NUCLEIC ACID CONSTRUCT

FIELD OF THE INVENTION

The present invention relates to constructs and methods for modulating the relative expression of transgenes.

BACKGROUND TO THE INVENTION

Expression of multiple transgenes

Gene therapy involves the modification of cells to express biological molecules to treat or correct a pathological condition. Obtaining either a physiological or a therapeutically relevant level of the biological molecule is key to successful gene therapy.

Current methods for modulating gene expression generally rely on modifying promoter regions to either increase or decrease the rate of transcription or to insert regulatory elements in them to render their expression inducible through the action of engineered transcription factors and small molecules.

Gene therapy approaches often involve the expression of more than one transgene. Transduction of a cell with multiple vectors in order to produce multiple products is difficult, expensive and unpredictable. For this reason, various methods have been developed to allow co-expression of two proteins from a single vector.

Initial attempts used two different promoters within the same cassette which results in two separate transcripts each of which code for a separate protein. This is a difficult approach for a number of reasons. A key problem is “promoter interference” whereby one promoter dominates and causes silencing of the second promoter. In addition, different promoters work differently in different cellular contexts and this makes consistent “tuning” of the relative expression of each transgene difficult to achieve.

An alternative approach is to use an Internal Ribosome Entry sequence (IRES). Here, a single transcript is generated. The IRES sequence in the transcript is placed between the open reading frames for the two transgenes and mimics an mRNA cap structure. Hence, the ribosome either initiates translation at the 5’ cap or the IRES, resulting in expression of two separate proteins. A key limitation with this method is the inability to control relative expression. The 3’ transcript is typically expressed less than the 5’ one, but the ratio of expression is difficult to predict and tune.

A further approach has been developed following characterization of the role of foot- and-mouth-disease virus (FMDV) 2A peptide in allowing FMDV (and related viruses) to express multiple proteins from a single open reading frame (ORF) (Donnelly et al; J. Gen. Virol.; 82, 1027-1041 (2001)). The 2A peptide (and homologs) cleaves at very high efficiency immediately after translation of the ORF, enabling the expression of multiple peptides from a single ORF. The use of self-cleaving peptides such as the 2A peptide results in expression of the transgenes at a 1:1 ratio.

W02020/025953 describes a system which uses frame-slip or translational readthrough as a means of regulating the expression of a transgene. In particular, the system can be used to control the relative expression of two or more transgenes expressed from a single mRNA transcript. When a ribosome encounters a stop codon (TGA, TAG or TAA), translation is usually terminated by release factors but if a translational readthrough motif (TRM) is positioned immediately 3’ of the stop codon, continued translation can occur in a proportion of the transcripts, leading to a low level of expression of the second transgene.

Chimeric antigen receptors (CARs)

Chimeric antigen receptor T cell (CAR-T cell) therapy redirects cytolytic T cells to target tumour cells through the expression of a chimeric antigen receptor (CAR) recognising a tumour-specific antigen on their cell surface. A chimeric antigen receptor consists of an antibody or ligand binding motif, a spacer domain and a transmembrane domain, fused to the intracellular signalling domains from the CD3 chain of the T cell receptor (TCR). Signalling domains from co-receptors, such as CD28, 0X40 or 4-1 BB, may also be included with CD3 signalling domain in the CAR 1.

CAR-T cell therapy has demonstrated itself to be an effective treatment for haematological malignancies, such as B cell leukaemia where it has achieved impressive response rates. However, CAR-T cell therapy has failed to achieve the same success in the treatment of solid tumours, where a number of factors compound to limit CAR-T cell activity. These include the expression of immuno- suppressive ligands, such as PD-L1 , secretion of cytokines that dampen favourable inflammatory responses, the expression of ligands that induce apoptosis by the tumour cells and nutrient depletion. To overcome the immuno-suppressive tumour microenvironment, CAR-T cells are being engineered to express molecules that block immune-suppressive signalling or enable T cells to survive in the hostile tumour microenvironment.

Cytokines that enhance the inflammatory response can increase the efficacy of CAR- T cell therapy. These include IL-7, IL-12, IL15, IL-17A, IL-18 and IL-21 , which have either been shown to enhance the response of CAR-T cells when provided exogenously or be released during CAR T-cell therapy.

IL-12 is a potent immunodulatory cytokine that is normally secreted by phagocytes and dendritic cells in response to: pathogens; T- and natural killer (NK) cell signals; and extracellular matrix components. CAR-T cells have been engineered to express IL- 12 either constitutively or from an inducible promoter, and this has been shown to improve the efficacy of CAR-T cell therapy when targeting solid tumours. IL-12 systemically is toxic so CAR T-cells or other immune cells have been engineered to release IL-12 into the tumour microenvironment 1. However, transgenic T-cell IL-12 secretion can result in highly toxic systemic levels.

While the expression of cytokines, chemokines or toxins may improve the efficacy of CAR-T cell therapy, the secretion of these biological molecules must be tightly regulated to limit their toxicity to within safe levels that offer therapeutic benefit and minimal side effects. This could be achieved by regulating the rate of gene transcription from promoter regions by either inserting or deleting cis-acting sequences to obtain the desired level of transcription or introducing inducible elements into promoter region. Notable inducible promoter systems include the tetracycline off and tetracycline off systems, which utilise the tetracycline-responsive element (TRE) in conjunction with an engineered form of the tetracycline repressor protein (TetR), which binds to the 19 bp nucleotide sequence of the TRE 2. In the tetracycline off system, the TetR is fused to the transactivating domain of viron protein 16 (VP16) from herpes simplex virus to generate a tetracycline transactivator (tTA). In the absence of tetracycline, tTA binds to the TRE in the promoter region and potentiates gene transcription; conversely, in the presence of tetracycline, tTA is unable to bind to the TRE and transcription does not occur. Mutation of the TetR domain in tTA to render it dependent on tetracycline for binding to the TRE created a reverse tTA (rtTA) and the tetracycline on system. In the presence of tetracycline the rtTA is able to bind to the TRE and stimulate transcription, but not in its absence. While the tetracycline inducible system enables the control of transgene expression, it relies on the use of a small molecule, the insertion of the TRE into promoter sequences and the expression of either tTA or rtTA to do so, placing a significant burden on the cell.

An alternative approach to controlling gene expression is to use an internal ribosome entry site (IRES). As explained above, an IRES is placed downstream of the primary transgene and facilitates the constitutive expression of an additional transgene, usually at lower levels than the primary transgene. Such an approach has been used to express IL-12 constitutively in CAR-T cells in an attempt to enhance the immune response and circumvent the need for host pre-conditioning prior to infusion of CAR-T cells. However, the level of expression of IL-12 is unpredictable and still likely to be toxic in vivo.

DESCRIPTION OF THE FIGURES

Figure 1 : Translational readthrough motifs and construct design.

(A) Examples of known translational readthrough motifs. (B-G) Structure of translational readthrough motif constructs. (B) Translational readthrough construct consisting of two transgenes where the translational readthrough motif is placed 3’ of the first transgene and 5’ of 2A self-cleaving peptide sequence and transgene 2. Expression of transgene 1 will be significantly higher than transgene 2. (B’) Double stop translational readthrough construct where two stop codons are incorporated into the translational readthrough motif to reduce expression levels even further than those achieved with a single stop codon. (C) Universal translational readthrough construct consisting of a translational readthrough motif flanked by two 2A selfcleaving peptide sequences. This construct mitigates sequence-dependent effects that can result in unpredictable levels of translational readthrough. (D) Combinatorial approach where a translational readthrough motif is combined with an attenuated signal peptide sequence to reduce the level of expression of transgene 2 even further. (E and E’) Compound translational readthrough motifs where multiple motifs are placed in series to produce a cascade of reduced transgene expression. (E) Multiple transgene are expressed from a single cassette using translational readthrough motifs and 2A self-cleaving peptide sequences. (E’) Self-cleaving peptide sequences are used to separate the translational readthrough motifs, which are 5’ of transgene 2. (F and F’) Functional translational readthrough constructs. (F) Secretion of an antigen-binding domain (scFv/VHH) and a functional CAR can be achieved by placing a translational readthrough motif 3’ of the spacer domain of the CAR. Functional readthrough results in the expression of a functional CAR, while termination of translation at the stop codon produces secreted antibody. (F’) Combinations of first, second and third generation CARs can be produced by placing a translational readthrough motif 3’ of the first endodomain (either a CD3 endodomain or a co-receptor endodomain). Functional translational readthrough will switch expression from a first generation CAR to a second or third generation CAR.

Figure 2: Determination of translational readthrough activity using secreted embryonic alkaline phosphatase (SEAP).

A) Diagram illustrating the constructs generated to determine translational readthrough motif activity. A stop codon and translational readthrough motif were placed at the 3’ end of the RQR8 sort-select coding sequence (5’ to the SEAP open reading frame). B) SEAP activity was measured in supernatants harvested from 293T cells transfected with the TRM constructs and the data were normalized to the appropriate no stop (TGG) control. The introduction of a stop codon and TRM reduced SEAP expression between 7.9-fold (TGA-CTAGCA) and 111.4-fold (TAA- CTAGCA) depending on the stop codon used, with TGA being the leakiest codon and TAG and TAA being more stringent. Data represent duplicates from three independent experiments. C) TRMs are functional in HeLa (ovarian adenocarcinoma), SupT1 (T cell lymphoma) and SCLC-21 H (small cell lung carcinoma) lines transduced with the SEAP. Data represent triplicates from two independent experiments. D) Comparison of SEAP expression from TRMs and attenuated EMCV IRESs in transfected 293T cells showed that a combination of the more stringent stop codons TAG and TAA and TRMs yielded similar expression levels to those obtained using attenuated IRESs. Data represent triplicates from a single experiment. In A to C, mean is shown, and statistical analysis carried out using an unpaired t-test with Welch’s correction: **** p<0.0001 ; ** p<0.01; * p<0.05.

Figure 3: TRMs enable differential expression of transmembrane proteins.

A) A chimera of the ectodomain of human CD22 and the transmembrane and truncated cytoplasmic domain of human CD19 was cloned 3’ to RQR8, a stop codon, a TRM and a self-cleaving peptide sequence. B) Transfected HEK293T cells were stained with antibodies to anti-CD34 (recognizing the Q-epitope in RQR8) and anti- CD22 and analysed by flow cytometry. C) Histograms of anti-CD22-PE fluorescence intensity of transfected (RQR8+ cells). The TGA stop codon is the leakiest and permits the highest level of translational readthrough when coupled with the CTAGCA TRM. D). Normalised anti-CD22-PE median fluorescence intensity and fold reduction. Data represent triplicates from three independent experiments. Mean is shown, and statistical analysis was carried using an unpaired t-test with Welch’s correction: *** p=0.003; ** p<0.01.

Figure 4: Compound TRMs enable fine tuning of transgene expression.

A) The fluorescent proteins eBFP2 and tandem mClover were cloned downstream of the cell surface marker HA8 (HA epitope presented on a human CD8D stalk). Stop codons, TRMs and self-cleaving peptide sequences were introduced to place either a single stop and TRM or two before the tandem mClover sequence to enable assessment of the impact of placing TRMs in a series on transgene expression. B) Flow cytometry plots of transfected 293T cells. C) Histograms showing tandem mClover fluorescence intensity. A decrease in mClover MFI is observed when two TGA-CTAGCA motifs are placed in series. D) Quantification of eBFP2 and tandem mClover MFIs showing that it is possible to fine tune transgene levels. Data were normalized to the TGG-CTAGCA no stop control and show the mean and standard deviation of duplicate transfections from three independent experiments.

Figure 5: TRMs facilitate therapeutically beneficial levels of IL-12 secretion in an aggressive immunocompetent melanoma mouse model.

A) Timeline of tumour and CAR-T cell engraftment in C57BI/6 immunocompetent mice. Cohorts of ten mice were subcutaneously injected with 1x105 GD2-expressing B16.F10 cells, which were allowed to engraft for 6 days prior to total body irradiation (TBI) on day 7. A 3x106 dose of non-transduced T cells or CAR-T cells was intravenously injected, via the tail vein, and tumour volume and cytokine secretion monitored. B) Tumour volume measurements. Mice injected with anti-GD2 CAR-T cells expressing IL-12 from a T2A self-cleaving peptide sequence (aGD2-T2A-IL-12 CAR-T) were culled on day 8 post CAR T injection because of cytokine-related toxicity. C) Body weight measurements. The aGD2-T2A-IL-12 CAR-T group rapidly lost body weight and were culled early, while the other groups maintained or increased body weight. D and E) Secreted IL-12 and IFNy measurements on day 6 post-CAR T injection. High serum levels of IL-12 and IFNy were observed in mice injected with the aGD2-T2A-IL-12 CAR-T cells, while considerably lower levels of cytokine secretion were observed from the aGD2-TGA-CAATAA-IL-12 CAR-T cells. Importantly, serum IFNy levels in the aGD2-TGA-CAATAA-IL-12 CAR-T cell cohort of mice were approximately 8.5-fold lower than those of the aGD2-T2A-IL-12 CAR-T cell group, but higher than those of the aEGFRvlll-TAGA-CAATTA CAR-T cell group, indicating that T cell activation and IL-12 stimulation led to increased IFNy secretion. Data analysed using ordinary one-way ANOVA with multiple comparisons; **** p<0.0001.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have found that the stop codon and translational readthrough motif UGA-CUAGCA gives the highest level of transcriptional readthrough, when compared with other stop codon/translational readthrough motif combinations.

Thus, in a first aspect, the present invention provides a nucleic acid construct comprising: a first nucleotide sequence of interest (NOI1); a UGA-CUAGCA motif (SEQ ID No. 1); and a second nucleotide sequence of interest (NOI2).

The nucleic acid construct may also comprise a nucleotide sequence encoding a cleavage site, so that NOI1 and NOI2 are expressed as separate proteins.

The cleavage site may comprise, for example, a self-cleaving peptide, a furin cleavage site or a Tobacco Etch Virus cleavage site.

The cleavage site may comprise, for example, a 2A self-cleaving peptide from an aphtho- or a cardiovirus or a 2A-like peptide.

The first nucleotide sequence of interest, NOI1, may encode a chimeric antigen receptor (CAR) or transgenic T-cell receptor (TOR).

The second nucleotide sequence of interest, NOI2, may encode a cytokine, chemokine or toxin. It may, for example, encode IL-12 or flexi-l L-12.

The nucleic acid construct may be capable of producing two products when expressed in a cell: a) a first product encoded by NOI1 alone; and b) a second product, encoded by NOI1 and NOI2, which is produced when translational readthrough occurs. The second product may, for example, be a chimeric antigen receptor (CAR) and the first product may be a truncated version of the CAR, incapable of inducing CAR- mediated cell signalling.

Alternatively, the first product may be a chimeric antigen receptor (CAR) comprising an intracellular signalling domain and the second product may be a CAR comprising an intracellular signalling domain and one or more co-stimulatory domain(s).

In a second aspect, the invention provides a vector comprising a nucleic acid construct according to the first aspect of the invention. The vector may, for example, be a retroviral vector or a lentiviral vector.

In a third aspect, the invention provides a cell comprising a nucleic acid construct according to the first aspect of the invention or a vector according to the second aspect of the invention.

In a fourth aspect, the invention provides a method for making a cell according to the third aspect of the invention which comprises the step of introducing a nucleic acid construct according to the first aspect of the invention or a vector the second aspect of the invention into a cell ex vivo.

In a fifth aspect, the invention provides a pharmaceutical composition comprising a plurality of cells according to the third aspect of the invention.

In a sixth aspect, the invention provides a pharmaceutical composition according to the fifth aspect of the invention for use in treating cancer.

In a seventh aspect, the invention provides a method for treating cancer which comprises the step of administration of a pharmaceutical composition according to the fifth aspect of the invention to a subject in need thereof.

In an eighth aspect, the invention provides the use of a cell according to the third aspect of the invention in the manufacture of a medicament for use in the treatment of cancer.

FURTHER ASPECTS OF THE INVENTION Further aspects of the invention are summarised in the following numbered paragraphs. Information provided in the Detailed Description below, for example on nucleic acid constructs, vectors, cells, and methods for their use and manufacture applies to the following aspects as well as the aspects presented in the claims.

1. A nucleic acid construct comprising: a sequence element which comprises at least one stop codon and at least one translational readthrough motif (TRM); and a nucleotide sequence of interest (NOI).

2. A nucleic acid construct according to paragraph 1, wherein the stop codon is selected from one of the following sequences: UGA, UAA and UAG.

3. A nucleic acid construct according to paragraph 1 or 2, wherein the TRM is selected from one of the following sequences:

CUACGA (SEQ ID No. 20)

CUAGGC (SEQ ID No. 21)

CAAUUA (SEQ ID No. 22)

GAGAGU (SEQ ID No. 23).

4. A nucleic acid construct according to any preceding paragraph, wherein the sequence element has the general structure SC-TRM in which SC is a stop codon and TRM is a translational readthrough motif.

5. A nucleic acid construct according to any preceding paragraph, wherein the stop-codon/translational readthrough motif is selected from the following:

UGA-CUAGCA (SEQ ID No. 1)

UAG-CUAGCA (SEQ ID No. 2)

UAA-CUAGCA (SEQ ID No. 3)

UGA-CUAGGC (SEQ ID No. 4)

UAG-CUAGGC (SEQ ID No. 5)

UAA-CUAGGC (SEQ ID No. 6)

UGA-CAAUUA (SEQ ID No. 24)

UAG-CAAUUA (SEQ ID No. 25)

UAA-CAAUUA (SEQ ID No. 26)

UGA-GAGAGU (SEQ ID No. 27) UAG-GAGAGU (SEQ ID No. 28)

UAA-GAGAGU (SEQ ID No. 29)

6. A nucleic acid construct according to any preceding paragraph, wherein the sequence element comprises two or more stop codons.

7. A nucleic acid construct according to paragraph 6 wherein the sequence element has the general structure SC1-SC2-TRM in which:

SC1 and SC2, which may be the same or different, are stop codons and TRM is a translational readthrough motif.

8. A nucleic acid construct according to any of paragraphs 1 to 6, wherein the sequence element comprises two or more stop codons/translational readthrough motifs arranged in series.

9. A nucleic acid construct according to paragraph 8 wherein the sequence element has the general structure SC1-TRM1-SC2-TRM2 in which:

SC1 and SC2, which may be the same or different, are stop codons; and

TRM1 and TRM2, which may be the same or different, are translational readthrough motifs.

10. A nucleic acid construct according to any preceding paragraph which also comprises a tRNA sequence.

11. A nucleic acid construct according to any preceding paragraph which comprises a tryptophan- or a tyrosine-encoding tRNA sequence.

12. A nucleic acid construct according to any preceding paragraph, wherein the NOI encodes an antigen for a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

13. A nucleic acid construct according to any preceding paragraph comprising: a first nucleotide sequence of interest (NOI1); a sequence element which comprises at least one stop codon and at least one translational readthrough motif (TRM); and a second nucleotide sequence of interest (NOI2). 14. A nucleic acid construct according to paragraph 13 which also comprises a nucleotide sequence encoding a cleavage site, so that NOI1 and NOI2 are expressed as separate proteins.

15. A nucleic acid construct according to paragraph 14 wherein the cleavage site comprises a self-cleaving peptide, a furin cleavage site or a Tobacco Etch Virus cleavage site.

16. A nucleic acid construct according to paragraph 15, wherein the cleavage site comprises a 2A self-cleaving peptide from an aphtho- or a cardiovirus or a 2A-like peptide.

17. A nucleic acid construct according to any of paragraphs 13 to 16, wherein NOI1 encodes a chimeric antigen receptor (CAR) or transgenic T-cell receptor (TCR).

18. A nucleic acid construct according to any of paragraphs 13 to 17, wherein NOI2 encodes a cytokine, chemokine or toxin.

19. A nucleic acid construct according to paragraph 18, wherein NOI2 encodes IL-12 or flexi-IL-12.

20. A nucleic acid construct according to paragraph 13 which is capable of producing two products when expressed in a cell: a) a first product encoded by NOI1 alone; and b) a second product, encoded by NOI1 and NOI2, which is produced when translational readthrough occurs.

21. A nucleic acid construct according to paragraph 20, wherein the second product is a chimeric antigen receptor (CAR) and the first product is a truncated version of the CAR, incapable of inducing CAR-mediated cell signalling.

22. A nucleic acid construct according to paragraph 20, wherein the first product is a chimeric antigen receptor (CAR) comprising an intracellular signalling domain and the second product is a CAR comprising an intracellular signalling domain and one or more co-stimulatory domain(s). 23. A vector comprising a nucleic acid construct according to any preceding paragraph.

24. A retroviral vector or a lentiviral vector according to paragraph 23.

25. A cell comprising a nucleic acid construct according to any one of paragraphs 1 to 22 or a vector according to paragraph 23 or 24.

26. A method for making a cell according to paragraph 25 which comprises the step of introducing a nucleic acid construct according to any of paragraphs 1 to 22 or a vector according to paragraph 23 or 24 into a cell ex vivo.

27. A pharmaceutical composition comprising a plurality of cells according to paragraph 25.

28. A pharmaceutical composition according to paragraph 27 for use in treating cancer.

29. A method for treating cancer which comprises the step of administration of a pharmaceutical composition according to paragraph 27 to a subject in need thereof.

30. The use of a cell according to paragraph 25 in the manufacture of a medicament for use in the treatment of cancer.

The Examples show that TRMs can be exploited to facilitate differential expression of transgenes, and that their use is broadly applicable to enable the defined expression of secreted (Figure 2), transmembrane (Figure 3) and cytosolic (Figure 4) proteins. Furthermore, it is demonstrated in a clinically relevant aggressive melanoma model that TRMs can be harnessed to control the expression of potent cytokines, such as IL-12, leading the way to their adoption in other gene and cell-based therapies (Figure 5). Achieving the desired level of transgene expression can be achieved by comparative studies followed by testing different TRMs in the applicable system.

The TRM-containing nucleic acid constructs can be exploited to facilitate differential transgene expression in multiple systems and applications. These include bioengineering processes, such as the purification of multi-subunit recombinant proteins complexes where a defined stoichiometry is required; the development of stable producer lines where a TRM could be used to downregulate the expression of a resistance gene and bias for the selection of high expressing producer lines; and in assay development where a range of ligand expression is desired.

DETAILED DESCRIPTION

The present invention relates to a nucleic acid construct comprising a stop/translational readthrough motif.

TRANSLATIONAL READTHROUGH

Translation terminates when the ribosome encounters a UGA, UAG or UAA stop codon. At this point, release factor recognises the stop codon and facilitates dissociation and recycling of the ribosome. Termination of translation usually occurs with high fidelity, with recoding of the stop codon, due to competition between release factor and a near cognate tRNA, and continued extension of the polypeptide only occurring 0.1% of the time. However, an elevated level of stop codon of recoding, which results in translational readthrough, has been reported in certain genes. In some cases, this has resulted in the generation of a longer polypeptide with additional functional motifs, in a process referred to as functional readthrough.

Translational readthrough occurs when release factor 1 (RF1) fails to recognise a stop codon and a near cognate aa-tRNA inserts an amino acid into the extending polypeptide, thereby suppressing the stop codon. The local concentration of release factor and the aa-tRNAs affects the level of stop codon suppression and translational readthrough, with low concentrations of release factor promoting translational readthrough. In mammals suppression of the UAG stop codon results in the insertion of a tryptophan, arginine or cysteine residue.

One of the earliest discovered examples of stop codon suppression is the rabbit beta globin gene, where translational readthrough results in the addition of 22 amino acids to the C-terminus of the protein 3.

The frequency of translational readthrough depends on a number of factors including: 1) the stop codon used (UGA, UAG or UAA); 2) the immediate sequence flanking the stop codon, with the six nucleotides upstream and downstream of the stop codon being particularly important and; 3) the presence of cis-acting sequences in the 3’ end of the mRNA.

The termination efficiency of the three stop codons varies, with UAA being the strongest stop codon and UGA being the weakest and hierarchy of termination efficiency is defined as UAA>UAG>UGA. Consequently, the highest level of translational readthrough is exhibited with the UGA stop codon and the lowest with the UAA stop codon. The nucleic acid construct of the invention may comprise any of these stop codons or a combination thereof.

Sequence analysis of genes exhibiting translational readthrough has identified at least five different sequence elements that promote stop codon suppression and sustained translation:

STOP-CUAG

STOP-CUACGA

STOP-CUAGGC

STOP-CAAUUA STOP-GAGAGU where stop can be UGA, UAG or UAA.

The sequence element may have the general structure SC-TRM in which SC is a stop codon and TRM is a translational readthrough motif.

The nucleic acid construct of the present invention may comprise one or more of the following sequence elements:

UGA-CUAGCA (SEQ ID No. 1)

UAG-CUAGCA (SEQ ID No. 2)

UAA-CUAGCA (SEQ ID No. 3)

UGA-CUAGGC (SEQ ID No. 4)

UAG-CUAGGC (SEQ ID No. 5)

UAA-CUAGGC (SEQ ID No. 6)

UGA-CAAUUA (SEQ ID No. 24)

UAG-CAAUUA (SEQ ID No. 25)

UAA-CAAUUA (SEQ ID No. 26)

UGA-GAGAGU (SEQ ID No. 27) In particular, the nucleic acid construct of the present invention may comprise one of the sequence elements shown as SEQ ID No. 1 to 6.

UGA-CUAGCA (SEQ ID No. 1)

UAG-CUAGCA (SEQ ID No. 2)

UAA-CUAGCA (SEQ ID No. 3)

UGA-CUAGGC (SEQ ID No. 4)

UAG-CUAGGC (SEQ ID No. 5)

UAA-CUAGGC (SEQ ID No. 6)

Where the nucleic acid construct is DNA rather than RNA, the motifs will be as follows:

TGA-CTAGCA (SEQ ID No. 7)

TAG-CTAGCA (SEQ ID No. 8)

TAA-CTAGCA (SEQ ID No. 9)

TGA-CTAGGC (SEQ ID No. 10)

TAG-CTAGGC (SEQ ID No. 11)

TAA-CTAGGC (SEQ ID No. 12)

In particular, the nucleic acid construct of the present invention may comprise UGA- CUAGCA (SEQ ID No. 1) or the equivalent DNA form TGA-CTAGCA (SEQ ID No. 7).

The sequence element may comprise two or more stop codons. As shown in the Examples, the use of two stop codons reduces the level of translational readthrough further than a single stop codon.

The sequence element may have the general structure SC1-SC2-TRM in which:

SC1 and SC2, which may be the same or different, are stop codons and TRM is a translational readthrough motif.

The sequence element may comprise two or more stop codons/translational readthrough motifs arranged in series. As shown in the examples, placing two TRMs in a series can be used to compound reductions in transgene expression.

The sequence element may have the general structure SC1-TRM1-SC2-TRM2 in which: SC1 and SC2, which may be the same or different, are stop codons; and

TRM1 and TRM2, which may be the same or different, are translational readthrough motifs.

FINE TUNING READTHROUGH/NOI EXPRESSION

Each stop codon/TRM sequence element is associated with a different level of transcriptional readthrough.

The Examples describe a secreted embryonic alkaline phosphatase (SEAP) assay which may be used to compare different stop codon/TRM sequence elements. Using this assay, the fold-reduction in SEAP activity for each sequence element is as shown in Table 1.

Table 1 - TRM driven fold reduction in SEAP expression

It is possible to reduce the level of transcriptional readthrough further by using two (or more) stop codons. For example, a construct containing two TGA stop codons and the CTAGCA TRM gives a level of translational readthrough 139.6-fold lower than an equivalent sequence which lacks a stop codon (Figure 2B), indicating that it provided increased termination stringency compared with a single stop and TRM. It is also possible to reduce the level of transcriptional readthrough further by using two (or more) stop codons/TRM sequence elements in series. For example it is shown in the Examples below that when placed in series, the TGA-CTAGCA stop codon and TRM reduced mClover MFI 90.9-fold lower compared to the no stop control.

It is therefore possible to fine tune transgene expression levels by a) choosing a particular stop codon/TRM combination and optionally b) using more than one stop codon and/or c) using more than one stop codon/TRM combination in series.

A sequence element may be selected which gives a fold-reduction in expression of between 5 and 10. This includes the elements UGA-CUAGCA (SEQ ID No. 1) and UGA-CUAGGC (SEQ ID No. 4).

A sequence element may be selected which gives a fold-reduction in expression of between 10 and 20. This includes the elements UGA-CAAUUA (SEQ ID No. 24) and UAG-CAAUUA (SEQ ID No. 25).

A sequence element may be selected which gives a fold-reduction in expression of between 20 and 40.

A sequence element may be selected which gives a fold-reduction in expression of between 40 and 60. This includes the element UAG-CUAGCA (SEQ ID No. 2).

A sequence element may be selected which gives a fold-reduction in expression of between 60 and 80. This includes the elements UAG-CUAGGC (SEQ ID No. 5) and UAA-CUAGGC (SEQ ID No. 6).

A sequence element may be selected which gives a fold-reduction in expression of between 80 and 100. This includes the element UAA-CAAUUA (SEQ ID No. 26).

A sequence element may be selected which gives a fold-reduction in expression of between 100 and 120. This includes the elements UAA-CUAGCA (SEQ ID No. 3), UGA-GAGAGU (SEQ ID No. 27) and UAA-GAGAGU (SEQ ID No. 29).

A sequence element may be selected which gives a fold-reduction in expression of between 120 and 400. This includes the element UAG-GAGAGU (SEQ ID No. 28). The fold-reduction may be measured using an suitable assay, for example an assay measuring the expression of a secreted protein, a cytosolic protein or a transmembrane protein, using any suitable marker.

The Examples describe a secreted embryonic alkaline phosphatase (SEAP) assay, assays looking at CD34, CD22 and HA8 expression using labelled antibodies and assays looking at the fluorescence intensities of the marker genes, eBFP2 and mClover.

ALTERED SIGNAL PEPTIDES

While translational readthrough motifs significantly decrease transgene expression, in certain situations it might be necessary to reduce the expression levels even further. For type I transmembrane and secreted proteins a further reduction in expression can be achieved by combining a translational readthrough motif with an altered signal peptide sequence. Such an approach is amenable to type I transmembrane proteins, which have a signal peptide sequence, and secreted proteins. In this case, the altered signal peptide and second transgene are placed 3’ of the translational readthrough motif and self-cleaving peptide sequence (Figure 1D).

A signal peptide is a short peptide, commonly 5-30 amino acids long, present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway. These proteins include those that reside either inside certain organelles (for example, the endoplasmic reticulum, golgi or endosomes), are secreted from the cell, and transmembrane proteins.

Signal peptides commonly contain a core sequence which is a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. The signal peptide is commonly positioned at the amino terminus of the molecule, although some carboxy-terminal signal peptides are known.

Altered signal peptides are described in detail in WO2016/174409, which is herein incorporated by reference. The altered signal peptide may comprise one or more mutation(s), such as substitutions or deletions, such that it has fewer hydrophobic amino acids than the wild-type signal peptide from which it is derived. The term “wild type” means the sequence of the signal peptide which occurs in the natural protein from which it is derived.

Where the nucleic acid construct comprises two transgenes both encoding transmembrane proteins, the protein encoded by the downstream transgene (which has lower relative expression) may comprise fewer hydrophobic amino acids than the protein encoded by the upstream transgene (which has higher relative expression).

The hydrophobic amino acids mutated in order to alter signal peptide efficiency may be: Alanine (A); Valine (V); Isoleucine (I); Leucine (L); Methionine (M); Phenylalanine (P); Tyrosine (Y); or Tryptophan (W).

The altered signal peptide may comprise 1 , 2, 3, 4 or 5 amino acid deletions or substitutions of hydrophobic amino acids. Hydrophobic amino acids may be replaced with non-hydrophobic amino acids, such as hydrophilic or neutral amino acids.

NUCLEIC ACID CONSTRUCT

The nucleic acid construct comprises: a sequence element which comprises at least one stop codon and at least one translational readthrough motif (TRM); and a nucleotide sequence of interest (NOI).

The NOI may encode an antigen for a chimeric antigen receptor (CAR) or T-cell receptor (TOR). The antigen may, for example be CD22. As shown in the Examples, using the technology of the invention it is possible to engineer cell lines expressing a range of target antigen densities. These may be used, for example, in cytotoxicity assays to assist with the identification and development of high sensitivity CARs.

The nucleic acid construct according may also comprise a tRNA sequence to promote readthrough. It has recently been suggested that overexpression of tryptophan and tyrosine encoding tRNAs in a cell can increase the efficiency of translational readth rough.

The nucleic acid construct may comprise: a first nucleotide sequence of interest (NOI1); a sequence element which comprises at least one stop codon and at least one translational readthrough motif (TRM); and a second nucleotide sequence of interest (NOI2).

The translational readthrough site may be located between first and second transgenes in the nucleic acid construct. The translational readthrough site may be placed upstream and/or downstream of a cleavage site in the nucleic acid construct. The translational readthrough site may be flanked by cleavage sites (Figure 1C). Two or more translational readthrough site may be used, for example, either positioned next to each other (Figure 1 B'), or flanking a cleavage site (Figure E').

The nucleic acid construct may have the structure:

NOI1-TRM-CL-NOI2 in which:

NOI1 and NOI2 are nucleotide sequences of interest;

TRM is a translational readthrough motifs; and

CL1 is a nucleic acid sequence encoding a cleavage site.

To reduce the expression level of a downstream transgene further, multiple stop codons can be inserted 5’ of the translational readthrough motif (Figure 1 B).

Where a nucleic acid construct comprises more than two transgenes, compound translational readthrough motifs may be placed in series, 5' for sequences encoding cleavage sites. This enables multiple transgenes to be expressed at different ratios. With each additional readthrough motif the level of expression should be reduced 10 to 50-fold relative to the upstream transgene.

A nucleic acid construct with compound translational readthrough motifs may have the structure:

NOI1-TRM1-CL1-NOI2-TRM2-CL2-NOI3

The readthrough site may be positioned within a coding sequence, so that two versions of a protein are made: a short version, made by translation of the transcript up to the readthrough site (i.e. where no readthrough occurs) and a long version, made by translation of the transcript beyond and downstream of the readthrough site (where readthrough occurs). Examples of such arrangements are shown in Figure 1 , F and F'.

A "nucleotide of interest" may be RNA or DNA. A nucleotide of interest (NOI) encodes a polypeptide of interest (POI) which may be all or part of a protein.

NOI1 and NOI2 (optionally together with subsequent NOI(s)) may encode a protein when transcribed and translated together. For example, The nucleic acid construct may be capable of producing two products when expressed in a cell: a) a first product encoded by NOI1 alone; and b) a second product, encoded by NOI1 and NOI2, which is produced when frame-slip or translational readthrough occurs.

The relative level of expression of the full length product, encoded by NOI1 and NOI2 may be less than the level of expression of the truncated product, encoded by NOI1 alone.

Alternatively, the nucleic acid construct may comprise one or more cleavage site(s) so that the nucleotide sequences of interest are expressed as separate proteins.

The nucleic acid construct of the present invention may encode a polyprotein, which comprises first and second polypeptides. The polyprotein may be cleaved at a cleavage site to produce two discrete polypeptides.

An NOI may encode an intracellular, a transmembrane or a secreted protein.

An NOI may, for example, encode a chimeric antigen receptor (CAR) or part thereof, or an agent which affects the activity of a CAR or CAR-expressing cell, such as a cytokine.

The transgene may encode a target antigen. In this respect, the technology may be used to produce target antigens with varying and very low levels of target antigen. These may be used in functional assays for, for example, T cells expressing CARs or engineered T-cell receptors (TCRs). The NOI may encode a protein involved in the synthesis of another entity by the cell. For example a cell can be induced to express the cancer antigen disialoganglioside (GD2) by the transgenic expression of two enzymes: GM3synthase and GD2synthase (WO2015/132604). By lowering the expression levels of these enzymes in the cell, it is possible to create GD2 ^|OW target cells.

The NOI may encode a cytokine such as a cytokine which enhances the inflammatory response and/or increases the efficacy of CAR-T cell therapy. The cytokine may be selected from the following: IL-7, IL-12, IL15, IL-17A, IL-18 and IL-21. In particular, the cytokine may be IL-12.

Interleukin 12 (IL-12) is an interleukin that is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells in response to antigenic stimulation. IL-12 is involved in the differentiation of naive T cells into Th1 cells. It is known as a T cell-stimulating factor, which can stimulate the growth and function of T cells. It stimulates the production of interferon-gamma (IFN-y) and tumor necrosis factor-alpha (TNF-a) from T cells and natural killer (NK) cells, and reduces IL-4 mediated suppression of IFN-y.

IL-12 plays an important role in the activities of natural killer cells and T lymphocytes. IL- 12 mediates enhancement of the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes.

IL-12 is a potent immunomodulatory cytokine of particular interest for modulating the tumour microenvironment redirecting the immune response against cancer. IL-12 is systemically toxic therefore methods for producing IL-12 locally are of interest.

IL-12 is a heterodimeric cytokine encoded by two separate genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (referred to as 'p70'), is formed following protein synthesis.

The NOI may encode IL-12A and/or IL-12B. The sequence for human IL-12A is available from Uniprot Accession number P29459. A portion of this sequence, lacking the signal peptide, is shown below as SEQ ID No. 13. The sequence for human IL-12B is available from Uniprot Accession number P29460. A portion of this sequence, lacking the signal peptide, is shown below as SEQ ID No. 14. SEQ ID No. 13 (human IL-12A)

RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKT STVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVE FKTM NAKLLMDPKRQIFLDQNMIJWIDELMQALNFNSETVPQKSSLEEPDFYKTKI KI- CI LLHAFRI RAVTI DRVMSYLNAS

SEQ ID No. 14 (human IL-12B)

WELKKDVYWELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQV KEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNY SGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVE CQEDSACPAAEESLPI EVMVDAVH KLKYENYTSSFFI RDI I KPDPPKN LQLKPLKNSR QVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASI SVRAQDRYYSSSWSEWASVPC

The NOI may encode "flexi-IL-12", which is a fusion between the human IL-12a (p35) and IL-12P (p40) subunits, joined by a linker. A suitable flexi-IL-12 sequence is shown below as SEQ ID No. 15.

SEQ ID No. 15 (a flexi-IL-12 sequence)

METDTLLLWVLLLWVPGSTGMWIWELKKDVYWELDWYPDAPGEMVVLTCDTPEE DGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIW STDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPI EVMVDAVH KLKYENYTS SFFI RDI I KPDPPKN LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSK REKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGG SGGGGSRNLPLA TPDPGMFPCLHHSQNLLRA VSNMLQKARQTLEFYPCTSEEIDHE DITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYED SKMYQ VEFKTMNAKLLMDPKRQIFLDQNMLA VIDELMQALNFNSETVPQKSSLEEP

DFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

In SEQ ID No. 15, the signal peptide, which is the signal peptide from Murine kappa chain V-lll region MOPC 63 (Uniprot P01661), is shown in bold; and the serineglycine linker is in bold and underlined.

The NOI may comprise one of the sequence shown as SEQ ID No. 13, 14 or 15 or a variant thereof. The variant sequence may have at least 80, 85, 90, 95, 98 or 99% sequence identity to SEQ ID No. 13, 14 or 15, provided that the variant sequence retains IL-12 function when expressed in vivo. For example, the variant sequence may retain the capacity to enhance the activity of cytotoxic T cells in vivo and/or the variant sequence may stimulate the production of interferon-gamma (IFN-y) by T cells.

A sequence encoding IL-12 or fliexi-IL12 may be placed downstream of the translational readthrough motif. This provides a means of controlling cytokine expression and reducing the level of expression of cytokine relative to the CAR.

The NOI may encode a chemokine, for example a chemokine which improves the efficacy of CAR-T cell therapy. The chemokine may be CCL19. In particular, the nucleic acid construct may co-express CCL19 and IL-7.

The NOI may encode an antibody or part thereof. For example, the NOI may encode an immunomodulatory antibodies or antibody fragment. The antibody may block inhibitory signals (like PD1) or activate the immune system (such as 0X40 agonistic agents, 41 BB agonistic agents or ICOS agonistic agents).

The transgene may encode a toxic compound such as Botulinum, Diptheria, or Pseudomonal toxin.

CHIMERIC ANTIGEN RECEPTORS

A classical chimeric antigen receptor (CAR) is a chimeric type I trans-membrane protein which connects an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site. A spacer domain is usually necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of I gG 1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen. A trans-membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.

Early CAR designs had endodomains derived from the intracellular parts of either the y chain of the FCER1 or CD3 Consequently, these first generation receptors transmitted immunological signal 1 , which was sufficient to trigger T-cell killing of cognate target cells but failed to fully activate the T-cell to proliferate and survive. To overcome this limitation, compound endodomains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of CD3 results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co-stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.

When the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on. Thus the CAR directs the specificity and cytotoxicity of the T cell towards tumour cells expressing the targeted antigen.

CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a transmembrane domain; and (iii) an intracellular domain which comprises or associates with a signalling domain.

A CAR may have the general structure:

Antigen binding domain - spacer domain - transmembrane domain - intracellular signaling domain (endodomain).

An NOI, or a combination of NOIs, of a nucleic acid construct of the invention may encode all or part of a CAR.

Functional readthrough can be exploited to increase the functionality of CARs. This can be achieved by inserting a translational readthrough motif downstream of the antigen-binding domain or between the signalling domains of the CAR (Figure 3F and F').

In some situations, it might be desirable to secrete an antigen-binding domain (scFv/VHH) to mitigate on-target off tumour effects where CAR T cells target normal tissue expressing low levels of a target antigen. The secreted antigen-binding domain would bind to the antigen expressed on the surface of the normal cell, thereby preventing recognition of the normal cell by the CAR T cell. Functional readthrough can be used to engineer T cells to secrete an antigen-binding domain by placing a translational readthrough motif immediately upstream of the transmembrane domain of the CAR.

The nucleic acid construct may have the general structure: scFvA/HH - TRM - spacer - TM domain - endodomain, or scFvA/HH - spacer - TRM - TM domain - endodomain in which: scFv/VHH is a nucleotide sequence encoding an antigen-binding domain spacer is a nucleotide sequence encoding a spacer

TM domain is a nucleotide sequence encoding a TM domain, and endodomain is a nucleotide sequence encoding an endodomain, which may, for example, be a first, second or third generation endodomain.

Functional readthrough can also be used to generate combinations of CARs that are first, second or third generation. In this situation the translational readthrough motif may be placed in between the CD3z and the co-receptor endodomains, which results in a high level of expression of a first generation CAR (CD3 signalling domain alone) and a substantially lower level of the second or third generation CAR.

The nucleic acid construct may have the general structure: scFvA/HH - spacer - TM domain - CD3 endodomain TRM - co-stimulatory domain, or scFv/VHH - spacer - TM domain - CD3 endodomain TRM1 - co-stimulatory domainl - TRM2 - costimulatory domain 2 in which: scFv/VHH is a nucleotide sequence encoding an antigen-binding domain spacer is a nucleotide sequence encoding a spacer

TM domain is a nucleotide sequence encoding a TM domain, and

CD3 endodomain is a nucleotide sequence encoding a CD3 endodomain, and Co-stimulatory domain is a nucleotide sequence encoding a co-stimulatory domain, such as the endodomain from a co-receptor such as CD28 or a member of the TNF receptor superfamily. Different iterations can be generated by switching the position of the CD3 and coreceptor endodomains so that engagement of the CAR will provide predominantly a co-stimulatory signal (signal 2) to the cell and a reduced antigen signal (signal 1) to the cell, because fewer CARs would have both the co-receptor and CD3 signalling domains.

CLEAVAGE SITE

The nucleic acid construct of the first aspect of the invention may comprise a sequence encoding a cleavage site positioned between nucleic acid sequences which encode first and second polypeptides, such that first and second polypeptides can be expressed as separate entities.

The cleavage site may be any sequence which enables the polypeptide comprising the first and second polypeptides to become separated.

The term “cleavage” is used herein for convenience, but the cleavage site may cause the first and second polypeptidess to separate into individual entities by a mechanism other than classical cleavage. For example, for the Foot-and-Mouth disease virus (FMDV) 2A self-cleaving peptide (see below), various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027- 1041). The exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode first and second polypeptides, causes the first and second polypeptides to be expressed as separate entities.

The cleavage site may be a furin cleavage site.

Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products. Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor. Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg' (SEQ ID No. 16)) and is enriched in the Golgi apparatus.

The cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.

TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin- like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TEV cleavage site is ENLYFQ\S (SEQ ID No. 17) (where ‘V denotes the cleaved peptide bond). Mammalian cells, such as human cells, do not express TEV protease. Thus in embodiments in which the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.

The cleavage site may encode a self-cleaving peptide.

A ‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the first and second polypeptides and the self-cleaving peptide is produced, it is immediately “cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.

The self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus. The primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus. In apthoviruses, such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus, the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating “cleavage” at its own C-terminus.

The C-terminal 19 amino acids of the longer cardiovirus protein, together with the N- terminal proline of 2B mediate “cleavage” with an efficiency approximately equal to the apthovirus FMDV 2a sequence. Cardioviruses include encephalomyocarditis virus (EMCV) and Theiler’s murine encephalitis virus (TMEV).

Mutational analysis of EMCV and FMDV 2A has revealed that the motif DxExNPGP (SEQ ID No. 18) is intimately involved in “cleavage” activity (Donelly et al (2001) as above). The cleavage site of the present invention may comprise the amino acid sequence: DX1EX2NPGP, where xi and X2 are any amino acid. Xi may be selected from the following group: I, V, M and S. X2 may be selected from the following group: T, M, S, L, E, Q and F.

The cleavage site, based on a 2A sequence may be, for example 15-22 amino acids in length. The sequence may comprise the C-terminus of a 2A protein, followed by a proline residue (which corresponds to the N-terminal proline of 2B).

The cleavage site may comprise the 2A-like sequence shown as SEQ ID No. 19 (EGRGSLLTCGDVEENPGP).

VECTOR

The present invention also provides a vector comprising a nucleic acid construct according to the first aspect of the invention.

Such a vector may be used to introduce the nucleic acid construct into a host cell so that it expresses the polypeptide(s) encoded by NOI1 and NOI2.

The vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.

The vector may be capable of transfecting or transducing a mammalian cell, for example a cytolytic immune cell such as a T-cell.

CELL

The present invention furthers provides a cell comprising a nucleic acid construct or vector of the present invention which expresses the polypeptide(s) encoded by NOI1 and NOI2.

The cell may be any eukaryotic cell such as an immunological cell.

The cell may be a cytolytic immune cell, such as a T cell or natural killer cell. The cell composition may comprise cytolytic immune cells such as a T cells and/or or NK cells.

T cells or T lymphocytes are a type of lymphocyte that play a central role in cell- mediated immunity. They can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of a T-cell receptor (TCR) on the cell surface. There are various types of T cell, as summarised below.

Helper T helper cells (TH cells) assist other white blood cells in immunologic processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface. TH cells become activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1 , TH2, TH3, TH17, Th9, or TFH, which secrete different cytokines to facilitate different types of immune responses.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumor cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells. Through IL-10, adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persist long-term after an infection has resolved. They quickly expand to large numbers of effector T cells upon re-exposure to their cognate antigen, thus providing the immune system with "memory" against past infections. Memory T cells comprise three subtypes: central memory T cells (TCM cells) and two types of effector memory T cells (TEM cells and TEMRA cells). Memory cells may be either CD4+ or CD8+. Memory T cells typically express the cell surface protein CD45RO.

Regulatory T cells (Treg cells), formerly known as suppressor T cells, are crucial for the maintenance of immunological tolerance. Their major role is to shut down T cell- mediated immunity toward the end of an immune reaction and to suppress auto- reactive T cells that escaped the process of negative selection in the thymus. Two major classes of CD4+ Treg cells have been described — naturally occurring Treg cells and adaptive Treg cells.

Naturally occurring Treg cells (also known as CD4+CD25+FoxP3+ Treg cells) arise in the thymus and have been linked to interactions between developing T cells with both myeloid (CD11c+) and plasmacytoid (CD123+) dendritic cells that have been activated with TSLP. Naturally occurring Treg cells can be distinguished from other T cells by the presence of an intracellular molecule called FoxP3. Mutations of the FOXP3 gene can prevent regulatory T cell development, causing the fatal autoimmune disease IPEX.

Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a normal immune response.

Natural Killer cells (or NK cells) form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner

NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.

The cells of the invention may be any of the cell types mentioned above.

The cells to be transduced with a method of the invention may be derived from a blood sample, for example from a leukapheresate. The cells may be or comprise peripheral blood mononuclear cells (PBMCs).

Cells may either be created 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).

Alternatively, cells may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to, for example, T or NK cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.

The cells may be activated and/or expanded prior to being transduced with nucleic acid encoding the molecules providing the chimeric polypeptide according to the first aspect of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

After transduction, the cells may then by purified, for example, selected on the basis of expression of the CAR or marker gene, such as RQR8.

PHARMACEUTICAL COMPOSITION

The invention provides a pharmaceutical composition comprising a plurality of cells which contain the nucleic acid construct.

The pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient. The pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds. Such a formulation may, for example, be in a form suitable for intravenous infusion.

METHOD OF TREATMENT

The present invention provides a method for treating a disease which comprises the step of administering the pharmaceutical composition of the present invention to a subject.

A method for treating a disease relates to the therapeutic use of the composition of the present invention. The composition may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.

The method for preventing a disease relates to the prophylactic use of the composition of the present invention. The composition may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease. The subject may have a predisposition for, or be thought to be at risk of developing, the disease.

The method may involve the steps of:

(i) isolating a cell-containing sample;

(ii) transducing the such cells with a vector of the invention;

(iii) administering the cells from (ii) to a subject.

The present invention also provides a pharmaceutical composition of the present invention for use in treating and/or preventing a disease.

The invention also relates to the use of a cell of the present invention in the manufacture of a pharmaceutical composition for the treatment of a disease.

The disease to be treated by the methods of the present invention may be a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

The disease may be Multiple Myeloma (MM), B-cell Acute Lymphoblastic Leukaemia (B-ALL), Chronic Lymphocytic Leukaemia (CLL), Neuroblastoma, T-cell acute Lymphoblastic Leukaema (T-ALL) or diffuse large B-cell lymphoma (DLBCL).

The disease may be a plasma cell disorder such as plasmacytoma, plasma cell leukemia, multiple myeloma, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain diseases, monoclonal gammopathy of undetermined significance or smoldering multiple myeloma.

The cells of the composition of the present invention may be capable of killing target cells, such as cancer cells. The target cell may be characterised by the presence of a tumour secreted ligand or chemokine ligand in the vicinity of the target cell. The target cell may be characterised by the presence of a soluble ligand together with the expression of a tumour-associated antigen (TAA) at the target cell surface. METHOD

In a further aspect, the present invention provides a method for making a cell according to the invention which comprises the step of introducing a nucleic acid construct or a vector of the invention into a cell.

The nucleic acid construct may be introduced by transduction or transfection.

The cell may be a cell isolated from a subject, for example a T cell or an NK cell isolated from a subject.

The invention will now be further described by way of Examples, which are meant to serve to assist one of ordinary skill in the art in carrying out the invention and are not intended in any way to limit the scope of the invention.

EXAMPLES

Example 1 - Determination of translational readthrough activity using a secreted alkaline phosphatase reporter system.

A secreted embryonic alkaline phosphatase (SEAP) assay was established to investigate the use of translational readthrough as a means of facilitating differential transgene expression. Constructs were cloned where the SEAP sequence was inserted 3’ to the sorting marker gene RQR8 which is described in WO2013/153391 , with an intervening stop codon, TRM and self-cleaving peptide sequence (Figure 2A). TRMs derived from the opioid receptor (OPRL1) (CTAGGC) and tobacco mosaic virus (TMV) (CAATTA) were selected for assessment as these TRMs are known to be functional, along with a new TRM sequence (CTAGCA) that we predicted to be functional. The length of the cis-acting TRM was limited to six bases to reduce transcriptional burden and minimize the introduction of foreign sequences to the retroviral genome constructs. As controls, constructs lacking TRMs were cloned along with a construct containing the first six bases (GAGAGT) 3’ to the TAG stop codon in feline leukemia virus (FeLV). This hexanucleotide sequence is predicted to be a nonfunctional TRM by itself, as suppression of the TAG stop codon in FeLV is dependent on an RNA pseudoknot located 3’ to the stop codon. The SEAP constructs were transfected into 293T cells and downstream analyses carried out 48 hours later. The activity of the TRMs was assessed by carrying out SEAP assays on collected supernatant, while the transfection efficiency was determined by staining the cells with anti-CD34 antibody to detect RQR8 and analysing them by flow cytometry (Figure 2B and data not shown). Quantification of SEAP activity, and normalization of the data to the appropriate no stop (TGG) control, demonstrated that the OPRL1 and TMV TRMs were functional, leading to decreased expression ranging from 7.9-fold (TGA-CTGACA motif) to 111.4-fold (TAA-CTAGCA) lower than the no stop controls (Figure 2B). The lowest level of translational readthrough from the TAA-CTAGCA TRM was comparable to the no TRM controls (TGA, 150.2-fold; TAG, 107.4-fold; and TAA, 123.6-fold decrease). In agreement with previously published results, the TGA stop codon was observed to be the leakiest, while the TAG and TAA codons were more stringent in nearly all cases. A notable exception was the TAG-CAATTA TRM, which yielded a similar level of translational readthrough as the TGA equivalent (TGA-CAATTA, 15.6-fold and TAG-CAATTA, 17.7-fold decrease in SEAP activity). As the TAG stop codon is present in TMV, this result suggests that the CAATTA TRM may have evolved to promote a higher level of translational readthrough. The TRM derived from FeLV was, as predicted, found to be non-functional and promoted background levels of translational readthrough comparable to the no TRM controls (Figure 2B). The highest level of translational readthrough was obtained with the TGA-CTAGCA, which gave a 7.9-fold decrease in expression.

To investigate if the use of two stop codons would reduce the level of translational readthrough further than a single stop codon, a construct containing two TGA stop codons and the CTAGCA TRM was generated along with its equivalent no stop control. SEAP assays showed that the level of translational readthrough from this motif was 139.6-fold lower than its no stop control (Figure 2B), indicating that it provided increased termination stringency compared with a single stop and TRM.

The above results data indicated that the TRMs were functional in 293T epithelial cells. To investigate if they were functional in different cell types, SCLC-21 H (small cell lung carcinoma), SupT1 (T cell lymphoma) and HeLa (ovarian/cervical adenocarcinoma) cells were transduced with the SEAP TRM constructs and the activity of the secreted enzyme determined (Figure 2C). Similar reductions in SEAP expression were observed as in 293T cells, except for SCLC-21 H cells, where the TAA-CTAGCA TRM produced barely detectable SEAP expression (Figure 2C). However, the TGA and TAG-CTAGCA TRMs were functional in SCLC-21 H cells and, overall, the activity of the TRMs and the hierarchy of stop codon stringency was comparable between the cell lines. Together, these results indicated that TRMs are functional in different cell types and can be used to modulate transgene expression. In all cell types tested, the highest level of translational readthrough was obtained with the TGA-CTAGCA.

Attenuated mutant IRESs have been used to regulate transgene expression. To compare the activity of attenuated IRESs to TRMs, we cloned two IRESs derived from the encephalomyocarditis virus (EMCV) IRES upstream of SEAP and determined their activity in transfected 293T cells (Figure 2D). The first mutant IRES, IRES mutant 1, contained an additional A in the JK bifurcation loop, while the second mutant, IRES mutant 2, bore the same insertion and six additional mutations (see Materials & Methods). Assessment of SEAP activity from transfected 293T cells indicated that IRES mutant 1 reduced expression 12.8-fold, while IRES mutant 2 exhibited a 99.1 -fold reduction in SEAP expression (Figure 2D). The motif TGA- CTAGCA gave higher expression than both the IRES mutants, giving a 6.1 fold reduction in expression.

Together, these results demonstrated that TRMs can be exploited to facilitate differential transgene expression, and, depending on the TRM utilized, can provide a reduction in expression ranging from 7.9-fold to 139.6-fold lower than multi-cistronic cassettes lacking a stop codon.

Example 2: TRMs enable differential expression of transmembrane proteins

CARs are usually directed to tumor-associated antigens that are expressed on the surface of cancer cells. Antigen density on the surface of tumors can vary between patients and it is desirable to develop CARs capable of recognizing ultra-low levels of target antigen (<200 copies/cell) to improve the efficacy of CAR-T therapy. In this study, TRMs were employed to develop target cell lines expressing low levels of antigen, to facilitate the design or identification of CARs sensitive to low levels of tumour antigen. Constructs were generated consisting of the ectodomain of human CD22 fused to the transmembrane and truncated cytoplasmic domain of human CD19, which was cloned downstream of RQR8, a stop codon, a TRM and a selfcleaving peptide sequence (Figure 3A). The constructs were transfected into 293T cells, which were stained 48 hours later with anti-CD34 and anti-CD22 antibodies and analysed by flow cytometry (Figure 3B and C). Quantification and normalization of anti-CD22-PE fluorescence to that of the RQR8 sort select marker indicated that the TRMs reduced the median fluorescence intensity by 5.5-fold to 17.3-fold, depending on the TRM used (Figure 3D). Consistent with the results obtained from the SEAP assays, the highest level of translational readthrough was observed with the TGA- CTAGCA stop codon and TRM, which reduced CD22 MFI 5.5-fold compared with the no stop control (Figure 3D). Moreover, when the more stringent TAG and TAA stop codons were used in conjunction with the CTAGCA TRM, CD22 MFI was reduced 10.9-fold and 9.7-fold, respectively. The level of translational readthrough observed from the CAATAA TRM was similar, with the TGA, TAG and TAA stop codons reducing CD22 MFI 10.1-fold, 10.3-fold, and 11.9-fold, respectively (Figure 3D). The TGATGA-CTAGCA double stop and TRM reduced CD22 MFI even further, to 17.3- fold less than its no stop control, demonstrating that the double stop and TRM offered increased termination stringency and lower expression levels.

These data indicated that TRMs can be used to regulate the expression of transmembrane proteins and it is possible to engineer cell lines expressing a range of target antigen densities for use in cytotoxicity assays to assist with the identification and development of high sensitivity CARs.

Example 3: Compound TRMs fine tune transgene expression

To investigate if placing two TRMs in a series could be used to compound reductions in transgene expression, constructs were generated where enhanced blue fluorescent protein 2 (eBFP2) and two copies of mClover fluorescent protein fused by a linker (tandem mClover) were cloned downstream of the cell surface marker HA8 (Figure 4A). Stop codons and the CTAGCA TRM were introduced upstream of the fluorescent protein coding sequences to generate constructs with either a single stop and TRM before tandem mClover or two. HEK293T cells were transfected with the constructs and were stained 48 hours later with anti-HA antibody and analysed by flow cytometry (Figure 4B and 4C). Quantification of anti-HA, eBFP2 and mClover fluorescence intensities indicated that a single stop and TRM placed upstream of mClover reduced MFI by 52.6-fold with the TGA stop codon and 100.0-fold with both the TAG and TAA stop codons, compared to the no stop control (Figure 4D). When placed in series, the TGA-CTAGCA stop codon and TRM reduced mClover MFI 90.9-fold lower compared to the no stop control, demonstrating that it is possible to fine tune transgene expression levels. Interestingly, the addition of a second TAA or TAG stop codon and TRM had no or only a marginal impact on mClover MFI, with 100.0-fold and 111.1- fold reductions, respectively, compared to the no stop control. The advantage of using two stop codons and TRMs in tandem is that is possible to achieve a range of transgene expression; accordingly, eBFP2 MFI was reduced 5.8-fold, 20.4-fold, and 20.0-fold with the TGA, TAG and TAA stop codons, respectively, in the constructs containing stop codons and TRMs in series (Figure 4D).

Together these data show that the TGA-CTAGCA stop codon and TRM are potentially useful for achieving a range of transgene expression levels. Such an approach could be relevant to the purification of multi-subunit complexes where a defined stoichiometry is required.

Example 4: TRMs enable therapeutically beneficial control of IL-12 secretion

The above data indicated that TRMs can be used to facilitate differential transgene expression and it was next sought to demonstrate their utility in a clinically relevant model. IL-12 is a potent cytokine with therapeutic potential for the treatment of cancer, as it activates macrophages, cytotoxic T cells and natural killer (NK) cells, and leads to an enhanced immune response. However, clinical studies have shown that its intravenous administration can result in severe toxicity, highlighting the need to develop strategies to regulate its expression. To date, approaches to control IL-12 administration include: local delivery at the tumour site, inducible promoters, T cell activation-induced promoters, and attenuated internal ribosome entry sites and transient mRNA expression. This study compared the use of the sequence element TGA-CAATTA with the T2A self-cleaving peptide sequence alone to control the secretion of murine flexi IL-12, consisting of the p35 and p40 subunits fused together via a flexible linker, in an aggressive immunocompetent mouse model of GD2+ B16.F10 melanoma, to evaluate both efficacy and toxicity. Cohorts of mice were injected with B16.F10 cells transduced with GD2 and GD3 synthases to express the ganglioside GD2 on their cell surface and allowed to engraft for 6 days. The mice were subjected to 5 Gy total body irradiation, to promote CAR-T cell engraftment, and intravenously injected the following day with a 3x10 ⁶ dose of anti-GD2, anti-GD2-T2A- IL-12, anti-GD2-TGA-CAATTA-IL-12 or anti-EGFRvlll-TGA-CAATTA-IL12 CAR-T cells (Figure 5A). All CARs had identical murine CD8a spacer and transmembrane, and CD28 and CD3 signalling endodomains. The anti-EGFRvlll CAR was a negative control included in the study to enable the assessment of the effects of IL-12 secretion in the absence of target recognition. CAR dose was adjusted based on expression of the marker gene Thy1.1.

Tumour volumes were measured for 2 weeks after the injection of CAR-T cells (Figure 5B). Mice injected with non-transduced T cells or the control anti-EGFRvlll- TGA-CAATTA-IL12 CAR failed to control growth of the GD2+ melanoma cells, and only limited control was observed in the cohort injected with the anti-GD2 CAR alone. Robust tumour control was observed in the anti-GD2-T2A-IL-12 and anti-GD2-TGA- CAATTA-IL-12 cohorts; however, mice in the anti-GD2-T2A-IL-12 cohort exhibited dramatic weight loss associated with the over-expression of IL- 12 and were culled 8 days post CAR-T cell injection (Figure 5C). In contrast, little to no weight loss was observed in the anti-GD2-TGA-CAATTA-IL-12 cohort, indicating that the TRM reduced IL-12 expression, resulting in lower levels of toxicity.

Serum IL-12 and IFNy levels were measured in all cohorts 6 days post CAR-T cell injection (Figure 5D and E). The anti-GD2-T2A-IL-12 cohort possessed high levels of IL-12 (mean 19328 pg/mL +/-9068) and IFNy (mean 4528 pg/mL +/-2409), while in the anti-GD2-TGA-CAATTA-IL-12 cohort had considerably lower levels of IL-12 (mean 84 pg/mL +/-18) and IFNy (mean 531.2 pg/mL +/-333). IL-12 levels in the anti- EGFRvlll-TGA-CAATTA-IL-12 cohort were comparable to those of non-transduced T cells or anti-GD2 CAR alone T cells, indicating that it the absence of T cell activation IL-12 secretion remained at background levels. Similarly, no induction of IFNy secretion was observed in the anti-EGFRvlll-TGA-CAATTA-IL-12 cohort, whereas IFNy levels rose in the serum of mice injected with anti-GD2-TGA-CAATTA-IL-12 CAR-T cells, indicating that sufficient IL-12 was being produced to induce IFNy secretion. These results were consistent with previously published data showing that IL-12 secretion leads to enhanced tumour clearance in the B16 melanoma mouse model due to reprogramming of myeloid-derived cells in the tumour microenviroment (TME) and the induction of an inflammatory response. Exposure of myeloid-derived suppressor cells (MDSCs), macrophages and dendritic cells in the TME results in their reprogramming and activation, which is partly mediated through IFNy, leading to antigen cross-presentation and the activation of CD8+ cells that mediate tumour clearance. Together, these results demonstrate that TRMs can be used to control the expression of potent cytokines and deliver them at levels offering therapeutic benefit for cell-based cancer immunotherapies.

Materials & Methods Constructs

All open reading frames were cloned into the MoMLV-based retroviral genome construct SFG. Cloning was carried out using Q5 DNA polymerase (NEB; M0491 L) and oligonucleotides (IDT) to amplify the coding sequences and to incorporate stop codons, TRMs and self-cleaving peptide sequences derived from Thosea asigna virus 2A (T2A), equine rhinitis A virus polyprotein (E2A) or porcine teschovirus-1 2A (P2A). Type IIS restriction enzyme sites were incorporated to the oligonucleotides, the resulting PCR products digested with Esp3l or Bsal-HF v2 (NEB; R0734L and R3733L, respectively), purified and ligated together using T4 DNA ligase (Roche; 10799009001). Secreted embryonic alkaline phosphatase (SEAP) constructs were generated by cloning its coding sequence 3’ to the sort select maker RQR8. Human CD22/CD19 chimera constructs were generated by fusing residues 1 to 20 of the murine immunoglobulin kappa chain V-lll (representing signal peptide sequence) to residues 20 to 687 of human CD22 and residues 294 to 332 of human CD19. The CD22/CD19 chimeric sequence was cloned 3’ to the RQR8, TRM and self-cleaving peptide sequences. Enhanced blue fluorescent 2 (eBFP2) and mClover fluorescent protein constructs were generated by cloning the sequences 3’ of the marker gene HA8, consisting of residues 98-106 of human influenza hemagglutinin (HA) fused to residues 141 to 222 of human CD8a. The IRES from encephalomyocarditis virus isolate JZ1202 (sequence ID: KF836387) was cloned from base 148 to 734. IRES mutant 1 contained an additional A at 661 in the A6 bifurcation loop and IRES mutant 2 possessed mutations at 150 C>G, 162 Odeleted, 337 A>G, 456 G>A, 462 A>G, 630 G>A and 661 insertion A. Chimeric antigen receptors recognizing EGFRvlll and the ganglioside GD2 with murine CD8 spacer and transmembrane domains, and murine CD28 and CD3 intracellular domains were cloned into SFG with an upstream Thy1.1 (murine CD90 marker). Murine flexi-12, consisting of the p35 and p40 subunits of IL-12 fused together via an S(GGGGS)3 flexible linker, was cloned downstream of the CARs. Murine GD2 and GD3 synthase were cloned into SFG with an intervening E2A self-cleaving peptide sequence.

Cell culture and Transfections

Cell lines were cultured at 37oC in a humified incubator with 5% CO2. HeLa (ATCC; CCL-2), 293T (ATCC-CRL-11268), Phoenix-ECO (ATCC; CRL-3214) and SCLC-21 H (DSMZ; ACC 372) cells were cultured in IMDM (Sigma; I3390-500ML) and SupT1 cells (ECACC; 95013123) in RMPI-1640 (Sigma R5886-500ML). All media were supplemented with 10% fetal bovine serum (BioSera; FB-1001/500) and 2 mM GlutaMAX (Gibco; 35050061). For 293T transfections, cells were plated in 6-well plates (3x105 cells/well) and were transfected 24 hours later with 2 pg of plasmid DNA using GeneJuice transfection reagent (Merck Millipore; 70967-3) at a 3:1 ratio (transfection reagent to DNA) according to the manufacturer’s instructions. The cells were cultured for 48 hours prior to carrying out enzyme-based or flow cytometric analyses.

Production of retroviral supernatants and cell transduction

Retroviral supernatants were produced by plating 2.4x10 ⁶ 293T cells in 10 cm plates and transfecting 24 hours later with 4.7 pg of □-retroviral genome plasmid, 3.1 pg RD114 (RDF glycoprotein), and 4.7 pg of PeqPam-env (Moloney murine leukaemia virus gagpol), using GeneJuice transfection reagent at a ratio of 3:1 (transfection reagent to DNA. For the transduction of murine splenocytes, supernatants were produced by transfecting Phoenix Eco cells with 4.68 pg DNA of y-retroviral genome plasmid and 2.6 pg of pCL-ECO, encoding gagpol and Eco envelope glycoprotein, using GeneJuice transfection reagent as described above). Cell culture media, containing viral particles, were collected at 48 and 72 hours after transfection, replacing the harvested media with fresh at 48 hours. Retroviral transductions were carried by plating 0.3x106 cells/well in 24-well non-tissue culture plates (Corning Falcon; 351147), previously coated with retronectin (TaKaRa; T100B) for 16-24 hours at 4oC, adding the appropriate volume of retroviral supernatant and centrifuging at 1000 x g for 20 minutes at ambient temperature. The cells were returned to the incubator for 72 hours prior to analysis.

Secreted embryonic alkaline phosphatase assays

Supernatant was collected from cells 48 hours after transfection and 20 pL analysed for SEAP activity by adding 180 pL of Quanti-blue solution (Invivogen; rep-qbs) in a flat bottom 96-well plate and incubating at 37oC for 2 hours. The optical density at 620 nm was measured using a Multiskan plate reader (Thermo Fisher Scientific).

Antibodies

Antibodies used in this study were: anti-human CD34 APC conjugated (R&D Systems; FAB7227A), anti-CD22 PE conjugated (Biolegend; 302506) and anti-HA PE-Cy7 conjugated (Biolegend; 901528). All antibodies were diluted 1 :50 in PBS (Gibco; 14190144).

Cell staining and flow cytometry Cells were harvested for flow cytometry using trypsin/EDTA (Life Technologies; 25200056) and stained with antibodies at ambient temperature for 10 minutes before washing the cells once with PBS and resuspending in Sytox Blue cell viability dye (Life Technologies; S34857). Stained cells were analysed by flow cytometry using a MACSQuant 10 flow cytometer (Miltenyi) and downstream analyses were carried out using FlowJo software. For fluorescence intensity measurements, a gate was drawn around transfected cell population (Figure 3) or a narrow gate was applied at 104 on the marker gene (Figure 4) and the median fluorescence intensity calculated.

GD2+ B16.F10 melanoma immunocompetent mouse model

Splenocytes from C57BI/6 mice were harvested, stimulated with 2 Dg/mL concanavalin A (Sigma Aldrich; C5275-5MG) and 1 ng/mL murine IL-7 (Peprotech; 217-17) and transduced with y-retrovirus pseudotyped with Eco glycoprotein. The cells were cultured for up to 4 days in the presence of 50 U/mL human IL-2 (Genscript; 200368). Cohorts of ten C57BI/6 mice (Charles River Laboratories) were injected subcutaneously with 1x105 B16.F10 melanoma cells transduced with a y- retroviral vector encoding the enzymes GD2- and GD3-synthases to synthesise the ganglioside GD2 and allowed to engraft for 6 days before subjecting the mice to 5 Gy total body irradiation. The following day, mice were intravenously injected with 3x106 non-transduced T cells or CAR-T-cells, and tumour growth and cytokine release monitored for up to 22 days after tumour cell engraftment.

Cytokine secretion

Blood was collected, via tail vein bleed, in heparin-coated tubes (Starsted), spun at 2000 x g for 10 min at 4 °C and stored at -80 °C. Secreted cytokine levels in blood collected from C57BI/6 mice measured using a custom Luminex cytokine bead assay (R&D Systems).

Statistical analysis

Data were analysed in GraphPad Prism (Windows 64-bit, version 9.2.0) using an unpaired t-test with Welch’s correction or ordinary one-way ANOVA with multiple comparisons.

All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.