: 15 (human NKG2D aa73-216 - extracellular domain)
• SEQ ID NO: 16 (human NKG2D aa82-216 - extracellular domain)
• SEQ ID NO: 17 (human NKG2D aa52-216 - transmembrane and extracellular domain)
• SEQ ID NO: 55 CD8a leader sequence
• SEQ ID NO: 58 human IgGl hinge - aa 218-229 of UniProt: PODOX5
• SEQ ID NO: 70 (encoding polypeptide of SEQ ID NO: 60)
• SEQ ID NO: 72 (encoding polypeptide of SEQ ID NO: 62)
• SEQ ID NO: 73 (encoding polypeptide of SEQ ID NO: 63)
• SEQ ID NO: 75 (encoding polypeptide of SEQ ID NO: 65/construct 3)
• SEQ ID NO:77 (encoding polypeptide of SEQ ID NO: 67/construct 9)
• SEQ ID NO:78 (encoding polypeptide of SEQ ID NO: 68/construct 10)
• SEQ ID NO:79 (encoding polypeptide of SEQ ID NO: 69/Construct 11)
• SEQ ID NO: 80 (A20FMDV2 peptide)
• SEQ ID NO: 82 (CD28 aall4-220) lEVMYPPPYLDNEKSNGTI IHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFI I FWVRSKRSRLLHSD YMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
• SEQ ID NO: 84 (SEQ ID NO: 1 of WO 2019/182425)
• SEQ ID NO: 87 sequence of human CXCR2 polypeptide expressed in N1012_CXCR2
• SEQ ID NO: 88 nucleic acid sequence encoding polypeptide of SEQ ID NO: 87 atggaggatttcaatatggagagcgactccttcgaggatttttggaagggcgaggacctgtctaactacagctatag ctccacactgccccttttctgctggatgccgccccttgtgagccagagtccctggagatcaacaagtacttcgtgg tcatcatctatgccctggtgtttctgctgtctgggcaatagcctggtcatgctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatctggtcatc
• SEQ ID NO: 90 protein encoded by SEQ ID NO: 89 (includes: (i) fusion of full length DAP10/DAP12 intracellular domain; (ii) furin cleavage site (RRKR); (iii) SGSG linker; (iv)
• P2A skip sequence (v) NKG2D; (vi) furin cleavage site (RRKR); (vii) SGSG linker; (viii) T2A skip sequence; (ix) CXCR2)
• SEQ ID NO: 91 (encoding the polypeptide of SEQ ID NO: 90)
• SEQ ID NO: 92 amino acid sequence of a replica of the Cyad-01 CAR (NKG2D fused to the intracellular domain of CD3O
• SEQ ID NO: 93 nucleic acid sequence encoding polypeptide of SEQ ID NO: 92
• SEQ ID NO: 95 human extracellular NKG2D domain
• SEQ ID NO: 96 human extracellular NKG2D domain
• SEQ ID NO: 100 (rat NKG2D)
• SEQ ID NO: 101 (rat NKG2D TM domain; UniProt accession no: 070215 aa 52-74)
• SEQ ID NO: 103 nucleic acid encoding polypeptide of SEQ ID NO: 102
• SEQ ID NO: 104 human IgGl hinge - aa 218-232 of UniProt: PODOX5
• SEQ ID NO: 105 amino acid sequence of CYAD-01_10 (NKG2D-CD3£ + ribosomal skip peptide + DAP10)

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