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1. WO2020222007 - CHIMERIC PROTEIN COMPRISING A CASPASE 1 DOMAIN

Note: Text based on automatic Optical Character Recognition processes. Please use the PDF version for legal matters

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CHIMERIC PROTEIN COMPRISING A CASPASE 1 DOMAIN

FIELD OF THE INVENTION

The present invention relates to a chimeric protein useful in adoptive cell therapy (ACT). The chimeric protein can act as a suicide gene enabling cells expressing the chimeric protein to be deleted. The present invention also provides a nucleic acid encoding such a chimeric protein, a cell comprising such a nucleic acid and therapeutic uses thereof.

BACKGROUND TO THE INVENTION

Adoptive Cell Therapy

Adoptive immunotherapy is an established and evolving therapeutic approach. In the setting of allogeneic haematopoietic stem cell transplantation (HSCT), donor lymphocyte infusions (DLI) are frequently given to treat relapse of haematological malignancies. Tumour infiltrating lymphocytes (TILs) are effective in treating metastatic melanoma. Genetic engineering of T-cells greatly increases the scope and potency of T-cell therapy: T-cell receptor transfer allows targeting of intracellular cancer antigens, while chimeric antigen receptors (CAR) allow targeting of surface cancer or lineage specific antigens. Clinical responses have been observed with both approaches, and numerous further trials are underway.

Acute adverse events can occur following adoptive immunotherapy. Graft-versus-host disease (GvHD) is a common and serious complication of DLI. Administration of engineered T-cells has also resulted in toxicity. For instance, on-target off-tumour toxicity has been reported in native T-cell receptor transfer studies against melanoma antigens; T-cells re-directed to the renal cell carcinoma antigen carbonic anhydrase IX (CAIX) produced unexpected hepatotoxicity. Immune activation syndromes have been reported after CD19 CAR therapy. Finally vector- induced insertional mutagenesis results in a theoretical risk of lymphoproliferative disorders. The incidence and severity of these toxicities is unpredictable. Further, in contrast to a therapeutic protein or small molecules whose adverse events usually abate with the half-life of the therapeutic, T-cells engraft and replicate, potentially resulting in escalating and fulminant toxicity.

Suicide Genes

A suicide-gene is a genetically encoded mechanism which allows selective destruction of adoptively transferred cells, such as T-cells, in the face of unacceptable toxicity. Two suicide-genes have been tested in clinical studies: Herpes Simplex Virus thymidine kinase (HSV-TK) and inducible caspase 9 (iCasp9).

The herpes simplex virus l-derived thymidine kinase (HSV-TK) gene has been used as an in vivo suicide switch in donor T-cell infusions to treat recurrent malignancy and Epstein Barr virus (EBV) lymphoproliferation after hemopoietic stem cell transplantation. However, destruction of T cells causing graft-versus-host disease was incomplete, and the use of ganciclovir (or analogs) as a pro-drug to activate HSV-TK precludes administration of ganciclovir as an antiviral drug for cytomegalovirus infections. Moreover, HSV-TK-directed immune responses have resulted in elimination of HSV-TK-transduced cells, even in immunosuppressed human immunodeficiency virus and bone marrow transplant patients, compromising the persistence and hence efficacy of the infused T cells.

The activation mechanism behind Caspase 9 was exploited in the original iCasp9 molecule. All that is needed for Caspase 9 to become activated, is overcoming the energic barrier for Caspase 9 to homodimerize. The homodimer undergoes a conformational change and the proteolytic domain of one of a pair of dimers becomes active. Physiologically, this occurs by binding of the CARD domain of Caspase 9 to APAF-1. In iCasp9, the APAF-1 domain is replaced with a modified FKBP12 which has been mutated to selectively bind a chemical inducer of dimerization (CID). Presence of the CID results in homodimerization and activation. iCasp9 is based on a modified human caspase 9 fused to a human FK506 binding protein (FKBP) (Straathof et al (2005) Blood 105:4247-4254). It enables conditional dimerization in the presence of a small molecule CID, known as AP1903. AP1903 is an experimental drug and is considered biologically inert since it does not interact with wild-type FKBP12. However clinical experience with this agent is limited to a very small number of patients (Di Stasi, A. et al. (2011) N. Engl. J. Med. 365, 1673-1683; and luliucci, J. D. et al. (2001) J. Clin. Pharmacol. 41 , 870-879). AP1903 is also a relatively large and polar molecule and unlikely to cross the blood-brain barrier.

WO2016/135470 describes a suicide gene based on Caspase 9 which is a multi-domain molecule, comprising (i) the FRB domain of mTOR; (ii) FKBP12; and (iii) caspase 9. Heterodimerization between the FRB domain of one copy of the molecule

and the FKB12 domain of another copy of the molecule causes homodimerization of the caspase domains. T cells expressing this FKBP12-FRB-Casp9 construct are shown to be deleted in the presence of rapamycin.

DESCRIPTION OF THE FIGURES

Figure 1 - Rapamycin and rapalogs. A) Rapamycin; B) C-20-methyllyrlrapamycin (MaRap); C) C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap); D) C16-(S)-3-mehylindolerapamycin (C16-iRap); and E) C16-(S)-7-methylindolerapamycin (AP21976/C16-AiRap).

Figure 2 - AP1903

Figure 3 - Cartoons showing different approaches to RapCaspl (A) Double construct where two molecules are expressed separately. One molecule has the catalytic domain of Caspl fused with FKBP12 and one molecule has the catalytic domain of Caspl fused with FRB. (B) Single construct where FKBP12 and FRB are fused together (optionally via a linker) and then fused to the catalytic domain of Caspl by a flexible linker. (C) Double construct where the catalytic domain of Caspase 1 is fused to FKBP12 and a separate small protein which is a fusion of two copies of FRB is co expressed. For approaches A to C, the CID is rapamycin or a rapalog. (D) Single construct where FKBP12 having a mutation F36V is fused to the catalytic domain of Caspl For approach D, the CID is AP1903.

Figures 4 - Flow cytometric analysis of T cells transfected with constructs expressing either Rapcasp9 or Rapcaspl , following 24 hours incubation with rapamycin from OnM to 1000nM concentration.

Figure 5 - Summary data for killing assays from three donors. The percentage of killing was calculated based on the percentage of remaining live cells expressing the suicide construct after drug administration and upon normalisation to the respective untreated control.

Figure 6 - Introduction of a STOP codon upstream of RapCaspl RapCasp9, RapCaspl , SKIP_RapCasp1 and STOPSKIP_RapCasp1 were plated at a concentration of 105 cells in a range of rapamycin concentrations. The MFI of eGFP was measured 24h later. The MFI normalised to OnM rapamycin is shown. The range

of rapamycin concentrations was 0.1-100nM, diluted by a 10-factor step. RapCasp9 constitutes the positive control for the eGFP MFI. SKIP_RapCasp1 bore the skip sequence upstream of RapCaspl without a STOP codon, thus being comparable to the original RapCaspl The STOPSKIP_RapCasp1 on the contrary bore the STOP and then SKIP sequence upstream of RapCaspl , which leads to a reduced expression of RapCaspl At the lowest concentration of rapamycin, 0.1 nM, STOPSKIP_RapCasp1 was more sensitive than RapaCasp9 with a decrease in normalised MFI of 2.4-fold. However, STOPSKIP_RapCasp1 was comparable to RapCaspl with normalised MFI of 0.152 and 0.116, respectively.

SUMMARY OF ASPECTS OF THE INVENTION

The present inventors have developed a new, improved, suicide gene, which dimerizes in the presence of a chemical inducer of dimerization (CID) such as rapamycin or a rapamycin analogue.

The present inventors have previously described a suicide gene having the structure FKBP12-FRB-Casp9 which has the following domains (i) FKBP12, (ii) the FRB domain of mTOR; and (iii) Caspase9. Heterodimerization between the FRB domain of one copy of the molecule and the FKB12 domain of another copy of the molecule causes homodimerization of the caspase 9 domains and triggers apoptosis.

The present inventors have now found that using Caspase 1 in the construct rather than Caspase 9 leads to a suicide gene with improved properties. Specifically, they have found that T cells expressing a suicide gene comprising a caspase-1 domain gave greater killing efficiency in the presence of rapamycin than cells expressing a suicide gene comprising a caspase-9 domain. Moreover, T cells expressing a suicide gene comprising a caspase-1 domain showed a greater sensitivity to rapamycin than cells expressing a suicide gene comprising a caspase-9 domain, giving significant killing even at rapamycin concentrations as low as 0.1 nM.

Thus in a first aspect, the present invention provides a chimeric protein having the formula:

Ht1-Ht2-Casp1

wherein

Caspl is a caspase 1 domain;

Ht1 is a first heterodimerization domain; and

Ht2 is a second heterodimerization domain.

Ht1 from one chimeric protein molecule dimerises with Ht2 from another chimeric protein molecule in the presence a chemical inducer of dimerization (CID). In other words, in the presence of the CID an identical pair of the chimeric proteins interact such that Ht1 from one chimeric protein heterodimerizes with Ht2 from the other chimeric protein, causing homodimerization of the two caspase domains.

One heterodimerization domain (i.e. Ht1 or Ht2) may comprise an FK506-binding protein (FKBP) and the other heterodimerization domain (i.e. Ht2 or Ht1) may comprises an FRB domain of mTOR. For example, Ht1 may comprise FRB and Ht2 may comprise FKBP. In such embodiments the CID may be rapamycin or a rapamycin analogue.

In a second aspect, the present invention provides a nucleic acid sequence which encodes a chimeric protein according to the first aspect of the invention.

In a third aspect, the present invention provides a nucleic acid construct which comprises a nucleic acid sequence according to the second aspect of the invention and a nucleic acid sequence encoding a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

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

In a fifth aspect, the present invention provides a kit of vectors which comprises first vector comprising a nucleic acid sequence according to the second aspect of the invention and a second vector comprising a nucleic acid sequence which encodes a chimeric antigen receptor or a T-cell receptor.

In a sixth aspect there is provided a cell which expresses a chimeric protein according to the first aspect of the invention.

The cell may, for example be a haematopoietic stem cell, a lymphocyte or a T cell.

In a seventh aspect, the present invention provides a method for making a cell according to the sixth aspect of the invention which comprises the step of transducing or transfecting a cell with a vector according to the fourth aspect of the invention or a kit of vectors according to the fifth aspect of the invention.

In an eighth aspect, there is provided a method for deleting a cell according to the sixth aspect of the invention, which comprises the step of exposing the cells to a chemical inducer of dimerization (CID).

The CID may, for example, be rapamycin or a rapamycin analogue.

In a ninth aspect, the present invention provides a chemical inducer of dimerization (CID) for use in deleting a cell according to the sixth aspect of the invention in vivo.

In a ninth aspect, there is provided a method for treating a disease in a subject, which comprises the step of administering a cell according to the sixth aspect of the invention to the subject.

The method may comprise the following steps:

(i) transducing or transfecting a sample of cells isolated from a subject with a vector according to the fourth aspect of the invention or a kit of vectors according to the fifth aspect of the invention, and

(ii) administering the transduced/transfected cells to a patient.

The method may be for treating cancer.

In a tenth aspect, the present invention provides a cell according to the sixth aspect of the invention for use in a method for treating a disease in a subject

In an eleventh aspect, there is provided a method for preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to the sixth aspect of the invention to the subject, which comprises the step of administering a chemical inducer of dimerization (CID) to the subject.

The pathological immune reaction may be selected from the following group: graft-versus-host disease; on-target, off-tumour toxicity; immune activation syndrome; and lymphoproliferative disorders.

In a twelfth aspect, there is provided a chemical inducer of dimerization (CID) for use in preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to the sixth aspect of the invention to the subject.

The method of the ninth aspect of the invention may comprise the following steps:

(i) administering a cell according to the sixth aspect of the invention to the subject;

(ii) monitoring the subject for the development of a pathological immune reaction; and

(iii) administering chemical inducer of dimerization (CID) to the subject if the subject shows signs of developing or having developed a pathological immune reaction.

The invention also provides a suicide gene in which caspase 1 and caspase 9-based systems are provided in trans or in cis. In this respect, the present invention provides a chimeric protein having the formula:

Ht1-Ht2-Casp1-Casp9 or Ht1-Ht2-Casp9-Casp1

wherein

Caspl is a caspase 1 domain;

Casp9 is a caspase 9 domain;

Ht1 is a first heterodimerization domain; and

Ht2 is a second heterodimerization domain.

There is also provided a cell which comprises a first chimeric protein having the formula: Ht1-Ht2-Casp1 ; and a second chimeric protein having the formula: Ht1-Ht2-Casp9

wherein

Caspl is a caspase 1 domain;

Casp9 is a caspase 9 domain;

Ht1 is a first heterodimerization domain; and

Ht2 is a second heterodimerization domain.

Aspects of the invention relating to nucleic acid constructs, vectors, cells and methods of making and using such cells apply equally to these Caspase1/Caspase9 combination embodiments of the invention.

Thus, the present invention provides a suicide gene which allows the selective destruction of adoptively infused cells in the face of unacceptable toxicity, and which is activated by rapamycin and/or its analogues.

Rapamycin is standard pharmaceutical with well understood properties, excellent bioavailability and volume of distribution and which is widely available. Rapamycin also does not aggravate the condition being treated, in fact, as it is an immunosuppressant it is likely to have a beneficial effect on unwanted toxicity as well as its suicide gene function.

The suicide genes of the present invention, based on triggering apoptosis by dimerization of caspase 1 domains, have improved properties over suicide genes based on triggering apoptosis by dimerization of caspase 1 domains both in terms of killing efficiency and sensitivity to rapamycin. Caspase-1 is also capable of initiating cell death by pyroptosis via the cleavage of Gasdermin D (GsdmD) (Tsuchiya et ai, 2019 Nat Commun 10, 1-19).

Additional aspect of the invention are summarised in the following numbered paragraphs:

A. Paragraphs relating to the embodiment of the invention shown in Figure 3D

A1. A chimeric protein comprising a caspase 1 domain and a dimerisation domain.

A2. A chimeric protein according to paragraph A1 , wherein the dimerization domain comprises FK506-binding protein 12 (FKBP12) with an F36V mutation.

A3. A chimeric protein according to paragraph A1 or A2, wherein, in the presence of a chemical inducer of dimerization (CID), the dimerization domains from a pair of chimeric proteins interact, causing homodimerization of the caspase domains.

A4. A chimeric protein according to paragraph A3, wherein the CID is AP1903.

A5. A chimeric protein according to any preceding paragraph, which comprises a linker between the caspase 1 domain and the dimerization domain.

A6. A chimeric protein according to paragraph A5, wherein the linker comprises or consists of the sequence SGGGS (SEQ ID No. 13).

A7. A nucleic acid sequence which encodes a chimeric protein according to any preceding paragraph.

A8. A nucleic acid construct which comprises a nucleic acid sequence according to paragraph 7 and a nucleic acid sequence encoding a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

A9. A vector which comprises a nucleic acid sequence according to paragraph A5 or a nucleic acid construct according to paragraph A6.

A10. A kit of vectors which comprises first vector comprising a nucleic acid sequence according to paragraph A7 and a second vector comprising a nucleic acid sequence which encodes a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

A11. A cell which expresses a chimeric protein according to any of paragraphs A1 to A6.

A12. A method for making a cell according to paragraph AA11 which comprises the step of transducing or transfecting a cell with a vector according to paragraph 9 or a kit of vectors according to paragraph A10.

A13. A method for deleting a cell according to paragraph A11 , which comprises the step of exposing the cells to a chemical inducer of dimerization (CID)

A14. A method according to paragraph A13, wherein the CID is AP1903.

A15. A chemical inducer of dimerization (CID) for use in deleting a cell according to paragraph 11 in vivo.

A16. A method for treating a disease in a subject, which comprises the step of administering a cell according to paragraph A11 to the subject.

A17. A cell according to paragraph A11 for use in a method for treating a disease in a subject.

A18. A method for preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to any of paragraph A11 to the subject, which comprises the step of administering a chemical inducer of dimerization (CID) to the subject.

A19. A chemical inducer of dimerization (CID) for use in preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to paragraph A11 to the subject.

A20. A method for treating a disease in a subject according to paragraph 16, which comprises the following steps:

(i) administering a cell according to paragraph A11 to the subject;

(ii) monitoring the subject for the development of a pathological immune reaction; and

(iii) administering chemical inducer of dimerization (CID) to the subject if the subject shows signs of developing or having developed a pathological immune reaction.

A21. A chimeric protein according to any of paragraphs A1 to A6, which has the structure D-Casp1-Casp9 or D-Casp9-Casp1 , in which

D is a dimerization domain

Caspl is a caspase 1 domain

Casp9 is a caspase 9 domain.

A22 A cell which comprises a first chimeric protein according to any of paragraphs A1 to A6 and a second chimeric protein which comprises a dimerization domain as defined in any of paragraphs A1 to A6 and a caspase 9 domain.

B. Paragraphs relating to the embodiment of the invention shown in Figure 3C

B1. A chimeric protein comprising a caspase 1 domain and a dimerisation domain.

B2. A cell which co-expresses two proteins:

i) a chimeric protein according to paragraph B1 having the structure: Ht1-Caspl ; and

ii) an interfacing protein having the structure Ht2-Ht2

in which

Caspl is a caspase 1 domain

Ht1 is a first heterodimerisation domain; and

Ht2 is a second heterodimerisation domain.

B3. A cell according to paragraph B2, wherein Ht1 comprises an FK506-binding protein (FKBP) and Ht2 comprises an FRB domain of mTOR.

B4. A cell according to paragraph B2 or B3 wherein, in the presence of a chemical inducer of dimerization (CID), a pair of the chimeric proteins Ht1 -Caspl interact such that Ht1 from each chimeric protein heterodimerizes with an Ht2 domain from the interfacing protein, causing homodimerization of the two caspase domains.

B5. A cell according to paragraph B4, wherein the CID is rapamycin or an analog thereof.

B6. A nucleic acid sequence which encodes a chimeric protein according to paragraph 1.

B7. A nucleic acid construct which comprises a first nucleic acid sequence according to paragraph B6 and a second nucleic acid sequence encoding an interfacing protein as defined in paragraph B2.

B8. A nucleic acid construct according to paragraph B7, having the structure:

Ht1-Casp1-coexpr-Ht2-Ht2

wherein:

Casp is a nucleic acid sequence encoding a caspase 1 domain;

Ht1 is a nucleic acid sequence encoding a first heterodimerization domain;

Ht2 is a nucleic acid sequence encoding a second heterodimerization domain; and

coexpr is a nucleic acid sequence allowing co-expression of Ht1-Casp and Ht2-Ht2,

B9. A nucleic acid construct according to paragraph B7 or B8, which comprises a third nucleic acid sequence encoding a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

B10. A vector which comprises a nucleic acid sequence according to paragraph 6 or a nucleic acid construct according to any of paragraphs B6 to B9.

B11. A kit of vectors which comprises first vector comprising a first nucleic acid sequence which encodes a chimeric protein according to paragraph B1 ; and a second vector comprising a second nucleic acid sequence which encodes an interfacing protein as defined in paragraph B2.

B12. A method for making a cell according to any of paragraphs B1 to B5 which comprises the step of transducing or transfecting a cell with a vector according to paragraph B10 or a kit of vectors according to paragraph B11.

B13. A method for deleting a cell according to any of paragraphs B1 to B5, which comprises the step of exposing the cells to a chemical inducer of dimerization (CID)

B14. A method according to paragraph B13, wherein the CID is rapamycin or an analogue thereof.

B15. A chemical inducer of dimerization (CID) for use in deleting a cell according to any of paragraphs B1 to B5 in vivo.

B16. A method for treating a disease in a subject, which comprises the step of administering a cell according to any of paragraphs B1 to B5 to the subject.

B17. A cell according to any of paragraphs B1 to B5 for use in a method for treating a disease in a subject.

B18. A method for preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to any of paragraphs B1 to B5 to the subject, which comprises the step of administering a chemical inducer of dimerization (CID) to the subject.

B19. A chemical inducer of dimerization (CID) for use in preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to any of paragraphs B1 to B5 to the subject.

B20. A method for treating a disease in a subject according to paragraph B16, which comprises the following steps:

(i) administering a cell according to any of paragraphs B1 to B5 to the subject;

(ii) monitoring the subject for the development of a pathological immune reaction; and

(iii) administering chemical inducer of dimerization (CID) to the subject if the subject shows signs of developing or having developed a pathological immune reaction.

B21. A cell which co-expresses two proteins:

i) a chimeric protein according to paragraph B1 having the structure: Ht1-Casp1-Casp9 or Ht1-Casp9-Casp1 ; and

ii) an interfacing protein having the structure Ht2-Ht2

in which

Caspl is a caspase 1 domain

Casp9 is a caspase 9 domain

Ht1 is a first heterodimerisation domain; and

Ht2 is a second heterodimerisation domain.

B22. A cell which co-expresses three proteins:

i) a chimeric protein according to paragraph B1 having the structure: Ht1- Caspl ;

ii) a chimeric protein having the structure: Ht1-Casp9; and

iii) an interfacing protein having the structure Ht2-Ht2

in which

Caspl is a caspase 1 domain

Casp9 is a caspase 9 domain

Ht1 is a first heterodimerisation domain; and

Ht2 is a second heterodimerisation domain.

C. Paragraphs relating to the embodiment of the invention shown in Figure 3A

C1. A chimeric protein comprising a caspase 1 domain and a dimerisation domain.

C2. A chimeric protein according to paragraph C1 , wherein the dimerization domain comprises FKBP12.

C3. A chimeric protein according to paragraph C1 , wherein the dimerization domain comprises FRB.

C4. A nucleic acid sequence which encodes a chimeric protein according to any preceding paragraph.

C5. A nucleic acid construct comprising a first nucleic acid sequence which encodes a chimeric protein according to paragraph C2; and a second nucleic acid sequence which encodes a chimeric protein according to paragraph C3.

C6. A nucleic acid construct according to paragraph C5, which comprises a third nucleic acid sequence encoding a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

C7. A cell comprising a first chimeric protein according to paragraph C2 and a second chimeric protein according to paragraph C3

C8. A cell according to paragraph C6, wherein, in the presence of a chemical inducer of dimerization (CID), the dimerization domains from the first and second chimeric proteins interact, causing homodimerization of the caspase domains.

C9. A cell according to paragraph C7, wherein the CID is rapamycin or an analog thereof.

C10. A vector which comprises a nucleic acid sequence according to paragraph 4 or a nucleic acid construct according to paragraph C5 or C6.

C11. A kit of vectors which comprises first vector comprising a first nucleic acid sequence which encodes a chimeric protein according to paragraph C2; and a second vector comprising a second nucleic acid sequence which encodes a chimeric protein according to paragraph C3.

C12. A method for making a cell according to any of paragraphs 7 to 9 which comprises the step of transducing or transfecting a cell with a vector according to paragraph C10 or a kit of vectors according to paragraph C11.

C13. A method for deleting a cell according to any of paragraphs C7 to C9, which comprises the step of exposing the cells to a chemical inducer of dimerization (CID)

C14. A method according to paragraph C13, wherein the CID is rapamycin or an analogue thereof.

C15. A chemical inducer of dimerization (CID) for use in deleting a cell according to any of paragraphs C7 to C9 in vivo.

C16. A method for treating a disease in a subject, which comprises the step of administering a cell according to any of paragraphs C7 to C9 to the subject.

C17. A cell according to any of paragraphs C7 to C9 for use in a method for treating a disease in a subject.

C18. A method for preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to any of paragraphs C7 to C9 to the subject, which comprises the step of administering a chemical inducer of dimerization (CID) to the subject.

C19. A chemical inducer of dimerization (CID) for use in preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to any of paragraphs C7 to C9 to the subject.

C20. A method for treating a disease in a subject according to paragraph 16, which comprises the following steps:

(i) administering a cell according to any of paragraphs C7 to C9 to the subject;

(ii) monitoring the subject for the development of a pathological immune reaction; and

(iii) administering chemical inducer of dimerization (CID) to the subject if the subject shows signs of developing or having developed a pathological immune reaction.

C21. A cell which comprises:

i) a first chimeric protein having the structure FRB-Casp1-Casp9 or FRB-Casp9-Casp1 ; and

ii) a second chimeric protein having the structure FKBP12-Casp1-Casp9 or FKBP12-Casp1-Casp9

in which Caspl is a caspase 1 domain, and

Casp9 is a caspase 9 domain.

C22. A cell which comprises:

i) a first chimeric protein having the structure FRB-Casp1 ;

ii) a second chimeric protein having the structure FKBP12-Casp1 ;

iii) a third chimeric protein having the structure FRB-Casp9; and

iv) a fourth chimeric protein having the structure FKBP12-Casp9

in which Caspl is a caspase 1 domain, and

Casp9 is a caspase 9 domain.

The additional information provided in the Detailed Description relating to for example chimeric proteins, nucleic acid sequences, nucleic acid constructs, vectors, cells and methods of the invention and CIDs used in the invention applies to the aspects of the invention presented as the claims and equally to the aspects of the invention presented above as numbered paragraphs.

DETAILED DESCRIPTION

CASPASE

Caspases are a family of endoproteases that provide critical links in cell regulatory networks controlling inflammation and cell death.

There are two types of caspase: apoptotic caspases and inflammatory caspases. Activation of apoptotic caspases results in inactivation or activation of substrates, and the generation of a cascade of signaling events permitting the controlled demolition of cellular components. Activation of inflammatory caspases results in the production of active proinflammatory cytokines and the promotion of innate immune responses to various internal and external insults.

Human caspases 2, 3, 6, 7, 8, 9 and 10 are apoptotic caspases. There are two types of apoptotic caspases: initiator caspases and executioner caspases. Initiator caspases, such as caspase-2, caspase-8, caspase-9, and caspase- 10, cleave inactive pro-forms of effector caspases, thereby activating them. Executioner caspases, such as caspase-3, caspase-6 and caspase-7, then cleave other protein substrates within the cell, to trigger the apoptotic process.

In vivo, the protease caspase 9 is the central participant in a multi-component pathway known as the apoptosome, which controls cell deletion during embryogenesis, and physiological responses that trigger cell death as well as lethal cellular insults such as ionizing radiation or chemotherapeutic drugs. The function of caspase 9 is to generate the active forms of caspases 3 and 7 by limited proteolysis, and thereby transmit the apoptotic signal to the execution phase. However, caspase 9 is unusual among its close relatives in that proteolysis between the large and small subunit does not convert the latent zymogen to the catalytic form. In fact, it is homodimerization which is required for activation.

WO2016135470 describes a suicide gene based on Caspase 9. As caspase 9 is the key initiator caspase its activation is a very sensitive trigger for apoptosis induction. Another attractive feature of Caspase 9 is that, homodimerization is all that is required for activation, rather than homodimerization and proteolytic cleavage.

Human caspases 1 , 4 and 5 are inflammatory caspases, acting as critical mediators of innate immune responses rather than proapoptotic factors.

Inflammasome formation has been best studied for the nucleotide-binding domain, leucine-rich repeat-containing (NLR) proteins, which are a family of pattern-recognition receptors (PRRs). In a resting cell, NLR monomers are held in an inactive conformation until an external or internal stimulus promotes their assembly. NLR

monomers interact through their NACHT domains and bind to the adapter protein apoptosis-associated speck-like protein containing a CARD (ASC/PYCARD). The presence of ASC permits the recruitment of an inactive inflammatory procaspase, typically procaspase-1 , to the inflammasome, followed by cleavage and activation of caspase-1 through induced proximity autocatalysis. Activated capsase-1 initiates a proinflammatory response through the cleavage and thus activation of the two inflammatory cytokines, interleukin 1 b (I L-1 b) and interleukin 18 (IL-18). The two inflammatory cytokines activated by Caspase-1 are excreted from the cell to further induce the inflammatory response in neighbouring cells.

The present inventors have surprisingly found that, even though Caspase-1 is an inflammatory caspase rather than an apoptotic caspase, Caspase-1 has highly beneficial properties when used are part of a caspase-based suicide gene, and is capable of triggering apoptosis of a cell following induced dimerization of caspase-1 domains. Caspase-1 is also capable of triggering cell death by pyroptosis.

Caspase-1 is produced as a zymogen that can then be cleaved into 20 kDa (p20) and 10 kDa (p10) subunits that become part of the active enzyme. Active Caspase 1 contains two heterodimers of p20 and p10. It contains a catalytic domain with an active site that spans both the p20 and p10 subunits, as well as a noncatalytic Caspase Activation and Recruitment Domain (CARD). The amino acid sequence of human procaspase 1 is available on UniProt under Accession Number P29466 and shown below as SEQ ID No. 1.

SEQ ID No. 1 (human Casase-1)

MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATV

MDKTRALIDSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLN

MQDSQGVLSSFPAPQAVQD NPAMPTSSGSEGNVKLCSLEEAQRIWKQKSA

EIYPIMDKSSRTRLALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSV

DVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSHGIREGICGKKHSE

QVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGWWFKDSVG

VSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFI

GRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYL

FPGH

In this sequence residues 1-119 correspond to a propeptide; 120-297 is the Caspase-1 subunit p20; 298-316 is a propeptide and 317-404 is Caspase-1 subunit p10.

The chimeric protein of the present invention may comprise a truncated version of Caspase-1 which lacksthe first 1 19 amino acids.

The chimeric protein of the invention may comprise a truncated version of caspase-1 having the sequence shown as SEQ ID No. 2.

SEQ ID No. 2 (truncated human Caspase-1)

NPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIP RRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVF MSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGV VWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVF IGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH

The chimeric protein of the first aspect of the invention may comprise SEQ ID No. 2 or a fragment or a variant thereof which retains the capacity to homodimerize and thus trigger apoptosis.

A variant caspase-1 sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 2.

The percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST which is freely available at http://blast.ncbi.njm.njh.gov.

CHIMERIC PROTEIN

The present invention relates to a chimeric protein which acts as a suicide gene. Cells expressing the chimeric protein may be deleted in vivo or in vitro by administration of a chemical inducer of dimerization (CID) such as rapamycin or a rapamycin analogue.

The chimeric protein may comprise a caspase-1 domain and a dimerisation domain.

The chimeric protein may have the formula:

Ht1-Ht2-Casp1

in which

Caspl is a caspase domain;

Ht1 is a first heterodimerization domain; and

Ht2 is a second heterodimerization domain.

The chimeric protein may have the formula:

Ht1-Ht2-L-Casp1

in which Caspl , Ht1 and Ht2 are as defined above and L is an optional linker.

The configuration should be such that Ht1 does not significantly heterodimerize with Ht2 within the same chimeric protein molecule, but when two chimeric proteins come together in the presence of a chemical inducer of dimerization (CID) Ht1 from one chimeric protein heterodimerizes with Ht2 from the other chimeric protein, causing homodimerization of the two caspase domains.

The configuration is such that Ht1 does not heterodimerize to any significant extent with Ht2 within the same chimeric protein. For example, in a cell expressing a chimeric protein according to this embodiment of the first aspect of the invention, the presence of the CID should cause a greater proportion of dimerization between two chimeric proteins, than heterodimerization within the same chimeric protein. The amount of chimeric proteins which are heterodimerized within the same molecule in a cell or cell population, or in solution, may be less than 50%, 40%, 30%, 20%, 10%, 5% or 1% of the amount of chimeric proteins which are heterdomerized with a separate chimeric protein molecule, in the presence of the CID.

The chimeric protein may comprise the sequence shown as SEQ ID No. 3.

SEQ ID No. 3 (FRB-L1-FKBP12-L2-dCasp1)

MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN

QAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGV

QVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIR

GWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGS

GGGGSGGGGSGNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTR

LALIICNEEFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHR

PEHKTSDSTFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKV

IIIQACRGDSPGWWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNV

SWRHPTMGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTL

TRCFYLFPGH

In the above sequence L1 and L2 are in bold and underlined.

In a second embodiment, the invention provides a “two-molecule” suicide gene system, in which the CID is rapamycin or a rapamycin analogue.

Thus, the present invention also provides i) a chimeric protein which comprises a caspase-1 domain and a heterodimerization domain which comprises an FK506-binding protein (FKBP12); and ii) a chimeric protein which comprises a caspase-1 domain and a heterodimerization domain which comprises an FRB domain of mTOR.

When a cell, such as a T-cell, expresses both these chimeric proteins, the presence of rapamycin or a rapamycin analogue causes the FKBP-comprising domain or i) to heterodimerise with the FRB-comprising domain or ii), thus causing homodimerization of the caspase domains from i) and ii).

In a third embodiment, the invention provides an alternative“two molecule” approach, with a smaller footprint than the second embodiment. Here, Ht1 is fused with Caspase-1 , and a second molecule comprises of Ht2-Ht2 fusion is co-expressed. In the presence of CID, Ht2-Ht2 brings together two Ht1-Casp molecules. In practise, this can be implemented by co-expressing FKBP12-Casp9 with FRB-FRB and activating with Rapamycin. Conveniently, these components can be co-expressed with a foot-and-mouth disease 2A like peptide. The second Ht2 (for example FRB) encoding sequence may be codon wobbled to prevent recombination.

In a fourth embodiment, the invention provides an alternative "one molecule" approach. In this embodiment, the chimeric protein comprises a caspase 1 and a homodimerisation domain.

The dimerization domain may, for example, comprise FK506-binding protein 12 (FKBP12) with an F36V mutation. This domain homodimerizes in the presence of the CID AP1903.

The amino acid sequence of FKBP12 with an F36V mutation is shown as SEQ ID No.

4.

SEQ ID No. 4 (FKBP12 with an F36V mutation)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQE

VIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

The structure of AP1903 is shown in Figure 2.

HETERODIMERIZATION DOMAINS

The macrolides rapamycin and FK506 act by inducing the heterodimerization of cellular proteins. Each drug binds with a high affinity to the FKBP12 protein, creating a drug-protein complex that subsequently binds and inactivates mTOR/FRAP and calcineurin, respectively. The FKBP-rapamycin binding (FRB) domain of mTOR has been defined and applied as an isolated 89 amino acid protein moiety that can be fused to a protein of interest. Rapamycin can then induce the approximation of FRB fusions to FKBP12 or proteins fused with FKBP 12.

In the context of the first embodiment of the present invention, one of the heterodimerization domains (Ht1 or Ht2) may be or comprise FRB, or a variant thereof and the other heterodimerization domain (Ht2 or Ht1) may be or comprise FKBP12 or a variant thereof.

Rapamycin has several properties of an ideal dimerizer: it has a high affinity (KD<1 nM) for FRB when bound to FKBP12, and is highly specific for the FRB domain of mTOR. Rapamycin is an effective therapeutic immunosuppressant with a favourable pharmacokinetic and pharmacodynamics profile in mammals. Pharmacological analogues of Rapamycin with different pharmacokinetic and dynamic properties such as Everolimus, Temsirolimus and Deforolimus (Benjamin et al, Nature Reviews, Drug Discovery, 2011) may also be used according to the clinical setting.

In order to prevent rapamycin binding and inactivating endogenous mTOR, the surface of rapamycin which contacts FRB may be modified. Compensatory mutation of the FRB domain to form a burface that accommodates the“bumped” rapamycin restores dimerizing interactions only with the FRB mutant and not to the endogenous mTOR protein.

Bayle et al. (Chem Bio; 2006; 13; 99-107) describes various rapamycin analogs, or “rapalogs” and their corresponding modified FRB binding domains. For example, Bayle et al. (2006) describes the rapalogs: C-20-methyllyrlrapamycin (MaRap), C16(S)-Butylsulfonamidorapamycin (C16-BS-Rap) and C16-(S)-7-methylindolerapamycin (AP21976/C 16-Ai Rap), as shown in Figure 3, in combination with the respective complementary binding domains for each. Other rapamycins/rapalogs include sirolimus and tacrolimus.

The heterodimerization domains of the chimeric protein may be or comprise one the sequences shown as SEQ ID NO: 5 to SEQ ID NO: 9, or a variant thereof.

SEQ ID No 5 - FKBP12 domain

LEGVQVETISPGDGRTFPKRGQTCWHYTGMLEDGKKFDSSRDRNKPFKFMLGKQ

EVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

SEQ ID No 6 - wild-type FRB segment of mTOR

MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN

QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISKLE

SEQ ID No 7 - FRB with T to L substitution at 2098 which allows binding to AP21967 MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLE

SEQ ID No 8 - FRB segment of mTOR with T to H substitution at 2098 and to W at F at residue 2101 of the full mTOR which binds Rapamycin with reduced affinity to wild type

MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN

QAYGRDLMEAQEWCRKYMKSGNVKDLHQAFDLYYHVFRRISKLE

SEQ ID No 9 - FRB segment of mTOR with K to P substitution at residue 2095 of the full mTOR which binds Rapamycin with reduced affinity

MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN

QAYGRDLMEAQEWCRKYMKSGNVPDLTQAWDLYYHVFRRISKLES

Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID No. 5 to 9, provided that the sequences provide an effective dimerization system. That is, provided that the sequences facilitate sufficient co localisation of the two chimeric proteins to allow homodimerization of the two caspase domains.

The“wild-type” FRB domain shown as SEQ ID No. 6 comprises amino acids 2025-21 14 of human mTOR. Using the amino acid numbering system of human mTOR, the FRB sequence of the chimeric protein of the invention may comprise an amino acid substitution at one of more of the following positions: 2095, 2098, 2101.

The variant FRB used in the chimeric protein of the invention may comprise one of the following amino acids at positions 2095, 2098 and 2101 :

2095: K, P, T or A

2098: T, L, H or F

2101 : W or F

Bayle et al (as above) describe the following FRB variants, annotated according to the amino acids at positions 2095, 2098 and 2101 (see Table 1): KTW, PLF, KLW, PLW, TLW, ALW, PTF, ATF, TTF, KLF, PLF, TLF, ALF, KTF, KHF, KFF, KLF. These variants are capable of binding rapamycin and rapalogs to varying extents, as shown in Table 1 and Figure 5A of Bayle et al. The chimeric protein of the invention may comprise one of these FRB variants.

LINKER

A linker may be included to spatially separate the caspase-1 domain and the heterodimerization domain(s).

In the first embodiment of the first aspect of the present invention, the chimeric protein comprises two heterodimerization domains which are held in a configuration such that they cannot heterodimerize with each other in the presence of the CID in a single molecule, but Ht1 on one molecule can heterodimerise with Ht2 on another chimeric molecule having the same heterodimerization domains (Figure 3B).

The molecule may have the general formula

Ht1-L1-HT2-L2-Casp1

The linker L1 may typically be fairly short in length, for example between 0-10 amino acids, such as 2-8, 4-6 or about 5 amino acids in length. The linker (L2) should provide sufficient flexibility so that the catalytic domains can homodimerize, but not so much flexibility that the energic barrier to homodimerization is not overcome (Figure 3B). For example, the linker L2 may be less than 25, less than 20, less than 15, less than 10 or between 5-25 or 10-20 amino acids in length.

Either or both L1 and L2 may be serine-glycine linkers.

In the second embodiment of the first aspect of the present invention, the chimeric protein comprises a single heterodimerization domain, which is capable of heterodimerization with a complementary heterodimerization domain on a second chimeric protein in the presence of a Cl D (Figure 3A). There may be a linker between the dimerization domain and the caspase-1 domain. The linker may be fairly short in length, for example between 0-10 amino acids, such as 2-8, 4-6 or about 5 amino acids in length.

In an alternative configuration, the two heterodimerisation domains may be provided on a single molecule with a long linker (L2), providing a construct having the formula:

Ht1-Casp1-L3-Ht2-Casp1

The HT and Caspl domains may be in either order on each side of the linker.

In this embodiment, the linker L3 may confer sufficient flexibility so the first heterodimerization domain can heterodimerize with the second heterodimerization domain; and so that the caspase domain in the part of the molecule corresponding to the‘first chimeric protein’ can homodimerize with the caspase domain in the part of the molecule corresponding to the‘second chimeric protein’.

In the third embodiment of the first aspect of the invention, Casp is linked to a single heterodimerization domain, and co-expressed with a second molecule which is a fusion of two or more copies of the other heterodimerization domain (Figure 3C). There may be a linker between the dimerization domain and the caspase-1 domain in the first molecul. The linker may be fairly short in length, for example between 0-10 amino acids, such as 2-8, 4-6 or about 5 amino acids in length. The second molecule acts as an interface bringing two or more Casp domains together in the presence of CID. In this molecule, the two or more copies of heterodimerization domains must be fused in such a way to allow approximation of the Caspl domains sufficiently to activate them, which may involve the use of a linker

The interfacing protein may be multimeric, comprising more than two Ht2 domains. For example, it is possible to combine a plurality of Ht2 domains in a single interfacing protein using a multimerising linker such as a coiled coil domain.

In this embodiment the interfacing protein may have the formula Ht2-L2-Ht2, or Ht2 -L2 in which L2 is a coiled-coil domain.

A coiled coil is a structural motif in which two to seven alpha-helices are wrapped together like the strands of a rope. The structure of coiled coil domains is well known in the art. For example as described by Lupas & Gruber (Advances in Protein Chemistry; 2007; 70; 37-38).

Coiled coils usually contain a repeated pattern, hxxhcxc, of hydrophobic (h) and charged (c) amino-acid residues, referred to as a heptad repeat. The positions in the heptad repeat are usually labeled abcdefg, where a and d are the hydrophobic positions, often being occupied by isoleucine, leucine, or valine. Folding a sequence with this repeating pattern into an alpha-helical secondary structure causes the hydrophobic residues to be presented as a 'stripe' that coils gently around the helix in left-handed fashion, forming an amphipathic structure. The most favourable way for two such helices to arrange themselves in the cytoplasm is to wrap the hydrophobic strands against each other sandwiched between the hydrophilic amino acids. Thus, it is the burial of hydrophobic surfaces that provides the thermodynamic driving force for the oligomerization. The packing in a coiled-coil interface is exceptionally tight, with almost complete van der Waals contact between the side-chains of the a and d residues.

Examples of proteins which contain a coiled coil domain include, but are not limited to, kinesin motor protein, hepatitis D delta antigen, archaeal box C/D sRNP core protein, cartilage-oligomeric matrix protein (COMP), mannose-binding protein A, coiled-coil serine-rich protein 1 , polypeptide release factor 2, SNAP-25, SNARE, Lac repressor or apolipoprotein E.

In the fourth embodiment of the first aspect of the present invention, the chimeric protein comprises a single homodimerization domain, which is capable of homodimerization with a complementary dimerization domain on a second chimeric protein in the presence of a CID (Figure 3D). There may be a linker between the homodimerization domain and the caspase-1 domain. The linker may be fairly short in length, for example between 0-10 amino acids, such as 2-8, 4-6 or about 5 amino acids in length.

In an alternative configuration, the two homodimerisation domains may be provided on a single molecule with a long linker (L), providing a construct having the formula:

Hm-Casp1-L-Hm-Casp1

The homodimerization (Hm) and Caspl domains may be in either order on each side of the linker. In this embodiment, the linker L may confer sufficient flexibility so the first homodimerization domain can dimerize with the second homodimerization domain; and so that the caspase domain in the part of the molecule corresponding to the‘first chimeric protein’ can homodimerize with the caspase domain in the part of the molecule corresponding to the‘second chimeric protein’.

CHEMICAL INDUCER OF DIMERIZATION (CID)

The chemical inducer of dimerization (CID) may be any molecule which induces heterodimerization between Ht1 and Ht2 on separate chimeric molecules having the same Ht1 and Ht2 domains.

The CID may be rapamycin or a rapamycin analog (“rapalogs”) which have improved or differing pharmadynamic or pharmacokinetic properties to rapamycin but have the same broad mechanism of action. The CID may be an altered rapamycin with engineered specificity for complementary FKBP12 or FRB - for example as shown in Figure 4. Bayle et al (2006, as above) describes various rapalogs functionalised at C16 and/or C20.

Examples of such rapalogs in the first category include Sirolimus, Everolimus, Temsirolimus and Deforolimus. Examples of rapalogs in the second category include C-20-methyllyrlrapamycin (MaRap); C16(S)-Butylsulfonamidorapamycin (C16-BS-

Rap); C16-(S)-3-mehylindolerapamycin (C16-iRap); and C16-(S)-7-methylindolerapamycin (AP21976/C 16-Ai Rap).

Homodimerisation of the caspase domains in the presence of CID may result in caspase activation which is 2, 5, 10, 50, 100, 1 ,000 or 10,000-fold higher than the caspase activity which occurs in the absence of CID.

Rapamycin is a potent immunsuppressive agent. Analogues of rapamycin (rapalogues) are in every day clinical use. Modern rapalogues have excellent bioavailability and volumes of distribution. Although they are potent immunsuppressive agents, a short dose (to activate a suicide gene) should have minimal side-effects. Further, unlike administration of a mAb, the pharmacological effects of rapamycin and analogues may well be advantageous in clinical scenarios where suicide genes require activation, such as off-tumour toxicity or immune hyperactivation syndromes.

In the chimeric protein shown in Figure 3D, the CID may be the dimerizer drug AP1903, also known as Rimiducid. AP1903 is a lipid-permeable tacrolimus analogue with homodimerizing activity. It homodimerizes an analogue of human protein FKBP12 (Fv) which contains a single acid substitution (Phe36Val). AP1903 binds to wild-type FKBP12 with 1000-fold lower affinity. The structure of AP1903 is shown in Figure 2.

NUCLEIC ACID SEQUENCES

The second aspect of the invention provides a nucleic acid sequence which encodes a chimeric protein according to the invention.

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 second aspect of 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 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 first embodiment of this aspect of the invention there is provided a nucleic acid which encodes a chimeric protein having the formula:

Ht1-Ht2-L-Casp1

wherein

Ht1 is a first heterodimerization domain; and

Ht2 is a second heterodimerization domain.

L is an optional linker;

Caspl is a caspasel domain;

The nucleic acid sequence may encode the chimeric protein sequence shown as SEQ ID No. 3 or a variant thereof.

In the embodiment shown in Figure 3A, the nucleic acid sequences may be provided in the form of a construct which encodes both chimeric proteins.

The construct may encode a polyprotein having the formula:

Ht1-L1-Casp-coexpr-Ht2-L2-Casp

wherein

Ht1 is a first heterodimerization domain;

L1 and L2 are optional linkers which may be the same or different;

Coexpr is a sequence enabling coexpression of the two proteins: Ht1-L1-Casp and Ht2-L2-Casp;

Ht2 is a second heterodimerization domain; and

Casp is a caspasel domain.

Where there are nucleic acid sequences encoding the same or similar sequences, such as the two caspase domains, one of the sequences may be codon wobbled to avoid homologous recombination.

For the embodiment shown in Figure 3C, a nucleic acid construct may be used which encodes a sequence with the following formula:

Ht1-Casp-coexpr-Ht2-Ht2

wherein

Casp is a caspasel domain;

Ht1 is a first heterodimerization domain;

Coexpr is a sequence enabling coexpression of the proteins Ht1-Casp and Ht2-Ht2, such as a cleavage site; and

Ht2 is a second heterodimerisation domain, which heterodimerises with Ht1 in the presence of a chemical inducer of dimerization (CID).

In the sequence encoding the second protein, Ht2-Ht2, one of the sequences encoding Ht2 may be codon wobbled, in order to avoid homologous recombination.

For the embodiment shown as Figure 3D, a nucleic acd sequence may be used which comprises a homodimerization domain, such as FKBP12 having a F36V mutation, and a caspase 1 domain.

NUCLEIC ACID CONSTRUCT

The invention also provides a nucleic acid construct which comprises:

i) a first nucleic acid sequence encoding a chimeric protein which comprises a caspase domain and a heterodimerization domain which comprises an FK506-binding protein (FKBP); and

ii) a second nucleic acid sequence encoding a chimeric protein which comprises a caspase domain and a heterodimerization domain which comprises an FRB domain of mTOR.

The invention also provides a nucleic acid construct which comprises a nucleic acid sequence encoding one or more chimeric protein(s) and a further nucleic acid sequence of interest (NOI). The NOI may, for example encode a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

The nucleic acid sequences may be joined by a sequence allowing co-expression of the two or more nucleic acid sequences. For example, the construct may comprise an internal promoter, an internal ribosome entry sequence (IRES) sequence or a sequence encoding a cleavage site. The cleavage site may be self-cleaving, such that when the polypeptide is produced, it is immediately cleaved into the discrete proteins without the need for any external cleavage activity.

Various self-cleaving sites are known, including the Foot-and-Mouth disease virus (FMDV) 2a self-cleaving peptide, which has the sequence shown as SEQ ID No. 10 or 11 :

SEQ ID No. 10

RAEGRGSLLTCGDVEENPGP.

or

SEQ ID No 11

QCTNYALLKLAGDVESNPGP

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

T-CELL RECEPTOR (TCR)

The T cell receptor or TCR is a molecule found on the surface of T cells that is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The binding between TCR and antigen is of relatively low affinity and is degenerate: many TCR recognize the same antigen and many antigens are recognized by the same TCR.

The TCR is composed of two different protein chains, i.e. it is a heterodimer. In 95% of T cells, this consists of an alpha (a) and beta (b) chain, whereas in 5% of T cells this consists of gamma and delta (g/d) chains. This ratio changes during ontogeny and in diseased states.

When the TCR engages with antigenic peptide and MHC (peptide/M HC), the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.

The nucleic acid construct or vector of the present invention may comprise a nucleic acid sequence encoding a TCR a chain, a TCR b chain, a TCRy chain or a TCR d chain. It may, for example, comprise a nucleic acid sequence encoding a TCR a chain and a nucleic acid sequence encoding a TCR b chain; or a a nucleic acid sequence encoding a TCRy chain or a nucleic acid sequence encoding a TCR d chain. The two nucleic acid sequences may be joined by a sequence enabling co expression of the two TCR chains, such as an internal promoter, an IRES sequence or a cleavage site such as a self-cleaving site.

Nucleic acid constructs or vectors of the present invention may also comprise sequences that affect the expression of the other sequences in the construct, for example different promotor sequences. These sequences may also include a frame-slip motif or a translational readthrough motif, such as the sequences discussed in detail in W02020/025953 (the contents of which are hereby incorporated by reference). These sequences may also be referred to as SKIP sequences or STOPSKIP sequences if a stop codon is included. By including a frame-slip motif or a translational readthrough motif the expression of a downstream transgene (such as RapCaspl) may be reduced.

A frame-slip motif (FSM) may comprise a repeat of uracil, thymine or guanine bases, such as the sequences UUUUUUU or TTTTTTT.

A frame-slip motif may also comprise a stop codon. For example, a FSM may comprise one of the following sequences:

UUUUUUUGA or TTTTTTTGA

U U U U U U U AG or TTTTTTTAG

U U U U U U U AA or TTTTTTTAA

A translational readthrough motif (TRM) may comprise the sequence STOP-CUAG or STOP-CAAUUA, in which "STOP" is a stop codon. For example, a translational readthrough motif may comprise one of the following sequences:

UGA-CUAG or TGA-CTAG

UAG-CUAG or TAG-CTAG

UAA-CUAG or TAA-CTAG

UGA-CAAUUA or TGA-CAATTA

UAG-CAAUUA or TAG-CAATTA

UAA-CAAUUA or TAA-CAATTA

CHIMERIC ANTIGEN RECEPTORS (CARs)

The nucleic acid sequence of interest (NOI) may encode a 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 antigen binding site such as a ligand. 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 which may comprise or associate with an intracellular signalling domain.

Early CAR designs had intracellular signalling domains 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 signalling domains have been constructed: fusion of the intracellular part of a T-cell co-stimulatory molecule to that of Oϋ3z results in second generation receptors which can transmit an activating and co-stimulatory signal simultaneously after antigen recognition. The co-stimulatory domain most commonly used is that of CD28. This supplies the most potent co stimulatory signal - namely immunological signal 2, which triggers T-cell proliferation. Some receptors have also been described which include TNF receptor family endodomains, such as the closely related 0X40 and 41 BB which transmit survival signals. Even more potent third generation CARs have now been described which have intracellular signalling domains 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.

VECTOR

In a third aspect, the present invention provides a vector which comprises a nucleic acid sequence or nucleic acid construct of the invention.

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 and optionally one of more additions nucleic acid sequences of interest (NOI). Such a vector may be used to introduce the nucleic acid sequence(s) or nucleic acid construct(s) into a host cell so that it expresses one or more chimeric protein(s) according to the first aspect of the invention and optionally one or more other proteins of interest (POI). The kit may also comprise a CID.

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 T cell.

The NOI may, for example encode a chimeric antigen receptor or a T-cell receptor, such that when the vector is used to transduce a target cell, the target cell co expresses a chimeric protein and a chimeric antigen receptor or T-cell receptor.

CELL

The present invention also relates to a cell comprising a chimeric protein according to the first aspect of the invention.

The cell may express a chimeric protein having two heterodimerization domains, according to the first embodiment of the first aspect of the present invention.

The cell may express two chimeric proteins; one which comprises a caspasel domain and a heterodimerization domain which comprises an FK506-binding protein (FKBP); and one which comprises a caspasel domain and a heterodimerization domain which comprises an FRB domain of mTOR, according to the second embodiment of the first aspect of the invention.

There is also provided a cell which expresses two proteins:

Ht1-Casp1 and Ht2-Ht2

in which Ht1-Casp is a chimeric protein comprising a caspasel domain (Caspl) and a first heterodimerization domain (Ht1); and Ht2-Ht2 is an interfacing protein comprising two second heterodimerization domains (Ht2)

such that, in the presence of a chemical inducer of dimerization (CID), a pair of the chimeric proteins Ht1-Casp1 interact such that Ht1 from each chimeric protein heterodimerizes with an Ht2 domain from the interfacing protein, causing homodimerization of the two caspase domains (see Figure 3C).

There is also provided a cell which expresses a chimeric protein having a homodimerization domain and a caspase 1 domain (see Figure 3D).

The cell may, for example, be an immune cell such as a T-cell or a natural killer (NK) cell.

The cell may be a stem cell such as a haematopoietic stem cell.

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

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

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

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

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

Two major classes of CD4+ Treg cells have been described— naturally occurring Treg cells and adaptive Treg cells.

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

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

Natural Killer Cells (or NK cells) 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.

Stem cells are undifferentiated cells which can differentiate into specialized cells. In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adult organisms, stem cells and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the specialized cells— ectoderm, endoderm and mesoderm (see induced pluripotent stem cells)— but also maintain the normal turnover of regenerative organs, such as blood, skin, or intestinal tissues.

There are three known accessible sources of autologous adult stem cells in humans:

1. Bone marrow, which requires extraction by harvesting, i.e. drilling into bone.

2. Adipose tissue, which requires extraction by liposuction.

3. Blood, which requires extraction through apheresis, wherein blood is drawn from the donor and passed through a machine that extracts the stem cells and returns other portions of the blood to the donor.

Adult stem cells are frequently used in medical therapies, for example in bone marrow transplantation. Stem cells can now be artificially grown and transformed (differentiated) into specialized cell types with characteristics consistent with cells of various tissues such as muscles or nerves. Embryonic cell lines and autologous embryonic stem cells generated through Somatic-cell nuclear transfer or dedifferentiation can also be used to generate specialised cell types for cell therapy.

Hematopoietic stem cells (HSCs) are the blood cells that give rise to all the other blood cells and are derived from mesoderm. They are located in the red bone marrow, which is contained in the core of most bones.

They give rise to the myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). The hematopoietic tissue contains cells with long term and short-term regeneration capacities and committed multipotent, oligopotent, and unipotent progenitors.

HSCs are a heterogeneous population. Three classes of stem cells exist, distinguished by their ratio of lymphoid to myeloid progeny (L/M) in blood. Myeloid-biased (My-bi) HSC have low L/M ratio (between 0 and 3), whereas lymphoid-biased (Ly-bi) HSC show a large ratio (>10). The third category consists of the balanced (Bala) HSC, whose L/M ratio is between 3 and 10. Only the myeloid-biased and balanced HSCs have durable self-renewal properties.

The chimeric protein-expressing cells of the invention may be any of the cell types mentioned above.

T or NK cells expressing one or more chimeric protein(s) according to the first aspect of the invention may either be created ex vivo either from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).

Alternatively, T or NK cells expressing one or more chimeric protein(s) according to the first aspect of the invention 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 and could act as a therapeutic may be used.

In all these embodiments, chimeric protein(s)-expressing cells are generated by introducing DNA or RNA coding for the, or each, chimeric protein, and optionally an NOI by means such as transduction with a viral vector or transfection with DNA or RNA.

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

The T or NK cell of the invention may be made by:

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

(ii) transduction or transfection of the T or NK cells with one or more a nucleic acid sequence(s) according to the second aspect of the invention.

The present invention also provides a kit which comprises a T or NK cell comprising one or more chimeric protein(s) according to the first aspect of the invention and a CID.

PHARMACEUTICAL COMPOSITION

The present invention also relates to a pharmaceutical composition containing a plurality of cells according to the fourth aspect of 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.

METHODS

The invention also provides a method for making a cell according to the fourth aspect of the invention which comprises the step of transducing or transfecting a cell with a vector according to the third aspect of the invention.

The vector may, for example, be a retroviral or lentiviral vector.

The invention also provides a method for deleting a cell according to the fourth aspect of the invention, which comprises the step of exposing the cells to the CID, such as rapamycin or a rapamycin analog or AP1903. The cells may be exposed to the CID in vivo or in vitro. Deletion of the cell may be caused by apoptosis induced by caspase activation, following CID-induced homodimerization of the caspase domains.

The CID 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 invention also provides a method for preventing and/or treating a pathological immune reaction in a subject caused by administration of a cell according to the fourth aspect of the invention to the subject, which comprises the step of administering a CID, such as rapamycin or a rapamycin analog or AP1903 to the subject.

The pathological immune reaction may be selected from the following group: graft-versus-host disease; on-target, off-tumour toxicity; immune activation syndrome; and lymphoproliferative disorders.

The invention also provides a method for treating or preventing a disease in a subject, which comprises the step of administering a cell according to the fourth aspect of the invention to the subject. The cell may be in the form of a pharmaceutical composition as defined above.

The method may comprises the following steps:

(i) transducing or transfecting a sample of cells isolated from a subject with a vector according to the third aspect of the invention, and

(ii) administering the transduced/transfected cells to a patient.

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 immune 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 methods for treating a disease provided by the present invention may involve monitoring the progression of the disease and monitoring any toxic activity and adjusting the dose of the CID administered to the subject to provide acceptable levels of disease progression and toxic activity.

Monitoring the progression of the disease means to assess the symptoms associated with the disease over time to determine if they are reducing/improving or increasing/worsening.

Toxic activities relate to adverse effects caused by the cells of the invention following their administration to a subject. Toxic activities may include, for example, immunological toxicity, biliary toxicity and respiratory distress syndrome.

In particular the invention provides a method for treating a disease in a subject, which comprises the following steps:

(i) administering a cell according to the fourth aspect of the invention to the subject;

(ii) monitoring the subject for the development of a pathological immune reaction; and

(iii) administering rapamycin or a rapamycin analogue or AP1903 to the subject if the subject shows signs of developing or having developed a pathological immune reaction.

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

The cell may, for example, be for use in haematopoietic stem cell transplantation, lymphocyte infusion or adoptive cell transfer.

The invention also relates to the use of a cell of the present invention in the manufacture of a medicament for the treatment and/or prevention of a disease.

The present invention also provides a CID agent capable inducing dimerizing a chimeric protein according to the first aspect of the invention for use in treating and/or preventing a toxic activity.

The present invention also provides a CID agent for use in activating a pair of caspase domains of chimeric proteins according to the first aspect of the invention in a cell.

The disease to be treated and/or prevented by the cells and methods of the present invention may be an infection, such as a viral infection.

The methods of the invention may also be for the control of pathogenic immune responses, for example in autoimmune diseases, allergies and graft-vs-host rejection.

Where the cells of the invention express a TCR or CAR, they may be useful for the treatment of a cancerous disease, such as bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, prostate cancer and thyroid cancer.

The TCR/CAR-expressing cells of the present invention may be capable of killing target cells, such as cancer cells.

The invention also provides rapamycin or a rapamycin analogue for use in preventing or treating a pathological immune reaction caused by administration of a cell according to the fourth aspect of the invention to a subject.

The cells of the present invention may be used in any cellular therapy in which modified or unmodified cells are administered to a patient. An example of a cellular therapy is adoptive T cell transfer after CD34+ stem cell transplantation. Administering T cells after stem cell transfer helps to accelerate the reconstitution of an immune system in the patient recipient. When a matched related or unrelated donor is not available, or the disease is too aggressive for an extensive donor search, the use of an HLA haploidentical family donor may be effective. Such donors may be parents, siblings, or second-degree relatives. Such infusions may enhance immune recovery and thereby reduce virus infections and eliminate relapsing leukemia cells. However, the coexistence of alloreactive T cells in a donor stem cell graft may cause graft-versus-host disease (GvHD) in which the donor cells react against the recipient, which may progressively damage the skin, gut, liver, and other organs of the recipient.

Other examples of cell therapies include using native cells or cells genetically engineered to express a heterologous gene. These treatments are used for many disorders, including blood disorders, but these therapies may have negative side effects. In another method, immature progenitor cells that can differentiate into many types of mature cells, such as, for example, mesenchymal stromal cells, may be used to treat disorders by replacing the function of diseased cells. There present invention provides a rapid and effective mechanism to remove possible negative effects of donor cells used in cellular therapy.

The present invention provides a method of reducing the effect of graft versus host disease in a human patient following donor T cell transplantation, comprising transfecting or transducing human donor T cells in a donor cell culture with vector according to the present invention; administering the transduced or transfected donor T cells to the patient; subsequently detecting the presence or absence of graft versus host disease in the patient; and administering a chemical inducer of dimerization (CID) to a patient for whom the presence of graft versus host disease is detected. The T cells may be non-allodepleted.

The present invention provides a method of stem cell transplantation, comprising administering a haploidentical stem cell transplant to a human patient; and administering haploidentical donor T cells to the patient, wherein the T cells are transfected or transduced in a haploidentical donor cell culture with a vector according to the invention.

The cells may be non-allodepleted human donor T cells in a donor cell culture.

The present invention also provides a method of stem cell transplantation, comprising administering a haploidentical stem cell transplant to a human patient; and administering non-allodepleted haploidentical donor T cells to the patient, wherein the T cells are transfected or transduced in a haploidentical donor cell culture with vector according to the invention.

The haploidentical stem cell transplant may be a CD34+ haploididentical stem cell transplant. The human donor T cells may be haploidentical to the patient's T cells. The patient may any disease or disorder which may be alleviated by stem cell transplantation. The patient may have cancer, such as a solid tumour or cancer of the blood or bone marrow. The patient may have a blood or bone marrow disease. The patient may have sickle cell anemia or metachromatic leukodystrophy.

The donor cell culture may be prepared from a bone marrow sample or from peripheral blood. The donor cell culture may be prepared from donor peripheral blood mononuclear cells. In some embodiments, the donor T cells are allodepleted from the donor cell culture before transfection or transduction. Transduced or transfected T cells may be cultured in the presence of IL-2 before administration to the patient.

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 - Comparing the activity to RaoCasp9 and RaoCaspl suicide genes

The RapCasp9 suicide gene is described in WO2016/135470 and has the sequence shown as SEQ ID No. 12.

SEQ ID No. 12 (FRB-FKBP12-L3-dCasp9)

<- FRB- MASRILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR

- FRB- XL1-X—FKBP12 - DLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKLEYSGGGSLEGVQVETISPGDGR

- FKBP12 - TFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK


LTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGGSGGGGSGGGGSGVDGFGDVGA

- dCasp9- LESLRGNADLAYILSMEPCGHCLI INNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMV

- dCasp9- EVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVE

- dCasp9- KIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQE

- dCasp9- GLRTFDQLDAISSLPTPSDI FVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQ

- dCasp9- >

SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS

The RapCaspl suicide gene has the sequence shown as SEQ ID No. 3 above.

T-cells were transduced a vector coding for the respective rapCasp9 together with eGFP. The T-cells were intentionally only partially transduced so within the cell culture a proportion of cells remained non-transduced to act as an internal negative control.

Cells were treated with different concentrations of rapamycin (0.1 , 1 , 10, 100 and 1000nM) or were left untreated.

Upon 24h incubation period, the cells were stained with 7-AAD and Annexin V and the percentage of live cells expressing GFP (therefore, the suicide construct) was assessed by flow-cytometry. By gating on the live cells, and interrogating the population of cells expressing fluorescent proteins, survival of the transduced and non-transduced populations could be measured. The experiment was run with T cells from three different Donors and the results are shown in Figures 4 and 5.

It was found that Rapcasp9 killed transfected cells at an efficiency of about 50-60% at concentrations of rapamycin above and including 1nM. However, Rapcaspl showed superior killing to Rapcasp9, both in terms of percentage of killing and sensitivity to rapamycin. Rapcaspl give a killing efficiency of about 90% at concentrations of rapamycin above and including 1nM and a killing efficiency of more than 60% (averaged over the three donors) at a rapamycin concentration of 0.1 nM (Figure 5).

Example 2 - Control of RapCaspl with STOPSKIP

RapCaspl is shown in Example 1 to be a sensitive suicide gene. The sensitivity of RapCaspl was further investigated via the use of a STOPSKIP sequence, which are discussed in detail in W02020/025953 (the contents of which are hereby incorporated by reference).

We introduced a STOP (TGA), SKIP (CTAG), or STOPSKIP (TGACTAG) sequence between the marker gene eGFP and RapCaspl to reduce the expression level of RapCaspl Our aim was to determine whether the reduction of RapCaspl level increases the MFI and whether it abrogates sensitivity to induce cell death.

The data acquired from the STOP codon introduction in RapCaspl are shown in Figure 6, wherein cells bearing the suicide genes were cultured for 24h in the presence of a rapamycin concentration range. The figure shows the MFI normalised to the OnM rapamycin condition for each suicide gene.

Despite the reduced expression of RapCaspl in the STOPSKIP_RapCasp1 , the sensitivity of the suicide gene was unaffected (Figure 6). At the lowest concentration of rapamycin, 0.1 nM, STOPSKIP_RapCasp1 was more sensitive than RapCasp9 with a decrease in normalised MFI of 2.4-fold. However, STOPSKIP_RapCasp1 was comparable to RapCaspl with normalised MFI of 0.152 and 0.116, respectively.

Example 3 - Role of Apoptosis versus Pyroptosis

Caspase-1 initiates cell death by the cleavage of Gasdermin D (GsdmD), which consequently leads to death by pyroptosis. In the absence of GsdmD, caspase-1 induced caspase-3 dependent apoptosis (Tsuchiya et ai, 2019 Nat Commun 10, 1-19). In order to determine whether the RapCaspl suicide initiates cell death in T-cells by pyroptosis or apoptosis, cells undergoing RapCaplinduced cell death are interrogated for the presence of cleaved GsdmD or caspase3 by western blot.

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.