NZ737662B2 - Cell - Google Patents
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- NZ737662B2 NZ737662B2 NZ737662A NZ73766216A NZ737662B2 NZ 737662 B2 NZ737662 B2 NZ 737662B2 NZ 737662 A NZ737662 A NZ 737662A NZ 73766216 A NZ73766216 A NZ 73766216A NZ 737662 B2 NZ737662 B2 NZ 737662B2
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- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K16/2815—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD8
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C12N2510/00—Genetically modified cells
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Abstract
The present invention relates to a cell which comprises a chimeric antigen receptor (CAR) and a signal transduction modifying protein, selected from one of the following: (i) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase domain; (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain; (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain. In a particular embodiment the invention related to a T cell which comprises a CAR and a truncated protein which comprises: i) A PTPN6 SH2 domain which binds a phosphorylated ITIM but lacks a PTPN6 phosphatase domain; or a SHP-2 SH2 domain which binds a phosphorylated ITIM but lacks a SHP-2 phosphatase domain. ctivation motif (ITAM), but lacks a kinase domain; (ii) a truncated protein which comprises an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase domain; (iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a heterologous domain. In a particular embodiment the invention related to a T cell which comprises a CAR and a truncated protein which comprises: i) A PTPN6 SH2 domain which binds a phosphorylated ITIM but lacks a PTPN6 phosphatase domain; or a SHP-2 SH2 domain which binds a phosphorylated ITIM but lacks a SHP-2 phosphatase domain.
Description
P106294PCT 1
CELL
FIELD OF THE INVENTION
The present invention relates to fusion proteins and truncated proteins which enable the signalling
pathways which are propagated following immune cell activation to be manipulated or modulated.
BACKGROUND TO THE INVENTION
Adoptive immunotherapy with autologous T cells involves the isolation of T cells from the patient
followed by their stimulation, modification and/or expansion ex vivo in order to generate a population
of T cells which display anti-tumour specificities. Once re-infused into the patient these cells are
capable of recognizing tumour-expressed antigens and mediating tumour rejection.
This approach has already been shown in a number of trials in different settings to have the potential
to be a powerful, effective and long-lasting treatment for cancer. For instance, EBV-driven tumours,
such as lymphoproliferative disease following solid organ transplant can be effectively treated by
ex vivo expanded EBV specific T-cells.
A similar therapy for non-viral malignancies involves tumour infiltrating lymphocytes (TILs) which
are isolated from resected fragments of tumour and then subjected to stimulation and expansion
with autologous tumour samples. Expanded T cell cultures which show tumour reactivity can then
be re-infused into the patient.
Rather than selecting and refining T cell specificities with repeated exposure to antigens, the desired
anti-tumour specificity can be conferred onto the T cells through gene modification and the
introduction of either a tumour-specific T cell receptor (TCR) or a chimeric antigen receptor (CAR).
These cells are expanded ex vivo in order to produce sufficient numbers of cells to achieve
meaningful clinical responses within the patient.
However, the approaches detailed above have limitations. For instance, adoptively transferred T-
cells may show limited persistence and expansion in vivo due to insufficient signalling, lack of IL2
or differentiation. By way of further example, adoptively transferred T-cells may succumb to
inhibitory stimuli within the tumour microenvironment. For example they may become exhausted,
undergo activation induced cell death consequent to over activation, or may cause on-target off-
tumour effects.
P106294PCT 2
Another promising approach to activating therapeutic anti-tumour immunity is the blockade of
immune checkpoints. Immune checkpoints refer to the various inhibitory pathways of the immune
system that are important for maintaining self-tolerance and modulating the duration and amplitude
of physiological immune responses in peripheral tissues.
It is known that tumours exploit certain immune-checkpoint pathways as a major mechanism of
immune resistance, particularly against T cells that are specific for tumour antigens. Many of the
immune checkpoints are initiated by ligand–receptor interactions, meaning that they can be blocked
by antibodies or modulated by recombinant forms of ligands or receptors. Cytotoxic T-lymphocyte-
associated antigen 4 (CTLA4) antibodies were the first of this class of immunotherapeutics to
achieve US Food and Drug Administration (FDA) approval. More recently, blockers of additional
immune-checkpoint proteins, such as programmed cell death protein 1 (PD1), have been
developed and shown to enhance anti-tumour immunity.
One problem with the use of immune checkpoint inhibitors is that there are a multitude of inhibitory
pathways triggered by a multitude of ligand:receptor interactions. The use of an antibody or a
recombinant form of the ligand/receptor will only block one such inhibitory pathway, leaving the
possibility open that the tumour can compensate for the specific immune checkpoint block using
other molecules.
SUMMARY OF ASPECTS OF THE INVENTION
The present inventors have developed a system for modulating and/or manipulating signal
transduction pathways in immune cells, such as T cells and natural killer (NK) cells.
Intracellular signalling pathways are initiated and controlled by the reversible post-translational
modification of proteins. The present inventors have determined that activating and inhibitory
signalling pathways in T cells can be modulated and/or manipulated by fusion proteins or
truncated proteins comprising SH2 domains from immediate T-cell signal transduction proteins.
In other words, activating and inhibitory signalling pathways in T cells can be modulated and/or
manipulated by fusion proteins or truncated proteins comprising SH2 domains from proteins
which are capable of binding phosphorylated immunoreceptor tyrosine-based activation motifs
(ITAM) or phosphorylated immunoreceptor tyrosine-based inhibition motifs (ITIM).
Thus in a first aspect, the present invention provides a cell which comprises a chimeric antigen
receptor (CAR) and a signal transduction modifying protein, selected from one of the following:
P106294PCT 3
(i) a truncated protein which comprises an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based activation motif (ITAM), but lacks a kinase
domain;
(ii) a truncated protein which comprises an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase
domain;
(iii) a fusion protein which comprises (a) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which
binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a
heterologous domain.
The signal transduction modifying protein may be a truncated protein which comprises a ZAP70
SH2 domain but lacks a ZAP70 kinase domain.
The signal transduction modifying protein may be a truncated protein which comprises an PTPN6
SH2 but lacks a PTPN6 phosphatase domain.
The signal transduction modifying protein may be a truncated protein which comprises a SHP-2
SH2 domain but lacks a SHP-2 phosphatase domain.
The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2
domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation
motif (ITAM); and (ii) a phosphatase domain.
The fusion protein may, for example, comprise a ZAP70 SH2 domain, a PTPN6 or an SHP-2
phosphatase domain.
The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2
domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition
motif (ITIM); and (ii) a kinase domain.
The fusion protein may comprise an SH2 domain from PTPN6 or SHP-2.
The fusion protein may comprise a Zap70 kinase domain
The fusion protein may comprise an AKT or JAK kinase domain.
P106294PCT 4
The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2
domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation
motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based
inhibition motif (ITIM); and (ii) a heterologous signalling domain.
The fusion protein may comprise an SH2 domain from ZAP70, PTPN6 or SHP-2.
The heterologous signalling domain may be from a signalling molecule which is not usually
activated by an ITAM or ITIM containing receptor.
The heterologous signalling domain may be a co-stimulatory domain. In this respect, the fusion
protein may comprise a CD28, OX40 or 41BB co-stimulatory domain.
The heterologous signalling domain may be an inhibitory domain. In this respect, the inhibitory
domain may be or comprise the endodomain of CD148 or CD45. Alternatively, the heterologous
signalling domain is or comprises the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.
The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2
domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation
motif (ITAM); and (ii) an ITAM-containing domain.
The fusion protein may comprises a ZAP70 SH2 domain.
The ITAM-containing domain may be or comprise the endodomain of CD3-Zeta.
The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2
domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition
motif (ITIM); and (ii) an ITIM-containing domain.
The fusion protein may comprise an SH2 domain from PTPN6 or SHP-2.
The ITIM-containing domain may be or comprise the endodomain from PD1, PDCD1, BTLA4,
LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.
The signal transduction modifying protein may be a fusion protein which comprises (i) an SH2
domain from a protein which binds a phosphorylated immunoreceptor tyrosine-based activation
P106294PCT 5
motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor tyrosine-based
inhibition motif (ITIM); and (ii) a protease domain.
The fusion protein may comprise an SH2 domain from ZAP70, PTPN6 or SHP-2.
The protease domain may be or comprise Tobacco Etch Virus Protease (TeV).
The cell may also comprises a membrane-tethered transcription factor having a protease
cleavage site. Cleavage at the protease cleavage site may release the transcription factor leading
to increased expression of a target gene.
The target gene encodes a cytokine, for example a cytokine selected from the following group:
IL-2, IL-7, IL-15 and IL-12.
In this embodiment, the chimeric antigen receptor (CAR) may be a target CAR which comprises
an intracellular protease cleavage site.
The target CAR may comprise an activatory or co-stimulatory endodomain and cleavage at the
protease cleavage site removes the endodomain from the target CAR.
Alternatively, the target CAR may comprise an inhibitory endodomain and cleavage at the
protease cleavage site removes the inhibitory endodomain from the target CAR. The inhibitory
endodomain may comprise a CD148 or CD45 endodomain.
The cell of the present invention may comprise two CARs: an activating CAR comprising an ITAM-
containing endodomain; and a target CAR as defined above.
Alternatively, the cell of the present invention may comprise two CARs: an inhibitory CAR
comprising an ITIM-containing endodomain; and a target CAR as defined above.
In a second aspect, the present invention provides a nucleic acid construct, which comprises:
a first nucleic acid sequence encoding a chimeric antigen receptor; and
a second nucleic acid sequence encoding a truncated protein or a fusion protein as
defined in connection with the first aspect of the invention.
The nucleic acid construct may also comprise a third nucleic acid sequence encoding a
membrane-tethered transcription factor as defined above.
P106294PCT 6
The nucleic acid construct may also comprise a third nucleic acid sequence encoding a target
CAR as defined above.
The nucleic acid construct may also comprise a fourth nucleic acid sequence encoding an
activating CAR or an inhibitory CAR as defined above.
In a third aspect, the vector which comprises a nucleic acid construct according to the second
aspect of the invention or first and second, and optionally third and/or fouth, nucleic acid
sequences as defined above.
There is also provided a set of vectors which comprises first and second, and optionally third
and/or fourth, nucleic acid sequences as defined above.
The vector or set of vectors may be retroviral or lentiviral vector(s).
In a fourth aspect, there is provided a pharmaceutical composition comprising a plurality of cells
according to the first aspect of the invention.
In a fifth aspect, there is provided a pharmaceutical composition according to the fourth aspect of
the invention for use in treating and/or preventing a disease.
In a sixth aspect, there is provided method for treating and/or preventing a disease, which
comprises the step of administering a pharmaceutical composition according to the fourth aspect
of the invention to a subject.
The method may comprise the following steps:
(i) isolation of a cell containing sample from a subject;
(ii) transduction or transfection of the cells with a nucleic acid construct according to the
second aspect of the invention, a vector or set of vectors according to the third aspect of the
invention; and
(iii) administering the cells from (ii) to the subject.
In a seventh aspect there is provided the use of a pharmaceutical composition according to the
fourth aspect of the invention in the manufacture of a medicament for the treatment and/or
prevention of a disease.
P106294PCT 7
The disease may be cancer.
In an eighth aspect, there is provided a method for making a cell according to the first aspect of
the invention, which comprises the step of introducing: a nucleic acid construct according to the
second aspect of the invention, a vector or set of vectors according to the third aspect of the
invention, into the cell.
The cell may be from a sample isolated from a subject.
In a first further aspect, the present invention also provides a fusion protein which comprises: (i)
a ZAP70 or PTPN6 SH2 domain; and (ii) a heterologous domain.
The fusion protein may comprise a ZAP70 SH2 domain and an ITAM-containing domain. The
ITAM-containing domain may be or comprise the endodomain of CD3-Zeta.
The fusion protein may comprise a PTPN6 SH2 domain and an ITIM-containing domain. The
ITIM-containing domain may be or comprise the endodomain from PDCD1, BTLA4, LILRB1,
LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.
The fusion protein may comprise a PTPN6 SH2 domain and fused to a ZAP70 kinase domain.
The fusion protein may comprise a ZAP70 SH2 domain fused to a PTPN6 kinase domain.
The fusion protein may comprise: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a heterologous
signalling domain.
The heterologous signalling domain may be from a signalling molecule which is not usually
activated by an ITAM containing receptor. The heterologous signalling domain may be or
comprise the endodomain of CD28, 41BB or OX40. The heterologous signalling domain may be
or comprise the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.
The fusion protein may comprise: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a kinase domain.
The kinase domain may be or comprise an AKT or JAK kinase domain.
The fusion protein may comprise: (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a protease domain.
P106294PCT 8
The protease domain may be or comprise Tobacco Etch Virus Protease (TeV).
In a second further aspect the present invention provides a truncated protein which comprises
the ZAP70 SH2 domain but lacks the ZAP70 kinase domain.
In a third further aspect the present invention provides a truncated protein which comprises the
PTPN6 SH2 domain but lacks the PTPN6 kinase domain.
The present invention also provides a signalling system comprising:
(i) a receptor comprising an antigen-binding domain, a transmembrane domain and an
intracellular signalling domain which comprises a CD3 zeta endodomain; and
(ii) a fusion protein according to the first further aspect of the invention which comprises
a ZAP70 SH2 domain; or a truncated protein according to the second further aspect of the
invention;
wherein binding of antigen to the antigen-binding domain results in binding between the
CD3 zeta endodomain and the fusion/truncated protein.
The present invention also provides a signalling system comprising:
(i) a receptor comprising an antigen-binding domain, a transmembrane domain and an
intracellular signalling domain which comprises a PTPN6 binding domain; and
(ii) a fusion protein according to the first further aspect of the invention which comprises
a PTPN6 SH2 domain; or a truncated protein according to the third further aspect of the
invention
wherein binding of antigen to the antigen-binding domain results in binding between the
PTPN6 binding domain and the fusion/truncated protein.
The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).
In a fourth further aspect the present invention provides a nucleic acid which encodes a fusion
protein according to the first further aspect of the present invention or a truncated protein
according to the second or third further aspects of the present invention.
In a fifth further aspect the present invention provides a nucleic acid construct which comprises a
nucleic acid sequence encoding a fusion protein which comprises (i) a ZAP70 or PTPN6 SH2
domain; and (ii) a protease domain (e.g. a TeV domain) and a nucleic acid sequence encoding a
membrane tethered transcription factor which comprises:
(i) a membrane tether;
P106294PCT 9
(ii) a protease recognition site; and
(iii) a transcription factor.
In a sixth further aspect the present invention provides a nucleic acid construct which comprises
(a) a nucleic acid sequence encoding a fusion protein according to the first further aspect
of the present invention which comprises a PTPN6 SH2 domain, or a truncated protein according
to the third further aspect of the present invention; and
(b) a nucleic acid sequence encoding a receptor comprising an ITIM containing
endodomain.
In a seventh further aspect the present invention provides a nucleic acid construct which
comprises a nucleic acid sequence encoding a fusion protein which comprises (i) a ZAP70 or
PTPN6 SH2 domain; and (ii) a protease domain (e.g. a TeV domain) and a nucleic acid sequence
encoding a receptor which comprises a protease cleavage site.
In an eighth further aspect the present invention provides a nucleic acid construct which
comprises:
(a) a nucleic acid sequence encoding a fusion protein which comprises (i) a PTPN6 SH2
domain; and (ii) a protease domain (e.g. a TeV domain);
(b) a nucleic acid sequence encoding a receptor which comprises a protease cleavage
site; and
(c) a nucleic acid sequence encoding a receptor comprising an ITIM containing
endodomain.
The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).
Suitably, in the nucleic acid construct according to the eighth aspect of the present invention, the
nucleic acid sequence (b) may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR)
which comprises:
(i) a protease cleavage site between a transmembrane domain and an activating
endodomain; or
(ii) an activating endodomain fused to an inhibitory endodomain via a protease cleavage
site.
In a ninth further aspect the present invention provides a vector comprising a nucleic acid
according to the fourth further aspect of the present invention or a nucleic acid construct according
to any of fifth to the ninth further aspects of the present invention.
P106294PCT 10
The vector may be a retroviral vector or a lentiviral vector.
In a tenth further aspect the present invention provides a cell comprising a fusion protein
according to the first further aspect of the present invention or a truncated protein according to
the second or third further aspects of the present invention.
In an eleventh further aspect the present invention provides a cell which comprises (a) a fusion
protein according to the first further aspect of the present invention which comprises a PTPN6
SH2 domain, or a truncated protein according to the third further aspect of the present invention;
and (b) a receptor comprising an ITIM containing endodomain.
The cell may be an immune cell, such as a T cell or a natural killer (NK) cell.
In a twelfth further aspect the present invention provides a cell which comprises a fusion protein
which comprises (i) a ZAP70 or PTPN6 SH2 domain; and (ii) a protease domain(e.g. a TeV
domain) and a receptor which comprises a protease cleavage site.
In a thirteenth further aspect the present invention provides a cell which comprises:
(a) a fusion protein which comprises (i) a PTPN6 SH2 domain; and (ii) a protease domain (e.g. a
TeV domain);
(b) a receptor which comprises a protease cleavage site; and
(c) a receptor comprising an ITIM containing endodomain.
The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).
The receptor (b) may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR) which
comprises:
(i) a protease cleavage site between a transmembrane domain and an activating
endodomain; or
(ii) an activating endodomain fused to an inhibitory endodomain via a protease cleavage
site.
In a fourteenth further aspect the present invention provides a cell which comprises a nucleic acid
according to the fourth further aspect of the present invention or a nucleic acid construct according
to any of the fifth to the ninth further aspects of the present invention.
P106294PCT 11
In a fifteenth further aspect the present invention provides a pharmaceutical composition
comprising a plurality of cells according to any of the tenth to the fourteenth further aspects of the
present invention.
In a sixteenth further aspect the present invention provides a pharmaceutical composition
according to the fifteenth further aspect of the present invention for use in treating and/or
preventing a disease.
In a seventeenth further aspect the present invention relates to a method for treating and/or
preventing a disease, which comprises the step of administering a pharmaceutical composition
according the fifteenth further aspect to a subject.
The method may comprise the following steps:
(i) isolation of a T cell or NK cell containing sample from a subject;
(ii) transduction or transfection of the T cells or NK cells with a nucleic acid according to
any of the fourth to the ninth further aspects of the present invention or a vector according
to the tenth further aspect of the present invention; and
(iii) administering the T cells or NK cells from (ii) to the subject.
In an eighteenth further aspect the present invention relates to the use of a pharmaceutical
composition according to the fifteenth further aspect of the present invention in the manufacture
of a medicament for the treatment and/or prevention of a disease.
The disease may be cancer.
In a nineteenth further aspect the present invention provides a kit which comprises a nucleic acid
according to the fourth further aspect of the present invention or a nucleic acid construct according
to any of the fifth to the eighth further aspects of the present invention or a vector according to
the ninth further aspect of the present invention.
In a twentieth further aspect the present invention relates to a kit which comprises a cell according
to any of the tenth to the fourteenth further aspects of the present invention.
In a twenty-first further aspect the present invention relates to a method for making a cell
according to any of the tenth to the fourteenth further aspects of the present invention, which
comprises the step of introducing: a nucleic acid sequence according to any of the fourth to the
P106294PCT 12
eighth further aspects of the present invention or the vector according to the ninth further aspect
of the present invention, into the cell.
The cell may be from a sample isolated from a subject.
Yet further aspect of the invention are summarised in the following paragraphs:
A1. A truncated protein which comprises an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) but lacks a kinase
domain.
A2. A truncated protein according to paragraph A1, which comprises the ZAP70 SH2 domain
but lacks the ZAP70 kinase domain.
B1. A truncated protein which comprises an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a phosphatase
domain.
B2. A truncated protein according to paragraph B1, which comprises the PTPN6 SH2
domain but lacks the PTPN6 phosphatase domain.
B3. A truncated protein according to paragraph B1, which comprises the SHP-2 SH2 domain
but lacks the SHP-2 phosphatase domain.
C1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); and (ii) a phosphatase
domain.
C2. A fusion protein according to paragraph C1, which comprises a ZAP70 SH2 domain.
C3. A fusion protein according to paragraph C1 or C2, which comprises a PTPN6 or SHP-2
phosphatase domain.
D1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a kinase domain.
P106294PCT 13
D2. A fusion protein according to paragraph D1, which comprises an SH2 domain from
PTPN6 or SHP-2.
D3. A fusion protein according to paragraph D1 or D2, which comprises a Zap70 kinase
domain
D4. A fusion protein according to paragraph D1 or D2, which comprises an AKT or JAK
kinase domain.
E1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) or from a protein which
binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a
heterologous signalling domain.
E2. A fusion protein according to paragraph E1, which comprises an SH2 domain from
ZAP70, PTPN6 or SHP-2.
E3. A fusion protein according to paragraph E1 or E2, wherein the heterologous signalling
domain is from a signalling molecule which is not usually activated by an ITAM or ITIM
containing receptor.
E4. A fusion protein according to paragraph E1, E2 or E3, wherein the heterologous
signalling domain is a co-stimulatory domain.
E5. A fusion protein according to paragraph E4 which comprises a CD28, OX40 or 41BB co-
stimulatory domain.
E6. A fusion protein according to paragraph E1, E2 or E3, wherein the co-stimulatory domain
is an inhibitory domain.
E7. A fusion protein according to paragraph E6, wherein the inhibitory domain comprises the
endodomain of CD148 or CD45.
E8. A fusion protein according to paragraph E6, wherein the heterologous signalling domain
is or comprises the endodomain of ICOS, CD27, BTLA, CD30, GITR or HVEM.
P106294PCT 14
F1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based activation motif (ITAM); and (ii) an ITAM-
containing domain.
F2. A fusion protein according to paragraph F1, which comprises a ZAP70 SH2 domain.
F3. A fusion protein according to paragraph F1 or F2, wherein the ITAM-containing domain
is or comprises the endodomain of CD3-Zeta.
G1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) an ITIM-
containing domain.
G2. A fusion protein according to paragraph G1, which comprises an SH2 domain from
PTPN6 or SHP-2.
G3. A fusion protein according to paragraph G1 or G2, wherein the ITIM-containing domain
is or comprises the endodomain from PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1,
KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.
H1. A fusion protein which comprises (i) an SH2 domain from a protein which binds a
phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) or from a protein which
binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM); and (ii) a
protease domain.
H2. A fusion protein according to paragraph H1, which comprises an SH2 domain from
ZAP70, PTPN6 or SHP-2.
H3. A fusion protein according to paragraph H1 or H2, wherein the protease domain is or
comprises Tobacco Etch Virus Protease (TeV).
I1. A nucleic acid sequence which encodes a truncated protein according to any of
paragraphs A or B, or a fusion protein according to any of paragraphs C, D, E, F, G or H.
J1. A nucleic acid construct which comprises a nucleic acid sequence according to
paragraph I and a nucleic acid sequence encoding a chimeric antigen receptor
P106294PCT 15
J2. A nucleic acid construct which comprises a nucleic acid sequence according to
paragraph I and a nucleic acid sequence encoding a membrane-tethered transcription factor
having a protease cleavage site.
J3. A nucleic acid construct which comprises a nucleic acid sequence according to
paragraph I and a nucleic acid sequence encoding a target CAR which comprises an
intracellular protease cleavage site.
K1. A vector comprising a nucleic acid sequence according to paragraph I or a nucleic acid
construct according to paragraphs J.
L1. A cell which comprises a truncated protein according to any of paragraphs A or B, or a
fusion protein according to any of paragraphs C, D, E, F, G or H.
M1. A cell which comprises a fusion protein according to any of paragraphs H and a
membrane-tethered transcription factor with a protease cleavage site.
M2. A cell according to paragraph M1, wherein cleavage at the protease cleavage site
releases the transcription factor leading to increased expression of a target gene.
M3. A cell according to paragraph M2, wherein the target gene encodes a cytokine.
M4. A cell according to paragraph M3, wherein the cytokine is selected from the following
group: IL-2, IL-7, IL-15 and IL-12.
M5. A cell which comprises a fusion protein according to any of paragraphs H and a target
receptor (CAR) which comprises an intracellular protease cleavage site.
M6. A cell according to claim M5, wherein the target CAR comprises an activatory or co-
stimulatory endodomain and cleavage at the protease cleavage site removes the endodomain
from the target CAR.
M7. A cell according to claim M5, wherein the target CAR comprises an inhibitory
endodomain and cleavage at the protease cleavage site removes the inhibitory endodomain
from the target CAR.
P106294PCT 16
M8. A cell according to paragraph M7, wherein the inhibitory endodomain comprises a
CD148 or CD45 endodomain.
M9. A cell which comprises a fusion protein according to any of paragraphs H and two CARs:
an activating CAR comprising an ITAM-containing endodomain; and a target CAR as defined in
any of paragraphs M5 to M8.
M10. A cell which comprises a fusion protein according to any of paragraphs H and two CARs:
an inhibitory CAR comprising an ITIM-containing endodomain; and a target CAR as defined in
any of paragraphs M5 to M8.
The aspects of the present invention described above enable T cell signalling pathways to be
modulated and altered by, for example, the mechanisms described in Table 1. Table 1 :
Application of signal modulation
Type Mechanism Application
Blocking signal ZAP70, SHP-2 or PTPN6 are Truncated ZAP70, SHP-2 or PTPN6
truncated - keeping SH2 domain competes with wild-type full-length
alone ZAP70, SHP-2 or PTPN6. Since this does
not signal, it will inhibit activation.
Applications include, for example, with
ZAP70 when a very strong activation
signal is deleterious, or with PTPN6 or
SHP-2 when the effect of an inhibitory
signal e.g. PD1/PDL1 needs to be
reduced.
Crosswire signal ZAP70 SH2 fused to PTPN6/SHP- In this embodiment, a ZAP70 SH2 is fused
2 phosphatase, or PTPN6/SHP-2 to the phosphatase from PTPN6/SHP-2,
SH2 fused to ZAP70 kinase for or the other way round, i.e. the
instance. PTPN6/SHP-2 SH2 domain is fused with
the ZAP70 kinase domain. When the T-
cell receives an inhibitory signal, it
interprets it as an excitatory signal or vice
versa.
Amplified signal ZAP70 fused to further ITAM A single phospho-ITAM or ITIM leads to a
domains or PTPN6/SHP-2 fused concatenation of ITAMs or ITIMs leading
to further ITIM domains. to augmented signal or increased
sensitivity to antigen.
Bypass signal ZAP70 SH2 or PTPN6/SHP-2 SH2 In this embodiment, a “non-
fused with e.g. CD28, 41BB physiological” signal can be attached to
endodomains or AKT kinase the ITAM/ITM pathway. In this way an
domain, a JAK kinase domain etc. ITAM/ITIM signal can lead to a co-
P106294PCT 17
stimulatory signal, or a signal such as AKT
or a cytokine type signal
Transcriptional signal ZAP70 SH2 or PTPN6/SHP-2 SH2 In this embodiment, a transcriptional
fused to protease domain along signal is transmitted upon immune
with co-expression of a receptor activation or inhibition. Such a
membrane tethered signal can, for example, result in the
transcription factor with a expression of a particular cytokine upon
liberating protease cleavage site T-cell activation or inhibition.
Castration signal ZAP70 SH2 domain or In this embodiment, activation or
PTPN6/SHP-2 SH2 domain fused inhibition of a receptor results in
to a protease domain; a inhibition or activation of another
reciprocal receptor has a receptor
protease cleavage site
DESCRIPTION OF THE FIGURES
Figure 1 (a) – Diagram of immediate T-cell activation pathways. T-cell receptor activation results
in phosphorylation of ITAMs. Phosphorylated ITAMs are recognized by the ZAP70 SH2 domains.
Upon recognition, ZAP70 is recruited to the juxta-membrane region and its kinase domain
subsequently phosphorylates LAT. Phosphorylated LAT is subsequently recognized by the SH2
domains of GRAP, GRB2 and PLC- . (b) – Diagram of immediate T-cell inhibition pathways.
Activation of an inhibitory immune-receptor such as PD1 results in phosphorylation of ITIM
domains. These are recognized by the SH2 domains of PTPN6. Upon recognition, PTPN6 is
recruited to the juxta-membrane region and its phosphatase domain subsequently de-
phosphorylates ITAM domains inhibiting immune activation.
Figure 2 – Diagram of a blocking signal system - a) A truncated ZAP70 which does not comprise
a kinase domain is over-expressed. Consequently, it competes with full-length ZAP70 for ITAMs
and reduces ITAM signalling. (b) A truncated PTPN6 which does not comprise a phosphatase
domain is over-expressed, competing for full-length PTPN6 reducing ITIM signalling.
Figure 3 – Diagram of a crosswire signal system: (a) ZAP70 SH2 is fused to PTPN6 phosphatase,
hence acts to dampen ITAM phosphorylation; (b) PTPN6 SH2 is fused to ZAP70 kinase resulting
in paradoxical activation in response to an inhibitory signal.
Figure 4 – Diagram of an amplified signal system: (a) full-length ZAP70 has a CD3 Zeta
endodomain attached to its amino terminus so a cascade of ITAMs assembles. (b) full-length
PTPN6 has PD1 endodomain attached to its amino terminus so a cascade of ITIMs assembles.
P106294PCT 18
Figure 5 – Diagram of examples of a bypass signal system: (a) ZAP70 fused with CD28
endodomain; (b) ZAP70 fused with 41BB endodomain; (c) ZAP70 fused with AKT kinase; (d)
PTPN6 SH2 domain is fused with 41BB endodomain
Figure 6 – Diagram of an illustrative transcriptional signal system: a) A ZAP-TeV fusion is co-
expressed with a membrane-tethered transcription factor which can be released from the
membrane by cleavage of its TeV recognition motif. This is shown co-expressed with a CD19
CAR. Hence, upon recognition of CD19 on a target cell, the T-cell becomes activated and in
addition, the transcription factor becomes active. (b) An alternative system using a PTPN6-TeV
fusion instead. Here the CAR consists of an ITIM-bearing endodomain. Hence, upon recognition
of CD19 by the CAR, the transcription factor becomes active but this is independent of T-cell
activation.
Figure 7 – Diagram of a castration signal system: two CARs are shown – one which recognizes
CD19 and is activating and one which recognizes CD33 and is inhibiting - these specificities are
for illustration only (a) AND NOT signal castration; here an SH2-Tev fusion protein is recruited to
activated ITIM CAR upon its activation. This results in cleavage of ITAMs from an activating CAR
which is constructed such that a TeV cleavage site connects the transmembrane-domain to the
ITAM domain. Hence, the activating CAR is inhibited. (b) AND signal castration: Here, an SH2-
Tev fusion protein is recruited to an ITIM CAR upon its activation. This results in release of a
phosphatase domain from an activating CAR which is constructed so that a phosphatase is
connected to its carboxy-terminus via a TeV cleavage domain. This results in release of
constitutive inhibition, allowing the CAR to activate in the presence of cognate antigen.
Figure 8 – Several fusions of different SH2 domains and AKT kinase domain were constructed:
ZAP-AKT, GRAP-AKT, GRB-AKT and PLC- .
Figure 9 - (a) Phospho-AKT staining of T-cells transduced with the different SH2/AKT fusions
with and without activation with the mitogenic antibody OKT3. (b) Phospho-AKT staining of T-
cells transduced with ZAP-AKT fusion, an improved ZAP-AKT fusion where ZAP and AKT are
connected via a flexible linker, and a control ZAP-AKT where R190K substitution removes ability
of ZAP to bind ITAMs. T-cells were either stimulated with OKT3 or not-stimulated with OKT3. The
facs plots are overlaid over that of non-transduced T-cells.
Figure 10 - (a) Phospho-AKT blot of T-cells activated with increasing amounts of OKT3. (b)
Microscopy of ZAP-AKT or control T-cells unstimulated, stimulated with just OKT3 or stimulated
with both OKT3 and IL-2. ZAP-AKT T-cells stimulated with just OKT3 resemble non-transduced
T-cells stimulated with both OKT3 and IL2.
P106294PCT 19
Figure 11 - (a) Implementation of direct TeV transcriptional switch. A CD19 CAR’s endodomain
is replaced with the TeV protease. A membrane tethered VP16/GAL4 transcription factor is also
co-expressed. A Luciferase reporter detects VP16/GAL5 activity. (b) Implementation with ZAP-
TeV. A standard CD19 CAR is co-expressed with ZAP-TeV fusion along with the membrane
tethered transcription factor.
Figure 12 - Activity of ZAP-TeV based transcriptional switches and control expressing T-cells
after exposure to CD19 negative (left), or CD19 positive (right) targets. Activity is measured by
light output after adding Luciferase. In order the conditions tested are: (a) aCD19 CAR co-
expressed with ZAP-TeV; (b) aCD19 CAR co-expressed with inactive (R190K); (c) aCD19 CAR
co-expressed with ZAP-TeV and the membrane tethered transcription factor; (d) aCD19 CAR co-
expressed with inactive (R190K) ZAP-TEV co-expressed with the membrane tethered
transcription factor; (e) aCD19 CAR / TeV fusion co-expressed with the membrane-tethered
transcription factor; (f) constitutively active GAL4/VP16 transcription factor.
Figure 13 - (a) Generalized architecture of a CAR: A binding domain recognizes antigen; the
spacer elevates the binding domain from the cell surface; the trans-membrane domain anchors
the protein to the membrane and the endodomain transmits signals. (b) to (d): Different
generations and permutations of CAR endodomains: (b) initial designs transmitted ITAM signals
alone through FcεR1-γ or CD3ζ endodomain, while later designs transmitted additional (c) one or
(d) two co-stimulatory signals in cis.
Figure 14 - Illustrative protein sequences of the present invention
Figure 15 – PD-1 signal blockade using truncated SHP-1 (PTPN6) or truncated SHP-2
PBMC cells were cotransduced with PD1 and either CAR alone (FMC63); or a bicistronic
construct containing CAR and truncated SHP-1, or CAR and truncated SHP-2. These cells were
co-cultured for 48 hours with SupT1 cells transduced with CD19, PDL1 or both and IFNγ release
measured by ELISA.
Figure 16 – PD-1 signal hijack using a fusion of SHP-2 SH2 domains and Zap70 kinase
PBMC cells were cotransduced with PD1 and either CAR alone (FMC63); or a bicistronic
construct containing CAR and a fusion protein comprising SHP-2 SH2 domains and the ZAP70
kinase. These cells were co-cultured in a 1:1 ratio for 24 hours with SupT1 cells transduced with
CD19 or PDL1. IFNγ release was measured by ELISA (A) and killing of SupT1 cells was
quantified by FACS (B).
P106294PCT 20
DETAILED DESCRIPTION
PROTEIN
The present invention provides a truncated protein which comprises an SH2 domain.
The present invention also provides a fusion protein comprising (i) an SH2 domain; and (ii) a
heterologous domain.
The SH2 domain may be from a protein which binds a phosphorylated immunoreceptor tyrosine-
based activation motif (ITAM) or from a protein which binds a phosphorylated immunoreceptor
tyrosine-based inhibition motif (ITIM).
An example of a protein which binds an ITAM is ZAP70. Examples of proteins which bind ITIMs
include PTPN6 and SHP-2
The fusion protein of the invention therefore comprises an SH2 domain and at least one further
domain which is not present in a wild-type protein from which the SH2 domain was derived.
SRC HOMOLOGY 2 (SH2) DOMAIN
Intracellular signalling pathways are initiated and controlled by the reversible post-translational
modification of proteins including phosphorylation, ubiquitinylation and acetylation.
SH2 domains are modular protein domains that serve as adaptors and mediate protein-protein
interactions by binding to phosphorylated peptides in their respective protein binding partners,
often cell surface receptors. SH2 domains typically bind a phosphorylated tyrosine residue in the
context of a longer peptide motif within a target protein, and SH2 domains represent the largest
class of known pTyr-recognition domains
Although SH2 domains lack any intrinsic catalytic activity they are frequently coupled to
independent catalytic domains and thus, in response to a specific input signal, serve to localize
these catalytic domains so particular sub-cellular locations or to the vicinity of appropriate
substrates, activators or inhibitors. In addition SH2 domains can also be found linked to adaptor
protein domains and so can serve in the formation of large multi-protein complexes.
ZETA-CHAIN-ASSOCIATED PROTEIN KINASE 70 (ZAP70)
P106294PCT 21
ZAP70 is a protein normally expressed near the surface membrane of T cells and natural killer
cells. It is part of the T cell receptor (TCR), and plays a critical role in T-cell signalling. Its molecular
weight is 70 kDa, and is composed of 2 N-terminal SH2 domains and a C-terminal kinase domain.
It is a member of the protein-tyrosine kinase family.
The earliest step in T cell activation is the recognition of a peptide MHC-complex on the target
cell by the TCR. This initial event causes the close association of Lck kinase with the cytoplasmic
tail of CD3-zeta in the TCR complex. Lck then phosphorylates tyrosine residues in the
cytoplasmic tail of CD3-zeta which allows the recruitment of ZAP70. ZAP70 is an SH2 containing
kinase that plays a pivotal role in T cell activation following engagement of the TCR. Tandem
SH2 domains in ZAP70 bind to the phosphorylated CD3 resulting in ZAP70 being phosphorylated
and activated by Lck or by other ZAP70 molecules in trans. Active ZAP70 is then able to
phosphorylate downstream membrane proteins, key among them the linker of activated T cells
(LAT) protein. LAT is a scaffold protein and its phosphorylation on multiple residues allows it to
interact with several other SH2 domain-containing proteins including Grb2, PLC-g and Grap which
recognize the phosphorylated peptides in LAT and transmit the T cell activation signal
downstream ultimately resulting in a range of T cell responses. This process is summarized in
Figure 1.
Human ZAP70 protein has the UniProtKB accession number P43403. This sequence is 619
amino acids in length and is shown as SEQ ID NO: 1.
ZAP70 amino acid sequence (SEQ ID NO: 1)
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
QRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFG
SVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGG
PLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADD
SYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMEC
PPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA
The fusion protein of the invention may comprise a ZAP70 SH2 domain. The truncated protein of
the invention may comprise or consist of a ZAP70 SH2 domain. In this respect, the fusion or
truncated protein may comprise or consist of the sequence shown as SEQ ID NO: 2.
ZAP70 complete SH2 domain (SEQ ID NO: 2)
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
P106294PCT 22
ZAP70 has two SH2 domains at the N-terminal end of the sequence, at residues 10-102 and 163-
254 of the sequence shown as SEQ ID No. 1. The truncated protein or fusion protein of the
invention may therefor comprise one or both of the sequences shown as SEQ ID No. 3 and 4.
ZAP70 SH2 1 (SEQ ID NO: 3)
FFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCG
PAELCEFYSRDPDGLPCNLRKPC
ZAP70 SH2 2 (SEQ ID NO: 4)
WYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTL
WQLVEYLKLKADGLIYCLKEAC
The fusion protein may comprise a variant of SEQ ID NO: 2, 3 or 4 having at least 80, 85, 90, 95,
98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence has
the required properties. In other words, the variant sequence should be capable of binding to the
phosphorylated tyrosine residues in the cytoplasmic tail of CD3-zeta which allow the recruitment
of ZAP70.
Methods of sequence alignment are well known in the art and are accomplished using suitable
alignment programs. The % sequence identity refers to the percentage of amino acid or
nucleotide residues that are identical in the two sequences when they are optimally aligned.
Nucleotide and protein sequence homology or identity may be determined using standard
algorithms such as a BLAST program (Basic Local Alignment Search Tool at the National Center
for Biotechnology Information) using default parameters, which is publicly available at
http://blast.ncbi.nlm.nih.gov . Other algorithms for determining sequence identity or homology
include: LALIGN (http://www.ebi.ac.uk/Tools/psa/lalign/ and
http://www.ebi.ac.uk/Tools/psa/lalign/nucleotide.html), AMAS (Analysis of Multiply Aligned
Sequences, at http://www.compbio.dundee.ac.uk/Software/Amas/amas.html), FASTA
(http://www.ebi.ac.uk/Tools/sss/fasta/) , Clustal Omega
(http://www.ebi.ac.uk/Tools/msa/clustalo/), SIM (http://web.expasy.org/sim/), and EMBOSS
Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html).
In certain embodiments, the fusion protein may comprise the ZAP70 SH2 domain and the ZAP70
kinase domain. For example, the fusion protein may comprise the sequence shown as SEQ ID
NO: 1 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.
TYROSINE-PROTEIN PHOSPHATASE NON-RECEPTOR TYPE 6 (PTPN6)
P106294PCT 23
PTPN6 is also known as Src homology region 2 domain-containing phosphatase-1 (SHP-1). It is
a member of the protein tyrosine phosphatase family.
The N-terminal region of PTPN6 contains two tandem SH2 domains which mediate the interaction
of PTPN6 and its substrates. The C-terminal region contains a tyrosine-protein phosphatase
domain.
PTPN6 is capable of binding to, and propagating signals from, a number of inhibitory immune
receptors or ITIM containing receptors. Examples of such receptors include, but are not limited
to, PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 and
KIR3DL3.
Human PTPN6 protein has the UniProtKB accession number P29350. This sequence is 595
amino acids in length and is shown as SEQ ID NO: 5.
PTPN6 amino acid sequence (SEQ ID NO: 5)
MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFA
TLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLS
QPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLR
QPYYATRVNAADIENRVLELNKKQESEDTAKAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNIL
PFDHSRVILQGRDSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMT
TREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDH
GVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQM
VRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHK
EDVYENLHTKNKREEKVKKQRSADKEKSKGSLKRK
The fusion protein of the invention may comprise a PTPN6 SH2 domain. The truncated protein of
the invention may comprise or consist of a PTPN6 SH2 domain. In this respect, the fusion or
truncated protein may comprise or consist of the sequence shown as SEQ ID NO: 6.
PTPN6 SH2 complete domain (SEQ ID NO: 6)
MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFA
TLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLS
QPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLR
QPYY
PTPN6 has two SH2 domains at the N-terminal end of the sequence, at residues 4-100 and 110-
213 of the sequence shown as SEQ ID No. 5. The truncated protein or fusion protein of the
invention may therefor comprise one or both of the sequences shown as SEQ ID No. 3 and 4.
PTPN6 SH2 1 (SEQ ID NO: 7)
P106294PCT 24
WFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLT
ELVEYYTQQQGVLQDRDGTIIHLKYPL
PTPN6 SH2 2 (SEQ ID No. 8)
WYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVG
GLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPY
The fusion protein may comprise a variant of SEQ ID NO: 6, 7 or 8 having at least 80, 85, 90, 95,
98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence has
the required properties. In other words, the variant sequence should be capable of binding to the
phosphorylated tyrosine residues in the cytoplasmic tail of at least one of PD1, PDCD1, BTLA4,
LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3 which allow the
recruitment of PTPN6.
In certain embodiments, the fusion protein may comprise the PTPN6 SH2 domain and the PTPN6
phosphatase domain. For example, the fusion protein may comprise the sequence shown as
SEQ ID NO: 5 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.
SHP-2
SHP-2, also known as PTPN11, PTP-1D and PTP-2C is is a member of the protein tyrosine
phosphatase (PTP) family. Like PTPN6, SHP-2 has a domain structure that consists of two
tandem SH2 domains in its N-terminus followed by a protein tyrosine phosphatase (PTP) domain.
In the inactive state, the N-terminal SH2 domain binds the PTP domain and blocks access of
potential substrates to the active site. Thus, SHP-2 is auto-inhibited. Upon binding to target
phospho-tyrosyl residues, the N-terminal SH2 domain is released from the PTP domain,
catalytically activating the enzyme by relieving the auto-inhibition.
Human SHP-2 has the UniProtKB accession number P35235-1. This sequence is 597 amino
acids in length and is shown as SEQ ID NO: 9.
SHP-2 amino acid sequence (SEQ ID NO: 9)
MTSRRWFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEK
FATLAELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRES
QSHPGDFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVL
QLKQPLNTTRINAAEIESRVRELSKLAETTDKVKQGFWEEFETLQQQECKLLYSRKEGQRQENKNKNRYK
NILPFDHTRVVLHDGDPNEPVSDYINANIIMPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQENS
RVIVMTTKEVERGKSKCVKYWPDEYALKEYGVMRVRNVKESAAHDYTLRELKLSKVGQALLQGNTERTVW
P106294PCT 25
QYHFRTWPDHGVPSDPGGVLDFLEEVHHKQESIVDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDC
DIDVPKTIQMVRSQRSGMVQTEAQYRFIYMAVQHYIETLQRRIEEEQKSKRKGHEYTNIKYSLVDQTSGD
QSPLPPCTPTPPCAEMREDSARVYENVGLMQQQRSFR
The fusion protein of the invention may comprise a SHP-2 SH2 domain. The truncated protein of
the invention may comprise or consist of a SHP-2 SH2 domain. In this respect, the fusion or
truncated protein may comprise or consist of the first SH2 domain of SHP-2, for example
comprising amino acids 6-102 of SEQ ID NO. 9 or the second SH2 domain of SHP-2, for example
comprising amino acids 112-216 of SHP-2. The fusion or truncated protein may comprise or
consist of the sequence shown as SEQ ID NO: 10, 11 or 12.
SHP-2 first SH2 domain (SEQ ID NO: 10)
WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEKFATLA
ELVQYYMEHHGQLKEKNGDVIELKYPL
SHP-2 second SH2 domain (SEQ ID No. 11)
WFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPGDFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDV
GGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQPL
SHP-2 both SH2 domains (SEQ ID No. 12)
WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEKFATLA
ELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPG
DFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQP
The fusion protein may comprise a variant of SEQ ID NO: 10, 11 or 12 having at least 80, 85, 90,
95, 98 or 99% sequence identity, provided that the variant sequence is a SH2 domain sequence
capable of binding an ITIM-containing domain. For example, the variant sequence may be
capable of binding to the phosphorylated tyrosine residues in the cytoplasmic tail of PD1, PDCD1,
BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.
HETEROLOGOUS DOMAIN
As used herein, the term ‘heterologous domain’ refers to any protein domain which is not present
i) wild type ZAP70 (see SEQ ID NO: 1) for fusion proteins comprising a ZAP70 SH2
domain;
P106294PCT 26
ii) wild type PTPN6 (see SEQ ID NO: 5) for fusion proteins comprising a PTPN6 SH2
domain; or
iii) wild-type SHP-2 (see SEQ ID No. 9) for fusion proteins comprising a SHP-2 SH2
domain.
The heterologous domain may be or be derivable from (e.g. part of) a different protein from ZAP70,
SHP-2 or PTPN6.
Alternatively the fusion protein may comprise a fusion of ZAP70 SH2 domain and a domain from
PTPN6, such as the PTPN6 kinase domain. By the same token the fusion protein may comprise
a fusion of PTPN6 SH2 domain and a domain from ZAP70, such as the ZAP70 kinase domain.
AMPLIFIED SIGNAL
The present invention provides a fusion protein which comprises: an SH2 domain from an ITAM-
binding protein; and an ITAM-containing domain.
The present invention also provide a fusion protein which comprises: an SH2 domain from an
ITIM-binding protein; and an ITIM-containing domain.
These “amplified” signalling molecules will amplify an excitatory or inhibitory signal inside an
immune cell such as a T cell.
As shown in Figure 4, the presence of such molecules will lead to a concatenation of either ITAMs
or ITIMs leading to an augmented activatory or inhibitory signal, respectively.
Amplification of an activatory signal is useful in situations where it is desirable to increase the
sensitivity of the immune cell (such as a CAR-T cell) to antigen. This may be the case when, for
example, the target antigen is expressed at low levels on the target cells.
Amplification of an inhibitory systems in situations where it is desirable to reduce or prevent T
cell activation. WO2015/075469 describes a panel of “logic gate” chimeric antigen receptor pairs
which, when expressed by a cell, such as a T cell, are capable of detecting a particular pattern of
expression of at least two target antigens A and B). The “AND NOT gate” described in this
application comprises a pair of CARs such that the T cell triggers only when antigen A but not
antigen B is present on the target cell. In this AND NOT gate, one CAR (recognising antigen A)
has an activating endodomain comprising and ITAM, whereas the other CAR (recognising antigen
B) has an inhibitory endodomain which may comprise an ITIM. In the presence of antigen A alone,
P106294PCT 27
the presence of unligated inhibitory CAR is insufficient to prevent T cell activation, so activation
occurs. However, in the presence of both antigens, areas of membrane form with high
concentrations of both activatory and inhibitory CARs. Since both endodomains are concentrated,
T-cell activation is prevented or reduced.
Amplification of the inhibitory signal using an amplified signalling molecule of the present invention
could be used in an AND gate to reduce or remove any residual signalling which occurs in the
presence of both antigens i.e. from incomplete inhibition of the activatory CAR by the inhibitory
CAR.
ITAM-CONTAINING DOMAIN
In one embodiment, the fusion protein comprises a ZAP70 SH2 domain and an immunoreceptor
tyrosine-based activation motif (ITAM)-containing domain.
A fusion of full-length ZAP70 with an ITAM containing domain results in a structure which amplifies
an activating immune signal. Here, the fusion protein is recruited to a phospho-ITAM immune-
receptor endodomain. ZAP70 functions normally to propagate the signal but also provides
another set of ITAMs which become phosphorylated and recruit more ZAP70. This may be useful
to increase signal strength and may increase sensitivity to low-density antigens, for example. In
some embodiments, the fusion may include only the ZAP70 SH2 domain with an ITAM containing
endodomain (e.g. the fusion does not contain a ZAP70 kinase domain). In other embodiments,
the ratio of ZAP70 catalytic domains (kinase domains) with ITAMs may be varied to affect the
kinetics of activation in response to dynamics of the activating receptor interactions with cognate
target.
An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic
tails of certain cell surface proteins of the immune system. The motif contains a tyrosine
separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/I.
Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the
molecule (YxxL/Ix(6-8)YxxL/I).
ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of
important cell signalling molecules such as the CD3 and ζ-chains of the T cell receptor complex,
the CD79-alpha and -beta chains of the B cell receptor complex, and certain Fc receptors. The
tyrosine residues within these motifs become phosphorylated following interaction of the receptor
P106294PCT 28
molecules with their ligands and form docking sites for other proteins involved in the signalling
pathways of the cell.
Several proteins are known to contain endodomains with one or more ITAM motifs. Examples of
such proteins include the CD3 epsilon chain, the CD3 gamma chain and the CD3 delta chain to
name a few. The ITAM motif can be easily recognized as a tyrosine separated from a leucine or
isoleucine by any two other amino acids, giving the signature YxxL/I. Typically, but not always,
two of these motifs are separated by between 6 and 8 amino acids in the tail of the molecule
(YxxL/Ix(6-8)YxxL/I). Hence, one skilled in the art can readily find existing proteins which contain
one or more ITAM to transmit an activation signal. Further, given the motif is simple and a complex
secondary structure is not required, one skilled in the art can design polypeptides containing
artificial ITAMs to transmit an activation signal (see WO 2000063372, which relates to synthetic
signalling molecules).
The ITAM-containing domain may be or comprise a CD3-zeta endodomain. Suitably, the ITAM-
containing domain may comprise the sequence shown as SEQ ID NO: 13 or a variant thereof
having at least 80, 85, 90, 95, 98 or 99% sequence identity which retains the capacity to be
phosphorylated and recruit ZAP70.
SEQ ID NO: 13 (CD3-zeta endodomain)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA
YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
By way of example, the fusion protein may be or comprise the sequence shown as SEQ ID NO:
14, which contains a ZAP70-SH2 domain fused to a CD3-zeta endodomain.
SEQ ID NO: 14
MRRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA
EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRSGGGGSGGGGSGGGGSGGGGSMPDPA
AHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGK
A
APQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQ
DKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRID
TLNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQG
VYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKF
LVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTA
RSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECP
PELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA
Suitably, the fusion protein may comprise the sequence shown as SEQ ID NO: 14 or a variant
thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.
P106294PCT 29
ITIM-CONTAINING DOMAIN
In one embodiment, the fusion protein comprises a PTPN6 SH2 domain and an immunoreceptor
tyrosine-based inhibition motif (ITIM)-containing domain
A fusion of full-length PTPN6 with an ITIM containing domain results in a structure which amplifies
an inhibitory immune signal. Here, the fusion protein is recruited to a phospho-ITIM immune-
receptor endodomain. PTPN6 functions normally to propagate the signal but also provides
another set of ITIMs which become phosphorylated and recruit more PTPN6. In some
embodiments, the fusion may include only the PTPN6 SH2 domain with an ITIM containing
endodomain (e.g. the fusion does not contain a PTPN6 phosphatase domain). In other
embodiments, the ratio of PTPN6 catalytic domains (phosphatase domains) with ITIMs may be
varied to affect the kinetics of activation in response to dynamics of the inhibitory receptor
interactions with cognate target.
An ITIM, is a conserved sequence of amino acids (S/I/V/LxYxxI/V/L) that is found in the
cytoplasmic tails of many inhibitory receptors of the immune system. After ITIM-possessing
inhibitory receptors interact with their ligand, their ITIM motif becomes phosphorylated by
enzymes of the Src kinases, allowing them to recruit PTPN6 via interactions between the PTPN6
SH2 domain and the phosphorylated ITIM domains.
ITIM containing endodomains include those from CD22, LAIR-1, the Killer inhibitory receptor
family (KIR), LILRB1, CTLA4, PD-1, BTLA, for example.
ITIM endodomains from PDCD1, BTLA4, LILRB1, LAIR1, CTLA4, KIR2DL1, KIR2DL4, KIR2DL5,
KIR3DL1 and KIR3DL3 are shown in SEQ ID NO: 15 to 24 respectively
SEQ ID NO: 15 PDCD1 endodomain
CSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATI
VFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL
SEQ ID NO: 16 BTLA4
KLQRRWKRTQSQQGLQENSSGQSFFVRNKKVRRAPLSEGPHSLGCYNPMMEDGISYTTLRFPEMNIPRTG
DAESSEMQRPPPDCDDTVTYSALHKRQVGDYENVIPDFPEDEGIHYSELI
QFGVGERPQAQENVDYVILKH
SEQ ID NO: 17 LILRB1
LRHRRQGKHWTSTQRKADFQHPAGAVGPEPTDRGLQWRSSPAADAQEENLYAAVKHTQPEDGVEMDTRSP
HDEDPQAVTYAEVKHSRPRREMASPPSPLSGEFLDTKDRQAEEDRQMDTEAAASEAPQDVTYAQLHSLTL
RREATEPPPSQEGPSPAVPSIYATLAIH
P106294PCT 30
SEQ ID NO: 18 LAIR1
HRQNQIKQGPPRSKDEEQKPQQRPDLAVDVLERTADKATVNGLPEKDRETDTSALAAGSSQEVTYAQLDH
WALTQRTARAVSPQSTKPMAESITYAAVARH
SEQ ID NO: 19 CTLA4
FLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN
SEQ ID NO: 20 KIR2DL1
GNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYTQLNH
CVFTQRKITRPSQRPKTPPTDIIVYTELPNAESRSKVVSCP
SEQ ID NO: 21 KIR2DL4
GIARHLHAVIRYSVAIILFTILPFFLLHRWCSKKKENAAVMNQEPAGHRTVNREDSDEQDPQEVTYAQLD
HCIFTQRKITGPSQRSKRPSTDTSVCIELPNAEPRALSPAHEHHSQALMGSSRETTALSQTQLASSNVPA
SEQ ID NO: 22 KIR2DL5
TGIRRHLHILIGTSVAIILFIILFFFLLHCCCSNKKNAAVMDQEPAGDRTVNREDSDDQDPQEVTYAQLD
HCVFTQTKITSPSQRPKTPPTDTTMYMELPNAKPRSLSPAHKHHSQALRGSSRETTALSQNRVASSHVPA
SEQ ID NO: 23 KIR3DL1
KDPRHLHILIGTSVVIILFILLLFFLLHLWCSNKKNAAVMDQEPAGNRTANSEDSDEQDPEEVTYAQLDH
CVFTQRKITRPSQRPKTPPTDTILYTELPNAKPRSKVVSCP
SEQ ID NO: 24 KIR3DL3
KDPGNSRHLHVLIGTSVVIIPFAILLFFLLHRWCANKKNAVVMDQEPAGNRTVNREDSDEQDPQEVTYAQ
LNHCVFTQRKITRPSQRPKTPPTDTSV
The ITIM-containing domain may be or comprise a PDCD1, BTLA4, LILRB1, LAIR1, CTLA4,
KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3 endodomain. Suitably, the ITIM-containing
domain may comprise the sequence shown any of SEQ ID NO: 15 to 24 or a variant thereof
having at least 80, 85, 90, 95, 98 or 99% sequence identity which retains the capacity to be
phosphorylated by Src kinases and amplify an inhibitory immune signal.
By way of example, the fusion protein may be or comprise the sequence shown as SEQ ID NO:
, which contains a PTPN6-SH2 domain fused to a PD1 endodomain.
SEQ ID NO: 25
MTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRS
AQPLRPEDGHCSWPLSGGGGSGGGGSGGGGSGGGGSMVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRK
NQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSD
P106294PCT 31
PTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGG
RYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTAKAGF
WEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLGP
DENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCGE
HDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCS
AGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLE
VLQSQKGQESEYGNITYPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKR
Suitably, the fusion protein may comprise the sequence shown as SEQ ID NO: 25 or a variant
thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity
CROSSWIRE SIGNAL
The present invention provides a fusion protein which comprises: an SH2 domain from an ITAM-
binding protein; and a phosphatase domain.
The present invention also provide a fusion protein which comprises: an SH2 domain from an
ITIM-binding protein; and a kinase domain.
These “crosswire” signalling molecules will reverse an excitatory or inhibitory signal inside an
immune cell such as a T cell. When a T-cell receives an excitatory signal, for example following
recognition of a target antigen by a CAR, or MHC:peptide by a TCR, the presence of the first type
of crosswire molecule will result in the cell interpreting the excitatory signal as an inhibitory signal.
Dampening down or revising T-cell activation may be useful in a variety of situations, for example,
it may be used for CAR-expressing T cells where there is a high level of expression of the target
antigen on the target cell. It may be used to prevent T-cell over-activation which may lead to T
cell exhaustion and/or activation-induced cell death. Preventing a T-cell becoming activated too
much or too quickly may also prevent or reduce pathological side effects of CAR-T cell treatment
such as cytokine release syndrome (CRS).
The reverse situation is when a T-cell receives an inhibitory signal, for example following ligation
of PD1, and the presence of the second type of crosswire molecule results in the cell interpreting
the inhibitory signal as an excitatory signal.
P106294PCT 32
Reducing or reversing T-cell inhibition will help the cell overcome the inhibitory stimuli within the
hostile tumour microenvironment and should therefore increase T-cell persistence and expansion
in vivo.
In one embodiment, the fusion protein comprises a PTPN6 SH2 domain and a ZAP70 kinase
domain. In another embodiment the present fusion protein comprises a ZAP70 SH2 domain fused
to a PTPN6 kinase domain.
In embodiments relating to a ZAP70 SH2 domain fused to the phosphatase domain from PTPN6,
when the T cell receives an excitatory signal it interprets it as an inhibitory signal because the
PTPN6 phosphatase domain is recruited to the activated ITAM via the ZAP70 SH2 domain.
In embodiments relating to a PTPN6 SH2 domain fused to the kinase domain from ZAP70, when
the T cell receives an inhibitory signal it interprets it as an excitatory signal because the ZAP70
kinase domain is recruited to the activated ITIM via the PTPN6 domain. A fusion between PTPN6
SH2 domain and ZAP70 kinase domain will result in competition for phosphorylated ITIMs by
wild-type PTPN6 blocking inhibitory signals, but in addition will transmit a paradoxical activation
signal. This may have application in over-coming checkpoint blockade signals in a tumour
microenvironment.
The sequence of human ZAP70 kinase, PTPN6 phosphatase and SHP-2 phosphatase domains
domains are shown as SEQ ID NO: 26, 27 and 28 respectively.
SEQ ID NO: 26 – ZAP70 kinase domain
DPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIM
HQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHR
DLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMW
EALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSL
ASKVEGPPGSTQKAEAACA
SEQ ID NO: 27 – PTPN6 phosphatase domain
FWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSDYINANYIKNQLLG
PDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCVPYWPEVGMQRAYGPYSVTNCG
EHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSFLDQINQRQESLPHAGPIIVHC
SAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKL
SEQ ID NO: 28 – SHP-2 phosphatase domain
WEEFETLQQQECKLLYSRKEGQRQENKNKNRYKNILPFDHTRVVLHDGDPNEPVSDYINANII
MPEFETKCNNSKPKKSYIATQGCLQNTVNDFWRMVFQENSRVIVMTTKEVERGKSKCVKYWPDEYALKE
YGVMRVRNVKESAAHDYTLRELKLSKVGQALLQGNTERTVWQYHFRTWPDHGVPSDPGGVLDFLEEVH
HKQESIMDAGPVVVHCSAGIGRTGTFIVIDILIDIIREKGVDCDIDVPKTIQMVRSQRSGMVQTEAQYRFIYM
P106294PCT 33
The ZAP70 kinase domain, PTPN6 phosphatase domain or SHP-2 phosphatase domain may be
or comprise the sequence shown as SEQ ID NO: 26, SEQ ID NO: 27 or SEQ ID NO: 28,
respectively; or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity which
retains the capacity to phosphorylate or dephosphorylate downstream proteins in the same
manner as the wild-type kinase/phosphatase domains.
Examples of fusion protein comprising a PTPN6 SH2 domain fused to a ZAP70 kinase domain;
a ZAP70 SH2 domain fused to a PTPN6 kinase domain; and a SHP-2 SH2 domain fused to a
ZAP70 kinase domain are shown as SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID No. 61,
respectively.
SEQ ID NO: 29 – PTPN6 SH2 domain fusion: ZAP70 kinase domain
MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFA
TLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLS
QPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLR
QPYYSGGGGSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADT
EEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMK
YLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRS
DVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVE
QRMRACYYSLASKVEGPPGSTQKAEAACA
SEQ ID NO: 30 – ZAP70 SH2 domain fusion: PTPN6 phosphatase domain
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
SGGGGSGGGGSGGGGSGGGGSFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGR
DSNIPGSDYINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNKCV
PYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDHGVPSEPGGVLSF
LDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDCDIDIQKTIQMVRAQRSGMVQTE
AQYKFIYVAIAQFIETTKKKL
SEQ ID NO. 61 – dual SH2 domains from SHP-2 fused to ZAP70 kinase domain
WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEKFATLA
ELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPG
DFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQP
LNTTRINPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYES
PYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKKQIDVAIKVLKQGTEKADTEEMMREA
QIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREEIPVSNVAELLHQVSMGMKYLEEKNF
VHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFRKFSSRSDVWSYGV
TMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRACY
The fusion protein may be or comprise the sequence shown as SEQ ID NO: 29, SEQ ID NO: 30
or SEQ ID No. 61 or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.
P106294PCT 34
HETEROLOGOUS SIGNALLING DOMAIN
The present fusion protein may comprise (i) a ZAP70, PTPN6 or SHP-2 SH2 domain; and (ii) a
heterologous signalling domain.
As used herein, the term “heterologous signalling domain” refers to a signalling domain which is
not present in the wild type ZAP70, PTPN6 or SHP-2 protein. As such, where the fusion protein
comprises a ZAP70 SH2 domain, it comprises a signalling domain which is not the ZAP70 kinase
domain. Alternatively, where the fusion protein comprises a PTPN6 SH2 domain, it comprises a
signalling domain which is not the PTPN6 phosphatase domain.
BYPASS SIGNAL
The heterologous signalling domain may be from a signalling molecule which is not usually
activated by an ITAM containing receptor. In other words, the heterologous signalling domain
may be from a signalling molecule which is not involved in the propagation of immunological signal
1 following the binding of antigen to the TCR. Immunological signal 1 is sufficient to trigger T-cell
killing of cognate target cells but does not fully activate the T-cell to proliferate and survive.
In one embodiment of this aspect of the invention, the present invention provides a fusion protein
which comprises (i) an SH2 domain from a protein which binds an ITAM; and (ii) a heterologous
signalling domain.
A fusion between, for example, ZAP70 and another signaling molecule not typically activated with
an ITAM containing receptor may act to bypass signal from one pathway into another. One
example is co-stimulation. A fusion between ZAP70 and the endodomain of CD28 may transmit
a CD28 co-stimulatory signal as well as an ITAM activatory signal. Similarly, a fusion between
ZAP70 and the endodomain of 41BB or OX40 may transmit a 41BB or OX40 co-stimulatory signal.
Other pathways may also be recruited, for instance a fusion between ZAP70 and AKT kinase
domain may result in transmission of an AKT signal upon ITAM phosphorylation. Other examples
might include Kinase domain from JAK. In this way, a T-cell may interpret a simple antigen
recognition signal as transmitting a co-stimulatory or even a cytokine type signal.
Such fusion proteins may be useful, for example, in approaches where repeated ex vivo
stimulations of T cells can result in populations which lack costimulatory surface antigens and
which have limited proliferative capacity in vivo resulting in limited persistence and efficacy. The
loss of costimulatory surface antigens leading to activation of T cells solely through the TCR has
P106294PCT 35
been linked to a greater degree of activation induced cell death which would negatively impact in
vivo efficacy and persistence. The effect can be reversed by the activation of surface-expressed
4-1BB and OX40 demonstrating that costimulation can prevent activation induced cell death and
can support greater expansion of tumour specific T cells.
In another embodiment of this aspect of the invention, the present invention provides a fusion
protein which comprises (i) an SH2 domain from a protein which binds an ITIM; and (ii) a
heterologous signalling domain.
For example, a PTPN6 SH2 domain or SHP-2 SH2 domain may be fused to a co-stimulatory
endodomain so a T-cell interprets an inhibitory signal as a co-stimulatory one.
The heterologous signalling domain may be from, for example, CD28, 41BB or OX40.
CD28 provides a potent co-stimulatory signal - namely immunological signal 2, which triggers T-
cell proliferation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2) proteins.
41BB (CD137) is a type 2 transmembrane glycoprotein belonging to the TNF superfamily,
expressed on activated T cells. Crosslinking of 41BB enhances T cell proliferation, IL-2 secretion
survival and cytolytic activity.
OX40 (CD134) is a secondary co-stimulatory molecule, expressed after 24 to 72 hours following
activation; its ligand, OX40L, is also not expressed on resting antigen presenting cells, but is
following their activation. Expression of OX40 is dependent on full activation of the T cell; without
CD28, expression of OX40 is delayed and of fourfold lower levels. Signalling through OX40 is
required for prolonged T cell survival following initial activation and proliferation.
The CD28, 41BB and OX40 signalling domains (endodomains) are shown as SEQ ID NO: 31, 32
and 33, respectively.
SEQ ID NO: 31 – CD28 endodomain
MRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS
SEQ ID NO: 32 – 41BB endodomain
MKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL
SEQ ID NO: 33 – OX40 endodomain
MRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI
P106294PCT 36
The heterologous signalling domain may be or comprise the sequence shown as SEQ ID NO: 31;
SEQ ID NO: 32 or SEQ ID NO: 33, respectively, or a variant thereof having at least 80, 85, 90,
95, 98 or 99% sequence identity.
The heterologous signalling domain may be or comprise an inhibitory signalling domain.
For example, the inhibitory signalling domain may comprise the endodomain of CD148 or CD45.
CD148 and CD45 have been shown to act naturally on the phosphorylated tyrosines up-stream
of TCR signalling.
CD148 is a receptor-like protein tyrosine phosphatase which negatively regulates TCR signaling
by interfering with the phosphorylation and function of PLCγ1 and LAT.
CD45 present on all hematopoetic cells, is a protein tyrosine phosphatase which is capable of
regulating signal transduction and functional responses, again by phosphorylating PLC γ1.
An inhibitory signalling domain may comprise all of part of a receptor-like tyrosine phosphatase.
The phospatase may interfere with the phosphorylation and/or function of elements involved in T-
cell signalling, such as PLCγ1 and/or LAT.
The inhibitory signalling domain may be or comprise the endodomain of ICOS, CD27, BTLA,
CD30, GITR or HVEM.
The inhibitory signalling domain may comprise the sequence shown as SEQ ID NO: 34 to 39 or
a variant thereof having at least 80% sequence identity.
SEQ ID NO: 34 - ICOS endodomain
CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL
SEQ ID NO: 35 - CD27 endodomain
QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP
SEQ ID NO: 36 - BTLA endodomain
RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCL
EENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS
SEQ ID NO: 37 - CD30 endodomain
HRRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLRSGASVTEPVAEERGLMSQPLMETCHSVGA
AYLESLPLQDASPAGGPSSPRDLPEPRVSTEHTNNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPE
LEEELEADHTPHYPEQETEPPLGSCSDVMLSVEEEGKEDPLPTAASGK
P106294PCT 37
SEQ ID NO: 38 - GITR endodomain
QLGLHIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLGDLWV
SEQ ID NO: 39 - HVEM endodomain
CVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH
A variant sequence may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to
SEQ ID NO: 34 to 39 provided that the sequence provides an effective intracellular signalling
domain.
Suitably, the fusion protein may be or comprise any of the sequences shown as SEQ ID NOs: 40
to 45.
(SEQ ID NO: 40) - CD28 endodomain fused to amino-terminus of full-length ZAP
MRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSGGGGSGGGGSGGGGSGGGGSMPDPAAH
LPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAH
CGPAELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAP
QVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDK
AGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTL
NSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVY
RMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLV
GKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARS
AGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPE
LYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA
(SEQ ID NO: 41) - 41BB endodomain fused to amino-terminus of full-length ZAP
MKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSGGGGSGGGGSGGGGSGGGGSMPDPAA
HLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKA
PQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQD
KAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDT
LNSDGYTPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGV
YRMRKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFL
VGKREEIPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTAR
SAGKWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPP
ELYALMSDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA
(SEQ ID NO: 42) - OX40 endodomain fused to amino-terminus of full-length ZAP
MRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKISGGGGSGGGGSGGGGSGGGGSMPDPAAHLPFFY
GSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGPAE
LCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVEKL
IATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYHYLISQDKAGKYC
IPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGY
TPEPARITSPDKPRPMPMDTSVYESPYSDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRMRKK
QIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYIVRLIGVCQAEALMLVMEMAGGGPLHKFLVGKREE
IPVSNVAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAGKWP
LKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEVMAFIEQGKRMECPPECPPELYALM
SDCWIYKWEDRPDFLTVEQRMRACYYSLASKVEGPPGSTQKAEAACA
P106294PCT 38
(SEQ ID NO: 43) - CD28 endodomain fused to the amino-terminus of PTPN6 SH2 domain.
MRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSSGGGGSGGGGSGGGGSGGGGSMVRWFHR
DLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVE
YYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVL
SVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPY
(SEQ ID NO: 44) - 41BB endodomain fused to the amino-terminus of PTPN6 SH2 domain
MKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSGGGGSGGGGSGGGGSGGGGSMVRWFH
RDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELV
EYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFV
LSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPY
(SEQ ID NO: 45) - OX40 endodomain fused to the amino-terminus of PTPN6 SH2 domain
MRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKISGGGGSGGGGSGGGGSGGGGSMVRWFHRDLSGL
DAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFATLTELVEYYTQQ
QGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLSQPGDFVLSVLSD
QPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLRQPY
Suitably, the fusion protein may comprise the sequence shown as any of SEQ ID NOs: 40 to 45
or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity
KINASE DOMAIN
The heterologous signalling domain may be a kinase domain. For example, the heterologous
signalling domain may comprise an AKT kinase domain or a JAK kinase domain.
Akt, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase that plays
a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation,
transcription and cell migration.
Following activation of the TCR, T cells secrete IL2 which supports survival and proliferation.
However this secretion is transient and T cells that are activated and expanded in vitro become
dependent on exogenous IL2 for survival. By increasing AKT phosphorylation following ITAM
phosphorylation associated with TCR or CAR activation, the dependence of activated T cells on
exogenous IL2 may be reduced or removed and their proliferation and survival enhanced.
The Akt kinase domain is shown as SEQ ID NO: 46.
SEQ ID NO: 46 – Akt kinase domain
AEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENR
VLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNV
P106294PCT 39
VYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAG
IVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA
The heterologous signalling domain may be or comprise the sequence shown as SEQ ID NO: 46,
or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity provided that the
sequence provides an effective kinase domain.
By way of example, the fusion protein may be or comprise the any of the sequences shown as
SEQ ID NO: 47 to 49 and 62 which contain a ZAP70-SH2 domain fused directly to an Akt kinase
domain, a ZAP70-SH2 domain fused to an Akt kinase domain via a linker a ZAP70 mutated to
be non-functional and fused to an Akt kinase domain; and a dual SHP-2 SH2 domain fused to an
Akt kinase domain, respectively.
SEQ ID NO: 47 - ZAP70-SH2 domain fused directly to an Akt kinase domain
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
AEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENR
VLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNV
VYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAG
IVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA
SEQ ID NO: 48 - ZAP70-SH2 domain fused to an Akt kinase domain via a linker
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
SGGGGSGGGGSGGGGSGGGGSAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMK
ILKKEVIVAKDEVAHTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDR
ARFYGAEIVSALDYLHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPE
VLEDNDYGRAVDWWGLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPK
QRLGGGSEDAKEIMQHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSME
CVDSERRPHFPQFSYSASGTA
SEQ ID NO: 49 - ZAP70 mutated to be non-functional and fused to an Akt kinase domain
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLKPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
AEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVAHTLTENR
VLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDYLHSEKNV
VYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWWGLGVVMY
EMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIMQHRFFAG
IVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFSYSASGTA
SEQ ID No. 62 - dual SHP-2 SH2 domain fused to an Akt kinase domain
P106294PCT 40
WFHPNITGVEAENLLLTRGVDGSFLARPSKSNPGDFTLSVRRNGAVTHIKIQNTGDYYDLYGGEKFATLA
ELVQYYMEHHGQLKEKNGDVIELKYPLNCADPTSERWFHGHLSGKEAEKLLTEKGKHGSFLVRESQSHPG
DFVLSVRTGDDKGESNDGKSKVTHVMIRCQELKYDVGGGERFDSLTDLVEHYKKNPMVETLGTVLQLKQP
LNTTRINAEEMEVSLAKPKHRVTMNEFEYLKLLGKGTFGKVILVKEKATGRYYAMKILKKEVIVAKDEVA
HTLTENRVLQNSRHPFLTALKYSFQTHDRLCFVMEYANGGELFFHLSRERVFSEDRARFYGAEIVSALDY
LHSEKNVVYRDLKLENLMLDKDGHIKITDFGLCKEGIKDGATMKTFCGTPEYLAPEVLEDNDYGRAVDWW
GLGVVMYEMMCGRLPFYNQDHEKLFELILMEEIRFPRTLGPEAKSLLSGLLKKDPKQRLGGGSEDAKEIM
QHRFFAGIVWQHVYEKKLSPPFKPQVTSETDTRYFDEEFTAQMITITPPDQDDSMECVDSERRPHFPQFS
YSASGTA
The fusion protein may comprise the sequence shown as any of SEQ ID NO: 47 to 49 or 62 or a
variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.
Janus kinase (JAK) is a family of intracellular, nonreceptor tyrosine kinases that transduce
cytokine-mediated signals via the JAK-STAT pathway. The four JAK family members are: Janus
kinase 1 (JAK1); Janus kinase 2 (JAK2); Janus kinase 3 (JAK3); and Tyrosine kinase 2 (TYK2).
SEQ ID NO: 50 – Kinase containing domain of JAK2
RNEDLIFNESLGQGTFTKIFKGVRREVGDYGQLHETEVLLKVLDKAHRNYSESFFEAASMMSKLSHKHLV
LNYGVCVCGDENILVQEFVKFGSLDTYLKKNKNCINILWKLEVAKQLAWAMHFLEENTLIHGNVCAKNIL
LIREEDRKTGNPPFIKLSDPGISITVLPKDILQERIPWVPPECIENPKNLNLATDKWSFGTTLWEICSGG
DKPLSALDSQRKLQFYEDRHQLPAPKWAELANLINNCMDYEPDFRPSFRAIIRDLNSLFTPDYELLTEND
MLPNMRIGALGFSGAFEDRDPTQFEERHLKFLQQLGKGNFGSVEMCRYDPLQDNTGEVVAVKKLQHSTEE
HLRDFEREIEILKSLQHDNIVKYKGVCYSAGRRNLKLIMEYLPYGSLRDYLQKHKERIDHIKLLQYTSQI
CKGMEYLGTKRYIHRDLATRNILVENENRVKIGDFGLTKVLPQDKEYYKVKEPGESPIFWYAPESLTESK
FSVASDVWSFGVVLYELFTYIEKSKSPPAEFMRMIGNDKQGQMIVFHLIELLKNNGRLPRPDGCPDEIYM
IMTECWNNNVNQRPSFRDLALRVDQIRDNM
PROTEASE DOMAIN
The present invention also provides a fusion protein which comprises (i) an SH2 domain from a
protein which binds an ITAM or ITIM-containing protein and (ii) a protease domain.
The protease domain may be any protease which is capable of cleaving at a specific recognition
sequence. As such the protease domain may be any protease which enables the separation of
a single target polypeptide into two distinct polypeptides via cleavage at a specific target
sequence.
The protease domain may be a Tobacco Etch Virus (TeV) protease domain.
TeV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like
proteases. It is very specific for its target cleavage site and is therefore frequently used for the
P106294PCT 41
controlled cleavage of fusion proteins both in vitro and in vivo. The consensus TeV cleavage site
is ENLYFQ\S (where ‘\’ denotes the cleaved peptide bond). Mammalian cells, such as human
cells, do not express endogenous TeV protease.
Accordingly, the TeV cleavage recognition site is shown as SEQ ID NO: 51.
SEQ ID NO: 51 – Tev cleavage site
ENLYFQS
The TeV protease domain is shown as SEQ ID NO: 52.
SEQ ID NO: 52
SLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHGVFKVKNTTTL
QQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTFPSSDGIFWKH
WIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGWRLNADSVLWG
GHKVFMSKPEEPFQPVKEATQLMNELVYSQ
Accordingly, the protease domain may be or comprise the sequence shown as SEQ ID NO: 52,
or a variant thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity provided that the
sequence provides an effective protease function.
By way of example, the fusion protein may be or comprise the sequence shown as SEQ ID NO:
53 or 54, which contains a ZAP70-SH2 domain fused to a TEV protease sequence or a PTPN6-
SH2 domain fused to a TEV protease sequence; respectively.
SEQ ID NO: 53
MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCLRSLGGYVLSLVHDVRFHHFPIERQLNGTYA
IAGGKA
AIISQAPQVEKLIATTAHERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYALSLIYGKTVYH
YLISQDKAGKYCIPEGTKFDTLWQLVEYLKLKADGLIYCLKEACPNSSASNASGAAAPTLPAHPSTLTHP
SGGGGSGGGGSGGGGSGGGGSSLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRN
NGTLLVQSLHGVFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSM
SSMVSDTSCTFPSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLT
NQEAQQWVSGWRLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQ
SEQ ID NO: 54
MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDFYDLYGGEKFA
TLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQAETLLQAKGEPWTFLVRESLS
QPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGGLETFDSLTDLVEHFKKTGIEEASGAFVYLR
QPYYSGGGGSSLFKGPRDYNPISSTICHLTNESDGHTTSLYGIGFGPFIITNKHLFRRNNGTLLVQSLHG
VFKVKNTTTLQQHLIDGRDMIIIRMPKDFPPFPQKLKFREPQREERICLVTTNFQTKSMSSMVSDTSCTF
PSSDGIFWKHWIQTKDGQCGSPLVSTRDGFIVGIHSASNFTNTNNYFTSVPKNFMELLTNQEAQQWVSGW
RLNADSVLWGGHKVFMSKPEEPFQPVKEATQLMNELVYSQ
P106294PCT 42
The fusion protein may comprise the sequence shown as SEQ ID NO: 53 or 54; or a variant
thereof having at least 80, 85, 90, 95, 98 or 99% sequence identity.
The SH2 domain and heterologous domain of the fusion protein may be separated by a linker in
order to spatially separate the SH2 domain and the heterologous domain.
TRANSCRIPTIONAL SIGNAL
A fusion protein which comprises a protease, as described in the previous section, may be co-
expressed in a cell with a membrane-tethered protein having a protease cleavage site. Cleavage
of the membrane-tethered protein at the protease site will release the membrane-distal part of the
protein.
The membrane tethered protein may, for example, be a membrane-tethered transcription factor.
When cleavage occurs, the transcription is released from its tether and free to transit to the
nucleus.
A fusion between ZAP70 SH2 or PTPN6 SH2 domain and a protease domain will result in
membrane-proximal recruitment of the protease following ITAM or ITIM phosphorylation,
respectively.
Phosphorylation of ITAM or ITIM domains results in recruitment of the ZAP70 SH2 or PTPN6 SH2
fused with the protease domain, respectively, to the membrane-proximal area. This results in the
transcription factor being cleaved from its tether and transferred to the nucleus. This may have
many applications: for example upon activation the T-cell may be programmed to express
transcription factors which act to prevent the T-cell from differentiating. For instance, upon
activation the T-cell may be programmed to express a cytokine such as IL2, IL7 or IL15 which
may act to stimulate proliferation and survival of the T-cell, or IL12 which may convert a hostile
tumour microenvironment to one which more favours immune rejection of a tumour.
In particular, there is provided a cell which co-expresses:
(i) a fusion protein comprising an SH2 domain from a protein which binds a phosphorylated
ITAM; and
(ii) a membrane tethered transcription factor
wherein the transcription factor, when released from the membrane tether, increases the
expression of IL2, Il7 and/or IL15 in the cell.
P106294PCT 43
There is also provided a cell which co-expresses:
(i) a fusion protein comprising an SH2 domain from a protein which binds a phosphorylated ITIM;
(ii) a membrane tethered transcription factor
wherein the transcription factor, when released from the membrane tether, increases the
expression of IL12 in the cell.
Protease recognition site
The protease recognition site may be any amino acid sequence which enables the protease
domain of the fusion protein to specifically cleave the membrane tethered transcription factor
between the membrane tether and the transcription factor. For example, in one embodiment the
protease domain is a TeV protease domain and the protease recognition site is a TeV protease
recognition site.
Membrane tether
The membrane tether may be any sequence, signal or domain which is capable of localising the
transcription factor and protease recognition site proximal to a membrane. For example, the
membrane tether may be a myrsitylation signal or a transmembrane domain.
Suitably, a transmembrane domain may be any protein structure which is thermodynamically
stable in a membrane. This is typically an alpha helix comprising of several hydrophobic residues.
The transmembrane domain of any transmembrane protein can be used to supply the
transmembrane portion. The presence and span of a transmembrane domain of a protein can
be determined by those skilled in the art using the TMHMM algorithm
(http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of
a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a
hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM
domain may also be used (US 7052906 B1 describes synthetic transmembrane components).
The transmembrane domain may be derived from CD28, which gives good stability.
Transcription factor
P106294PCT 44
The transcription factor may be any transcription factor chosen to stimulate a desired response
following phosphorylation of the relevant ITAM or ITIM motifs.
The transcripton factor can be natural or artificial. Artificial transcription factors may be derived
from, for example, TALENs, zinc-finger assemblies or CrispR/CAS9, the latter co-expressed with
a guide mRNA.
Preferably, the transcription factor will contain a nuclear localization signal to aid its transportation
to the nuclease following cleavage by the protease domain.
By way of example, nucleic acid sequence (ii) (which encodes a protein comprising a membrane
tethered transcription factor which comprises: (i) a membrane tether; (ii) a protease recognition
site; and (iii) a transcription factor) may encode a protein which consists of or comprises the
sequence shown as SEQ ID NO: 55, which contains a RQR8 domain; a CD4-Endotox1
transmembrane domain, a TEV protease recognition site and a VP16-GAL4 transcription factor.
SEQ ID NO: 55
MGTSLLCWMALCLLGADHADACPYSNPSLCSGGGGSELPTQGTFSNVSTNVSPAKPTTTACPYSNPSLCS
GGGGSPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDMALIVLGGVAGLLLFIGLGIFFC
VRCRHRRRQAERMAQIKRVVSEKKTAQAPHRFQKTCSPISGGGGSENLYFQMPKKKRKVAPPTDVSLGDE
LHLDGEDVAMAHADALDDFDLDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGGSGG
GSMQILVASDATMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTKRSPLTRAHLTEVE
SRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVETDMPLTLRQHRIS
ATSSSEESSNKGQRQLTV
Suitably, the protein may comprise the sequence shown as SEQ ID NO: 55; or a variant thereof
having at least 80, 85, 90, 95, 98 or 99% sequence identity
RECEPTOR
The present invention further provides a nucleic acid construct which comprises (a) a nucleic acid
sequence encoding a fusion protein according to the first aspect of the present invention which
comprises a PTPN6 SH2 domain, or a truncated protein according to the third aspect of the
present invention; and (b) a nucleic acid sequence encoding a receptor comprising an ITIM
containing endodomain.
CASTRATION SIGNAL
P106294PCT 45
A fusion protein which comprises a protease, as described above, may be co-expressed in a cell
with a target receptor which comprises an intracellular protease cleavage site. Cleavage of the
target receptor at the protease site will release an intracellular, membrane-distal part of the target
receptor.
The target receptor may, for example, be a T-cell receptor (TCR), or a chimeric antigen receptor
(CAR).
The receptor may comprise an activatory or co-stimulatory endodomain positioned at the end of
the intracellular part of the protein. Cleavage at the protease cleavage site then removes the
activatory or co-stimulatory endodomain from the target CAR, reducing or preventing target
receptor-mediated T cell activation.
Alternatively, the target receptor may comprise an inhibitory endodomain positioned at the end of
the intracellular part of the protein. Cleavage at the protease cleavage site then removes the
inhibitory endodomain from the target CAR, “switching-on” the potential for target receptor
mediated T cell activation.
The inhibitory endodomain may, for example, comprise a CD148 or CD45 endodomain or an
ITIM-containing endodomain from a protein such a PD1, PDCD1, BTLA4, LILRB1, LAIR1, CTLA4,
KIR2DL1, KIR2DL4, KIR2DL5, KIR3DL1 or KIR3DL3.
By way of example, the target receptor may comprise the sequence shown as SEQ ID NO: 56,
which contains a CAR against CD33 containing an ITIM endodomain from PD-1.
SEQ ID NO: 56
MAVPTQVLGLLLLWLTDARCDIQMTQSPSSLSASVGDRVTITCRASEDIYFNLVWYQQKPGKAPKLLIYD
TNRLADGVPSRFSGSGSGTQYTLTISSLQPEDFATYYCQHYKNYPLTFGQGTKLEIKRSGGGGSGGGGSG
GGGSGGGGSRSEVQLVESGGGLVQPGGSLRLSCAASGFTLSNYGMHWIRQAPGKGLEWVSSISLNGGSTY
YRDSVKGRFTISRDNAKSTLYLQMNSLRAEDTAVYYCAAQDAYTGGYFDYWGQGTLVTVSSMDPATTTKP
VLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIYVGVVGGLLGSLVLLVWVLAVICSRAARGTI
GARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADG
PRSAQPLRPEDGHCSWPL
Suitably, the protein may comprise the sequence shown as SEQ ID NO: 56; or a variant thereof
having at least 80, 85, 90, 95, 98 or 99% sequence identity.
Where the receptor comprises a protease cleavage site between a transmembrane domain and
an activating endodomain, the castration signal fusion protein may be used to inhibit the receptor.
For instance, a first CAR might be constructed whereby its endodomain is separated from the
P106294PCT 46
transmembrane domain by a protease cleavage site. A second CAR recognizing a different
antigen might comprise of an ITIM containing endodomain. Recognition of the cognate antigen of
the second receptor would result in recruitment of the castration signal fusion protein to the
membrane and subsequent cleavage at the protease recognition site. Such cleavage would
separate the activating endodomain from the first receptor and prevent activation and signal
propagation from said receptor.
This would result in an "AND NOT" type logic gate where a sustained signal would be transmitted
only if the first CAR was activated in isolation (i.e. when the first CAR bound its cognate antigen
but the second CAR did not bind its cognate antigen). Such ‘logic gates’ may be useful, for
example, because it is relatively rare for the presence (or absence) of a single antigen to
effectively describe a cancer, which can lead to a lack of specificity. Targeting antigen expression
on normal cells leads to on-target, off-tumour toxicity. In some cancers, a tumour is best defined
by presence of one antigen (typically a tissue-specific antigen) and the absence of another
antigen which is present on normal cells. For example, acute myeloid leukaemia (AML) cells
express CD33. Normal stem cells express CD33 but also express CD34, while AML cells are
typically CD34 negative. Targeting CD33 alone to treat AML is associated with significant toxicity
as it depletes normal stem cells. However, specifically targeting cells which are CD33 positive
but not CD34 positive would avoid this considerable off-target toxicity.
Potential pairs of antigens for such an ‘AND NOT’ gate are shown in Table 2.
TABLE 2
Disease TAA Normal cell which Antigen expressed by normal
expresses TAA cell but not cancer cell
AML CD33 stem cells CD34
Myeloma BCMA Dendritic cells CD1c
B-CLL CD160 Natural Killer cells CD56
Prostate PSMA Neural Tissue NCAM
cancer
Bowel cancer A33 Normal bowel HLA class I
epithelium
By way of example, the receptor which comprises a protease cleavage site between a
transmembrane domain and an activating endodomain may be the sequence shown as SEQ ID
NO: 57, which contains a CAR against CD19 with a cleavable CD3-zeta endodomain.
P106294PCT 47
SEQ ID NO: 57
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY
HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGS
GGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYN
SALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRP
PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRCRHR
RRQAERMAQIKRVVSEKKTAQAPHRFQKTCSPISGGGGSENLYFQMRRVKFSRSADAPAYQQGQNQLYNE
LNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGL
STATKDTYDALHMQALPPR
Suitably, the receptor may comprise the sequence shown as SEQ ID NO: 57; or a variant thereof
having at least 80, 85, 90, 95, 98 or 99% sequence identity.
Where the receptor comprises an activating endodomain fused to an inhibitory endodomain via a
protease cleavage site, a castration signal fusion protein can be used to activate artificial
signalling domains. For instance, a first CAR might be constructed whereby its endodomain
comprises an activating endodomain fused to an inhibitory endodomain via a protease cleavage
site. A second CAR recognizing a different antigen might comprise of an ITIM containing
endodomain. Recognition of the cognate antigen of the second receptor would result in
recruitment of the castration signal fusion protein to the membrane and subsequent cleavage of
inhibitory endodomain from the activating endodomain of the first receptor. Cleavage, and thus
separation, of the inhibitory domain from the activating domain would allow activation of the first
CAR following antigen binding and hence activation of signalling via the first receptor.
This would result in an "AND" type CAR logic gate where productive signalling would occur only
if both the first and second receptors were activated. Such ‘logic gates’ are useful, for example,
because most cancers cannot be differentiated from normal tissues on the basis of a single antigen.
Hence, considerable “on-target off-tumour” toxicity occurs whereby normal tissues are damaged by
the therapy. For some cancers, targeting the presence of two cancer antigens may be more
selective and therefore effective than targeting one. For example, B-chronic lymphocytic leukaemia
(B-CLL) is a common leukaemia which is currently treated by targeting CD19. This treats the
lymphoma but also depletes the entire B-cell compartment such that the treatment has a
considerable toxic effect. B-CLL has an unusual phenotype in that CD5 and CD19 are co-
expressed. By targeting only cells which express CD5 and CD19, it would be possible to
considerably reduce on-target off-tumour toxicity.
Potential pairs of antigens for such an ‘AND’ logic gate are shown in Table 3.
Table 3
Cancer Type Antigens
P106294PCT 48
Chronic Lymphocytic Leukaemia CD5, CD19
Neuroblastoma ALK, GD2
Glioma EGFR, Vimentin
Multiple myeloma BCMA, CD138
Renal Cell Carcinoma Carbonic anhydrase IX, G250
T-ALL CD2, N-Cadherin
Prostate Cancer PSMA, hepsin (or others)
By way of example, the receptor which comprises an activating endodomain fused to an inhibitory
endodomain via a protease cleavage site may be the sequence shown as SEQ ID NO: 58, which
contains a CAR against CD19 with a CD3-zeta endodomain and a cleavable CD148 endodomain.
SEQ ID NO: 58
MSLPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIY
HTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITKAGGGGSGGGGS
GGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYN
SALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSDPTTTPAPRP
PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIFWVLVVVGGVLACYSLLVTVAFIIFWVRRVKF
SRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI
GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRENLYFQMAVFGCIFGALVIVTVGGFIFWRKKR
KDAKNNEVSFSQIKPKKSKLIRVENFEAYFKKQQADSNCGFAEEYEDLKLVGISQPKYAAELAENRGKNR
YNNVLPYDISRVKLSVQTHSTDDYINANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTK
CVEQGRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPLRQFHFTSWPDHGVPD
TTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGRTGTFIAIDRLIYQIENENTVDVYGIVYDLRMHRPL
MVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTAMTIYENLAPVTTFGKTNGYIA
Suitably, the receptor may comprise the sequence shown as SEQ ID NO: 58; or a variant thereof
having at least 80, 85, 90, 95, 98 or 99% sequence identity.
BLOCKING SIGNAL
The present invention provides a truncated protein which comprises an SH2 domain from a
protein which binds a phosphorylated immunoreceptor tyrosine-based activation motif (ITAM) but
lacks a kinase domain
For example, the truncated protein may comprise the ZAP70 SH2 domain but lack the ZAP70
kinase domain. In other words, the present invention provides a truncated protein which: (i)
comprises the sequence shown as SEQ ID NO: 2 but does not comprise the sequence shown as
SEQ ID NO: 26.
P106294PCT 49
Over-expression of the ZAP70 SH2 domain results in competition with full-length / wild-type
ZAP70. Since the truncated ZAP70 cannot propagate signals, signal transmission is reduced in
proportion to the ratio between wild-type ZAP70 and the truncated protein. This may be useful to
reduce strength of T-cell activation for instance to prevent T-cell over-activation which can result
in T-cell exhaustion, activation induced cell death and in a clinical setting can result in cytokine
storms.
The present invention also provide a truncated protein which comprises an SH2 domain from a
protein which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but
lacks a phosphatase domain
For example, the truncated protein may comprise the PTPN6 SH2 domain but lack the PTPN6
phosphatase domain. In other words, the present invention provides a truncated protein which:
(i) comprises the sequence shown as SEQ ID NO: 6 but does not comprise the sequence shown
as SEQ ID NO: 27.
In this case, ITIM signalling can be reduced in proportion to the ratio between wild-type PTPN6
and the truncated protein. This may be useful to reduce inhibitory signals such as PD1 signalling.
This may have application when T-cells are targeting a tumour which over-expresses PDL1 (or
similar inhibitory receptors) to evade immune rejection.
The use of a blocking signal or a cross-wire signal as described above, offers a significant
advantage over traditional immune checkpoint blockade approaches which typically block a single
ligand/receptor interaction, such as PD-L1/PD1, with an antibody. As explained above, the
inhibitory immune receptor class contains many members with redundancies and expression
patterns which fluctuate with T-cell state. The use of an antibody or a recombinant ligand/receptor
may effectively block one inhibitory receptor, but will not affect inhibitory signals transmitted from
the rest. Genomic editing of individual inhibitory receptors (Menger et al, Cancer Res. 2016 Apr
;76(8):2087-93) has a similar limitation. Strategies of fusions between individual inhibitory
receptors and co-stimulatory domains also suffer from similar limitations (Liu et al, Cancer Res.
2016 Mar 15;76(6):1578-90).
The method of the present invention will block (and depending on the strategy re-interpret)
inhibitory signals transmitted via an ITIM. Hence an entire class of inhibitory signals are modulated.
A list of inhibitory receptors which signal through ITIMs is provided in Table II of Odorizzi and
Wherry (2012) J. Immunol. 188:2957-2965. They include: PD1, BTLA, 2B4, CTLA-4, GP49B,
Lair-1, Pir-B, PECAM-1, CD22, Siglec 7, Siglec 9, KLRG1, ILT2, CD94-NKG2A and CD5.
P106294PCT 50
NUCLEIC ACID
In one aspect the present invention provides a nucleic acid which encodes a fusion protein or a
truncated protein according to the present 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 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.
NUCLEIC ACID CONSTRUCT
In one aspect the present invention provides a nucleic acid construct which co-expresses a
truncated protein or fusion protein of the present invention with another protein. The nucleic acid
construct may comprise: a nucleic acid sequence encoding a truncated protein or a fusion protein
of the present invention; and a nucleic acid encoding another protein.
P106294PCT 51
The present invention provides a nucleic acid construct which co-expresses a truncated protein
or fusion protein of the present invention with a chimeric antigen receptor. The nucleic acid
construct may comprise: (i) a nucleic acid sequence encoding a truncated protein or a fusion
protein of the present invention; and (ii) a nucleic acid encoding a chimeric antigen receptor.
The chimeric antigen receptor (CAR) may be an activatory CAR comprising an ITAM-containing
endodomain, such as CD3 zeta. The CAR may be an inhibitory CAR comprising a “ligation-off”
endodomain, as described in WO2015/075469 which may comprise all or part of the endodomain
from a receptor-like tyrosine phosphatase, such as CD148 or CD45. The CAR may be an
inhibitory CAR comprising a “ligation-on” endodomain, as described in WO2015/075470 which
may comprise an ITIM domain.
The fusion proteins and truncated proteins of the invention may be used together with a cell
expressing a “logic gate” combination of two or more CARs. An OR gate comprises two activatory
CARs as described in WO2015/075468. An AND gate comprises an activatory CAR and a
“ligation off” inhibitory CAR, as described in WO2015/075469. An AND not comprises an
activatory CAR and a “ligation on” inhibitory CAR, as described in WO2015/075470.
Thus the present invention provides a nucleic acid construct which comprises:
(i) a nucleic acid sequence encoding a truncated protein or fusion protein of the invention;
(ii) a first chimeric antigen receptor (CAR); and
(iii) a second chimeric antigen receptor.
With reference to the transcription signal aspect of the invention, there is provided a nucleic acid
construct which comprises (i) a nucleic acid sequence encoding a fusion protein comprising a
SH2 domain; and a protease domain; and (ii) a nucleic acid sequence encoding a membrane
tethered transcription factor which comprises: a membrane tether; a protease recognition site;
and a transcription factor.
With reference to the castration signal aspect of the invention, there is provided a nucleic acid
construct which comprises (i) a nucleic acid sequence encoding a fusion protein which comprises
an SH2 domain and a protease domain (e.g. a TeV domain); and (ii) a nucleic acid sequence
encoding a receptor which comprises a protease cleavage site.
For example, the present invention provides a nucleic acid construct which comprises: (a) a
nucleic acid sequence encoding a fusion protein which comprises (i) PTPN6 SH2 domain; and
(ii) a protease domain (e.g. a TeV domain); (b) a nucleic acid sequence encoding a receptor which
P106294PCT 52
comprises a protease cleavage site; and (c) a nucleic acid sequence encoding a receptor
comprising an ITIM containing endodomain.
The receptor may be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR).
Suitably, the protein encoded by nucleic acid sequence (b) may be a T-cell receptor (TCR) or a
chimeric antigen receptor (CAR) which comprises: (i) a protease cleavage site between a
transmembrane domain and an activating endodomain; or (ii) an activating endodomain fused to
an inhibitory endodomain via a protease cleavage site.
Where the nucleic acid construct of the invention, produces discrete polypeptides, such when it
coexpresses a fusion protein of the invention and a CAR, it may also comprise a nucleic acid
sequence enabling expression of both proteins. For example, it may comprise a sequence
encoding a cleavage site between the two nucleic acid sequences. The cleavage site may be
self-cleaving, such that when the nascent polypeptide is produced, it is immediately cleaved into
the two 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:
SEQ ID NO: 59
RAEGRGSLLTCGDVEENPGP
SEQ ID NO: 60
QCTNYALLKLAGDVESNPGP
The co-expressing sequence may be an internal ribosome entry sequence (IRES). The co-
expressing sequence may be an internal promoter.
CHIMERIC ANTIGEN RECEPTOR (CAR)
CARs, which are shown schematically in Figure 13, are chimeric type I trans-membrane proteins
which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling
domain (endodomain). The binder is typically a single-chain variable fragment (scFv) derived from
a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-
like antigen binding site. A spacer domain is usually necessary to isolate the binder from the
P106294PCT 53
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.
Early CAR designs had endodomains 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 endodomains
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 endodomains capable of transmitting activation, proliferation and survival
signals.
CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
Lentiviral vectors may be employed. In this way, a large number of cancer-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 tumour cells expressing the targeted antigen.
CARs typically therefore comprise: (i) an antigen-binding domain; (ii) a spacer; (iii) a
transmembrane domain; and (iii) an intracellular domain which comprises or associates with a
signalling domain.
ANTIGEN BINDING DOMAIN
The antigen binding domain is the portion of the CAR which recognizes antigen. Numerous
antigen-binding domains are known in the art, including those based on the antigen binding site
of an antibody, antibody mimetics, and T-cell receptors. For example, the antigen-binding domain
may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a
natural ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain
P106294PCT 54
antibody; an artificial single binder such as a Darpin (designed ankyrin repeat protein); or a single-
chain derived from a T-cell receptor.
The antigen binding domain may comprise a domain which is not based on the antigen binding
site of an antibody. For example the antigen binding domain may comprise a domain based on
a protein/peptide which is a soluble ligand for a tumour cell surface receptor (e.g. a soluble peptide
such as a cytokine or a chemokine); or an extracellular domain of a membrane anchored ligand
or a receptor for which the binding pair counterpart is expressed on the tumour cell.
The antigen binding domain may be based on a natural ligand of the antigen.
The antigen binding domain may comprise an affinity peptide from a combinatorial library or a de
novo designed affinity protein/peptide.
SPACER DOMAIN
CARs comprise a spacer sequence to connect the antigen-binding domain with the
transmembrane domain and spatially separate the antigen-binding domain from the endodomain.
A flexible spacer allows the antigen-binding domain to orient in different directions to facilitate
binding.
In aspects of the present invention which require two CARs, the first and second CARs may
comprise different spacer molecules. For example, the spacer sequence may, for example,
comprise an IgG1 Fc region, an IgG1 hinge or a human CD8 stalk or the mouse CD8 stalk. The
spacer may alternatively comprise an alternative linker sequence which has similar length and/or
domain spacing properties as an IgG1 Fc region, an IgG1 hinge or a CD8 stalk. A human IgG1
spacer may be altered to remove Fc binding motifs.
All the spacer domains mentioned above form homodimers. However the mechanism is not
limited to using homodimeric receptors and should work with monomeric receptors as long as the
spacer is sufficiently rigid. An example of such a spacer is CD2 or truncated CD22.
Since CARs are typically homodimers (see Figure 13a), cross-pairing may result in a
heterodimeric chimeric antigen receptor. This is undesirable for various reasons, for example:
(1) the epitope may not be at the same "level" on the target cell so that a cross-paired CAR may
only be able to bind to one antigen; (2) the VH and VL from the two different scFv could swap
over and either fail to recognize target or worse recognize an unexpected and unpredicted antigen.
P106294PCT 55
For the “AND” and “AND NOT” gates described above, the spacer of the first CAR may be
sufficiently different from the spacer of the second CAR in order to avoid cross-pairing but
sufficiently similar to co-localise. Pairs of orthologous spacer sequences may be employed.
Examples are murine and human CD8 stalks, or alternatively spacer domains which are
monomeric – for instance the ectodomain of CD2.
Examples of spacer pairs which co-localise are shown in the following Table:
Stimulatory CAR spacer Inhibitory CAR spacer
Human-CD8aSTK Mouse CD8aSTK
Human-CD28STK Mouse CD8aSTK
Human-IgG-Hinge Human-CD3z ectodomain
Human-CD8aSTK Mouse CD28STK
Human-CD28STK Mouse CD28STK
Human-IgG-Hinge-CH2CH3 Human-IgM-Hinge-CH2CH3CD4
TRANSMEMBRANE DOMAIN
The transmembrane domain is the sequence of the CAR that spans the membrane.
A transmembrane domain may be any protein structure which is thermodynamically stable in a
membrane. This is typically an alpha helix comprising of several hydrophobic residues. The
transmembrane domain of any transmembrane protein can be used to supply the transmembrane
portion of the invention. The presence and span of a transmembrane domain of a protein can be
determined by those skilled in the art using the TMHMM algorithm
(http://www.cbs.dtu.dk/services/TMHMM-2.0/). Further, given that the transmembrane domain of
a protein is a relatively simple structure, i.e a polypeptide sequence predicted to form a
hydrophobic alpha helix of sufficient length to span the membrane, an artificially designed TM
domain may also be used (US 7052906 B1 describes synthetic transmembrane components).
The transmembrane domain may be derived from CD28, which gives good receptor stability.
ACTIVATING ENDODOMAIN
The endodomain is the signal-transmission portion of the CAR. It may be part of or associate
with the intracellular domain of the CAR. After antigen recognition, receptors cluster, native CD45
and CD148 are excluded from the synapse and a signal is transmitted to the cell. The most
commonly used endodomain component is that of CD3-zeta which contains 3 ITAMs. This
transmits an activation signal to the T cell after antigen is bound. CD3-zeta may not provide a
P106294PCT 56
fully competent activation signal and additional co-stimulatory signaling may be needed. For
example, chimeric CD28 and OX40 can be used with CD3-Zeta to transmit a proliferative / survival
signal, or all three can be used together.
Where a CAR comprises an activating endodomain, it may comprise the CD3-Zeta endodomain
alone, the CD3-Zeta endodomain with that of either CD28 or OX40 or the CD28 endodomain and
OX40 and CD3-Zeta endodomain.
Any endodomain which contains an ITAM motif can act as an activation endodomain in this
invention. Suitable endodomains which contain an ITAM motif are described herein.
INHIBITORY DOMAIN
In embodiments referred to above as the “AND” gate, the first CAR may comprise an activating
endodomain fused to an inhibitory endodomain via a protease cleavage site. As such the
inhibitory endodomain inhibits T-cell activation by the first CAR in the absence of activation of the
second CAR. Upon activation of the second CAR, the ITIM in the endodomain of the second
CAR is phosphorylated and the PTPN6/protease domain fusion protein is recruited to the
membrane. This results in cleavage of the first CAR between the activating endodomain and
inhibitory endodomain, thus enabling T-cell activation.
The inhibitory endodomains may comprise any sequence which inhibits T-cell signalling by the
activating CAR when it is in the same endodomain.
The inhibitory endodomain may be or comprise a tyrosine phosphatase, such as a receptor-like
tyrosine phosphatase. An inhibitory endodomain may be or comprise any tyrosine phosphatase
that is capable of inhibiting the TCR signalling when co-localised with the activating endodomain
of the CAR. An inhibitory endodomain may be or comprise any tyrosine phosphatase with a
sufficiently fast catalytic rate for phosphorylated ITAMs that is capable of inhibiting the TCR
signalling when co-localised with the activating endodomain of the CAR.
VECTOR
The present invention also provides a vector, or kit of vectors which comprises one or more
nucleic acid sequence(s) or construct(s) according to the present invention. Such a vector may
be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it
expresses the proteins encoded by the nucleic acid sequence or construct.
P106294PCT 57
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.
CELL
The present invention also relates to an immune cell comprising the fusion protein, truncated
protein, nucleic acid and/or nucleic acid construct of the present invention.
The cell may be a cytolytic immune cell.
Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play
a central role in cell-mediated immunity. 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
P106294PCT 58
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.
The cells of the invention may be any of the cell types mentioned above.
T or NK cells expressing the molecules 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).
P106294PCT 59
Alternatively, T or NK cells expressing the molecules 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, cells are generated by introducing DNA or RNA coding for the receptor
component and signalling component by one of many means including transduction with a viral
vector, transfection with DNA or RNA.
The cell of the invention may be an ex vivo 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 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 invention.
The T or NK cells may then by purified, for example, selected on the basis of expression of the
antigen-binding domain of the antigen-binding polypeptide.
The present invention also provides a cell which comprises a fusion protein or a truncated protein
of the invention and a chimeric antigen receptor (CAR).
The chimeric antigen receptor (CAR) may be an activatory CAR comprising an ITAM-containing
endodomain, such as CD3 zeta. The CAR may be an inhibitory CAR comprising a “ligation-off”
endodomain, as described in WO2015/075469 which may comprise all or part of the endodomain
from a receptor-like tyrosine phosphatase, such as CD148 or CD45. The CAR may be an
inhibitory CAR comprising a “ligation-on” endodomain, as described in WO2015/075470 which
may comprise an ITIM domain.
The fusion proteins and truncated proteins of the invention may be used together with a cell
expressing a “logic gate” combination of two or more CARs. An OR gate comprises two activatory
CARs as described in WO2015/075468. An AND gate comprises an activatory CAR and a
P106294PCT 60
“ligation off” inhibitory CAR, as described in WO2015/075469. An AND not comprises an
activatory CAR and a “ligation on” inhibitory CAR, as described in WO2015/075470.
Thus the present invention provides a cell which comprises:
(i) a nucleic acid sequence encoding a truncated protein or fusion protein of the invention;
(ii) a first chimeric antigen receptor (CAR); and
(iii) a second chimeric antigen receptor.
With reference to the transcription signal aspect of the invention, there is provided a cell which
comprises (i) a fusion protein comprising an SH2 domain and a protease; and (ii) a membrane
tethered transcription factor which comprises: a membrane tether, a protease recognition site;
and a transcription factor.
With reference to the castration signal aspect of the invention there is provided a cell which
comprises (i) a fusion protein comprising an SH2 domain and a protease; and (ii) receptor which
comprises a protease cleavage site.
The receptor may, for example be a T-cell receptor (TCR) or a chimeric antigen receptor (CAR)
which comprises: (i) a protease cleavage site between a transmembrane domain and an
activating endodomain; or (ii) an activating endodomain fused to an inhibitory endodomain via a
protease cleavage site.
COMPOSITION
The present invention also relates to a pharmaceutical composition containing a plurality of cells
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.
METHOD OF TREATMENT
The cells of the present invention may be capable of killing target cells, such as cancer cells.
The cells of the present invention may be used for the treatment of an infection, such as a viral
infection.
P106294PCT 61
The cells of the invention may also be used for the control of pathogenic immune responses, for
example in autoimmune diseases, allergies and graft-vs-host rejection.
The cells of the invention may be used 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 cells of the invention may be used to treat: cancers of the oral cavity and pharynx which
includes cancer of the tongue, mouth and pharynx; cancers of the digestive system which includes
oesophageal, gastric and colorectal cancers; cancers of the liver and biliary tree which includes
hepatocellular carcinomas and cholangiocarcinomas; cancers of the respiratory system which
includes bronchogenic cancers and cancers of the larynx; cancers of bone and joints which
includes osteosarcoma; cancers of the skin which includes melanoma; breast cancer; cancers of
the genital tract which include uterine, ovarian and cervical cancer in women, prostate and
testicular cancer in men; cancers of the renal tract which include renal cell carcinoma and
transitional cell carcinomas of the utterers or bladder; brain cancers including gliomas,
glioblastoma multiforme and medullobastomas; cancers of the endocrine system including thyroid
cancer, adrenal carcinoma and cancers associated with multiple endocrine neoplasm syndromes;
lymphomas including Hodgkin's lymphoma and non-Hodgkin lymphoma; Multiple Myeloma and
plasmacytomas; leukaemias both acute and chronic, myeloid or lymphoid; and cancers of other
and unspecified sites including neuroblastoma.
Treatment with the cells of the invention may help prevent the escape or release of tumour cells
which often occurs with standard approaches.
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 – Subjugation of the T cell activation pathway to augmented or non-
physiological signals
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A number of SH2 domains involved in early T cell signal activation were tested to determine
whether T cell activation signals could be subjugated or “hijacked”, such that when a T cell was
activated the signal could be modulated or re-transmitted.
The inventors generated several chimeric AKT constructs by linking the kinase domain of AKT to
SH2 domains from Zap70, Grap, Grb2 and PLCγ (Figure 8).
In non-transduced (NT) T cells, very low levels of phosphorylation of the endogenous AKT were
detectable following treatment with OKT3 to induce cross-linking and activation of the TCR (Figure
10b:top panel). However, in cells expression the Zap-AKT construct, significant levels of
phospho-AKT were observed (Figure 10b:bottom panel).
Linker for activation of T cell (LAT) is a downstream target of ZAP70 and is bound by several
SH2-containing proteins, such as Grb2, Grap and PLCγ. It was anticipated that the SH2 domains
from each of these LAT-binders would also allow the activation signal from CD3-zeta to be
hijacked. However, this was not the case.
No TCR-dependent phosphorylation of the AKT kinase domain was observed above levels
observed for NT T cells when the AKT kinase domain was linked to the SH2 domains from Grb2,
Grap or PLCγ (Figure 9a).
This demonstrates that this system of T cell signalling hijacking specifically requires the tandem
SH2 domain from a very early T cell signalling molecule, such as Zap70 or Tyrosine-protein
phosphatase non-receptor type 6 (PTPN6).
Example 2 – Transcriptional Control
The TeV protease was fused to the Zap70 SH2 domain. A membrane-bound transcription factor
was also generated as follows: RQR8 was cloned in frame with the VP16/GAL4 transcription
factor separated by a TeV cleavage site. This fusion protein allows release of the VP16/GAL4
transcription factor (which contains a nuclear localizing signal) upon TeV cleavage.
These proteins were both expressed in a T-cell which also expressed a CD19-specific chimeric
antigen receptor. To demonstrate that the ZAP70-TeV approach is needed, the transcription
factor was co-expressed with a CD19 CAR whose endodomain was replaced by TeV (Figure 11).
P106294PCT 63
T-cells were exposed to CD19 negative and positive targets. Transcriptional activation was
measured by a Luciferase cassette responsive to GALv/VP16. Only the condition where a
standard CD19 CAR was co-expressed with ZAP-TeV and the membrane tethered transcription
factor resulted in selective transcriptional activation upon CD19 recognition. The CD19 CAR fused
directly to TeV resulted in constitutive transcriptional activation (Figure 12).
Example 3 - PD-1 signal blockade using truncated SHP-1 (PTPN6) or truncated SHP-2
PBMC cells were transduced as shown in the following table:
Name on Figure Description Construct(s)
key
NT Untransduced -
FMC63 Transduced with CD19 CAR only SFG.aCD19_fmc63-HCH2CH3w-
CD28tmZw
PD1 Transduced with PD1 only pDual-PD1-GFP
FMC63+PD1 Co-transduced with CD19CAR SFG.aCD19_fmc63-HCH2CH3w-
and PD1 CD28tmZw and pDual-PD1-GFP
FMC63- Co-transduced with a) bicistronic SFG.aCD19_fmc63-HCH2CH3w-
SHP1+PD1 construct encoding CD19CAR CD28tm-Zeta_w-2A-dualSH2_SHP-1
and truncated SHP1, and b) PD1 and pDual-PD1-GFP
FMC63- Co-transduced with a) bicistronic SFG.aCD19_fmc63-HCH2CH3w-
SHP2+PD1 construct encoding CD19CAR CD28tm-Zeta_w-2A-dualSH2_SHP-2
and truncated SHP1, and b) PD1 and pDual-PD1-GFP
The cells were co-cultured for 48 hours with SupT1 cells transduced with CD19, PDL1 or both
and IFNγ release measured by ELISA. The results are shown in Figure 15.
The presence of PDL1 on SupT1 target cells caused a reduction in IFNγ release. There was
increased IFNγ release with PBMC which expressed CAR together with the truncated SHP-1 or
truncated SHP-2 construct compared with those which expressed CAR alone. This indicates that
the truncated SHp-1 and SHP-2 constructs successfully inhibited the PDL1 inhibitory signal from
the target cells.
Example 4 – PD-1 signal hijack using a fusion of SHP-2 SH2 domains and Zap70 kinase
P106294PCT 64
PBMC cells were transduced as shown in the following table:
Name on Figure Description Construct(s)
16 key
NT Untransduced -
FMC63 Transduced with CD19 CAR only SFG.aCD19_fmc63-HCH2CH3w-
CD28tmZw
PD1 Transduced with PD1 only pDual-PD1-GFP
FMC63+PD1 Co-transduced with CD19CAR SFG.aCD19_fmc63-HCH2CH3w-
and PD1 CD28tmZw and pDual-PD1-GFP
FMC63- Co-transduced with a) bicistronic SFG.aCD19_fmc63-HCH2CH3w-
SHP2Zap70+PD1 construct encoding CD19CAR CD28tm-Zeta_w-2A-dualSH2_SHP-
and fusion of SHP2 SH2domains 2-Zap70_Kinase and pDual-PD1-GFP
and Zap70 kinase, and b) PD1
The cells were co-cultured in a 1:1 ratio for 24 hours with SupT1 cells transduced with CD19 or
PDL1. IFNγ release was measured by ELISA (Figure 16A). An increase in IFN-γ production in
was seen co-cultures of CAR-SHP2.Zap70 + PD1 transduced T cells with PDL1 SupT1 target
cells compared with CAR + PD1 transduced T cells.
A cytotoxicity assay was also conducted in which killing of SupT1 cells was quantified by FACS
(Figure 16B). Near complete killing of PDL1 SupT1 targets was observed in co-cultures of PDL1
positive target cells with CAR-SHP2.Zap70 + PD1 transduced T cells. By contrast, killing was not
seen with CAR + PD1 alone construct. This indicated that replacing the phosphatase domain of
SHP2 with the kinase domain of Zap70 successfully converted the inhibitory PD1 signal to an
activatory signal. The SHPZap70kinase fusion protein therefore successfully hijacked the
inhibitory PDL1-PD1 signal and turned it into a T-cell activation signal.
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, cellular immunology or related fields are
intended to be within the scope of the following claims
Claims (13)
1. A T cell which comprises a chimeric antigen receptor (CAR) and a truncated protein which comprises: (i) a PTPN6 SH2 domain which binds a phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) but lacks a PTPN6 phosphatase domain; or (ii) a SHP-2 SH2 domain which binds a phosphorylated ITIM but lacks a SHP-2 phosphatase domain.
2. A T cell according to claim 1, wherein the truncated protein comprises the sequence shown as SEQ ID No. 6 or one or both of the sequences shown as SEQ ID No. 7 and 8.
3. A T-cell according to claim 1, wherein the truncated protein comprises or consists of the sequence shown as SEQ ID No. 10, 11 or 12.
4. A nucleic acid construct which comprises a first nucleic acid sequence encoding a chimeric antigen receptor; and a second nucleic acid sequence encoding a truncated protein as defined in claim 1.
5. A vector which comprises a nucleic acid construct according to claim 4.
6. A vector according to claim 5, which is a retroviral or lentiviral vector.
7. A pharmaceutical composition comprising a plurality of T cells according claim 1.
8. Use of a pharmaceutical composition according to claim 7 in the manufacture of a medicament for the treatment and/or prevention of a disease.
9. A pharmaceutical composition according to claim 7, in which: (i) a cell containing sample isolated from a subject; (ii) the cells are transduced or transfected with a nucleic acid construct according to claim 4, or a vector according to claim 5 or 6; and (iii) the cells from (ii) are to be administered to the subject.
10. Use of a pharmaceutical composition according to claim 7 in the manufacture of a medicament for the treatment of cancer.
11. A pharmaceutical composition according to claim 9, wherein the subject has cancer.
12. A method for making a cell according to claim 1, which comprises the step of introducing: a nucleic acid construct according to claim 4, or a vector according to claim 5 or 6, into the cell ex vivo.
13. A method according to claim 12, wherein the cell is from a sample isolated from a subject.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB1509413.9A GB201509413D0 (en) | 2015-06-01 | 2015-06-01 | Fusion protein |
GB1509413.9 | 2015-06-01 | ||
PCT/GB2016/051576 WO2016193696A1 (en) | 2015-06-01 | 2016-05-31 | Cell |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ737662A NZ737662A (en) | 2021-10-29 |
NZ737662B2 true NZ737662B2 (en) | 2022-02-01 |
Family
ID=
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