EP3864034A1 - Cell - Google Patents

Abstract

The present invention relates to a cell which co-expresses (i) a dominant-negative SMAD (dnSMAD); and (ii).a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

Description

CELL

FIELD OF THE INVENTION

The present invention relates to a cell which expresses a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR). The cells may be engineered to be resistant to the immunosuppressive effects of cytokines such as TQRb which signal through the Smad family of transcription factors.

BACKGROUND TO THE INVENTION

Adoptive immunotherapy of cancer involves the ex vivo generation of cancer-antigen specific cells and their administration. Adoptively transferred immune effector cells also activate existing adaptive and innate immune cells within the tumour once they activate and start causing inflammation.

The native specificity of immune effector cells can be exploited in adoptive immunotherapy - for example during the generation of melanoma specific T-cells from expansion of tumour infiltrating lymphocytes in tumour resections. Otherwise a specificity can be grafted onto a T- cell using genetic engineering. Two common methods for achieving this are using chimeric antigen receptors or transgenic T-cell receptors. Different kinds of immune effector cells can also be used. For example, alpha/beta T-cells, NK cells, gamma delta T-cells or macrophages can be used.

Adoptive immunotherapy has been successful in treating a number of lymphoid malignancies, such as B-cell Acute Lymphoblastic Leukaemia (B-ALL), Diffuse Large B-cell Lymphoma (DLBCL) and Multiple Myeloma (MM), however there has been relatively little success in the treatment of other cancers.

Engineered cells face hostile microenvironments which limit adoptive immunotherapy. Modulating the tumour microenvironment may convert the microenvironment into a more favourable environment which enables the engineered immune effector cells to proliferate, survive and/or engraft thereby providing a more effective engineered cell therapy. One of the main inhibitory mechanisms within the tumour microenvironment is transforming growth factor beta (TQRb).

The use of systemic therapeutic agents which block TQRb has been tested. For example, Fresolimumab is a neutralizing antibody which blocks TQRb1-3. Fresolimumab has been tested in metastatic melanoma and high-grade glioma. This showed some effectiveness in the enhancement of a tumour-specific immune response but failed to eradicate the tumour.

Other approaches include small molecules which inhibit SMAD signalling, downstream of transforming growth factor beta receptor (TbR). The best characterized example is Galunisertib which has been tested as a monotherapy or in combination with alkylating agents, Lomustine or temozolamide for glioblastoma and other combinations. These approaches have focussed on the inhibitory microenvironment and have not been particularly effective.

Accordingly, there remains a need for approaches to produce immune effector cells which are capable of tolerating the tumour microenvironment e.g. which are less susceptible to TQRb, which may improve the effectiveness of engineered immune effector cells to proliferate, survive and/or engraft in the microenvironment.

SUMMARY OF THE INVENTION

The present inventors have designed and generated cells with an in-built mechanism to reduce immunosupprossive signalling by molecules such as TQRb. The present invention provides engineered cells comprising a dominant negative SMAD which renders the cells less susceptible (i.e. more resistant) to TGFb-mediated signalling.

Accordingly, in a first aspect, the present invention provides a cell which co-expresses (i) a dominant-negative SMAD (dnSMAD); and (ii).a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

The dnSMAD may be a dominant negative SMAD2, SMAD3 or SMAD4 polypeptide.

The dnSMAD may lack a functional MH1 domain and/or a functional nuclear localization signal (NLS) domain.

In a second aspect, the present invention provides a chimeric dominant negative SMAD (dnSMAD) which comprises at least two dnSMAD polypeptides. The at least two dnSMAD polypeptides may be joined by a linker domain.

The chimeric dnSMAD may comprise: (i) a dnSMAD2 polypeptide and a dnSMAD3 polypeptide; (ii) a dnSMAD2 polypeptide and a dnSMAD4 polypeptide; or (iii) a dnSMAD3 polypeptide and a dnSMAD4 polypeptide. In a third aspect, there is provided a polynucleotide which encodes a chimeric dnSMAD according to the second aspect of the invention.

In a fourth aspect, there is provided a nucleic acid construct which comprises: (i) a first polynucleotide according to the third aspect of the invention or which encodes a dnSMAD as defined in the first aspect of the invention; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

The first and second polynucleotides may be separated by a co-expression site.

In a fifth aspect, the present invention provides a kit of polynucleotides comprising: (i) a first polynucleotide according to the third aspect of the invention or which encodes a dnSMAD as defined in the first aspect of the invention; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

In a sixth aspect, the present invention provides a vector which comprises a polynucleotide according to the second aspect of the invention or a nucleic acid construct according to the third aspect of the invention.

In a seventh aspect, there is provided a kit of vectors which comprises: (i) a first vector which comprises a polynucleotide which encodes a dnSMAD as defined above or a chimeric dnSMAD according to the second aspect of the invention; and (ii) a second vector which comprises a polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

In an eighth aspect, the present invention provides a pharmaceutical composition which comprises a plurality of cells according to the first aspect of the invention.

In a ninth aspect, the present invention provides a pharmaceutical composition according to the eighth aspect of the invention, for use in treating a disease.

In a tenth aspect, the present invention provides a method for treating a disease, which comprises the step of administering a pharmaceutical composition according to the eighth aspect of the invention to a subject in need thereof. In an eleventh aspect, the present invention provides the use of a cell according to the first aspect of the invention in the manufacture of a pharmaceutical composition for the treatment of a disease.

The disease may be cancer.

In a twelfth aspect, there is provided a method for making a cell according to the first aspect of the invention, which comprises the step of introducing: a polynucleotide according to the third aspect of the invention, a nucleic acid construct according to the fourth aspect of the invention, a kit of polynucleotides according to the fifth aspect of the invention, a vector according to the sixth aspect of the invention, or a kit of vectors according to the seventh aspect of the invention into a cell ex vivo.

Advantageously, the present inventors provide engineered immune effector cells which counteract the inhibitory microenvironment and prevent inhibition of immune effector cells, thereby augmenting the ability of intrinsic tumour-specific T cells or engineered immune effector cells to attack the tumour.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 - Schematic diagram showing different generations of chimeric antigen receptors. The basic architecture of a canonical CAR is shown as well as different iterations of the three generations of this form of receptor.

Figure 2 - Schematic diagram showing the domain structure of SMAD2, SMAD3, SMAD4 and SMAD7.

Figure 3 - Schematic diagram showing a dnSMAD2/3 preventing signalling through the TQRb receptor.

Figure 4 - Schematic diagram showing a dnSMAD4 preventing signalling through the TQRb receptor.

Figure 5 - Cytotoxicity assay. Graphs showing the % live target cells where T cells expressing a second generation anti-GD2 CAR with a CD28-CD3z endodomain either alone or in combination with a truncated SMAD2 (dnSMAD2-MH2) or truncated SMAD3 (dnSMAD3-MH2) molecule were co-cultured with GD2-expressing SupT1 target cells at a 2:1 and 4:1 target: effector cell ratio. TQRb was spiked into the co-culture at 10ng/ml on day 0 and target cell killing was investigated at day 5 and 7 by flow cytometry.

DETAILED DESCRIPTION OF THE INVENTION

Transforming growth factor beta (TGF-b) is a cytokine belonging to the transforming growth factor superfamily.

The transforming growth factor beta receptors are a superfamily of serine/threonine kinase receptors. These receptors bind members of the TΰRb superfamily of growth factor and cytokine signalling proteins. There are five type II receptors (which are activatory receptors) and seven type I receptors (which are signalling propagating receptors). Type I receptors are also known as activin receptor-like kinases (ALKS).

Auxiliary co-receptors (also known as type III receptors) also exist. Each subfamily of the TΰRb superfamily of ligands binds to type I and type II receptors.

The amino acid sequence of human TGF-beta receptor type-1 O^RI) is available from UniProt accession P36897. The amino acid sequence of human TGF-beta receptor type-2 (TbRII) is available from UniProt accession P37173.

The three transforming growth factors have many activities. TΰRb1 and 2 are implicated in cancer, where they may stimulate the cancer stem cell, increase fibrosis / desmoplastic reactions and suppress immune recognition of the tumour.

A variety of cancerous tumour cells are known to produce TΰRb directly. In addition to the TΰRb production by cancerous cells, TΰRb can be produced by the wide variety of non- cancerous cells present at the tumour site. Specifically, tumour-associated T cells, natural killer (NK) cells, macrophages, epithelial cells and stromal cells have all been shown to produce TGF-b in various tumour models.

TΰRb1 , 2 and 3 signal via binding to receptors TbRII and then association to TbRI and in the case of TΰRb2 also to TbRIII. This leads to subsequent signalling through SMADs via TbRI.

The signalling pathway is activated following the formation of a hetero-tetramer consisting of TGF-b and the receptors TGFBRI and TGFBRII. Structurally TGFBRI and TGFBRII are composed of a short disulphide rich endodomain that is heavily bonded in a three-finger toxin conformation. These domains contain binding sites for serine/threonine kinases. Upon TGF-b ligation to TGFBRII, the endodomain is auto-phosphorylated enabling the recruitment and subsequent cross-linking with TGFBRI.

TGF-b receptors are serine/threonine kinases. Following TGF-b ligation, TGFBRII is auto- phosphorylated enabling the recruitment and cross-linking with TGFBRI. Activation of TGFBRI kinases phosphorylates receptor regulated SMAD (R-SMAD) proteins.

R-SMADs are transcription factors specific for the TGF-b family. Upon receptor ligation, serine residues in the C-terminus of R-SMAD are phosphorylated resulting in their detachment from the TGFBRI- TGFBRII receptor complex. R-SMADs exist in two subtypes with SMAD 2 and SMAD 3 phosphorylated upon TGF-b receptor ligation. Once phosphorylated, both R-SMAD subclasses form a complex with SMAD 4 which is a common mediator SMAD (co-SMAD).

SMAD proteins consist of two globular domains connected by a linker region. The main function of the SMAD N-terminal domain, or“Mad homology 1” (MH1 ) domain, is to bind DNA. The C-terminal domain, or MFI2 domain, mediates protein-protein interaction with numerous regulator and effector proteins, including the TbB receptors, certain cytoplasmic anchor proteins, lineage-specific DNA-binding cofactors, and chromatin modifiers. In the presence of TΰBb, R-SMAD is phosphorylated by TΰBb receptor. This phosphorylation targets two serine residues in the SMAD C terminus sequence, pSer-X-pSer, creating an acidic tail that drives the formation of SMAD transcriptional complexes.

Phosphorylated R-SMAD proteins bind the MH2 domain of SMAD 4 enabling accumulation of this complex at the nucleus, where SMAD complexes act as DNA site-specific transcriptional regulators.

Complexes containing SMAD 3 and SMAD 4 recognise 5’CAGAC3’ binding elements. The regulation of gene expression is achieved by SMAD proteins interacting with transcriptional activators and repressors to initiate the functions associated with TGF-b ligands.

DOMINANT NEGATIVE SMAD (dnSMAD)

As used herein“dominant-negative SMAD (dnSMAD) polypeptide” refers to a SMAD which acts antagonistically to the wild-type SMAD polypeptide. For example, a dnSMAD polypeptide may be capable of binding to TQBbR and/or to other SMADs but be incapable of modulating or have a reduced capacity to modulate (e.g. inhibit/augment) transcriptional activity.

For example, a dnSMAD polypeptide may have an MH2 domain which is capable of binding to TQBbR and to other SMADs but is incapable of modulating or have a reduced capacity to modulate (e.g. inhibit/augment) transcriptional activity.

A dnSMAD or chimeric dnSMAD as disclosed herein may inhibit the function of its natural or wild-type counterpart. The dnSMAD or chimeric dnSMADs may inhibit signalling induced by wild-type TQRb and thus neutralise its biological effects.

Binding of the dnSMAD to Tbί^ or to other SMADs may inhibit signalling downstream of TbίT

Any method known in the art for determining protei protein interactions may be used to determine whether a dnSMAD is capable of binding to a TQBbR or to other SMAD(s). For example, co-immunoprecipitation followed by western blot.

Any method known in the art may be used to determine whether a SMAD is capable of modulating (e.g. initiating or augmenting) transcriptional activity. For example, a transcriptional response assay may be used wherein cells are transfected with a promoter reporter construct e.g. luciferase in the presence of various combinations of SMAD plasmids. The activity of the reporter gene may be measured as an indicator of SMAD ability to initiate transcriptional activity.

A dnSMAD may be identified or characterised using a TQBb cytotoxicity assay. An example of methodology for such an assay is as follows: PBMCs depleted of CD56+ cells and transfected with nucleic acids encoding dnSMADs or wild-type SMADs may be co-cultured with target cells in e.g. a 1 :2 effector: target cell ratio in the presence and absence of TQBb. Cytotoxicity may be assessed by flow cytometry e.g. after 1 , 5 and 7 days. The dnSMAD may show increased (e.g. at least 1.2 fold, or at least 1 .5 fold, or at least 2 fold, or at least 3 fold, or at least 4 fold, or at least 5 fold) cytotoxicity compared with a wild-type SMAD in the presence of TQBb, the dnSMAD reduces (e.g. inhibits) the effects of TQBb.

A dnSMAD may be identified or characterised using a proliferation assay. An example of methodology for such an assay is as follows: T cells transfected with nucleic acids encoding dnSMADs or wild-type SMADs may be co-cultured with target cells in e.g. a 1 :2 effector: target cell ratio in the presence and absence of TQRb. The dnSMAD may restore proliferation compared with a wild-type SMAD in the presence of TQRb. For example the proliferation rate of the cell comprising the dnSMAD in the presence of TQRb may be essentially the same as the proliferation rate of a cell comprising the dnSMAD in the absence of TQRb e.g. the difference in proliferation rate between a cell comprising a dnSMAD in the presence of TQRb compared with proliferation rate of a cell comprising a dnSMAD the absence of TQRb may be less than 40% difference, less than 30% difference, less than 20% difference, less than 20% difference, less than 10% difference, less than 5% difference or less than 3% difference.

The dnSMAD may be a dominant negative version of SMAD2, SMAD3 or SMAD4.

The dnSMAD may be a dominant negative SMAD2 polypeptide. The dnSMAD may be a dominant negative SMAD3 polypeptide. The dnSMAD may be a dominant negative SMAD4 polypeptide.

Various dominant negative SMADs have been described, as summarised in Table 1.

Table 1 - Dominant negative SMADs

The dnSMAD may lack a functional MH1 domain. An“MH1 domain” as used herein refers to a conserved MAD homology domain of at the N terminus of a SMAD protein. The MH1 domain is capable of DNA binding. The MH1 domain negatively regulates the functions of the MH2 domain.

DNA binding may be determined using any method known in the art, for example quantitative-electrophoretic mobility shift assay (EMSA) with labelled DNA and purified protein e.g. purified MH1 protein. Other methods for measuring DNA binding include isothermal titration calorimetry which titrates small volumes of concentrated ligand (e.g. MH1 ) into the DNA and measures the amount of heat which needs to be added or subtracted to return it to a reference temperature.

The MH1 domain of SMAD2 corresponds to amino acids 10-176 of SEQ ID NO: 1. SEQ ID NO:1 , which is UniProtKB accession Q15796 (SMAD2 Human).

MSSILPFTPPVVKRLLGWKKSAGGSGGAGGGEQNGQEEKWCEKAVKSLVKKLKKTGRLD

ELEKAITTQNCNTKCVTIPSTCSEIWGLSTPNTIDQWDTTGLYSFSEQTRSLDGRLQVSHRK

GLPHVIYCRLWRWPDLHSHHELKAIENCEYAFNLKKDEVCVNPYHYQRVETPVLPPVLVPR

HTEILTELPPLDDYTHSIPENTNFPAGIEPQSNYIPETPPPGYISEDGETSDQQLNQSMDTGS

PAELSPTTLSPVNHSLDLQPVTYSEPAFWCSIAYYELNQRVGETFHASQPSLTVDGFTDPS

NSERFCLGLLSNVNRNATVEMTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYG

WHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYR

RQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSVRCSSMS (SEQ ID NO: 1 ).

The MH1 domain of SMAD3 corresponds to amino acids 10-136 of SEQ ID NO: 2. SEQ ID NO: 2 is UniProtKB accession P84022 (SMAD3_Human).

MSSILPFTPPIVKRLLGWKKGEQNGQEEKWCEKAVKSLVKKLKKTGQLDELEKAITTQNVNT

KCITIPRSLDGRLQVSHRKGLPHVIYCRLWRWPDLHSHHELRAMELCEFAFNMKKDEVCVN

PYHYQRVETPVLPPVLVPRHTEIPAEFPPLDDYSHSIPENTNFPAGIEPQSNIPETPPPGYLS

EDGETSDHQMNHSMDAGSPNLSPNPMSPAHNNLDLQPVTYCEPAFWCSISYYELNQRVG

ETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAE

CLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLT

RMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSSVS

(SEQ ID NO: 2).

The MH1 domain of SMAD 4 corresponds to amino acids 18-142 of SEQ ID NO: 3. SEQ ID NO: 3 is UniProtKB accession Q13485 (SMAD4_Human). MDNMSITNTPTSNDACLSIVHSLMCHRQGGESETFAKRAIESLVKKLKEKKDELDSLITAITT

NGAHPSKCVTIQRTLDGRLQVAGRKGFPHVIYARLWRWPDLHKNELKHVKYCQYAFDLKC

DSVCVNPYHYERVVSPGIDLSGLTLQSNAPSSMMVKDEYVHDFEGQPSLSTEGHSIQTIQH

PPSNRASTETYSTPALLAPSESNATSTANFPNIPVASTSQPASILGGSHSEGLLQIASGPQP

GQQQNGFTGQPATYHHNSTTTWTGSRTAPYTPNLPHHQNGHLQHHPPMPPHPGH

YWPVHNELAFQPPISNHPAPEYWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGD

RFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGR

APG DAVFI Kl YPSAYI KVFDLRQCFI RQMQQQAAT AQAAAAAQAAAVAGN I PG PGS VGG I APA

ISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMP

IADPQPLD (SEQ ID NO: 3).

The phrase“corresponds to” indicates that the amino acid position is equivalent to the one shown for the recited amino acid sequence e.g. the amino acid sequence shown in SEQ ID NO: 1 . It will be appreciated that the actual number of amino acids from the N-terminus of the protein may vary between different proteins. However, it is clear from, an alignment of the dnSMAD protein or chimeric dnSMAD protein with the sequence of the recited amino acid sequence (e.g. SEQ ID NO: 1 ) which is the“equivalent” amino acid position.

For example, the S-X-S motif at amino acid positions 465-467 of SEQ ID NO: 1 correspond to amino acid positions ! 92-194 of SEQ ID NO: 4 and SEQ ID NO: 8.

A functional MH1 domain is capable of DNA binding. A polypeptide which“lacks a functional MH1 domain” as used herein exhibits reduced DNA binding or DNA binding is completely eliminated when compared with a wild type MH1 domain. Suitably, a polypeptide which lacks a functional MH1 domain may exhibit less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% of the DNA binding compared to a wild type MH1 polypeptide. In one embodiment, a polypeptide which lacks a functional MH1 domain may be unable to bind to DNA.

In one embodiment, the dnSMAD essentially lacks a MH1 domain compared to a corresponding wild-type SMAD polypeptide.

In one embodiment, the nucleic acid sequence which encodes the MH1 domain may be wholly or partially deleted. The deletion may be continuous, or may comprise a plurality of sections of sequence. The deletion preferably removes a sufficient amount of nucleotide sequence such that the nucleic acid sequence no longer encodes a functional protein. The deletion may be total, in which case 100% of the coding portion of the nucleic acid sequence is absent, when compared to the corresponding nucleic acid sequence of a wild type MH1 . The deletion may, for example, remove at least 70%, 80%, 90% or 95% of the MH1 -coding portion of the nucleic acid sequence.

The dnSMAD may lack a functional nuclear localization signal (NLS) domain.

As used herein“nuclear localisation signal” has its normal meaning in the art and refers to an amino acid sequence which tags a protein for nuclear transport for import into the cell nucleus. NLS typically comprise one or more short sequences of charged lysines or arginines.

A polypeptide which lacks a functional NLS is not capable of import to the cell nucleus. Any methods known in the art for monitoring translocation of proteins may be used to determine whether a polypeptide lacks a functional NLS. For example, nuclear import may be monitored using immunofluorescence confocal microscopy or by subcellular fractionation and organelle isolation of the nucleus followed by western blot to determine the subcellular localisation of the polypeptide.

The wild-type SMAD NLS may have the sequence: Lys-Lys-Leu-Lys-Lys. For example the NLS sequence in SMAD3 spans residues 40-44 and has the sequence: KKLKK

In one embodiment, the dnSMAD lacks a NLS domain compared to a corresponding wild- type SMAD polypeptide.

The dnSMAD may comprise a deletion or substitution in the NLS. For example, one or more of the lysine residues in the NLS may be deleted or substituted with another amino acid such as glutamine. All four lysine residues may be substituted with an alternative amino acid.

An“MFI2 domain” as used herein refers to a conserved MAD homology domain of at the C terminus of a SMAD protein. The MFI2 domain contain a central b-sandwich with a conserved loop-helix is capable of binding phosphor-serine residues. The MFI2 domain mediates protein: protein interactions with regulator and effector proteins, including the Tbί^ receptors, cytoplasmic anchor proteins, lineage-specific DNA-binding cofactor and chromatin modifiers.

Examples of MFI2 binding partners are shown in Table 2. Table 2 - MHS binding partners

The dnSMAD may comprise, consist essentially of, or consist of a wild-type MH2 domain.

For example, the dnSMAD may consist essentially of or consist of the MH2 domain of wild- type SMAD2 which has the sequence shown as SEQ ID No. 4.

WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM GSPSVRCSSMS (SEQ ID NO: 4).

Alternatively, the dnSMAD may consist essentially of or consist of the MH2 domain of wild- type SMAD3 which has the sequence shown as SEQ ID No. 8.

WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM GSPSIRCSSVS (SEQ ID NO: 8).

Alternatively, the dnSMAD may consist essentially of or consist of the MH2 domain of wild- type SMAD4 which has the sequence shown as SEQ ID No. 12.

WCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLSNVHRTEAIERARLHI GKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHKIYPSAYIKVFDLRQC HRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGIGVDDLRRLCILRMS FVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD (SEQ ID NO: 12). The dnSMAD may comprise, consist essentially of or consist of a truncated MH2 domain of a wild type SMAD polypeptide, such as a truncated version of SEQ ID NO: 4, 8 or 12.

The truncation may be at the C-terminus, for example, Yuan and Varga (2001 , as above) describe a dnSMAD4 with a C-terminal truncation of 51 amino acids.

The truncated MH2 domain may:

a) retain a Helix5 sequence which contains a SARA binding site which corresponds to amino acid residues 171 -183 of SEQ ID NO: 4; or

b) lack a Helix5 sequence which contains a SARA binding site which corresponds to amino acid residues 171 -183 of SEQ ID NO: 4.

The truncated MH2 domain may:

a) lack at least the amino acids which correspond to position 457 to 467 of SEQ ID NO: 1 or which correspond to position 184 to 194 of SEQ ID NO: 4; or

b) lack at least the amino acids which correspond to position 444 to 467 of SEQ ID NO: 1 or which correspond to position 171 to 194 of SEQ ID NO: 4.

A dnSMAD which lacks a functional MH1 domain may further:

a) lack at least the amino acids which correspond to position 457 to 467 of SEQ ID NO: 1 ; or b) lack at least the amino acids which correspond to position 444 to 467 of SEQ ID NO: 1.

A dnSMAD4 may comprise the mutation R497H, K507Q and/or R515G where the amino acid numbering corresponds to the numbering set forth in SEQ ID NO: 3.

A dnSMAD3 may comprise a mutation K378R and/or K314R where the amino acid numbering corresponds to the numbering set forth in SEQ ID NO: 2.

Where the dnSMAD has a SMAD2 MH2 domain comprising a C terminal truncation, the truncation may be a deletion of at least 1 1 amino acids from the C terminus, wherein the amino acid numbering corresponds to SEQ ID NO: 4. In this respect, the dnSMAD may consist essentially of or consist of SEQ ID NO: 5 or a variant of SEQ ID NO: 5 which has at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity thereto.

WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM (SEQ ID NO: 5).

The truncation may be a deletion of at least 24 amino acids from the C terminus, wherein the amino acid numbering corresponds to SEQ ID NO: 4. In this respect, the dnSMAD may consist essentially of or consist of SEQ ID NO: 6 or a variant of SEQ ID NO: 6 which has at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity thereto.

WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLN (SEQ ID NO: 6)

Where the dnSMAD has a SMAD3 MH2 domain comprising a C terminal truncation, the truncation may be a deletion of at least 1 1 amino acids from the C terminus, wherein the amino acid numbering corresponds to SEQ ID NO: 8. In this respect, the dnSMAD may consist essentially of or consist of SEQ ID NO: 9 or a variant of SEQ ID NO: 9 which has at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity thereto.

WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

(SEQ ID NO: 9).

The truncation may be a deletion of at least 24 amino acids from the C terminus, wherein the amino acid numbering corresponds to SEQ ID NO: 8. In this respect, the dnSMAD may consist essentially of or consist of SEQ ID NO: 10 or a variant of SEQ ID NO: 10 which has at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity thereto.

WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLN (SEQ ID NO: 10). The dnSMAD may further comprise a mutation in the MH2 domain which increases the binding affinity of the dnSMAD for a phosphorylated TQEb receptor compared to a corresponding MH2 domain which does not comprise said mutation.

An increase in binding to the phosphorylated TQEb may be determined using any method known in the art or described herein; for example cells may be transfected with a nucleic acid encoding a dnSMAD comprising a mutation in the MH2 domain or with a nucleic acid encoding a corresponding MH2 domain which does not comprise said mutation. A comparison of the binding to phosphorylated TQRb may be determined by co- immunoprecipitation and western blotting of the phosphorylated TQRb receptor and the dnSMAD versus co-immunoprecipitation of the phosphorylated TQRb receptor an MH2 domain which does not comprise the mutation.

Other assays for measuring binding are known in the art such as radioactive ligand binding assays (including saturation binding, scatchard plot), non-radioactive ligand binding assays (including fluorescence polarization, fluorescence resonance energy transfer and surface plasmon resonance/Biacore, and solid phase ligand binding assays.

The dnSMAD may comprises a substitution of one or both amino acids which correspond to positions 465 and 467 of SEQ ID NO: 1 . The substitution of the amino acids which correspond to positions 465 and 467 of SEQ ID NO: 1 may prevent phosphorylation of the SMAD C terminus sequence. The substitution of the amino acids which correspond to positions 465 and 467 of SEQ ID NO: 1 may replace one or both of the serine residues in the Ser-X-Ser motif with an amino acid which cannot be phosphorylated. Suitably, one or both serine residues are not substituted for threonine, tyrosine, histidine, lysine, arginine, aspartic acid or glutamic acid. In one embodiment, one or both serine residues are substituted for alanine.

The dnSMAD may consist essentially of or consist of SEQ ID NO: 7 or a variant of SEQ ID NO: 7 which has at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity thereto and comprises amino acid substitutions at positions which correspond to positions 465 and 467 of SEQ ID NO: 1. One or both of the substitutions may be the substitution of serine for an amino acid which cannot be phosphorylated (e.g. alanine).

In SEQ ID NO: 7, the residues in bold highlight substitutions in the in the Ser-X-Ser motif relative to SEQ ID NO: 1. WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM GSPSVRCSAMA (SEQ ID NO: 7).

The dnSMAD may consist essentially of or consist of SEQ ID NO: 1 1 or a variant of SEQ ID NO: 1 1 which has at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity thereto and comprises amino acid substitutions at positions which correspond to positions 465 and 467 of SEQ ID NO: 1. Suitably, one or both of the substitutions may be the substitution of serine for an amino acid which cannot be phosphorylated (e.g. alanine).

In SEQ ID NO: 1 1 , the residues in bold highlight substitutions in the in the Ser-X-Ser motif relative to SEQ ID NO: 1.

WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGR GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM GSPSIRCSAVA (SEQ ID NO: 1 1 ).

The terms protein and polypeptide are used synonymously herein.

CHIMERIC dnSMAD

In one embodiment the present invention provides a chimeric dnSMAD which comprises at least two dnSMAD polypeptides as defined herein. In one embodiment, the chimeic dnSMAD may comprise at least two, at least 3 at least 4, at least five dnSMADs as defined herein.

In one embodiment the chimeric dnSMAD comprises a dnSMAD2 and a dnSMAD3. In one embodiment the chimeric dnSMAD comprises a dnSMAD2 and a dnSMAD4. In one embodiment the chimeric dnSMAD comprises a dnSMAD3 and a dnSMAD4. In one embodiment the chimeric dnSMAD comprises at least two dnSMAD2s. In one embodiment the chimeric dnSMAD comprises at least two dnSMAD2s. In one embodiment the chimeric dnSMAD comprises at least two dnSMAD4s. In one embodiment the chimeric dnSMAD comprises a dnSMAD2, a dnSMAD3 and a dnSMAD4. Suitably, the dnSMAD polypeptides of the chimeric dnSMAD may be connected by a linker domain.

Linkers (or spacers) are short sequences which separate multiple domains in a protein. A linker may comprise flexible residues such as glycine and serine repeats which allow the adjacent protein domains to move relative to one another.

Suitably, the chimeric dnSMAD may comprise one or more serine-glycine linker domains. The linker may be of any length with provides sufficient flexibility. Suitably, the linker may be 2, 3, 4, 5, 10, 15, 20, 25, 30 or more residues long. In one embodiment, the chimeric dnSMAD comprises a linker comprising or consisting essentially of or consisting of the following sequence: LEYSGGGSGGGSLE (SEQ ID NO: 19).

In one embodiment, the chimeric dnSMAD comprises a dnSMAD2 polypeptide and a dnSMAD3 polypeptide.

Suitably, the chimeric dnSMAD may comprise an SMAD2-MH2 domain and a SMAD3-MH2 domain. Suitably, one or more of the SMAD-MH2 domains may be a truncated MH2 domain. Suitably, the dnSMAD may also comprise a linker domain.

In one embodiment, the dnSMAD chimeric molecule comprises or consists essentially of or consists of a sequence set forth in SEQ ID NO: 13. The linker sequence is shown in bold.

WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ

SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

LEYSGGGSGGGSLEWCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNV

NRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGC

NLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELH

LNGPLQWLDKVLTQM (SEQ ID NO: 13)

Suitably, the chimeric dnSMAD may comprise a dnSMAD2 polypeptide and a dnSMAD4 polypeptide. Suitably, one or more of the SMAD-MH2 domains may be a truncated MH2 domain. Suitably, the dnSMAD may also comprise a linker domain.

In one embodiment, the dnSMAD chimeric molecule comprises or consists essentially of or consists of a sequence set forth in SEQ ID NO: 14. The linker sequence is shown in bold. WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ

SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

LEYSGGGSGGGSLEWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLS

NVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHK

IYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGI

GVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD

(SEQ ID NO: 14)

Suitably, the chimeric dnSMAD may comprise a dnSMAD3 polypeptide and a dnSMAD4 polypeptide. Suitably, one or more of the SMAD-MH2 domains may be a truncated MH2 domain. Suitably, the dnSMAD may also comprise a linker domain.

In one embodiment, the dnSMAD chimeric molecule comprises or consists essentially of or consists of a sequence set forth in SEQ ID NO: 15. The linker sequence is shown in bold.

WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ

SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

LEYSGGGSGGGSLEWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPSGGDRFCLGQLS

NVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDREAGRAPGDAVHK

IYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGGIAPAISLSAAAGI

GVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLHTMPIADPQPLD

(SEQ ID NO: 15)

Suitably, the chimeric dnSMAD may comprise a dnSMAD2 polypeptide and a dnSMAD3 polypeptide. Suitably, one or more of the SMAD-MH2 domains may comprise a mutation in the SXS motif of the MH2 domain. Suitably, the dnSMAD may also comprise a linker domain.

In one embodiment, the dnSMAD chimeric molecule comprises or consists essentially of or consists of a sequence set forth in SEQ ID NO: 16. The linker sequence is shown in bold.

WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ

SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

GSPSVRCSAMALEYSGGGSGGGSLEWCSISYYELNQRVGETFHASQPSMTVDGFTDPSN SERFCLGLLSNVNRNAAVELTRRHIGRGVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGW HPATVCKIPPGCNLKIFNNQEFAALLAQSVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQ TVTSTPCWIELHLNGPLQWLDKVLTQMGSPSIRCSAVA (SEQ ID NO:16)

Suitably, the chimeric dnSMAD may comprise a dnSMAD2 polypeptide and a dnSMAD4 polypeptide. Suitably, the SMAD2-MH2 domain may comprise a mutation in the SXS motif of the MH2 domain. Suitably, the dnSMAD may also comprise a linker domain.

In one embodiment, the dnSMAD chimeric molecule comprises or consists essentially of or consists of a sequence set forth in SEQ ID NO: 17. The linker sequence is shown in bold.

WCSIAYYELNQRVGETFHASQPSLTVDGFTDPSNSERFCLGLLSNVNRNATVEMTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ

SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

GSPSVRCSAMALEYSGGGSGGGSLEWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPS

GGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDRE

AGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGG

IAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLH

TMPIADPQPLD (SEQ ID NO: 17)

Suitably, the chimeric dnSMAD may comprise a dnSMAD3 polypeptide and a dnSMAD4 polypeptide. Suitably, the SMAD3-MH2 domain may comprise a mutation in the SXS motif of the MH2 domain. Suitably, the dnSMAD may also comprise a linker domain.

In one embodiment, the dnSMAD chimeric molecule comprises or consists essentially of or consists of a sequence set forth in SEQ ID NO: 18. The linker sequence is shown in bold.

WCSISYYELNQRVGETFHASQPSMTVDGFTDPSNSERFCLGLLSNVNRNAAVELTRRHIGR

GVRLYYIGGEVFAECLSDSAIFVQSPNCNQRYGWHPATVCKIPPGCNLKIFNNQEFAALLAQ

SVNQGFEAVYQLTRMCTIRMSFVKGWGAEYRRQTVTSTPCWIELHLNGPLQWLDKVLTQM

GSPSIRCSAVALEYSGGGSGGGSLEWCSIAYFEMDVQVGETFKVPSSCPIVTVDGYVDPS

GGDRFCLGQLSNVHRTEAIERARLHIGKGVQLECKGEGDVWVRCLSDHAVFVQSYYLDRE

AGRAPGDAVHKIYPSAYIKVFDLRQCHRQMQQQAATAQAAAAAQAAAVAGNIPGPGSVGG

IAPAISLSAAAGIGVDDLRRLCILRMSFVKGWGPDYPRQSIKETPCWIEIHLHRALQLLDEVLH

TMPIADPQPLD (SEQ ID NO: 18) A chimeric dnSMAD according to the present invention may comprise at least one dnSMAD according to the present invention. Suitably, a dnSMAD may comprise at least one sequence selected from: SEQ ID NO: 4 to 18; or a variant having at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 4 to 18. Suitably, the chimeric dnSMAD may comprise two, or three or four or five or more sequences selected from SEQ ID NO: 4 to 18; or a variant having at least 80% (preferably at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%) sequence identity to SEQ ID NO: 4 to 18.

TUNING

The present invention provides a cell which encodes a dnSMAD, wherein the expression of said dnSMAD may be“tunable”.

As used herein,“tunable” means that it is possible to increase, decrease, turn on or turn off the expression of the dnSMAD in the engineered immune effector cell.

Suitably, expression of the dnSMAD may be controlled or tuned by an inducible promoter. Suitably, the dnSMAD may be regulated by Nuclear factor of activated T cells (NFAT) response element.

An NFAT response element may comprise the nucleotide sequence set forth in SEQ ID NO: 20 or a variant thereof.

GGAGGAAAAACT GTTT CAT ACAGAAGGCGT (SEQ ID NO: 20).

Variant sequences of SEQ ID NO: 20 may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 20. Suitably, the variant sequence is able to function as a NFAT response element.

The NFAT response element may comprise repeat units such as 3, 4, 5 or 6 repeat units. Suitably, the NFAT response element may comprise 3, 4, 5 or 6 repeat units of SEQ ID NO: 20. The NFAT response element may be positioned in front of a promoter (e.g. a CMV promoter).

The expression or activity of the dnSMAD may be controlled or tuned through interaction with an intracellular retention domain. For example, the dnSMAD may be retained within a cellular compartment by interaction with an intracellular retention domain. An agent may be used to disrupt the interaction with the intracellular retention domain, thereby allowing translocation of the dnSMAD and expression of the dnSMAD in the appropriate cellular localisation of the engineered immune effector cell.

The activity of the dnSMAD factor may be controlled or tuned.

The dnSMAD or chimeric dnSMAD according to the present invention may compete with the wild type SMAD protein for the receptor-docking domain on TbR or for competes with the wild type SMAD protein for binding with partner proteins.

The dnSMAD or chimeric dnSMAD according to the present invention may reduce or completely inhibit TQRb signalling in the engineered immune effector cell.

The dnSMAD or chimeric snSMAD according to the present invention may reduce or eliminate signalling downstream of TbίT

Assays for measuring downstream signalling of Tbί^ are known in the art such as luminescent kinase assays which measure ADP formed from the kinase reaction or measuring the proportion of cytoplasmic signalling molecules such as SMAD/SMAD2 phosphorylation. Any method known in the art may be used to measure downstream signalling of TbίT dnSMADs or chimeric SMADs according to the present invention may maintain their ability to bind to TbR and/or to other SMADs. The ability of a variant TQRb to bind to a TbR or to another SMAD may be measured by any means known in the art for example by an ELISA assay, to detect TbR receptor chain or other SMADs. dnSMADs (or chimeric dnSMADs) according to the present invention may block the binding of wild-type SMADs to TbR or to other wild type SMADs. This may be measured by a competitive ELISA for example, by covering the plates with TbR or wild type SMAD and assessing the ability of the dnSMAD (or chimeric dnSMAD) to inhibit binding of wild type TbR or wild type SMADs to their wild type binders. dnSMADs may be tested for in vitro binding to consensus binding sequences for example using GST fusion proteins. dnSMADs (or chimeric dnSMADs) according to the present invention may have reduced ability to signal through TbR or may not be capable of signalling through TbίT This may be measure by measuring the transcription of genes induced by TQRb for example by qPCR. A dnSMAD (or chimeric dnSMADs) according to the present invention may exhibit reduced phosphorylation of the wild type SMAD2/3. This may be measured by western blot or flow- cytometry using antibodies specific for the phosphorylated tyrosine of the wild-type SMAD.

dnSMADs (or chimeric dnSMADs) according to the present invention may be capable of inhibiting signalling induced by wild-type TQRb. This may be measured by Western immunoblotting assays and quantifying the levels of phosphorylated SMAD2 and SMAD3 in cell lysates treated with the mutant or wild-type TQRb.

CELL

The present invention relates to a cell which expresses a dominant-negative SMAD (dnSMAD). The cell may also express. a chimeric antigen receptor (CAR) or a transgenic T- cell receptor (TCR). The cell may be engineered to express the dnSMAD and/or CAR/TCR.

An“engineered cell” as used herein means a cell which has been modified to comprise or express a nucleic acid sequence which is not naturally encoded by the cell. 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 sequence into a cell.

Accordingly, the nucleic acid sequence encoding the dnSMAD is not naturally expressed by a corresponding, unmodified cell.

An engineered cell is a cell whose genome has been modified e.g. by transduction or by transfection, such as retroviral or lentiviral transduction.

As used herein, the term“introduced” refers to methods for inserting foreign DNA or RNA into a cell. As used herein the term introduced includes both transduction and transfection methods. Transfection is the process of introducing nucleic acids into a cell by non-viral methods. Transduction is the process of introducing foreign DNA or RNA into a cell via a viral vector. Engineered cells according to the present invention may be generated by introducing DNA or RNA coding a dnSMAD by one of many means including transduction with a viral vector, transfection with DNA or RNA.

Cells may be activated and/or expanded prior to the introduction of a nucleic acid sequence encoding the dnSMAD, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies. As used herein“activated” means that a cell has been stimulated, causing the cell to proliferate, differentiate or initiate an effector function.

Methods for measuring cell activation are known in the art and include, for example, measuring the expression of activation markers by flow cytometry, such as the expression of CD69, CD25, CD38 or HLA-DR or measuring intracellular cytokines.

As used herein“expanded” means that a cell or population of cells 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.

An“immune effector cell” as used herein is a cell of the immune system which responds to a stimulus and effects a change.

Suitably, an immune effector cell may a T cell (such as an alpha-beta T cell or a gamma- delta T cell), a B cell (such as a plasma cell), a Natural Killer (NK) cell or a macrophage.

“Cytolytic immune cell” as used herein is a cell which directly kills other cells. Cytolytic cells may kill cancerous cells; virally infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural killer (NK) cells.

Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a TCR on their cell surface.

Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells, and are also implicated in transplant rejection. CTLs express the CD8 at their surface. CTLs may be known as CD8+ T cells. 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.

The cell of the present invention may be a T -cell, such as an alpha-beta T cell or a gamma- delta T cell.

Natural Killer Cells (or NK cells) are a type of cytolytic cell which 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 cell of the present invention may be a wild-type killer (NK) cell or a cytokine induced killer cell.

The cell may be derived from a patient’s own peripheral blood (1 st 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). T or NK cells, for example, may be activated and/or expanded prior to being transduced with nucleic acid molecule(s) encoding the polypeptides of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

Alternatively, the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells. Alternatively, an immortalized T-cell line which retains its lytic function may be used.

The cell may be a haematopoietic stem cell (HSC). HSCs can be obtained for transplant from the bone marrow of a suitably matched donor, by leukapheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF [peripheral blood stem cells (PBSCs)], or from the umbilical cord blood (UCB) collected from the placenta after delivery. The marrow, PBSCs, or UCB may be transplanted without processing, or the HSCs may be enriched by immune selection with a monoclonal antibody to the CD34 surface antigen.

CHIMERIC ANTIGEN RECEPTOR (CAR)

Classical CARs are chimeric type I trans-membrane proteins which connect 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 or on a ligand for the target antigen. A spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation. A common spacer domain used is the Fc of lgG1. 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 g chain of the FcsR1 or Oϋ3z. 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 ΰϋ3z 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 4-1 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.

CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors. In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer. 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 cells expressing the targeted antigen.

ANTIGEN BINDING DOMAIN

The antigen-binding domain is the portion of a classical CAR which recognizes antigen. Numerous antigen-binding domains are known in the art, including those based on the antigen binding site of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-type ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.

Various tumour associated antigens (TAA) are known, as shown in the following Table 3. The antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.

Table 3

TRANSMEMBRANE DOMAIN

The transmembrane domain is the sequence of a classical CAR that spans the membrane. It may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which gives good receptor stability. CAR OR TCR SIGNAL PEPTIDE

The CAR or transgenic TCR for use in the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed. The core of the signal peptide may contain 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.

SPACER DOMAIN

The receptor may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain. A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.

The spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk. A human lgG1 spacer may be altered to remove Fc binding motifs.

INTRACELLULAR SIGNALLING DOMAIN

The intracellular signalling domain is the signal-transmission portion of a classical CAR.

The most commonly used signalling domain component is that of CD3-zeta endodomain, which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a fully competent activation signal and additional co stimulatory signalling may be needed. For example, chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together.

The intracellular signalling domain may be or comprise a T cell signalling domain.

The intracellular signalling domain may comprise one or more immunoreceptor tyrosine- based activation motifs (ITAMs). An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system. The motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/l. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (YxxL/lx_(6.8)YxxL/l). ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CD3 and z-chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors. The tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the cell.

The intracellular signalling domain component may comprise, consist essentially of, or consist of the Oϋ3-z endodomain, which contains three ITAMs. Classically, the Oϋ3-z endodomain transmits an activation signal to the T cell after antigen is bound.

The intracellular signalling domain may comprise additional co-stimulatory signalling. For example, 4-1 BB (also known as CD137) can be used with Oϋ3-z, or CD28 and 0X40 can be used with Oϋ3-z to transmit a proliferative / survival signal.

The CAR may have the general format: antigen-binding domain-TCR element.

As used herein“TCR element” means a domain or portion thereof of a component of the TCR receptor complex. The TCR element may comprise (e.g. have) an extracellular domain and/or a transmembrane domain and/or an intracellular domain e.g. intracellular signalling domain of a TCR element.

The TCR element may selected from TCR alpha chain, TCR beta chain, a CD3 epsilon chain, a CD3 gamma chain, a CD3 delta chain, CD3 epsilon chain.

TRANSGENIC T-CELL RECEPTOR (TCR)

The T-cell receptor (TCR) is a molecule found on the surface of T cells which is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.

The TCR is a heterodimer composed of two different protein chains. In humans, in 95% of T cells the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD, respectively). When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction.

In contrast to conventional antibody-directed target antigens, antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex.

It is possible to engineer cells to express heterologous (i.e. non-native) TCR molecules by artificially introducing the TRA and TRB genes; or TRG and TRD genes into the cell using a vector. For example the genes for engineered TCRs may be reintroduced into autologous T cells and transferred back into patients for T cell adoptive therapies. Such‘heterologous’ TCRs may also be referred to herein as‘transgenic TCRs’.

The transgenic TCR for use in the present invention may recognise a tumour associated antigen (TAA) when fragments of the antigen are complexed with major histocompatibility complex (MHC) molecules on the surface of another cell.

The transgenic TCR for use in the present invention may recognise a TAA listed in Table 3. NUCLEIC ACID CONSTRUCT / KIT OF NUCLEIC ACID SEQUENCES

The present invention provides a nucleic acid construct which comprises:

(i) a first nucleic acid sequence encoding a dnSMAD or a chimeric dnSMAD according to the present invention; and

(ii) a second nucleic acid sequence which encodes a CAR or transgenic TCR.

The present invention also provides a kit comprising nucleic acid sequences according to the present invention. For example, the kit may comprise

(i) a first nucleic acid sequence encoding a a dnSMAD or a chimeric dnSMAD according to the present invention.; and

(ii) a second nucleic acid sequence which encodes a CAR or transgenic TCR.

As used herein, the terms“polynucleotide”,“nucleotide”, and“nucleic acid” are intended to be synonymous with each other.

The nucleic acid construct may comprise a plurality of nucleic acid sequences which encode a dnSMAD or a chimeric dnSMAD according to the present invention; and a CAR or transgenic TCR. For example, the nucleic acid construct may comprise two, three, four or more nucleic acid sequences which encode different components of the invention. The plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.

It will be understood by a skilled person that numerous different polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed. Suitably, the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein.

Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides 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 life span of polynucleotides of interest.

The terms“variant”, “homologue” or“derivative” in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.

CO-EXPRESSION SITE

A co-expression site is used herein to refer to a nucleic acid sequence enabling co expression of nucleic acid sequences encoding the dnSMAD and a CAR or transgenic TCR according to the present invention.

There may be a co-expression site between the first nucleic acid sequence and the second nucleic acid sequence. Suitably, there may be a co-expression site between the nucleic acid sequence encoding the dnSMAD and the nucleic acid sequence which encodes the CAR or transgenic TCR. In embodiments where a plurality of co-expression sites is present in the engineered polynucleotide, the same co-expression site may be used.

The co-expression site may be a cleavage site. The cleavage site may be any sequence which enables the two polypeptides to become separated. The cleavage site may be self cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.

The term“cleavage” is used herein for convenience, but the cleavage site may cause the peptides 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 proteins, causes the proteins 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') 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 (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 proteins 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 (Donelly et al (2001 ) as above).

“2A-like” sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al., 2001 ) as above.

The co-expression sequence may be an internal ribosome entry sequence (IRES). The co expressing sequence may be an internal promoter.

VECTOR

The present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) or nucleic acid construct(s) of the invention. Such a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses a dnSMAD as defined herein.

The vector may comprise a plurality of nucleic acid sequences which encode different components as provided by the present invention. For example, the vector may comprise two, three, four or more nucleic acid sequences which encode different components, such as the dnSMAD and a CAR or transgenic TCR. Suitably, the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.

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 cell.

PHARMACEUTICAL COMPOSITION

The present invention also relates to a pharmaceutical composition comprising an engineered immune effector cell according to the present invention or a cell obtainable (e.g. obtained) by a method according to the present invention.

The present invention also provides a pharmaceutical composition comprising a nucleic acid construct according to the present invention, a first and second polynucleotide as defined herein, or a vector according to the present invention or a first and second vector as defined herein.

In particular, the invention relates to a pharmaceutical composition containing a cell according to the present invention.

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 and/or preventing a disease which comprises the step of administering an engineered immune effector cell according to the invention, or obtainable (e.g. obtained) by a method according to the present invention, or a nucleic acid construct according to the present invention, or a first and second nucleic acid sequence as defined herein; a vector according to the present invention or a first and second vector as described herein (for example in a pharmaceutical composition as described above) to a subject.

Suitably, the present methods for treating and/or preventing a disease may comprise administering an engineered immune effector cell according to the present invention (for example in a pharmaceutical composition as described above) to a subject.

The present invention also provides a method for treating and/or preventing a disease in a subject which subject comprises cells of the invention, which method comprises the step of administering an agent to the subject wherein the agent is capable of controlling the release or expression of the dnSMAD or chimeric dnSMAD. As such, this method involves administering an agent to a subject which already comprises cells of the present invention.

Suitably, the present methods for treating and/or preventing a disease may comprise administering an agent which increases the expression or activity of the dnSMAD or chimeric dnSMAD to a subject to which the engineered immune cell according to the present invention has been administered.

A method for treating a disease relates to the therapeutic use of the cells of the present invention. In this respect, the cells 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 may involve the steps of:

(i) isolating a cell-containing sample;

(ii) introducing the nucleic acid construct according to the present invention, a first and second nucleic acid sequence as defined herein, a vector according to the present invention or a first and second vector as herein to the cell; and

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

The engineered immune effector cell may be administered in the form of a pharmaceutical composition. 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.

The present invention provides a cell according to the present invention for use in treating a disease.

The present invention also relates to the use of a cell according to the present invention for the manufacture of a medicament for the treatment of a disease.

The disease to be treated and/or prevented by the method of the present invention may be cancer.

The cancer may be a cancer such as neuroblastoma, multiple myeloma, prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, and thyroid cancer. Suitably, the cancer may be neuroblastoma. Suitably, the cancer may be multiple myeloma. Suitably, the cancer may be prostate cancer.

The cell of the present invention may be capable of killing target cells, such as cancer cells. The target cell may be recognisable by expression of a TAA, for example the expression of a TAA provided above in Table 3. The cancer may be a cancer listed in Table 3.

The administration of a cell according to the present invention can be accomplished using any of a variety of routes, such as intraperitoneally, intravenously, subcutaneously, transcutaneously or intramuscularly.

METHOD OF MAKING A CELL

Cells of the present invention may be generated by introducing DNA or RNA coding for dnSMAD or chimeric dnSMAD, as defined herein, by one of many means including transduction with a viral vector, transfection with DNA or RNA.

The cell of the invention may be made by:

introducing to a cell (e.g. by transduction or transfection) the polynucleotide according to the present invention, the nucleic acid construct or vector according to the present invention, or a first and second nucleic acid sequence as defined herein, or a first and second vector as defined herein.

The cell may be from a sample isolated from a subject.

METHOD OF RENDERING A CELL LESS SUSCEPTIBLE TO TGF

The present invention also provides a method of rendering an immune effector cell less susceptible to TΰRb by introducing a polynucleotide coding for a dnSMAD or chimeric dnSMAD to said immune effector cell.

The method may comprise:

introducing to a cell (e.g. by transduction or transfection) the polynucleotide according to the present invention, the nucleic acid construct or vector according to the present invention, or a first and second nucleic acid sequence as defined herein, or a first and second vector as defined herein. The method of rendering an immune effector cell less susceptible to TQRb signalling may comprise maintaining the cell under conditions which allow the expression of the dnSMAD.

The present invention further relates to the use of a dnSMAD to render an immune effector cell less susceptible to TQRb.

This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.

It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

The terms "comprising", "comprises" and "comprised of as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of also include the term "consisting of.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. 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 - Investigating the capacity of truncated SMAD2 and truncated SMAD3 to block TGFp signalling in T cells

T cells were generated expressing a second generation anti-GD2 CAR with a CD28-CD3z endodomain either alone or in combination with a truncated SMAD2 (dnSMAD2-MH2) or truncated SMAD3 (dnSMAD3-MH2) molecule.

The cells were co-cultured with GD2-expressing SupT1 target cells at a 2:1 and 4:1 target: effector cell ratio. TGFp was spiked into the co-culture at 10ng/ml on day 0 and target cell killing was investigated at day 5 and 7 by flow cytometry. The results are shown in Figure 5.

In the absence of dnSMADs, the addition of TGFp inhibited CAR mediated killing of GD2+ target cells. The expression of dnSMAD2 or dnSMAD3 reduced this TGF mediated inhibition of killing. This was observed at both a 2:1 and 4:1 T:E ratios and on both 5 and 7 days of co-culture. Interestingly, the presence of dnSMADs enhanced CAR-mediated target cell killing even in the absence of exogenously added TGFp, with the amount of live target cells being virtually undetectable for the cells expressing dnSMAD2-MH2t or dnSMAD3- MH2t after 5 days of culture at both T :E ratios.

Example 2 - In vitro testing of a dnSMAD

In vitro Cytotoxicity Assays

T cells expressing the CAR and dnSMAD2/SMAD3 or dnSMAD4 proteins (or CAR only) are depleted of CD56-expressing natural killer cells using the EasySep human CD56 positive selection kit (STEMCELL Technologies) according to the manufacturer’s instructions. Cells are then used in cytotoxicity assays after 1 , 5 and 7 days. Cytotoxicity assays are set up at a 1 :2 effector: target (E:T) cell ratio using SupT 1 expressing the target in presence/absence of human TGF-bI cells in 96-well plates. SupT1 WT cells are used in the same conditions as control. Not transduced T cells (NT T cells) are used in co-cultures with targets as a negative control. CAR-mediated cytotoxicity is assessed by flow cytometry after 1 , 5 and 7 days. T cells are identified from target cells by CD3 and staining. 7-AAD viability dye is used for exclusion of dead cells. T cell Proliferation Assay

To assess the proliferation of T cells expressing the CAR and dnSMAD2/SMAD3 or dnSMAD4 proteins (or CAR only) in co-cultures with target cells in presence of TGF-bI , Cell Trace Violet (CTV) staining is carried out. T cells expressing the different CAR constructs (NT T cells used as controls) are labelled with CTV before setup of co-cultures with target cells. Staining is performed by re-suspending the T cells at in fresh PBS containing CTV dye. Co-cultures are then set up with target cells expressing TGF-bI (and wild type cell as control), at an E:T ratio of 1 :2 and 1 :8 using. Proliferation is assessed by flow cytometry 5 and 7 days later. Cells are stained with 7-AAD and CD3 for exclusion of dead cells and detection of T cells, respectively, and the CTV-stained cells are used to measure proliferation by the extent of dye dilution of dead cells. Viable target cells are enumerated for each co-culture condition. The percentage of remaining target cells is calculated by normalizing the number of viable target cells of each condition to that recovered from co cultures carried out with NT T cells (100%).

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.

Claims

1. A cell which co-expresses (i) a dominant-negative SMAD (dnSMAD); and (ii).a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

2. A cell according to claim 1 , wherein said dnSMAD is a dominant negative SMAD2, SMAD3 or SMAD4 polypeptide.

3. A cell according to claim 1 or 2, wherein said dnSMAD lacks a functional MH1 domain.

4. A cell according to any of claims 1 to 3, wherein said dnSMAD lacks a functional nuclear localization signal (NLS) domain.

5. A chimeric dominant negative SMAD (dnSMAD) which comprises at least two dnSMAD polypeptides as defined in any of claims 1 to 4 connected by a linker domain.

6. A chimeric dnSMAD according to claim 5 which comprises (i) a dnSMAD2 polypeptide and a dnSMAD3 polypeptide; (ii) a dnSMAD2 polypeptide and a dnSMAD4 polypeptide; or (iii) a dnSMAD3 polypeptide and a dnSMAD4 polypeptide.

7. A polynucleotide which encodes a chimeric dnSMAD according to claim 5 or 6.

8. A nucleic acid construct which comprises: (i) a first polynucleotide which encodes a dnSMAD as defined in any of claims 1 to 4 or a chimeric dnSMAD according to claim 5 or 6; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

9. A nucleic acid construct according to aspect 17 wherein the first and second polynucleotides are separated by a co-expression site.

10. A kit of polynucleotides comprising: (i) a first polynucleotide which encodes a dnSMAD as defined in any of claims 1 to 4 or a chimeric dnSMAD according to claim 5 or 6; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

1 1. A vector which comprises a polynucleotide according to claim 7 or a nucleic acid construct according to claim 9.

12. A kit of vectors which comprises: (i) a first vector which comprises a polynucleotide which encodes a dnSMAD as defined in any of claims 1 to 4 or a chimeric dnSMAD according to claim 5 or 6; and (ii) a second vector which comprises a polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).

13. A pharmaceutical composition which comprises a plurality of cells according to any of claims 1 to 4.

14. A pharmaceutical composition according to claim 13, for use in treating a disease.

15. A method for treating a disease, which comprises the step of administering a pharmaceutical composition according to claim 13 to a subject in need thereof.

16. The use of a cell according to any of claims 1 to 4 in the manufacture of a medicament for use in the treatment of a disease.

17. A method for making a cell according to any of claims 1 to 4, which comprises the step of introducing: a polynucleotide according to claim 7; a nucleic acid construct according to claim 8 or 9; a kit of polynucleotides according to claim 10; a vector according to claim 1 1 ; or a kit of vectors according to claim 12, into a cell ex vivo.