HYPOXIA-RESPONSIVE CHIMERIC ANTIGEN RECEPTORS
20220195009 · 2022-06-23
Inventors
- James Noble ARNOLD (Strand, London, GB)
- John MAHER (Strand, London, GB)
- Paraskevas KOSTI (Strand, London, GB)
Cpc classification
C07K14/705
CHEMISTRY; METALLURGY
A61K35/17
HUMAN NECESSITIES
C07K2319/33
CHEMISTRY; METALLURGY
C07K14/70567
CHEMISTRY; METALLURGY
C07K2319/30
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07K14/705
CHEMISTRY; METALLURGY
A61K35/17
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
Abstract
The present invention relates to therapeutic agents, particularly to therapeutic polypeptides and nucleic acids having the capacity for selective expression under conditions of hypoxia, cells incorporating the nucleic acids and their use in therapy, in particular in methods requiring selective expression under conditions of hypoxia, such as typically found in solid cancers. The nucleic acids encode novel hypoxia-responsive chimeric antigen receptors (CARs). The invention also relates to hypoxia-responsive regulatory nucleic acids.
Claims
1. A nucleic acid molecule comprising: a. a polynucleotide encoding a Chimeric Antigen Receptor (CAR), wherein the CAR comprises: (i) one or more Oxygen-Dependent Degradation Domains (ODD); and (ii) at least one polypeptide with anti-tumour properties; and b. a hypoxia-responsive regulatory nucleic acid, wherein said CAR-encoding polynucleotide is operably linked to said hypoxia-responsive regulatory nucleic acid.
2. The nucleic acid molecule of claim 1, wherein said hypoxia-responsive regulatory nucleic acid comprises a plurality of hypoxia-responsive elements (HREs), wherein each individual HRE of said plurality of HREs independently comprises (i) an HIF binding site (HBS): 5′-(A/G)CGT(G/C)-3′ (SEQ ID NO: 1); and optionally (ii) an HIF ancillary site (HAS): 5′-CA(C/G)(G/A)(T/C/G)-3′ (SEQ ID NO: 2); or (iii) an HNF-4 site: 5′-TGACCT-3′ (SEQ ID NO: 3).
3. The nucleic acid molecule of claim 2, wherein said HBS and HAS if present are separated by a linker, optionally wherein said linker is at least 6 nucleotides in length.
4. The nucleic acid molecule of claim 2, wherein said plurality of HREs comprises at least one or a plurality of sequences selected from SEQ ID NOs 5-17 or sequences having at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to any of SEQ ID NOs 5-17.
5. The nucleic acid molecule of claim 2, wherein said plurality is at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more individual HREs, which may be sequentially positioned or which may be spatially separate.
6. (canceled)
7. The nucleic acid molecule of claim 2, wherein said hypoxia-responsive regulatory nucleic acid comprises a sequence of SEQ ID NOs 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 or functional fragment thereof or homologues thereof.
8. The nucleic acid molecule of claim 2, wherein said hypoxia-responsive regulatory nucleic acid comprises a sequence of SEQ ID NO 19 or 26 or a homologue thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity thereto.
9. The nucleic acid molecule of claim 2, wherein the hypoxia responsive regulatory nucleic acid is comprised in a retroviral or lentiviral vector, optionally an SFG retroviral vector.
10. The nucleic acid molecule of claim 9, wherein the retroviral or lentiviral vector comprises an enhancer region, wherein the enhancer region comprises a plurality of HREs, optionally wherein the plurality is nine HREs which may be sequentially positioned or which may be spatially separate.
11. (canceled)
12. The nucleic acid molecule of claim 2, wherein said HREs are derived from any one or more of the following oxygen-responsive genes or from orthologues or paralogues thereof: erythropoietin (EPO), vascular endothelial growth factor (VEGF), phosphoglycerate kinase (PGK), glucose transporters (e.g. Glut-1), lactate dehydrogenase (LDH), aldolase (ALD), Enolase (e.g. ENO3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nitric oxide synthetase (NOS), Heme oxygenase, muscle glycolytic enzyme pyruvate kinase (PKM), endothelin-1 (ET 1).
13. The nucleic acid molecule of claim 1, wherein said ODD has the sequence of SEQ ID NO: 28: X.sup.1X.sup.2LEMLAPYIXMDDDX.sup.3X.sup.4X.sup.5, where “X.sup.1-5” can be any amino acid residue, optionally wherein X.sup.1 is “L” or any conservative substitution; X.sup.2 is “D” or any conservative substitution, X.sup.3 is “F” or any conservative substitution, X.sup.4 is “Q” or any conservative substitution, X.sup.5 is “L” or any conservative substitution.
14. The nucleic acid molecule of claim 1, wherein said ODD has the sequence of SEQ ID NO: 29, 30 or 31 or homologue thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity thereto and comprising SEQ ID NO: 28 or the sequence of SEQ ID NO: 5 or variant thereof having at least 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to SEQ ID NO: 5, wherein said variant comprises SEQ ID NO: 4.
15. (canceled)
16. The nucleic acid molecule of claim 1, wherein said polypeptide with an anti-tumour property comprises: a. an extracellular antigen-specific targeting region, or b. a protein for delivery to a tumour, selected from immune stimulating antibodies; surface or intracellular receptors that confer cell activation and tumour-killing capability; T-cell Receptor (TCR); immunomodulatory cytokines (for example, IL-12, IL-15), decoy antibodies (for example, PD axis-interacting antibodies), and a protein that alters host cell function (for example, Lck, TCR zeta chain, ZAP70).
17. (canceled)
18. (canceled)
19. The nucleic acid molecule of claim 1, wherein hypoxia is a condition with O.sub.2 concentration below 5%, preferably below 3%, or reduced O.sub.2 availability relative to O.sub.2 availability or partial pressure of the corresponding non-cancerous organ, tissue or cells.
20. (canceled)
21. The nucleic acid molecule of claim 1, wherein said CAR is selected from a first, second, third, fourth generation CAR, a split CAR design, and armoured CAR.
22. The nucleic acid molecule of claim 1, wherein said CAR has specificity towards the ErbB family of receptors.
23. An immunoresponsive cell comprising said nucleic acid molecule of claim 1.
24. (canceled)
25. (canceled)
26. A method for the preparation of a modified immunoresponsive cell, comprising: a isolating lymphoid or myeloid-derived cells from a subject; b. modifying said cells to introduce the nucleic acid molecule of claim 1; c. expanding said modified cells ex-vivo; and d. obtaining expanded cells capable of expressing said nucleic acid molecule under conditions of hypoxia.
27. (canceled)
28. (canceled)
29. A method for treatment of haematological or solid cancer, comprising administering the immunoresponsive cell of claim 23 to a patient in need thereof.
30. (canceled)
31. (canceled)
32. (canceled)
33. A pharmaceutical composition comprising the immunoresponsive cell of claim 23.
34. (canceled)
35. (canceled)
36. (canceled)
37. (canceled)
38. (canceled)
39. (canceled)
40. (canceled)
41. The method of treatment of claim 29, further comprising a preceding step of: a monitoring co-expression of at least two, three, four or all five the following genes: PGK1, SLC2A1, CA9, ALDOA and VEGFA, wherein co-expression of said genes in said subject is indicative of the subject's suitability for treatment, b. immunohistochemical staining of a tumour biopsy from the subject and assessing HIF stabilisation in the tumour or stoma, or c. monitoring T-cell infiltration (and/or of other immunoresponsive cells) to HIF stabilised regions of the tumour, wherein infiltration of the immunoresponsive cells to HIF stabilised regions of the tumour is indicative of a subject's suitability for treatment.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0247] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:
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[0265] 17A: Constructs containing variable ODDs fused on the C-terminus of Click Beetle luciferase.
[0266] 17B: T47D cells transduced with constructs shown in A, non-transduced (NT) or constitutive transduced (wild type non ODD modified Click Beetle luciferase) were exposed in hypoxia (0.1% oxygen) for 18 h. Fold induction is the luciferase expression induction seen in hypoxia in relative to the normoxic expression in each construct. N=3 Line=mean and error bars SEM.
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[0269] (a) Shows the detected light (shown in blue/green on the picture) from the luciferase that is expressed within the T4-CAR T-cells in three mice bearing established SKOV3 human ovarian tumours implanted subcutaneously (left) and the dissected organs/tumour from a representative mouse (right), 4 days post infusion.
[0270] (b) Quantitation of the luciferase signal in each indicated organ (n=6 individual mice). As can be seen at the 4 day timepoint post infusion, these cells preferentially reside in the lung and liver rather than the tumour.
[0271] (c) T4-CAR T-cells have specificity for 8 homo- and heterodimers formed by the Erbb receptor family, which are expressed by most, if not all, epithelial cells. Analysis of the vital organs for mRNA expression of the Erbb family (presented relative to the housekeeping gene Tbp), demonstrated that both the lung and liver, where T4-CAR T-cells initially accumulate, are both rich sources of the CAR ligands. N=6 (biological replicates combined).
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EXAMPLES
[0282] The invention will now be described with reference to the following examples.
[0283] Materials and Methods
[0284] Constructs
[0285] Three HRE sequences, each containing three in tandem HBS from human EPO, VEGFA and GLUT3, were synthesized by GeneArt (ThermoFisher Scientific) and flanked by a NheI and an XbaI restriction sites. These sequences were sub-cloned and replaced the natural NheI/XhoI sequence within the 3′ LTR of the SFG Moloney murine leukemia virus plasmid. Specific modification of the 3′ LTR was achieved by the synthesis of a XhoI/EcoRI-flanked intermediate fragment, which contained the HREs, achieved using primers that contained the restriction enzyme sites and complementary sequences to the respective HRE cassettes. Overlapping PCR and sub-cloning of the fragment achieved insertion into the SFG vector. Next, a protein-coding sequence coding for green-emitting variant of click beetle luciferase and green fluorescent protein separated by a P2A was cloned into NcoI/XhoI site of the SFG. Restriction digestions were performed at 37° C. using enzymes and buffers purchased from New England Biolab. DNA was detected in ethidium bromide stained 1.2% agarose gels and bands of appropriate sizes as assessed according to the DNA ladder were excised and extracted from gels using QIAquick Gel Extraction Kit (Qiagen). Sticky end ligations were catalysed by T4 DNA ligase (ThermoFisher Scientific) at 16° C. for 1 hour.
[0286] CAR/Reporter Construct Cloning
[0287] Human T1E CAR containing SFG retroviral vector was modified to generate the constructs utilized in this study. The full-length ODD cDNA encoding amino acids 401-603 (SEQ ID NO: 29) from human HIF1α was synthesis as a gBlock® (Integrated DNA Technologies) and was appended onto the C-terminus of the CD3ζ within the T1E CAR through overlap PCR using Platinum Pfx DNA polymerase (Thermo Fisher Scientific) according to the manufacturer's instructions with the primers; 5′-TCCAGCGGCTGGGGCGCGAGGGGGCAGGGCC-3′ (SEQ ID NO: 38) and 5′-GGCCCTGCCCCCTCGCGCCCCAGCCGCTGGA-3′ (SEQ ID NO: 39). PCR products were run on 1.2% Agarose (Sigma-Aldrich) gels and product size was estimated against a 1 kb Plus DNA ladder (Thermo Fisher Scientific). Fragments of the expected size were excised and purified using the QIAquick® Gel Extraction kit. T1E CAR-ODD was cloned into the SFG vector using AgeI and XhoI restriction endonucleases (New England Biolabs) to cleave AgeI and XhoI restriction enzyme sites in the SFG plasmid and those which had been built into the T1E CAR-ODD cDNA. Vector and constructs that had been restriction endonuclease digested were purified using QIAquick PCR purification kit (QIGEN) and ligated using T4 ligase (Thermo Fisher scientific) prior to transformation into One Shot Stb13™ chemically competent E. coli (Thermo Fisher Scientific).
[0288] Transformed E. coli were selected using ampicillin (Santa Cruz Biotechnology) containing Luria Bertani (LB) Agar (Sigma-Aldrich) plates. Transformed colonies were there grown up in LB broth (Sigma-Aldrich) with 100 μg/ml ampicillin and then purified using either QIAGEN Plasmid Midi or Maxi kits. Final constructs were sequence verified (Source BioScience). Using a similar approach, the following additional modifications were made: The constitutive reporter construct was generated using a Click Beetle Luciferase (Luc) and eGFP, separated by a viral P2A sequence, reporter construct previously generated in the lab. This was achieved by PCR amplification using Platinum Pfx DNA polymerase (Thermo Fisher Scientific) according to the manufacturer's protocol with the forward primer 5′-CCATGGTGAAGCGTGAGAAAAATG-3′ (SEQ ID NO: 40) and the reverse primer 5′-CTCGAGTTACTTGTACAGCTCGTCCATGC-3′ (SEQ ID NO: 41). The amplified product was digested with NcoI and XhoI (New England Biolabs) and cloned into the SFG vector using the NcoI and XhoI and T4 DNA ligase (Thermo Fisher Scientific). Full length ODD (as described above) was also appended onto the C-terminus of Luc from the reporter construct by overlap PCR using the primers: forward 5′-GAGAAGGCCGGCGGTGCCCCAGCCGCTGGA-3′ (SEQ ID NO: 42) and reverse 5′-CCTCAAAGCACAGTTACAGTATTCCAGGGAAGCGGAGCTACTAACTTCAG-3′ (SEQ ID NO: 43) to amplify the ODD flanked with complimentary overhangs. Subsequently, overlapping fusion PCR using primers: forward 5′-CCATGGTGAAGCGTGAGAAAAATG-3′ (SEQ ID NO: 44) and reverse 5′-CTCGAGTTACTTGTACAGCTCGTCCATGC-3′ (SEQ ID NO: 45) was performed to generate a fragment encoding Luciferase-ODD-P2A-eGFP flanked by NcoI and XhoI restriction sites, which were used to insert Luciferase-ODD-P2A-eGFP into the SFG vector. The HRE modification was targeted in the 3′ LTR of the SFG retroviral vector, as the 3′ LTR region gets copied to the 5′ LTR upon integration. DNA containing 9 tandem 5′-GGCCCTACGTGCTGTCTCACACAGCCTGTCTGAC-3′ (SEQ ID NO: 27) HRE motifs containing both HIF-binding and ancillary site was synthesized as a gBlock® (Integrated DNA Technologies) and sub-cloned into the 3′ LTR of the SFG vector between the NheI and XbaI restriction endonuclease sites using the NheI and Xba1 restriction endonucleases (New England Biolabs). The T1E CAR CD3.sup.− truncated control construct was synthesized as a gBlock® (Integrated DNA Technologies) with flanking SbfI and XhoI restriction sites and sub-cloned into the HRE-modified SFG vector using SbfI and XhoI restriction endonucleases (New England Biolabs). To generate the bicistronic Luciferase-T2A-CAR construct, a gBlock® (Integrated DNA Technologies), which was designed to include Luciferase-T2A-T1E peptide binder flanked with AgeI and NotI restriction sites, was inserted into the T1E CAR construct.
[0289] Bacterial Transformation
[0290] One Shot Stb13 Chemically Competent E. coli (ThermoFisher Scientific) were used for transformations. 5 μl of the ligation mixture was added into a vial of One Shot Stb13 cells that were thawed on ice. Cells were subsequently incubated on ice for 30 minutes. Next, the cells were heat-shocked (45 seconds, 42° C.), placed on ice for 2 minutes then 250 μl of S.O.C. Media was added and the vial incubated in a 37° C. bacterial shaker. The cells were spread on ampicillin (100 μg/ml) agar plates and incubated overnight at 37° C. in a humidified bacterial incubator. Colonies were picked and grown in 3 ml LB broth containing 100 μg/ml ampicillin. DNA was extracted from bacteria using QIAprep Miniprep Kit (Qiagen) according to the manufacturers protocol. DNA was quantified by nanodrop spectrophotometer at 280 nm and sequenced by Source BioScience. SnapGene software was used for sequencing alignments and verification.
[0291] Cell Lines
[0292] All cell lines were grown at 37° C. and 5% CO.sub.2 in a humidified incubator. Human embryonic kidney (HEK) 293, Phoenix-ECO (gift from Sandra Diebold), human fibrosarcoma cell line HT1080, BW5147.G.1.4 (purchased from ATCC), Jurkat (Clone E6-1) (ATCC) were maintained in RPMI 1640 medium (Gibco) supplemented with 10% foetal calf serum (FCS; Thermo Fisher Scientific). T47D cells were maintained in RPMI 1640 medium (Gibco) supplemented with 10% FCS and insulin (0.2 U/ml).
[0293] SKOV3 human ovarian adenocarcinoma cells were originally purchased from ATCC and were re-authenticated for this study by ATCC. HN3 human head and neck adenocarcinoma were acquired from Ludwig Institute for Cancer Research, London and grown in D10 medium, Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% FCS and GlutaMAX (Thermo Fisher Scientific). Murine Lewis Lung carcinoma (LL2) cells were purchased from ATCC and were cultured in RPMI 1640 supplemented with 10% FCS. Cell lines were confirmed to be free of Mycoplasma for this study using the MycoAlert® Mycoplasma Detection Kit (Lonza).
[0294] Mice
[0295] NSG (NOD-scid IL2Rgamma.sup.null) mice were purchased from Charles River and bred internally. Balb/c Rag2.sup.−/− mice were a gift from Professor Adrian Hayday (KCL). Male mice were used for studies involving HN3 and female mice were used for studies involving SKOV3 and LL2 studies. All mice used for ectopic tumor studies were 6-8 weeks old and approximately 22 g in weight.
[0296] Generation of Retrovirus
[0297] To produce retrovirus with tropism for human cells, RD114 pseudotyped transient retroviral particles were generated by triple transfection using (per well of a six well plate) 1.5 μg of Peq-Pam plasmid (Moloney GagPol), 1 μg RDF plasmid (RD114 envelope) and 1.5 μg of the SFG plasmids using FuGENE HD transfection reagent into 50%-60% confluent HEK 293T cells (Promega, US). Peq-Pam, RDF and SFG plasmids were incubated in plain RPMI 1640 media (Gibco) for 15 minutes at room temperature (RT) and then added drop-wise onto the 293T cells. Retrovirus-containing supernatant was harvested after 48 hours and used to transduce human cell lines.
[0298] Hypoxic Conditions
[0299] A hypoxia chamber was purchased from STEMCELL Technologies (Canada) and purged with certified gas supplied by BOC containing 0.1%, 1% or 5% O.sub.2, with constant 5% CO.sub.2 and using N2 as a balance. The chamber was re-purged 1 hour after the first purge according to the manufacturer's protocol. Equal numbers of cells plated on two parallel plates where one was exposed to hypoxic conditions and the other maintained at normoxia for 18 hours. Luciferase activity was then measured using a luciferase assay (Promega, US) according to the manufacturer's protocol on a Perkin Elmer Fusion α-FP plate reader (Life Sciences). Incubation time for assessing hypoxia responsive gene expression was based on known studies. Hypoxic conditions were also mimicked using cobalt (II) chloride (Sigma-Aldrich, US) (PHD inhibitor) at a final concentration of 100 μM.
[0300] Western Blot Analysis
[0301] Cells were lysed in Western lysis buffer (2.5 ml 1M Tris pH 6.8, 1 g SDS, 5 ml glycerol, 17.5 ml water) containing a 1× concentration of a protease inhibitor cocktail (Thermo Scientific). Total protein in cell lysate was quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific). 10 ug of protein from each lysate alongside with SeeBlue pre-stained protein ladder (ThermoFisher Scientific) were separated using 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) at 150V and transferred onto an activated PVDF nitrocellulose membrane (Thermo Scientific, Pierce) at 30V for 2 hours. The membrane was blocked with 1% milk in PBS 0.1% Tween-20 for 1 h at RT and then incubated with rabbit anti-HIF1α antibody (Novus Biologicals, Littleton, Colo.) in 1% milk (1:2000) overnight at 4° C. or polyclonal anti-β-Actin (1:5000; Abcam). After washing, the membrane was incubated with a secondary anti-rabbit horseradish peroxidase (HRP) goat anti-rabbit IgG antibody in 1% milk (1:5000; Invitrogen). Next, the HRP substrate 3,3′,5,5′ tetramethylbenzidine (TMB) was added to the PVDF membrane and the signal was read using a CL-XPosure Film (Thermo Scientific) and Western blot X-ray analyser.
[0302] Quantitative PCR
[0303] Genomic DNA was extracted from cell lines using a DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to manufacturer's protocol and measured with nanodrop spectrophotometer at 280 nm absorbance. qPCR was performed using KiCqStart SYBR Green qPCR ReadyMix with ROX (purchased from Sigma-Aldrich, US) according to the manufacturer's protocol using custom designed primers to generate amplicons from Tbp, Luc or T2A sequences in the genome. The primers used were: murine Tbp 5′-TGTCTGTCGCAGTAAGAATGGA-3′ (SEQ ID NO: 46) and 5′-AAAATCCCAGACACGGTGGG-3′ (SEQ ID NO: 47), human Tbp 5′-TTTGGTGTTTGCTTCAGTCAG-3′ (SEQ ID NO: 48) and 5′-ATACCTAGAAAACAGGAGTTGCTCA-3′ (SEQ ID NO: 49), Luc 5′-ATTTGACTGCCGGCGAAATG-3′ (SEQ ID NO: 50) and 5′-AAGATTCATCGCCGACCACAT-3′ (SEQ ID NO: 51), T2A 5′-CGGAGAAAGCGCAGC-3′ (SEQ ID NO: 52) and 5′-GGGTCCGGGGTTCTCTT-3′ (SEQ ID NO: 53). Amplifications of the genes of interest were detected on an ABI 7900HT Fast Real Time PCR instrument (ThermoFisher Scientific).
[0304] Quantitative Reverse Transcription PCR
[0305] Healthy female C57BL/6 mice were sacrificed and the following organs were extracted: mammary gland, fat, liver, kidneys, colon, small intestine, stomach, skeletal muscle, lung, heart, brain, olfactory bulb and eyes (n=13). Organs were submerged in RNAlater (Sigma-Aldrich, US) reagent to stabilise and protect cellular RNA and kept overnight at 4° C. RNA was isolated from the tissues using PrepEase RNA Spin Kit (Affymetrix, US) according to the manufacturer's protocol and quantified using NanoDrop spectrophotometer at 280 nm. Erbb1-4 and Integrin β-6 mRNA expression was analyzed in purified mRNA by quantitative reverse transcriptase PCR using the EXPRESS One-step Superscript qRT-PCR kit (ThermoFisher Scientific), alongside assays on demand for the genes of interest which included: Egfr Mm01187858_m1, Erbb2 Mm00658541_m1 Erbb3 Mm01159999_m1, Erbb4 Mm01256793_m1, Itgb6 Mm01269869_m1, Tbp Mm01277042_m1. qRT PCR was performed using an ABI 7900HT Fast Real Time PCR instrument (ThermoFisher Scientific) and data analysis was done in Excel. RNA was stored at −80° C. Expression of all genes is represented relative to the house-keeping gene Tata-binding protein (Tbp).
[0306] List of Primers Used:
TABLE-US-00017 Primer name Sequence Fwd EPO HRE 5′-CCA CCT GTA GGT TTG GCA AGC TAG CGT CCG GGA AAC-3′ (SEQ ID NO: 54) Fwd GLUT3 HRE 5′-CCA CCT GTA GGT TTG GCA AGC TAG CCA CGC CTG TAA TC-3′ (SEQ ID NO: 55) fwd VEGFA HRE 5′-CCA CCT GTA GGT TTG GCA AGC TAG CCC CCC TTT GGG-3′ (SEQ ID NO: 56) Fwd frag 3 5′-GAA CCA TCA GAT downstream GTT TCC AGG-3′ Xba HRE (SEQ ID NO: 57) Fwd frag A 5′-ATC CGC CAC AAC binds in eGFP ATC GAG-3’ (SEQ ID NO: 58) Rev EPO HRE 5′-CCT GGA AAC ATC TGA TGG TTC TCT AGA CCT CAG GCC CGG-3′ (SEQ ID NO: 59) Rev frag 3 5′-GCG GGC CTC TTC downstream GCT ATT A-3′ EcoRI (SEQ ID NO: 60) Rev frag A 5′-TTG CCA AAC CTA upstream Nhe CAG GTG G-3′ HRE (SEQ ID NO: 61) fwd HRE from 5’-GGT GGT ACC GGT p3 p4 p5 CTG TAG GTT TGG CAA GCT AGC-3′ (SEQ ID NO: 62) fwd primer 5′-GAA AGA CCC CAC seq genome to CTG TAG GTT T-3′ verify (SEQ ID NO: 63) orientation of HRE fwd puro plus 5′-GCC ACG ACC GGT AgeI plus GCC GCC ACC ATC CCC buffering TGA CCC ACG CC-3′ (SEQ ID NO: 64) fwd tataa 5′-GGG TAT ATA ATG linker gilbert GAA GCT CGA ATT CTA overlap GCG-3′ (SEQ ID NO: 65) fwr HRE overlap 5′-CGA AAG GAG CGC and skip ACG ACC AAT TCA ATT Nco GGC CCT ACG TG-3′ (SEQ ID NO: 66) gagSFG seq primer 5′-CGG ATG GCC GCG AGA-3′ (SEQ ID NO: 67) qPCRfwd Luc 5′-ATT TGA CTG CCG GCG AAA TG-3′ (SEQ ID NO: 68) qPCRfwdrefmouseTBP 5’-TGT CTG TCG CAG TAA GAA TGG A-3′ (SEQ ID NO: 46) qPCRreffwdhumanTBP 5′-TTT GGT GTT TGC TTC AGT CAG-3′ (SEQ ID NO: 48) qPCRrefrevhumanTBP 5′-ATA CCT AGA AAA CAG GAG TTG CTC A-3′ (SEQ ID NO: 49) qPCRrefrevmouseTBP 5′-AAA ATC CCA GAC ACG GTG GG-3’ (SEQ ID NO: 47) qPCRrev Luc 5′-AAG ATT CAT CGC CGA CCA CAT-3′ (SEQ ID NO: 69) rev GLUT3 HRE 5′-CCT GGA AAC ATC TGA TGG TTC TCT AGA TTT GGC CAT GTT GAC TAG-3′ (SEQ ID NO: 70) rev VEGFA HRE 5′-CCT GGA AAC ATC TGA TGG TTC TCT AGA GTT CCG GGG TTA GTC AGT-3′ (SEQ ID NO: 71) rev primer seq 5′-CAC CAA AGA GTC orientation CTA AAC GAT C-3′ HRE (SEQ ID NO: 72) rev puro skip 5′-CAC GTA GGG CCA Nco site ATT GAA TTG GTC GTG CGC TCC TTT CG-3′ (SEQ ID NO: 73)
[0307] Cell Viability
[0308] Cells were washed twice with cold Dulbecco's Phosphate Buffered Saline (DPBS) (Gibco) and resuspended in 1× Binding Buffer supplied in the PE Annexin V Apoptosis Detection Kit (BD Biosciences). Cells were then stained with PE Annexin V and 7-Amino-Actinomycin (7-AAD) according to PE Annexin V Apoptosis Detection Kit protocol (BD Biosciences) for 15 minutes at RT in the dark, washed and resuspended in 1× Binding Buffer and analysed by flow cytometry (FACSCanto II Flow cytometer, BD Biosciences). Flow data were analysed using FlowJo software. PE Annexin V and 7-AAD negative cells are considered viable, PE Annexin V positive and 7-AAD negative cells are in early apoptosis and PE Annexin V and 7-AAD positive cells are in late apoptosis or dead.
[0309] T-Cell Isolation
[0310] For isolating human T-cells; blood was obtained from healthy volunteers under approval of the Guy's and St Thomas' Research Ethics Committee (REC reference 09/H0804/92). Blood was collected into Falcon tubes containing anti-coagulant (10% Citrate), mixed at 1:1 with RPMI 1640 and layered over Ficoll-Paque Plus (GE Healthcare). Samples were centrifuged at 750 g for 30 mins at 20° C. to separate the peripheral blood mononuclear (PBMC) cell fraction. The interface between the plasma and the Ficoll layer, which contained the PBMCs, was harvested using a sterile Pasteur pipette and washed in RPMI 1640. T-cells were purified from the PBMC fraction using human Pan T-cell isolation kit (Miltenyi Biotec) and isolated using a MidiMACs™ separator and LS columns (Miltenyi Biotec) according to the manufacturer's protocol. Purified human T-cells were activated using CD3/CD28 Human T-Activator Dynabeads (Gibco) at a 1:1 cell to bead ratio and seeded in tissue culture plates at 3×10.sup.6 in RPMI 1640 supplemented with 5% human serum (Sigma-Aldrich) and 1× penicillin/streptomycin. The following day, 100 IU/ml recombinant human IL-2 (PROLEUKIN) was added to the cultures.
[0311] T-Cell and Cell Line Transduction
[0312] To produce retrovirus with tropism for human cells, RD114 pseudotyped retroviral particles were generated by triple transfection, using Peq-Pam plasmid (Moloney GagPol), RDF plasmid (RD114 envelope) and the SFG plasmid of interest, using FuGENE HD transfection reagent (Promega), of HEK 293T cells as previously described. To produce retrovirus with murine cell tropism, Phoenix-ECO retrovirus producer cells were transfected using FuGENE HD (Promega) with the relevant plasmid. Supernatant containing viral particles were harvested and incubated with the cells of interest for at least 48 h to allow their transduction. T-cells were transduced in non-tissue culture treated plates that were pre-coated with 4 μg/cm.sup.2 RetroNectin (Takara Bio) overnight at 4° C. Prior to the retroviral transduction of human T-cells, CD3/CD28 Human T-Activator Dynabeads (Gibco) were removed and fresh IL-2 was added as stated in the T-cell isolation section. In the case of T-cell transduction with the bicistronic 4αβ-T2A-CAR construct, following T-cell transduction, human IL-4 (Peprotech) at 30 ng/ml final concentration was added to the culture to enrich the transduced T-cell population. Adherent cell lines, including SKOV-3 and HN3, were transduced with retrovirus, produced as indicated before, in media solution containing Polybrene (Santa Cruz Biotechnology Inc) at 4 μg/ml final concentration to increase infection efficiency. Cells modified to express Luc/eGFP were purified by cell sorting using BD FACSAria III (BD Biosciences) based on their eGFP fluorescence.
[0313] In Vitro Studies
[0314] In vitro hypoxia was achieved using a hypoxia incubator chamber (Stemcell Technologies) purged at 25 L/min for 4 mins with gas containing either; 0.1, 1, 5% O.sub.2, 5% CO.sub.2 and nitrogen as a balance (BOC), after which the chamber was sealed. This process was repeated again after 1 h. Hypoxia-mediated HIF1α stabilization was, in some cases, mimicked by using the chemical CoCl.sub.2 (Sigma-Aldrich), which inhibits HIF1α hydroxylation, at 100 μM final concentration, unless otherwise stated. In vitro cytotoxicity assays 1×10.sup.4 Luc/eGFP-expressing SKOV3 cells were seeded in 96-well tissue culture plates and transduced or non-transduced T-cells were added in the well at the indicated effector to target ratios. Co-cultures were incubated for 24, 48 and 72 h time points and target cell viability was determined by luciferase quantification (in normoxic conditions, following the addition of 1 μl of 15 mg/ml XenoLight D-luciferin (PerkinElmer) in PBS per 100 μl of media. Luminescence was quantified using a FLUOstar Omega plate reader (BMG Labtech). At the 24 and 48 h co-culture time points a sample of media was taken from the co-culture and subsequently used for IL-2 and IFNγ quantification, respectively. IL-2 was quantified using Human IL-2 ELISA Ready-SET-Go! Kit, 2nd Generation (eBioscience) as per manufacturer's protocol. IFNγ was quantified using Human IFN-gamma DuoSet ELISA kit (Bio-Techne) as per manufacturer's protocol. In both ELISAs cytokine concentration was determined by absorbance measurements at 450 nm on a Fusion alpha-FP spectrophotometer (Perkin-Elmer).
[0315] In Vivo Studies
[0316] Tumour cell lines (2.5×10.sup.5 cells in PBS) were inoculated by subcutaneous (s.c.) injection into female (for SKOV3 and LL2) and male (for HN3) mice that were six to eight weeks of age. Once tumours were palpable, digital caliper measurements of the long (L) and short (S) dimensions of the tumour were performed every 2 or 3 days. Tumour volume was established using the following equation: Volume=(S.sup.2×L)/2. Blood samples were taken from mice in EDTA-coated Microvette™ tubes (Sarstedt) and plasma was extracted by centrifugation of these samples at 2,000 g for 5 mins. The indicated doses of CAR T-cells were injected in 200 μl PBS through the tail vein using a 30 G needle. Tumour tissue, and other organs, for flow cytometry analyses were enzyme-digested to release single cells as previously described. In brief, tissues were minced using scalpels, and then single cells were liberated by incubation for 60 mins at 37° C. with 1 mg/ml Collagenase 1, from Clostridium Histolyticum (Sigma-Aldrich) and 0.1 mg/ml Deoxyribonuclese I (AppliChem) in RPMI (Gibco). Released cells were then passed through a 70 μm cell strainer prior to staining for flow cytometry analyses. Viable cells were numerated using a haemocytometer with trypan blue (Sigma-Aldrich) exclusion.
[0317] Bioluminescence Imaging
[0318] To assess luciferase bio-distribution in vivo, mice were injected intraperitoneally (i.p.) with 200 μl (15 mg/ml) XenoLight D-luciferin (PerkinElmer) in sterile PBS 10 mins prior to imaging. Animals were anesthetized for imaging and emitted light was detected using the In vivo Imaging System (IVIS®) Lumina Series III (PerkinElmer) and data analysed using the Living Image software (Perkin Elmer). Light was quantified in photons/second/unit area.
[0319] Flow Cytometry
[0320] Flow cytometry was performed as previously described. The following antibodies were purchased from eBioscience and were used at 1 μg/ml unless stated otherwise: anti-human CD3ε Brilliant Violet 421™ (SK7; Biolegend®), anti-human CD8α Alexa Fluor 488 (RPA-T8), anti-human CD4 PE (RPA-T4), anti-human CD45 Brilliant Violet 510™ (H130 Biolegend®), anti-mouse CD4 FITC (Clone: RM4-5), anti-mouse CD8α eFluor®450 (Clone: 53-6.7), anti-mouse CD3ε PE (Clone: 145-2C11), neutralizing anti-mouse CD16/CD32 (Clone: 2.4G2). Background staining was established using fluorescence minus one stained samples. T1E CAR was stained with a biotinylated anti-human EGF antibody (Bio-Techne: BAF236) and detected using Streptavidin APC. eGFP was detected by its native fluorescence. Dead cells and red blood cells were excluded using 1 μg/ml 7-amino actinomycin D (Cayman Chemical Company) alongside anti-Ter-119 PerCP-Cy5.5 (Ter-119; eBioscience). Data were collected on a BD FACS Canto II (BD Biosciences). Data was analyzed using FlowJo software (Freestar Inc.).
[0321] Statistics
[0322] Normality and homogeneity of variance were determined using a Shapiro-Wilk normality test and an F-test respectively. Statistical significance was then determined using a two-sided unpaired Students t test for parametric or Mann-Whitney test for nonparametric data using GraphPad Prism 6 software. When comparing paired data, a paired ratio Students t test was performed. A Welch's correction was applied when comparing groups with unequal variances. Statistical analysis of tumour growth curves was performed using the “CompareGrowthCurves” function of the statmod software package. No outliers were excluded from any data presented.
[0323] Results
[0324] HRE Design
[0325] Based on analysis of genomic data obtained from the Ensembl database, putative HIF1-binding site (HBS), which is conserved between species and between hypoxia-induced genes, were identified. We compared the putative 6 nucleotide (nt)-long HBS from different oxygen-sensitive genes in human, mouse and rat based on the frequency of each nucleotide in each position in the 6-nt sequence, which binds HIF, and a sequence logo was constructed for human and mouse HBS (
[0326] The HRE design included an HBS and a HAS site separate by a 8 nt linker region taken from the genomic sequence. In the first instance, 3 sequential HBS-HAS sequences were used. Also, to see whether different HBS sequences have different sensitivities to HIF, three constructs were initially designed, each containing 3 sequential HBS-HAS (HRE for simplicity) sequences. The difference between these constructs was that the HBS in each construct was derived from different genes (
[0327] HREs in the LTR
[0328] To stably integrate the construct into the host cell's genome we used the SFG retroviral vector with modified LTRs as previously described. The SFG vector is derived from the Moloney murine leukaemia virus (MMLV). We attempted to modify the retroviral enhancer region within the LTRs without affecting the integration of the transgene into the host cell genome. This has previously been achieved by cloning HREs in to the NheI/XbaI site of the LTR, which is upstream the viral promoter. In order to avoid inactivating the vector or its ability to integrate into the host genome, we replaced the NheI/XbaI region with a fragment of similar length.
[0329] DNA sequences containing our HREs sequences that include 5′ NheI and 3′ XbaI restriction sites were synthesized by GeneArt. These sequences were sub-cloned in the NheI/XbaI site in the 3′ LTR of the SFG MMLV vector. We modified the 3′ LTR but not the 5′ LTR as, when reverse transcription occurs, the modified 3′ LTR U3 region is copied to the 5′ LTR. Due to the fact that NheI/XbaI were not unique restriction sites in the SFG, we synthesised a fragment in several steps using sequential overlapping PCR, which contained unique restriction sites (XhoI/EcoRI) in order to achieve specific modification of the NheI/XbaI site in the 3′ LTR. To make an oxygen-sensing reporter construct, green-emitting variant of click beetle luciferase and green fluorescent protein separated by a P2A peptide (self-cleaving peptide) were cloned into NcoI/XhoI site of the SFG vector. The resulting constructs are shown in
[0330] ODD Addition
[0331] We simultaneously cloned an additional set of vectors that had an ODD domain attached to the luciferase reporter to facilitate protein degradation under conditions of normoxia. HIF1α stability is controlled by oxygen-dependent hydroxylation of prolines (p402 and p564) in the ODD. This sequence was fused with a protein of interest to make the degradation of the protein oxygen-dependent. Based on the UniProt database, the ODD domain (highlighted in
[0332] In subsequent experiments we fused SEQ ID Nos 29, 30, 31. All three SEQ ID Nos conferred oxygen sensitivity to the fusion partner, with optimal results being obtained with SEQ ID NO: 29, i.e. whole ODD (401-603) (
[0333] HIF1α Stability Under Normoxia or Hypoxia in Different Cell Lines
[0334] Cell lines were cultured for 18 hours in normoxic or hypoxic conditions, 20% or 0.1% O.sub.2, respectively. The following human cell lines were screened under these conditions: HEK293 T, HT1080, T47D and Jurkat (Clone E6-1). Immediately after the 18-hour exposure, cells were lysed and a Western blot was performed to quantify HIF1α as described in the methods. In all cell lines tested, HIF1α was found to be stabilised under hypoxic conditions (0.1% O.sub.2), when compared to normoxia (20% O.sub.2) (
[0335] Cell Choice
[0336] We chose to use 293T cells in initial experiments for three reasons. First, HIF1α Western blot analysis showed that 293T cells had strong expression of HIF1α protein under hypoxic conditions, at levels 5-fold higher than found in normoxia. Second, we observed that 293T are fast-growing cells when compared to T47D, allowing multiple experiments to be performed in a short time period. Third, 293T cells are the packaging cell lines that we use to produce the retrovirus. Therefore, transfection of 293T cells to produce retrovirus results in an auto-transduction of the 293T cells themselves.
[0337] Transduction Efficiency Based on Flow Cytometry
[0338] Since the expression of transgene in our constructs is oxygen-sensitive, we cannot rely on flow cytometry to determine accurate transduction efficiency. Flow cytometry analysis of 293T cells, which had been transduced with the constitutive luciferase-P2A-GFP construct (SFG Reporter construct), revealed a transduction efficiency in the live cell population (7-AAD negative) of 83% (
[0339] Sequencing to Verify Post-Integration HRE Orientation within the LTR
[0340] To confirm that the modifications in the 3′ LTR had been duplicated to the 5′ LTR and were correctly orientated in the integrated provirus we sequenced the 5′ LTR region after transduction. Genomic DNA was isolated from transduced 293T cells and the 5′ LTR region was amplified via PCR and run on a 1.2% agarose gel. The band of the correct length was excised, gel purified and then sequenced. Sequence analysis revealed that the HRE modifications to the 3′ LTR were correctly copied and had the correct orientation in the 5′ LTR.
[0341] Establishment of Copy Number Assay/qPCR (Copy Number) Assay Validation
[0342] For our assay in which we would quantitate luciferase expression under hypoxic conditions, we need to normalise our data, as not every cell would be transduced and some cells may have contained multiple copies of the reporter construct. To permit this we utilised quantitative PCR (qPCR) using the amplification of a reference gene (TBP), which is present as 2 copies in every cell (native genomic DNA), as well as that of the transgene (luciferase) to allow us to calculate the number of integrated transgenes. To design the qPCR primers, we screened multiple possible primer sequences in silico using the Ensembl database to ensure high specificity of binding. We chose primers that bind to unique sites in the genes of interest so that the amplicons produced by PCR would be indicative of reference and transgene gene amount. We designed a primer set that binds to click beetle luciferase and human and mouse TBP (since we are using both human and mouse cell lines). Using this approach, the following three sets of primers were designed: forward mouse TBP (5′-TGT CTG TCG CAG TAA GAA TGG A-3′ (SEQ ID NO: 46)) and reverse mouse TBP (5′-AAA ATC CCA GAC ACG GTG GG-3′ (SEQ ID NO: 47)) that amplify a 94 nt fragment specifically from the mouse TBP gene, forward human TBP (5′-TTT GGT GTT TGC TTC AGT CAG-3′ (SEQ ID NO: 48)) and reverse human TBP (5′-ATA CCT AGA AAA CAG GAG TTG CTC A-3′ (SEQ ID NO: 49)) that amplify a 103 nt fragment specifically from the human TBP, and forward luciferase (5′-ATT TGA CTG CCG GCG AAA TG-3′ (SEQ ID NO: 68)) and reverse luciferase (5′-AAG ATT CAT CGC CGA CCA CAT-3′ (SEQ ID NO: 69)), which amplify specifically a 90 nt fragment from luciferase transgene.
[0343] To determine primer binding specificity (a single amplified product), we performed qPCR on genomic DNA extracted from cells and run the PCR product on an agarose gel. All PCR products gave a single band of appropriate length demonstrating that the primers were specific.
[0344] To validate the copy number assay, genomic DNA was extracted from non-transduced cells and from cells transduced with the construct containing the click beetle luciferase. 200 ng of DNA was serially diluted (1:2) and qPCR was performed using the designed primers. Each reaction was performed in triplicate. As expected, no luciferase amplicon was detected in the DNA extracted from non-transduced cells. qPCR data generated using DNA extracted from the transduced cells demonstrated that there was a linear relationship between the qPCR signal from both luciferase and TBP primer sets and the cycle number of the reaction, validating the assay. 18-hour incubation of 293T cells in 20%, 5%, 1% and 0.1% oxygen 293T cells were transduced with retrovirus and transduction efficiency was determined by qPCR. Non-transduced 293T cells and 293T cells transduced with luciferase constructs 1-8 (A, B, C and D from
[0345] The effect of adding the ODD domain within the construct is best assessed by comparing the constitutively expressing unmodified LTR construct +/−ODD. See
[0346]
[0347] Hypoxia Inducibility in the Presence of Increasing Numbers of HRE Elements in the Promoter
[0348] As shown in
[0349] Luciferase Stability in Normoxia (+/−ODD)
[0350] A variety of ODD segments were fused to the C-terminus of luciferase and the results are shown in
[0351] In Vitro and In Vivo T4-CAR Results
[0352] We utilised a pan-ErbB CAR T1E28z which has specificity towards 8/10 of the possible ErbB homo- and hetero-dimers in both mice and humans. We modified the CAR construct to concurrently co-express a reporter Click Beetle luciferase (Luc) to permit in vivo tracking once transduced into T-cells. ErbB-CAR/Luc T-cells were i.v. infused into immunocompromised NSG mice bearing subcutaneous SKOV3 ovarian cancer xenografts. The bio-distribution of the CAR T-cells was analysed 4 days post infusion. At this early time point, the majority of cells were seen to reside in the lungs and liver, while there was minimal uptake in the tumour (
[0353] As hypoxia differentiates the tumour microenvironment from healthy tissues, we sought to exploit this to create a hypoxia-sensing T4-CAR. T4 is a next generation anti-ErbB CAR co-expressed with a chimeric IL-4 receptor delivering an intracellular IL-2/IL-15 signal upon binding of IL-4 to the extracellular domain, thereby providing a means to selectively enrich CAR T-cells during ex vivo expansion without affecting the CAR-dependent killing capacity of the T-cells. We engineered the anti-ErbB CAR to contain a C-terminal 203 amino acid ODD and modified the CAR promoter in the long terminal repeat to contain a series of 9 HREs, rendering the CAR selectively responsive to hypoxia when transduced into T-cells (Schematic
[0354] CAR expression was highly dynamic and represented a switch that could be turned ‘on’ and ‘off’ in an O.sub.2-dependent manner (
[0355] Having validated HypoxiCAR's ability to sense hypoxia, we sought to investigate its ability to elicit hypoxia-dependent killing of target cells. For this, SKOV3 ovarian cancer cells were used which express ErbB1-4. Cells were seeded onto culture plates and co-incubated with T4-CAR or HypoxiCAR under normoxic (20% O.sub.2) and hypoxic (0.1% O.sub.2) conditions. Despite equivalent transduction efficiencies, HypoxiCAR displayed efficient hypoxia-dependent killing of the SKOV3 cells with no significant killing under normoxic conditions. Target-cell killing was CAR-mediated as when HypoxiCAR's intracellular tail was truncated to prevent signalling (CD3.sup.−), killing was abrogated (
[0356] To translate these observations in vivo, we evaluated whether HypoxiCAR could circumvent off-tumour toxicity of ErbB-CAR T-cells. This is a major hurdle that precludes their systemic administration in the clinic. To evaluate this technology in the tumour setting, HypoxiCAR T-cells were injected concurrently i.v. and i.t. in HN3 tumour-bearing NSG mice. By this means, we achieved a rapid accumulation of these cells in tumour and vital organs for ex vivo investigation (
[0357] The results show a stringent hypoxia-sensing CAR T-cell approach which achieves selective expression of a panErbB-targeted CAR within a solid tumour, a microenvironment characterized by an inadequate oxygen supply. Despite widespread expression of ErbB receptors in healthy organs, the approach provides anti-tumour efficacy without off-tumour toxicity in murine xenograft models. This dynamic oxygen-sensing safety switch potentially facilitates unlimited expansion of the CAR T-cell target repertoire for treating solid malignancies.
[0358] Identifying approaches to circumvent off-tumour toxicity has the potential to unlock an entirely new repertoire of CAR antigen targets for carcinomas, which are currently limited.
[0359] To investigate this issue, we utilized a 2.sup.nd generation pan-anti-ErbB CAR T1E28z which has specificity towards 8/10 of the possible ErbB receptor homo- and hetero-dimers and crosses the species barrier binding both mice and human receptors equivalently. This CAR is currently undergoing Phase I evaluation by intra-tumoural (i.t.) delivery in patients with SCCHN. The CAR is co-expressed with a chimeric cytokine receptor (4αβ) which delivers an intracellular IL-2/IL-15 signal upon binding of IL-4 to the extracellular domain (
[0360] Hypoxia is a characteristic of most solid tumours. The proliferative and high metabolic demands of the tumour cells, alongside inefficient tumour vasculature, result in a state of inadequate oxygen supply (<2% O.sub.2) compared to that of healthy organs/tissues (5-10% O.sub.2) (
[0361] Having validated HypoxiCAR's ability to sense hypoxia, we sought to investigate its ability to elicit hypoxia-dependent killing of tumour target cells. SKOV3 ovarian cancer cells were seeded onto culture plates and co-incubated with T4-CAR or HypoxiCAR under normoxic and hypoxic (0.1% O.sub.2) conditions. Despite equivalent transduction efficiencies and CD4.sup.+:CD8.sup.+ T-cells ratios, HypoxiCAR T-cells displayed efficient hypoxia-dependent killing of the SKOV3 cells, almost equivalent to T4-CAR T-cells, with no significant killing observed under normoxic conditions (
[0362] To evaluate whether HypoxiCAR could provide tumour-restricted CAR expression in vivo, human HypoxiCAR T-cells were injected concurrently i.v. and i.t. in NSG mice bearing HN3 tumours. These tumours had an approximate volume of 500 mm.sup.3 (
[0363] Hypoxia has been extensively studied in SCCHN. To assess which patients might be most appropriate for HypoxiCAR T-cell immunotherapy, we firstly generated an HRE-regulated gene signature using patient tumour transcriptomic data. Known HRE-regulated genes were analyzed for co-expression, and a refined signature utilizing the genes PGK1, SLC2A1, CA9, ALDOA and VEGFA was chosen as we observed a significant positive correlation between these genes (
[0364] Immunohistochemistry staining of SCCHN tumour sections for stabilized HIF1α, the master transcription factor for HypoxiCAR's CAR expression, revealed large regions of the tumours where HIF1α had become stabilized (
[0365] Approaches to improve tumour-specificity of CAR T-cells have been developed, such as T-cell receptor-mimetic CARs with specificity for HLA-presented antigens, combined targeting of tumour antigens, or tuning of CAR affinity to preferentially target high density antigens. This study demonstrates an alternative approach to achieve cancer-selective immunotherapy, exploiting one of the most innate characteristics of the tumour microenvironment. The ‘dual hypoxia-sensing’ system described here achieves compelling anti-tumour efficacy while abrogating off-tumour toxicity of a CAR that recognizes multiple targets in normal tissues. The hypoxia-sensing HRE module and the ODD appended onto the CAR act synergistically to provide stringent hypoxia-specific target killing (
[0366] The hypoxic tumour microenvironment is not conducive to efficient immune reactions. Hypoxia can activate immune-suppressive programmes in stromal cells such as macrophages, regulate the expression of immune checkpoint molecules and promote a more aggressive tumour cell phenotype. However, encouragingly we found that hypoxia did not negatively affect T-cell effector function directly in vitro (
[0367] T-cells could take place. As such, a suicide switch could be incorporated into HypoxiCAR to provide an additional level of safety for the most pervasive CARs. Although the ‘HypoxiCAR’ dual oxygen sensing system was exemplified using a pan-ErbB-targeted CAR, the broadly applicable strategy may be used to overcome the paucity of safe targets available for the treatment of solid malignancies.