WO2025003651A1

WO2025003651A1 - Chimeric transmembrane protein

Info

Publication number: WO2025003651A1
Application number: PCT/GB2024/051623
Authority: WO
Inventors: Matteo Righi; Isaac GANNON; Simon Thomas; Martin PULÉ
Original assignee: Autolus Ltd
Current assignee: Autolus Ltd
Priority date: 2023-06-27
Filing date: 2024-06-26
Publication date: 2025-01-02
Anticipated expiration: 2025-12-27
Also published as: GB202309704D0

Abstract

The present invention relates to a chimeric transmembrane protein which provides a cytokine signal to a cell in which it is expressed. The chimeric transmembrane protein comprises an endodomain having a plurality of cytokine-receptor derived tyrosine or threonine motifs, joined together by one or more linker(s). The invention also provides a cell which co-expresses such a chimeric transmembrane protein and a chimeric cytokine receptor (CAR), and its use in the treatment of diseases such as cancer and autoimmune diseases.

Description

CHIMERIC TRANSMEMBRANE PROTEIN

FIELD OF THE INVENTION

The present invention relates to chimeric transmembrane proteins. In particular, the present invention relates to constitutively active chimeric transmembrane proteins which produce cytokine signal when expressed in a cell.

BACKGROUND TO THE INVENTION

Chimeric antigen receptors (CARs)

A number of immunotherapeutic agents have been described for use in cancer treatment, including therapeutic monoclonal antibodies (mAbs), bi-specific T-cell engagers and chimeric antigen receptors (CARs).

Chimeric antigen receptors are proteins which graft the specificity of a binding portion such as a monoclonal antibody (mAb) to the effector function of a T-cell. Their usual form is that of a type I transmembrane domain protein with an antigen recognizing amino terminus, a spacer, a transmembrane domain all connected to a compound endodomain which transmits T-cell survival and activation signals.

The most common form of these molecules are fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies which recognize a target antigen, fused via a spacer and a trans-membrane domain to a signaling endodomain. Such molecules result in activation of the T-cell in response to recognition by the scFv of its target. When T cells express such a CAR, they recognize and kill target cells that express the target antigen. Several CARs have been developed against tumour associated antigens, and adoptive transfer approaches using such CAR-expressing T cells are currently in clinical trial for the treatment of various cancers.

While CAR-T cell therapies have had considerable success in the treatment of haematological malignancies, it has been found that not all patients respond to CAR-T cell therapies and CAR- T cells directed against solid tumours have provided limited therapeutic benefit to date. This may be because, in vivo, CAR T-cells struggle to overcome the hostile tumour microenvironment mediated by inhibitory ligands, such as PD-L1 ; soluble factors, such as TGFp; and suppressive cell types such as MDSCs and regulatory T cells.

CAR T-cell persistence and activity can be enhanced by administration of cytokines, or by the CAR T-cells producing cytokines constitutively. However, these approaches have limitations: systemic administration of cytokines can be toxic; constitutive production of cytokines may lead to uncontrolled proliferation and transformation.

In addition to safety concerns, the systemic coadministration or extracellular secretion of cytokines may accelerate host rejection of allogeneic CAR-T cells and lead to reduced overall efficacy.

There is therefore a need for alternative CAR T-cell approaches, which facilitate engraftment and expansion of T cells but without the associated disadvantages of systemic coadministration or extracellular secretion of cytokines.

Chimeric cytokine receptors (OCRs)

A chimeric cytokine receptor is a molecule having a cytokine receptor endodomain fused to an extracellular domain which is not derived from a cytokine receptor and which causes dimerization of the cytokine receptor endodomain.

WO2017/029512 describes two types of chimeric cytokine receptor (CCR). The first type of CCR grafts the binding specificity of a non-cytokine binding molecule on to the endodomain of a cytokine receptor. In the presence of the ligand for the CCR, a cytokine signal is delivered to the CCR-expressing cell. The second type of CCR comprises a dimerization domain and a cytokine receptor endodomain. Dimerisation may occur spontaneously, in which case the chimeric transmembrane protein will be constitutively active. Alternatively, dimerization may occur only in the presence of a chemical inducer of dimerization (CID) in which case the transmembrane protein only causes cytokine-type signalling in the presence of the CID.

The co-expression of such a CCR with a chimeric antigen receptor (CAR) helps a CAR T-cell to engraft and expand in the hostile tumour microenvironment.

WO2021/023987 describes chimeric cytokine receptors having a series of C-terminal truncations in one of the chains of the cytokine receptor endodomain. It was found that the initial deletion improved cellular proliferation and subsequent longer deletions cause cytokine signalling to be reduced in an analog manner, so it is possible to choose the desired level of cytokine signalling by selecting the appropriate truncation.

CAR-T cells are usually made using viral gene delivery. The need to co-express a chimeric cytokine receptor with a CAR and possibly other modules (such as a suicide gene) may make the vector cargo prohibitively large and compromise manufacturability. Even with the truncations described in WO2021/023987, heterodimeric CCR take up a considerable proportion of the cargo capacity of the viral vector.

There is therefore a need for alternative molecules capable of providing a cytokine signal to a cell which are encoded by gene(s) with a reduced insert size. It would also be advantageous to be able to combine signalling domains from different cytokine receptors, whilst maintaining a compact cargo size. This would permit a tailored and complete cytokine signal.

DESCRIPTION OF THE FIGURES

Figure 1 : Schematic diagram summarising the structure of various cytokine receptors, the cell types which produce the cytokines and the cell types which express the cytokine receptors.

Figure 2: The general structure of a receptor from the type I cytokine receptor family. In the extracellular cytokine receptor module, four conserved cysteine residues exist and are involved in disulfide bonds. A WSXWS (Tre, Ser, any, Tre, Ser) motif that is essential for receptor processing, ligand binding, and activation of the receptor is also located in the extracellular domain. In the intracellular portion, two short domains termed Box 1 and Box 2 are important for JAK binding. Tyrosine residues are present on the intracellular part which are phosphorylated upon receptor activation.

Figure 3: Schematic diagram illustrating a I L2-signalling chimeric transmembrane protein of the invention. One polypeptide has an extracellular domain comprising an antibody-type light chain constant region and a truncated IL2 receptor common y chain endodomain. The other polypeptide of the molecule has an extracellular domain comprising an antibody-type heavy chain constant region and an endodomain which comprises the Box 1 and Box 2 motifs from IL2 receptor chain joined to four tyrosine/threonine motifs from IL2 receptor p chain (Y364, Y418, T477 and Y536) each joined together by either G4S or EA3K linkers. Constant dimerization between antibody-type heavy and light chain constant regions in the extracellular domains brings together the truncated IL2 receptor common y chain with the modified IL-2 receptor p chain, leading to constitutive cytokine signalling.

Figure 4: Schematic diagram illustrating a I L7-signalling chimeric transmembrane protein of the invention. One polypeptide has an extracellular domain comprising an antibody-type light chain constant region and a truncated common y chain endodomain. The other polypeptide of the molecule has an extracellular domain comprising an antibody-type heavy chain constant region and an endodomain which comprises the Box 1 and Box 2 motifs from IL7 receptor a chain joined to three two motifs from IL7 receptor a chain (Y401 , Y449) joined together by either G4S or EA3K linkers. The tyrosine residue Y456 was mutated to phenylalanine. Constant dimerization between antibody-type heavy and light chain constant regions in the extracellular domains brings together the truncated common y chain with the modified IL-7 receptor a chain, leading to constitutive cytokine signalling.

Figure 5: SpYced_CCR components - Expression

T cells were transduced with vectors expressing the constructs described in Example 1 and stained for expression of RQR8 (the marker gene) and chimeric transmembrane receptor components Kappa light chain and heavy chain constant region (CH1).

Figure 6: SpYced_CCR - functionality

T cells were transduced with vectors expressing the constructs described in Example 1 and cultured for 5 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable, transduced cells was assessed by flow cytometry. A non-transduced T cell population (NT) was used as a control.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have found that it is possible to “stitch together” key tyrosine/threonine containing motifs from cytokine receptor endodomains with linkers and produce a molecule capable of cytokine signalling. Because large portions of the wild-type cytokine receptor endodomain are left out, the resulting molecule has a reduced size in terms of vector capacity.

The present inventors have termed these molecules “Stitched pTYrosine spaced” or “SpYced” chimeric transmembrane proteins. Chimeric transmembrane proteins with SpYced endodomain(s) enable the integration of multiple signaling domains from distinct cytokine receptors while maintaining a compact cargo size, making it possible to provide a tailored and multidimensional cytokine signal.

Thus, in a first aspect, the present invention provides a chimeric transmembrane protein which comprises two polypeptides:

(a) a first polypeptide which comprises:

(i) a first dimerization domain;

(ii) a first endodomain which comprises a first Janus Kinase (JAK)-binding domain; and

(b) a second polypeptide which comprises:

(i) a second dimerization domain which spontaneously dimerises with the first dimerization domain;

(ii) a second endodomain which comprises a second JAK-binding domain and a signalling domain having the general formula:

(L-Y)n in which L is a linker;

Y is a cytokine-receptor derived tyrosine or threonine motif; and n is an integer from 2 to 6.

The dimerisation domains may, for example, be leucine zipper domains or may comprise a heavy chain constant domain (CH) on one polypeptide and a light chain constant domain (CL) on the other polypeptide.

The first JAK-binding domain may bind JAK3 and the second JAK binding domain may bind JAK1. Alternatively, both the first JAK-binding domain and the second JAK binding domain may bind JAK2.

The first endodomain may comprise: GM-CSF receptor a-chain endodomain, common y-chain endodomain, IL12 receptor pi subunit, IFNAR1 , IFNGR2 or a truncated version thereof.

The linkers, L, may be the same or different. The, some, or each linker may, for example, be an G4S or EA3K linker.

The tyrosine/threonine motifs, Y, may be derived from the same or different cytokine receptor endodomain(s). The some or each tyrosine/threonine motif(s) may be derived from the endodomain of one or more of the following cytokine receptor chains: IL2Rp, IL7Ra; GMCSFRp, IL9R, IL21 R, IL12R 2 subunit, IFNAR2, IFNGR1. The some or each Y may be selected from the tyrosine/threonine motifs listed in Table 1.

There is provided a chimeric transmembrane protein wherein the first polypeptide comprises:

(i) a light chain constant domain (CL);

(ii) a truncated common y-chain endodomain having the sequence shown as SEQ ID No. 19; and the second polypeptide comprises:

(i) a heavy chain constant domain (CH);

(ii) a second endodomain which comprises the JAK-binding domain from IL2RP chain and a signalling domain having the general formula:

L-Y1-L-Y2-L-Y3-L-Y4 in which

L is a G4S or EA3K linker;

Y1 has the sequence shown as SEQ ID No. 24

Y2 has the sequence shown as SEQ ID No. 25

Y3 has the sequence shown as SEQ ID No. 26

Y4 has the sequence shown as SEQ ID No. 27.

There is also provided a chimeric transmembrane protein wherein the first polypeptide comprises:

(i) a light chain constant domain (CL);

(i) a heavy chain constant domain (CH);

(ii) a second endodomain which comprises the JAK-binding domain from IL7Ra chain and a signalling domain having the general formula:

L-Y1-L-Y2 in which

L is a G4S or EA3K linker;

Y1 has the sequence shown as SEQ ID No. 28

Y2 has the sequence shown as SEQ ID No. 29.

In a second aspect, the invention provides a cell which comprises a chimeric transmembrane protein according to the first aspect of the invention. The cell may also comprise a chimeric antigen receptor (CAR).

In a third aspect, there is provided a nucleic acid construct encoding a chimeric transmembrane protein according to the first aspect of the invention, which comprises a nucleic acid sequence encoding the first polypeptide; a nucleic acid sequence encoding a selfcleaving peptide; and a nucleic acid sequence encoding the second polypeptide.

Alternative codons may be used in regions of sequence encoding the linkers, to avoid homologous recombination.

The nucleic acid construct may also comprise a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

In a fourth aspect, there is provided a vector comprising a nucleic acid construct according to the third aspect of the invention.

In a fifth aspect, there is provided a kit of vectors for expressing a chimeric transmembrane protein according to the first aspect of the invention in a cell, which kit comprises: i) a vector comprising a nucleic acid sequence encoding the first polypeptide; ii) a vector comprising a nucleic acid sequence encoding the second polypeptide; and optionally iii) a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

In a sixth aspect, there is provided a method for making a cell according to the second aspect of the invention, which comprises the step of introducing: a nucleic acid construct according to the third aspect of the invention; a vector according to the fourth aspect of the invention; or a kit of vectors according to the fifth aspect of the invention into a cell, wherein the cell is from a sample isolated from a subject.

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

In an eighth aspect, the invention provides a method for treating a cancerous disease or an autoimmune disease in a subject, which comprises the step of administering a pharmaceutical composition according to the seventh aspect of the invention to the subject. In a ninth aspect there is provided a pharmaceutical composition according to the seventh aspect of the invention for treating a cancerous disease or an autoimmune disease.

In a tenth aspect, there is provided the use of a cell according to the second aspect of the invention in the manufacture of a medicament for treating a cancerous disease or an autoimmune disease.

DETAILED DESCRIPTION

CHIMERIC TRANSMEMBRANE PROTEINS

Cytokine signalling chimeric transmembrane proteins are described in WO2017/029512. They comprise a cytokine receptor endodomain and a dimerization domain, which brings the two chains of the cytokine receptor endodomain together.

The chimeric transmembrane proteins comprise two polypeptides, each of which comprise the following domains:

(i) a dimerising exodomain;

(ii) an optional spacer;

(iii) a transmembrane domain; and

(iv) a cytokine-receptor endodomain.

In the chimeric transmembrane proteins of the present invention, dimerization occurs spontaneously giving constitutively active cytokine signalling.

The chimeric transmembrane protein may comprise two polypeptides:

(i) a first polypeptide which comprises:

(a) a first dimerisation domain; and

(b) a first chain of the cytokine receptor endodomain or a modified version thereof; and

(ii) a second polypeptide which comprises:

(a) a second dimerization domain, which dimerises with the first dimerization domain; and

(b) a second chain of the cytokine-receptor endodomain or a modified version thereof. Dimerization of the chimeric transmembrane protein may be based on the dimerization domain of an antibody. In this respect, the dimerisation domains of the chimeric transmembrane protein may comprise the dimerization portion of a heavy chain constant domain (CH) and a light chain constant domain (CL). The “dimerization portion” of a constant domain is the part of the sequence which forms the inter-chain disulphide bond.

Alternatively, the chimeric transmembrane protein may comprise a coiled coil domain giving spontaneous dimerization or multimerization (e.g. tetramerization).

For example, one polypeptide of the chimeric transmembrane protein may comprise the first pair of an alpha-helices coiled coil (such as an Acid Zipper) and the other polypeptide may comprise the second pair (such as a Base Zipper). These domains spontaneously dimerise, bringing together the cytokine receptor endodomains.

Two give a tetrameric arrangement, one polypeptide of the chimeric transmembrane protein may comprise Chain A; one polypeptide may comprise Chain B; one polypeptide may comprise Chain C; and one polypeptide may comprise Chain D of the SNAP-25/SNARE heterotetrametric complex. These domains spontaneously hetero-dimerise, bringing together two copies of the cytokine receptor endodomains, giving constitutive cytokine signalling.

CYTOKINE RECEPTORS AND SIGNALLING

Many cell functions are regulated by members of the cytokine receptor superfamily. Signalling by these receptors depends upon their association with Janus kinases (JAKs), which couple ligand binding to tyrosine phosphorylation of signalling proteins recruited to the receptor complex. Among these are the signal transducers and activators of transcription (STATs), a family of transcription factors that contribute to the diversity of cytokine responses.

When the chimeric transmembrane protein of the invention dimerises, one or more of the following intracellular signaling pathways may be initiated:

(i) the JAK-STAT pathway

(ii) the MAP kinase pathway; and

(iii) the Phosphoinositide 3-kinase (PI3K) pathway.

The JAK-STAT system consists of three main components: (1) a receptor (2) Janus kinase (JAK) and (3) Signal Transducer and Activator of Transcription (STAT). JAKs, which have tyrosine kinase activity, bind to cell surface cytokine receptors. The binding of the ligand to the receptor triggers activation of JAKs. With increased kinase activity, they phosphorylate tyrosine residues on the receptor and create sites for interaction with proteins that contain phosphotyrosine-binding SH2 domains. STATs possessing SH2 domains capable of binding these phosphotyrosine residues are recruited to the receptors and are themselves tyrosine-phosphorylated by JAKs. These phosphotyrosines then act as binding sites for SH2 domains of other STATs, mediating their dimerization. Different STATs form hetero- or homodimers. Activated STAT dimers accumulate in the cell nucleus and activate transcription of their target genes.

A schematic diagram illustrating the general structure of a cytokine receptor endodomain is shown in Figure 2. The endodomain contains elements know as Box 1 and Box 2 which are important for JAK binding. JAK association with cytokine receptors is facilitated by N-terminal FERM and SH2 domains. Together, the JAK FERM and SH2 domains mediate a bipartite interaction with two distinct receptor peptide motifs, the proline-rich “Box1” and hydrophobic “Box 2” which are present in the intracellular domain of cytokine receptors. The sequences and position of Box 1 and Box 2 motifs of several class I and class II are given in Figure 4 of Ferraro and Luparpus (2017) Front. Endocrinol. 8:71 , which is herein incorporated by reference.

In the chimeric transmembrane protein of the present invention, one or both polypeptides comprise a JAK-binding domain. The JAK-binding domain may comprise a Box1 and optionally Box 2 motif from a cytokine receptor endodomain, such as a type I or type II cytokine receptor endodomain.

CYTOKINE RECEPTOR ENDODOMAIN

The chimeric transmembrane protein of the present invention comprises an endodomain which causes “cytokine-type” cell signalling.

The endodomain may be derived from a type I cytokine receptor. Type I cytokine receptors share a common amino acid motif (WSXWS) in the extracellular portion adjacent to the cell membrane. The endodomain may be derived from a type II cytokine receptor. Type II cytokine receptors include those that bind type I and type II interferons, and those that bind members of the interleukin-10 family (interleukin-10, interleukin-20 and interleukin-22).

The term “derived from” means that the endodomain of the chimeric transmembrane protein of the invention has a portion or portions of the wild-type sequence of the endogenous molecule, and retains the ability to form a complex with JAK-1 or JAK-3 and activate one of the signalling pathways mentioned above. The endodomain may, for example, be a truncated version of the wild-type endodomain, or it may comprise a plurality of portions of the wild-type sequence joined together.

Type I cytokine receptors include:

(i) Interleukin receptors, such as the receptors for IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11 , IL-12, IL13, IL-15, IL-21 , IL-23 and IL-27;

(ii) Colony stimulating factor receptors, such as the receptors for erythropoietin, GM- CSF, and G-CSF; and

(iii) Hormone receptor/neuropeptide receptor, such as hormone receptor and prolactin receptor

Members of the type I cytokine receptor family comprise different chains, some of which are involved in ligand/cytokine interaction and others that are involved in signal transduction. For example the IL-2 receptor comprises an a-chain, a p-chain and a y-chain.

There are three subclasses of type I cytokine receptors:

• type I cytokine receptors which use the common gamma chain (yc) such as I L2R, IL- 7R, IL-4R, IL-9R, IL-13R, IL-15R and IL-21 R;

• type I cytokine receptors which use the common beta chain ( c) such as IL-3R, IL-5R and GM-CSFR;

• type I cytokine receptors which use gp130 such as IL-27R and IL-6R; and

• type I cytokine receptors which use IL12R-P1 such as IL-12R and IL-23R.

IL-2

IL-2 binds to the IL-2 receptor, which has three forms, generated by different combinations of three different proteins, often referred to as "chains": a, p and y; these subunits are also parts of receptors for other cytokines. The p and y chains of the IL-2R are members of the type I cytokine receptor family. The three receptor chains are expressed separately and differently on various cell types and can assemble in different combinations and orders to generate low, intermediate, and high affinity IL-2 receptors.

The a chain binds IL-2 with low affinity, the combination of p and y together form a complex that binds IL-2 with intermediate affinity, primarily on memory T cells and NK cells; and all three receptor chains form a complex that binds IL-2 with high affinity (Kd ~ 10-11 M) on activated T cells and regulatory T cells.

The three IL-2 receptor chains span the cell membrane and extend into the cell, thereby delivering biochemical signals to the cell interior. The alpha chain does not participate in signalling, but the beta chain is complexed with the tyrosine phosphatase JAK1. Similarly the gamma chain complexes with another tyrosine kinase called JAK3. These enzymes are activated by IL-2 binding to the external domains of the IL-2R.

IL-2 signalling promotes the differentiation of T cells into effector T cells and into memory T cells when the initial T cells are also stimulated by an antigen. Through their role in the development of T cell immunologic memory, which depends upon the expansion of the number and function of antigen-selected T cell clones, they also have a key role in long-term cell-mediated immunity.

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IL-2 receptor p-chain endodomain. It may comprise a full length, truncated or SpYced version of the IL-2 receptor (i.e. common) y-chain endodomain.

The amino acid sequence of the endodomain of human IL-2R common y-chain is shown below as SEQ ID No. 1.

I L2y chain endodomain, showing Box 1 and Box 2 motifs (SEQ ID No. 1)

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE GPGASPCNQHSPYWAPPCYTLKPET

The sequence of the endodomain from human IL-2RP is shown below as SEQ ID No. 2. The Box 1 motif is from amino acids 278-286 in the full length sequence and has the sequence KCNTPDPS (SEQ ID No. 3). The Box 2 motif is from amino acids 323-333 in the full length sequence and has the sequence SPLEVLERDKV (SEQ ID No. 4). IL2BR endodomain, showing Box 1 and Box 2 motifs and key tyrosine/threonine residues (SEQ ID No. 2) NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPL EVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPY SEEDPDEGVAGAPTGSSPQPLQPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGG SGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPR EGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

IL-7

The interleukin-7 receptor is made up of two chains: the interleukin-7 receptor-a chain (CD127) and common-y chain receptor (CD132). The common-y chain receptors is shared with various cytokines, including interleukin-2, -4, -9, and -15. Interleukin-7 receptor is expressed on various cell types, including naive and memory T cells.

The interleukin-7 receptor plays a critical role in the development of lymphocytes, especially in V(D)J recombination. IL-7R also controls the accessibility of a region of the genome that contains the T-cell receptor gamma gene, by STAT5 and histone acetylation. Knockout studies in mice suggest that blocking apoptosis is an essential function of this protein during differentiation and activation of T lymphocytes.

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IL-7 receptor a-chain endodomain. It may comprise a full length, truncated or SpYced version of the IL-7 receptor (i.e. common) y-chain endodomain.

The sequence of the endodomain of the IL-7 a-chain endodomain is shown below as SEQ ID No. 5. The Box 1 motif has the sequence VWPSLPDHK (SEQ ID No. 6). The Box 2 motif has the sequence KNLNVSFNPESFLDCQIHRVDDIQ (SEQ ID No. 7).

SEQ ID No. 5 - Endodomain derived from human IL-7Ra showing Box 1 and Box 2 motifs and key tyrosine residues:

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQD TFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRS LDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAY VTMSSFYQNQ IL-9

Interleukin 9 receptor (IL9R) also known as CD129 (Cluster of Differentiation 129) is a type I cytokine receptor.

The protein encoded by this gene is a cytokine receptor that specifically mediates the biological effects of interleukin 9 (IL9). The functional IL9 receptor complex requires this protein as well as the interleukin 2 receptor, gamma (IL2RG), a common gamma subunit shared by the receptors of many different cytokines. The ligand binding of this receptor leads to the activation of various JAK kinases and STAT proteins, which connect to different biologic responses.

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IL-9 receptor endodomain. It may comprise a full length, truncated or SpYced version of the common y-chain endodomain.

The sequence of the endodomain of the IL-9 receptor endodomain is shown below as SEQ ID No. 8.

SEQ ID No. 8 - Endodomain derived from IL-9 receptor endodomain showing Box 1 and Box 2 motifs and key tyrosine residue: KLSPRVKRIFYQNVPSPAMFFQPLYSVHNGNFQTWMGAHGAGVLLSQDCAGTPQGALEP CVQEATALLTCGPARPWKSVALEEEQEGPGTRLPGNLSSEDVLPAGCTEWRVQTLAYLPQ EDWAPTSLTRPAPPDSEGSRSSSSSSSSNNNNYCALGCYGGWHLSALPGNTQSSGPIPAL ACGLSCDHQGLETQQGVAWVLAGHCQRPGLHEDLQGMLLPSVLSKARSWTF

IL-21

Interleukin 21 receptor is a type I cytokine receptor. It forms a heterodimeric receptor complex with the common gamma chain (yc), a receptor subunit also shared by the receptors for interleukin 2 (IL2), interleukin 7 (IL7) and interleukin 15 (IL15). This receptor transduces the growth promoting signal of IL21 and is important for the proliferation and differentiation of T cells, B cells, and natural killer (NK) cells. The ligand binding of this receptor leads to the activation of multiple downstream signaling molecules, including JAK1 , JAK3, STAT1 , and STAT3. The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IL-21 receptor endodomain. It may comprise a full length, truncated or SpYced version of the common y-chain endodomain.

The sequence of the endodomain of the IL-21 receptor endodomain is shown below as SEQ ID No. 9.

SEQ ID No. 9 - Endodomain derived from IL-21 receptor endodomain showing Box 1 and Box 2 motifs and key tyrosine residue:

SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPS TLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYG LVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSA GSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDS GFVGSDCSSPVECDFTSPGDEGPPRSYLRQWWIPPPLSSPGPQAS

GM-CSF

The granulocyte-macrophage colony-stimulating factor receptor also known as CD116 (Cluster of Differentiation 116), is a receptor for granulocyte-macrophage colony-stimulating factor, which stimulates the production of white blood cells.

The granulocyte-macrophage colony-stimulating factor receptor is a heterodimer composed of at least two different subunits; an a chain, and a p chain which is also present in the receptors for IL-3 and IL-5. The a subunit contains a binding site for granulocyte macrophage colony-stimulating factor, but associates with the ligand only with low affinity. The chain is involved in signal transduction and formation of high affinity receptor complex together with a chain. Association of the a and p subunits results in receptor activation.

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the GM-CSF receptor p-chain endodomain. It may comprise a full length, truncated or SpYced version of the GM-CSF receptor a-chain endodomain.

GM-CSF receptor a-chain endodomain, showing Box 1 and Box 2 motifs (SEQ ID No. 10) KRFLRIQRLFPPVPQIKDKLNDNH EVEDEIIWEEFTPEEGKGYREEVLTVKEIT The sequence of the endodomain from human GM-CSF receptor chain (i.e. common p chain endodomain) is shown below as SEQ ID No. 11.

GM-CSF receptor p-chain endodomain, showing Box 1 and Box 2 motifs and key tyrosine residues (SEQ ID No. 11) RFCGIYGYRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFP ELEGVFPVGFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTP EKQASSFDFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLV PLAQAMGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSS GDTEDPGVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPV KSGFEGYVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQV GDYCFLPGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQ DYLSLPPWEVNKPGEVC

IL12

Interleukin 12 receptor is a type I cytokine receptor, binding interleukin 12. It consists of beta 1 and beta 2 subunits.

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IL12 receptor p2-subunit endodomain. It may comprise a full length, truncated or SpYced version of the IL12 receptor pi -subunit endodomain.

IL12 receptor pi-subunit endodomain, showing Box 1 and Box 2 motifs (SEQ ID No. 12) NRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALWEMSWDKGERT EPLEKTELPEGAPELALDTELSLEDGDRCKAKM

The sequence of the endodomain from human I L12 receptor p2-subunit endodomain is shown below as SEQ ID No. 13.

IL12 receptor p2-subunit endodomain, showing Box 1 and Box 2 motifs and key tyrosine residue (SEQ ID No. 13)

HYFQQKVFVLLAALRPQWCSREIPDPANSTCAKKYPIAEEKTQLPLDRLLIDWPTPEDPEP

LVISEVLHQVTPVFRHPPCSNWPQREKGIQGHQASEKDMMHSASSPPPPRALQAESRQLV DLYKVLESRGSDPKPENPACPWTVLPAGDLPTHDGYLPSNIDDLPSHEAPLADSLEELEPQ HISLSVFPSSSLHPLTFSCGDKLTLDQLKMRCDSLML Type II cytokine receptors, also commonly known as class II cytokine receptors, bind and respond to a select group of cytokines including interferon type I, interferon type II, interferon type III and members of the interleukin-10 family (IL-10, IL-20, IL-22, and IL-28). Type II cytokine receptors are characterized by the lack of a WSXWS motif which differentiates them from type I cytokine receptors.

Typically type II cytokine receptors are heterodimers or multimers with a high and a low affinity component. These receptors are related by sequence similarities in their extracellular portions that are composed of tandem Ig-like domains.

IFNa

The interferon-a/p receptor (IFNAR) is a virtually ubiquitous membrane receptor which binds endogenous type I interferon (IFN) cytokines. Endogenous human type I IFNs include many subtypes, such as interferons-a, - , -E, -K, -CO, and - .

IFNAR is a heteromeric cell surface receptor composed of two subunits, referred to as the low affinity subunit, IFNAR1 , and the high affinity subunit, IFNAR2

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IFNAR2 endodomain. It may comprise a full length, truncated or SpYced version of the IFNAR1 endodomain.

IFNAR1 endodomain, showing Box 1 and Box 2 motifs (SEQ ID No. 14) KVFLRCINYVFFPSLKPSSSIDEYFSEQPLKNLLLSTSEEQIEKCFIIENISTIATVEETNQTDE DHKKYSSQTSQDSGNYSNEDESESKTSEELQQDFV

The sequence of the endodomain from human IFNAR2 endodomain is shown below as SEQ ID No. 15.

IFNAR2 endodomain, showing Box 1 and Box 2 motifs and key tyrosine residue (SEQ ID No. 15) KWIGYICLRNSLPKVLNFHNFLAWPFPNLPPLEAMDMVEVIYINRKKKVWDYNYDDESDSD TEAAPRTSGGGYTMHGLTVRPLGQASATSTESQLIDPESEEEPDLPEVDVELPTMPKDSPQ QLELLSGPCERRKSPLQDPFPEEDYSSTEGSGGRITFNVDLNSVFLRVLDDEDSDDLEAPL MLSSHLEEMVDPEDPDNVQSNHLLASGEGTQPTFPSPSSEGLWSEDAPSDQSDTSESDV

DLGDGYIMR

IFNY

The interferon-gamma receptor (IFNGR) protein complex is a heterodimer of two chains: IFNGR1 and IFNGR2. It binds interferon-y, the sole member of interferon type II.

The chimeric transmembrane protein of the present invention may comprise a SpYced version of the IFNGR1 endodomain. It may comprise a full length, truncated or SpYced version of the IFNGR2 endodomain.

IFNGR2 endodomain, showing Box 1 and Box 2 motifs (SEQ ID No. 16)

LVLKYRGLIKYWFHTPPSIPLQIEEYLKDPTQPILEALDKDSSPKDDVWDSVSIISFPEKEQE DVLQTL

The sequence of the endodomain from human IFNGR1 endodomain is shown below as SEQ ID No. 17.

IFNGR1 endodomain, showing Box 1 and Box 2 motifs and key tyrosine residue (SEQ ID No. 17)

CFYI KKI NPLKEKSIILPKSLISWRSATLETKPESKYVSLITSYQPFSLEKEWCEEPLSPATV PGMHTEDNPGKVEHTEELSSITEVVTTEENIPDWPGSHLTPIERESSSPLSSNQSEPGSIAL NSYHSRNCSESDHSRNGFDTDSSCLESHSSLSDSEFPPNNKGEIKTEGQELITVIKAPTSFG YDKPHVLVDLLVDDSGKESLIGYRPTEDSKEFS

TRUNCATED CYTOKINE RECEPTOR ENDODOMAINS

In the chimeric transmembrane protein of the present invention, one of the cytokine receptor endodomain chains may be truncated. WO2021/023987, which is incorporated by reference herein, describes chimeric cytokine receptors having a series of C-terminal truncations in one of the chains of the cytokine receptor endodomain.

A schematic diagram illustrating the general structure of a cytokine receptor endodomain is shown in Figure 2. The endodomain contains elements know as Box 1 and Box 2 which are important for JAK binding. A series of tyrosine residues are present on the intracellular part which are phosphorylated upon receptor activation. The chimeric transmembrane protein may comprise a type 1 cytokine receptor endodomain which is truncated at the C-terminus but which retains the Box 1 and Box 2 motif.

The endodomain derived from human common gamma chain has the sequence shown above as SEQ ID No. 1 above, which has 86 amino acids.

A truncated version of this sequence may, for example, have a C-terminal truncation of up to 60, up to 50, up to 40, up to 30, up to 20 or up to 10 amino acids.

A truncated version of human common gamma chain may have one of the sequences shown as SEQ ID No. 18 to 23. A truncated version of human common gamma chain may have a sequence "between" two of the truncated sequences shown as SEQ ID No. 18 to 23, for example, a sequence "between” IL2Ry aa284-359 (SEQ ID NO. 18) and IL2Ry aa284-349 (SEQ ID NO. 19) may be aa284-358, aa284-357, etc... until aa284-350, aa284-349.

IL2Ry aa284-359 (SEQ ID NO. 18):

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE GPGASPCNQHSPYWA

IL2Ry aa284-349 (SEQ ID NO. 19):

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE GPGAS

IL2Ry aa284-339 (SEQ ID NO. 20):

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKG

IL2Ry aa284-329 (SEQ ID NO. 21):

ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERL

IL2Ry aa284-309(SEQ ID NO. 22):

ERTMPRIPTLKNLEDLVTEYHGNFSA

IL2Ry aa284-289 (SEQ ID NO. 23):

ERTMPR

The common gamma chain has four tyrosine residues in the endodomain. A truncated version of common gamma chain endodomain may lack one or more tyrosine residues compared to the wild-type sequence. A truncated version of common gamma chain endodomain may lack 1 , 2, 3, or all 4 tyrosine residues compared to the wild-type sequence. A truncated version of common gamma chain endodomain may retain the Box 1 and optionally Box 2 motif(s).

STITCHED PHOSPHOTYROSINE SPACED (SPYCED) ENDODOMAINS

The chimeric transmembrane protein of the invention may comprise a cytokine receptor endodomain which comprises a plurality of cytokine receptor derived tyrosine/threonine motifs joined together by one or more linkers.

The present inventors have termed these molecules “Stitched pTYrosine spaced” or “SpYced” chimeric transmembrane proteins.

A SpYced endodomain may comprise a JAK binding domain (such as a Box1 and/or Box2 motif) and a signalling domain having the general formula:

(L-Y)n in which L is a linker;

Y is a cytokine-receptor derived tyrosine or threonine motif; and n is an integer.

A SpYced endodomain may comprise a plurality of tyrosine/threonine motifs derived from the same cytokine receptor endodomain. Alternatively, a SpYced endodomain may comprise a plurality of tyrosine/threonine motifs derived from different cytokine receptor endodomains.

A list of cytokine-receptor tyrosine/threonine motifs is given in Table 1. The chimeric transmembrane protein of the present invention may comprise 2 or more such motifs derived from the same or different cytokine receptor endodomains. A SpYced endodomain may comprise about 2 to about 10, about 2 to about 8, about 2 to about 6; or about 2 to about 4 cytokine-receptor tyrosine/threonine motifs.

In one embodiment, the chimeric transmembrane protein comprises a SpYced endodomain with the motifs shown as SEQ ID Nos. 24, 25, 26 and 27 from IL2 receptor endodomain.

In another embodiment, the chimeric transmembrane protein comprises a SpYced endodomain with the motifs shown as SEQ ID Nos. 28 and 29 from IL7 receptor a endodomain. Table 1 - Cytokine-receptor tyrosine/threonine motifs (Key tyrosine/threonine in bold and underlined)

LINKER

A SpYced endodomain comprises a plurality of tyrosine/threonine motifs joined together by a linker. A linker may also be used to connect the JAK binding domain (such as a Box1 and/or Box2 motif) to the first tyrosine/threonine motif. The linkers in a SpYced endodomain may be the same or different.

The linker may be any amino acid sequence which spatially separates the JAK binding domain from the first tyrosine/threonine motif or spatially separates the tyrosine/threonine motifs from each other, facilitating the binding of JAK and STAT proteins. The linkers may be from about 3 to about 20, about 4 to about 15, or about 5 to about 10 amino acids in length. The or leach linker may be 5 or 6 amino acids in length.

One or more linkers in a SpYced endodomain may be a GS linker, such as a G4S linker. A G4S linker may have the sequence SGGGGS (SEQ ID No. 41) or it/they may be made of multiples of the G4S sequence. One or more linkers in a SpYced endodomain may be an EA3K linker. An EA3K linker may have the sequence EAAAK (SEQ ID No. 42) or it may be made of multiples of the EA3K sequence. EA3K linkers are more rigid than G4S linkers.

TRANSMEMBRANE DOMAIN

The transmembrane domain is the sequence of the chimeric transmembrane polypeptides which span the membrane. It may comprise a hydrophobic alpha helix. The transmembrane domain may be derived from CD28, which gives good receptor stability.

Alternatively the transmembrane domain may be derived from a cytokine receptor, for example the same cytokine from which the endodomain is derived.

The transmembrane domain may, for example be derived from IL-2R, IL-7R or IL-15R.

SEQ ID No. 43 - Transmembrane derived from human common gamma chain: VVISVGSMGLIISLLCVYFWL

SEQ ID No. 44 - Transmembrane derived from human IL-2RP: IPWLGHLLVGLSGAFGFIILVYLLI

SEQ ID No. 45 - Transmembrane derived from human IL-7Rcc

PI LLTISI LSFFSVALLVI LACVLW

SEQ ID No. 46 - Transmembrane derived from human IL-15Rcc

AISTSTVLLCGLSAVSLLACYL

The chimeric transmembrane protein of the invention may have a SpYced endodomain having one of the amino acid sequences shown below as SEQ ID No. 47 to 50. In each sequence, the transmembrane domain is underlined, the Box1/Box2 motifs are in bold an underlined, the linkers are in italics and the tyrosine/threonine motifs are in bold

SEQ ID No. 47 - SpYced IL2RP TM domain and endodomain with Y364, Y418, T477 and Y536 motifs and SG4S linkers

IPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQK WLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLSGGGGSTNQGYFFFHSGGGGSDDAY

CTFPSGGGGSGPPTPGVPDSGGGGSLNTDAYLSLQELQGQDPTHLV SEQ ID No. 48 - SpYced IL2R TM domain and endodomain with Y364, Y418, T477 and Y536 motifs and EA3K linkers

IPWLGHLLVGLSGAFGFIILVYLLINCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQK

WLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLSGGGGSTNQGYFFFHEAAAKDDAYC

TFPEAAAKGPPTPGVPDEAAAKLNTDAYLSLQELQGQDPTHLV

SEQ ID No. 49 - SpYced IL7Ra TM domain and endodomain with Y401 and Y449 motifs,

Y456F mutation and SG4S linkers

PILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKN LNVSFN PESFL

DCQIHRVDDIQARDEVEGFLQDTFPQQLESGGGGSGPHVYQDLLLSGGGGSQEEAYVTM SSFFQNQ

SEQ ID No. 50 - SpYced IL7Ra TM domain and endodomain with Y401 and Y449 motifs, Y456F mutation and EA3K linker

PILLTISILSFFSVALLVILACVLWKKRIKPIVWPSLPDHKKTLEHLCKKPRKN LNVSFN PESFL

DCQIHRVDDIQARDEVEGFLQDTFPQQLESGGGGSGPHVYQDLLLEAAAKQEEAYVTMSS

FFQNQ

The amino acid sequence for the truncated CyC chain illustrated schematically in Figures 3 and 4 and tested in the Examples is given below:

Signal peptide (SEQ ID No. 51)

MGWSCIILFLVATATGVHS

CL domain (SEQ ID No. 52)

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD

SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Hinge (SEQ ID No. 53)

EPKSCDKTHTCPPCPKDPK

CyC TM domain (SEQ ID No. 43)

VVISVGSMGLIISLLCVYFWL

CyC truncated endodomain (SEQ ID No. 19) ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGE GPGAS

The amino acid sequence for the truncated SpYced IL2RP chain illustrated schematically in Figure 3 and tested in the Examples is given below. The construct may have either “IL2Rbeta SpYced CCR endodomain SG4S linker” (SEQ ID No. 56) or “IL2Rbeta SpYced CCR endodomain EA3K linker” (SEQ ID No. 57)

Mouse Kappa chain Signal peptide (SEQ ID No. 54)

M ETDTLI LWVLLLLVPGSTG

CH1 domain (SEQ ID No. 55)

STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGL YSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRV

Hinge (SEQ ID No. 53)

EPKSCDKTHTCPPCPKDPK

IL2R beta transmembrane (SEQ ID No. 44)

IPWLGHLLVGLSGAFGFIILVYLLI

IL2Rbeta SpYced CCR endodomain SG4S linker (SEQ ID No. 56)

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPL EVLERDKVTQLLSGGGGSTNQGYFFFHSGGGGSDDAYCTFPSGGGGSGPPTPGVPDSG GGGSLNTDAYLSLQELQGQDPTHLV

IL2Rbeta SpYced CCR endodomain EA3K linker (SEQ ID No. 57)

NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPL EVLERDKVTQLLSGGGGSTNQGYFFFHEAAAKDDAYCTFPEAAAKGPPTPGVPDEAAAKL NTDAYLSLQELQGQDPTHLV

The amino acid sequence for the truncated SpYced IL7Ra chains illustrated schematically in Figure 4 and tested in the Examples is given below. The construct may have either “I L7Ralpha SpYced CCR endodomain SGGGGS linker” (SEQ ID No. 58) or “IL7Ralpha SpYced CCR endodomain EAAAK linker” (SEQ ID No. 59)

Mouse Kappa chain Signal peptide (SEQ ID No. 54) M ETDTLI LWVLLLLVPGSTG

CH1 domain (SEQ ID No. 55)

Hinge (SEQ ID No. 53)

EPKSCDKTHTCPPCPKDPK

IL7Ralpha transmembrane (SEQ ID No. 45)

PI LLTISI LSFFSVALLVI LACVLW

IL7Ralpha SpYced CCR endodomain SG4S linker (SEQ ID No. 58)

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQD TFPQQLESGGGGSGPHVYQDLLLSGGGGSQEEAYVTMSSFFQNQ*

IL7Ralpha SpYced CCR endodomain EA3K linker (SEQ ID No. 59)

KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQD TFPQQLESGGGGSGPHVYQDLLLEAAAKQEEAYVTMSSFFQNQ*

CHIMERIC ANTIGEN RECEPTORS (CAR)

The cell of the present invention may also comprise one or more chimeric antigen receptor(s). The CAR(s) may be specific for a tumour-associated antigen or an autoantibody.

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 or ligand-based antigen binding site. 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 costimulatory domain most commonly used is that of CD28. This supplies the most potent costimulatory 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.

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.

The cell of the present invention may comprise one or more CAR(s).

The CAR(s) may comprise an antigen-binding domain, a spacer domain, a transmembrane domain and an endodomain. The endodomain may comprise or associate with a domain which transmit T-cell activation signals.

CAR ANTIGEN BINDING DOMAIN

The antigen-binding domain is the portion of a 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 antigenbinding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a natural 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.

The term “ligand” is used synonymously with “antigen” to mean an entity which is specifically recognised and bound by the antigen-binding domain of a CAR.

CARs have also been described for the elimination of pathogenic B cells which comprise an autoantigen as the extracellular domain. In autoimmune diseases, pathogenic autoreactive B cells express autoantibodies on their cell surface. By designing the CAR to express the autoantigen itself (or an autoantibody binding portion thereof) as the extracellular domain, the CAR will specifically bind autoantibodies expressed in the surface of autoreactive B cells and will selectively kill those cells.

CELL SURFACE ANTIGEN

The CAR may recognise a cell-surface antigen, i.e. an entity, such as a transmembrane protein which is expressed on the surface of a target cell, such as a tumour cell.

The CAR may specifically bind a tumour-associated cell-surface antigen.

Various tumour associated antigens (TAA) are known, some of which are shown in Table 2. The antigen-binding domain used in the present invention may be a domain which is capable of binding a TAA as indicated therein.

Table 2

For treating autoimmune diseases, the CAR may target a B-cell antigen, such as CD19.

Alternatively, the CAR may target an autoantibody expressed on the surface of an autoreactive B cell. In order to do this, the CAR may express an anti-idiotype binder for the autoantibody, or may express the autoantigen itself (or an autoantigen-binding portion thereof) as extracellular domain.

CAR TRANSMEMBRANE DOMAIN

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

SIGNAL PEPTIDE

The CAR and chimeric transmembrane polypeptides described herein may comprise a signal peptide so that when it/they 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.

The signal peptide may be at the amino terminus of the molecule.

The signal peptide may comprise the sequence shown as SEQ ID No. 60, 61 or 62 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause cell surface expression of the CAR.

SEQ ID No. 60: MGTSLLCWMALCLLGADHADG

The signal peptide of SEQ ID No. 60 is compact and highly efficient and is derived from TCR beta chain. It is predicted to give about 95% cleavage after the terminal glycine, giving efficient removal by signal peptidase.

SEQ ID No. 61 : MSLPVTALLLPLALLLHAARP The signal peptide of SEQ ID No. 61 is derived from lgG1.

SEQ ID No. 62: MAVPTQVLGLLLLWLTDARC

The signal peptide of SEQ ID No. 62 is derived from CD8a.

SPACER

The CAR and chimeric transmembrane polypeptides described herein may comprise a to connect the dimerization/antigen-binding domain with the transmembrane domain and spatially separate the dimerization/antigen-binding domain from the endodomain. A flexible spacer allows to the dimerization/antigen-binding domain to orient in different directions.

The spacer sequence may, for example, comprise an I gG 1 Fc region, an lgG1 hinge or a CD8 stalk, or a combination thereof. The spacer may alternatively comprise an alternative sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an IgG 1 hinge or a CD8 stalk.

A human I gG 1 spacer may be altered to remove Fc binding motifs.

Examples of amino acid sequences for these spacers are given below:

SEQ ID No. 63 (hinge-CH2CH3 of human lgG1)

AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCWVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD

SEQ ID No. 64 (human CD8 stalk):

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI

SEQ ID No. 65 (human lgG1 hinge):

AEPKSPDKTHTCPPCPKDPK

CAR ENDODOMAIN The endodomain is the portion of a classical CAR which is located on the intracellular side of the membrane.

The endodomain is the signal-transmission portion of a classical CAR. After antigen recognition by the antigen binding domain, individual CAR molecules cluster, native CD45 and CD148 are excluded from the synapse and a signal is transmitted to the cell.

The CAR endodomain may be or comprise an intracellular signalling domain. In an alternative embodiment, the endodomain of the present CAR may be capable of interacting with an intracellular signalling molecule which is present in the cytoplasm, leading to signalling.

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

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 I survival signal, or all three can be used together.

The CAR may comprise the CD3-Zeta endodomain alone, the CD3-Zeta endodomain with that of either CD28 or 0X40 or the CD28 endodomain and 0X40 and CD3-Zeta endodomain.

The CAR endodomain may comprise one or more of the following: an ICOS endodomain, a CD27 endodomain, a BTLA endodomain, a CD30 endodomain, a GITR endodomain and an HVEM endodomain.

The endomain may comprise the sequence shown as SEQ I D No. 66 to 74 or a variant thereof having at least 80% sequence identity.

SEQ ID No. 66 - CD3 Z endodomain

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 67 - CD28 and CD3 Zeta endodomains SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKG ERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID No. 68 - CD28, 0X40 and CD3 Zeta endodomains

SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKPPGGGSFR TPIQEEQADAHSTLAKIRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR

SEQ ID No. 69 - ICOS endodomain

CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL

SEQ ID No. 70 - CD27 endodomain

QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP

SEQ ID No. 71 - BTLA endodomain

RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQE GSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS

SEQ ID No. 72 - CD30 endodomain

HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQP LMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGT VKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPT AASGK

SEQ ID No. 73 - GITR endodomain

QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV

SEQ ID No. 74 - HVEM endodomain CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH

A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 66 to 74, provided that the sequence provides an effective intracellular signalling domain.

NUCLEIC ACID The present invention also provides a nucleic acid encoding one or both polypeptides of a chimeric transmembrane protein of the invention.

The nucleic acid may have the structure:

Dim-spacer-TM-endo in which

Dim is a nucleic acid sequence encoding the dimerisation domain of a polypeptide of the chimeric transmembrane protein; spacer 1 is a nucleic acid sequence encoding the spacer of a polypeptide of the chimeric transmembrane protein;

TM1 is a nucleic acid sequence encoding the transmembrane domain of a polypeptide of the chimeric transmembrane protein; endo 1 is a nucleic acid sequence encoding the endodomain of a polypeptide of the chimeric transmembrane protein.

NUCLEIC ACID CONSTRUCT

The present invention further provides a nucleic acid construct encoding a chimeric transmembrane protein which comprises a first nucleic acid sequence encoding the first polypeptide; and a second nucleic acid sequence encoding the second polypeptide, the nucleic acid construct having the structure:

Dim1 -TM1-endo1-coexpr-Dim2 -TM2-endo2 in which

Dim1 is a nucleic acid sequence encoding the first dimerisation domain;

TM1 is a a nucleic acid sequence encoding the transmembrane domain of the first polypeptide; endo 1 is a nucleic acid sequence encoding the endodomain of the first polypeptide; coexpr is a nucleic acid sequence enabling co-expression of both polypeptides

Dim2 is a nucleic acid sequence encoding the second dimerization domain;

TM2 is a a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo 2 is a nucleic acid sequence encoding the endodomain of the second polypeptide. A nucleic acid construct encoding a chimeric transmembrane protein of the invention may comprise a first nucleic acid sequence encoding the first polypeptide and a second nucleic acid sequence encoding the second polypeptide, the nucleic acid construct having the structure:

CH-spacer1-TM1-endo1-coexpr-CL-spacer2-TM2-endo2 in which

CH is a nucleic acid sequence encoding the CH domain of the first polypeptide; spacer 1 is a nucleic acid sequence encoding the spacer of the first polypeptide;

CL is a nucleic acid sequence encoding the CL domain of the second polypeptide; spacer 2 is a nucleic acid sequence encoding the spacer of the second polypeptide;

TM2 is a a nucleic acid sequence encoding the transmembrane domain of the second polypeptide; endo 2 is a nucleic acid sequence encoding the endodomain of the second polypeptide.

When the nucleic acid construct is expressed in a cell, such as a T-cell, it encodes a polypeptide which is cleaved at the cleavage site such that the first and second polypeptides are co-expressed at the cell surface.

The first and second polypeptides may have complementary endodomains e.g. one derived from the a or chain of a cytokine receptor and one derived from the y chain of the same cytokine receptor.

The present invention also provides a nucleic acid construct encoding a chimeric transmembrane protein of the invention and a CAR.

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

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 here to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.

Nucleic acids according to the invention may comprise DNA or RNA. They may be singlestranded 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 include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.

In the structure above, “coexpr” is a nucleic acid sequence enabling co-expression of both first and second polypeptides. It may be a sequence encoding a cleavage site, such that the nucleic acid construct produces comprises two or more CCR-forming polypeptides, or a CCR and a CAR, joined by a cleavage site(s). 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 cleavage site may be any sequence which enables the first and second polypeptides, or chimeric transmembrane protein and CAR, to become separated.

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 cleavage site may comprise one of these 2A-like sequences, such as:

YHADYYKQRLIHDVEMNPGP (SEQ ID No. 75)

HYAGYFADLLIHDIETNPGP (SEQ ID No. 76)

QCTNYALLKLAGDVESNPGP (SEQ ID No. 77)

ATNFSLLKQAGDVEENPGP (SEQ ID No. 78)

AARQMLLLLSGDVETNPGP (SEQ ID No. 79)

RAEGRGSLLTCGDVEENPGP (SEQ ID No. 80)

TRAEIEDELIRAGIESNPGP (SEQ ID No. 81)

TRAEIEDELIRADIESNPGP (SEQ ID No. 82)

AKFQIDKILISGDVELNPGP (SEQ ID No. 83)

SSIIRTKMLVSGDVEENPGP (SEQ ID No. 84)

CDAQRQKLLLSGDIEQNPGP (SEQ ID No. 85)

YPIDFGGFLVKADSEFNPGP (SEQ ID No. 86)

The cleavage site may comprise the 2A-like sequence shown as SEQ ID No. 80 (RAEGRGSLLTCGDVEENPGP).

The present invention also provides a kit comprising one or more nucleic acid sequence(s) encoding first and second polypeptides of the chimeric transmembrane protein of the invention, or a chimeric transmembrane protein according to the invention and one or more CAR(s).

VECTOR

The present invention also provides a vector, or kit of vectors, which comprises one or more nucleic acid sequence(s) encoding a one or more chimeric transmembrane protein(s) according to the first aspect of the invention and optionally one or more CAR(s). Such a vector may be used to introduce the nucleic acid sequence(s) into a host cell so that it expresses the chimeric transmembrane protein(s) and optionally one or more CAR(s)

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.

CELL

The present invention provides a cell which comprises one or more chimeric transmembrane protein(s) of the invention and optionally one of more CAR(s).

The cell may comprise a nucleic acid or a vector of the present invention.

The cell may be a cytolytic immune cell such as a T cell or an NK cell.

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, TH 17, 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.

The cell may be a Natural Killer cell (or NK cell). 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.

Invariant natural killer T (iNKT) cells, also known as type I or classical NKT cells, are a distinct population of T cells that express an invariant a T-cell receptor (TCR) and a number of cell surface molecules in common with natural killer (NK) cells. NKT cells express a restricted TCR repertoire that, in humans, is composed of a Va24-Ja18 TCRa chain preferentially coupled with a V i 1 TCR chain. Unlike conventional T cells, which mostly recognise peptide antigens presented by MHC molecules, iNKT cells recognise glycolipid antigens presented by the non-polymorphic MHC class l-like molecule, CD1d. The chimeric transmembrane protein-expressing cells of the invention may be any of the cell types mentioned above.

Chimeric transmembrane protein-expressing cells of the invention may either be created in vitro or 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, according may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T or NK cells. Alternatively, an immortalized T-cell line which retains its lytic function and could act as a therapeutic may be used.

In all these embodiments, chimeric transmembrane-protein -expressing cells are generated by introducing DNA or RNA coding for the or each polypeptide(s) by one of many means including transduction with a viral vector, transfection with DNA or RNA.

The cell of the invention may be an ex vivo cell, such as a T or NK cell from a subject. The cell may be from a peripheral blood mononuclear cell (PBMC) sample. Cells may be activated and/or expanded prior to being transduced with nucleic acid encoding the chimeric transmembrane protein of the invention, for example by treatment with an anti-CD3 monoclonal antibody.

A cell of the invention may be made by:

(i) isolation of a cell-containing sample from a subject or other sources listed above; and

(ii) transduction or transfection of the cells with one or more a nucleic acid sequence(s) encoding a chimeric transmembrane protein.

The cells may then by purified, for example, selected on the basis of expression of the chimeric transmembrane protein, CAR or a marker gene.

PHARMACEUTICAL COMPOSITION

The present invention also relates to a pharmaceutical composition containing a plurality of cells according to the 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 the cells of the present invention (for example in a pharmaceutical composition as described above) to a subject.

A method for treating a disease relates to the therapeutic use of the cells of the present invention. Herein 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 for preventing a disease relates to the prophylactic use of the cells of the present invention. Herein such cells 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 or transfecting such cells with a nucleic acid sequence or vector provided by the present invention;

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

The cell-containing sample may be isolated from a subject or from other sources, for example as described above.

The present invention provides a chimeric transmembrane protein-expressing cell of the present invention for use in treating and/or preventing a disease. The invention also relates to the use of a chimeric transmembrane protein -expressing cell of the present invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

The disease to be treated and/or prevented 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 cells 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.

The disease to be treated and/or prevented by the methods of the present invention may be an autoimmune disease such as Addison disease, Dermatomyositis, Graves disease, Hashimoto thyroiditis, Multiple sclerosis, Myasthenia gravis, Pernicious anemia, Reactive arthritis, Rheumatoid arthritis, Sjogren syndrome, Systemic lupus erythematosus, Type I diabetes, Mucosal or mucocutaneous pemphigus vulgaris or Membranous nephropathy.

The disease or disorder may be characterised by an inappropriate or undesirable immune response such as graft rejection, GvHD or hemophilia.

The cells and pharmaceutical compositions of present invention may be for use in the treatment and/or prevention of the diseases described above.

Where the disease or disorder is characterised by the presence of autoantibodies and/or the activation of autoreactive B cells (such as in Systemic lupus erythematosus (SLE)), the cell of the invention may target a B-cell antigen such as CD19.

The cells and pharmaceutical compositions of present invention may be for use in any of the methods described above.

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 - Generation and testing of a panel of chimeric transmembrane proteins with SpYced endodomains

Constructs were created having the general structure:

RQR8-2A-CL-TM1-tCyC-2Aw-CH-TM2-SpYIL2/7R in which:

RQR8 is a marker gene described in WO2013/153391

2A and 2Aw are self-cleaving peptides: the sequence encoding 2Aw is codon wobbled to prevent homologous recombination

CL is Light kappa chain

SP1 and SP2 are spacers

TM1 and TM2 are transmembrane domains tCyC is a truncated version of the common y-chain endodomain having amino acids 284-349 of the full length sequence (SEQ ID NO. 19)

CH is heavy chain constant region

SpYIL2Ra/7Rp is a SpYced version of IL-2 receptor p-chain endodomain or IL-7 receptor a-chain endodomain

Constructs were generated with the SpYced versions of IL-2 receptor p-chain endodomain or IL-7 receptor a-chain endodomain having flexible SG4S and rigid alpha-helical EA3S shown as SEQ ID Nos 56 to 59.

As a control, constructs were generated and tested having an unSpYced IL-2 receptor p-chain endodomain or IL-7 receptor a-chain endodomain (IL2-CCR and IL7-CCR).

T cells were transduced with vectors expressing each construct and stained for expression of RQR8 (the marker gene) and endodomain components Kappa light chain and heavy chain constant region (CH1). The results are shown in Figure 5. Cells transduced with vectors expressing chimeric transmembrane proteins with SpYced endodomains had equivalent if not slightly higher expression that cells transduced with vectors expressing chimeric transmembrane proteins with wild-type endodomains. This was true whether G4S or EA3K linkers were used, and was true for both chimeric transmembrane proteins with endodomains derived from IL-2 receptor p-chain endodomain and chimeric transmembrane proteins with endodomains derived from IL-7 receptor a-chain endodomain.

In order to test functionality, cells were cultured for 7 days in the absence of exogenous cytokines (starvation assay). The absolute number of viable, transduced cells was assessed by flow cytometry. A non-transduced T cell population (NT) was used as a negative control. The results are shown in Figure 6.

Expression of a chimeric transmembrane protein with either a wild-type IL-2 receptor p-chain endodomain (IL2-CCR) or wild-type IL-7 receptor a-chain endodomain (IL7-CCR) increased T-cell expansion compared to an equivalent population of cells which did not express a chimeric transmembrane protein (NT). Surprisingly, cell expressing a chimeric transmembrane protein with a SpYced endodomain gave greater proliferation than the wildtype counterpart, both for chimeric transmembrane proteins with endodomains derived from IL-2 receptor p-chain endodomain and chimeric transmembrane proteins with endodomains derived from IL-7 receptor a-chain endodomain. Of the two linker types tested, constructs with G4S linkers gave higher T-cell expansion that equivalent constructs with EA3K linkers, suggesting that linker flexibility is important.

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 chimeric transmembrane protein which comprises two polypeptides:

(a) a first polypeptide which comprises:

(i) a first dimerization domain;

(b) a second polypeptide which comprises:

(L-Y)n in which L is a linker;

2. A chimeric transmembrane protein according to claim 1 wherein the first and second dimerisation domains are leucine zipper domains.

3. A chimeric transmembrane protein according to claim 1 , wherein the first dimerization domain comprises a heavy chain constant domain (CH) and the second dimerization domain comprises a light chain constant domain (CL); or first dimerization domain comprises a light chain constant domain (CL) and the second dimerization domain comprises a heavy chain constant domain (CH).

4. A chimeric transmembrane protein according to any preceding claim, wherein: the first JAK-binding domain binds JAK3 and the second JAK binding domain binds JAK1; or the first JAK-binding domain binds JAK2 and the second JAK binding domain binds JAK2.

5. A chimeric transmembrane protein according to any preceding claim, wherein the first endodomain comprises: GM-CSF receptor a-chain endodomain, common y-chain endodomain, IL12 receptor pi subunit, IFNAR1, IFNGR2 or a truncated version thereof.

6. A chimeric transmembrane protein according to any preceding claim, wherein L is an G4S or EA3K linker.

7. A chimeric transmembrane protein according to any preceding claim, wherein Y is derived from the endodomain of one or more of the following cytokine receptor chains: I L2R|3, IL7Ra; GMCSFR , IL9R, IL21 R, IL12R p2 subunit, IFNAR2 and IFNGR1.

8. A chimeric transmembrane protein according to any preceding claim, wherein Y is selected from the tyrosine/threonine motifs listed in Table 1.

9. A chimeric transmembrane protein according to any preceding claim wherein the first polypeptide comprises:

(i) a light chain constant domain (CL);

(i) a heavy chain constant domain (CH);

(ii) a second endodomain which comprises the JAK-binding domain from IL2R chain and a signalling domain having the general formula:

L-Y1-L-Y2-L-Y3-L-Y4 in which

L is a G4S or EA3K linker;

Y1 has the sequence shown as SEQ ID No. 24

Y2 has the sequence shown as SEQ ID No. 25

Y3 has the sequence shown as SEQ ID No. 26

Y4 has the sequence shown as SEQ ID No. 27

10. A chimeric transmembrane protein according to any preceding claim wherein the first polypeptide comprises:

(i) a light chain constant domain (CL);

(i) a heavy chain constant domain (CH);

L-Y1-L-Y2 in which

L is a G4S or EA3K linker;

Y1 has the sequence shown as SEQ ID No. 28 Y2 has the sequence shown as SEQ ID No. 29

11. A cell which comprises a chimeric transmembrane protein according to any preceding claim.

12. A cell according to claim 11 , which also comprises a chimeric antigen receptor (CAR).

13. A nucleic acid construct encoding a chimeric transmembrane protein according to any of claims 1 to 10, which comprises a nucleic acid sequence encoding the first polypeptide; a nucleic acid sequence encoding a self-cleaving peptide; and a nucleic acid sequence encoding the second polypeptide.

14. A nucleic acid construct according to claim 13, wherein alternative codons are used in regions of sequence encoding the linkers, to avoid homologous recombination.

15. A nucleic acid construct according to claim 13 or 14 which also comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR).

16. A vector comprising a nucleic acid construct according to any of claims 13 to 15.

17. A kit of vectors for expressing a chimeric transmembrane protein according to any of claims 1 to 10 in a cell, which kit comprises: i) a vector comprising a nucleic acid sequence encoding the first polypeptide; ii) a vector comprising a nucleic acid sequence encoding the second polypeptide; and optionally iii) a vector comprising a nucleic acid sequence encoding a chimeric antigen receptor.

18. A method for making a cell according to claim 11 or 12, which comprises the step of introducing: a nucleic acid construct according to any of claims 13 to 15; a vector according to claim 16; or a kit of vectors according to claim 17 into a cell, wherein the cell is from a sample isolated from a subject.

19. A pharmaceutical composition comprising a plurality of cells according to claim 11 or

20. A method for treating a cancerous disease or an autoimmune disease in a subject, which comprises the step of administering a pharmaceutical composition according to claim 19 to the subject.

21. A pharmaceutical composition according to claim 19 for treating a cancerous disease or an autoimmune disease.

22. The use of a cell according to claim 11 or 12 in the manufacture of a medicament for treating a cancerous disease or an autoimmune disease.