Hypoxia responsive therapeutic agents

Abstract

The present invention relates to therapeutic agents, particularly to nucleic acids capable of hypoxia-responsive expression of armouring proteins and cells incorporating the same, and their use in therapeutic or prophylactic treatment, especially of cancer. The nucleic acids may further encode novel hypoxia-responsive chimeric antigen receptors (CARs).

Description

[0001] P5598PC00

[0002] THERAPEUTIC AGENTS TECHNICAL FIELD

[0003] The present invention relates to therapeutic agents, particularly to nucleic acids capable of hypoxia-responsive expression of armouring proteins and cells incorporating the same, and their use in therapeutic or prophylactic treatment, especially of cancer. The nucleic acids may further encode novel hypoxia-responsive chimeric antigen receptors (CARs).

[0004] BACKGROUND

[0005] T cell therapies are a type of adoptive cell therapy involving genetic modification of T cells. T cell therapies include Chimeric Antigen Receptor T Cell (CAR-T) therapies and T Cell Receptor (TCR T-cell) therapies.

[0006] CAR-T therapies involve genetically modifying T cells to express a CAR that recognises a specific tumour-associated antigen (TAA), allowing the CAR-T cells to kill cells bearing that antigen. TCR therapies involve genetically modifying T cells to express a modified T cell receptor (TCR) that recognises intracellular cancer antigens presented by MHC molecules, also allowing the T cell to kill cells bearing that antigen. Harnessing the immune system to target cancer in this way is a highly promising treatment modality, given the potent cytotoxic capacity of T cells, and the potential for the immune system to form memory against target cells.

[0007] Whilst some CAR-T cell therapies are now clinically approved for the treatment of haematological cancers, clinical translation of CAR-T cell therapy to effectively treat solid tumours has not yet been achieved. There are thought to be a variety of barriers that have hindered the effective and safe application of CAR-T cell therapy against solid tumours, including: on-target off-tumour effects that arise due to target antigen expression on both tumour and healthy cells, which can lead to serious toxicities; antigen escape; and both physical and biochemical barriers within the immunosuppressive tumour microenvironment (TME) that can prevent CAR-T cell trafficking to tumour cells and induce rapid exhaustion of CAR-T cells, significantly reducing their antitumour efficacy and persistence.

[0008] One strategy to try to improve the effector function of engineered T cells, such as CAR-T cells and TCR-engineered T-cells, is to "armour" them. " Armoured" T-cells express, alongside the CAR or the engineered TCR, a protein payload that serves to counteract some immunosuppressive aspect of the TME. An armouring approach is able to potentiate the anti-tumour efficacy of the engineered T cell by, for instance, altering the other cells that are present in the TME. One particular aim of expressing an armouring protein is to convert tumour-associated macrophages to a pro-inflammatory phenotype, which allows for the formation of a positive feedback loop that enhances T cell effector function and survival.P5598PC00

[0009] However, since engineered T cells circulate through the blood and healthy organs before reaching the tumour, armouring payloads may also induce toxicities, especially when constitutively expressed. For instance, IL-12 is a pro-inflammatory cytokine with pleiotropic effects on immune cells which has garnered significant interest in the field as a potential armouring protein, but this payload has fallen out of favour due to toxicities when expressed in its constitutive form in CAR T-cells.

[0010] Furthermore, it is not always the case that improvements in anti-tumour activity correlate with the amount of an armouring protein being expressed by an engineered T-cell. For example, Type I interferons (IFN-Is) are a class of pro-inflammatory cytokines which have the potential to mediate antitumour immunity, but excessive IFN-I signalling leads to T cell dysfunction and apoptosis. This is a major factor in why attempts at armouring CAR-T cells with IFN-Is thus far have not shown sustained improvement in anti-tumour efficacy.

[0011] As such, there is a need to develop approaches for armouring engineered T-cells that result in tumour-selective expression of armouring proteins, at a level that allows for anti-tumour activity.

[0012] Strategies that have been explored thus far include placing the armouring protein under the control of a nuclear factor of activated T-cells (NFAT)-responsive promoter or tethering the armouring protein to the membrane of the CAR-T cell. NFATs are transcriptional factors that play an important role in gene transcription in activated T cells and such promoters are induced following activation of T cells triggered by TCR engagement. However, NFAT-inducible or membrane-tethered armouring payloads would still require targeting of the CAR to an appropriate antigen. Identifying bona fide tumour-associated antigens that are present only on tumour tissues and absent on healthy tissues has been challenging, and targeting non-specific antigens can lead to on-target off-tumour toxicities. Utilising a platform that exploits the low oxygen tensions, or hypoxic conditions, of solid tumours to target both CAR activation and armouring payload selectively to the tumour may be a way of avoiding toxicities arising from off-tumour antigen recognition.

[0013] SUMMARY OF THE INVENTION

[0014] The present inventors sought to solve the problem of achieving tumour-selective expression of armouring proteins, in particular IFN-Is, at a level that allows for anti-tumour activity by restricting the expression of the armouring protein to hypoxia.

[0015] Tumour-selective expression was achieved by restricting the expression of the armouring protein to hypoxia. Tight hypoxia-specific regulation was achieved by both (i) fusing the armouring protein to one or more ODDs and (ii) placing the armouring protein under the transcriptional control of one or more HREs.P5598PC00

[0016] Furthermore, surprisingly, the present inventors found that this hypoxia-specific regulation of armouring proteins, in particular IFN-p, allows for anti-tumour effects to be achieved. Without wishing to be bound by theory, it is believed that, by fusing the armouring protein to an ODD and placing it under the transcriptional control of one or more HREs, IFN-I is expressed in a hypoxic TME at a level that is sufficient to allow for anti-tumour activity, but is not at a level high enough to induce significant T-cell dysregulation or apoptosis.

[0017] Furthermore, fusing the IFN-I to an ODD and placing it under the transcriptional control of HREs restricts expression of the IFN-I in healthy, normoxic tissues, thus allowing for side effects and toxicities to be reduced.

[0018] Furthermore, the present inventors found that armoured T-cells have the ability to mediate the killing of tumour cells that do not express the CAR-targeted antigen. Without wishing to be bound by theory, it is believed that this is because the armouring protein can have both direct and indirect cytotoxic or cytostatic effects on tumour cells. These results indicate that the armoured T-cells of the present invention could be particularly useful in the treatment of solid tumours, which are heterogeneous and may include cells which do not express the CAR-targeted antigen.

[0019] Expression of some armouring proteins, especially interferons, only under hypoxic conditions and not during manufacture of therapeutic cells avoids the armouring protein leading to dysfunction and apoptosis.

[0020] In one aspect, the present invention provides a nucleic acid molecule comprising

[0021] a. a first polynucleotide encoding an armouring protein fused to one or more Oxygen-Dependent Degradation Domains (ODDs); and

[0022] b. a second polynucleotide comprising a regulatory region comprising one or more hypoxia-responsive elements (HREs);

[0023] wherein the first polynucleotide is operably linked to the second polynucleotide.

[0024] In a further aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention.

[0025] In a further aspect, the present invention provides a cell, especially an immunoresponsive cell, comprising a nucleic acid molecule of the present invention or a vector of the present invention.

[0026] In a further aspect, the present invention provides a population of cells of the present invention or a population of immunoresponsive cells of the present invention.P5598PC00

[0027] In a further aspect, the present invention provides a method of preparing a cell, especially an immunoresponsive cell, comprising

[0028] a. Isolating a cell from a subject;

[0029] b. Modifying the cell to introduce a nucleic acid molecule of the present invention or a vector of the present invention;

[0030] c. Expanding the modified cell ex-vivo; and

[0031] d. Obtaining an expanded cell capable of expressing the nucleic acid molecule under conditions of hypoxia.

[0032] In a further aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid molecule of the present invention, a vector of the present invention, a cell or an immunoresponsive cell of the present invention or a population of the present invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0033] In a further aspect, the present invention provides a nucleic acid molecule of the present invention, a vector of the present invention, a cell or an immunoresponsive cell of the present invention, a population of the present invention or a pharmaceutical composition of the present invention for use in the treatment or prevention of a disease or disorder, preferably wherein the disease or disorder is a cancer, most preferably a solid cancer.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0036] Figure 1 shows a schematic of an exemplary construct of the present invention. The construct includes a polynucleotide encoding an IFN-p (an IFN-I) fused to an ODD and a polynucleotide encoding a CAR fused to an ODD. The two polynucleotides are separated by a T2A sequence and both are under the transcriptional control of HREs.

[0037] Figure 2 shows a graph of IFN-p secretion over time, as detected by an ELISA assay, from CAR-T cells expressing the construct shown in Figure 1. For the first 48 hours, the CAR-T cells were cultured in hypoxic conditions and for the next 48 hours, the CAR-T cells were cultured under normoxia. The graph shows IFN-p was secreted under hypoxic conditions only.

[0038] Figure 3 demonstrates that constructs of the present invention result in both hypoxia-restricted CAR expression and hypoxia-restricted armouring protein secretion. Figure 3A is a schematic of an exemplary construct of the present invention, where sequences encodingP5598PC00

[0039] mouse IFN-p fused to an ODD and the TIE binder-containing CAR fused to an ODD are separated by a self-cleaving T2A peptide. H = Hinge domain, TM = transmembrane domain, Endo = endodomain. Figure 3B shows flow cytometry histograms of CAR expression, comparing untransfected control cells to cells transfected with the mIFNp-TlE-HypoxiCAR construct, after 24 hours in normoxia or hypoxia. Figure 3C shows the concentration of mIFNP secreted by untransfected control HEK293T cells or HEK293T cells transfected with the mIFN[3-TlE-HypoxiCAR construct, cultured in normoxia or hypoxia. Figure 3D shows a schematic of an alternative exemplary construct of the present invention, where sequences encoding mouse IFN-p fused to an ODD and the TE9-containing CAR fused to an ODD are separated by a self-cleaving T2A peptide. H = Hinge domain, TM = transmembrane domain, Endo = endodomain. Figure 3E shows a schematic of an alternative construct which lacks the armouring protein. Figures 3F-G show the surface CAR expression in T-cells transduced with either the TE9-HypoxiCAR (Figures 3F) or the mIFNp-TE9-HypoxiCAR (Figure 3G) construct at the indicated time points in hypoxia and upon re-exposure to normoxia. Figure 3H shows the secretion of mouse IFN-p by T-cells transduced with the mIFNp-TE9-HypoxiCAR construct at the indicated time points in hypoxia and upon re-exposure to normoxia.

[0040] Figure 4 shows that T-cells transduced with constructs of the present invention produce bioactive armouring proteins. Figure 4A-D show the expression of key costimulatory and antigen-presenting molecules and interferon-stimulated genes (Sca-1, MHC class II, CD86 and MHC class I H-2Kb) in murine BMDMs treated with cell culture supernatant from untransduced control T-cells (UTD) or T-cells transduced with either the mIFNp-TE9-HypoxiCAR construct or the TE9-HypoxiCAR construct, under conditions of normoxia (left column) or hypoxia (right column). Figure 4E shows representative flow cytometry contour plots illustrating the gating of high (hi), low (Io), and negative (neg) MHCI H-2Kbexpression of BMDMs treated with conditioned media from T-cells transduced with either the mlFNp-TE9-HypoxiCAR construct or the TE9-HypoxiCAR construct, gated on live F4 / 80+macrophages.

[0041] Figure 5 shows that T-cells transduced with constructs of the present invention polarise macrophages to pro-inflammatory states in vivo and exhibit improved anti-tumour effects. Figure 5A is a schematic showing the workflow for the in vivo experiments. After 3 days, the mice were killed and tumours were harvested for flow cytometry. Figure 5B shows the proportions of different immune cell subsets expressed as percentage of total live cells in the tumour (left column is UTD, middle column is TE9 and right column is hTE9-mIFNb T-cells). Figure 5C-D show MHCI H-2Kb+tumour-associated macrophages (5C) and Sca-1+tumour-associated macrophages (5D) as a proportion of F4 / 80+macrophages from mice treated with hTE9 T-cells or hTE9-mIFNb T-cells. Figure 5E shows in vitro Py8119-eGFP / Luc tumour cell killing under normoxic and hypoxic conditions by untransduced control T-cellsP5598PC00

[0042] (UTD - left column) or T-cells transduced with either the T4-HypoxiCAR construct (hT4 -middle column) or the mIFNp-TlE-HypoxiCAR construct (hTlE-mIFNb - right column). Figure 5F shows the quantification of mouse IFN-p and human IFN-y secreted by nontransduced control T-cells (UTD - left column) or T-cells transduced with either the T4-HypoxiCAR construct (hT4 - middle column) or the mIFNp-TlE-HypoxiCAR construct (hTlE-mlFNb - right column).

[0043] Figure 6 shows that hypoxia-specific expression and activity can also be achieved when IL12 and IL23 are fused to an ODD and under the transcriptional control of HREs. Figure 6A shows the surface CAR expression in T-cells transduced with either the hTE9 or the hTE9-mIL12 / 23 construct at the indicated time points in hypoxia and upon re-exposure to normoxia. Figure 6B shows the secretion of IL12 and IL23 by T-cells transduced with the hTE9-mIL12 / 23 construct at the indicated time points in hypoxia and upon re-exposure to normoxia. Figure 6C shows viability of LAN-1 tumour cells co-cultured with untransduced control T-cells (left column), T-cells transduced with the hTE9 construct (middle column) or T-cells transduced with the hTE9-mIL12 / 23 construct (right column).

[0044] Figure 7 shows that hypoxia-specific expression of IL12 and IL23 also results in anti-tumour activity. Figure 7A shows the percentage of CAR+ T-cells recovered from the lungs, liver and tumours of mice treated with either hTE9 CAR-T cells (left column) or hTE9-mIL12 / 23-CAR-T cells (right column). Figure 7B shows the proportion of Ml-like MHCII+CD206" tumour associated macrophages (TAMs) and the proportion of immunosuppressive M2-like MHCIT CD206+TAMs in the tumour microenvironment of mice treated with either hTE9 CAR-T cells or hTE9-mIL12 / 23-CAR T cells. Figures 7C and 7E show the tumour volume in mice bearing established (C) LAN-1 tumours or (E) SK-N-SH tumours following treatment with either a vehicle control (line with highest tumour volume at 14 days), untransduced T-cells (line with second highest tumour volume at 14 days), hTE9 CAR-T cells (line with second lowest tumour volume at 14 days) or hTE9-mIL12 / 23-CAR T cells (line with lowest tumour volume at 14 days). Figures 7D and 7F show the percentage weight change in mice bearing established (D) LAN-1 tumours or (F) SK-N-SH tumours treated with either a vehicle control, untransduced T-cells, hTE9 CAR-T cells or hTE9-mIL12 / 23-CAR T cells.

[0045] Figure 8 shows that hypoxia-specific expression and anti-tumour effects can also be achieved when IL-27 and IL-39 are fused to an ODD and under the transcriptional control of HREs. Figure 8A shows a construct of the present invention. Figures 8B and 8C show (B) CAR expression and (C) IL-27 and IL-39 concentration over time from T-cells expressing the construct of Figure 8A. For the first 48 hours the CAR-T cells were cultured in hypoxic conditions and in the subsequent 48 hours when the CAR-T cells were cultured under normoxia. Figure 8D shows tumour viability following a three-round rechallenge assay with LAN-1 tumour cells co-cultured with T-cells transduced with the hTE9 construct or theP5598PC00

[0046] construct of Figure 8A. Figure 8E shows the expression of granzyme B in the cell culture supernatant of LAN-1 tumour cells co-cultured with T-cells transduced with the hTE9 construct or the construct of Figure 8A.

[0047] DETAILED DESCRIPTION

[0048] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0049] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0050] The present invention is described with respect to particular aspects and embodiments and with reference to certain Figures but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

[0051] The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying Figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "some embodiments" or a "preferred embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in some embodiments" or "in a preferred embodiment" in various places throughout thisP5598PC00

[0052] specification are not necessarily all referring to the same embodiment, but may do so. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0053] In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a nucleic acid molecule" includes two or more nucleic acid molecules, reference to "a vector" includes two or more such vectors, reference to "a cell" includes two or more cells, reference to "a subject" refers to two or more subjects, reference to "a method" includes two or more methods and the like.

[0054] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0055] The invention uses the terms nucleic acid molecule and polynucleotide to refer to different parts of the nucleic acid molecule of the invention even though the terms are interchangeable. A "nucleic acid molecule" and a "polynucleotide" is a macromolecule comprising two or more nucleotides. The nucleic acid molecule or polynucleotide may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the nucleic acid may be modified, for instance with a label or a tag, for which suitable examples are known by a skilled person. A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar.

[0056] Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably a deoxyribose. The nucleic acid preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC). The nucleotide is typically a ribonucleotide or deoxyribonucleotide. TheP5598PC00

[0057] nucleotide typically contains a monophosphate, diphosphate, or triphosphate. The nucleotide may comprise more than three phosphates, such as 4 or 5 phosphates.

[0058] Phosphates may be attached on the 5' or 3' side of a nucleotide. The nucleotides in the nucleic acid molecule or polynucleotide may be attached to each other in any manner. The nucleic acid molecule or polynucleotide may be single stranded or double stranded. The nucleic acid molecule or polynucleotide is most preferably ribonucleic nucleic acid (RIMA) or deoxyribonucleic acid (DNA).

[0059] Suitable nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate. The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.

[0060] Nucleic acid molecules or polynucleotides may also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) and (poly)ADPribose modified DNA. Sizes of nucleic acid molecules or polynucleotides are typically expressed as the number of base pairs (bp) or nucleotide pairs for double stranded nucleic acids, or in the case of single stranded nucleic acids as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). In all instances herein, the term "nucleic acid molecule" is interchangeable with "polynucleotide", "polynucleotide sequence", " DNA sequence" or "nucleic acid sequence". In all instances herein, the term "polynucleotide" is interchangeable with "nucleic acid molecule", "polynucleotide sequence", " DNA sequence" or "nucleic acid sequence. The sequence of the nucleotides in the nucleic acid molecule or polynucleotide is described herein as the "nucleotide sequence".

[0061] A "variant" of a nucleic acid molecule or polynucleotide encompasses nucleic acid molecules and polynucleotides having nucleotide substitutions, deletions and / or insertions relative to the unmodified or wild-type nucleic acid molecule or polynucleotide in question and having similar biological and functional activity as the nucleic acid molecule or polynucleotide from which they are derived. The variant may encode the same (or an identical) polypeptide or protein as the nucleic acid molecule or polynucleotide from which its derived. The variant may encode a variant polypeptide or protein as discussed below. Variants typically share sequence identity and / or homology with the unmodified nucleic acid molecule or polynucleotide from which they are derived. Methods for measuring identity and / orP5598PC00

[0062] homology are discussed below. Variant may include codon optimised nucleic acid molecules and polynucleotides.

[0063] The term "amino acid" in the context of the present disclosure is used in its broadest sense and is meant to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, along with a side chain (e.g., a R group) specific to each amino acid. The amino acids typically refer to naturally occurring L o-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M = Met;

[0064] N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, A. L., (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogues, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as P-amino acids. For example, analogues or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as do natural Phe or Pro, are included within the definition of amino acid. Such analogues and mimetics are referred to herein as "functional equivalents" of the respective amino acid. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N. Y. 1983, which is incorporated herein by reference.

[0065] The terms "polypeptide", and "peptide" are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can also undergo maturation or post-translational modification processes that may include, but are not limited to glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like. A peptide can be made using recombinant techniques, e.g., through the expression of a recombinant or synthetic polynucleotide. A recombinantly produced peptide is typically substantially free of culture medium, e.g., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0066] The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. The protein may be composed of a single polypeptide or may comprise multiple polypepties that are assembled to form a multimer. The multimer may be a homooligomer,P5598PC00

[0067] or a heterooligmer. The protein may be a naturally occurring or wild type protein, or a modified, or non-naturally, occurring protein. The protein may, for example, differ from a wild type protein by the addition, substitution, or deletion of one or more amino acids.

[0068] The sequence of the amino acids in the protein, polypeptide or variant is described herein as the "amino acid sequence".

[0069] A "variant" of a polypeptide or protein encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type polypeptide or protein in question and having similar biological and functional activity as the unmodified polypeptide or protein from which they are derived.

[0070] A variant is preferably a functional variant or a biologically active variant. This means the variant carries out the same, or a similar, function or activity as a polypeptide or polynucleotide having the reference sequence. The function or activity may be the same. The function or activity may be increased. The function or activity may be decreased.

[0071] Methods are known in the art for measuring the activity of variants.

[0072] Variants includes mutational variants, substitutional variants, insertional variants, derivatives, variants including intervening sequences, splice variants and allelic variants.

[0073] Variants typically share sequence identity and / or homology with the unmodified polypeptide or protein from which they are derived. Standard methods in the art may be used to determine identity and / or homology. The identity and / or homology may be calculated using an alignment program. Preferably a pair wise global alignment program may be used, which implements the algorithm of Needleman-Wunsch (J. Mol. Biol. 48: 443-453, 1970). This algorithm maximizes the number of matches and minimizes the number of gaps. Such programs are for example GAP, Needle (EMBOSS package), stretcher (EMBOSS package) or Align X (Vector NTI suite 5.5) and may use the standard parameters (for example gap opening penalty 15 and gap extension penalty 6.66). Alternatively, a local alignment program implementing the algorithm of Smith-Waterman (Advances in Applied Mathematics 2, 482-489 (1981)) may be used. Such programs are for example Water (EMBOSS package) or matcher (EMBOSS package). The UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).P5598PC00

[0074] In all instances herein, identity or homology is typically measured over the entire length of the reference sequence.

[0075] The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "variant" refers to a gene or gene product that displays modifications in sequence (e.g., substitutions, truncations, or insertions), post-translational modifications and / or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For instance, methionine (M) may be substituted with arginine (R) by replacing the codon for methionine (ATG) with a codon for arginine (CGT) at the relevant position in a polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art. The term "variant" is interchangeable with "modified" or "mutant".

[0076] The protein or polypeptide variant may include conservative substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art.

[0077] Amino acids may be replaced with amino acids in the same class defined as follows:

[0078] Class Amino acid examples

[0079] Nonpolar: A, V, L, I, P, M, F, W

[0080] Uncharged polar: G, S, T, C, Y, N, Q

[0081] Acidic: D, E

[0082] Basic: K, R, H.

[0083] As is well known to those skilled in the art, altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the function or activity of that polypeptide because the side-chain of the amino acid which is inserted into the amino acid sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out.P5598PC00

[0084] Non-conservative substitutions may also be possible provided that these do not interrupt the function or activity of the polypeptide as described above.

[0085] In all instances, the variant may be a naturally occurring variant or a "homologue". The homologue may be an orthologue or a paralogue. " Orthologues" and "paralogues" are two forms of homology which encompass evolutionary concepts used to describe ancestral relationships of genes. The term "paralogue" relates to gene duplications within the genome of a species leading to paralogous genes. The term "orthologue" relates to homologous genes in different organisms due to speciation. Orthologues and paralogues may readily be identified by a person skilled in the art using a (reciprocal) blast search.

[0086] Variants include functional fragments of the nucleic acid molecule, polynucleotide, polypeptide, protein, nucleic acid molecule or polynucleotide. The fragment may be any length as long as it retains the function and / or activity of the original sequence. Suitable fragment lengths are discussed in more detail below.

[0087] " About" as used herein when referring to a measurable value such as a percentage, an amount or a number and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any statement herein including the term "about" includes the same feature without the term. For instance, "about 70%" includes "70%".

[0088] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers, or steps. The terms "comprise" and "comprising" can be replaced with "consists essentially of" and "consisting essentially of" or with "consists of" or "consisting of".

[0089] Nucleic acid molecule

[0090] The present invention provides a nucleic acid molecule. The nucleic acid molecule comprises a first polynucleotide encoding an armouring protein fused to one or more Oxygen-Dependent Degradation Domains (ODDs). The nucleic acid molecule also comprises a second polynucleotide comprising a regulatory region comprising one or more hypoxia-responsive elements (HREs). The first polynucleotide is operably linked to the second polynucleotide. Nucleic acid molecules and polynucleotides are described above.

[0091] The nucleic acid molecule may also be called a nucleic acid transgene.

[0092] Armouring ProteinP5598PC00

[0093] The term "armouring protein" as defined herein refers to a protein or polypeptide, or a homologue, variant or functional fragment thereof, that is capable of enhancing one or more effects of a cell or an immunoresponsive cell in which it is expressed. Armouring proteins are known in the art.

[0094] In a preferred embodiment, the one or more effects comprise a homing effect. In a preferred embodiment, the one or more effects comprise one or more anti-tumour effects. The one or more anti-tumour effects may occur through one or more direct effects of the armouring protein, cell or immunoresponsive cell on tumour cells and / or through one or more indirect effects of the armouring protein, cell or immunoresponsive cell. Indirect effects include altering the phenotype or characteristics of other host cells present in the tumour microenvironment (TME). Indirect effects also include beneficial changes in the phenotype or characteristics of other host cells in other parts of the body outside of the TME. In a preferred embodiment, the indirect effect is an effect on macrophages, preferably tumour associated macrophages. In a preferred embodiment, expression of the armouring protein induces increased expression of key costimulatory and / or antigen-presenting molecules and / or interferon-stimulated genes on macrophages, preferably tumour associated macrophages. In a particularly preferred embodiment, the key costimulatory and / or antigen-presenting molecule and / or interferon-stimulated gene are Sca-1, MHCII, CD86 and / or MHCI H-2Kb. In one embodiment, the one or more anti-tumour effects comprise one or more of a reduction in the number of tumour cells, a reduction in proliferation of tumour cells, a reduction in the growth of a tumour, an increase in death of tumour cells, a reduction in size of the tumour, a reduction in angiogenesis, a reduction in dissemination of tumour cells, a reduction in tumour relapse or reoccurrence and / or a reduction in incidence of metastasis.

[0095] In another preferred embodiment, the one or more effects comprise the ability to counteract an immunosuppressive aspect of the tumour microenvironment (TME) and / or the ability to increases the resistance of the cell or immunoresponsive cell to an immunosuppressive aspect of the TME. The armouring protein is preferably capable of increasing the visibility of the tumour cells to the immune system. The armouring protein is preferably capable of increasing the resistance of the cell or immunoresponsive cell to exhaustion in the tumour microenvironment (TME) and / or reducing the amount and / or rate of apoptosis of the cell or immunoresponsive cell in the tumour microenvironment (TME). In one embodiment, the armouring protein is capable of altering the number and / or the composition of cells or immunoresponsive cells present in the tumour microenvironment (TME). For example, the armouring protein may one or more of (a) increase the overall number or proportion of cells or immunoresponsive cells in the TME, (b) increase the number or proportion of cells or immunoresponsive cells with pro-inflammatory characteristics in the TME and (c) decrease the number or proportion of cells orP5598PC00

[0096] immunoresponsive cells with anti-inflammatory characteristics in the TME. The armouring protein may (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). The armouring protein may also be capable of altering the number and / or the composition of other cells or immune cells, such as the subject's cells or immune cells, present in the tumour microenvironment (TME). For example, the armouring protein may one or more of (a) increase the overall number or proportion of other cells or immune cells in the TME, (b) increase the number or proportion of other cells or immune cells with pro-inflammatory characteristics in the TME and (c) decrease the number or proportion of other cells or immune cells with anti-inflammatory characteristics in the TME. The armouring protein may (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). In all instances, the other immune cells may the subject's own immune cells. The immune cells may be any of the immunoresponsive cell types described below. The other cells may be any other cells found in the TME, including cancer associated fibroblasts or mesenchymal populations or tumour cells.

[0097] In one embodiment, the armouring protein may alter the phenotype of the cell or the immunoresponsive cells to one that is considered to be more favourable within the specific context. The phenotype may be an effector phenotype, memory phenotype, or stem celllike phenotype.

[0098] In one embodiment, the armouring protein may increase the survival or persistence of the cell or immunoresponsive cell in the TME and / or in the body, especially the subject's body.

[0099] If the cell or immunoresponsive cell is used in regenerative medicine or ischemic diseases, one or more the effects discussed above for tumours would be reversed. For example, the one or effects comprise increased immune suppression and / or increased angiogenesis. The skilled person is capable of tailoring the armouring protein to the use of the cell or immunoresponsive cell.

[0100] In one embodiment, the armouring protein is a cytokine or chemokine. The armouring protein may be an armouring cytokine or armouring chemokine. Cytokines are signalling proteins that help control inflammation and the immune system. Chemokines are small, secreted proteins (including cytokines) that play a crucial role in cell migration, particularly of immune cells, by inducing directed movement (chemotaxis) in response to a chemical gradient. Cytokines or chemokines that may be used as armouring proteins are known in the art. The cytokine or chemokine may be any cytokine or chemokine. The cytokine or chemokine may be selected from IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-15, IL-18, IL-21, IL-12, IL-23, IL-17A, IL-18, IL-10, TNF-alpha, GM-CSF, HMGB1, IL-27, IL-33, IL-35 and IL-39. The IL-1 may be IL-1α or IL-1β. In a preferred embodiment the cytokine is IL-12, IL-23, IL-27 or IL-39. In a more preferred embodiment, the cytokine is IL-12 or IL-23.P5598PC00

[0101] In an alternative embodiment, the armouring protein is an armouring receptor. Armouring receptors are known in the art. The armouring receptor may be a cytokine or chemokine receptor that enhances the response of the cell or immunoresponsive cell to the cytokine or chemokine, for instance in the TME. The cytokine or chemokine may be any of those described above.

[0102] In an alternative embodiment, the armouring protein is a protein that is capable of neutralising immunosuppressive cytokines. The immunosuppressive cytokine may be one or more of TGFβ, PD-L1 and PD-1. The armouring protein may be capable of neutralising one or more of (a) TGFβ immunosuppressive signals or signalling pathways, (b) PD-L1 immunosuppressive signals or signalling pathways and (c) PD-1 immunosuppressive signals or signalling pathways. The armouring protein may be capable of neutralising (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). The armouring protein may be capable of neutralising TGFβ immunosuppressive signals or signalling pathways. The armouring protein may be capable of neutralising PD-L1 immunosuppressive signals or signalling pathways. The armouring protein may be capable of neutralising PD-1 immunosuppressive signals or signalling pathways.

[0103] In one embodiment, the armouring protein is a protein that is capable of driving mitochondrial function in a nutrient-poor tumour microenvironment (TME). The armouring protein may be c-MYC.

[0104] In a preferred embodiment, the armouring protein is an interferon. In one embodiment, the interferon is a type I interferon (IFN-I). The IFN-I may be selected from IFN-α, IFN-β, IFN-ε, IFN-κ and IFN-ω. The IFN-I is preferably interferon-alpha (IFN-α) or interferon-beta (IFN-β). The IFN-α may be IFN-α1, IFN-α2, IFN-α4, IFN-α5, IFN-α6, IFN-α7, IFN-α8, IFN-α10, IFN-α14, IFN-α16, IFN-α17 or IFN-α21. The IFN-I is most preferably interferon-beta (IFN-β).

[0105] In one embodiment, the interferon is a type II interferon (IFN-II). The IFN-II is preferably interferon-gamma (IFN-γ).

[0106] The armouring protein may be from or derived from any of the species described below with reference to the subject being treated in accordance with the invention. The armouring protein is preferably human. The amino acid sequences of the armouring proteins, including the armouring cytokines or chemokines, discussed above are known in the art and are shown in SEQ ID NOs: 39-84. The armouring protein may be naturally-occurring. The armouring protein may be artificially created.

[0107] The armouring protein may be a homologue or a variant of any of these proteins, cytokines or chemokines. Homologues and variants are defined above. The homologue or variant preferably comprises or consists of an amino acid sequence having at least about 70%, atP5598PC00

[0108] least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the wild-type protein, cytokine or chemokine.

[0109] The armouring protein may be a functional fragment of any of these proteins, cytokines or chemokines. Suitable functional fragments may be tested using routine methods for their capability to have any of the effects discussed herein. Functional fragments preferably comprise at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, at least 500 or more contiguous amino acids of the protein, cytokine or chemokine from which they are derived. The functional fragment is preferably capable of having one or more of the effects discussed above for armouring proteins. The functional fragment is preferably capable of one or more anti-tumour effects. The functional fragment is preferably capable of counteracting an immunosuppressive aspect of the tumour microenvironment (TME) and / or increasing the resistance of a cell or immunoresponsive cell to an immunosuppressive aspect of the TME, and / or increasing the visibility of the tumour cells to the immune system.

[0110] In a preferred embodiment, the interferon-alpha (IFN-α) comprises or consists of an amino acid sequence having a sequence selected from SEQ ID NOs: 39 to 50, a homologue or variant thereof, or a functional fragment thereof. In a preferred embodiment, the interferon-alpha (IFN-α) comprises or consists of an amino acid sequence having about 100% identity and / or homology to a sequence selected from SEQ ID NOs: 39 to 50, a homologue or variant thereof, or a functional fragment thereof. The homologue or variant thereof preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least aboutP5598PC00

[0111] 99% identity and / or homology to a sequence selected from SEQ ID NOs: 39 to 50. The functional fragment may have any of the lengths discussed above. Preferably, the homologue, variant or functional fragment is capable of binding to a Type I Interferon Receptor (IFNAR). The IFNAR is preferably present on the surface of a target cell. The homologue, variant or functional fragment is preferably capable of triggering JAK-STAT signalling within the target cell. In one embodiment, the homologue, variant or functional fragment is capable of having one or more of the effects discussed above for armouring proteins. The homologue, variant or functional fragment is preferably capable of one or more anti-tumour effects. The homologue, variant or functional fragment is preferably capable of counteracting an immunosuppressive aspect of the tumour microenvironment (TME) and / or increasing the resistance of the cell or immunoresponsive cell to an immunosuppressive aspect of the TME and / or increasing the visibility of the tumour cells to the immune system.

[0112] In a preferred embodiment, the interferon-beta (IFN-β) comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 51 or 52, a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the interferon-beta (IFN-β) comprises or consists of an amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 51 or 52, a homologue or variant thereof or a functional fragment thereof. The homologue or variant thereof preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 51 or 52. In a preferred embodiment, the interferon-beta (IFN-β) comprises or consists of an amino acid sequence having the sequence of SEQ ID NO: 52, a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the interferon-beta (IFN-β) comprises or consists of an amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 52, a homologue or variant thereof or a functional fragment thereof. The homologue or variant thereof preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 52. The functional fragment may have any of the lengths described above. Preferably, the homologue, variant or functional fragment is capable of binding to a Type I Interferon Receptor (IFNAR). The IFNAR is preferably present on the surface of a target cell. The homologue, variant or functional fragment is preferably capable of triggering JAK-STAT signalling within the target cell. In one embodiment, the homologue, variant or functional fragment is capable of having one or more of the effectsP5598PC00

[0113] discussed above for armouring proteins. The homologue, variant or functional fragment is preferably capable of one or more anti-tumour effects. The homologue, variant or functional fragment is preferably capable of counteracting an immunosuppressive aspect of the tumour microenvironment (TME) and / or increasing the resistance of the cell or immunoresponsive cell to an immunosuppressive aspect of the TME and / or increasing the visibility of the tumour cells to the immune system.

[0114] In a preferred embodiment, the armouring protein comprises or consists of an amino acid sequence having a sequence selected from SEQ ID NOs: 53 to 84, a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the armouring protein comprises or consists of an amino acid sequence having about 100% identity and / or homology to a sequence selected from SEQ ID NOs: 53 to 84, a homologue or variant thereof or a functional fragment thereof. The homologue or variant thereof preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to a sequence selected from SEQ ID NOs: 53 to 84. The functional fragment may have any of the lengths described above. Preferably, the homologue, variant or functional fragment is capable of binding to the relevant receptor (e.g., the IL-2 receptor). The receptor is preferably present on the surface of a target cell. The homologue, variant or functional fragment is preferably capable of triggering intracellular signalling within the target cell. In one embodiment, the homologue, variant or functional fragment is capable of having one or more of the effects discussed above for armouring proteins. The homologue, variant or functional fragment is preferably capable of one or more anti-tumour effects. The homologue, variant or functional fragment is preferably capable of counteracting an immunosuppressive aspect of the tumour microenvironment (TME) and / or increasing the resistance of the cell or immunoresponsive cell to an immunosuppressive aspect of the TME and / or increasing the visibility of the tumour cells to the immune system.

[0115] In a preferred embodiments, the armouring protein is a dimer comprising or consisting of:

[0116] • an amino acid sequence having the sequence of SEQ ID NO: 67, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 69, a homologue or variant thereof or a functional fragment thereof;

[0117] • an amino acid sequence having the sequence of SEQ ID NO: 68, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 69, a homologue or variant thereof or a functional fragment thereof;P5598PC00

[0118] • an amino acid sequence having the sequence of SEQ ID NO: 70, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 72, a homologue or variant thereof or a functional fragment thereof;

[0119] • an amino acid sequence having the sequence of SEQ ID NO: 71, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 72, a homologue or variant thereof or a functional fragment thereof;

[0120] • an amino acid sequence having the sequence of SEQ ID NO: 73, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 74, a homologue or variant thereof or a functional fragment thereof;

[0121] • an amino acid sequence having the sequence of SEQ ID NO: 75, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 76, a homologue or variant thereof or a functional fragment thereof;

[0122] • an amino acid sequence having the sequence of SEQ ID NO: 84, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 76, a homologue or variant thereof or a functional fragment thereof; or

[0123] • an amino acid sequence having the sequence of SEQ ID NO: 70, a homologue or variant thereof or a functional fragment thereof, and amino acid sequence having the sequence of SEQ ID NO: 76, a homologue or variant thereof or a functional fragment thereof.

[0124] The homologue, variant or functional fragment may be any of those described above.

[0125] Oxygen-Dependent Degradation Domains (ODDs)

[0126] The term "oxygen-dependent degradation domain" or " ODD" as used herein refers to a protein or polypeptide whose stability in a cell is regulated by oxygen levels. In one embodiment, the ODD has a reduced level and / or rate of degradation under conditions of hypoxia. This is typically when compared with its level and / or rate of degradation under conditions of normoxia. In a preferred embodiment, the ODD is capable of increasing the level and / or rate of degradation of a protein or polypeptide to which it is fused under conditions of normoxia. This is typically compared with the rate of degradation of the protein or polypeptide when it is not fused to the ODD. In a further preferred embodiment,P5598PC00

[0127] the ODD is capable of reducing the level and / or rate of degradation of a protein or polypeptide to which it is fused under conditions of hypoxia. This is typically compared with its levels of degradation under conditions of normoxia.

[0128] In the context of the invention, the protein or polypeptide fused to the one or more ODDs is an armouring protein, preferably an IFN-I, and most preferably IFN-p. The armouring protein, IFN-I or IFN-p may be any of those described above.

[0129] The term "hypoxia" as used herein refers to an oxygen level that is lower than that of healthy organs and tissues in the body. This includes the oxygen level found in solid tumours. In a preferred embodiment, "hypoxia" refers to (i) an oxygen concentration of below about 5%, such as less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.5%, less than about 0.25% or less than about 0.1%, or the mmHg and / or (ii) reduced O₂ availability relative to O₂ availability or partial pressure of a corresponding non-cancerous organ, tissue or cells.

[0130] As used herein, the term "normoxia" refers to an oxygen concentration of above about 5% or O₂ availability associated with healthy tissues and / or organs.

[0131] As used herein, a reduced level and / or rate of degradation refers to a reduction in degradation in a cell, preferably an immunoresponsive cell. In a preferred embodiment, this reduction in degradation leads to an increase in the amount or levels of the armouring protein present in a cell, preferably an immunoresponsive cell.

[0132] As used herein, an increased level and / or rate of degradation refers to an increase in degradation in a cell, preferably an immunoresponsive cell. In a preferred embodiment, this increase in degradation leads to a decrease in the amount or levels of the armouring protein in a cell, preferably an immunoresponsive cell.

[0133] As used herein, the term "decrease", "lower" or "reduced" preferably refers to a reduction of more than about 1%, more than about 5%, more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than about 70%, more than about 80%, more than about 90%, more than about 100%, more than about 150%, more than about 200%, more than about 250%, more than about 300%, more than about 400%, more than about 500%, more than about 1,000% (i.e. 10-fold), more than about 15,000%, more than about 20,000% (i.e. 20-fold) or more than about 50,000% (i.e. 50-fold) and includes 0 or a level below the rate of detection.

[0134] As used herein, the term "increase" preferably refers to an increase of more than about 1%, more than about 5%, more than about 10%, more than about 20%, more than about 30%, more than about 40%, more than about 50%, more than about 60%, more than aboutP5598PC00

[0135] 70%, more than about 80%, more than about 90%, more than about 100%, more than about 150%, more than about 200%, more than about 250%, more than about 300%, more than about 400%, more than about 500%, more than about 1,000% (i.e. 10-fold), more than about 15,000%, more than about 20,000% (i.e. 20-fold) or more than about 50,000% (i.e. 50-fold).

[0136] The one or more ODDs or each ODD may be from or derived from any ODD-containing protein. The one or more ODDs or each ODD may be from or derived from ATF-4, HIF2-alpha, HIF3-alpha or HIFl-alpha. The one or more ODDs or each ODD may be from or derived from HIFl-alpha.

[0137] The one or more ODDs or each ODD may be from or derived from any of the species described below with reference to the subject being treated in accordance with the invention. The one or more ODDs or each ODD is / are preferably human. The amino acid sequences of ODDs are known in the art. The one or more ODDs or each ODD may be naturally occurring. The one or more ODDs or each ODD may be artificially created.

[0138] Preferably, the one or more ODDs or each ODD independently comprise(s) or consist(s) of an amino acid sequence having the sequence of SEQ ID NO: 1 (X1X2LEMLAPYIX3MDDDX4X5X6), where " X1’6" can be any amino acid residue, optionally wherein X1is " L" or any conservative substitution; X2is " D" or any conservative substitution, X3is " P" or any conservative substitution, X4" F” or any conservative substitution, X5is " Q" or any conservative substitution and X6is " L" or any conservative substitution, or a homologue or variant thereof or a functional fragment thereof. Preferably, the one or more ODDs or each ODD independently comprise(s) or consist(s) of an amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 1 (X1X2LEMLAPYIX3MDDDX4X5X6), where " X1’6" can be any amino acid residue, optionally wherein X1is " L" or any conservative substitution; X2is " D" or any conservative substitution, X3is " P" or any conservative substitution, X4" F” or any conservative substitution, X5is " Q" or any conservative substitution and X6is " L" or any conservative substitution, or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 1 (X1X2LEMLAPYIX3MDDDX4X5X6), where " X1’6" can be any amino acid residue, optionally wherein X1is " L" or any conservative substitution; X2is " D" or any conservative substitution, X3is " P" or any conservative substitution, X4" F” or any conservativeP5598PC00

[0139] substitution, X5is " Q" or any conservative substitution and X6is " L" or any conservative substitution.

[0140] In a preferred embodiment, the one or more ODDs or each ODD independently comprise(s) or consist(s) of an amino acid sequence having a sequence selected from SEQ ID NOs: 2 to 4, or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the one or more ODDs or each ODD independently comprise(s) or consist(s) of an amino acid sequence having about 100% identity and / or homology to a sequence selected from SEQ ID NOs: 2 to 4, or a homologue or variant thereof or a functional fragment thereof. The homologue or variant thereof preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to a sequence selected from SEQ ID NOs: 2 to 4. Preferably, the homologue or variant comprises an amino acid sequence having the sequence of SEQ ID NO: 1 as defined above.

[0141] In relation to any one of SEQ ID NOs: 1-4, the functional fragment preferably comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, at least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, at least 500 or more contiguous nucleotides or amino acids of the sequence from which they are derived. Suitable functional fragments may be tested using routine methods for their capability to have any of the effects discussed herein. The functional fragment may have any of the effects discussed above. The functional fragment preferably has a reduced level and / or rate of degradation under conditions of hypoxia and / or is capable of reducing the level and / or rate of degradation of a protein or polypeptide to which it is fused under conditions of hypoxia.P5598PC00

[0142] First polynucleotide

[0143] The first polynucleotide encodes an armouring protein fused to one or more Oxygen-Dependent Degradation Domains (ODDs). The armouring protein may be any of those described above. The one or more ODDs or each ODD independently may be any of those described above.

[0144] In one embodiment of the first polynucleotide, where the armouring protein comprises a plurality of subunits, the first polynucleotide comprises a nucleotide sequence that is transcribed into a single RNA molecule encoding for the plurality of subunits and is translated into a single contiguous polypeptide comprising the plurality of subunits. In such embodiments, the first polynucleotide does not comprise a nucleotide sequence that results in the individual subunits of the armouring protein being transcribed as discrete RNA molecules or separated into discrete RNA molecules. In such embodiments, the first polynucleotide also does not comprise a nucleotide sequence that results in the individual subunits of the armouring protein being translated into discrete polypeptides or separated into discrete polypeptides. Thus, in such embodiments, the first polynucleotide does not comprise separate promoter sequences for the different subunits, a ribosomal skipping element between different subunits, an internal translation initiation element (IRES) between different subunits or a protease cleavage site between different subunits. In a preferred embodiment, the one or more ODDs is fused to one of the subunits of the armouring protein.

[0145] In an alternative embodiment of the first polynucleotide, where the armouring protein comprises a plurality of subunits, the first polynucleotide comprises a nucleotide sequence that is expressed into two or more discrete polypeptides corresponding to the two or more subunits of the armouring protein. In a preferred embodiment, the plurality of subunits encoded for by the first polynucleotide sequence are capable of associating post-translation to form a functional multimeric armouring protein. In one embodiment, the first polynucleotide comprises a nucleotide sequence that is transcribed into two or more discrete RNA molecules or is separated into two or more discrete RNA molecules, wherein each RNA molecule encodes a different subunit of the armouring protein. In such embodiments, the first polynucleotide may comprise a nucleotide sequence that results in the individual subunits of the armouring protein being transcribed as discrete RNA molecules or separated into discrete RNA molecules. In one embodiment, the first polynucleotide comprises a nucleotide sequence that is transcribed into a single RNA molecule encoding for the plurality of subunits and is translated into two or more discrete polypeptides or is separated into two or more discrete polypeptides, wherein each polypeptide comprises a different subunit of the armouring protein. In such embodiments, the first polynucleotide does not comprise a nucleotide sequence that results in the individual subunits of the armouring protein beingP5598PC00

[0146] transcribed as discrete RNA molecules or separated into discrete RNA molecules but may comprise a nucleotide sequence that results in the individual subunits of the armouring protein being translated into discrete polypeptides or separated into discrete polypeptides. Thus, the first polynucleotide may comprise a ribosomal skipping element preferably a 2A peptide between different subunits, an internal translation initiation element (IRES) between different subunits or a protease cleavage site between different subunits. In a preferred embodiment, the one or more ODDs is fused to one of the subunits of the armouring protein.

[0147] In one embodiment, the first polynucleotide encodes an armouring protein fused to more than one ODD. In one embodiment, the first polynucleotide encodes an armouring protein fused to two or more ODDs. Preferably, the armouring protein is fused to two, three, four or five or more ODDs. More preferably, the armouring protein is fused to two ODDs.

[0148] The two or more ODDs may be the same ODDs or may be different ODDs. The two or more ODDs may all have different amino acid sequences. Alternatively, the two or more ODDs may all have the same amino acid sequence. In a further alternative embodiment, some or two or more of the ODDs (e.g. at least two of the ODDs) may have the same amino acid sequence and some or two or more of the ODDs (e.g. at least two of the ODDs) may have different amino acid sequences.

[0149] In a preferred embodiment, the two or more ODDs are identical ODDs or have the same amino acid sequence and each comprise or consist of any of the sequences of SEQ ID NO: 1-4, or a homologue or variant thereof or a functional fragment thereof. Such ODDs are described in detail above.

[0150] It will be understood by a skilled person that numerous different nucleotide sequences can encode the same amino acid sequence as a result of the degeneracy of the genetic code. In addition, it is to be understood that the skilled person may, using routine techniques, make nucleotide substitutions that do not affect the amino acid sequence encoded by the nucleotide sequence to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

[0151] In a preferred embodiment, the two or more ODDs are identical ODDs or have the same amino acid sequence and each ODD is encoded by a different nucleotide sequence in the first polynucleotide. In a preferred embodiment, the two or more ODDs are identical ODDs or have the same amino acid sequence and each ODD comprises or consists of any of the sequences of SEQ ID NO: 1-4, or a homologue or variant thereof or a functional fragment thereof, and each ODD is encoded by a different nucleotide sequence in the first polynucleotide. In one embodiment, the different nucleotide sequences encoding the two or more identical ODDs have less than about 99%, less than about 98%, less than about 97%,P5598PC00

[0152] less than about 96%, less than about 95%, less than about 90%, less than about 85% or less than about 80% sequence identity and / or homology with each other.

[0153] The two or more ODDs may be sequential or may be spatially separate. The two or more ODDs may be directly fused or indirectly fused. These terms are defined below. The two or more ODDs may be fused via any of the linkers discussed below. The linkers preferably do not affect the function of the two or more ODDs.

[0154] In a preferred embodiment, the first polynucleotide encodes an armouring protein fused to one ODD. The ODD preferably comprises or consists of any of the sequences of SEQ ID NO: 1-4, or a homologue or variant thereof or a functional fragment thereof. Such ODDs are described in detail above.

[0155] As used herein, the term "fused" includes where proteins or polypeptides are directly or indirectly fused together. The armouring protein may be directly fused to one or more ODDs. The term "directly fused" as defined herein refers to where there are no intervening amino acids between the proteins or polypeptides that are fused together. The armouring protein may be indirectly fused to one or more ODDs. The term "indirectly fused" as defined herein refers to where there intervening amino acids, such as a linker, are present between the proteins or polypeptides that are fused together. When the proteins or polypeptides are indirectly fused together, the intervening amino acids do not impact the function of fused proteins or polypeptides. Suitable linkers are well known in the art and may be rigid or flexible. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine amino acids. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5and (SG)8wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P)12wherein P is proline.

[0156] The nucleotide sequence(s) encoding the one or more ODDs may be positioned anywhere in the first polynucleotide. For instance, the nucleotide sequence encoding the two or more ODDs may be fused to the 3' end of the nucleotide sequence encoding the armouring protein. In an alternative embodiment, the nucleotide sequence encoding the two or more ODDs may be fused to the 5' end of the nucleotide sequence encoding the armouring protein. In a further alternative embodiment, the nucleotide sequence encoding one or more of the two or more ODDs may be fused to the 3' end of the nucleotide sequence encoding the armouring protein and the nucleotide sequence encoding the one or more of the two or more ODDs may be fused to the 5' end of the nucleotide sequence encoding the armouring protein.

[0157] The nucleotide sequence encoding a single (or individual) ODD may be fused to the 5' end of the nucleotide sequence encoding the armouring protein. In an alternative embodiment,P5598PC00

[0158] the nucleotide sequence encoding a single (or individual) ODD may be fused to the 3' end of the nucleotide sequence encoding the armouring protein.

[0159] In one embodiment, the first polynucleotide encodes an armouring protein having one or more ODDs fused at its C-terminus, one or more ODDs fused at its N-terminus or one or more ODDs fused at both its C-terminus and its N-terminus. The armouring protein and one or more ODDs may be directly fused or indirectly fused. Alternatively, the amino acid sequence encoded by the first polynucleotide may include additional amino acids, such as one or more linkers. These may be present (a) within the amino acid sequence, (b) at the C-terminus of the polypeptide and / or (c) at the N-terminus of the amino acid sequence. These may be present at (a), (b), (c), (a) and (b), (b) and (c), (a) and (c) or (a), (b) and (c).

[0160] In a preferred embodiment, the first polynucleotide encodes an amino acid sequence having the sequence of SEQ ID NO: 35, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide encodes an amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 35, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 35.

[0161] In a preferred embodiment, the first polynucleotide comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 36, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 36, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 36.

[0162] Hypoxia-responsive elements (HREs)

[0163] The second polynucleotide comprises one or more HREs. The term "hypoxia-responsive element" (HRE) as used herein refers to a nucleotide sequence that is capable of regulating the expression of a nucleic acid molecule in which it is contained under conditions of hypoxia and / or regulating the expression of a polynucleotide to which the one or more HREsP5598PC00

[0164] are operably linked under conditions of hypoxia. In a preferred embodiment, the one or more HREs are capable of regulating the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia when comprised in a regulatory region.

[0165] Regulatory regions are described below.

[0166] In a preferred embodiment, the one or more HREs, and / or a regulatory region comprising the one or more HREs, is / are capable of driving the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia when with the expression of the nucleic acid molecule and / or polynucleotide under conditions of normoxia. Hypoxia and normoxia are defined above.

[0167] In a further preferred embodiment, the one or more HREs, and / or a regulatory region comprising the one or more HREs, is / are capable of driving the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia when compared with the expression of the nucleic acid molecule and / or polynucleotide in hypoxia if the one or more HREs, or the regulatory region comprising the one or more HREs, was / were not present.

[0168] As used herein, a drive in expression of a nucleic acid molecule and / or polynucleotide refers to an increase in the initiation of transcription of the nucleic acid molecule and / or polynucleotide in a cell, preferably an immunoresponsive cell. In a preferred embodiment, this increase in the initiation of transcription results in an increase in expression of the nucleic acid molecule and / or polynucleotide. This increase in expression may be an increase in the amount or levels in a cell, preferably an immunoresponsive cell, of the RNA(s) encoded for by the nucleic acid molecule and / or polynucleotide. Preferably, the increase in the initiation of transcription results in an increase in the amount or levels in a cell, preferably an immunoresponsive cell, of the protein or polypeptide encoded for by the nucleic acid molecule and / or polynucleotide. In the context of the invention, the protein or polypeptide is an armouring protein fused to an one or more Oxygen-Dependent Degradation Domains (ODDs). The protein or polypeptide may also be a cell surface receptor as discussed in more detail below. It may be any of the fusion constructs discussed above. The armouring protein is preferably an IFN-I or most preferably IFN-p.

[0169] In a further preferred embodiment, the one or more HREs, and / or a regulatory region comprising the one or more HREs, is / are capable of reducing the initiation of transcription of the nucleic acid molecule and / or polynucleotide under conditions of normoxia when compared with the initiation of transcription of the nucleic acid molecule and / or polynucleotide in normoxia if the one or more HREs, or the regulatory region comprising the one or more HREs, was / were not present. For instance, the one or more HREs and / or regulatory region is / are not present when a constitutive promoter is used instead.P5598PC00

[0170] In a preferred embodiment, this reduction in the initiation of transcription results in a decrease in expression of the nucleic acid molecule and / or polynucleotide in normoxia. This decrease in expression may be a decrease in the amount or levels in a cell, preferably an immunoresponsive cell, of the RNA(s) encoded for by the nucleic acid molecule and / or polynucleotide. Preferably, the reduction in the initiation of transcription is a decrease in the amount or levels in a cell, preferably an immunoresponsive cell, of a protein or polypeptide encoded for by the nucleic acid molecule and / or polynucleotide.

[0171] HREs are known in the art and are used to express a variety of nucleic acids molecules. Any HRE may be used in the invention.

[0172] In one embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of at least one HIF-binding site (HBS). In a preferred embodiment, the one or more HREs or each HRE independently further comprise(s) at least one HIF ancillary site (HAS) and / or at least one HNF-4 site.

[0173] In one embodiment, the at least one HBS comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 5. The at least one HBS more preferably comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 6.

[0174] In one embodiment, the HAS comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 7. The HAS more preferably comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 8.

[0175] In one embodiment, the HNF-4 site comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 9.

[0176] The HBS and HAS (if present) may be separated by a linker which may be rigid or flexible. Suitably, the linker is at least 6 nucleotides in length, optionally more than 8 nucleotides in length. In a preferred embodiment, the linker is 6 or 8 nucleotides in length. The linker may correspond to linkers naturally found in the promoter region of oxygen-responsive genes. An example of a suitable linker is the sequence of SEQ ID NO: 28 or 29. Other suitable linkers are well known in the art.

[0177] The parts making up each individual HRE element, i.e. the HBS, and optionally the HAS and further optionally HNF-4, may be in any order. The parts may be positioned sequentially and / or spatially separate, such as through the use of suitable linkers, intervening sequences etc. Sequential positioning is also referred to herein as "in tandem" or "stacked".

[0178] The one or more HREs or the parts making up the one or more HRE (i.e. the HBS, and optionally HAS and further optionally HNF-4) may suitably be from or derived from any oxygen-responsive gene. The oxygen-responsive gene may be from or derived from any ofP5598PC00

[0179] the species discussed below. The oxygen-responsive gene is preferably mammalian. The oxygen-responsive gene is more preferably human. The oxygen-responsive gene may be artificially synthesised. Examples of such oxygen-responsive genes include, but are not limited to, erythropoietin (EPO) such as hEPO or mEPO, vascular endothelial growth factor (VEGF) such as hVEGF, mVEGF or rVAGF, phosphoglycerate kinase (PGK) such as hPGK or mPGK, glucose transporters (e.g. Glut-1), Glucose trpt, lactate dehydrogenase (LDH) such as hLDH or mLDH, aldolase (ALD) such as hAldolase, enolase (e.g. ENO3 and hEnolase), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), nitric oxide synthetase (NOS) such as hNOS, heme oxygenase such as mHeme oxygenase and hHeme oxygenase, muscle glycolytic enzyme pyruvate kinase (PKM), endothelin-1 (ET-1), including orthologues or paralogues of any of the listed genes. In addition, other oxygen-responsive gene include, but are not limited to, aldolase A, aldolase C, HIF-113, HIF-213, CTLA-4, PHD2, PHD3, enolase 1, enolase 2, glyceraldehyde-3-phosphate dehydrogenase, glucose phosphate isomerase 1, HIF-3o, IL-10, interferon-7, lymphocyte activation gene 3, mitochondrially encoded 12S rRNA, 6-phosphofructo-2-kinase / fructose-2,6-biphosphatase 3, phosphofructokinase; phosphoglycerate kinase 1, phosphoglucomutase 2, pyruvate kinase, perforin 1, glutl, glut3, triosephosphate isomerase 1, vascular endothelial growth factor A, Von Hippel-Lindautumour suppressor, including orthologues or paralogues of any of the listed. These genes were shown by Gropper et al., 2017 (Cell Reports 20, 2547-2555) to be upregulated in T-cells upon exposure to hypoxia. The one or more HREs may be from or derived from any of the listed genes. Alternative, the one or more HREs may be artificially synthesised.

[0180] The one or more HREs or each HRE independently may be a homologue or a variant of any of the HREs from or derived from these genes. Homologues and variants are defined above. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the wild-type HRE.

[0181] The one or more HREs or each HRE independently may be a functional fragment of any of HREs from or derived from these genes. Suitable functional fragments may be tested using routine methods for their capability to have any of the effects discussed herein. Functional fragments preferably comprise at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, atP5598PC00

[0182] least 130, at least 135, at least 140, at least 145, at least 150, at least 155, at least 160, at least 165, at least 170, at least 175, at least 180, at least 185, at least 190, at least 195, at least 200, at least 205, at least 210, at least 215, at least 220, at least 225, at least 230, at least 235, at least 240, at least 245, at least 250, at least 255, at least 260, at least 265, at least 270, at least 275, at least 280, at least 285, at least 290, at least 295, at least 300, at least 305, at least 310, at least 315, at least 320, at least 325, at least 330, at least 335, at least 340, at least 345, at least 350, at least 355, at least 360, at least 365, at least 370, at least 375, at least 380, at least 385, at least 390, at least 395, at least 400, at least 405, at least 410, at least 415, at least 420, at least 425, at least 430, at least 435, at least 440, at least 445, at least 450, at least 455, at least 460, at least 465, at least 470, at least 475, at least 480, at least 485, at least 490, at least 495, at least 500 or more contiguous nucleotides of the sequence from which they are derived. The functional fragment is preferably capable of having one or more of the effects discussed above for HREs. In a preferred embodiment, the functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotide under conditions of normoxia and / or if the functional fragment, and / or a regulatory region comprising functional fragment, was / were not present.

[0183] In a preferred embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of a nucleotide sequence having a sequence selected from SEQ ID NO: 10 to 22, or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of a nucleotide sequence having about 100% identity and / or homology to a sequence selected from SEQ ID NO: 10 to 22, or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to a sequence selected from SEQ ID NO: 10 to 22. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotide under conditions of normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present.P5598PC00

[0184] In a preferred embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of a nucleotide sequence having the sequence of SEQ ID NO: 10, or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 10, or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 10. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotide under conditions of normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present.

[0185] In a preferred embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of a nucleotide sequence having the sequence of SEQ ID NO: 23, or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the one or more HREs or each HRE independently comprise(s) or consist(s) of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 23, or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 23. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotide under conditions ofP5598PC00

[0186] normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present. Regulatory Region

[0187] The second polynucleotide comprises a regulatory region comprising one or more HREs. The term "regulatory region" as defined herein refers to a nucleotide sequence capable of regulating the expression of a nucleic acid molecule within which it is contained and / or a polynucleotide to which it is operably linked. In a preferred embodiment, the regulatory region drives the expression of the nucleic acid molecule within which it is contained and / or the polynucleotide to which it is operably linked. In a preferred embodiment, the regulatory region drives the expression of the nucleic acid molecule of the invention and / or the first polynucleotide to which it is operably linked.

[0188] The regulatory region regulates expression of the nucleic acid molecule and / or polynucleotide via transcriptional control elements, which are generally embedded in the nucleotide sequence 5'-flanking or upstream of the expressed nucleic acid molecule and / or polynucleotide.

[0189] The one or more HREs may themselves be capable of driving expression of the nucleic acid molecule and / or polynucleotide. In other words, the one or more HREs may have sufficient regulatory function and / or promoter activity to initiate transcription. Thus, in one embodiment, the regulatory region does not comprise any further additional transcriptional control elements beyond the one or more HREs. In one embodiment, the regulatory region consists of the one or more HREs.

[0190] In an alternative embodiment, the regulatory region further comprises additional transcriptional control elements. In a preferred embodiment, the regulatory region further comprises a promoter. Promoters are well known to the skilled person.

[0191] The promoter may be any prokaryotic or eukaryotic promoter. Suitable promoters include, but are not limited to, SFG, hACTB, hEF-lalpha, CAG, CMV, HSV-TK, hACTB, hACTB-R, LTRs, EFla, SV40, PGK1, Ubc, human beta actin, TRE, UAS, Ac5, Polyhedrin, CaMKIIa, GALI, 10, TEF1, GDS, ADH1, Ca MV35S, Ubi, Hl, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, pL, and NFAT-interacting promoter (such as an IL-2 promoter), including functional fragments and minimal versions thereof. Other promoters which may be used include the promoters listed in Table 1 below from Powel et al., (Discov Med. 2015, 19 (102), 49-57), also including functional fragments and minimal versions of the promoters listed in Table 1.P5598PC00

[0192] Table 1

[0193] Comparison of Selected Ubiquitous and Cell-specific Promoters.

[0194] Promoter Specificity Relative Strength Size (bps) Referencefs)

[0195] CMV Ubiquitous +++ 750-800 Xu et al., 2001; Gray et al, 2011

[0196] CBA (including Ubiquitous +++ 248-1,600 Klein et al., 2002; Ohlfest et al, 2005; Gray et al., derivatives: CAG, 2011

[0197] CBh, etc.)

[0198] EF-1α Ubiquitous ++ 2,500 Gill et al., 2001; Xu et al., 2001; Ikeda et al.,

[0199] 2002; Gilham et al, 2010

[0200] PGK Ubiquitous ++ 426 Gilham et al., 2010

[0201] UBC Ubiquitous + 403 Gill et al.. 2001; Qin et al. 2010 GUSB (hGBp) Ubiquitous + 378 Husain etal., 2009

[0202] UCOE (Promoter of Ubiquitous ++ 600-2,500 Antoniou er al, 2013

[0203] HNRPA2B1-CBX3)

[0204] hAAT Liver ++ 347-1, 500 Van Linthout et al., 2002; Cunningham et aL,

[0205] 2008

[0206] TBG Liver ++ 400 Yan et al, 2012

[0207] Desmin Skeletal muscle +++ 1,700 Talbot etal., 2010

[0208] MCK Skeletal muscle ++ 595-1,089 Wang et al, 2008: Talbot etal, 2010; Katwal et al, 2013

[0209] C5-12 Skeletal, cardiac, and ++ 312 Wang et al., 2008

[0210] diaphragm

[0211] NSE Neuron +++ 300–2,200 Xu et al., 2001

[0212] Synapsin Neuron + 470 Kiigler et al, 2003; Hioki et al, 2007; Kuroda er ul. 2008

[0213] PDGF Neuron +++ 1,400 Patterna et al., 2000; Hioki et al., 2007 MecP2 Neuron + 229 Rastegar et al., 2009; Gray et al., 2011 CaMKII Neuron ++ 364 2,300 Hioki el al, 2007: Kuroda et al, 2008 mGiuR2 Neuron + 1,400 Brene et al, 2000; Kuroda et al., 2008 NFL Neuron + 650 Xu el ul. 2001

[0214] NFH Neuron + 920 Xu et al., 2001

[0215] nβ2 Neuron + 650 Xu et al., 2001

[0216] PPE Neuron + 2,700 Xu et al., 2001

[0217] Enk Neuron + 412 Xu et ul. 2001

[0218] EAAT2 Neuron and astrocyte ++ 966 Su et al., 2003; Kuroda et al., 2008 GFAP Astrocyte ++ 681–2,200 Brenner et al., 1994; Xu et al., 2001; Lee et al.,

[0219] 2008; Dirren et al, 2014

[0220] MBP Oligodendrocytes ++ 1,900 Chen et al., 1998

[0221]

[0222] Note: Cell type specificity, relative strength (+ being the weakest and +++ being the strongest), size, and relevant references for commonly used promoters.

[0223] The promoter is preferably the promoter region in the SFG retroviral vector. This vector contains Moloney murine leukaemia virus long terminal repeats (LTRs) which contain the promoter region.

[0224] An example of an arrangement of a regulatory region is shown in SEQ ID NO: 27.P5598PC00

[0225] The regulatory region may comprise, in addition to the one or more of HREs and optionally the promotor, a part or parts, preferably functional part(s), from a second promoter.

[0226] Examples of such parts include minimal promoters, additional regulatory elements to further enhance activity and / or to alter spatial and / or temporal expression pattern.

[0227] As described above, the regulatory region comprises or consists of one or more HREs. The regulatory region may comprise (i) one HRE or (ii) a plurality of HREs (also known as two or more HREs).

[0228] The "plurality" of HREs as defined herein is taken to mean more than one HRE. The plurality of HREs or two or more HREs may comprise or consist of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty HREs. In one embodiment, the plurality of HREs or two or more HREs may comprise or consist of two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more HREs. The HREs may be any of those described above.

[0229] The plurality of HREs or two or more HREs may be multiple copies of the same HRE. The plurality of HREs or two or more HREs may be different HREs. There may also be a mixture of both options. The plurality of HREs or two or more HREs may comprise multiple copies of the same HRE and one or more different HREs. In one embodiment, the plurality of HREs or two or more HREs all have different nucleotide sequences. In an alternative embodiment, some or two or more of the plurality of HREs or two or more HREs (e.g. at least two of the HREs) have the same nucleotide sequence and some or two or more of the plurality of HREs or two or more HREs (e.g. at least two of the HREs) have different nucleotide sequences. In a preferred alternative embodiment, the regulatory region comprises or consists of a plurality of HREs or two or more HREs and all the HREs have the same nucleotide sequence.

[0230] Single (or individual) HREs making up the plurality of HREs may be spatially separate. For instance, the single HREs may be separated by one or more elements such as one or more enhancers, one or more linkers, and / or one or more intervening sequences. The singe HREs may be sequential. The single HREs may be a combination of both spatially separate and sequential. In one embodiment where the regulatory region comprises or consists of a plurality of HREs, the HREs may be sequential or spatially separate. The single HREs may be separated by a linker which may be rigid or flexible. Suitably, the linker is at least 6 nucleotides in length, optionally more than 8 nucleotides in length. In a preferred embodiment, the linker is 6 or 8 nucleotides in length. The linker may correspond to linkers naturally found in the promoter region of oxygen-responsive genes. An example of a suitable linker is given in SEQ ID NO: 31. Other suitable linkers are well known in the art.P5598PC00

[0231] In one embodiment, each individual HRE comprises or consists of any combination of the following in any order:

[0232] (i) 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, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen or more HIF-binding sites (HBS), for example the sequence of SEQ ID NO: 5 or more preferably the sequence of SEQ ID NO 6, and optionally

[0233] (ii) 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, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen or more HIF ancillary sites (HAS), for example the sequence of SEQ ID NO: 7 or more preferably the sequence of SEQ ID NO 8, and optionally

[0234] (iii) 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, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen or more HNF-4 sites, for example the sequence of SEQ ID NO: 9.

[0235] In one embodiment, the plurality of HREs or two or more comprises or consists of multiple copies of the sequence of SEQ ID NO: 23 or a homologue or variant thereof or a functional fragment thereof. For example, the plurality of HREs or two or more comprises or consists of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or more copies of the sequence of SEQ ID NO: 23 or a homologue or variant thereof or a functional fragment thereof. Each individual HRE copy may be spatially separate (e.g. separated by elements such as enhancers, linkers, intervening sequences), or may be sequential (also referred to herein as "in tandem" or "stacked"), or a combination of both.

[0236] According to one embodiment, the plurality of HREs or the two or more HREs comprise or consist of three sequential " HBS-linker-HAS" sequences, i.e. HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS. The linker being as defined herein or any suitable linker. In an alternative embodiment, there is no linker or wherein not every HBS-HAS is separated by a linker. In an alternative embodiment, there is no HAS element.

[0237] In a preferred embodiment, the plurality of HREs or the two or more HREs comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 24 or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the plurality ofP5598PC00

[0238] HREs or the two or more HREs comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 24 or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 99% sequence identity to SEQ ID NO: 24. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotide under conditions of normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present.

[0239] According to an alternative one embodiment, the plurality of HREs or two or more HREs comprise or consist of five sequential " HBS-linker-HAS" sequences, i.e. HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS. The linker being as defined herein or any suitable linker. In an alternative embodiment, there is no linker or wherein not every HBS-HAS is separated by a linker. In an alternative embodiment, there is no HAS element.

[0240] In a preferred embodiment, the plurality of HREs or the two or more HREs comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 25 or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the plurality of HREs or the two or more HREs comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 25 or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 99% sequence identity to SEQ ID NO: 25. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotideP5598PC00

[0241] under conditions of normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present.

[0242] According to a further alternative one embodiment, the plurality of HREs or two or more HREs comprises or consists of nine sequential " HBS-linker-HAS" sequences, i.e. HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS-linker-HBS-linker-HAS. The linker as being defined herein or any suitable linker. In an alternative embodiment, there is no linker or wherein not every HBS-HAS is separated by a linker. In an alternative embodiment, there is no HAS element.

[0243] In a preferred embodiment, the plurality of HREs or the two or more HREs comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 26 or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the plurality of HREs or the two or more HREs comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 26 or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or 99% sequence identity to SEQ ID NO: 26. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotide under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotide under conditions of normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present.

[0244] In a preferred embodiment, the regulatory region comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 27 or a homologue or variant thereof or a functional fragment thereof. In a preferred embodiment, the regulatory region comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 27 or a homologue or variant thereof or a functional fragment thereof. The homologue or variant preferably comprises or consists of a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% orP5598PC00

[0245] 99% sequence identity to SEQ ID NO: 27. Preferably, the homologue, variant or functional fragment comprises the sequence of SEQ ID NO: 5 or SEQ ID NO 6. Preferably, the homologue, variant or functional fragment comprises (i) SEQ ID NO: 5 or SEQ ID NO 6 and (ii) SEQ ID NO: 7 or SEQ ID NO 8. The functional fragment may be any of the lengths described above. In a preferred embodiment, the homologue, variant or functional fragment retains the capability to drive the expression of the nucleic acid molecule and / or polynucleotides under conditions of hypoxia, such as when compared with the expression of the nucleic acid molecule and / or polynucleotides under conditions of normoxia and / or if the homologue, variant or functional fragment, and / or a regulatory region comprising the homologue, variant or functional fragment, was / were not present.

[0246] Optional third polynucleotide

[0247] In one embodiment, the nucleic acid molecule of the present invention further comprises a third polynucleotide encoding a cell surface receptor.

[0248] In one embodiment, the cell surface receptor is capable of binding specifically to an antigen that is overexpressed in a disease or disorder, preferably in a disease or disorder associated with hypoxia. In a preferred embodiment, the cell surface receptor is capable of specifically binding an antigen that is overexpressed in a cancer cell, preferably in a cancer cell of a solid tumour.

[0249] In one embodiment, the cell surface receptor is capable of delivering an activation signal, and optionally one or more co-stimulatory signals, to the cell or immunoresponsive cell when it specifically binds to the antigen. In a preferred embodiment, the cell-surface receptor is an engineered T-cell receptor (TCR) or, more preferably, a Chimeric Antigen Receptor (CAR).

[0250] The CAR preferably comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular signalling domain. The intracellular signalling domain leads to activation of the cell or immunoresponsive cell, preferably a T-cell, when the extracellular antigen binding domain specifically binds the antigen. The antigen is preferably present on the surface of a cell, such as a cancer cell. The CAR may be of any known design including, but not limited to, first, second, third, fourth or subsequent generation of CARs, split CAR systems and TRUCKS etc.

[0251] First generation CARs are composed of an extracellular binding domain, a hinge region, a transmembrane domain, and one or more intracellular signalling domains. Commonly, the extracellular binding domain comprises a single-chain variable fragment (scFv) from or derived from a tumour antigen-reactive antibody and usually has high specificity to tumour antigen. A first-generation CAR typically comprises the CD3ζ chain domain or a modifiedP5598PC00

[0252] derivative thereof as the intracellular signalling domain, which is the primary transmitter of signals.

[0253] Second generation CARs also contain a co-stimulatory domain, such as CD28 and / or 4-1BB. The inclusion of an intracellular co-stimulatory domain improves T-cell proliferation, cytokine secretion, resistance to apoptosis, and in vivo persistence. The co-stimulatory domain of a second-generation CAR is typically in cis with and upstream of the one or more intracellular signalling domains.

[0254] Third-generation CARs combine multiple co-stimulatory domains in cis with one or more intracellular signalling domains, to augment T-cell activity. For example, a third-generation CAR may comprise co-stimulatory domains from or derived from CD28 and 41BB, together with an intracellular signalling domain from or derived from CD3z. Other third-generation CARs may comprise co-stimulatory domains from or derived from CD28 and 0X40, together with an intracellular signalling domain from or derived from CD3z.

[0255] Fourth-generation CARs (also known as TRUCKS or armoured CARs), combine the expression of a second-generation CAR with factors that enhance anti-tumoural activity (e.g., cytokines, co-stimulatory ligands, chemokines receptors or further chimeric receptors of immune regulatory or cytokine receptors). The factors may be in trans or in cis with the CAR, typically in trans with the CAR.

[0256] The CAR or polynucleotide encoding the CAR may additionally include other mechanisms to deal with off target effects, dose control, location and timing of activation. For example, the polynucleotide encoding the CAR may include suicide gene(s), such as herpes simplex virus thymidine kinase (HSV-TK) or inducible caspase 9 (iCas9), or other means to control off target effects. Other means for control of CAR activity include, but are not limited to, the use of a small molecule agent (e.g. as reported in Giordano-Attinese et al., 2020, Nature Biotechnology Letters). These control systems may be activated by an extracellular molecule to induce apoptosis of the immunoresponsive cell.

[0257] Another example includes a CAR designed to express two or more antigen-specific targeting regions (as defined herein). The CAR may be a split CAR system in which the therapeutic function of the CAR requires the presence of both a tumour antigen and a benign exogenous molecule.

[0258] In various embodiments, the cell surface receptor is a first generation CAR, such as those described in Eshhar et al., Proc. Natl. Acad. Sci. USA (1993) 90(2):720-724.

[0259] In various embodiments, the cell surface receptor is a co-stimulatory chimeric receptor, such as those described in Krause etal., J. Exp. Med. (1998) 188(4):619-26.P5598PC00

[0260] In various embodiments, the cell surface receptor is a second generation CAR, such as those described in Finney etal., J. Immunol. (1998) 161(6):2791-7; Maher eta / ., Nat. Biotechnol. (2002) 20(l):70-75; Finney etal., J. Immunol. (2004) 172(1): 104-113; and Imai etal., Leukemia (2005) 18(4):676-84.

[0261] In various embodiments, the cell surface receptor is a third generation CAR, such as those described in Pule et al. (2005), Mol. Then. 12(5):933-941; Geiger et al., Blood (2001) 98:2364-71; and Wilkie etal. J. Immunol. (2008) 180(7):4901-9.

[0262] In various embodiments, the cell surface receptor is a tandem (Tan)CAR, as described in Ahmed etal., Mol. Then. Nucleic Acids (2013) 2:el05.

[0263] In various embodiments, the cell surface receptor is a TRUCK CAR, as described in Chmielewski etal., Cancer Res. (2011), 71:5697-5706 (2011).

[0264] In various embodiments, the cell surface receptor is an Armoured CAR, as described in Pegram etal., Blood (2012) 119:4133-4141 and Curran etal., Mol. Ther. (2015) 23(4):769-78.

[0265] In various embodiments, the cell surface receptor is a Switch Receptor, as described in WO 2013 / 019615.

[0266] In various embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs.

[0267] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide co-stimulation in cis and in trans, as described in Stephan etal. Nat. Med. (2007) 13(12): 1440-49.

[0268] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide dual-targeted CARs, such as those described in Wilkie etal., J. Clin. Immunol. (2012) 32(5): 1059-70.

[0269] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide inhibitory CARs (NOT gate), as described in Fedorov etal., Sci. Transl. Med. (2013) 5(215):215ral72.

[0270] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide combinatorial CARs (AND gates), as described in Kloss etal., Nat. Biotechnol. (2013) 31(l):71-5 and WO 2014 / 055668.P5598PC00

[0271] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide a Go-CAR T, as described in Foster et al., (2014), Abstract, http: / / www.bloodjournal.org / content / 124 / 21 / 1121?sso-checked=true.

[0272] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide engineered costimulation, as described in Zhao et al., Cancer Cell (2015) 28:415028.

[0273] In some embodiments, the cell surface receptor is expressed in the cell or immunoresponsive cell with other engineered constructs to provide SynNotch / sequential AND gate as described in Roybal et al., Cell (2016) 164:770-79.

[0274] In certain preferred embodiments, the cell surface receptor is expressed in the cell with other engineered constructs to provide a parallel CAR (pCAR), as described in

[0275] WO 2017 / 021701. A pCAR may comprise a second generation chimeric antigen receptor comprising:

[0276] (a) a signalling region;

[0277] (b) a co-stimulatory signalling region;

[0278] (c) a transmembrane domain; and

[0279] (d) a binding element that specifically interacts with a first epitope on a target antigen; and a chimeric costimulatory receptor comprising

[0280] (e) a co-stimulatory signalling region which is different to that of (b);

[0281] (f) a transmembrane domain; and

[0282] (g) a binding element that specifically interacts with a second epitope on a target antigen.

[0283] In various embodiments, the cell surface receptor is an engineered T-cell receptor. The engineered T-cell receptor may be any of those described in WO 2010 / 026377; WO 2010 / 133828; WO 2011 / 001152; WO 20123 / 013913; WO 2013 / 041865; WO 2017 / 109496; WO 2017 / 163064; and WO 2018 / 234319.

[0284] In some embodiments, the CAR comprises means to home to or infiltrate the tumour bed. For example, the CAR may comprise one or more chemokine receptors.

[0285] Further engineered receptors may be included. Additional cell surface receptors may be designed to include means to home to or infiltrate the tumour bed. For example, anP5598PC00

[0286] additional cell surface receptor may comprise a chimeric cytokine receptor or a chemokine receptor.

[0287] The CAR will typically include the following known components described below.

[0288] The CAR typically comprises at least one polypeptide with anti-tumour properties, also referred to herein as an extracellular antigen-specific targeting region. Such proteins for delivery to a tumour include but are not limited to any one or more of an immune stimulating antibodies, a surface or intracellular receptors that confer cell activation and tumour-killing capability and a T-cell Receptor (TCR).

[0289] The antigen-specific targeting region provides the CAR with the ability to bind a predetermined antigen of interest. The antigen-specific targeting region preferably targets an antigen of clinical interest. The antigen-specific targeting region may be any protein or peptide that possesses the ability to specifically recognise and bind to a biological molecule (e.g., a cell surface receptor or a component thereof). The antigen-specific targeting region includes any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule of interest. Illustrative antigen-specific targeting regions include antibodies or antibody fragments or derivatives, extracellular domains of receptors, ligands for cell surface molecules / receptors, or receptor binding domains thereof, and tumour binding proteins.

[0290] In a preferred embodiment, the antigen-specific targeting region is, or is from or derived from, an

[0291] antibody. An antibody-derived targeting domain can comprise a fragment of an antibody or a genetically engineered product of one or more fragments of the antibody, which fragment is involved in binding with the antigen. Examples include a variable region (Fv), a complementarity determining region (CDR), a Fab, a single chain antibody (scFv), a heavy chain variable region (VH), a light chain variable region (VL) and a single-domain antibody (VHH). The antigen-specific targeting region may additionally or alternatively comprise or consist of or be from or derived from monobodies. In a preferred embodiment, the binding domain is a single chain antibody (scFv). The scFv may be murine, human or humanized scFv.

[0292] " Complementarity determining region" or " CDR" with regard to an antibody or antigenbinding fragment thereof refers to a highly variable loop in the variable region of the heavy chain or the light chain of an antibody. CDRs can interact with the antigen conformation and largely determine binding to the antigen (although some framework regions are known to be involved in binding). The heavy chain variable region and the light chain variable region each contain 3 CDRs. " Heavy chain variable region" or " VH" refers to the fragment of the heavy chain of an antibody that contains three CDRs interposed between flanking stretchesP5598PC00

[0293] known as framework regions, which are more highly conserved than the CDRs and form a scaffold to support the CDRs. " Light chain variable region" or " VL" refers to the fragment of the light chain of an antibody that contains three CDRs interposed between framework regions.

[0294] " Fv" refers to the smallest fragment of an antibody to bear the complete antigen binding site. An Fv fragment consists of the variable region of a single light chain bound to the variable region of a single heavy chain. " Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to one another directly or via a peptide linker sequence.

[0295] Antigen binding regions of a CAR that specifically bind a predetermined antigen can be prepared using methods well known in the art. Such methods include phage display, methods to generate human or humanized antibodies, or methods using a transgenic animal or plant engineered to produce human antibodies. Phage display libraries of partially or fully synthetic antibodies are available and can be screened for an antibody or fragment thereof that can bind to the target molecule. Phage display libraries of human antibodies are also available. Once identified, the amino acid sequence or nucleotide sequence coding for the antibody can be isolated and / or determined.

[0296] Antigens which may be targeted by the present CAR include but are not limited to antigens expressed on cells associated with a solid cancer.

[0297] The antigen to target is not limited to but may be selected from one or more and any combination of the following and derivatives and variants thereof: extended ErbB family, Erbbl, Erbb3, Erbb4, Erbb2 / HER-2, mucins, PSMA, CEA, mesothelin, B7-H3, GD2, MUC1, folate receptor, GPC3, CAIX, FAP, NY-ESO-1, gplOO, PSCA, ROR1, PD-L1, PD-L2, EpCAM, EGFRvIII, CD19, CD20, GD3, CLL-1, ductal epithelial mucin, Gp36, TAG-72, glycosphingolipids, glioma-associated antigen, beta-hCG, AFP (alpha-fetoprotein) and lectinreactive AFP, thyroglobulin, receptor for advanced glycation end products (RAGE), TERT, telomerase, carboxylesterase, M-CSF, PSA, survivin, PCTA-1, MAGE, CD22, IGF-1, IGF-2, IGF-1 receptor, MHC-associated tumour peptide, 5T4, tumour stroma-associated antigens, WT1, MLANA, CA 19-9, BCMA, ov[36 integrin, NKG2D, and virus-specific antigens.

[0298] In a preferred embodiment, the antigen-specific targeting region is, or is from or derived from, a ligands for a cell surface molecule / receptor. Suitable binding ligands are known in the art.

[0299] A preferred extracellular antigen-specific targeting region is TIE (Davies et al., 2012, Mol Med 18:565-576) having about 100% identity and / or homology with the sequence of SEQ ID NO: 30 or a functional fragment and or variants thereof. The variant preferably has atP5598PC00

[0300] least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more identity and / or homology to the sequence of SEQ ID NO: 30.

[0301] A preferred extracellular antigen-specific targeting region is TE9 having about 100% identity and / or homology with the sequence of SEQ ID NO: 31 or a functional fragment and or variants thereof. The variant preferably has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more identity and / or homology to the sequence of SEQ ID NO: 31.

[0302] The CAR typically comprises an intracellular signalling domain (also referred to as an endodomain). Suitable intracellular signalling domains are known in the art and include, for example, any region comprising an Immune-receptor-Tyrosine-based-Activation-Motif (ITAM), as reviewed for example by Love et al. Cold Spring Harbor Perspect. Biol 2010 2(6):a002485. In a particular embodiment, the signalling region comprises the intracellular domain of human CD3 [zeta] (CDz) chain as described for example in US Patent No 7,446,190, or a variant thereof.

[0303] The intracellular signalling domain may also be a transcription factor for indirect signalling.

[0304] The intracellular domain comprises or consists of amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 32 or a functional fragment or variant thereof. The variant preferably has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more identity and / or homology to the sequence of SEQ ID NO: 32.

[0305] The CAR typically comprises a transmembrane domain. CARs are expressed on the surface of the cell membrane and therefore typically comprise transmembrane domains. Suitable transmembrane domains are known in the art and include for example, the transmembrane sequence from any protein which has a transmembrane domain, including any of the type I, type II or type III transmembrane proteins. The transmembrane domain of the CAR may also comprise an artificial hydrophobic sequence. The transmembrane domains of the CAR may be selected so as not to dimerize. Suitable transmembrane domains include CD8α, CD28, CD4 or CD3z transmembrane domains.

[0306] In one embodiment, the transmembrane domain comprises or consists of amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 33 or 34 or a functional fragment or variant thereof. The variant preferably has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or more identity and / or homology to the sequence of SEQ ID NO: 33 or 34.P5598PC00

[0307] The CAR typically comprises a co-stimulatory domain. Suitable co-stimulatory domains are also well known in the art and include, but are not limited to, members of the B7 / CD28 family such as B7-1, B7-2, B7-H1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA, CD28, CTLA-4, Gi24, ICOS, PD-1, PD-L2 or PDCD6; ILT / CD85 family proteins such as LILRA3, LILRA4, LILRB1, LILRB2, LILRB3 or LILRB4; tumour necrosis factor (TNF) superfamily members such as 4-1BB, BAFF, BAFF R, CD27, CD30, CD40, DR3, GITR, HVEM, LIGHT, Lymphotoxin-alpha, 0X40, RELT, TACI, TL1A, TNF-alpha or TNF RII; or members of the SLAM family such as 2B4, BLAME, CD2, CD2F-10, CD48, CD58, CD84, CD229, CRACC, NTB-A or SLAM; members of the TIM family such as TIM-1, TIM-3 or TIM-4; and other co-stimulatory molecules such as CD7, CD96, CD160, CD200, CD300a, CRTAM, DAP12, Dectin-1, DPPIV, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7 / LPAM-l, LAG-3 or TSLP R.

[0308] In some embodiments, the CAR comprises a plurality of co-stimulatory domains or two or more co-stimulatory domains. In some embodiments the co-stimulatory domain is from or derived from CD28, 4-1BB and / or 0X40.

[0309] In some embodiments, the co-stimulatory domain is CD28 or is from or derived from CD28. In some embodiments, the co-stimulatory domain is 4-1BB or is from or derived from 4-1BB.

[0310] In a preferred embodiment, the cell surface receptor is fused to one or more ODDs. The cell surface receptor may be any of those described above. The one or more ODDs may be any of those described above. The cell surface receptor may be fused to one or more ODDs in any of the formats discussed above. In any of the embodiments discussed above with reference to the first polynucleotide, the reference to the armouring protein may be replaced with cell surface receptor. In one embodiment, the cell surface receptor is fused to more than one ODD, such as two, three, four, five or more ODDs. The two or more ODDs may be sequential or spatially separate. In a preferred embodiment, the cell surface receptor is fused to one ODD.

[0311] In a preferred embodiment, the one or more ODDs are fused to an intracellular domain of the cell surface receptor. The one or more ODDs are preferably fused to the intracellular signalling domain of the CAR.

[0312] In a preferred embodiment, the third polynucleotide encodes an amino acid sequence having the sequence of SEQ ID NO: 37, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide encodes an amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 37, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least aboutP5598PC00

[0313] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 37.

[0314] In a further aspect, the present invention provides a polypeptide having the sequence of SEQ ID NO: 37, or a homologue or variant thereof. The homologue or variant may be any of those defined in the previous paragraph.

[0315] In a preferred embodiment, the third polynucleotide comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 38, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 38, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 38.

[0316] In an alternative preferred embodiment, the third polynucleotide encodes an amino acid sequence having the sequence of SEQ ID NO: 85, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide encodes an amino acid sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 85, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 85.

[0317] In a further aspect, the present invention provides a polypeptide having the sequence of SEQ ID NO: 85, or a homologue or variant thereof. The homologue or variant may be any of those defined in the previous paragraph.

[0318] In a preferred embodiment, the third polynucleotide comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 86, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 86, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least aboutP5598PC00

[0319] 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 86.

[0320] Nucleic Acid Molecule

[0321] The first polynucleotide is operably linked to the second polynucleotide. The first and second polynucleotides may be any of those described above. The nucleic acid molecule and polynucleotides may be any of the types described above.

[0322] As used herein, the term "operably linked" refers to the arrangement and relative positioning of the promoter / regulatory region in the second polynucleotide and the first polynucleotide, such that the promoter / regulatory region is able to drive expression of the first polynucleotide. The second polynucleotide is positioned such that is able to drive the expression of the first polynucleotide.

[0323] The nucleic acid molecule optionally further comprises a third polynucleotide encoding a cell surface receptor. The third polynucleotide may be any of those described above and may be any of the types described above.

[0324] In a further aspect, the present invention provides a nucleic acid molecule comprising a first polynucleotide encoding an armouring protein fused to an one or more Oxygen-Dependent Degradation Domains (ODDs), a second polynucleotide comprising a regulatory region comprising one or more hypoxia-responsive elements (HREs) and a third polynucleotide encoding a cell surface receptor, wherein the first polynucleotide is operably linked to the second polynucleotide. The first, second and third polynucleotides may be any of those described above.

[0325] The nucleic acid molecule may comprise the first polynucleotide and the third polynucleotide in any position relative to one another.

[0326] In one embodiment, the one or more ODDs of the third polynucleotide have different amino acid sequences from the one or more ODDs of the first polynucleotide. In an alternative embodiment, the one or more ODDs of the third polynucleotide have the same amino acid sequence(s) as the one or more ODDs of the first polynucleotide.

[0327] In a preferred embodiment, the one or more ODDs of the third polynucleotide and the one or more ODDs of the first polynucleotide are encoded by different nucleotide sequences but preferably have the same amino acid sequence. In one embodiment, the nucleotide sequence encoding the one or more ODDs of the third polynucleotide and the nucleotide sequence encoding the one or more ODDs of the first polynucleotide have less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than aboutP5598PC00

[0328] 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, or less than about 60% identity and / or homology with one another, but preferably encode the same amino acid sequence.

[0329] In a preferred embodiment, the first polynucleotide and the third polynucleotide are operably linked to the second polynucleotide.

[0330] In another embodiment, the third polynucleotide is operably linked to a fourth polynucleotide comprising or consisting of a regulatory region comprising or consisting of one or more HREs. The regulatory region and / or the one or more HREs may be any of those discussed above with reference to the second polynucleotide.

[0331] In a further aspect, the present invention provides a nucleic acid molecule comprising a first polynucleotide encoding an armouring protein fused to an one or more Oxygen-Dependent Degradation Domains (ODDs), a second polynucleotide comprising a regulatory region comprising one or more hypoxia-responsive elements (HREs), a third polynucleotide encoding a cell surface receptor and a fourth polynucleotide comprising a regulatory region comprising one or more hypoxia-responsive elements (HREs), wherein the first polynucleotide is operably linked to the second polynucleotide and the third polynucleotide is operably linked to the fourth polynucleotide. The first, second, third and fourth polynucleotides may be any of those described above.

[0332] In any of the embodiments above, the nucleic acid molecule of the present invention may comprise the first polynucleotide and the third polynucleotide in the form of a polycistronic nucleic acid construct.

[0333] In any of the embodiments above, the first polynucleotide and the third polynucleotide are separated by a nucleotide sequence that allows for two separate polypeptide products to be produced as discreet entities, one from the first polynucleotide and one from the third polynucleotide. In one embodiment, the nucleotide sequence comprises or consists of a nucleotide sequence that allows for cap-independent translation initiation. The nucleotide sequence is preferably an Internal Ribosome Entry Site (IRES) sequence. In a preferred embodiment, the nucleotide sequence comprises or consists of a nucleotide sequence which induces a ribosomal stall and skip, such as a self-cleaving ribozyme sequence. The nucleotide sequence may encode a 2A peptide. The 2A peptide may be P2A, E2A, F2A or T2A. The 2A peptide is preferably T2A. Further suitable nucleotide sequences comprise or consist of a nucleotide sequence that encode for self-cleaving or cleavage domains. Such sequences may either auto-cleave during protein production or may be cleaved by common enzymes present in the cell. Accordingly, inclusion of such self-cleaving or cleavage domains in the amino acid sequence enables a two polypeptides to be expressed as a singleP5598PC00

[0334] polypeptide, which is subsequently cleaved to provide discrete, separated functional polypeptides.

[0335] In a preferred embodiment, the T2A has about 100% identity and / or homology to the sequence of SEQ ID NO: 87, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 87.

[0336] In a preferred embodiment, the T2A has about comprises or consists of a nucleotide sequence having the sequence of SEQ ID NO: 88, or a homologue or variant thereof. In a preferred embodiment, the first polynucleotide comprises or consists of a nucleotide sequence having about 100% identity and / or homology to the sequence of SEQ ID NO: 88, or a homologue or variant thereof. The homologue or variant preferably comprises or consists of a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity and / or homology to the sequence of SEQ ID NO: 88.

[0337] The nucleic acid molecule may further comprise a nucleotide sequence encoding a selectable marker. Selectable markers are well known in the art and include, but are not limited to, fluorescent proteins, such as green fluorescent protein (GFP). The nucleotide sequence encoding a selectable marker may be provided in combination with the first polynucleotide in the form of a polycistronic nucleic acid construct. The use of a selectable marker is advantageous as it allows a cell in which the nucleic acid molecule has been successfully introduced (such that the encoded armouring protein and optionally CAR is expressed) to be selected and isolated from a starting cell population using common methods, e.g. flow cytometry. The first polynucleotide and the nucleotide sequence encoding the selectable marker may be separated by a nucleotide sequence that allows for two separate polypeptide products to be produced as discreet entities as described above.

[0338] Any of the nucleic acid molecules and polynucleotides used in the present invention may be codon-optimised. Codon optimisation has previously been described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which theP5598PC00

[0339] corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available.

[0340] The nucleic acid molecule may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the nucleic acid molecule.

[0341] The nucleic acid molecule, which may be DNA, may be produced recombinantly, synthetically or by any means available to those of skill in the art. It may also be cloned by standard techniques.

[0342] Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

[0343] The nucleic acid molecule of the present invention may comprise further polynucleotide sequences that encode for additional proteins. In one embodiment, the nucleic acid molecule further comprises a suicide gene or safety switch, which encodes for a polypeptide that allows for the T-cells to be killed. The suicide gene or safety switch can be any known in the art. In one embodiment, the suicide gene encodes for RQR8, which allows for selective elimination of cells expressing this gene when rituximab is administered.

[0344] Vectors

[0345] In a further aspect, the present invention provides vectors comprising the nucleic acid molecule of the present invention. The nucleic acid molecule may be any of those described above.

[0346] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the present invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. In a preferred embodiment, the vector allows the nucleic acid molecule to be transferred into a cell or immunoresponsive cell. The vector more preferably allows the nucleic acid molecule to be stably integrated into the genome of the cell or immunoresponsive cell.P5598PC00

[0347] Vectors may be introduced into cells using a variety of techniques known in the art, such as transformation and transduction. Several techniques are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors, or direct injection of nucleic acids and biolistic transformation. Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell.

[0348] Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques and suitable for use in the present invention include, but are not limited to, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be the nucleic acid molecule. The vectors used in the invention may be, for example, plasmid, mRNA or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter. In a preferred embodiment, the vector is a viral vector that is capable of stably integrating into the genome, for example a lentivirus vector (e.g., FIV), an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a Herpes Simplex Virus (HSV) vector, a Vaccinia Virus vector, a Sendai Virus vector, a Measles Virus vector, an Epstein-Barr Virus (EBV) vector, a Vesicular Stomatitis Virus (VSV) vector, Newcastle Disease Virus (NDV) vector, Coronavirus (e.g., SARS-CoV-2) vector, or a Baculovirus vector.

[0349] In one embodiment, the vector allows the nucleic acid molecule to be stably integrated into the genome of a target cell through any method are known in the art for causing knock-in or expression of a gene. Various nuclease systems are known in the art for editing nucleic acid, for example to cause gene knockout, knock-in or expression of a gene to be downregulated or overexpressed, or to introduce mutations in the form of one or more deletions, insertions or substitutions. Examples of such nuclease systems include zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), meganucleases, transposon systems (such as sleeping beauty transposons) or combinations thereof are known in the art may be used in the present invention. The clustered regularly interspersed short palindromic repeats (CRISPR) / CRISPR-associated (Cas) (CRISPR / Cas) nuclease system has become more commonly used for genome engineering. The CRISPR / Cas system is detailed in, for example WO2013 / 176772, WO2014 / 093635 and W02014 / 089290. For example, a CRISPR / Cas9 may include a guide RNA (gRNA) sequence with a binding site for Cas9 and a targeting sequence specific for the area to be modified. The Cas9 binds the gRNA to form a ribonucleoprotein that binds and cleaves the target area. In addition to the CRISPR / Cas 9 platform (which is a type II CRISPR / Cas system), alternative systems exist including type I CRISPR / Cas systems, type III CRISPR / Cas systems, and type V CRISPR / CasP5598PC00

[0350] systems. Any of the above CRISPR systems may be used to prepare vectors comprising the nucleotide sequences of the invention.

[0351] Thus, in one embodiment, the vector of the present invention is for use with a nuclease system for genetic knock-ins, such as for use with ZFNs, TALENs or a CRISPR / Cas system.

[0352] In particular, the nucleic acid molecule may be comprised in a donor DNA template, preferably for use with ZFNs, TALENs or a CRISPR / Cas system. Use of a donor DNA template with these systems allows for the nucleic acid molecule of the present invention to be knocked-in, for instance, stably integrated into the genome of an immunoresponsive cell. The donor DNA template comprises the nucleic acid molecule of the present invention, homology arms and optionally a selection marker.

[0353] Preferred vectors include, but are not limited to, DNA vectors, RNA vectors, plasmid vectors, cosmid vectors, herpes virus vectors, measles virus vectors, lentivirus vectors, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, Vaccinia Virus vectors, Sendai Virus vectors, Epstein-Barr Virus (EBV) vectors, Vesicular Stomatitis Virus (VSV) vectors, Newcastle Disease Virus (NDV) vectors, Coronavirus vectors, Baculovirus vectors, transposons or donor DNA templates for use with ZFNs, TALENs or a CRISPR / Cas system.

[0354] The vector is preferably the SFG retroviral vector. This vector contains Moloney murine leukaemia virus long terminal repeats (LTRs) which contain the promoter region.

[0355] Homology arms are defined herein as nucleotide sequences that flank the desired knock in sequence (i.e. the nucleic acid molecule of the present invention) and have sequence identity to the target site in the genome. The homology arms guide homology-directed repair and allow the cell to recognise the sequence they flank as a repair template.

[0356] Preferably the homology arms are 500 to 1000 base pairs in length, 1000 to 2000 base pairs in length or more than 2000 base pairs in length.

[0357] Cells of the invention

[0358] In a further aspect, the present invention provides a cell, preferably an immunoresponsive cell, comprising a nucleic acid molecule of the present invention or a vector of the present invention. In a further aspect, the present invention provides a cell, preferably an immunoresponsive cell, expressing a nucleic acid molecule of the present invention or a vector of the present invention.

[0359] The cell may be any type of cell. The cell may be a prokaryotic cell. The cell may be bacterial or archaeal. The cell are typically eukaryotic cell. The cell may be a protozoan, algal, fungal, plant or animal cell. The animal cell may be from or derived from the ectoderm, endoderm, or mesoderm. The cell may be from or derived from any of the species described below. The cell is preferably human.P5598PC00

[0360] The cell may be stem cell, such as an embryonic stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell or mesenchymal stem cell, a fibroblast, bone cell, such as osteoclasts, osteoblasts or osteocytes, tendon cell, such as tenoblasts or tenocytes, chondrocytes, synovial cell, vascular cell, blood cell, such as red blood cell, immune cell, platelet, neutrophils or basophils, muscle cell, such as skeletal muscle cell, cardiac muscle cell or smooth muscle cell, reproductive cell, such as sperm, oocytes, duct cell or epididymal cell, secretory cell, adipocytes, liver lipocytes, epithelial cell, odontoblasts, cementoblasts, hormone-secreting cell, barrier cell, exocrine secretory epithelial cell, nerve cell, astrocytes, oligodendrocytes, or neurons. The cell may be a tumour cell. The tumour cell may be from or derived from any of the listed cell types.

[0361] The immune cell may be neutrophil granulocyte and precursors, such as myeloblasts, promyelocytes, myelocytes, or metamyelocytes, eosinophil granulocyte and precursors, basophil granulocyte and precursors, mast cell, leukocytes, lymphocytes, helper T cell, regulatory T cell, cytotoxic T cell, natural killer T cell, B cell, macrophages, dendritic cell, plasma cell, neutrophils, or monocytes.

[0362] The cell may be wild-type or naturally occurring. The cell may be genetically modified or genetically engineered. For instance, the immune cell may be genetically engineered to express a recombinant chimeric antigen receptor (CAR) orT cell receptor (TCR). The cell may be genetically modified or genetically engineered using transduction or transfection or any other common techniques known to those skilled in the art.

[0363] The cell may be healthy cell or obtained from a healthy donor or source. The cell may be diseased or damaged, associated with a disease or damage or obtained from a diseased or damaged donor or source.

[0364] The cell is preferably an immunoresponsive cell. The immune responsive cell is preferably a lymphoid-derived cell, a T-cell, an αβ T-cell, a y6 T-cell, a B-cell, a myeloid-derived cell, a stem cell or an induced pluripotent stem cells (iPSC).

[0365] The lymphoid-derived cell is preferably a Natural Killer (NK) cell, NK T-cell, or an invariant NKT-cell.

[0366] The T-cell is preferably a cytotoxic T-cell, a helper T-cells or a regulatory T-cell.

[0367] The myeloid-derived cell is preferably as a macrophage or a neutrophil.

[0368] Any of the cells above, including the immunoresponsive cells, may be from or derived from a stem cell, such as a hematopoietic stem cell or iPSC.

[0369] In a further aspect, the present invention provides a population of cells or a population of immunoresponsive cells of the invention. In a further aspect, the present invention providesP5598PC00

[0370] a population of cells comprising a nucleic acid molecule of the present invention or a vector of the present invention. In a further aspect, the present invention provides a population of immunoresponsive cells comprising a nucleic acid molecule of the present invention or a vector of the present invention. The cells or immunoresponsive cells may be any of those described above. The cells or immunoresponsive cells typically express the nucleic acid molecule or vector.

[0371] The population may comprise any number of cells or immunoresponsive cells, such as at least about 5 x 105cells. The population preferably comprises at least about 1 x 106, at least about 2 x 106, at least about 2.5 x 106, at least about 5 x 106, at least about 1 x 107, at least about 2 x 107, at least about 5 x 107, at least about 1 x 108or at least about 2 x 108cells. In some instances, the population comprises at least about 1.0 x 107, at least about 1.0 x 108, at least about 1.0 x 109, at least about 1.0 x 1010, at least about 1.0 x 1011or at least about 1.0 x 1012cells or immunoresponsive cells or even more.

[0372] The cell, immunoresponsive cell or population of the invention may be isolated, substantially isolated, purified, or substantially purified. A cell, immunoresponsive cell or population is isolated or purified if it is completely free of any other components, such as culture medium. A cell, immunoresponsive cell or population is substantially isolated or substantially purified if it is mixed with carriers or diluents, such as a culture medium or a pharmaceutical composition, which will not interfere with its intended use. Other carriers and diluents are discussed in more detail below.

[0373] The cell, immunoresponsive cell or population may be in vitro, in vivo or ex vivo. The cell, immunoresponsive cell or population may optionally be allogenic. The cell, immunoresponsive cell or population may be an "off the shelf" CAR T-Cell, where the T cells are not necessarily from or derived from the subject with cancer (see for example Depil et al., 2020 (Nature Reviews Drug Discovery)).

[0374] The cell, immunoresponsive cell or population is preferably matched or is autologous to the subject. The cell may be generated ex vivo either from a patient's own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party). Suitably the cell is matched or autologous to the subject.

[0375] Methods of preparing cells

[0376] In a further aspect, the present invention provides a method of preparing a cell, preferably an immunoresponsive cell, comprising:

[0377] a) Isolating the cell, or a precursor of the cell, from a subject;P5598PC00

[0378] b) Modifying said cell to introduce a nucleic acid molecule of the present invention or a vector of the present invention,

[0379] c) Expanding said modified cell ex-vivo;

[0380] d) Obtaining expanded cell capable of expressing the nucleic acid molecule of the present invention under conditions of hypoxia.

[0381] In a further aspect, the present invention provides a method of preparing an immunoresponsive cell comprising:

[0382] a) Isolating lymphoid or myeloid-derived cells from a subject;

[0383] b) Modifying said cell to introduce a nucleic acid molecule of the present invention or a vector of the present invention,

[0384] c) Expanding said modified cell ex-vivo;

[0385] d) Obtaining expanded cell capable of expressing the nucleic acid molecule of the present invention under conditions of hypoxia.

[0386] The cell or immunoresponsive cell may be any of those described above. The subject may be any of those described above. These methods may be methods of preparing two or more cells or two or more immunoresponsive cells. These methods may be methods of preparing a population of cells or a population of immunoresponsive cells. Populations are defined above.

[0387] A further aspect of the invention provides a cell, immunoresponsive cell or population obtainable by or obtained from the method of the invention.

[0388] Step b) comprises modifying or engineering the cell to introduce a nucleic acid molecule of the present invention or a vector of the present invention. Methods for engineering cells are known in the art and include, but are not limited to, genetic modification of cells e.g. by transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection — DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation. Any suitable method may be used to introduce a nucleic acid molecule into a cell.

[0389] In a preferred embodiment, the nucleic acid molecule of the present invention is stably integrated into the genome of the cell or immunoresponsive cell. In one embodiment, the nucleic acid molecule is integrated into the genome through the use of a viral vector, preferably by retroviral transduction or lentiviral transduction. In an alternative embodiment, the nucleic acid molecule is integrated into the genome through the use of aP5598PC00

[0390] nuclease system, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), meganucleases, or preferably a CRISPR / Cas nuclease system in combination with a donor DNA template comprising the nucleic acid molecule of the present invention.

[0391] As used herein, the term "introduced" refers to methods for inserting foreign DNA or RNA into a cell and includes both transduction and transfection methods. As outlined above, the method of introducing the nucleic acid molecule into the cell preferably allows for the stable integration of the nucleic acid molecule into the cell's genome. Thus in a preferred embodiment, the method of introducing the nucleic acid molecule of the present invention into the cell is a viral method, such as retroviral transduction or lentiviral transduction, or through the use of a nuclease system, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), meganucleases, or preferably a CRISPR / Cas nuclease system in combination with a donor DNA template comprising the nucleic acid molecule of the present invention.

[0392] As used herein "expanding" means that a cell or population has been induced to proliferate. The expansion of a population of cells may be measured for example by counting the number of cells present in a population. The phenotype of the cells may be determined by methods known in the art such as flow cytometry.

[0393] The subject in step a) may be a cancer patient or a healthy donor. The subject may be an allogeneic or third party donor. The subject may be the subject which is treated using the cell or immunoresponsive cell. The subject may be an autologous donor.

[0394] In one embodiment, the cells are modified in step b) by the introduction of both the nucleic acid molecule of the present invention and a second nucleic acid molecule encoding a cell surface receptor. The cell surface receptor may be any of those described above, including a TCR or a CAR, preferably fused to one or more ODDs and / or operably linked to one or more HREs.

[0395] In the method, expression of the nucleic acid molecule of the present invention is typically driven by the second polynucleotide. In the method, expression of the nucleic acid molecule of the present invention may be driven by the second polynucleotide and the fourth polynucleotide.

[0396] Pharmaceutical Composition

[0397] In a further aspect, the present invention provides a pharmaceutical composition comprising one or more of (i) a nucleic acid of the present invention, (ii) a vector of the present invention, (iii) a cell or an immunoresponsive cell of the present invention and (iv) a population of the invention and a pharmaceutically or physiologically acceptable diluentP5598PC00

[0398] and / or carrier. The pharmaceutical composition may comprise (i); (ii); (iii); (iv); (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (iii) and (iv); or (i), (ii), (iii) and (iv).

[0399] In a further aspect, the present invention provides a pharmaceutical composition comprising a nucleic acid of the present invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0400] In a further aspect, the present invention provides a pharmaceutical composition comprising a vector of the present invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0401] In a further aspect, the present invention provides a pharmaceutical composition comprising a cell or an immunoresponsive cell of the present invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0402] In a further aspect, the present invention provides a pharmaceutical composition comprising a population of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0403] The nucleic acid, vector, cell or an immunoresponsive or population may be any of those described above. The pharmaceutical composition preferably comprises a therapeutically effective amount or a prophylactically effective amount of one or more of (i), (ii), (iii) or (iv). These terms are defined below.

[0404] The carrier and / or diluent 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. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media.

[0405] Suitable further agents for inclusion in the pharmaceutical composition 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 hydrogensulphite), 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 diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such asP5598PC00

[0406] sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0407] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable 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.

[0408] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include 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, 22ndEdition).

[0409] 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, intrapleural, subcutaneous, intratumoural, spinal, intra-bone marrow or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein 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, intratumoural, intrapleural and intra-sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptableP5598PC00

[0410] 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.

[0411] Alternatively, the pharmaceutical composition of the invention can be administered by a nonparenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0412] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, 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. Alternatively, the pharmaceutical composition may be for intra-bone marrow administration.

[0413] Therapy

[0414] In a further aspect, the present invention provides a method for treating or preventing a disease or disorder in a subject in need thereof comprising administering to the subject a therapeutically effective amount or prophylactically effective amount of (i) a nucleic acid of the present invention, (ii) a vector of the present invention, (iii) a cell or an immunoresponsive cell of the present invention, (iv) a population of the invention or (v) a pharmaceutical composition of the present invention.

[0415] A further aspect of the present invention provides a (i) a nucleic acid of the present invention, (ii) a vector of the present invention, (iii) a cell or an immunoresponsive cell of the present invention, (iv) a population of the invention or (v) a pharmaceutical composition of the present invention for use in the treatment or prevention of a disease or disorder.

[0416] A further aspect of the present invention provides the use of (i) a nucleic acid of the present invention, (ii) a vector of the present invention, (iii) a cell or an immunoresponsive cell of the present invention, (iv) a population of the invention or (v) a pharmaceutical composition of the present invention in the manufacture of a medicament for treatment or prevention of a disease or disorder in a subject.

[0417] The invention is also useful in therapy or in prophylactic treatment to stimulate a T-cell mediated immune response to a target cell population. The invention further provides a method for stimulating a T-cell mediated immune response to a target cell population in a subject in need thereof comprising administering to the subject (i) a nucleic acid of the present invention, (ii) a vector of the present invention, (iii) a cell or an immunoresponsive cell of the present invention, (iv) a population of the invention or (v) a pharmaceutical composition of the present invention.P5598PC00

[0418] The nucleic acid, the vector, the cell or immunoresponsive cell, the population or the pharmaceutical composition may be any of those described above.

[0419] The method or uses of the present invention need not be carried out using T-cells, but may also be carried out using other suitable immunoresponsive cells such as lymphoid-derived cells such as Natural Killer cell, B-cell, invariant NKT-cell or T-cell, such as cytotoxic T-cells, helper T-cells or regulatory T-cells; or myeloid-derived cells such as a macrophages or neutrophils.

[0420] Any of (i)-(v) may be administered to a subject that displays symptoms of disease or disorder. Any of (i)-(v) may be administered to a subject that is asymptomatic, i.e., does not display symptoms of disease or disorder. Any of (i)-(v) may be administered when the subject's disease status is unknown, or the subject is expected not to have a disease or disorder. Any of (i)-(v) may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease or disorder.

[0421] Any of (i)-(v) may be administered to a subject having an existing disease or disorder in order to lessen, reduce or improve at least one symptom associated with the disease or disorder and / or to slow down, reduce or block the progression of the disease or disorder. Any of (i)-(v) may be administered to a subject who has not yet contracted the disease or disorder and / or who is not showing any symptoms of the disease or disorder to prevent or impair the cause of the disease or disorder or to reduce or prevent development of at least one symptom associated with the disease or disorder.

[0422] Administration of any of (i)-(v) to the subject may reduce symptoms by at least 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 subject.

[0423] The disease or disorder is preferably a disease or disorder associated with hypoxia, preferably a tumour or a cancer. The present invention is particularly useful in the treatment of solid cancers. In one embodiment, the solid cancer has a proportion of cells that do not express the antigen targeted by the CAR-T cell and / or express low levels of the antigen targeted by the CAR-T cell. Other disease or disorders are discussed below.

[0424] The subject suitable for treatment include mammals, such as a human, non-human primate, cow, horse, pig, sheep, goat, dog, cat, rabbit, or rodent. In preferred embodiments, the subject is a human. Practice of methods described herein in other mammalian subjects, especially mammals that are conventionally used as models for demonstrating therapeutic efficacy in humans (e.g. murine, primate, porcine, canine, or rabbit animals), is also encompassed. Standard dose-response studies are used to optimise dosage and dosing schedule.P5598PC00

[0425] In embodiments where the subject is human, the subject may be a human adult or child. In the context of the present invention, an adult will be understood to be an at least 18-year-old human. A child will be understood to be a human less than 18 years old. In some embodiments, the adult is at least 60 years old, such as at least 70, at least 80 or at least 90 years old.

[0426] " Administering" refers to the physical introduction of the immunoresponsive cells to a subject using any of the various known methods and delivery systems. Examples include intratumoural (i.t.), intravenous (i.v.), intramuscular, subcutaneous, intraperitoneal, intrapleural, spinal, pleural effusion, or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intracavitary, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0427] In some embodiments, the cells can be autologous with respect to the subject. For example, cells can be removed from the subject, prepared as described above and returned to the subject. Preferably, the cells are allogeneic with respect to the subject. In this embodiment, the cells may be matched to the subject.

[0428] In the TCR or CAR-based anti-cancer immunotherapy of the invention, T-lymphocytes are isolated from a cancer patient (or healthy donor), modified and expanded ex-vivo by, for example, retro / lenti-viral vectors to constitutively express a TCR or a CAR molecule at the cell surface, which comprises a tumour-associated receptor with binding specificity for a tumour-associated antigen (TAA) expressed on the surface by the tumour cell, and then are re-infused back into the patient. As a result, a large population of patient autologous T-cells and / or non-patient derived allogenic T-cells is redirected towards killing cancerous cells.

[0429] The dual oxygen sensing properties of the nucleic acid of the present invention allows for off target effects to be reduced or eliminated (through the use of the hypoxia responsive promoter in conjunction with the activity of the ODD(s)), and furthermore, there is increased expression of the armouring protein, and optionally the TCR or the CAR, at the site of the tumour.

[0430] Any of (i)-(v) are useful for treating any disease or disorder associated with hypoxia. The disease or disorder may be selected from cancer, an autoimmune disease or disorder, anP5598PC00

[0431] immune deficiency disease or disorder, an ischemic disease or disorder and a disease or disorder requiring regenerative medicine.

[0432] Any of (i)-(v) are useful for treating or preventing cancer. Any of (i)-(v) are useful for, for example, inhibiting cancer growth, including complete cancer remission, for inhibiting cancer metastasis, and for promoting cancer resistance. The term "cancer growth" generally refers to any one of a number of indices that suggest change within the cancer to a more developed form. Indices for measuring an inhibition of cancer growth include but are not limited to a decrease in cancer cell survival, a decrease in tumour volume or morphology (for example, as determined using computed tomographic (CT), sonography, or other imaging method), a delayed tumour growth, a destruction of tumour vasculature, improved performance in delayed hypersensitivity skin test, an increase in the activity of cytolytic T-lymphocytes, and a decrease in levels of tumour-specific antigens. The term "cancer resistance" refers to an improved capacity of a subject to resist cancer growth, in particular growth of a cancer already had. In other words, the term "cancer resistance" refers to a decreased propensity for cancer growth in a subject.

[0433] Cancer cells in the individual with cancer may be immunologically distinct from normal somatic cells in the individual. For example, the cancer cells may express an antigen which is not expressed by normal somatic cells in the individual (i.e. a tumour antigen). Tumour antigens are well-known in the art and are described in more detail herein.

[0434] Various types of cancers are known in the art. The cancer may be metastatic or non-metastatic. The cancer may be familial or sporadic. In some embodiments, the cancer is selected from the group consisting of: leukaemia and multiple myeloma. Additional cancers that can be treated using the methods of the invention include, for example, benign and malignant solid tumours and benign and malignant non-solid tumours.

[0435] For example, a cancer may comprise a solid tumour, for example, a carcinoma or a sarcoma.

[0436] Carcinomas include malignant neoplasms from or derived from epithelial cells which infiltrate, for example, invade, surrounding tissues and give rise to metastases.

[0437] Adenocarcinomas are carcinomas from or derived from glandular tissue, or from tissues that form recognizable glandular structures.

[0438] Carcinomas that may be treated include adrenocortical, acinar, acinic cell, acinous, adenocystic, adenoid cystic, adenoid squamous cell, cancer adenomatosum, adenosquamous, adnexel, cancer of adrenal cortex, adrenocortical, aldosterone-producing, aldosterone-secreting, alveolar, alveolar cell, ameloblastic, ampullary, anaplastic cancer of thyroid gland, apocrine, basal cell, basal cell, alveolar, comedo basal cell, cystic basal cell, morphea-like basal cell, multicentric basal cell, nodulo-ulcerative basal cell, pigmented basalP5598PC00

[0439] cell, sclerosing basal cell, superficial basal cell, basaloid, basosquamous cell, bile duct, extrahepatic bile duct, intrahepatic bile duct, bronchioalveolar, bronchiolar, bronchioloalveolar, bronchoalveolar, bronchoalveolar cell, bronchogenic, cerebriform, cholangiocelluarl, chorionic, choroids plexus, clear cell, cloacogenic anal, colloid, comedo, corpus, cancer of corpus uteri, cortisol-producing, cribriform, cylindrical, cylindrical cell, duct, ductal, ductal cancer of the prostate, ductal cancer in situ (DCIS), eccrine, embryonal, cancer en cuirasse, endometrial, cancer of endometrium, endometroid, epidermoid, cancer ex mixed tumour, cancer ex pleomorphic adenoma, exophytic, fibrolamellar, cancer fibro ' sum, follicular cancer of thyroid gland, gastric, gelatinform, gelatinous, giant cell, giant cell cancer of thyroid gland, cancer gigantocellulare, glandular, granulose cell, hepatocellular, Hurthle cell, hypernephroid, infantile embryonal, islet cell carcinoma, inflammatory cancer of the breast, cancer in situ, intraductal, intraepidermal, intraepithelial, juvenile embryonal, Kulchitsky-cell, large cell, leptomeningeal, lobular, infiltrating lobular, invasive lobular, lobular cancer in situ (LCIS ), lymphoepithelial, cancer medullare, medullary, medullary cancer of thyroid gland, medullary thyroid, melanotic, meningeal, Merkel cell, metatypical cell, micropapillary, mucinous, cancer muciparum, cancer mucocellulare, mucoepidermoid, cancer mucosum, mucous, nasopharyngeal, neuroendocrine cancer of the skin, noninfiltrating, non-small cell, non-small cell lung cancer (NSCLC), oat cell, cancer ossificans, osteoid, Paget's, papillary, papillary cancer of thyroid gland, periampullary, preinvasive, prickle cell, primary intrasseous, renal cell, scar, schistosomal bladder, Schneiderian, scirrhous, sebaceous, signet-ring cell, cancer simplex, small cell, small cell lung cancer (SCLC), spindle cell, cancer spongiosum, squamous, squamous cell, terminal duct, anaplastic thyroid, follicular thyroid, medullary thyroid, papillary thyroid, trabecular cancer of the skin, transitional cell, tubular, undifferentiated cancer of thyroid gland, uterine corpus, verrucous, villous, cancer villosum, yolk sac, squamous cell particularly of the head and neck, oesophageal squamous cell, and oral cancers and carcinomas.

[0440] Another broad category of cancers includes sarcomas and fibrosarcomas, which are tumours whose cells are embedded in a fibrillar or homogeneous substance, such as embryonic connective tissue.

[0441] Sarcomas that may be targeted include adipose, alveolar soft part, ameloblastic, avian, botryoid, sarcoma botryoides, chicken, chloromatous, chondroblastic, clear cell sarcoma of kidney, embryonal, endometrial stromal, epithelioid, Ewing's, fascial, fibroblastic, fowl, giant cell, granulocytic, hemangioendothelial, Hodgkin's, idiopathic multiple pigmented hemorrhagic, immunoblastic sarcoma of B cells, immunoblastic sarcoma of T-cells, Jensen's, Kaposi's, Kupffer cell, leukocytic, lymphatic, melanotic, mixed cell, multiple, lymphangio, idiopathic haemorrhagic, multipotential primary sarcoma of bone, osteoblastic, osteogenic, parosteal, polymorphous, pseudo-Kaposi, reticulum cell, reticulum cell sarcoma of the brain,P5598PC00

[0442] rhabdomyosarcoma, Rous, soft tissue, spindle cell, synovial, telangiectatic, sarcoma (osteosarcoma) / malignant fibrous histiocytoma of bone, and soft tissue sarcomas.

[0443] Lymphomas that may be treated include Acquired Immune Deficiency Syndrome (AIDS)-related, non-Hodgkin's, Hodgkin's, T-cell, T-cell leukaemia / lymphoma, African, B-cell, B-cell monocytoid, bovine malignant, Burkitt's, centrocytic, lymphoma cutis, diffuse, diffuse, large cell, diffuse, mixed small and large cell, diffuse, small cleaved cell, follicular, follicular centre cell, follicular, mixed small cleaved and large cell, follicular, predominantly large cell, follicular, predominantly small cleaved cell, giant follicle, giant follicular, granulomatous, histiocytic, large cell, immunoblastic, large cleaved cell, large non-cleaved cell, Lennert's, lymphoblastic, lymphocytic, intermediate; lymphocytic, intermediately differentiated, plasmacytoid; poorly differentiated lymphocytic, small lymphocytic, well differentiated lymphocytic, lymphoma of cattle; Mucosa-Associated Lymphoid Tissue (MALT), mantle cell, mantle zone, marginal zone, Mediterranean lymphoma, mixed lymphocytic-histiocytic, nodular, plasmacytoid, pleomorphic, primary central nervous system, primary effusion, small B-cell, small cleaved cell, small non-cleaved cell, T-cell lymphomas; convoluted T-cell, cutaneous T-cell, small lymphocytic T-cell, undefined lymphoma, u-cell, undifferentiated, aids-related, central nervous system, cutaneous T-cell, effusion (body cavity based), thymic lymphoma, and cutaneous T-cell lymphomas.

[0444] Leukaemias and other blood cell malignancies that may be targeted include acute lymphoblastic, acute myeloid, acute lymphocytic, acute myelogenous leukaemia, chronic myelogenous, hairy cell, erythroleukaemia, lymphoblastic, myeloid, lymphocytic, myelogenous, leukaemia, hairy cell, T-cell, monocytic, myeloblastic, granulocytic, gross, hand mirror-cell, basophilic, haemoblastic, histiocytic, leukopenic, lymphatic, Schilling's, stem cell, myelomonocytic, monocytic, prolymphocytic, promyelocytic, micromyeloblastic, megakaryoblastic, megakaryoctyic, Rieder cell, bovine, aleukemic, mast cell, myelocytic, plasma cell, subleukaemic, multiple myeloma, nonlymphocytic, chronic myelogenous leukaemia, chronic lymphocytic leukaemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom's macroglobulinaemia, heavy chain disease, myelodysplastic syndrome, myelodysplasia and chronic myelocytic leukaemias.

[0445] Brain and central nervous system (CNS) cancers and tumours that may be treated include astrocytomas (including cerebellar and cerebral), brain stem glioma, brain tumours, malignant gliomas, ependymoma, glioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumours, visual pathway and hypothalamic gliomas, primary central nervous system lymphoma, ependymoma, brain stem glioma, visual pathway and hypothalamic glioma, extracranial germ cell tumour, medulloblastoma, myelodysplastic syndromes, oligodendroglioma, myelodysplastic / myeloproliferative diseases, myelogenousP5598PC00

[0446] leukaemia, myeloid leukaemia, multiple myeloma, myeloproliferative disorders, neuroblastoma, plasma cell neoplasm / multiple myeloma, central nervous system lymphoma, intrinsic brain tumours, astrocytic brain tumours, gliomas, and metastatic tumour cell invasion in the central nervous system.

[0447] Gastrointestinal cancers that may be treated include extrahepatic bile duct cancer, colon cancer, colon and rectum cancer, colorectal cancer, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumour, gastrointestinal carcinoid tumours, gastrointestinal stromal tumours, bladder cancers, islet cell carcinoma (endocrine pancreas), pancreatic cancer, islet cell pancreatic cancer, prostate cancer rectal cancer, salivary gland cancer, small intestine cancer, colon cancer, and polyps associated with colorectal neoplasia.

[0448] Lung and respiratory cancers that may be treated include bronchial adenomas / carcinoids, oesophageal cancer, hypopharyngeal cancer, laryngeal cancer, hypopharyngeal cancer, lung carcinoid tumour, non-small cell lung cancer, small cell lung cancer, small cell carcinoma of the lungs, mesothelioma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, nasopharyngeal cancer, oral cancer, oral cavity and lip cancer, oropharyngeal cancer; paranasal sinus and nasal cavity cancer, and pleuropulmonary blastoma.

[0449] Urinary tract and reproductive cancers that may be treated include cervical cancer, endometrial cancer, ovarian epithelial cancer, extragonadal germ cell tumour, extracranial germ cell tumour, extragonadal germ cell tumour, ovarian germ cell tumour, gestational trophoblastic tumour, spleen, kidney cancer, ovarian cancer, ovarian epithelial cancer, high grade serous ovarian cancer, ovarian germ cell tumour, ovarian low malignant potential tumour, penile cancer, renal cell cancer (including carcinomas ), renal cell cancer, renal pelvis and ureter (transitional cell cancer), transitional cell cancer of the renal pelvis and ureter, gestational trophoblastic tumour, testicular cancer, ureter and renal pelvis, transitional cell cancer, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, ovarian carcinoma, primary peritoneal epithelial neoplasms, cervical carcinoma, uterine cancer and solid tumours in the ovarian follicle), superficial bladder tumours, invasive transitional cell carcinoma of the bladder, and muscle-invasive bladder cancer.

[0450] Skin cancers and melanomas (as well as non-melanomas) that may be treated include cutaneous T-cell lymphoma, intraocular melanoma, tumour progression of human skin keratinocytes, basal cell carcinoma, and squamous cell cancer. Liver cancers that may be targeted include extrahepatic bile duct cancer, and hepatocellular cancers. Eye cancers that may be targeted include intraocular melanoma, retinoblastoma, and intraocular melanoma.P5598PC00

[0451] Hormonal cancers that may be treated include: parathyroid cancer, pineal and supratentorial primitive neuroectodermal tumours, pituitary tumour, thymoma and thymic carcinoma, thymoma, thymus cancer, thyroid cancer, cancer of the adrenal cortex, and adrenocorticotrophic hormone (ACTH)-producing tumours.

[0452] Miscellaneous other cancers that may be targeted include advanced cancers, AIDS-related, anal cancer adrenal cortical, aplastic anaemia, aniline-induced and betel-induced cancers, buyo cheek cancer, cerebriform, chimney-sweeps' carcinoma, clay pipe-induced cancer, colloid cancer, cystic, dendritic, cancer a deux, duct, dye workers, encephaloid, cancer en cuirasse, endometrial, endothelial, epithelial, glandular, cancer in situ, Kang cancer, Kangri cancer, latent, medullary, melanotic, mule-spinners’, occult cancer, paraffin, pitch workers', scar, schistosomal bladder, scirrhous, lymph node, soft, soot, spindle cell, swamp, tar, and tubular cancers.

[0453] Miscellaneous other cancers that may be targeted also include carcinoid (gastrointestinal and bronchial), Castleman's disease, chronic myeloproliferative disorders, clear cell sarcoma of tendon sheaths, Ewing's family of tumours, head and neck cancer, lip and oral cavity cancer, metastatic squamous neck cancer with occult primary, multiple endocrine neoplasia syndrome, Wilms' tumour, mycosis fungoides, pheochromocytoma, Sezary syndrome, supratentorial primitive neuroectodermal tumours, tumours of unknown primary site, peritoneal effusion, malignant pleural effusion, trophoblastic neoplasms, and hemangiopericytoma.

[0454] The cancer may particularly include but is not limited to any of the following: lung, breast, ovarian, head and neck, pancreatic, epithelioma, sarcoma, neuroblastoma, prostate, colorectal, gastric, small intestine, hepatic, bone, testicular, renal, thyroid cancers.

[0455] In a preferred embodiment, the cancer or tumour to be treated comprises hypoxic regions. Any of (i)-(v) are useful for treating or preventing an autoimmune disease or disorder, astroviruses. The autoimmune disease or disorder may be psoriatic arthritis, rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus (Lupus, SLE), Crohn's disease, celiac disease, ulcerative colitis, Graves' disease, Hashimoto's thyroiditis, Addison's disease, dermatomyositis, psoriasis, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barre syndrome, multiple sclerosis (MS), myasthenia gravis, autoimmune vasculitis, type 1 diabetes, pernicious anaemia or vasculitis.

[0456] Any of (i)-(v) are useful for treating or preventing an immune deficiency disease or disorder. The immunodeficiency disease or disorder may be Autoimmune Lymphoproliferative Syndrome (ALPS), APS-1 (APECED), CARD9, Chronic Granulomatous Disease (CGD), congenital neutropenia syndromes, Common Variable Immunodeficiency (CVID), CTLA4 deficiency, DOCK8 deficiency, glycosylation disorders withP5598PC00

[0457] immunodeficiency, Hyper-Immunoglobulin E Syndromes (HIES), PI3 kinase disease, PLAID, Severe Combined Immunodeficiency (SCID), STAT3 Dominant-Negative Disease, WHIM Syndrome, X-Linked Agammaglobulinemia (XLA) or X-Linked Lymphoproliferative Disease (XLP).

[0458] Any of (i)-(v) are useful for treating or preventing an ischemic disease or disorder. The ischemic disease or disorder may be coronary artery disease, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, congenital heart disease, valvular heart disease, carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, venous thrombosis, including deep vein thrombosis, or a thrombotic microangiopathy, such as thrombocytopenic purpura (TPP).

[0459] Any of (i)-(v) are useful for treating or preventing a disease or disorder requiring regenerative medicine. The disease or disorder may be a cardiovascular disease or disorder, including any of those described above, a neurological disease or disorder, such as Parkinson's disease, Alzheimer's disease, stroke, spinal cord injuries or multiple sclerosis, diabetes, osteoarthritis, wounds, organ damage, disorders requiring an organ transplant, cancer, infertility, blindness, an immunodeficiency disease or disorder, a metabolic disease or disorder, a hepatic disease or disorder, a renal disease or disorder, injury, thalassemia or sickle cell disease.

[0460] The therapy or method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the cells. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease or disorder and / or abolishes one or more symptoms, such as all the symptoms, of the disease or disorder. The therapeutically effective amount preferably cures the disease or disorder. A prophylactically effective amount is an amount which prevents the onset of the disease or disorder and / or prevents the onset of one or more symptoms, such as all the symptoms, of the disease or disorder. The prophylactically effective amount preferably prevents the subject from developing the disease or disorder. Suitable amounts are discussed in more detail below.

[0461] The number of cells administered to the subject should take into account the route of administration, the disease or disorder being treated, the weight of the subject and / or the age of the subject. In general, from about 1 x 106to about 1 x 1012cells are administered to the subject. In one embodiment, from about 1 x 107to about 1 x 1010cells, or from about 1 x 108to about 1 x 109cells are administered to the subject.

[0462] In some embodiments, from about 1 mg / ml to about 100 mg / ml of any of (i)-(v) may be administered to the subject. In some embodiments, from about 1 mg / ml to about 10 mg / ml of any of (i)-(v) are administered to the subject.P5598PC00

[0463] The invention may be used in combination with other means of, and substances for, treating disease or disorder. In some cases, any of any of (i)-(v) may be administered simultaneously, sequentially, or separately with other substances which are intended for treating the disease or disorder or ameliorating the symptoms of the disease or disorder, or for providing pain relief. Any of the populations or the pharmaceutical composition may be used in combination with existing treatments for disease or disorder and may, for example, be simply mixed with such treatments. Thus the invention may be used to increase the efficacy of existing treatments for disease.

[0464] EXAMPLES

[0465] Example 1

[0466] Previously described T4-CAR (which targets ErbB receptor dimers through the TIE binder) and HypoxiCAR-containing SFG retroviral vectors were used, and were also modified to generate new constructs. First, the complementary DNA (cDNA) sequence encoding an anti-B7-H3 CAR, featuring the previously described TE9 binder, was ordered as a cloned gene (GeneArt, Thermo Fisher Scientific). This cDNA sequence was sub-cloned into the T4-HypoxiCAR-containing SFG retroviral vector backbone using the Agel and Xhol restriction endonuclease cleavage sites, producing a hypoxia-restricted anti-B7-H3 CAR hereafter referred to as 'TE9-HypoxiCAR'.

[0467] A cDNA sequence encoding the armouring protein, interferon (IFN)-beta, fused to an ODD, was sub-cloned into the T4-HypoxiCAR-containing SFG retroviral vector backbone using the Agel and Notl restriction endonuclease cleavage sites, producing an IFN-beta-armoured T4 HypoxiCAR. PCR was then carried out to amplify the armour-encoding insert from the IFN-beta-armoured T4-HypoxiCAR and the vector backbone from the TE9-HypoxiCAR plasmid, and the PCR products were joined using HiFi DNA Assembly (New England Biolabs) to produce an IFN-beta-armoured TE9-HypoxiCAR.

[0468] The results are shown in Figure 1.

[0469] Example 2

[0470] In order to assess the expression of the armouring protein in conditions of hypoxia versus normoxia, IFN-beta-armoured TE9-HypoxiCAR-T cells were cultured for 48 hours in experimental hypoxia (0.1% O2), followed by 48 hours in normoxia (20% O2). T cells were seeded at 0.7 x 106cells in 1 mL media (RPMI supplemented with L-glutamine, 10% FBS, 1% penicillin / streptomycin, and 100 IU / mL IL-2) per well in a 12-well plate for each of the following time points: 0, 6, 12, 24, 48, 54, 60, 72, and 96 hours. At each time point, the cell culture supernatant was harvested for ELISA to assess armouring cytokine release. Media was changed for all cells at the 0-hour (start of hypoxia) and 48-hour (start of normoxia) time points.P5598PC00

[0471] The results are shown in Figure 2.

[0472] Example 3

[0473] To investigate the hypoxia-specific regulation of both the CAR and the armouring protein, further experiments were carried out.

[0474] The construct used in these experiments is based on the SFG vector, which retains the viral promoter and enhancer elements within the 5' and 3' long terminal repeats (LTRs) which drive constitutive gene expression, as well as the psi (ψ) packaging sequences required for encapsulation of the genome into viral particles. In the construct shown in Figure 3A, a portion of the 3' LTR of the SFG vector was replaced with a sequence comprising 9 tandem HRE motifs, each motif consisting of SEQ ID NO: 23. Further, the rest of the wild-type viral genome, including other genes required for viral packaging and transduction, are replaced by the transgene(s) of interest, rendering the virus replication-incompetent.

[0475] For these experiments, the transgene of interest from 5' to 3' includes:

[0476] • Mouse IFN-β (mIFNβ) fused to a first ODD having SEQ ID NO: 35;

[0477] • A T2A sequence having SEQ ID NO: 87; and

[0478] • A TIE binder-containing CAR fused to a second ODD having SEQ ID NO: 85.

[0479] This construct is referred to herein as "mIFNp-TlE-HypoxiCAR" or "or "hTlE-mIFNb".

[0480] Figure 3A shows the construct. It includes sequences encoding mouse IFN-β (mIFNβ) and for a TIE binder-containing CAR. These sequences are separated by a self-cleaving T2A peptide. Both the mIFNβ and CAR each have an ODD domain fused to them.

[0481] To assess whether the construct allows for hypoxia-restricted expression of the CAR and for hypoxia-restricted secretion of the mIFNβ, HEK293T cells were transfected with the construct and cultured in either normoxia or hypoxia (0.1% O2) conditions for 24 hours. Flow cytometry was performed to assess CAR expression, and an ELISA was carried out on the cell culture supernatant to assess mIFNβ secretion. The ELISA was performed using a kit as per the manufacturer's protocols (DuoSet; R& D Systems).

[0482] Figure 3B shows significantly increased CAR expression in hypoxia compared to normoxia.

[0483] Figure 3C shows significantly increased mIFNβ secretion in hypoxia compared to normoxia. Only a very low amount of IFNβ secretion was detected in the HEK293T cells cultured under conditions of normoxia.

[0484] Given the positive results obtained transfecting HEK293T cells, the inventors went on to test the effects of transducing human T-cells with a similar construct.P5598PC00

[0485] The construct used in these subsequent experiments, to transduce T-cells, is also based on the SFG vector, where a portion of the 3' LTR of the SFG vector has been replaced with a sequence comprising 9 tandem HRE motifs, each motif consisting of SEQ ID NO: 23. In this case the transgene of interest (which replaces the wild-type viral genome) from 5' to 3' includes:

[0486] • Mouse IFN-β (mIFNβ) fused to a first ODD having SEQ ID NO: 35

[0487] • A T2A having SEQ ID NO: 87

[0488] • A TE9 binder-containing CAR fused to a second ODD having SEQ ID NO: 37.

[0489] In this construct, whilst the second ODD has the same amino acid sequence as the first ODD, it is encoded for by a different nucleotide sequence. The first ODD is encoded for by the underlined portion of SEQ ID NO: 36 and the second ODD is encoded for by the underlined portion of SEQ ID NO: 38. This construct is referred to herein as "mIFN[3-TE9-HypoxiCAR" or "hTE9-mIFNb".

[0490] Figure 3D shows this construct. It includes sequences encoding mouse IFN-β (mIFNβ) and the TE9-containing CAR, separated by a self-cleaving T2A peptide. Both the mIFNβ and CAR each have an ODD domain attached.

[0491] As a control, the inventors also transduced human T-cells with a non-armoured construct shown in Figure 3E. This non-armoured construct includes the TE9 binder-containing CAR and the second ODD under the control of the 9 tandem HRE motifs but does not include the mIFNP fused to the first ODD or the T2A peptide. This construct is referred to herein as " TE9-HypoxiCAR' or "hTE9".

[0492] RD114-pseudotyped retrovirus particles were generated by triple transfection of HEK293T cells with RDF plasmid (RD114 envelope), Peq-Pam plasmid (Moloney Gag-Pol), and the SFG plasmid of interest. Retroviral supernatant was harvested 48 hours and 72 hours after transfection and either used fresh for transduction or snap-frozen and stored at -80°C.

[0493] Activated human T-cells were transduced in non-tissue culture-treated 24-well plates (Corning) that had been pre-coated with 0.5 mL / well of 20 pg / mL RetroNectin (Takara Bio) at 4°C overnight. The RetroNectin solution was aspirated from the 24-well plate, and the plate was blocked with 0.5 mL / well of 2% bovine serum albumin (BSA) for 30 minutes at room temperature. The plate was then washed once with DPBS. Next, 1.5 mL of either fresh or thawed retroviral supernatant was added per well, and the plate was centrifuged at 1000 x g for 2 hours at 32°C, with the brake off. The retroviral supernatant was then removed from the wells, and T-cells were added at 0.3-0.4 x 106cells per well. The plate was incubated at 37°C for 24-72 hours. The T-cells were then harvested, resuspended in freshP5598PC00

[0494] IL-2-supplemented media, and transferred to tissue culture-treated plates or flasks for ongoing culture.

[0495] To assess the dynamics of CAR expression, the CAR-T cells were cultured for 48 hours in experimental hypoxia, followed by 48 hours in normoxia. All in vitro experiments performed in hypoxia were performed at 0.1% oxygen. At each of the following time points, 0, 6, 12, 24, 48, 60, 72, 84 and 96 hours, CAR-T cells were harvested for assessment of CAR expression using flow cytometry, and the cell culture supernatant was harvested for ELISA to assess armouring cytokine release. Media was changed for all cells at the 0-hour (start of hypoxia) and 48-hour (start of normoxia) time points.

[0496] Figure 3F and 3G show more than half of CAR+T-cells expressed detectable CAR within the first 12 hours of hypoxia, and that CAR expression plateaued after 24 hours. After reestablishment of normoxia, CAR downregulation occurred most rapidly in the initial 12 hours and approached or reached baseline expression by 48 hours of normoxia. The expression dynamics of the CAR T-cells transduced with the armoured construct appeared similar to those transduced with the non-armoured construct, suggesting that co-expressing the armouring payload did not affect the stringency of hypoxia-restricted CAR expression.

[0497] Figure 3H shows that the secretion of mIFNP by armoured TE9-HypoxiCAR-T cells was also hypoxia-restricted, with accumulation of mIFNP in the cell culture medium over the course of 48 hours in hypoxia, compared with negligible levels of mIFNP detected over the subsequent 48 hours in normoxia.

[0498] Example 4

[0499] To assess whether the armouring protein is bioactive, T-cells were transduced with either the TE9-HypoxiCAR construct or the mIFN[3-TE9-HypoxiCAR, as outlined in example 3.

[0500] CAR-T cells were resuspended in fresh IL-2-supplemented media and cultured in either normoxia or hypoxia (0.1% oxygen) conditions. After 48 hours, cell culture supernatant was harvested and used to treat Murine bone marrow-derived macrophages (BMDMs). Following 24 hours of treatment, the BMDMs were harvested for flow cytometry staining and analysis to assess the expression of key costimulatory and antigen-presenting molecules (Sca-1, MHCII, CD86 and MHCI H-2Kb).

[0501] Figures 4A-D show that only the media conditioned by hTE9-mIFNb-CAR-T cells in hypoxia upregulate the expression of key costimulatory and antigen-presenting molecules, or interferon-stimulated genes, on BMDMs. An increase was observed in the proportions of BMDMs expressing Sca-1 and MHCII, as well as an increase in overall MHCI H-2Kband CD86 expression as determined by the median fluorescence intensity (MFI) of these markers on BMDMs. Figure 4E shows that although hTE9 conditioned media-treated BMDMs expressedP5598PC00

[0502] MHCI H-2Kb+, their expression was low compared to that of hTE9-mIFNb conditioned media-treated BMDMs.

[0503] Example 5

[0504] Having demonstrated the effects of mIFNP armouring on macrophages in vitro, the effects of the armoured CAR T-cells on other immune cells in the in vivo tumour microenvironment were explored.

[0505] A xenograft model of neuroblastoma was used. SCID / Beige {C^17. C. -PrkdccidLys^9] / C.v\) mice are B and T lymphocyte deficient and have impaired NK cells but have essentially normal myeloid compartments. These mice were inoculated subcutaneously (s.c.) in the right flank with human LAN-1 neuroblastoma cells. Once tumours reached a mean volume of 100-150 mm3, the mice were then treated with non-transduced control T-cells (UTD), hTE9 CAR-T cells or hTE9-mIFNb CAR-T cells by concurrent i.v. (3 x 106T-cells) and intratumoural (i.t.) injection (2 x 106T-cells). After 3 days, the mice were killed and the tumours were harvested for immunophenotyping by flow cytometry. Figure 5A is a schematic showing this workflow.

[0506] Figure 5B shows the proportions of different immune cell subsets expressed as percentage of total live cells in the tumour. There was no significant difference in abundance of macrophages in the tumour microenvironment between mice treated with unarmoured TE9-HypoxiCAR-T cells or armoured TE9-HypoxiCAR-T cells.

[0507] Figures 5C and 5D show that hTE9-mIFNb-CAR-T cell treatment was associated with an increased proportion of MHCI and Sca-l-expressing tumour associated macrophages, as compared with hTE9 CAR-T cells treatment, indicating reprogramming of the macrophages to more activated, pro-inflammatory states.

[0508] To investigate whether the mIFNP armoured CAR-T cells exhibit improved anti-tumour effects, effects against Py8119 cells were tested. In these experiments the T-cells were transduced with either the "mIFNp-TlE-HypoxiCAR" construct described above, or a " T4-HypoxiCAR" construct. The T4-HypoxiCAR includes only the TIE binder-containing CAR and the second ODD under the control of the 9 tandem HRE motifs, but not the mIFNP fused to the first ODD or the T2A.

[0509] Py8119 cells do not significantly express ErbBl, 3, or 4 (data not shown), which suggests that Py8119 cells would not be effectively targeted by TIE binder-containing CAR-T cells. Thus, this provided the opportunity to test the antitumour activity of mIFNp-armoured T1E-HypoxiCAR-T cells against tumour cells with non / low-expressing tumour associated antigens.P5598PC00

[0510] Luciferase-expressing Py8119 cells and the CAR T cells were co-cultured. The Py8119 cells were seeded in 96-well plates. The following day, CAR-T cells were added at a 1:1 effector to target (E: T) ratio. Co-cultures were incubated for 72 hours in normoxia or experimental hypoxia (0.1% oxygen), and tumour cell viability was determined by luciferase quantification following the addition of 1 pL of 15 mg / mL XenoLight D-Luciferin (PerkinElmer) per 100 pL of media. Results of tumour killing assays were normalised to the untransduced T-cell condition.

[0511] Figure 5E shows that, whilst the untransduced T cells (left column) and unarmoured CAR-T cells (middle column) did not elicit significant tumour killing, hTlE-mIFNb-CAR-T cells (right column) reduced tumour cell viability specifically in the hypoxic condition.

[0512] The amount of mouse IFN-p and human IFN-y was assessed using an ELISA kit as per the manufacturer's protocols (DuoSet; R& D Systems).

[0513] Figure 5F shows that hTlE-mIFNb-CAR-T cells (right column) secreted mIFNP specifically in the hypoxic condition. The unarmoured CAR-T cells (middle column) did not secrete mIFN-p under either normoxia or hypoxia. Additionally, the levels of hlFN-y detected for both the armoured and unarmoured CAR-T cells were far below those usually seen for antigen-activated CAR-T cells, further supporting that the decrease in tumour viability observed was predominantly mIFNp-mediated, and not CAR-mediated.

[0514] The ability of the armoured CAR-T cells to target tumour cells with non / low-expressing tumour associated antigens indicates that they could be particularly useful in the treatment of heterogeneous solid tumours.

[0515] Example 6

[0516] A new construct was generated to investigate whether other armouring proteins that are under the transcriptional control of HREs and have their degradation regulated through fusion to an ODD also exhibit effective hypoxia-specific expression and anti-tumour effects.

[0517] The construct includes the following transgene from 5' to 3':

[0518] • pl9 subunit;

[0519] • a P2A sequence;

[0520] • p35 subunit;

[0521] • an E2A sequence;

[0522] • p40 subunit fused to a first ODD;

[0523] • a T2A sequence; and

[0524] • a TE9 binder-containing CAR fused to a second ODD.P5598PC00

[0525] The transgene is under the transcriptional control of a promoter region containing 9 tandem HRE motifs, each motif consisting of SEQ ID NO: 23. This construct encodes for a TE9 binder-containing CAR fused to an ODD as well armouring proteins IL-12 and IL-23, both of which are also fused to an ODD. The construct is referred to herein as "hTE9-mIL12 / 23".

[0526] A non-armoured construct was also used as a comparison, which includes the T9 bindercontaining CAR fused to the second ODD, under the control of the 9 tandem HRE motifs, but does not include the pl9 subunit, the p35 subunit, the p40 subunit fused to the ODD or the 2A peptides. This construct is referred to herein as "hTE9".

[0527] Human T-cells were transduced with hTE9 and hTE9-mIL12 / 23 constructs using retroviruses. RD114-pseudotyped retrovirus particles were generated by triple transfection of HEK293T cells with RDF plasmid (RD114 envelope), Peq-Pam plasmid (Moloney Gag-Pol), and the SFG plasmid of interest. Retroviral supernatant was harvested 48 hours and 72 hours after transfection and either used fresh or snap-frozen and stored at -80°C.

[0528] To assess the dynamics of CAR expression, the CAR-T cells were cultured for 48 hours in experimental hypoxia (0.1% oxygen), followed by 48 hours in normoxia. At each of the following time points, 0, 12, 24, 36, 48, 60, 72, 84 and 96 hours, CAR-T cells were harvested for assessment of CAR expression using flow cytometry, and the cell culture supernatant was harvested for ELISA to assess armouring cytokine release. Media was changed for all cells at the 0-hour (start of hypoxia) and 48-hour (start of normoxia) time points.

[0529] Figure 6A shows similar dynamics of CAR induction and downregulation in hypoxia and normoxia, respectively, are observed between unarmoured and co-armoured TE9-HypoxiCAR T cells, indicating that the incorporation of armouring payloads does not negatively affect the stringency of hypoxia-restricted expression.

[0530] Figure 6B shows that secretion of IL-12 and IL-23 by the co-armoured hTE9-mIL12 / 23-CAR-T cells occurs solely under hypoxic conditions.

[0531] To assess anti-tumour activity, the viability of LAN-l-BFP / Luc tumour cells in vitro was quantified following co-culture with hTE9 and hTE9-mIL12 / 23-CAR T cells at a 1:1 effector to target (E: T) ratio for 72 h in either normoxia or hypoxia (0.1% oxygen).

[0532] Figure 6C shows that the oxygen-sensitive switch is functionally rigorous, as antitumor activity of hTE9-mIL12 / 23-CAR T cells against LAN-1 neuroblastoma cells remains highly specific to hypoxic conditions.

[0533] The T-cells were then tested in vivo, by injection into LAN-1 tumour-bearing SCID / Beige mice. Untransduced T-cells were used as a control. The tumours, lungs, and livers of theP5598PC00

[0534] mice were harvested and enzyme-dissociated for flow cytometry to detect T-cells and evidence of CAR expression.

[0535] Figure 7A shows that, for both hTE9 and hTE9-mIL12 / 23-CAR T cells, CAR expression was only found on the recovered T cells (hCD45+hCD3+) in the tumour, with negligible CAR expression in the lungs or liver. These data highlight that the hTE9-mIL12 / 23-CAR T cells are effectively able to differentiate between the solid tumour microenvironment and healthy tissues with high precision.

[0536] Having demonstrated that hypoxia-restricted expression leads to tumour-selective CAR expression in vivo, armouring-induced tumour microenvironment (TME) changes were explored. The CAR-T cells were concurrently intravenously (i.v.) and intratumourally (i.t.) injected into mice bearing established LAN-1 tumours. After 3 days, the mice were sacrificed, and the tumours were harvested and enzyme-digested to release single cells for immunophenotyping by flow cytometry.

[0537] Figure 7B shows that CAR-T cells that express the IL-12 and IL-23 armouring proteins induce a significant increase in Ml-like MHCII+CD206" tumour associated macrophages (TAMs), which are associated with immunostimulatory and tumouricidal functions. These CAR-T cells also led to a concurrent decrease in immunosuppressive M2-like MHCIT CD206+TAMs in the TME. These data suggest that the armouring proteins lead to functional rewiring of TAMs to antitumor states.

[0538] To explore the therapeutic potential of hTE9-mIL12 / 23-CAR-T cells, mice bearing established LAN-1 tumours were i.v. infused with a therapeutic dose of these cells.

[0539] Figure 7C demonstrates that, whilst unarmoured hTE9-CAR-T cells reduce tumour volume as compared with untransduced T-cells or the vehicle control, hTE9-mIL12 / 23-CAR-T cells deliver a far superior reduction.

[0540] Figure 7D shows that mice maintain their weight for the duration of the experiment, which acts as a strong surrogate for the health of the animal. This indicates the hTE9-mIL12 / 23-CAR-T cells do not cause significant toxicity and thus indicates that hypoxia-specific expression of the armouring proteins is being achieved.

[0541] For validation, the same experiment was performed in a different in vivo neuroblastoma model, SK-N-SH.

[0542] Figure 7E again shows that, whilst unarmoured hTE9-CAR-T cells reduce tumour volume as compared with untransduced T-cells or the vehicle control, hTE9-mIL12 / 23-CAR-T cells deliver a far superior reduction.P5598PC00

[0543] Figure 7F shows that this was achieved in the absence of apparent toxicity, as assessed by animal weight post-CAR T-cell infusion. This again indicates that hypoxia-specific expression of the armouring proteins is being achieved.

[0544] Example 7

[0545] A new construct was generated to further confirm that yet more armouring proteins display hypoxia-specific expression and anti-tumour effects when placed under the transcriptional control of HREs and have their degradation regulated through fusion to an ODD.

[0546] Human T-cells were generated by transduction with a construct that allows for expression of IL-27 and IL-39 alongside a TE9 binder-containing CAR.

[0547] Figure 8A shows the hypoxia-specific expression construct for dual IL-27 and IL-39 armouring. The construct includes the following transgene from 5' to 3':

[0548] • pl9 subunit;

[0549] • a P2A sequence;

[0550] • p28 subunit;

[0551] • a E2A sequence;

[0552] • Ebi3 subunit fused to a first ODD;

[0553] • a T2A sequence; and

[0554] • a TE9 binder-containing CAR fused to a second ODD.

[0555] The transgene is under the transcriptional control of a promoter region containing 9 tandem HRE motifs, each motif consisting of SEQ ID NO: 23. This construct encodes for a TE9 binder-containing CAR fused to an ODD as well armouring proteins IL-27 and IL-39, both of which are also fused to an ODD.

[0556] To assess the expression of the armouring proteins in conditions of hypoxia versus normoxia, the IL-27 / 39-armoured TE9-HypoxiCAR-T cells were cultured for 48 hours in experimental hypoxia (0.1% O2), followed by 48 hours in normoxia (20% O2). T-cells were seeded at 0.7 x 106cells in 1 mL media (RPMI supplemented with L-glutamine, 10% FBS, 1% penicillin / streptomycin, and 100 IU / mL IL-2) per well in a 12-well plate for each of the following time points: 0, 48 and 96 hours. At each time point, the cell culture supernatant was harvested for ELISA to assess IL-27 and IL-39 release. Media was changed for all cells at the 0-hour (start of hypoxia) and 48-hour (start of normoxia) time points.

[0557] Figure 8B and 8C show the successful hypoxia-specific expression of the CAR and the hypoxia-specific secretion of IL-27 and IL-39.

[0558] To investigate effector functions of hTE9-hIL27 / 39-CAR-T cells, an in vitro tumour rechallenge assay with LAN-1 tumour cells was performed. A non-armoured construct was also used as a comparison, which includes the T9 binder-containing CAR fused to the secondP5598PC00

[0559] ODD, under the control of the 9 tandem HRE motifs, but does not include the pl9 subunit, the p28 subunit, the Ebi3 subunit fused to the ODD or the 2A peptides. This construct is referred to herein as "hTE9".

[0560] LAN-l-BFP / Luc cells were seeded onto plates and the following day; CAR T cells were added at a 1:5 effector to target (E: T) ratio. For each subsequent round of tumour rechallenge, all cells in the well were harvested, washed with PBS, resuspended in fresh media, and added to a new plate that had been seeded with fresh tumour cells the day before. At the end of three rounds, cells from duplicate wells were harvested for flow cytometry, and cell culture supernatant was harvested to test for cytokine release using ELISA.

[0561] Figure 8D shows that the hTE9-hIL27 / 39-CAR-T cells demonstrate superior sustained tumour killing compared to hTE9-CAR-T cells.

[0562] Figures 8E shows that IL-27 / 39-armouring is associated with enhanced granzyme B expression.

[0563] These data indicate the hypoxia-specific expression and anti-tumour effects can be achieved when different armouring proteins are under the transcriptional control of HREs and have their degradation regulated through fusion to an ODD.P5598PC00

[0564] SEQUENCES SEQ ID NO: Sequence

[0565] SEQ ID NO: 1 XxX2LE M LA PYIX3M D D DX4X5X6

[0566] HIFl-alpha amino acids

[0567] 558-571

[0568] SEQ ID NO: 2 APAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNEKLQNINL HIFl-alpha amino acids AMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQ 401-603 TPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTE AKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESA SPQSTVTVFQ SEQ ID NO: 3 EFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSF HIFl-alpha amino acids DQLSPLESSSASPESASPQSTVTVFQ

[0569] 530-603

[0570] SEQ ID NO: 4 EFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSF HIFl-alpha amino acids DQLSPLESSSASPESASPQSTVTVFQQTQIQEPTANAI 1 1 IATTDELK 530-653 TVTKDRMEDIKILIASPSPTHIHKETTS

[0571] SEQ ID NO: 5 (A / G)CGT(G / C)

[0572] HIF binding site (HBS)

[0573] SEQ ID NO: 6 ACGTG

[0574] HIF binding site

[0575] SEQ ID NO: 7 CA(C / G)(G / A)(T / C / G)

[0576] HIF ancillary site (HAS)

[0577] SEQ ID NO: 8 CACAG

[0578] HIF ancillary site

[0579] SEQ ID NO: 9 TGACCT

[0580] HNF-4 site

[0581] SEQ ID NO: 10 GGGCCCTACGTGCTGTCTCACACAGC

[0582] HRE from hEPO gene

[0583] SEQ ID NO: 11 GGGCCCTACGTGCTGCCTCGCATGGC

[0584] HRE from mEPO gene

[0585] SEQ ID NO: 12 TGTCACGTCCTGCACGACGCGAGTA

[0586] HRE from hPGK gene

[0587] SEQ ID NO: 13 CGCGTCGTGCAGGACGTGACAAAT

[0588] HRE from mPGK gene

[0589] SEQ ID NO: 14 CCAGCGGACGTGCGGGAACCCACGTGTAGG

[0590] HRE from mLDH gene

[0591] SEQ ID NO: 15 TCCACAGGCGTGCCGTCTGACACGCA

[0592] HRE from Glucose trpt

[0593] gene

[0594] SEQ ID NO: 16 CCACAGTGCATACGTGGGCTCCAACAGGTCCTCTT

[0595] HRE from hVEGF gene

[0596]

[0597] SEQ ID NO: 17 TACGTGGGP5598PC00

[0598] HRE from mVEGF gene

[0599] SEQ ID NO: 18 ACAGTGCATACGTGGGCTTCCACA

[0600] HRE from rVEGF gene

[0601] SEQ ID NO: 19 ACTACGTGCTGCCTAGG

[0602] HRE from hNOS gene

[0603] SEQ ID NO: 20 CCCCTCGGACGTGACTCGGACCACAT

[0604] HRE from hAldolase

[0605] gene

[0606] SEQ ID NO: 21 ACGCTGAGTGCGTGCGGGACTCGGAGTACGTGACGGA

[0607] HRE from hEnolase

[0608] gene

[0609] SEQ ID NO: 22 CGGACGCTGGCGTGGCACGTCCTCTC

[0610] HRE from mHeme

[0611] Oxygenase gene

[0612] SEQ ID NO: 23 GGCCCTACGTGCTGTCTCACACAGCCTGTCTGAC

[0613] HRE sequence

[0614] SEQ ID NO: 24 GGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCCCTACGTGC Three sequential copes TGTCTCACACAGCCTGTCTGACGGCCCTACGTGCTGTCTCACACAG of SEQ ID NO: 23 CCTGTCTGAC

[0615] SEQ ID NO: 25 GGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCCCTACGTGC five sequential copes of TGTCTCACACAGCCTGTCTGACGGCCCTACGTGCTGTCTCACACAG SEQ ID NO: 23 CCTGTCTGACGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGG CCCTACGTGCTGTCTCACACAGCCTGTCTGAC SEQ ID NO: 26 GGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCCCTACGTGC nine sequential copes of TGTCTCACACAGCCTGTCTGACGGCCCTACGTGCTGTCTCACACAG SEQ ID NO: 23 CCTGTCTGACGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGG CCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCCCTACGTGCTG TCTCACACAGCCTGTCTGACGGCCCTACGTGCTGTCTCACACAGCC TGTCTGACGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCC CTACGTGCTGTCTCACACAGCCTGTCTGAC SEQ ID NO: 27 GGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCCCTACG Regulatory region TGCTGTCTCACACAGCCTGTCTGACGGCCCTACGTGCTGTCTCACA CAGCCTGTCTGACGGCCCTACGTGCTGTCTCACACAGCCTGTCTGA

[0616] Single HRE in bold CGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACGGCCCTACGTG Nine tandem HREs CTGTCTCACACAGCCTGTCTGACGGCCCTACGTGCTGTCTCACACA underlined GCCTGTCTGACGGCCCTACGTGCTGTCTCACACAGCCTGTCTGACG Unmodified portion of 3' GCCCTACGTGCTGTCTCACACAGCCTGTCTGACTCTAGAGA4CCAT long terminal repeat of CAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACCCTGTGCCT Moloney murine TATTTGAACTAACCAATCAGTTCGC 11 Cl CGC 1 1 Cl GTTCGCGCGCT leukaemia virus in TCTGCTCCCCGA GCTCAA TAAAA GA GCCCA CAA CCCCTCA CTCGGG italics GCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCC AA TAAACCC IC. I I GCAGTTGCA TCCGACTTGTGGTCTCGCTGTTCCT TGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTC TTTCA SEQ ID NO: 28 GTCTCA

[0617] Linker sequence

[0618] SEQ ID NO: 29 GGCCCT

[0619] Linker sequence

[0620]

[0621] P5598PC00

[0622] SEQ ID NO: 30 VVSHFNDCPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYR T1E sequence DLKWWELR

[0623] SEQ ID NO: 31 QVQLQQSGAALVKPGTSVKLSCKASGYTFTSYWMHWVKQRPGQGL TE9 sequence EWIGMIHPKSGSVDYNEKFTNKATLTGDKSSGTAYMQLSSLTSEDSA VYYCARGGYGSPFDYWGQGTTVTVSSGGGGSGGGGSGGGGSENVL TQSPAIMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYRTS NLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFG GGTKLEIKR SEQ ID NO: 32 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG CD3z or CD3 zeta GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG (intracellular domain) LSTATKDTYDALHMQALPPR

[0624] SEQ ID NO: 33 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGG CD28 VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPY (transmembrane APPRDFAAYRS

[0625] domain)

[0626] SEQ ID NO: 34 TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW CD8o (transmembrane APLAGTCGVLLLSLVITLYC

[0627] domain)

[0628] SEQ ID NO: 35 MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLE Mouse interferon-beta+ QLNGKINLTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFL ODD VFRNNFSSTGWNETIVVRLLDELHQQTVFLKTVLEEKQEERLT WEMSSTALHLKSYYWRVQRYLKLMKYNSYAWMWRAEIFRN

[0629] Armouring protein F LII RRLTRN FONAPAAGDTIISLDFGSNDTETDDOQLEEVPLYNDV (mouse IFN-beta) in MLPSPNEKLONINLAMSPLPTAETPKPLRSSADPALNOEVALKLEPNPE bold. SLELSFTMPOIODOTPSPSDGSTROSSPEPNSPSEYCFYVDSDMVNE ODD underlined. FKLELVEKLFAEDTEAKNPFSTODTDLDLEMLAPYIPMDDDFOLRSFD OLSPLESSSASPESASPQSTVTVFQ SEQ ID NO: 36 ATGAACAACAGGTGGATCCTGCACGCCGCCTTCCTGCTGTGCT Mouse interferon-beta+ TCAGCACCACCGCCCTGAGCATCAACTACAAGCAGCTGCAGCT ODD GCAGGAGAGGACCAACATCAGGAAGTGCCAGGAGCTGCTGG AGCAGCTGAACGGCAAGATCAACCTGACCTACAGGGCCGACT

[0630] Armouring protein TCAAGATCCCCATGGAGATGACCGAGAAGATGCAGAAGAGCT (mouse IFN-beta) in ACACCGCCTTCGCCATCCAGGAGATGCTGCAGAACGTGTTCCT bold. GGTGTTCAGGAACAACTTCAGCAGCACCGGCTGGAACGAGAC ODD underlined. CATCGTGGTGAGGCTGCTGGACGAGCTGCACCAGCAGACCGT GTTCCTGAAGACCGTGCTGGAGGAGAAGCAGGAGGAGAGGC TGACCTGGGAGATGAGCAGCACCGCCCTGCACCTGAAGAGCT ACTACTGGAGGGTGCAGAGGTACCTGAAGCTGATGAAGTACA ACAGCTACGCCTGGATGGTGGTGAGGGCCGAGATCTTCAGGA ACTTCCTGATCATCAGGAGGCTGACCAGGAACTTCCAGAACGC TCCTGCTGCAGGCGATACCATTATCAGCCTGGACTTCGGATCTAAT GATACCGAGACAGACGATCAACAGCTGGAAGAGGTGCCCCTGTAC AACGACGTGATGTTGCCTTCTCCAAATGAGAAGCTGCAAAACATCA ACCTCGCCATGAGCCCCCTGCCTACTGCCGAGACACCTAAACCTCT GAGGTCCTCTGCCGATCCCGCTCTGAACCAGGAGGTGGCCCTGAA GCTGGAGCCCAACCCTGAATCTCTTGAGCTGAGCTTCACAATGCCT CAAATCCAAGACCAAACCCCATCACCCAGCGACGGCTCTACGCGTC AGTCTAGCCCCGAACCAAACAGCCCTAGCGAGTACTGCTTCTACGT TGACAGCGACATGGTGAACGAGTTTAAACTGGAGCTCGTGGAGAA GCTGTTCGCCGAGGATACCGAGGCCAAAAATCCCTTCAGCACCCAA GATACCGACCTGGATCTTGAAATGCTGGCCCCTTACATTCCCATGG

[0631]

[0632] ACGACGATTTTCAACTGCGGTCTTTGACCAACTGAGCCCTCTGGAP5598PC00

[0633] GTCTAGCTCTGCCTCTCCAGAGTCCGCCAGCCCACAGTCTACCGTG ACCGTGTTTCAA SEQ ID NO: 37 METDTLLLWVLLLWVPGSTGQVQLQQSGAALVKPGTSVKLSC TE9-CAR targeting B7- KASGYTFTSYWMHWVKQRPGQGLEWIGMIHPKSGSVDYNEK H3 + ODD FTNKATLTGDKSSGTAYMQLSSLTSEDSAVYYCARGGYGSPFD YWGQGTTVTVSSGGGGSGGGGSGGGGSENVLTQSPAIMSAS CAR in bold PGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYRTSNLASG ODD underlined VPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPPTFG GGTKLEIKRTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAV HTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHS DYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KN PQ EG LYN E LQ KD KM AE AYSE IG M KG E RRRG KG H DG LYQG LSTATKDTYDALH MOALPPRAPAAGDTIISLDFGSNDTETDDQQL EEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQ SEQ ID NO: 38 ATGGAAACCGACACGCTACTGCTGTGGGTCCTCCTGCTGTGG TE9-CAR targeting B7- GTCCCTGGCTCCACTGGACAGGTGCAGCTGCAGCAGAGCGGA H3 + ODD GCGGCGCTGGTGAAGCCCGGCACCAGTGTCAAGCTGTCATGC AAAGCCAGCGGCTACACCTTTACGTCTTATTGGATGCACTGGG CAR in bold TGAAGCAGAGACCGGGCCAGGGCCTGGAGTGGATCGGTATG ODD underlined ATCCACCCCAAGTCCGGAAGCGTGGATTACAACGAGAAGTTC ACAAACAAGGCTACACTGACAGGCGACAAGAGCTCCGGAACA GCCTACATGCAGCTGTCCTCTCTGACCAGCGAGGATTCTGCCG TGTACTACTGCGCCAGAGGCGGCTACGGCTCGCCTTTCGACTA CTGGGGCCAGGGCACCACAGTCACCGTGTCTTCCGGGGGTGG TGGTAGTGGAGGCGGTGGCAGCGGAGGCGGTGGCTCAGAGA ACGTGCTGACCCAGAGTCCAGCCATCATGAGCGCTTCTCCTGG CGAAAAGGTGACCATGACCTGCTCTGCTTCTAGTTCCGTAAGC TACATGTATTGGTACCAACAGAAGCCAGGGAGCAGCCCCCGC CTGCTGATCTATAGAACTTCTAATCTGGCCTCTGGCGTGCCTG CCAGATTCAGCGGCTCCGGGTCAGGTACCAGCTACTCCTTGAC CATTAGCTCCATGGAGGCCGAGGACGCCGCCACTTACTACTGC CAGCAGTGGTCTTCTAATCCTCCTACATTCGGCGGGGGGACCA AGCTGGAAATCAAGCGGACCACTACTCCTGCTCCCCGGCCTCC AACCCCAGCCCCTACAATCGCCAGCCAGCCTCTGTCTCTGAGA CCCGAGGCATGCCGGCCAGCAGCTGGAGGAGCCGTGCACACA CGTGGCCTGGACTTTGCCTGCGACATCTATATTTGGGCTCCGT TGGCTGGCACTTGTGGCGTTCTTCTGCTGTCGCTGGTGATCAC CTTATACTGTCGGTCCAAGAGATCCCGCCTCCTGCATAGCGAC TATATGAACATGACACCGCGCCGGCCCGGCCCTACCCGCAAG CATTACCAGCCCTATGCCCCTCCCCGGGACTTCGCGGCCTACC GCAGCAGAGTGAAATTTAGCAGATCTGCTGACGCCCCTGCCT ACCAACAGGGACAGAACCAGCTGTACAACGAACTAAACCTGG GGCGGAGAGAGGAGTACGACGTCTTGGACAAGCGCAGGGGC CGCGACCCAGAGATGGGAGGGAAACCTAGAAGAAAGAACCC TCAGGAGGGCCTGTACAACGAGCTGCAGAAAGACAAGATGGC CGAAGCCTACTCCGAGATCGGCATGAAGGGCGAGCGGCGCAG AGGCAAGGGCCACGACGGTCTTTATCAGGGTCTTTCCACCGCC ACCAAAGACACTTACGATGCCCTGCACATGCAGGCCCTGCCCC CCAGAGCCCCAGCCGCTGGAGACACAATCATATCTTTAGA 1 1 1 1 GG CAGCAACGACACAGAAACTGATGACCAGCAACTTGAGGAAGTACCA TTATATAATGATGTAATGCTCCCCTCACCCAACGAAAAATTACAGAA TATAAATTTGGCAATGTCTCCATTACCCACCGCTGAAACGCCAAAG

[0634]

[0635] CCACTTCGAAGTAGTGCTGACCCTGCACTCAATCAAGAAGTTGCATP5598PC00

[0636] TAAAATTAGAACCAAATCCAGAGTCACTGGAAC 1 1 1 C 1 1 1 1 ACCATG CCCCAGATTCAGGATCAGACACCTAGTCCTTCCGATGGAAGCACTA GACAAAGTTCACCTGAGCCTAATAGTCCCAGTGAATATTG 1 1 1 1 1 A 1 GTGGATAGTGATATGGTCAATGAATTCAAGTTGGAATTGGTAGAAA AAC 1 1 1 1 1 GCTGAAGACACAGAAGCAAAGAACCCA 1 1 1 1 L 1 ACTCAG GACACAGATTTAGACTTGGAGATGTTAGCTCCCTATATCCCAATGG ATGATGACTTCCAGTTACGTTCCTTCGATCAGTTGTCACCATTAGAA AGCAGTTCCGCAAGCCCTGAAAGCGCAAGTCCTCAAAGCACAGTTA CAGTATTCCAG SEQ ID NO: 39 MASPFALLMVLVVLSCKSSCSLGCDLPETHSLDNRRTLMLLAQMSRIS Human interferon- PSSCLMDRHDFGFPQEEFDGNQFQKAPAISVLHELIQQIFNLFTTKDS alphal / 13 amino acid SAAWDEDLLDKFCTELYQQLNDLEACVMQEERVGETPLMNADSILAV sequence KKYFRRITLYLTEKKYSPCAWEVVRAEIM RSLSLSTN LQERLRRKE https: / / www.uniprot.org

[0637] / uniprotkb / P01562 / entr

[0638] SEQ ID NO: 40 MALTFALLVALLVLSCKSSCSVGCDLPQTHSLGSRRTLMLLAQMRRIS Human interferon- LFSCLKDRHDFGFPQEEFGNQFQKAETIPVLHEMIQQIFNLFSTKDSS alpha2 amino acid AAWDETLLDKFYTELYQQLNDLEACVIQGVGVTETPLMKEDSILAVRK sequence YFQRITLYLKEKKYSPCAWEVVRAEIMRSFSLSTNLQESLRSKE ttpsiZ / wwwx^

[0639] SEQ ID NO: 41 MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRIS Human interferon- HFSCLKDRHDFGFPEEEFDGHQFQKAQAISVLHEMIQQTFNLFSTED alpha4 amino acid SSAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAV sequence RKYFQRITLYLTEKKYSPCAWEVVRAEIMRSLSFSTNLQKRLRRKD

[0640] SEQ ID NO: 42 MALPFVLLMALVVLNCKSICSLGCDLPQTHSLSNRRTLMIMAQMGRIS Human interferon- PFSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKD alpha5 amino acid SSATWDETLLDKFYTELYQQLNDLEACMMQEVGVEDTPLMNVDSILT sequence VRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSAN LQERLRRKE https: / / www.uniprot.org

[0641] unipt otkb P01 bt>'9 >'ntt

[0642] SEQ ID NO: 43 MALPFALLMALVVLSCKSSCSLDCDLPQTHSLGHRRTMMLLAQMRRI Human interferon- SLFSCLKDRHDFRFPQEEFDGNQFQKAEAISVLHEVIQQTFNLFSTKD alpha6 amino acid SSVAWDERLLDKLYTELYQQLNDLEACVMQEVWVGGTPLMNEDSILA sequence VRKYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSSSRN LQERLRRKE ttpsiZ / wwwx^

[0643] SEQ ID NO: 44 MARSFSLLMVVLVLSYKSICSLGCDLPQTHSLRNRRALILLAQMGRISP

[0644]

[0645] FSCLKDRHEFRFPEEEFDGHQFQKTQAISVLHEMIQQTFNLFSTEDSSP5598PC00

[0646] Human interferonAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDFILAVRK alpha? amino acid YFQRITLYLMEKKYSPCAWEVVRAEIMRSFSFSTNLKKGLRRKD sequence

[0647] https: / / www.uniprot.org

[0648] / uniprotkb / P01567 / entr

[0649] SEQ ID NO: 45 MALTFYLLVALVVLSYKSFSSLGCDLPQTHSLGNRRALILLAQMRRISP Human interferon- FSCLKDRHDFEFPQEEFDDKQFQKAQAISVLHEMIQQTFNLFSTKDSS alpha8 amino acid AALDETLLDEFYIELDQQLNDLESCVMQEVGVIESPLMYEDSILAVRKY sequence FQRITLYLTEKKYSSCAWEVVRAEIMRSFSLSINLQKRLKSKE ttpsiZ / wwwx^

[0650] SEQ ID NO: 46 MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLGQMGRIS Human interferon- PFSCLKDRHDFRIPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTEDS alphalO amino acid SAAWEQSLLEKFSTELYQQLNDLEACVIQEVGVEETPLMNEDSILAVR sequence KYFQRITLYLIERKYSPCAWEVVRAEIM RSLSFSTN LQKRLRRKD https: / / www.uniprot.org

[0651] / uniprotkb / P01566 / entr

[0652] SEQ ID NO: 47 MALPFALMMALVVLSCKSSCSLGCNLSQTHSLNNRRTLMLMAQMRRI Human interferon- SPFSCLKDRHDFEFPQEEFDGNQFQKAQAISVLHEMMQQTFNLFSTK alphal4 amino acid NSSAAWDETLLEKFYIELFQQMNDLEACVIQEVGVEETPLMNEDSILA sequence VKKYFQRITLYLM EKKYSPCAWEVVRAEI M RSLSFSTN LQKRLRRKD https: / / www.uniprot.org

[0653] / uniprotkb / P01570 / entr

[0654] SEQ ID NO: 48 MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRIS Human interferon- HFSCLKDRYDFGFPQEVFDGNQFQKAQAISAFHEMIQQTFNLFSTKD alphal6 amino acid SSAAWDETLLDKFYIELFQQLNDLEACVTQEVGVEEIALMNEDSILAV sequence RKYFQRITLYLMGKKYSPCAWEVVRAEIMRSFSFSTNLQKGLRRKD

[0655] SEQ ID NO: 49 MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISP Human interferon- FSCLKDRHDFGLPQEEFDGNQFQKTQAISVLHEMIQQTFNLFSTEDS alphal7 amino acid SAAWEQSLLEKFSTELYQQLNNLEACVIQEVGMEETPLMNEDSILAVR sequence KYFQRITLYLTEKKYSPCAWEVVRAEIM RSLSFSTN LQKILRRKD https: / / www.uniprot.org

[0656] / uniprotkb / P01571 / entr

[0657] SEQ ID NO: 50 MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISP Human interferon- FSCLKDRHDFGFPQEEFDGNQFQKAQAISVLHEMIQQTFNLFSTKDS alpha21 amino acid SATWEQSLLEKFSTELNQQLNDLEACVIQEVGVEETPLMNVDSILAVK sequence KYFQRITLYLTEKKYSPCAWEVVRAEIMRSFSLSKIFQERLRRKE

[0658]

[0659] P5598PC00

[0660] unipt otkb I 01 >'ntt

[0661] SEQ ID NO: 51 MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKI Mouse interferon -beta N LTYRADFKI PM EMTEKMQKSYTAFAIQEM LQN VFLVFRN N FSSTG W amino acid sequence NETIVVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWR VQRYLKLMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQN

[0662] https: / / www.uniprot.org

[0663] / uniprotkb / P01575 / entr

[0664] SEQ ID NO: 52 MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGR Human interferon -beta LEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSS amino acid sequence TGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKR YYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

[0665] ttpsiZ / wwwx^

[0666] SEQ ID NO: 53 MIIKHFFGTVLVLLASTTIFSLDLKLIIFQQRQVNQESLKLLNKLQTLSI Human interferon- QQCLPHRKNFLLPQKSLSPQQYQKGHTLAILHEMLQQIFSLFRANISL epsilon amino acid DGWEENHTEKFLIQLHQQLEYLEALMGLEAEKLSGTLGSDNLRLQVK sequence: MYFRRIHDYLENQDYSTCAWAIVQVEISRCLFFVFSLTEKLSKQGRPL NDMKQELTTEFRSPR

[0667] https: / / www.uniprot.org

[0668] protkb / Q86WN2 / ent

[0669] SEQ ID NO: 54 MSTKPDMIQKCLWLEILMGIFIAGTLSLDCNLLNVHLRRVTWQNLRHL Human interferon- SSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNI kappa amino acid FSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMK sequence ENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIR RCLYYFYKFTALFRRK

[0670] SEQ ID NO: 55 MALLFPLLAALVMTSYSPVGSLGCDLPQNHGLLSRNTLVLLHQMRRIS Human interferon- PFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTER omegal amino acid SSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALT sequence LRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQERLRSKD RDLGSS

[0671] ttpsiZ / wwwx^

[0672] SEQ ID NO: 56 MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADN Human interferonGTLFLGILKNWKEESDRKIMQSQIVSFYFKLFKNFKDDQSIQKSVETI gamma amino acid KEDMNVKFFNSNKKKRDDFEKLTNYSVTDLNVQRKAIHELIQVMAEL sequence SPAAKTGKRKRSQMLFRGRRASQ

[0673] https: / / www.uniprot.org

[0674] / uniprotkb / P01579 / entr

[0675] SEQ ID NO: 57 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGIN Human interleukin-2 NYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF

[0676]

[0677] amino acid sequenceP5598PC00

[0678] HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSI

[0679] https: / / www.uniprot.org ISTLT

[0680] protkb / P60568 / entr

[0681] SEQ ID NO: 58 MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCT Human interleukin-4 ELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQF amino acid sequence HRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMR EKYSKCSS

[0682] https: / / www.uniprot.org

[0683] / uniprotkb / P05112 / entr

[0684] y

[0685] SEQ ID NO: 59 MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLL Human interleukin-7 DSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNS amino acid sequence TGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSL KEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH

[0686] SEQ ID NO: 60 MLLAMVLTSALLLCSVAGQGCPTLAGILDINFLINKMQEDPASKCHCS Human interleukin-9 ANVTSCLCLGIPSDNCTRPCFSERLSQMTNTTMQTRYPLIFSRVKKSV amino acid sequence EVLKNNKCPYFSCEQPCNQTTAGNALTFLKSLLEIFQKEKMRGMRGKI https: / / www.uniprot.org

[0687] / uniprotkb / P15248 / entr

[0688] SEQ ID NO: 61 MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEAN Human interleukin-15 WVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQV amino acid sequence ISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKE FLQSFVHIVQMFINTS

[0689] ttpsiZ / wwwx^

[0690] SEQ ID NO: 62 MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVD Human interleukin-21 QLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNER amino acid sequence IINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSL LQKMIHQHLSSRTHGSEDS

[0691] SEQ ID NO: 63 MAAEPVEDNCINFVAMKFIDNTLYFIAEDDENLESDYFGKLESKLSVIR Human interleukin-18 NLNDQVLFIDQGNRPLFEDMTDSDCRDNAPRTIFIISMYKDSQPRGM amino acid sequence AVTISVKCEKISTLSCENKIISFKEMNPPDNIKDTKSDIIFFQRSVPGH DNKMQFESSSYEGYFLACEKERDLFKLILKKEDELGDRSIMFTVQNED

[0692] https: / / www.uniprot.org

[0693] uniptotkb QI 4116 ontt

[0694] SEQ ID NO: 64 MHSSALLCCLVLLTGVRASPGQGTQSENSCTHFPGNLPNMLRDLRDA Human interleukin-10 FSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVM amino acid sequence PQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQV

[0695] KNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN

[0696]

[0697] P5598PC00

[0698] y

[0699] SEQ ID NO: 65 MSTESMIRDVELAEEALPKKTGGPQGSRRCLFLSLFSFLIVAGATTLFC Human tumour necrosis LLHFGVIGPQREEFPRDLSLISPLAQAVRSSSRTPSDKPVAHVVANPQ factor amino acid AEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIYSQVLFKGQG sequence CPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEP IYLGGVFQLEKGDRLSAEINRPDYLDFAESGQVYFGIIAL

[0700] Jlttpsi / 7w\w

[0701] SEQ ID NO: 66 MWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRD Human granulocyteTAAEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTM macrophage colonyMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE stimulating factor amino

[0702] acid sequence

[0703] ttpsiZ / wwwA^

[0704] SEQ ID NO: 67 MLDCRAVIMLWLLPWVTQGLAVPRSSSPDWAQCQQLSRNLCMLAW Mouse interleukin-23 NAHAPAGHMNLLREEEDEETKNNVPRIQCEDGCDPQGLKDNSQFCL subunit alpha IL-23 QRIRQGLAFYKHLLDSDIFKGEPALLPDSPMEQLHTSLLGLSQLLQPED pl9) amino acid HPRETQQMPSLSSSQQWQRPLLRSKILRSLQAFLAIAARVFAHGAATL sequence TEPLVPTA

[0705] Forms IL-23 with SEQ

[0706] ID NO: 69

[0707] SEQ ID NO: 68 MCQSRYLLFLATLALLNHLSLARVIPVSGPARCLSQSRNLLKTTDDMV Mouse interleukin-12 KTAREKLKHYSCTAEDIDHEDITRDQTSTLKTCLPLELHKNESCLATRE subunit alpha (IL-12 TSSTTRGSCLPPQKTSLMMTLCLGSIYEDLKMYQTEFQAINAALQNHN p35) amino acid HQQIILDKGMLVAIDELMQSLNHNGETLRQKPPVGEADPYRVKMKLC sequence ILLHAFSTRVVTINRVMGYLSSA

[0708] Forms IL-12 with SEQ

[0709] ID NO: 69

[0710] SEQ ID NO: 69 MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVN Mouse interleukin-12 LTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGG subunit beta (IL-12 ETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWL p40) amino acid VQRNMDLKFNIKSSSSSPDSRAVTCGMASLSAEKVTLDQRDYEKYSV sequence [335 amino SCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNL acids, 1005 base pairs]: QMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEE GCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPC RVRS SEQ ID NO: 70 MLGSRAVMLLLLLPWTAQGRAVPGGSSPAWTQCQQLSQKLCTLAWS Human interleukin-23 AHPLVGHMDLREEGDEETTNDVPHIQCGDGCDPQGLRDNSQFCLQR subunit alpha (IL-23 IHQGLIFYEKLLGSDIFTGEPSLLPDSPVGQLHASLLGLSQLLQPEGHH pl9) amino acid WETQQIPSLSPSQPWQRLLLRFKILRSLQAFVAVAARVFAHGAATLSP sequence

[0711] Forms IL-23 with SEQ

[0712] ID NO: 72

[0713]

[0714] P5598PC00

[0715] Forms IL-39 with SEQ

[0716] ID NO: 76

[0717] SEQ ID NO: 71 MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAV Human interleukin-12 SNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCL subunit alpha (IL-12 NSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKL p35) amino acid LMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKI sequence KLCILLHAFRIRAVTIDRVMSYLNAS

[0718] Forms IL-12 with SEQ

[0719] ID NO: 72

[0720] SEQ ID NO: 72 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVV Human interleukin-12 LTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGG subunit beta (IL-12 EVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTC p40) amino acid WWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEY sequence SVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPP KNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREK KDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS SEQ ID NO: 73 MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREF Human interleukin-27 TVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQ subunit alpha amino AWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMR acid sequence LDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQ GPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPT

[0721] https: / / www.uniprot.org LSPQP

[0722] uniptotkb QSNF\ '9 ><ntr

[0723] SEQ ID NO: 74 MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCS Human interleukin-27 WTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDV subunit beta amino acid QLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAER sequence QLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILR AVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK

[0724] https: www.uniptot.otQ

[0725] uniptotkb Q14211 entt

[0726] SEQ ID NO: 75 MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAV Human interleukin-12 SNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCL subunit alpha (IL-12 NSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKL p35) amino acid LMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKI sequence KLCILLHAFRIRAVTIDRVMSYLNAS

[0727] Forms IL-35 with SEQ

[0728] ID NO: 76

[0729] SEQ ID NO: 76 MTPQLLLALVLWASCPPCSGRKGPPAALTLPRVQCRASRYPIAVDCS Human interleukin-27 WTLPPAPNSTSPVSFIATYRLGMAARGHSWPCLQQTPTSTSCTITDV subunit beta amino acid QLFSMAPYVLNVTAVHPWGSSSSFVPFITEHIIKPDPPEGVRLSPLAER sequence QLQVQWEPPGSWPFPEIFSLKYWIRYKRQGAARFHRVGPIEATSFILR AVRPRARYYVQVAAQDLTDYGELSDWSLPATATMSLGK

[0730] https: / / www.uniprot.org

[0731] / uniprotkb / Q14213 / entr

[0732] Forms IL-27 with SEQ

[0733] ID NO: 84

[0734]

[0735] P5598PC00

[0736] Forms IL-35 with SEQ

[0737] ID NO: 75

[0738] Forms IL-39 with SEQ

[0739] ID NO: 70

[0740] SEQ ID NO: 77 MAKVPDMFEDLKNCYSENEEDSSSIDHLSLNQKSFYHVSYGPLHEGC Human interleukin- MDQSVSLSISETSKTSKLTFKESMVVVATNGKVLKKRRLSLSQSITDD lalpha amino acid DLEAIANDSEEEIIKPRSAPFSFLSNVKYNFMRIIKYEFILNDALNQSIIR sequence ANDQYLTAAALHNLDEAVKFDMGAYKSSKDDAKITVILRISKTQLYVT AQDEDQPVLLKEMPEIPKTITGSETNLLFFWETHGTKNYFTSVAHPNL

[0741] https: / / www.uniprot.org FIATKQDYWVCLAGGPPSITDFQILENQA / uniprotkb / P01583 / entr

[0742] y

[0743] SEQ ID NO: 78 MAEVPELASEMMAYYSGNEDDLFFEADGPKQMKCSFQDLDLCPLDG Human interleukin- GIQLRISDHHYSKGFRQAASVVVAMDKLRKMLVPCPQTFQENDLSTF Ibeta amino acid FPFIFEEEPIFFDTWDNEAYVHDAPVRSLNCTLRDSQQKSLVMSGPYE sequence LKALHLQGQDMEQQVVFSMSFVQGEESNDKIPVALGLKEKNLYLSCV LKDDKPTLQLESVDPKNYPKKKMEKRFVFNKIEINNKLEFESAQFPNW YISTSQAENMPVFLGGTKGGQDITDFTMQFVSS SEQ ID NO: 79 MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVPPGEDSKDVAAPHRQPLT Human interleukin-6 SSERIDKQIRYILDGISALRKETCNKSNMCESSKEALAENNLNLPKMA amino acid sequence EKDGCFQSGFNEETCLVKIITGLLEFEVYLEYLQNRFESSEEQARAVQ MSTKVLIQFLQKKAKNLDAITTPDPTTNASLLTKLQAQNQWLQDMTT

[0744] https: / / www.uniprot.org HLILRSFKEFLQSSLRALRQM

[0745] / uniprotkb / P05231 / entr

[0746] y

[0747] SEQ ID NO: 80 MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNL Human interleukin-17A NIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRH amino acid sequence LGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGC TCVTPIVHHVA

[0748] ttpsiZ / wwwx^

[0749] SEQ ID NO: 81 MTSKLAVALLAAFLISAALCEGAVLPRSAKELRCQCIKTYSKPFHPKFIK Human interleukin-8 ELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAEN amino acid sequence S

[0750] SEQ ID NO: 82 M KPKM KYSTN KISTAKWKNTASKALCFKLGKSQQKAKEVCPM YFM KL Human interleukin-33 RSGLMIKKEACYFRRETTKRPSLKTGRKHKRHLVLAACQQQSTVECFA amino acid sequence FGISGVQKYTRALHDSSITGISPITEYLASLSTYNDQSITFALEDESYEI YVEDLKKDEKKDKVLLSYYESQHPSNESGDGVDGKMLMVTLSPTKDF

[0751] https: / / www.uniprot.org WLHANNKEHSVELHKCEKPLPDQAFFVLHNMHSNCVSFECKTDPGVF / uniprotkb / O9576Q / entr IGVKDNHLALIKVDSSENLCTENILFKLSET

[0752] y

[0753] SEQ ID NO: 83 MGKGDPKKPRGKMSSYAFFVQTCREEHKKKHPDASVNFSEFSKKCS Human high mobility ERWKTMSAKEKGKFEDMAKADKARYEREMKTYIPPKGETKKKFKDPN group protein Bl amino APKRPPSAFFLFCSEYRPKIKGEHPGLSIGDVAKKLGEMWNNTAADD acid sequence KQPYEKKAAKLKEKYEKDIAAYRAKGKPDAAKKGVVKAEKSKKKKEE

[0754] EEDEEDEEDEEEEEDEEDEDEEEDDDDE

[0755]

[0756] P5598PC00

[0757] y

[0758] SEQ ID NO: 84 MGQTAGDLGWRLSLLLLPLLLVQAGVWGFPRPPGRPQLSLQELRREF Human p28 subunit TVSLHLARKLLSEVRGQAHRFAESHLPGVNLYLLPLGEQLPDVSLTFQ sequence AWRRLSDPERLCFISTTLQPFHALLGGLGTQGRWTNMERMQLWAMR LDLRDLQRHLRFQVLAAGFNLPEEEEEEEEEEEEERKGLLPGALGSALQ

[0759] https: / / www.uniprot.org GPAQVSWPQLLSTYRLLHSLELVLSRAVRELLLLSKAGHSVWPLGFPT protkb / Q8NEV9 / entr LSPQP

[0760] Forms IL-27 with SEQ

[0761] ID NO: 76

[0762] SEQ ID NO: 85 MGPGVLLLLLVATAWHGQGGVVSHFNDCPLSHDGYCLHDGV Amino acid sequence of CMYIEALDKYACNCVVGYIGERCQYRDLKWWELRAAAIEVMY TIE-CAR targeting ErbB PPPYLDNEKSNGTIIH VKGKHLCPSPLFPGPSKPFWVLVVVGG dimers + ODD VLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNL CAR in bold GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM ODD underlined AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPRAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNEKLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFTMPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKLFAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSASPESASPQSTVTVFQ SEQ ID NO: 86 ATGGGCCCAGGAGTTCTGCTGCTCCTGCTGGTGGCCACAGCTT Nucleotide sequence of GGCATGGTCAGGGAGGTGTGGTGTCGCACTTCAATGACTGTC TIE-CAR targeting ErbB CACTGTCGCACGATGGATACTGCCTCCATGATGGTGTGTGCAT dimers + ODD GTACATCGAGGCATTGGACAAGTATGCATGCAACTGTGTCGT CGGCTACATCGGAGAGCGATGTCAGTACCGAGACCTGAAGTG CAR in bold GTGGGAACTGAGAGCGGCCGCAATTGAAGTTATGTATCCTCC ODD underlined TCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCAT GTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGAC CTTCTAAGCCCTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCT GGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTC TGGGTGAGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTAC ATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATT ACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTC CAGAGTGAAGTTCAGCAGGAGCGCAGACgCCCCCGCGTACCA GCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACG AAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGG ACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAG GAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAG GCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGG CAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCAC CAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCT CGCGCCCCAGCCGCTGGAGACACAATCATATCTTTAGA I l l i GGCA GCAACGACACAGAAACTGATGACCAGCAACTTGAGGAAGTACCATT ATATAATGATGTAATGCTCCCCTCACCCAACGAAAAATTACAGAATA TAAATTTGGCAATGTCTCCATTACCCACCGCTGAAACGCCAAAGCC ACTTCGAAGTAGTGCTGACCCTGCACTCAATCAAGAAGTTGCATTA AAATTAGAACCAAATCCAGAGTCACTGGAAC 1 1 1 C 1 1 1 1 ACCATGCC CCAGATTCAGGATCAGACACCTAGTCCTTCCGATGGAAGCACTAGA CAAAGTTCACCTGAGCCTAATAGTCCCAGTGAATATTG 1 1 1 1 1 ATGT GGATAGTGATATGGTCAATGAATTCAAGTTGGAATTGGTAGAAAAA

[0763] C 1 1 1 1 1 GCTGAAGACACAGAAGCAAAGAACCCA 1 1 1 1 L 1 ACTCAGGA

[0764]

[0765] CACAGATTTAGACTTGGAGATGTTAGCTCCCTATATCCCAATGGATP5598PC00

[0766] GATGACTTCCAGTTACGTTCCTTCGATCAGTTGTCACCATTAGAAAG CAGTTCCGCAAGCCCTGAAAGCGCAAGTCCTCAAAGCACAGTTACA GTATTCCAGSEQ ID NO: 87 RRKRSGSGEGRGSLLTCGDVEENPGP

[0767] Amino acid sequence of

[0768] T2A sequence

[0769] SEQ ID NO: 88 CGGAGAAAGCGCAGCGGCTCCGGCGAGGGCCGGGGCAGCCTGCT Nucleotide sequence of GACCTGCGGCGACGTGGAAGAGAACCCCGGACCC

[0770]

[0771] T2A sequence

Claims

1. P5598PC00CLAIMS1. A nucleic acid molecule comprisinga. a first polynucleotide encoding an armouring protein fused to one or more Oxygen-Dependent Degradation Domains (ODDs); andb. a second polynucleotide comprising a regulatory region comprising one or more hypoxia-responsive elements (HREs);wherein the first polynucleotide is operably linked to the second polynucleotide.

2. The nucleic acid molecule of claim 1, wherein the armouring protein is:a. a cytokine or chemokine, preferably IL-1, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-12, IL-15, IL-18, IL-21, IL-12, IL-23, IL-17A, IL-18, IL-10, TNF-alpha, GM-CSF, HMGB1, IL-27, IL-33, IL-35 or IL-39;b. an armouring receptor;c. a protein that is capable of neutralising immunosuppressive cytokines, preferably one or more of TGF[3, PD-L1 and PD-1;d. a type II interferon (IFN-II), preferably interferon-gamma (IFN-γ); ore. preferably a type I interferon (IFN-I).

3. The nucleic acid molecule of claim 2, wherein the IFN-I is:a. interferon-alpha (IFN-α), preferably a sequence selected from SEQ ID NOs:39 to 50, a homologue or variant thereof or a functional fragment thereof; orb. preferably interferon-beta (IFN-β), preferably the sequence of SEQ ID NO: 51 or 52, a homologue or variant thereof or a functional fragment thereof.

4. The nucleic acid molecule of any preceding claim, wherein the first polynucleotide encodes one ODD.

5. The nucleic acid molecule of any preceding claim, wherein the one or more ODDs or each ODD independently comprise(s) an amino acid sequence having the sequence of SEQ ID NO: 1, where " X1-6" can be any amino acid residue, optionally wherein X1is " L" or any conservative substitution; X2is " D" or any conservative substitution, X3is " P" or any conservative substitution, X4" F” or any conservative substitution, X5is " Q" or any conservative substitution and X6is " L" or any conservative substitution, or a homologue or variant thereof or a functional fragment thereof.P5598PC006. The nucleic acid molecule of any preceding claim, wherein the one or more ODDs or each ODD independently comprise an amino acid sequence having a sequence selected from SEQ ID NOs: 2 to 4, or a homologue or variant thereof or a functional fragment thereof.

7. The nucleic acid molecule of any of the preceding claims, wherein the regulatory region comprises:a. one HRE; orb. a plurality of HREs or two or more HREs, which may be sequentially positioned and / or which may be spatially separate.

8. The nucleic acid molecule of claim 7, wherein the one or more HREs or each HRE independently comprise(s) at least one HIF binding site (HBS) which preferably comprises the sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

9. The nucleic acid molecule of claim 8, wherein the one or more HREs or each HRE independently comprise(s at least one HIF ancillary site (HAS) and preferably comprises the sequence of SEQ ID NO: 7 or SEQ ID NO: 8.

10. The nucleic acid molecule of claim 8 or claim 9, wherein the one or more HREs or each HRE independently comprise(s) at least one HNF-4 site and preferably comprises the sequence of SEQ ID NO: 9.

11. The nucleic acid molecule of any one of claims 7 to 10, wherein the one or more HREs comprise or each HRE independently comprise(s) a nucleotide sequence having a sequence selected from SEQ ID NO: 10 to 22, or a homologue or variant thereof or a functional fragment thereof.

12. The nucleic acid molecule of any preceding claim, wherein the regulatory region comprises a nucleotide sequence having a sequence selected from SEQ ID NO: 23 to 26, or a homologue or variant thereof or a functional fragment thereof.

13. The nucleic acid molecule of any preceding claim, wherein the regulatory region comprises the sequence of SEQ ID NO: 27, or a homologue or variant thereof or a functional fragment thereof.

14. The nucleic acid molecule of any preceding claim, further comprising a third polynucleotide encoding a cell surface receptor, preferably wherein the cell surface receptor is an engineered T-cell receptor (TCR) or more preferably, a Chimeric Antigen Receptor (CAR).P5598PC0015. The nucleic acid molecule of claim 14, wherein the cell surface receptor is fused to one or more ODDs, further wherein the third polynucleotide is operably linked to the second polynucleotide comprising a regulatory region.

16. The nucleic acid molecule of claim 14 or 15, wherein the first polynucleotide encoding an armouring protein fused to one or more ODDs and the third polynucleotide encoding the cell surface receptor are separated by a sequence encoding a 2A peptide or an IRES.

17. The nucleic acid molecule of any of claims 14 to 16, wherein the one or more ODDs are as defined in claim 5.

18. The nucleic acid molecule of any of claims 14 to 17, wherein the one or more ODDs are as defined in claim 6.

19. A vector comprising the nucleic acid molecule of any one of claims 1 to 18.

20. The vector of claim 19 wherein the vector is a DNA vector, RNA vector, plasmid vector, cosmid vector, herpes virus vector, measles virus vector, lentivirus vector, adenoviral vector, adeno-associated virus vector, retroviral vector, a Vaccinia Virus vector, a Sendai Virus vector, an Epstein-Barr Virus (EBV) vector, a Vesicular Stomatitis Virus (VSV) vector, a Newcastle Disease Virus (NDV) vector, a Coronavirus vector, a Baculovirus vector, a transposon, or a donor DNA template for use with ZFNs, TALENs or a CRISPR / Cas system.

21. A cell comprising a nucleic acid molecule of any one of claims 1 to 18 or a vector of claim 19 or claim 20, preferably wherein the cell comprises:a. a nucleic acid molecule of any one of claims 1 to 13 and a second nucleic acid molecule, wherein the second nucleic acid molecule comprises a polynucleotide encoding a CAR; orb. a nucleic acid molecule of any one of claims 14 to 18.

22. A method of preparing a cell comprisinga. Isolating a cell from a subject;b. Modifying said cell to introduce a nucleic acid molecule of any one of claims 1 to 18 or a vector of claim 19 or claim 20, preferably wherein:i.a nucleic acid molecule of any one of claims 1 to 13 and a second nucleic acid molecule is introduced, wherein the second nucleic acid molecule comprises a polynucleotide encoding a CAR; orii.a nucleic acid molecule of any one of claims 14 to 18 is introduced;P5598PC00c. Expanding said modified cell ex-vivo; andd. Obtaining an expanded cell capable of expressing the nucleic acid molecule under conditions of hypoxia.

23. The cell of claim 21 or the method of claim 22, wherein the cell is a fibroblast, a stem cell or an immunoresponsive cell, such as a Natural Killer cell, a B-cell, a T-cell or a macrophage.

24. A pharmaceutical composition comprising a nucleic acid molecule of any one of claims 1 to 18, a vector of claim 19 or 20, a cell of claim 21 or 23 or a population of cells of claim 21 or 23 and a pharmaceutically or physiologically acceptable diluent and / or carrier.

25. A nucleic acid molecule of any one of claims 1 to 18, a vector of claim 19 or 20, a cell of claim 21 to 23, a population of cells of claim 21 or 23 or a pharmaceutical composition of claim 24 for use in the treatment or prevention of a disease or disorder, preferably wherein the disease or disorder is a cancer, most preferably a solid cancer.

26. Use of a nucleic acid molecule of any one of claims 1 to 18, a vector of claim 19 or 20, a cell of claim 21 to 23, a population of cells of claim 21 or 23 or a pharmaceutical composition of claim 24 for the manufacture of a medicament for use in the treatment or prevention of a disease or disorder, preferably wherein the disease or disorder is a cancer, most preferably a solid cancer.

27. A method of treatment of a subject in need thereof, the method comprising administering a pharmaceutically effective amount of a nucleic acid molecule of any one of claims 1 to 18, a vector of claim 19 or 20, a cell of claim 21 to 23, a population of cells of claim 21 or 23 or a pharmaceutical composition of claim 24 to the subject, preferably wherein the disease or disorder is a cancer, most preferably a solid cancer.

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