FIELD OF THE INVENTION
[0001] 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
[0002] 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.
[0003] 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
[0004] 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).
[0005] 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.
[0006] 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.
[0007] In an alternative approach, executioner caspases can be activated by small molecules
using a complex strategy which involves introduction of tobacco etch virus (TeV) proteolysis
sites into Caspase 3 or 6 or 7 and co-expression with a split TEV protease which is
recombined in the presence of rapamycin (
Morgan et al (2014) Methods Enzymol. 544:179-213). This is an unsatisfactory strategy for a clinically useful suicide switch for a
number of reasons: firstly three separate proteins are required which is highly complex:
the modified caspase, and the two components of the split TeV protease respectively;
secondly, TeV components are xenogeneic and likely immunogenic; finally, this strategy
only activates protease sensitive caspase molecules which are downstream and less
sensitive than apical caspases.
[0008] A suicide gene based on CID activation of FAS has been described (
Amara et al (1999) Hum. Gene Ther. 10, 2651-2655). This also depends on this CID for activation, and since it does not directly activate
the apoptosis cascade, escape (through FAS resistance) is possible.
[0009] A homodimerization system based on a standard pharmaceutical which replaces the need
for an experimental CID would be an attractive alternative. However, no homodimerizing
small molecule pharmaceuticals are available.
[0010] Other suicide genes have been proposed for instance full-length CD20 when expressed
on a T-cell can render T-cells susceptible to lysis by the therapeutic anti-CD20 antibody
Rituximab (
Introna, M. et al. (2000) Hum. Gene Ther. 11, 611-620). Further suicide genes have also been described on this theme of antibody recognition,
for example: RQR8 renders T-cells susceptible to CD20 but is more compact than the
full-length CD20 molecule (
Philip, B. et al. (2014) Blood doi:10.1182/blood-2014-01-545020); a truncated version of EGFR (huEGFRt) renders cells susceptible to lysis by anti-EGFR
mAbs (
Wang, X. et al. (2011) Blood 118, 1255-1263); and a myc epitope tag expressed on a cell surface leaves cells susceptible to lysis
with an anti-myc antibody (
Kieback et al (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 623-628). A major limitation of these antibody dependent approaches is their dependence on
bioavailability of a therapeutic antibody at high local concentrations to act. It
is known for instance that lytic antibodies are not particularly effective against
bulky disease and a limitation of antibody based suicide genes is that cells resident
where high antibody concentrations are not reached would escape. Further, in certain
situations: for instance a severe macrophage activation syndrome or cytokine storm
induced by a CAR T-cells; the additional immune activation induced by a monoclonal
antibody may be deleterious to the clinical situation activation of the suicide gene
is trying to treat.
[0011] US 2004/040047 discloses artificial death switches (ADSs) based on chemically induced dimerization
of cysteine proteases, caspase -1 (ICE) and caspase-3 (YAMA).
[0013] There is thus a need for an alternative suicide gene which is not associated with
the disadvantages mentioned above.
DESCRIPTION OF THE FIGURES
[0014]
Figure 1 - Cartoons showing different approaches to RapCasp9. (a) Double construct where two
molecules are expressed separately. Each molecule has the catalytic domain of Casp9
fused with either FKBP12 or FRB respectively. (b) Single construct where FKBP12 and
FRB are directly fused together and then fused to the catalytic domain of Casp9 by
a flexible linker. Self heterodimerization should not be possible in this orientation.
(c) Single construct where the catalytic domain of Caspase 9 is flanked by FRB and
FKBP12. Here, self heterodimerization may occur so this iteration is not expected
to function well. (d) Double construct where the catalytic domain of Caspase 9 is
fused to FKBP12 and a separate small protein which is a fusion of two copies of FRB
is co-expressed.
Figure 2 - Demonstration that it is possible to activate Caspase 9 with a heterodimerizer.
T-cells were either transduced with eGFP alone (Figure 2a), or cotransduced with FKBP12-dCasp9
(co-expressing eGFP) and FRB-dCasp9 (co-expressing eBFP2) (Figure 2b). T-cells were
intentionally only partially transduced so that the non-transduced T-cells would act
as internal controls. T-cells were then exposed to decreasing concentrations of Rapamycin.
After 48 hours, cells were stained with Annexin-V and 7AAD and analysed by flow cytometry
looking at the proportion of live cells which were expressing fluorescent proteins.
T-cells expressing both eGFP and eBFP2 were very effectively deleted even in the presence
of the lowest concentration of Rapamycin.
Figure 3 - Function of RapCasp9 variants. T-cells were transduced with (a) eGFP alone; (b)
double transduced with FKBP12-Casp9 and FRB-Casp9 co-expressed with eGFP and eBFP2
respectively; (c) transduced with FRB-FKBP12-Casp9 and (d) transduced with FRB-Casp9-FKBP12
and (e) FBP12-Casp9-2A-FRB-FRBw. Only a proportion of cells were transduced, the negative
cells acted as an internal negative control. T-cells were exposed for 48 hours to
2.5nM Rapamycin. T-cells were then stained with Annexin-V and 7AAD and analysed by
flow-cytometry. eGFP vs eBFP2 is shown on live cells as determined by Annexin-V and
7AAD staining.
Figure 4 - 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 5 - Summary of the constructs tested in Example 3.
Figure 6 - Summary of gating strategy for Example 3.
Figures 7, 8 and 9 - Study showing the killing of Jurkat cells transfected with the constructs shown
in Figure 5 after incubation with various concentrations of rapamycin.
Figure 10 - Graph to summarise the FACS data shown in Figures 7, 8 and 9.
Figure 11 - Graph comparing Jurkat cell killing in the presence of rapamycin vs temsirolimus.
SUMMARY OF ASPECTS OF THE INVENTION
[0015] The present inventors have developed a new suicide gene, which dimerizes in the presence
of a chemical inducer of dimerization (CID) such as rapamycin or a rapamycin analogue.
[0016] Rapamycin and rapamycin analogues induce heterodimerisation by generating an interface
between the FRB domain of mTOR and FKBP12. This association results in FKBP12 blocking
access to the mTOR active site inhibiting its function. While mTOR is a very large
protein, the precise small segment of mTOR required for interaction with Rapamycin
is known and can be used.
[0017] The present inventors have shown that it is possible to use the heterodimerization
mediated by rapamycin to induce homodimerization of a caspase. In particular, they
have surprisingly shown that it is possible to create a multi-domain molecule, which
includes (i) the FRB domain of mTOR; (ii) FKBP12; and (iii) a caspase, and use heterodimerization
between the FRB domain of one copy of the molecule and the FKB12 domain of another
copy of the molecule to cause homodimerization of the caspase domains.
[0018] Thus in a first aspect of the invention, the present invention provides a soluble
chimeric protein having the formula:
Ht1-Ht2-Casp
wherein
Casp is a caspase domain;
Ht1 is a first heterodimerization domain; and
Ht2 is a second heterodimerization domain
and wherein, in the presence of a chemical inducer of dimerization (CID), an identical
pair of the soluble chimeric proteins interact such that Ht1 from one soluble chimeric
protein heterodimerizes with Ht2 from the other soluble chimeric protein, causing
homodimerization of the two caspase domains, wherein one heterodimerization domain
comprises an FK506-binding protein (FKBP) and the other heterodimerization domain
comprises an FRB domain of mTOR, and wherein the CID is rapamycin or a rapamycin analog.
[0019] The configuration is such that Ht1 does not heterodimerize to any significant extent
with Ht2 within the same chimeric protein.
[0020] The caspase domain may comprise an initiator caspase selected from the following
group: caspase-8, caspase-9 and caspase-10, or an executioner caspase selected from
caspase-3 and caspase-7.
[0021] In the present invention, where one heterodimerization domain comprises an FK506-binding
protein (FKBP) and the other heterodimerization domain comprises an FRB domain of
mTOR and the CID is rapamycin or a derivative thereof, then concentrations of less
that 5nm, for example 1-3nm or about 1nm may be used in order to cause homodimerisation
of the two caspase domains.
[0022] The chimeric protein may comprise a caspase domain fused to FKBP12 and is the interfacing
protein may be a fusion of two or more FRB domains. These two or more FRB domains
act as an interface, brining two FKBP12-Casp domains together.
[0023] In a second aspect, the present invention provides a nucleic acid sequence which
encodes a soluble chimeric protein according to the first aspect of the invention.
[0024] The nucleic acid may be in the form of a nucleic acid construct, which comprises
a plurality of nucleic acid sequences. For example, the construct may comprise one
or more nucleic acid sequence(s) according to the second aspect of the invention and
a nucleic acid sequence encoding a T-cell receptor (TCR) or chimeric antigen receptor
(CAR).
[0025] Ht1 may comprise an FK506-binding protein (FKBP) and Ht2 may comprise an FRB domain
of mTOR.
[0026] The nucleic acid construct may also comprise a nucleic acid sequence encoding a T-cell
receptor (TCR) or chimeric antigen receptor (CAR).
[0027] In a third aspect, the present invention provides a vector which comprises a nucleic
acid sequence or a nucleic acid construct according to the second aspect of the invention.
[0028] The vector which may also comprise a nucleotide of interest, such as a nucleotide
sequence encoding 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 soluble
chimeric protein according to the first aspect of the invention and a chimeric antigen
receptor or T-cell receptor.
[0029] In a fourth aspect the present invention provides a cell which expresses a soluble
chimeric protein according to the first aspect of the invention.
[0030] The cell may comprise a nucleic acid sequence or construct according to the second
aspect of the invention.
[0031] The cell may, for example, be a haematopoietic stem cell, a lymphocyte or a T cell.
[0032] There is also provided a method for making a cell according to the fourth aspect
of the invention which comprises the step of transducing or transfecting a cell ex
vivo with a vector according to the third aspect of the invention.
[0033] There is also provided a method for deleting a cell according to the fourth aspect
of the invention, which comprises the step of exposing the cells to a chemical inducer
of dimerization (CID) in vitro, wherein the CID is rapamycin or a rapamycin analog.
[0034] There is also provided a cell according to the present invention for use in a method
for treating a disease in a subject.
[0035] 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 third aspect of the invention, and
- (ii) administering the transduced/transfected cells to a patient.
[0036] The method may be for treating cancer.
[0037] Described herein is a method for preventing and/or treating an 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 rapamycin
or a rapamycin analog to the subject.
[0038] 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.
[0039] The method for treating a disease in a subject may comprise 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 analog to the subject if the subject
shows signs of developing or having developed a pathological immune reaction.
[0040] There is also provided a cell according to the present invention for use in a method
for treating a disease in a subject, wherein the method involves haematopoietic stem
cell transplantation, lymphocyte infusion or adoptive cell transfer.
[0041] 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.
DETAILED DESCRIPTION
CHIMERIC PROTEIN
[0042] The present invention relates to a soluble 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) which is rapamycin or a
rapamycin analogue.
[0043] The soluble chimeric protein has the formula:
Ht1-Ht2-Casp
in which
Casp is a caspase domain;
Ht1 is a first heterodimerization domain; and
Ht2 is a second heterodimerization domain
and wherein, in the presence of a chemical inducer of dimerization (CID), an identical
pair of the soluble chimeric proteins interact such that Ht1 from one soluble chimeric
protein heterodimerizes with Ht2 from the other soluble chimeric protein, causing
homodimerization of the two caspase domains, wherein one heterodimerization domain
comprises an FK506-binding protein (FKBP) and the other heterodimerization domain
comprises an FRB domain of mTOR, and wherein the CID is rapamycin or a rapamycin analog.
[0044] The chimeric protein may have the formula:
Ht1-Ht2-L-Casp
in which Casp, Ht1 and Ht2 are as defined above and L is an optional linker.
[0045] 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.
[0046] 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.
[0047] The chimeric protein may comprise the sequence shown as SEQ ID No. 1.
SEQ ID No. 1 (FRB-FKBP12-L3-dCasp9)

[0048] In the above sequence "FKBP12" refers to the sequence of FKBP12; "dCasp9" refers
to the catalytic domain of Casp9; "L1" is a one repeat linker; "FMD-2A" is a Foot
and mouth disease 2A like peptide ERAV; "FRB" is the FRB domain of mTOR; "L3" is a
two repeat linker; and "FRBw" is codon wobbled FRB
[0049] Described herein is a "two-molecule" suicide gene system, in which the CID is rapamycin
or a rapamycin analogue.
[0050] Thus, described herein is i) a chimeric protein which comprises a caspase domain
and a heterodimerization domain which comprises an FK506-binding protein (FKBP12);
and ii) a chimeric protein which comprises a caspase domain and a heterodimerization
domain which comprises an FRB domain of mTOR.
[0051] 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).
[0052] Herein, the chimeric protein described herein may comprise the sequence shown as
SEQ ID No. 2 or 3.
SEQ ID No. 2 (FKBP12-dCasp9)

SEQ ID No. 3 (FRB-dCasp9)


[0053] Described herein is an alternative "two molecule" approach, with a smaller footprint.
Here, Ht1 is fused with Caspase, and a second molecule comprises of Ht2-Ht2 fusion
is co-expressed. In the prescence 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.
SEQ ID No. 4 (FKBP12-dCasp9-2A-FRB-FRBw)

[0054] In the above sequence: "FKBP12" refers to FKBP12; "dCasp9" is the catalytic domain
of Casp9; "L1" is a one repeat linker; "FMD-2A" is a Foot and mouth disease 2A like
peptide ERAV; "FRB" is the FRB domain of mTOR; "L2" is a two repeat linker; and "FRBw"
is codon wobbled FRB.
CASPASE
[0055] Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed
proteases are a family of cysteine proteases that play essential roles in apoptosis.
[0056] Twelve caspases have been identified in humans. 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.
[0057] The caspase domain of the chimeric protein of the first aspect of the present invention
may comprise an initiator caspase selected from caspase-2; caspase-8, caspase-9 and
caspase-10; or an executioner caspase selected from caspase-3, caspase-6 and caspase-7.
[0058] In particular, the caspase domain of the chimeric protein of the first aspect of
the present invention may comprise caspase-9. Caspase 9 is the key initiator caspase
so its activation is a very sensitive trigger for apoptosis induction. Furthermore,
homodimerization is all that is required for activation, rather than homodimerization
and proteolytic cleavage.
[0059] Full length caspase-9 has the sequence shown as SEQ ID No. 5.
SEQ ID No. 5 (Caspase-9)

[0060] Caspase-9 may be truncated, for example to remove the caspase recruitment domain.
Truncated Caspase-9 is shown as SEQ ID No. 6
SEQ ID No. 6 (truncated Caspase-9, lacking the CARD domain)

[0061] The chimeric protein of the first aspect of the invention may comprise SEQ ID No.
5 or SEQ ID No. 6 or a fragment or a variant thereof which retains the capacity to
homodimerize and thus trigger apoptosis.
[0062] A variant caspase-9 sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence
identity to SEQ ID No. 5 or 6.
[0063] The percentage identity between two polypeptide sequences may be readily determined
by programs such as BLAST which is freely available at
http://blast.ncbi.nlm.nih.gov.
[0064] 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.
HETERODIMERIZATION DOMAINS
[0065] 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.
[0066] In the context of the present invention one of the heterodimerization domains (Ht1
or Ht2) comprises FRB, or a variant thereof and the other heterodimerization domain
(Ht2 or Ht1) comprises FKBP12 or a variant thereof.
[0067] 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.
[0068] 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.
[0069] 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/C16-AiRap), as shown in
Figure 3, in combination with the respective complementary binding domains for each.
Other rapamycins/rapalogs include sirolimus and tacrolimus.
[0070] The heterodimerization domains of the chimeric protein may be or comprise one the
sequences shown as SEQ ID NO: 7 to SEQ ID NO: 11, or a variant thereof.
SEQ ID No 7 - FKBP12 domain

SEQ ID No 8 - wild-type FRB segment of mTOR

SEQ ID No 9 - FRB with T to L substitution at 2098 which allows binding to AP21967

SEQ ID No 10 - 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

SEQ ID No 11 - FRB segment of mTOR with K to P substitution at residue 2095 of the
full mTOR which binds Rapamycin with reduced affinity

[0071] Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity
to SEQ ID No. 7 to 11, provided that the sequences provide an effective dimerization
system. That is, provided that the sequences facilitate sufficient colocalisation
of the two chimeric proteins to allow homodimerization of the two caspase domains.
[0072] The "wild-type" FRB domain shown as SEQ ID No. 8 comprises amino acids 2025-2114
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.
[0073] 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
[0074] 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
[0075] A linker may be included to spatially separate the caspase domain and the heterodimerization
domain(s).
[0076] In 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
1B). In a design where Ht1 and Ht2 flank the Caspase domain (Ht1-Casp-Ht2), activation
was inferior to designs where Ht1 and Ht2 were linked together, indicating the importance
of preventing non-productive binding of Ht1 and Ht2 from a single molecule to a single
CID.
[0077] In this embodiment, the linker (L1) 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 1). For example, the linker may
be less than 15, less than 10 or between 5-15 or 5-10 amino acids in length.
[0078] As described herein, a chimeric protein may comprise a single heterodimerization
domain, which is capable of heterodimerization with a complementary heterodimerization
domain on a second chimeric protein in the presence of a CID.
[0079] In an alternative configuration described herein for reference, the two heterodimerisation
domains may be provided on a single molecule with a long linker (L2), providing a
construct having the formula:
Ht1-Casp1-L2-Ht2-Casp2
[0080] The HT and Casp domains may be in either order on each side of the linker.
[0081] Described herein, the linker L2 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'.
[0082] Described herein, Casp is fused to a single heterodimerization domain, but a second
molecule which is a fusion of two or more copies of the other heterodimerization domain.
The two molecules may be co-expressed. In this case, the second molecule acts as an
interface bringing two or more Casp domains together in the presence of CID. In this
case, the two or more copies of heterodimerization domains must be fused in such a
way to allow approximation of the Casp9 domains sufficiently to activate them.
[0083] The interfacing protein described herein 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.
[0084] Described herein, the interfacing protein may have the formula Ht2-L2-Ht2, or Ht2
- L2 in which L2 is a coiled-coil domain.
[0086] 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.
[0087] 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.
CHEMICAL INDUCER OF DIMERIZATION (CID)
[0088] The chemical inducer of dimerization (CID) for use in the present invention is rapamycin
or a rapamycin analog which induces heterodimerization between Ht1 and Ht2 on separate
chimeric molecules having the same Ht1 and Ht2 domains.
[0089] The CID is 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.
[0090] 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/C16-AiRap).
[0091] 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.
[0092] 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.
NUCLEIC ACID SEQUENCES
[0093] The second aspect of the invention provides a nucleic acid sequence which encodes
a chimeric protein according to the invention.
[0094] As used herein, the terms "polynucleotide", "nucleotide", and "nucleic acid" are
intended to be synonymous with each other.
[0095] 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.
[0096] 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.
[0097] 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.
[0098] In the present invention there is provided a nucleic acid which encodes a chimeric
protein having the formula:
Ht1-Ht2-L-Casp
wherein
Ht1 is a first heterodimerization domain; and
Ht2 is a second heterodimerization domain.
L is an optional linker;
Casp is a caspase domain;
[0099] The nucleic acid sequence may encode the chimeric protein sequence shown as SEQ ID
No. 1 or a variant thereof.
[0100] For example the nucleotide sequence may comprise the sequence shown as SEQ ID No.
12
SEQ ID No. 12 (FRB-FKBP12-L3-Casp9)

[0101] Also described herein is a nucleic acid sequence encoding a chimeric protein having
the formula: Ht1-L-Casp
wherein
Ht1 is a heterodimerization domain.
L is an optional linker; and
Casp is a caspase domain;
[0102] Described herein is a nucleic acid sequence that encodes the chimeric protein sequence
shown as SEQ ID No. 2 or 3 or a variant thereof.
[0103] For example the nucleotide sequence may comprise the sequence shown as SEQ ID No.
13 or 14
SEQ ID No. 13 (FKBP12-dCasp9)

SEQ ID No. 14. (FRB-dCasp9)

[0104] Herein, the nucleic acid sequences may be described in the form of a construct which
encodes both chimeric proteins.
[0105] The construct described herein may encode a polyprotein having the formula:
Ht1-L2-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 caspase domain.
[0106] 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.
[0107] Described herein, a nucleic acid sequence is described which encodes a sequence with
the following formula:
Ht1-Casp-coexpr-Ht2-Ht2
wherein
Casp is a caspase 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).
[0108] 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.
[0109] The nucleic acid construct described herein may have the sequence shown as SEQ ID
No. 15.
SEQ ID No. 15 (FKBP12-Casp9-2A-FRB-FRBw)

[0110] Nucleic acid sequences with a high degree of similarity, such as the caspase sequence(s)
or FRB sequences may be codon wobbled to avoid recombination.
NUCLEIC ACID CONSTRUCT
[0111] Described herein is 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.
[0112] The invention also provides a nucleic acid construct which comprises a nucleic acid
sequence encoding one or more soluble chimeric protein(s) of the invention 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).
[0113] 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.
[0114] 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. 16 or
17:
SEQ ID No. 16
RAEGRGSLLTCGDVEENPGP.
or
SEQ ID No 17
QCTNYALLKLAGDVESNPGP
[0115] 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)
[0116] 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.
[0117] 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 (α) and beta (β) chain, whereas in 5% of
T cells this consists of gamma and delta (γ/δ) chains. This ratio changes during ontogeny
and in diseased states.
[0118] When the TCR engages with antigenic peptide and MHC (peptide/MHC), 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.
[0119] The nucleic acid construct or vector of the present invention may comprise a nucleic
acid sequence encoding a TCR α chain, a TCR β chain, a TCRγ chain or a TCR δ chain.
It may, for example, comprise a nucleic acid sequence encoding a TCR α chain and a
nucleic acid sequence encoding a TCR β chain; or a a nucleic acid sequence encoding
a TCRγ chain or a nucleic acid sequence encoding a TCR δ chain. The two nucleic acid
sequences may be joined by a sequence enabling coexpression of the two TCR chains,
such as an internal promoter, an IRES sequence or a cleavage site such as a self-cleaving
site.
CHIMERIC ANTIGEN RECEPTORS (CARs)
[0120] The nucleic acid sequence of interest (NOI) may encode a chimeric antigen receptor
(CAR).
[0121] 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 IgG1. More compact spacers can suffice e.g. the stalk from CD8α and even
just the IgG1 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.
[0122] Early CAR designs had intracellular signalling domains derived from the intracellular
parts of either the γ chain of the FcεR1 or CD3ζ. 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 CD3ζ
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 OX40 and 41BB 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.
[0123] 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
[0124] In a third aspect, the present invention provides a vector which comprises a nucleic
acid sequence or nucleic acid construct of the invention.
[0125] The present invention also provides a vector 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).
[0126] 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.
[0127] The vector may be capable of transfecting or transducing a T cell.
[0128] 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
[0129] The present invention also relates to a cell comprising a chimeric protein according
to the first aspect of the invention.
[0130] The cell expresses a chimeric protein having the two heterodimerization domains,
according to the first aspect of the present invention.
[0131] Also described herein is a cell that expresses two chimeric proteins; one which comprises
a caspase domain and a heterodimerization domain which comprises an FK506-binding
protein (FKBP); and one which comprises a caspase domain and a heterodimerization
domain which comprises an FRB domain of mTOR.
[0132] There is also described a cell which expresses two proteins:
Ht1-Casp and Ht2-Ht2 in which Ht1-Casp is a chimeric protein comprising a caspase
domain (Casp) 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-Casp9 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 1d).
[0133] The cell may, for example, be an immune cell such as a T-cell or a natural killer
(NK) cell.
[0134] The cell may be a stem cell such as a haematopoietic stem cell.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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 autoreactive
T cells that escaped the process of negative selection in the thymus.
[0140] Two major classes of CD4+ Treg cells have been described - naturally occurring Treg
cells and adaptive Treg cells.
[0141] 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.
[0142] Adaptive Treg cells (also known as Tr1 cells or Th3 cells) may originate during a
normal immune response.
[0143] 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
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] The chimeric protein-expressing cells of the invention may be any of the cell types
mentioned above.
[0152] 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 (1
st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral
blood (2
nd party), or peripheral blood from an unconnected donor (3
rd party).
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] Described herein is 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
[0158] Described herein is 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
[0159] 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 ex
vivo with a vector according to the third aspect of the invention.
[0160] The vector may, for example, be a retroviral or lentiviral vector.
[0161] 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
rapamycin or a rapamycin analog,
in vitro. Deletion of the cell may be caused by apoptosis induced by caspase activation, following
CID-induced homodimerization of the caspase domains.
[0162] 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.
[0163] Described herein is 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 to the subject.
[0164] 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.
[0165] Described herein is 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] The methods for treating a disease described herein 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.
[0170] 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.
[0171] 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.
[0172] In particular described herein is 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 to the subject if the subject
shows signs of developing or having developed a pathological immune reaction.
[0173] The present invention provides a cell of the present invention for use in treating
a disease.
[0174] The cell may, for example, be for use in haematopoietic stem cell transplantation,
lymphocyte infusion or adoptive cell transfer.
[0175] Described herein is the use of a cell of the present invention in the manufacture
of a medicament for the treatment and/or prevention of a disease.
[0176] Described herein is 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.
[0177] Described herein is 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.
[0178] The disease to be treated and/or prevented by the cells of the present invention
may be an infection, such as a viral infection.
[0179] The methods described herein may also be for the control of pathogenic immune responses,
for example in autoimmune diseases, allergies and graft-vs-host rejection.
[0180] 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.
[0181] The TCR/CAR-expressing cells of the present invention may be capable of killing target
cells, such as cancer cells.
[0182] Described herein is 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.
[0183] 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.
[0184] 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. The present invention provides
a rapid and effective mechanism to remove possible negative effects of donor cells
used in cellular therapy.
[0185] Described herein is 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.
[0186] Described herein is 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.
[0187] The cells may be non-allodepleted human donor T cells in a donor cell culture.
[0188] Described herein is 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.
[0189] 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.
[0190] 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.
[0191] The invention will now be further described by way of Examples.
EXAMPLES
Example 1 - Production of T-cells expressing chimeric proteins
[0192] T-cells were transduced with the different constructs. For the two-molecule rapCasp9
(Figure 1a), T-cells were transduced with two vectors: one coding for FKBP12-Casp9
co-expressed with the green fluorescent protein eGFP by means of an internal ribosome
entry sequence, and the other coding for FRB-Casp9 co-expressed with the blue fluorescent
protein eBFP2. For the one molecule rapCasp9 (Figure 1b), T-cells were transduced
with just one vector coding for the respective rapCasp9 which are co-expressed eGFP.
A construct which provided FKB12-Casp9 and FRB-FRBw was encoded in a tri-cistronic
cassette whereby the FKBP12-Casp9 and FRB-FRBw were co-expressed using a FMD-2A like
peptide and eGFP was co-expressed with an IRES. 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. As a further control, T-cells were transduced
with a vector which codes for eGFP alone to exclude non-specific effects of Rapamycin
on transduced cells.
Example 2 - Testing deletion of chimeric protein-expressing cells with rapamvcin
[0193] T-cells were exposed to different concentrations of Rapamycin and incubated for 48
hours. Following this, T-cells were stained with Annexin-V and 7AAD and analysed 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 clearly measured. The dual FRB-Casp9 and FKBP12-Casp9 approach resulted in
effective deletion of only double positive cells as expected. The FKBP12-FRB-Casp9
construct resulted in effective deletion of single positive cells. The FKBP12-Casp9-FRB
construct resulted in minimal deletion. The FKBP12-Casp9/FRB-FRBw resulted in effective
deletion of single positive cells. The control resulted in no specific deletion (Figures
2 and 3).
Example 3 - Testing an expanded set of constructs
[0194] The constructs shown in Figure 5 we generated and transduced into Jurkat cells. Transduced
cells were mixed with non-transduced (NT) cells to have both construct positive and
negative cells within the population. Rapamycin was added at a concentration of 0,
1, 10, 100 and 1000 nM and the cells were incubated for 24h. Following harvesting,
the cells were stained with PI and annexin V and analysed by FACS. The results are
shown in Figures 6 to 9 and summarised in Figure 10.
[0195] The construct which has a configuration as defined according to the invention, namely
MP20244, performed very well in this assay, giving very efficient killing of transfected
cells at all concentrations of rapamycin above and including 1nM.
[0196] The pair of constructs having a configuration as defined according to MP20206 and
MP20207 also performed very well, giving very efficient killing of transfected cells
at all concentrations of rapamycin above and including 1nM.
[0197] The construct having a configuration as defined according to MP20265, also performed
well, giving some killing at 1nM rapamycin and efficient killing at concentrations
of rapamycin of 10nM and above.
[0198] Constructs having a configuration as defined according to MP20263, MP20264 and MP21067
prefomed well at 1nM rapamycin, but at higher concentrations of rapamycin killing
was less efficient.
Example 4 - Testing the constructs with temsirolimus
[0199] In an equivalent experiment to the one described in Example 3, cells expressing the
constructs shown in Figure 5 were treated with both rapamycin and temsirolimus, a
rapamycin analogue.
[0200] As with the experiment outlined in Example 3, the transduced Jurkat cells were mixed
with non-transduced (NT) giving a population containing both cells expressing the
constructs and non-transduced cells.
[0201] Cells at a concentration of with 2×10
5 cells per well were either left untreated, or were treated with rapamycin or temsirolimus
at the following concentrations: 0.01, 0.1, 1, 10nM (of either rapamycin or temsirolimus)
[0202] Cells were incubated for 24h and were then stained for Annexin V and PI and were
analysed by FACS. The results are shown in Figure 11.
[0203] An equivalent pattern of Jurkat cell killing was observed with the various constructs
shown in Figure 5 in the presence of temsirolimus as had been previously observed
in the presence of rapamycin.
[0204] In particular, the construct MP20244, which has a configuration as defined according
to the invention; and the pair of constructs MP20206 and MP20207 both performed well.
Both gave efficient killing of transfected cells at all concentrations of temsirolimus
above and including 1nM.
SEQUENCE LISTING
[0205]
<110> UCL Business PLC Syncona Partners LLP
<120> CHIMERIC PROTEIN
<130> P106629PCT
<150> GB 1503133.9
<151> 2015-02-24
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 517
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<213> Artificial Sequence
<220>
<223> Chimeric protein FRB-FKBP12-L3-dCasp9
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<211> 402
<212> PRT
<213> Artificial Sequence
<220>
<223> Chimeric protein FKBP12-dCasp9
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Ala Ser
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<213> Artificial Sequence
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<223> Chimeric protein FRB-dCasp9
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<210> 4
<211> 651
<212> PRT
<213> Artificial Sequence
<220>
<223> Chimeric protein FKBP12-dCasp9-2A-FRB-FRBw
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<210> 5
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<212> PRT
<213> Homo sapiens
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<210> 6
<211> 282
<212> PRT
<213> Homo sapiens
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<210> 7
<211> 108
<212> PRT
<213> Artificial Sequence
<220>
<223> FKBP12 domain
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<210> 8
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<213> Artificial Sequence
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<223> wild-type FRB segment of mTOR
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<223> FRB with T to L substitution at 2098 which allows binding to AP21967
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<212> PRT
<213> Artificial Sequence
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<223> FRB segment of mTOR with T to H substitution at 2098 and to W at F at residue
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<223> FRB-dCasp9 nucleotide sequence
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<210> 16
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<212> PRT
<213> Foot-and-mouth disease virus
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<210> 17
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<212> PRT
<213> Foot-and-mouth disease virus
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